Graduate Theses and Dissertations
Graduate College
2014
Effect of oregano essential oil and tannic acid on
storage stability and quality of ground chicken meat
Marwan Alhijazeen
Iowa State University
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Alhijazeen, Marwan, "Effect of oregano essential oil and tannic acid on storage stability and quality of ground chicken meat" (2014).
Graduate Theses and Dissertations. Paper 13966.
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Effect of oregano essential oil and tannic acid on storage stability and quality of
ground chicken meat
by
Marwan Alhijazeen
A dissertation submitted to the graduate faculty
In partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Meat Science
Program of Study Committee:
Ahn Dong UK, Major Professor
Sebranek Joseph G
Cordray Joseph C
Dickson James S
Mendonca Aubrey F
Iowa State University
Ames, Iowa
2014
Copyright © Marwan Alhijazeen, 2014. All rights reserved.
ii
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS…………………………………………………………. iv
ABSTARCT…………………………………………………………………………...v
CHAPTER 1: INTRODUCTION……………………………………………………..1
CHAPTER 2: LITERATURE REVIEW……………………………………………...6
References…………………………………………………………………………....31
CHAPTER 3: THE EFFECT OF OREGANO ESSENTIAL OIL (origanuim
vulgare subsp.hirtum) ON THE STORAGE STABILITY AND QUAILTY
CHARACTERISTICS OF GROUND CHICKEN MEAT…………………………...51
Abstract………………………………………………………………………………52
Introduction…………………………………………………………………………..53
Materials and Methods……………………………………………………………….55
Result and Discussion………………………………………………………………..59
Conclusion…………………………………………………………………………...64
References……………………………………………………………………………65
CHAPTER 4: THE EFFECT OF ADDING DIFFRENT LEVEL OF TANNIC
ACID ON THE STORAGE STABILITY AND QUALITY
CHARACTERISTICS OF GROUND CHICKEN MEAT…………………………...81
Abstract………………………………………………………………………………82
Introduction…………………………………………………………………………..83
Materials and Methods……………………………………………………………….85
Result and Discussion………………………………………………………………..89
Conclusion……………………………………………………………………….…..94
References……………………………………………………………………………95
CHAPTER 5: THE EFFECT OF OREGANO OIL AND TANNIC ACID
COMBINATIONS ON THE QUALITY AND SENSORY CHARACTRISTICS
OF GROUND CHICKEN MEAT……………………………………..……………109
Abstract……………………………………………………………………………..110
Introduction…………………………………………………………………………111
Materials and Methods……………………………………………………………...113
Result and Discussion………………………………………………………………119
Conclusion………………………………………………………………………….124
References…………………………………………………………………………..125
iii
CHAPTER 6: SURVIVAL AND GROWTH OF NATURAL BACTERIAL
FLORA AND HUMAN ENTERIC PATHOGENS IN GROUND CHICKEN
MEAT WITH ADDED OREGANO ESSENTIAL OIL OR TANNIC ACID,
ALONE OR IN COMBINATION…………………………………………………..147
Abstract……………………………………………………………………………..148
Introduction…………………………………………………………………………149
Materials and Methods……………………………………………………………...152
Result and Discussion………………………………………………………………156
Conclusion………………………………………………………………………….160
References…………………………………………………………………………..161
CHAPTER 7: SUMMARY AND CONCLUSIONS……………………………….174
iv
AKNOWLEDGMENTS
My deepest gratitude goes to my major professor Dr. Dong Ahn for his
continuous encouragement, guidance, warmth and support through out my PhD
program at Iowa State University. Completing this dissertation would have not been
possible without his patience, kindness and help.
I am truly grateful to my POS committee members, Dr. Joseph G. Sebranek, Dr.
Aubrey F. Mendonca, Dr. Joseph C. Cordray, and Dr. James S. Dickson, for their
commitment, advice, and help through out my PhD program.
In addition, I would also like to thank my friends, colleagues, the department
faculty and staff for making my time at Iowa State University a wonderful experience.
I want to also offer my appreciation to those who were willing to participate in my
research observations, without whom, this thesis would not have been possible.
My deepest appreciation goes to Dr. Aubrey Mendonca and his research group
at the Department of Food Science and Human Nutrition (Food safety laboratory),
without his great commitment and help, completing the work related to the microbial
part of this desertion would not have been possible.
My deepest gratitude goes to my wife Rania Ibrahim for her encouragement,
hours of patience, respect and love. Thanks to my family and my relatives for being
with me. I’m very much grateful to all my friends in my life for all the love, care, and
help given to me and for always being with me. Finally I would like to thank my
sponsor Mu’tah University (Jordan) for their help and support during my study.
Thank you so much!!
v
ABSTRACT
Lipid and protein oxidations are most important factors that can cause quality
deterioration in fresh and cooked poultry meat during storage because many
secondary compounds formed by lipid and protein oxidation can cause off-odor and
off-flavor
production
in
meat.
Several
synthetic
antioxidants
such
as
butylatedhydroxyanisole (BHA), butylatedhydroxytoluene (BHT), and tertiary
butylhydroquinone (TBHQ) have been successfully used to prevent the oxidative
changes in meat. In recent years, however, growing number of consumers are more
interested in natural antioxidants than synthetic ones because of the potential
carcinogenic effects of the latter.
Plant polyphenol extracts are known to have strong antioxidant and
antimicrobial characteristics and have been used as preservatives in meat. Oregano
essential oil and tannic acid are two examples of these polyphenol plant extracts that
have strong antioxidant and antimicrobial effects. The aims of this study were: 1) to
evaluate the antioxidant effect of adding oregano essential oil, tannic acid, and/or their
diffrent combinations to ground chicken meat, 2) to evaluate the antimicrobial effect
of oregano essential oil, tannic acid, and/or their combinations in ground chicken meat,
and 3) to evaluate the sensory characteristics of combined oregano oil and tannic acid
in ground chicken meat.
Ground chicken meat was prepared to study the effect of adding oregano oil
and tannic acid in both raw and cooked meat patties. Several oregano oil and tannic
acid concentrations were prepared to study their single and combination effects on
meat quality. For the raw meat study, samples were individually packaged in
oxygen-permeable bags and stored in a 4 °C cooler for up to 7 days. On the other hand
cooked meat samples were packaged in oxygen-impermeable vacuum bags and then
cooked in-bag reaching the internal temperature of 75 °C. After cooling to room
temperature, the cooked meat was transferred to new oxygen-permeable bags and
stored at 4 °C for up to 7 days. Chicken meat patties were analyzed for lipid and protein
vi
oxidation, color, and volatiles at 0, 3, and 7 days of storage. Parts of the samples
prepared were also used for microbial studies.
Oregano essential oil at level 400 ppm significantly reduced (p < 0.05) lipid
oxidation, protein oxidation and off-odor volatiles, but improved color stability of raw
ground meat. The effects of oregano oil on cooked meat were similar to that of the raw
meat but the effects were greater than the raw meat. Hexanal was the major aldehyde
and was decreased significantly (P < 0.05) by oregano oil treatment in cooked meat and
the differences among the treatments were clearer in cooked meat than in raw meat.
Overall, the addition of oregano essential oil at 100 - 300 ppm was a good way of
preserving meat and could replace the synthetic antioxidant, BHA.
Both raw and cooked chicken breast meat added with 10 ppm of tannic acid had
significantly (P < 0.05) lower lipid and protein oxidation than other treatments during
storage. In addition, tannic acid at 10 ppm level maintained the highest color a*- and
L*-values during storage. Cooked chicken breast meat added with > 5 ppm tannic acid
had significantly (p < 0.05) lower amounts of off-odor volatiles than control. Among
the volatile compounds, the amount of hexanal in cooked meat increased rapidly during
storage. Therefore, tannic acid at > 5 ppm could be used as a natural preservative in
ground chicken meat to improve its quality during storage.
The combination of 200 ppm oregano oil and 10 ppm tannic acid showed the
highest effect (P < 0.05) on TBARS, total carbonyl, and off odor volatile formation in
both breast and thigh meat. The combination of 200 ppm of oregano oil and 10 ppm of
tannic acid also showed the highest effect in stabilizing color values of both raw
breast and thigh meats. Sensory evaluation of thigh chicken meat also indicated that
200 ppm of oregano oil and 10 ppm of tannic acid combination treatment had positive
effects on most of the attributes evaluated. In conclusion, the combination of 200 ppm
oregano oil and 10 ppm tannic acid could be a good replacement for the synthetic
antioxidant in ground chicken meat.
Oregano oil, tannic acid, BHA treatments reduced the APC significantly (P <
0.05) when used separately. However, combination of oregano + tannic acid showed
vii
the strongest (P < 0.05) effect in reducing the APC in raw chicken breast meat during
storage at 4 and 10 oC. The combination treatment also showed the strongest
antimicrobial effects of all the treatments against Salmonella enterica, Listeria
monocytogenes, Escherichia coli, and Enterobacteriacea (ENT) in raw meat during
storage. In cooked chicken breast meat, oregano oil and oregano + tannic acid
combination were efficient (P < 0.05) in suppressing the growth of aerobic bacteria in
meat at all temperatures (10, 25, and 43 oC) tested. Oregano essential oil treatment
was more effective than the BHA and tannic acid treatments in slowing down the
growth and survival of Stapylococcus aureus in cooked meat during storage at 10, 25,
and 43 oC, but oregano oil + tannic acid treatment was more effective than the
oregano oil alone. Based on these results it is suggested that oregano oil has a strong
antimicrobial activity but its effect can be further improved when it is combined with
tannic acid. This study demonstrated that oregano oil + tannic acid combination could
be a good natural antimicrobial treatment to suppress most of the food borne
pathogens in chicken meat.
In conclusion oregano essential oil at > 200 ppm significantly improved meat
quality and prevented meat deterioration during storage. Tannic acid at 10 ppm also
improved meat quality and safety significantly during storage. However, the
combination of 200 ppm oregano essential oil and 10 ppm tannic acid showed higher
effect on both meat quality and safety of ground chicken meat than using them singly.
This combination could be an excellent replacement for synthetic antioxidants such as
BHA, BHT and TBHQ, which are commonly used for meat preservation.
1
CHAPTER 1
INTRODUCTION
Oxidation process in fresh and cooked meats can be controlled by using artificial
antioxidants such as butylatedhydroxyanisole (BHA), butylatedhydroxytoluene (BHT)
and tertiary butylhydroquinone (TBHQ) (Monahan and Troy, 1997), but these could
cause negative effects on human health. Therefore, many studies tested
naturally-occurring antioxidants such as ascorbic acid, vitamin E and certain crude
plant extracts as a replacements for synthetic antioxidants. Recently, researchers have
been more interested in natural than synthetic antioxidants because consumers and
industry prefer foods with natural antioxidants to synthetic ones (Kosar et al., 2005;
Monahan and Troy, 1997; Solomakos et al., 2008). During past two decades, many
researchers tested herbs and plant extracts like oregano, sage, rosemary, and grape
seed (Economou et al., 1991; Man and Jaswir, 2000; Yanishlieva and Marinova, 1995)
because they contain chemical compounds that can increase or enhance shelf-life of
foods, make the them more resistant to oxidation, slow down the microbial or fungal
growth, and show positive effect on their physiochemical properties (Shahdidi et al.,
1992 ; Brannan, 2008; Abdel-Hamied et al., 2009).
Lipid oxidation is one the most important problems that affect the quality and
shelf life of meat, especially in poultry meat. It produces many volatile compounds
such as aldehydes, which are responsible for off-odor (Ahn et al., 1998, Ahn and Lee,
2002), off-flavor (Dietze et al., 2007) and rancidity (Campo et al., 2006; Ahn et al.,
2002; Byrne et al., 2002), and change meat color (Guillen and Guzman, 1998).
Oxidation of lipids can occur in both fresh and cooked meats (Min and Ahn, 2005; Jo
et al., 2006), and can have significant impact to meat industry. Many factors are
influencing the development of lipid oxidation in meat, but the amount of fat,
packaging, metal catalysts and degree of polyunsaturation in the lipids of meat play
the most important roles (Gray and Pearson, 1987; Calkins and Hodgen, 2007)
2
Protein oxidation is also considered as an important factor that can cause
changes in meat quality. As in lipid oxidation, protein oxidation is induced by several
free radical species such as hydroxyl radicals, peroxyl and alkoxyl radicals as well as
superoxide anions, hydrogen peroxide, and nitric oxide (Lawrie, 1998; Ouali et al.,
2006; Hopkins and Geesink, 2009). The oxidation process in proteins leads to the
destruction of amino acids and forms carbonyl compounds, which will affect protein
functionality such as gel-forming ability, water-binding capacity, emulsifying capacity,
solubility, and viscosity (Howell et al., 2001). The mechanisms of protein oxidation
are very similar to lipid oxidation, but protein oxidation produces more complex
oxidation products than lipid oxidation: In protein oxidation, hydrogen atom
abstraction by ROS produces a protein carbon-centered radical (P·). In the presence of
oxygen this carbonyl radical will be converted to peroxyl radical (POO·), and then
converted to peroxide (POOH), alkoxyl radical (PO·) and its derivatives (POH) which
are the final primary protein oxidation products (Lund et al., 2011; Estevez, 2011).
Finding natural antioxidants to resolve the lipid and protein oxidation is of great
interest recently (Brewer, 2011). Oregano essential oil is one of the most popular herb
extracts that can be used as a meat preservative. Oregano is commonly used as a spice
in the Mediterranean cuisine, and is produced by drying leaves and flowers of
Origanum vulgare subsp. Hirtum plants. Oregano is well known for its antioxidant
activity (Economou et al., 1991) because of its high phenolic content. There are two
major phenols in oregano: carvacrol and thymol, which constitute about 78-82% of
the essential oils in oregano, and are responsible for most of the antioxidant activity of
oregano oil (Adam et al., 1998; Yanishlieva et al., 1999). Other phenolic compounds
such as γ-terpinene and p-cymene take about 5 to 7 % of the total oil, and they also
have strong antioxidant activities.
The essential oil of oregano is also known for its antimicrobial effects.
Sivropoulou et al. (1996) reported that the three species of oregano essential oils they
tested showed strong antimicrobial activities against eight different strains of
gram-positive and negative bacteria. Chouliara et al. (2007) studied the combined
3
effect of oregano essential oil on the shelf-life of fresh chicken breast meat in
modified atmosphere packaging and found that the microbial populations (lactic acid
bacteria, TVC, pseudomonas spp., yeast) and lipid oxidation significantly decreased
by adding oregano oil. They indicated that thymol and carvacrol damaged bacterial
cell membranes, destabilized the operation of phospholipids bilayer of bacteria,
entered into the bacterial cell, and changed cell processes (Kalemba and Kunicha,
2003; Cristani et al., 2007; Xu et al., 2008; Di Pasqua et al., 2010).
Another natural antioxidant found in most plant foods is tannic acid or tannins.
Tannic acid is widely used in the process of converting animal skins to leather. Tannic
acid is a water–soluble polyphenol found in a wide variety of plants in different
concentrations (Chung et al., 1998a). For example, food grains such as millets, barley,
peas, fababean, legumes, sorghum, and millets (Chaven et al., 1977; Hulse, 1980;
Price and Butler, 1980; Salunkhe et al., 1982; Deshpande et al., 1984), fruits such as
bananas, apples, grapes, and strawberries have a good quantity of tannins (Lease and
mitchell, 1940; Reeve, 1959; Sanderson et al., 1975; Jones and Mangan, 1977). Wines
and tea also have tannins (Lyoyd, 1911; Reeve., 1959; Hoff and Singleton, 1977).
Tannins are classified into two categories: hydrolysable and non-hydrolysable
(condensed) tannins (Freudenberg, 1920). The hydrolysable types contain polyhydric
alcohol and hydroxyl groups, which are connected to gallic acid such as gallotannins
or hexahydroxydiphenic acid (ellagitannins) by ester bonds. After hydrolysis by
enzymes or acid/base conditions, gallotannins produce glucose and gallic acid
(Hagerman, 2010; Salminen et al., 2011).
The FDA categorized tannic acid as a generally recognized as safe (GRAS) food
additive, which is allowed to use at 400 ppm in frozen dairy desserts and mixes, 50
ppm in non-alcoholic beverages and beverage bases, 150 ppm in alcoholic beverages,
100 ppm in backed goods and backing mixes, and 10 ppm in meat products (FDA,
2011).
The radical scavenging and antioxidant mechanisms of tannic acid are still not
fully understood and need more investigations (Gulcin et al., 2010). It is generally
4
known that the antioxidant activity of tannic acid is coming through the inhibition of
hydroxyl radical formation from the Fenton reaction by complexing with ferrous ions
(Lopes et al., 1999). However tannic acid may act as a pro-oxidant in the presence of
copper ions, causing DNA damages (Freguson, 2001). Tannic acid can cause DNA
degradation or DNA base modification in the presence of Cu(II) due to the generation
of reactive oxygen species (Baht and Hadi, 1994a, 1994b). Glucin et al. (2010)
reported that the structural features of tannic acid are responsible for the antioxidant
activity, but it also can contribute to the pro-oxidant activity of tannic acid. It can
produce hydroxyl radical in the presence of metal catalyst such as Cu(II), but it also
can chelate large number of free radicals (Khan et al., 2000). Tannic acid is used in
many foods as flavoring agent and has pharmacological applications in medicinal
products to treat burns, diarrhea and chemical antidotes in poisoning and astringents
(Hirono and Yamada, 1987). Furthermore, tannic acid is used in brewing and wine
industries as a clarifying agent, and flavoring agent in frozen dairy desserts, baked
foods and meat products (Chung et al., 1998a).
Tannins and their derivatives showed significant antimicrobial activity against
several foodborne pathogens (Chung et al., 1998b; Kim et al., 2010). The
antimicrobial mechanisms of tannins are as follow: 1) through the astringent effect of
tannins to many microbial enzymes such as cellulase, pectinase and xylanase; 2) their
toxicity on the cell membrane of microorganisms; and 3) their binding ability to
metals (metal chelator), which reduce the availability of essential metal ions such as
iron, which is important for the metabolic activities of microorganisms (Scalbert,
1991). However, the antimicrobial mechanisms of tannins still need more studies, and
suitable levels that could be used in food processing also should be determined.
Strong evidence indicated that tannic acid has anti-carcinogenic effects
(Korpassy, 1961; Morton, 1972; Pamukcu et al., 1984; Salunkhe et al., 1990). Green
tea, fruit, and vegetables, which contain high levels of tannins and some other
phenolic compounds, inhibited the growth of cancer cells in the experimental animals
such as rats (Stocks, 1970; Stich and Rosin, 1984; Das et al., 1989; Shi et al., 1994).
5
Over all it is very rare to find research studies on using tannic acid as meat
preservative in the recent publications especially in poultry meat.
Poultry meat is considered as one of the most popular food commodities around
the world, and their consumption increased dramatically over the last two decades in
many countries (Chouliara et al., 2007). Recently, shelf-life extension of poultry meat
has become one of the main concerns in the industry (Chouliara and Kontominas,
2006; Chouliara et al., 2007). Use of multiple factors or technology combinations
called “hurdle technology’’ to suppress microorganism in food is of great interest for
many researchers (Jay, 2000). However, no study has been conducted using the
combination of both oregano oil and tannic acid to improve the safety and quality of
poultry meat. The over all objective of this study is to determine the suitable levels of
these two natural plant extracts (oregano oil, tannic acid, and their combinations) as
the replacement of synthetic antioxidants to improve the quality as well as the safety
of poultry meat products.
The specific objectives are: 1) to evaluate the antioxidant effect of adding
oregano essential oil, tannic acid, and/or their combinations to the ground chicken
meat, 2) to evaluate the antimicrobial effect of oregano essential oil, tannic acid,
and/or their combinations in ground chicken meat, and 3) to evaluate the sensory
characteristics of combined oregano oil and tannic acid in ground chicken meat.
6
CHAPTER 2
LITERATURE REVIEW
Oxidants and free radicals in body systems
Oxygen is essential to human life. Yet, paradoxically, oxygen is also involved in
toxic reactions, and thus is a constant threat to the well-being of all living things
(Halliwell and Gutteridge, 1989). Unfortunately, human natural defense systems are
imperfect; they limit the harm caused by oxygen but do not eliminate it completely.
Many evidences suggested that as years go by oxygen-induced damage to body
tissues may accumulate (Machlin and Bendich, 1987). This damage has been
hypothesized to be a major contributor to aging and many of the degenerative diseases
including cardiovascular diseases, cancers, cataracts, age-related decline in immune
system, and degenerative diseases of nervous system (Langseth, 1995). Most of the
potentially harmful effects of oxygen are due to the formation and activity of reactive
oxygen species. Reactive oxygen species are produced continuously in the human
body as a consequence of normal metabolic processes and act as oxidants. Many
reactive oxygen species are free radicals (Halliwell and Gutteridge, 1989). A free
radical is any chemical species that has one or more unpaired electrons. Many free
radicals are unstable and highly reactive. The chemical reactivity of hydroxyl radical
(OH.), superoxide radical (O2.-), nitric oxide radical (NO.) and lipid peroxyl radical
(LOO.) can damage all types of cellular macromolecules, including proteins,
carbohydrates, lipids, and nucleic acids (Dalle-Donne et al., 2003), But they are not
always harmful. For example, some free radicals by specialized blood cells like
phagocytes play a role in the destruction of disease-causing microbes (Langseth, 1995;
Hampton et al., 1998).
Lipid oxidation mechanism and its effect on meat quality
Lipid oxidation is considered as the most important factor that causes meat
deterioration in both fresh and cooked meat products (Ladikos and Lougovois, 1990,
7
Nam et al., 2002; Ahn et al., 2009). This process is responsible for the formation of
free radicals that leads to form various volatile compounds including aldehydes,
which participate in the development of rancid flavor, and change meat color (Guillen
and Guman, 1998; Ahn et al., 2009). Poultry meat tissues have low lipid content but
contain relatively high polyunsaturated fatty acids (Igene and pearson, 1979, Pikul et
al., 1984). Polyunsaturated fatty acids are highly susceptible to free radical attack that
leads to the production of hydroperoxides, which are responsible for the formation of
secondary by-products such as aldehydes, ketones and other compounds that affect
negatively on the over all food quality (Lund et al., 2011;Vercellotti et al., 1992). This
quality change will cause loss of protein solubility, loss of color, reduce nutritive
value of food, and loss of vitamins such as vitamin E (-tocopherol), D, A and C
(Madhavi et al., 1996; Min and Ahn, 2005). In addition, lipid oxidation is also
reported to affect meat texture and over all their safety (Gray and Monahan, 1992).
The rate of lipid oxidation in fresh and cooked meat products depends on
various internal factors such as fat content, fatty acid composition, and antioxidants,
heme pigments and iron content (Addis, 1986; Du et al., 2000; Choe and Min, 2006;
Min et al., 2008). The external conditions such as processing, packaging and storage
conditions also play important roles in lipid oxidation process (Ahn et al., 2009).
Lipid oxidation can occur by autoxidation, photoxidation, and enzymatic oxidation
processes. The enzymatic oxidation process is facilitated by cyclooxygenase and
lipoxygenase. Photoxidation is primarily found in milk powder and oils, and their
effect on meat quality is minimal (Choe and Min, 2006; Ahn et al., 2009). However
Autoxidation of oils requires radical forms of acylglycerols, on the other hand
photosensitized oxidation dose not require lipid radicals since 1O2 reacts directly with
double bonds. Autoxidation is considered the major mechanism that causes lipid
oxidation in food and meat products and is initiated by reactive oxygen species (ROS)
(Gray and Monhan, 1992; Kanner, 1994; Min and Ahn, 2005).
Autoxidation mechanism is well studied and summarized into three main steps initiation, propagation, termination (Gray and Monahan, 1992; Min and Ahn, 2005;
8
Laguerre et al., 2007). The initiation step is the rate-limiting, but very important in
lipid peroxidation process, this usually starts by reactive oxygen species that abstracts
hydrogen atoms from the site of a fatty acid chain and form a lipid free radical (L.).
This lipid free radical will react rapidly with oxygen to form a peroxyl radical (LOO.).
A more detailed explanation of the initiation step reported that the formation of lipid
free radicals increased with the increase in the total number of bis-allylic carbons,
where the energy between C-H bond between two double bonds position is the
weakest (75-90 Kcal/mol), and the other positions at allylic and alkyl C-H bond are
88 kcal/mol and 101 kcal/mol, respectively (Sevanian and Hochstein, 1985; Buettner,
1993; Wagner et al., 1994).
Therefore, C-H bond between two double bonds is the most reactive site for
hydrogen abstraction (Min and Ahn, 2005; Choe and Min, 2006). The peroxyl radical
(LOO.) formed in the presence of oxygen abstracts hydrogen atom from another
hydrocarbon chain, and yields hydroperoxide (LOOH) and a new free radical (L.),
which participate in propagation chain reaction (Pearson et al., 1977; Enser, 1987).
Lipid peroxyl radical and alkoxyl radical can also abstract hydrogen atom from
another lipid molecule and initiate and propagate the chain reaction (Addis, 1986;
Coyle and Puttfarcken, 1993; Min and Ahn, 2006). The final step is called termination
step in which free radicals such as lipid peroxyl radicals (LOO.) react with each other
to form non-radical products. Usually lipid hydroperoxide (LOOH) is stable at the
physiological temperature, but will be decomposed at high temperature or when it is
exposed to metal ions (Gruger and Tappel, 1970; Halliwell and Gutteridge, 1990;
Choe and Min, 2006). The decomposition of hydroperoxides (ROOH) through free
radicals by the homolytic and hetrolytic -scission, catalyzed by transitional metal
ions, produces a variety of volatile and nonvolatile compounds (Choe and Min., 2006).
The homolytic cleavage pathway of hydroperoxide is more likely to happen first due
to the low activation energy (46 kcal/mol) between oxygen-oxygen bonds compared
to the oxygen-hydrogen bonds (Hiatt et al., 1968). The homolytic -scission of
carbon-carbon bond is the next step for the alkoxy radical converting to
9
oxo-copmpounds and other alkyl radicals (Choe and Min, 2006). Formation of
peroxyl or an alkoxyl radical is considered the first step during the decomposition of
hydroperoxides (Gardner, 1989). However, peroxyl and alkoxyl radicals are relatively
difficult to generate from the structure of hydroperoxides: a ROO-H bond has bond
dissociation energy about 90 kcal/mol and a RO-OH bond of about 44 kcal/mol
(Gardner, 1989). Thus, the formation of peroxyl and alkoxyl radical is highly
depending on the presence of catalyst and other favorable conditions such as light,
oxygen availability, and heat (Schaich, 1992; Velasco et al., 2003). The decomposition
progress of hydroperoxides is very slow in the presence of antioxidants, low
temperature and the absence of catalyst (Gruger and Tappel, 1970). Hydroperoixde
compounds may be converted to different monomeric decomposition products which
are composed of different functional groups such as keto, hydroxyl and epoxy group
(Schieberle et al., 1979; Frankel, 1987). The low molecular weight of volatile
oxidation products are usually produced by the decomposition process of
hydroperoxides. These compounds play important roles on the flavor and odor of
foods (Grosch et al., 1981).
The formation of volatile and non-volatile oxidation secondary products is highly
dependant on the composition and isomeric distribution of monohydroperoxides and
cleavage pathway processes (Frankel, 1987). In addition, polymeric oxidation
products composed of two or more fatty acid units where they are connected together
by peroxide or carbon-carbon linkage are the products of the hydroperoxide
decomposition (Miyashita et al., 1984; Neff et al., 1988).
In the meat system, the secondary products of lipid oxidation such as
carbonyls (ketones and aldehydes), alcohols, hydrocarbons (alkane, alkene), and
furans play an important role in meat flavor and rancidity development in food
systems (Frankel, 1984; 1987). Lipid oxidation products also affect protein solubility,
emulsification, water-binding capacity, texture and the other rheological properties
through the interactions between lipid and protein oxidation products (Hall, 1987).
10
Protein oxidation and its effect on meat quality
The physical, chemical and functional properties of proteins mainly depend on
the amino acid structure and polypeptide backbone. These functional properties are
very important for protein quality in the final products of processed meat. Gelation,
solubility, viscosity, and emulsification properties can be altered when any changes in
amino acid side chain or conformational shape of the final protein structure occur
(Xiong, 2000). Furthermore, protein quality can be modified and changed by many
reactions with reactive oxygen species (Nystrom, 2005).
In meat tissues, myofibrillar proteins are the main components that affect
protein quality and functionality. These proteins can be oxidized by ROS (reactive
oxygen species) during the aging or storage period. The most common consequences
of protein oxidation are as follow (Lund et al., 2011, Estevez, 2011): first the reactive
oxygen species react with protein and peptides in the presence of oxygen from the
back bone of amino acid side chains. Protein exposure to ROS, irradiation, light,
metal catalyst, and peroxidation form protein radicals (P.). The results of these
reactions include protein cross linking, modification of amino acid side chains, and
protein fragmentation. These changes in proteins affect the hydrophobicity,
conformation, and solubility, and alter their susceptibility to proteolytic enzymes. In
addition, protein hydroperoxide (POOH), protein carbonyl (P=O), and protein
sulfoxide or sulfone are produced.
Lund et al. (2011) explained the mechanisms of protein oxidation in muscle as a
three step process: In the initial step, ROS leads to abstraction of a hydrogen atom
from a protein molecule and form a protein carbon-centered radical (p.). In the next
step, this protein radical is converted to a peroxyl radical (POO.) in the presence of
oxygen, and then to a hydroperoxide or alkyl peroxide (POOH) by abstracting a
hydrogen atom from a different molecule. Finally protein oxidation products such as
hydroxyl derivative (POH) and alkoxyl radical (PO.) are produced by the reactions of
POOH with ROS (HO2. radical) or catalyzed by reduced metals (Fe
2+
or Cu+).
Oxidation in meat tissues has been measured using different methods depending on
11
the chemical reactions and products such as loss of sulfhydyle groups, loss of
tryptophan fluorescence, formation of carbonyl derivatives, inter- and intra-molecular
cross links between amino acid residues. DNPH (2,4-dinitrophenylhydrazine) is an
earlier method that determines the carbonyl content (nmol/mg protein) in muscle
samples (Lund et al., 2008). This method has been used in protein oxidation studies in
foods, but is considered nonspecific for measuring the protein oxidation or the actual
level of carbonyls in meat tissues (Cao and Culter, 1995). For example, protein
oxidation reaction can form other compounds than carbonyls. In addition, the
carbonyl moieties could be formed by other mechanisms than amino acid oxidation in
their residues. Advanced methodologies to measure protein oxidation such as electron
spin resonance spectroscopy, fluorescence spectroscopy, and HPLC coupled to
fluorescence or MS were developed later. These methods are very useful to
understand the mechanisms of protein oxidation, role of iron, myoglobin, lipids, and
phenolic compounds through this process (Lund et al., 2011).
Carbonylation is considered as one of the most remarkable chemical changes
during protein oxidation. Using HPLC-MS coupled with flourcence, Estevies et al.
(2011) identified a specific carbonyl compounds in oxidized myofibril proteins. They
found that two semialdehydes, -aminoadpic (AAS) and -glutamic semialdehyde
(GGS), were produced mainly by protein carbonylation. These semialdehydes could
form around 70% of the total amount of protein carbonylation in oxidized animal
proteins. The AAS and GSS are usually formed in the presence of ferric iron (Fe+3)
and H2O2 by oxidizing amino acids such as lysine, praline and arginine in animal
tissues. Carbonyls such as ketones and aldehydes can be formed through four
pathways as reviewed by Eztevies (2011): 1) Directly by oxidation of side chain
amino acid (lysine, threonine and proline), 2) Glycation by a non-enzymatic reaction
in the presence of reducing sugars, 3) The oxidative cleavage of the peptide backbone
via the -amidation pathway or via oxidation of glutamyl side chain, and 4) Covalent
binding to non-protein carbonyl compounds such as 4-hydroxyl-2-nonenal or
malondialdehyde (MDA).
12
Protein is the major component in muscle tissue that affects their nutritional
value, functional properties, and sensory traits (Lawrie, 1998). Protein oxidation
process affects the amino acid binding ability, decrease protein solubility
(polymerization), decrease protolytic activity, and loss of their nutritional value (Lund
et al., 2011). Possible mechanism on how protein carbonylation could affect
technological and sensory traits of meat and their products were reviewed by Eztevez
(2011), Lund et al. (2011), and Xiong (2000). They showed that carbonylation
affected negatively on meat flavor, tenderness, and water holding capacity, juiciness,
and protein functionality. These changes in meat quality caused by carbonylation
decreased proteolytic enzyme activity (e.g., calpain system); cross linking which
stabilize the aggregation, loss of ε-NH3 group, and alteration of the electrical
arrangement of meat proteins. In addition, protein oxidation has a negative effect on
human health: carbonylation is associated with human diseases such as Alzheimer’s,
Parkinson’s, cancer, catacataractogenesis, diabetes, and sepsis (Levine, 2002;
Dalle-Donne et al., 2003).
Antioxidants
Halliwell and Gutteridge (1985, 1989) defined antioxidant as “any substance,
when present at much low concentrations compared with that of an oxidizable
substrate, that significantly delays or inhibits oxidation of that substrate”.
One important line of defense in biological systems is enzymes, including
glutathione peroxidases, superoxide dismutases and catalase, which decrease the
concentrations of the most harmful oxidants. Nutrition (several essential minerals
including selenium, copper, manganese and zinc) is involved in the structure or
catalytic activity of these enzymes and plays a role in maintaining the body's
enzymatic defense against free radicals. A second line of defense in human is
small-molecular-weight compounds which act as antioxidants. They react with
oxidizing chemicals, reducing their capacity for damaging effects. Compounds such
as glutathione, ubiquinol and uric acid are produced by normal metabolism. Other
13
small-molecular weight antioxidants are found in diet, which include vitamin E,
vitamin C, carotenoids, phenolic, and polyphenolic compounds (Langseth, 1995).
Synthetic
antioxidants
such
as
butylatedhydroxytoluene
(BHT),
butylatedhydroxyanisole (BHA) and tert-butylhydroquinone (TBHQ) have been
dominant (Dapkevicius et al., 1998), but they may be harmful to human health.
Therefore, many studies focused on testing the effects of naturally occurring
antioxidants such as ascorbic acid, vitamin E and certain crude plant extracts (Kosar
et al., 2003, 2005).
Using natural antioxidants as meat preservatives to prevent lipid and protein
oxidation
Recently there have been many researches done using natural antioxidant from
a plant origin. This growing interest was due to the possible carcinogenic effects from
using the synthetic antioxidants; the assumption that food contains certain amount of
phytochemicals can affect the etiology and pathology of chronic disease and aging
process in human body (Dorman and Hiltunen, 2004; Shahidi, 2008). In addition,
consumer demands of finding more natural and healthy food product and avoid using
the synthetic ones have been increased.
In the meat and chicken products, rosemary and sage extracts found to be
effective in reducing the rancidity and improve shelf-life (Murphy et al., 1998). Many
herbs such as oregano, sage, thyme, basil, black and white pepper have antioxidant
activity in porcine meat when used at 0.5% to 2.5% level (Fasseas et al., 2007). Tea
catechins and carotenoids also showed antioxidant effects (Tang et al., 2000, 2001;
Staszewski et al., 2011). Bostoglou et al. (2002) studied the effect of dietary oregano
oil on the performance of broiler chicken (1-day old) and on the iron-induced lipid
oxidation of breast, thigh and abdominal fat tissues, and found that the birds’
performance was unaffected by the experimental diet. However, the essential oil
showed a significant antioxidant effect in the tissues. Botsoglou et al. (2003c)
reported that dietary oregano oil had a significant effect in reducing lipid oxidation in
14
turkey meat during frozen storage.
Brannan (2008) reported that adding 1% of grape seed extract to the ground
chicken meat reduced lipid oxidation in processed meat. Abdel-Hamied et al. (2009)
reported that adding natural antioxidant (rosemary, sage and their combination)
extracts reduced oxidative rancidity and microbial growth in minced meat during
refrigerated storage. Du and Ahn (2002) studied the effect of adding natural
antioxidants (vitamin E, sesamol, rosemary, gallic acid) on the quality of irradiated
sausages prepared with turkey thigh meat, and found that the antioxidants reduced the
redness of meat and total volatiles. However, they did not show significant effect on
the off-flavor of turkey sausages induced by irradiation. Adding oregano essential oil
to beef Longissimus dorsi and Semimembranosus muscles decreased lipid oxidation
and extended their shelf life, but adding higher levels of oregano essential oil showed
negative effect on the palatability of the beef steaks because of the strong flavor from
the essential oil (Scramlin et al., 2010). Natural plant extracts such as rosemary
contain many phenolic compounds including rosmarinic acid, carnosic acid,
rosmaridiphenol, rosmariquinone, rosmanol and carnosot, which react with lipid
hydroxyl radicals, stabilize them and, chelate ionic ions such as Fe+2.
Protein oxidation also can cause changes in meat quality such as meat color
because of the conversion of myolglobin to oxymyoglobin or metmyoglobin. This
oxidation process is accelerated by free radical species such as hydroxyl radicals,
peroxyl radicals, superoxide anions, hydrogen peroxide, and nitric oxide (Lawrie,
1998; Ouali et al., 2006; Ahn et al., 2009). Protein oxidation can lead to the
destruction of amino acids and form a carbonyl compound which will affect the
protein functionality such as gel-forming ability, water-binding capacity, emulsifying
capacity, solubility, and viscosity (Howell et al., 2001). Several changes in muscle
fiber proteins during the post mortem phase can affect meat tenderness, pH, and color.
In addition proteolytic enzymes can be affected by the oxidation process, which may
inhibit the activity of these enzymes and affect the degradation process during the
postmortem (Guttmann and Johnson, 1998). Rowe et al. (2004) examined the effect of
15
early postmortem protein oxidation on the color and tenderness of beef steaks, and
found that increased oxidation of muscle proteins early postmortem could have
negative effects on fresh meat color and tenderness.
Finding natural antioxidants used to prevent the protein and lipid oxidation in the
meat system is well documented (Abdel-Hamied et al., 2009; Brewer, 2011). Dietary
supplementation of natural antioxidants such as tocopherols, rosemary, green tea,
grape seed, and tomato extracts increased the oxidative stability of broiler breast meat
and decreased the protein and lipid oxidation (Smet et al., 2008). Furthermore, the
natural antioxidants played important roles in inhibiting the oxidation process not
only during storage but also during cooking of pork (Salminen et al., 2006). The
mechanisms of these natural antioxidants are very similar to those of the synthetic
ones, which scavenge the free radicals, absorb light in ultraviolet (UV) region (100 to
400 nm), and form chelating complex with transition metals. All of these properties
can decrease the autoxidation process, off-odors, and off-flavor production (Brewer,
2011). In the meat industry, recently finding new combinations of natural plant
extracts have been of great interest. These natural antioxidants were used directly
adding to the meat product (Jo et al., 2006; Abdel-Hamied et al., 2009; Glucin et al.,
2010) or dietary supplement to the animal diet (Botsoglou et al., 2003a,b,c; Jamilah et
al., 2009; Ariza-Nieto et al., 2011). Due to their positive effects on the food quality,
safety, and over all animal health researchers found them as good replacements for the
synthetic antioxidants.
Oregano
Oregano has been used for flavoring fish, meat and sauces since ancient times
(Sahin et al., 2004). In the Middle East where it grows abundantly, it has the common
name of Za'atar and is usually collected for human consumption (Daouk et al., 1995).
There are two major phenolics in oregano: carvacrol and thymol, which constitute
about 78-82% of the essential oil, and are responsible for the antioxidant activity
(Adam et al., 1998; Yanishlieva et al., 1999). γ-Terpenene and p-cymene constitute
16
about 5 to 7% of the total oregano essential oil, and also have antioxidant activity.
It is important to know that the composition, production and quality of oregano
essential oil can vary depending upon many factors including genetic, seasonal,
temperature, moisture, soil, and daylight length (Farooqi et al., 1999; Russo et al.,
1998; Gonuz and Ozorgucu, 1999; Ceylan et al., 2003; oncer et al., 2009;
Viuda-Martos et al., 2010). Their antioxidant (Burt, 2004; Skerget et al., 2005) or
antimicrobial activity (Sivropoulou et al., 1996; Adam et al., 1998) are also highly
dependent on their chemical phenolic composition mainly carvacrol and thymol.
Essential oil composition of Origanum
The essential oil in the Lebanese Za’atar (Origanum syriacum L.) is
characterized for its high thymol and carvacrol content (Daouk et al., 1995; Tepe et al.,
2004). On the other hand, the essential oil (EO) of Origanum majorana L. is
characterized by the dominant occurrence of linalool, terpinen-4-ol, and sabiene
hydrate (Berna’th, 1997). It is used as a folk remedy against asthma, indigestion,
headache and rheumatism (Jun et al., 2001). Origanum syriacum var. bevanii
collected from Turkey contains 43% – 79% of carvacrol (Baser, 2002). Alma et al.
(2003) found that the major components in the essential oil of Origanum syriacum
grown in Turkey are high in -terpenene, carvacrol, paracymene and -caryophyllene.
Thymol or 5-methyl-2-(1-methylethyl) phenol (Lee et al., 2003) is a phenolic
compound and is one of the predominant components of oil derived from the thyme
plant, Thymus vulgari, and from Oregano, Origanum vulgare (Hazzit et al., 2006). It
has antimicrobial (Lamber et al., 2001), antioxidant, astringent and antibiotic
properties (Braga et al., 2006), and has been used as an oral care product. Because of
its distinctive sharp odor and pungent flavor, thymol has adverse effect on the sensory
characteristics of products when used in high levels (Lee et al., 2008) (Figure 1).
Carvacrol or 2-methyl-5-(1-methylethyl) phenol is a phenolic compound found in
certain essential oils (EOs) and shows antimicrobial, antioxidant and antiviral activity.
It is abundant in some oils and its level can reach to 75% of essential oil (Sokmen et
17
al., 2004) and shows highly specific activities as compared to other EO components
(Dorman et al., 2000).
p-Cymene is an aromatic hydrocarbon that occurs widely in tree leaf oils (Yatagai
and Sato, 1985). Its chemical structure is similar to that of the styrene. Generally, a
good solvent for a certain polymer has a solubility parameter value close to that of the
polymer. p-Cymene can be used as an alternative shrinking agent for expanded
polystyrene (Hattori et al., 2010).
Terpinen-4-ol is widely distributed in various plants such as oranges and
mandarins, origanum, the New Zealand lemonwood tree and the Japanese cedar to
black pepper. With other terpenes, it is thought to contribute to the unique taste of the
Cuban bullock’s heart fruit (Pino et al., 2003). Terpinen-4-ol has shown a strong
antimicrobial and anti-inflammatory property (Hart et al., 2000).
Carvone is known as a component of spearmint (Egawa et al., 2003; Roohi and
Imanpoor, 2014). Carvone is a member of terpenoids (Simonsen et al., 1953) found in
a small amount in oregano vulgare spp. hirtum (Russo et al., 1998). Carvone is found
naturally in many essential oils but is the most abundant in the oils from seeds of
caraway (Carum carvi) and dill. Carvone has two mirror image forms or enantiomers:
S-(+)-carvone smells like caraway. Its mirror image, R-(–)-carvone, smells like
spearmint (Figure 1). Both carvones are used in the food and flavor industry (De
Carvalho and Da Fonesca, 2006). R-(-)-carvone is also used for air freshening
products, and like many essential oils, oils containing carvones are used in
aromatherapy and alternative medicines (Leitereg et al., 1971).
The amounts and kinds of other constituents in Oreganuim depend on the species
and method of analysis (GC, HPLC): many other terpenoids including
gamma-terpenene, borneol, linalool, and alpha-terpenene are found in O. onites
(Ceylan et al., 1999; Baydar et al., 2004; Kacar et al., 2006; Toncer et al., 2009).
Sabinene, limonene, chrysoeriol, diosmetin, quercetin, eriodictyol, cosmoside,
vicenin-2, thymoquinol glycosides, neoisodihydrocarvyl acetate, caryophyllene oxide,
oleanolic acid, trans-carveol, bornyl acetate, lithospermic acid, aristolochic acids, and
18
alph-pinene are also found in Origanum vulgare Species (Adam et al., 1998; Antuono
et al., 2000). Some minor components such as aristolochic acid are still under
investigation (Brewer, 2011).
Antioxidant activities of oregano
The antioxidant activity of natural sources, including Origanum species, has
been investigated by many researchers. Al-Bandak (2007) investigated the antioxidant
activity of O. syriacum in corn oil at concentrations of 200, 500 and 1000 ppm, and
found that diethyl ether (D) and ethanol (E) extracts of O. syriacum have the highest
protection against oxidation followed by ethyl acetate (EAc) and petroleum ether (P).
Tepe et al. (2004) suggested that the essential oil and extracts from the herbal parts of
O. syriacum could be used as natural preservatives in food industry. They found that
the inhibitive effect of Origanum on linoleic acid oxidation might be promoted by the
presence of non-polar phenolics because both hexane and dichloromethane extracts
showed high antioxidant activities. Sahin et al. (2004) suggested that the essential oil
of Origanum vulgare ssp. vulgare possesses compounds with antioxidant activity, and
therefore can be used as a natural preservative in food and/or pharmaceutical industry.
Capecka et al. (2005) found that the strongest inhibition of linolenic acid (LA)
peroxidation was found in both fresh and dried O. vulgare.
Kosar et al. (2003) investigated antioxidant compounds in Origanum vulgare L.,
O. onites L., O. minutiflorum and O. syriacum L., and found that rosmaric acid and
carnosic acid were the dominant radical scavengers in the extracts. Purified
compounds from O. majorana L. possessed both O2-scavenging activity and an
inhibitory effect against TPA-induced O2 generation (Jun et al., 2001).
The antioxidant capacity of essential oils and aqueous tea obtained from
Origanum vulgare, Thymus vulgaris and Thymus serpyllum showed a dose-dependent
protective effect against copper-induced LDL oxidation (Kulisic et al., 2007). The
protective effect of essential oils was due to the presence of phenolic monoterpenes,
thymol and carvacrol, which are identified as the dominant compounds in these
19
essential oils. The strong protective effect of aqueous tea was proposed to be the
consequence of large amounts of polyphenols, namely rosmarinic acid and flavonoids.
Tsimogiannis et al. (2006) examined Origanum heracleoticum as a potential source of
phenolic antioxidants. They extracted the component from plant using a Soxhlet
apparatus sequentially using solvents with different polarity and found that the
activity of the extracts widely varied depending upon the solvents used.
The prime justification for using an antioxidant in frying oils is to delay lipid
oxidation during frying. Antioxidants retard oxidation and prolong the shelf-life of the
product. Recently, research on natural antioxidants for frying purposes has increased.
Plant extracts, especially those obtained from herbs and spices, have been proposed
for stabilizing frying oils and fried products (Houhoula et al., 2003). The effect of
Origanum vulgare on the oxidative stability of cottonseed oil during frying of potato
chips and on the storage stability of the produced chips indicated that the oxidation
rates of both frying oil and the fried product were decreased when Origanum vulgare
was added to the frying oil (Houhoula et al., 2003). Tsimidou et al. (1995) tested dry
Origanum vulgare for its antioxidant activity in mackerel oil stored at 40 °C in the
dark and found that its effectiveness at 0.5% level was comparable to that of 200 ppm
BHA and 0.5% (w/w) dry rosemary, and stronger than that of red chillies and bay leaf
which did not improve the stability of the fish oil.
In the meat products area, researchers extensively worked on two strategies to
improve antioxidant stability of meat and their products. The first strategy was adding
this natural antioxidant through the animal diet (Botsoglou et al., 2003a; Simitzis et al.,
2008) and this has been confirmed in a broiler research experiments (Wong and
Shibamoto, 1995; Sarraga and Reguerio, 1999; Botsoglou et al., 2003b). In this case
they were trying to use this natural antioxidant through the incorporation into
phospholipids membrane layer where it can work effectively at their reaction site
location (Gary and Pearson, 1987; De Winne and Dirinck, 1996). The second strategy
was adding the oregano essential oil directly in the meat surface (Goulas and
Kontominas, 2007; Martin-Sanchez et al., 2011), ground meat (Fasseas et al., 2007;
20
Oral et al., 2009; Mastromatteo et al., 2009) or their brine solution (Scramlin et al.,
2010) prior to processing in order to improve meat quality and extend their shelf-life.
In addition, oregano essential oils have medical properties which help to
improve human health. Origanum spp. has been reported to have several
pharmaceutical uses such as an antibacterial, fungicidal, antiviral, biocidal,
antioxidant (Berna’th, 1997), antimicrobial (Sahin et al., 2004), anti-hyperglycemic
(Lemhadri et al., 2004) and antithrombic activities, and possess activity against cancer
(Goun et al., 2002). It also has a nematicidal (Berna’th, 1997) and insecticidal
activities (Traboulsi et al., 2002). In some countries such as Jordan, Origanum
syriacum leaves are used to cure eye ailments, burns and stomach troubles, and the
seeds are used as sedative (Oran and Al-Eisawi, 1998).
Antimicrobial Activity of Oregano
Origanum essential oils have high levels of carvacrol, thymol, γ-terpenene and
p-cymene, and showed a high level of antimicrobial activity against eight different
strains of gram-positive and gram negative bacteria (Sivropoulou et al., 1996). Among
the essential oil components, carvacrol and thymol have the highest activity against
bacterial growth, but the other two pheonolics (γ-terpenene and p-cymene) have no
activity against bacterial strains. In addition, essential oils from different strains of
oregano showed different antimicrobial activities. Horosova et al. (2006) found that
oregano oils showed strong antibiotic effects on lactobacilli and E. coli when
combined with antibiotics such as apramycin, streptomycin, and neomycin. Chouliara
et al. (2007) reported that oregano essential oil showed a shelf-life extension effect on
fresh chicken breast meat when combined with modified atmosphere packaging.
Recently researchers are studying the antimicrobial effects of essential oils and
their active components, including thymol and carvacrol. Thymol and carvacrol are
both effective against E. coli (Xu et al., 2008), but carvacrol is more effective than
thymol (Cristani et al., 2007). They both appeared to harm bacterial cell wall, and
enter into bacteria and change cell processes. These effects may account for their
21
antibacterial properties (Cristani et al., 2007; Xu et al., 2008; Di Pasqua et al., 2010).
Different hypothesis were suggested about the antimicrobial mechanism of the
phenolic compounds or the essential oil components on microorganisms. Ultee et al.
(2002) found that both carvacrol and cymene caused destabilization of the membrane
of bacteria cells (Bacillus cereus), and decreased membrane potential. So, the most
probable mechanism for the antimicrobial activity of carvacrol could be through the
decrease in pH gradient across the cytoplasmic membrane due to hydroxyl groups and
delocalized electrons. So if the metabolism is aerobic, this will reduce a proton motive
force and effect on ATP synthesis. The absence of ATP inside the microorganism will
lead to change in the cell process and may cause cell death. In addition, researchers
found that increasing in the membrane fluidity and leakage of protons and ions were
observed when bacteria were exposed to antimicrobial compounds (Sikkema et al.,
1995; Heipieper et al., 1996). However, certain concentration of these compounds is
needed to cause membrane leakage (Mendoza-Yepes et al., 1997). The mode of action
of these phenolic compounds still needs more investigation to understand their
antimicrobial effects. Recently, food industry has been showing a great interest in
using plant derived extract as antimicrobial compounds in the food to suppress the
growth of foodborne pathogens. Carvacrol and thymol are the main components of
the oregano essential oil responsible for their antimicrobial activity (Ultee et al., 2002;
Nostro et al., 2007). Combination studies using more than one phenolic compound are
conducted to maximize their effect through the synergistic philosophy. For example,
the combination action of nisin and carvacrol were found to have a synergistic effect
against Bacillus cereus and listeria monocytogenes (Pol and Smid, 1999). Oregano
essential oil has been well documented as an antimicrobial agent for many foodborne
pathogens such as Staphylococcus aureus, Vibrio parahaemolyticus, Salmonella
typhimurium, Listeria monocytogenes and Escherichia coli (Sivropouluo et al., 1996;
Ozcan, 2001; Lin et al., 2004). However, Lambert et al. (2001) reported that the
mixture of carvacrol and thymol at certain concentration increased the total inhibition
of Stapylococcus aureus and Pseudomonas aeruginosa. Similar results were found on
22
the growth of Enterobacteriaceae, lactic acid bacteria, and pseudomonas spp when
thymol and carvacrol were added to the poultry meat patties (Mastromatteo et al.,
2009). The inhibition may be related to the cell membrane damage, pH homeostasis,
and inorganic ions equilibrium. Carvacrol is a major component of essential oils that
were extracted from plant sources such as (Thymus vulgaris) and oregano (Oreganum
vulgare) (Adam et al., 1998; Burt, 2004). Its percentage in the oregano essential oil is
highly dependent upon the Oreganuim species, the extraction method, storage
conditions, and laboratory method analysis. Carvacrol [2-methyl-5-(1-methlethyl)
phenol is a hydrophobic phenolic compound synthesized from p-cymene and
-terpinene (Poulose and Rodney, 1978). Their antimicrobial activity against several
types of pathogens such as Bacillus cereus (Beuchat et al., 1994; Ultee et al., 1998,
1999; 2000), Escherichia coli O157:H7 (Friedman et al., 2004; Perez-Conessa et al.,
2011), Staphylococcus aureus (Lambert et al., 2001; Knowles et al., 2005; Nostro et
al., 2004, 2007), Salmonella enterica (Friedman et al., 2002; Nazer et al., 2005;
Gutierrez et al., 2008) are well documented.
Tannic Acid
Plants produce a wide variety of “secondary compounds” including alkaloids,
terpenes, and phenolics in their cells. These compounds play important roles in
protecting plants from herbivores and diseases (Hagerman, 1987, 2002, 2010;
Getachew et al., 2000). Tannin is one of these compounds that have metal ion
chelating and antioxidant activities, influence protein digestibility, and precipitate
proteins through the tannin protein interactions (Hagerman and Butler, 1980;
Hagerman, 1992; Hagerman et al., 1998; Okuda and Ito, 2011). Tannins were defined
by Bate-Smith and Swain (1962) as “water soluble phenolic compounds that have
molecular weights between 500 and 3000 Da, and have special properties such as the
ability to precipitate alkaloids, gelatin, and other proteins’’. These properties of tannin
are based on their chemical structures which have two or three phenolic hydroxyl
groups on phenyl ring (Fig.2). Tannins can be classified into two groups: pyrogallol
23
type and catechol types (or certain catechin type) according to the polyphenol group
in their structure. However, tannins can also be classified into hydrolysable tannin and
condensed tannins (Okuda et al., 1985; Haslam, 1989; Okuda, 1999; 2005), or
polyphenols of constant chemical structure (Type A) and polyphenols of variable
composition (Type B).
Tannins or plant polyphenols are distinguished from other polyphenols found in
plants by their characteristics such as protein interaction ability, antioxidant ability,
metal chelating ability, basic compounds, large molecular weight compounds, and
pigments attached (Haslam, 1989; 1996).
The United State Department of Health and Human Services (Food and Drug
Administration) defined tannic acid or hydrolysable gallotannins as a complex
polyphenolic compound that yields gallic acid and either glucose or quinic acid as
hydrolysis products. It has yellowish-white to light brown color, odorless or has a
faint characteristic odor, and has astringent taste. Tannic acid can be obtained in two
different ways: solvent extraction of nutgalls or excrescences from young twigs of
Quercus infectoria Oliver, from solvent extraction of the seed pod of Tara
(Caesalpinia spinosa), or the nutgalls of various sumac species, including Rhus
semialata, R. coriaria, R. galabra, and R. typhia (FDA, 2011).
The hydrolysable types of tannins contain polyhydric alcohol and hydroxyl
groups, which are connected to gallic acid such as gallotannins or to hexahydroxy
diphenic acid (ellagitannins) by ester bond. After hydrolysis by enzyme or acid/base
conditions, gallotannins produce glucose and gallic acid. Example on the gallotannins
is Chinese tannin, Turkish tannin, Tara tannin, Acer tannin, and hamaelis tannin.
Ellagitannin (corilagin), Chebulagic acid, Chebulinic acid, and Chebulic acid are also
examples of ellagatanins (Chung et al., 1998a).
Condensed tannin types are more complex in their structure compared to
hydrolysable tannins and they are mainly produced by two main polymerized
products of flavan-3-ols and flavan-3,4-diols, or mixture of both (Hagerman and
Butler, 1994; Hagerman et al., 1997; Chung et al., 1998a). Flavan-3-ols are
24
considered as catechins which have four possible isomers due to two asymmetrical
carbon atoms at carbon number 2 and 3. These isomers are formed into (+) and (-)
catechins, (-) epicatechin, (-) epigallocatechin, (+)-gallocatechin, and (+)-catechin
(Chung et al., 1993, 1998a). Falvan-3,4-diols are classified as a type of compounds
called leucoanthocyanins due to their properties after exposure to heat and acid
conditions since they polymerize to phlobaphene-like products which will produce the
anthocyanidines (Haslam, 1981; Zucker, 1983). Falvan-3,4 diols can form eight
isomers from their molecule structure which depend on three asymmetric carbon
atoms at C-2, C-3, and C-4; leucocyanidin, leucopelargonidin, leucodelphinidin,
guibourtacacidin, (+)-leucorobinetinidin, (-)–melacacidin, and (-)–teracacidin (Chung
et al., 1998a).
Tannic acid as used in many studies is a specific commercial type of tannin with
pKa around 6 due to their polyphenol structure (Fig. 2). It is also classified as an
important gallotannins which belong to the hydrolysable class (Scalbert, 1991;
Miranda et al., 1996). The commercial formula for tannic acid is given as C 76H52O46
and is composed of a mixture of polygalloyl glucose and polygalloyl quinnic acid
ester depending on the plant extract source. The commercial form of tannic acid can
be fractionated chromatographically to yield a specific galloyl ester or can be
methanolyzed which produce homogenous pentagalloyl glucose (Hagerman, 2002;
Salminen et al., 2011).
Tannin purification method is also important because it affects product purity
and chemical content. The percentage of condensed tannins and hydrolysable tannins
highly depend on the method of purification from plant tissues such as tissue
preservation, grinding and extraction. The purity of tannic acid products can be
assessed by different methods such as TLC and HPLC. In addition, their structures
can be studied by degradative functional methods or by spectroscopic methods
(Hagerman, 2002).
25
Antioxidant Activity of Tannic acid
Polyphenol tannic acid inhibits hydroxyl radical formation from the Fenton
reaction by complexing with ferrous ions. When ferric ion (Fe3+) is added to sample
containing tannic acid, they form Fe(II)n-TA complexes, which mean tannic acid is
capable of reducing ferric ion (Fe3+) to ferrous ion (Fe2+). So, researchers proposed
that Fe3+ complexes to tannic acid converts Fe3+ to Fe2+ and prevents ferrous ion (Fe2+)
from participating in the Fenton reaction and so the hydroxyl radical formation (Lopes
et al., 1999). Tannic acid chelates iron due to polyphenol groups (ten galloyl group)
and decreases the intestinal non-heme iron absorption (Brune et al., 1989; Geisser,
1990). Tannic acid also protects DNA from oxidative damage by chelating Fe(II) and
so prevents .OH formation in the presence of H2O2 (Fenton Reaction) (Toyokuni,
1996; Meneghini, 1997). Therefore, the antimutagenic and anticarcinogenic ability of
tannic acid could come from its ability to prevent or delay the Fenton reaction and
hydroxyl radical formation (Lopes et al., 1999). In addition, lipid peroxidation could
be decreased by tannic acid through its ability to chelate iron ions (Afanas’ev et al.,
1989; Guo et al., 1996; Moran et al., 1997).
Tannic acid showed antioxidant activity when added to samples containing
copper due to the ability of tannic acid to complexes with copper, and this complex is
less capable of inducing ascorbate oxidation (Khan et al., 2000; Andrade et al., 2005).
Okuda et al. (1992) found that adding green tea (rich in tannins) increased ascorbic
acid stability in the presence of oxygen and metal catalyst. In a recent study about the
enhancing antioxidant capacity of tannic acid by using thermal processing,
researchers found that thermal processing to hydrolysable tannins increased their
antioxidant and antimicrobial activities (Kim et al., 2010). This improvement on the
antioxidant capacity could be due to the hydroxyl groups of flavonoids and tocopherol
formed by heat treatment (Kwon et al., 2003). The increase in the antioxidant capacity
may also be explained by the hydrolysis of tannic acid, which produced one free
gallic acid and a galloyl group on the remaining gallotanins (Kim et al., 2010).
Gulcin et al. (2010) studied the radical scavenging and antioxidant activity of
26
tannic acid and found that tannic acid inhibited oxidation of linoleic acid emulsion by
97.7 % at 15 g/mL. In addition, they found that tannic acid was effective in DPPH
(1,1-diphenyl-2-picry-hydrazyl free radical) scavenging, ABTS.+ 2,2-azino-bis
(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging, superoxide anion radical
scavenging and hydrogen peroxide scavenging, and had Fe+3-reducing power and
metal chelating activities. Ho et al. (1999) concluded that the antioxidant activity of
tannin components from Vaccinium vitis-idaea L. exhibited multiple antioxidant
activity, and could be used to treat periodontal disease due to the complex interactions
between pathogenic bacteria and the host’s immune response.
Antimicrobial activity of tannic acid
The antimicrobial effect of tannins is well studied and documented. Many
studies showed that tannins have the ability to inhibit the growth of many
microorganisms such as fungi, yeast, bacteria, and viruses. The antimicrobial
characteristics of tannins are related to the hydrolysis of ester linkage between gallic
acid and polyols especially in the edible fruits. This natural defense by the tannins can
be used in food processing to improve their shelf life and prevent microbial growth
(Chung et al., 1993, 1998b).
Tannin with unknown origin showed inhibitory effects on the growth of
filamentous fungi Fomes annosus with a minimum inhibitory concentration (MCI)
(Haars et al., 1981). The same effect was found by Wehmer (1912) on the growth of
merulius lacrymans and penicillium species at 10 to 20 mg/L tannin concentration.
Condensed tannins were found to have inhibitory effects on the growth of
Colletorichum graminicola (Nicholson et al., 1986). In addition, many studies showed
the inhibitory effects of different tannin sources on the filamentous fungi such as
Coniophora olivaccea (Nicholson et al., 1986), Coriolus versicolor (Hart and Hilis,
1972), Crinipellis perniciosa (Brownlee et al., 1990), Trametes hirsute (Arora and
Sandhu., 1984) and Trichoderma Viride (Arriet-escobar and Belin., 1982).
Tannins also showed inhibitory effects on the growth of various yeast species
27
(Jacob and Pignal, 1972) and bacteria such as Staphylococus aureus, Streptocoucus
punumonia, Bacilus anthracis, Shigella dysenteriae, and Salmonella senftenberg
(Beuchat and Heaton, 1975; Kau and Wu, 1980; Akiyama et al., 2001). Tannic Acid
and propyl gallate also showed inhibitory effects on the growth of food borne bacteria
such as E. coli, Alcaligenes faecalis, Listeria monocytogeneses, Salmonella entritidis,
S. paratyhi, Staphyl. Aureus, Strep. Faecalis, Strep. Pyogenes, and Yersinia
enterocolitica (Scalbert et al., 1991; Chung et al., 1993).
Tannins give more benefits to human health from their effect on the survival of
viruses and their activities. For example, tobacco mosaic virus (Bawden and
Kleczkowski, 1945; Thresh, 1956) and influenza virus (Carson and Frisch, 1953)
were affected negatively by adding tannic acid to their environment. Tannic acid also
has an inhibitory effect on many virus species such as echovirus, coxsackievrus,
reoviruses, polioviruses, and herpes viruses (Kucera et al., 1967; Konowalchuk and
Spiers, 1976). Also, tannic acid sulfate has inhibitory activity to the cytopathic effect
of HIV (human immunodeficiency virus) at 6 g/ml level (Mizumo et al., 1992).
The antimicrobial mechanisms of tannins are through the astringent effect of
tannins by complex formation with enzymes and other substrates (Mason and
Wasserman., 1987; Chung et al., 1998a), their effect on the membranes of
microorganisms, metal complex formation (Chang et al., 1998b; Engels et al. 2011),
and the hydrolysis of ester linkage between gallic acid and glucose.
Chung et al. (1998b) reported that tannic acid works as a siderphore produced
by bacteria to bind irons (Neilands, 1995) in the media which make it unavailable for
microorganism to use in their metabolism and enzyme activity. The siderphores are
low-molecular-weight carriers that have high iron (Fe+3) affinity, which help bacteria
to make iron biologically available (Neiland, 1982). This iron complex structure is
highly contributed in the antimicrobial activity of several gallatonnins (Engels et al.,
2009; Cho et al., 2010). Other tannins compounds such as proyl gallate and methyl
gallate (ester of gallic acid) are unable to bind iron efficiently. Therefore, their effects
on bacterial growth should be explained by other mechanisms. In addition, tannic acid
28
does not affect lactic acid bacteria effectively compared to other intestinal bacteria
because they do not have heme enzymes (Kabuki et al., 2000). However, the bacterial
response to the gallotannins also depends on their membrane structure. The outside
membrane of Gram-negative bacteria makes it more resistant to gallotannins
compared to the Gram-postive bacteria (Tian et al., 2009; Engels et al., 2009, 2011).
29
Figure 1. Structure of A. thymol, B. carvacrol, C. p-cymene, D.
terpenene-4-ol, E. the two mirror image form S and R-carvone, F.
aristolochic acid.
Figure 2. Structure of tannic acid.
T.J. Kim et al. Food Chem. (2010). 118: 740–746
30
Figure 3. The hydrolysis of tannic acid at an ester linkage between two gallic groups.
T.J. Kim et al. Food Chem. (2010). 118: 740–746.
Figure 4. Chemical structure of gallic acid (3,4,5-trihydrox-ybenzoic acid)
containing a galloyl group.
K. South, and D. D. Miller, 1998. Food Chem.. 63 (2): 169.
31
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51
CHAPTER 3
THE EFFECT OF OREGANO ESSENTIAL OIL (ORIGANUIM VULGARE
SUBSP. HIRTUM) ON THE STORAGE STABILITY AND QUALITY
CHARACTERISTICS OF GROUND CHICKEN MEAT
Marwan Al-Hijazeen1, Aubrey Mendonca2,* and Dong Uk Ahn1,*
1
Department of Animal Science, Iowa State University, Ames, Iowa 50011, USA, and
2
Department of Food Science and Human Nutrition, and 2Department of Animal
Science, Iowa State University, Ames, IA 50011, USA.
*Corresponding author: D. U. Ahn
Phone: (515) 294-6595, Fax: (515) 294-6595
Email: [email protected]
52
Abstract
The objective of this study was to investigate the effect of different levels of
oregano essential oil on important meat quality parameters, including oxidative
storage stability, of ground chicken meat. Five different treatments including 1)
control (none added), 2) 100 ppm oregano essential oil, 3) 300 oregano essential oil, 4)
400 ppm oregano essential oil, and 5) 5 ppm butylatedhydroxyanisole (BHA) were
added to ground boneless, skinless breast meat and used for both raw and cooked
meat studies. For raw meat study, samples were individually packaged in
oxygen-permeable bags and stored at 4 ℃ cooler for up to 7 days. For cooked meat study,
the raw meat samples were packaged in oxygen impermeable vacuum bags and then
cooked in-bag to the internal temperature of 75 °C. After cooling to room temperature,
the cooked meat was transferred to a new oxygen-permeable bag and stored at 4 oC
for up to 7 days. Both raw and cooked meats were analyzed for lipid and protein
oxidation, color at 0, 3, and 7 days of storage. Volatiles profile of cooked meat was
reported during storage time. Oregano essential oil treatments significantly reduced (p
< 0.05) lipid oxidation, protein oxidation, and improved color stability of raw meat.
However, oregano oil at 400 ppm showed the highest effect for all these parameters.
No significant difference (P > 0.05) was seen on the a*-value of meat during storage.
Cooked meat showed similar results to raw meat when oregano oil was added.
Hexanal was the major aldehyde which decreased significantly (P < 0.05) by oregano
oil treatment in cooked meat. The significant differences in the aldehydes formation
among the treatments were clearer in cooked meat than in raw meat. Overall, oregano
essential oil at level between 100-300 ppm could be a good meat preservative that can
replace the synthetic antioxidant, BHA.
Key Words: Oregano essential oil, Butylatedhydroxyanisole, Ground meat, Lipid
oxidation
53
Introduction
Poultry meat is among the most popular meats in the world because of its low
price, short production time, and ease of preparation (Chouliara et al., 2007). Chicken
meat contains high percentage of polyunsaturated fatty acids, which make it more
susceptible to oxidative deterioration during storage (Ahn et al., 2002). Both raw and
cooked poultry meats can be oxidized, but cooked meat is more sensitive to lipid
oxidation than raw meat (Gray et al., 1996; Lee et al., 2003). Lipid oxidation produces
many volatile compounds responsible for the off-flavor and rancidity in the meat
(Ahn et al., 2009). Other quality characteristics, such as coloration and texture of meat,
can also be influenced by the oxidation of meat (Jensen et al., 1997; Du et al., 2000).
Several synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT) and propyl gallate (PG) have been used widely in the food
industry to prevent the deteriorative changes. However, the use of synthetic
antioxidants are discouraged due to their negative effects on human health, and the
food industry is interested in finding new replacements for the synthetic ones
(Solomakos et al., 2008).
Natural antioxidants from plant origin are safe and can replace the synthetic
ones. Herbs, spices and many plant extracts have been widely used in foods to
improve their flavor, quality, and to extend their shelf-life (Abdel-hamied et al., 2009).
Oregano essential oil is one of many plant extracts that have antioxidant effects when
added to meat (Fasseas et al., 2007; Goulas and Kontominas, 2007; Scramlin et al.,
2010). The antioxidant effect of oregano essential oil is due to its high poly-phenol
content; Carvacrol, thymol, p-cymene and γ-terpinene are the major components
responsible for the antioxidant activity of oregano essential oil (Kosar et al., 2003;
Capecka et al., 2005; Al-Bandak, 2007). This type of plant extract could be used as a
good replacement for the synthetic antioxidant in meat products. Most of the research
that had been done on oregano essential oil were adding the oil directly to the diets of
poultry (Botsoglou et al., 2003a,b;c), lamb (Simitizis et al., 2008), and swine
54
(Ariza-Nieto et al., 2011) to stabilize the meats from those animals.
However, very little work has been done to add the oregano essential oil
directly to meat to prevent oxidative changes in it (Fasseas et al., 2007; Chouliara et
al., 2007; Scramlin et al., 2010). Many previous works showed that adding oregano
essential oil directly into the meat products had positive effects on meat quality. Some
research investigated the combination effects of carvacrol and thymol on the poultry
meat patties and showed positive effect on their color and reduced off-odor formation
(Mastromatteo et al., 2009). Sullivan et al. (2004) studied the effect of natural
antioxidants such as rosemary, sage, and tea catechins, on the chicken nugget and
found that the addition of these antioxidants decreased total lipid oxidation, and
increased color stability and storage stability of the product. Keokamnerd et al. (2008)
studied the effect of adding commercial rosemary oleoresin preparations on ground
chicken meat which were packaged in a high-oxygen atmosphere and found a positive
overall effect on raw meat appearance during storage and cooked meat flavor.
Oxidation process was decreased in ground meat with added rosemary, and the color
was more stable. Oregano essential oil containing high concentration of carvacrol
(major antioxidant) showed higher antioxidant activity than other herbs extracts
(Fasseas et al., 2007; Brewer et al., 2011). Oregano essential oil is one of the natural
antioxidants that needs more research to determine its effect on food quality and
preservation, especially for its optimal level to prevent oxidative changes while
maintaining other quality parameters such as flavor.
The objective of this study was to investigate the effect of different levels of
oregano essential oil on important meat quality parameters, including storage stability,
of ground chicken meat. It is also beneficial to compare the effect of oregano essential
oil with a synthetic antioxidant BHA, in both raw and cooked meat.
55
Materials and Methods
1. Sample preparation: One hundred and twenty, 6-wk-old broilers raised on a
corn-soybean meal diet were slaughtered using the USDA guidelines (USDA, 1982).
The chicken carcass were chilled in ice water for 2 h and drained in a cold room, and
the breast muscles were separated from the carcasses at 24h after slaughter. Boneless
breast muscles were cleaned, skins removed, external fats trimmed off, and stored at
-20 oC freezer until used.
The frozen meats were thawed in a walk-in cooler (4 oC), ground twice a through
a 10-mm and a 3-mm plates (Kitchen Aid, Inc., St. Joseph, MI, USA) before use. Five
different treatments including 1) (none added), 2) 100 ppm oregano essential oil, 3)
300 oregano essential oil, 4) 400 ppm oregano essential oil, and 5) 5 ppm
butylatedhydroxy anisole (BHA) were prepared. The oregano essential oil was
obtained from a certified company in Turkey (Healthy-Health, Oil of Oregano,
Turkey). The GC/MS analysis of the oregano essential oil indicated that 80.12% of
the essential oil was carvacrol. BHA powder (0.1 g) and oregano essential oil (1.25 g)
were dissolved in 10 ml of 100% ethanol, and then mixed with 50 ml mineral oil to
make their stock solutions. The ethanol added was removed using a rotary evaporator
(BUCH Rotavapor, Model R-200) at (70 °C, 175 mbar vacuum pressure) before
adding the stock solution to meat samples. Each additive treatment was added to the
ground breast meat and then mixed for 2 min in a bowl mixer (Model KSM 90;
Kitchen Aid Inc., St. Joseph, MI, USA). All treatments were added with the same
amounts of mineral oil to provide the same conditions.
For raw-meat study, the prepared meat samples (approximately 100 g each) were
individually packaged in oxygen-permeable bags (polyethylene, 4 x 6.2 mil, Associated
Bag Co., Milwaukee, WI), stored at 4 °C cooler for up to 7 days, and analyzed for lipid
and protein oxidation, and color at 0, 3, and 7 days of storage.
The same preparation method was used for cooked meat study, but the raw meat
samples were packaged in oxygen impermeable vacuum bags (O2 permeability, 9.3
mL O2/m2/24 h at 0 °C, Koch, Koch, Kansas City, MO, USA), and the meats were
56
cooked in-bag in a 90 °C water bath (Isotemp®, Fisher Scientific Inc., Pittsburgh, PA,
USA) until the internal temperature of the meat reached to 75 °C. After cooling to
room temperature, the cooked meat was transferred to a new oxygen-permeable bag
(polyethylene, 4 x 6.2 mil, Associated Bag Co., Milwaukee, WI, USA), and stored at
4 °C for up to 7 days, and analyzed for lipid and protein oxidation and volatiles at 0, 3,
and 7 days of storage.
2. 2-Thiobarbituric acid-reactive substances (TBARS) measurement: Lipid
oxidation was determined using a TBARS method (Ahn et al., 1998). Five grams of
ground chicken meat were weighed into a 50-mL test tube and homogenized with
50μL BHT (7.2%) and 15 mL of deionized distilled water (DDW) using a Polytron
homogenizer (Type PT 10/35, Brinkman Instruments Inc., Westbury, NY, USA) for
15s at high
speed. One milliliter of the meat homogenate was transferred to a
disposable test tube (13 × 100 mm), and thiobarbituric acid (15 mM TBA/15% TCA,
2 mL) was added. The mixture was vortex mixed and incubated in a boiling water
bath for 15 min to develop color. Then samples were cooled in the ice-water for 10
min, mixed again, and centrifuged for 15 min at 2,500 x g at 4 oC. The absorbance of
the resulting supernatant solution was determined at 532 nm against a blank
containing 1 mL of DDW and 2 mL of TBA/TCA solution. The amounts of TBARS
were expressed as mg of malondialdehyde (MDA) per kg of meat.
3. Color measurement (Konioka Minolta): The color of meat was measured on the
surface of meat samples using a Konica Minolta Color Meter (CR-410, Konioka
Minolta, Osaka, Japan). The colorimeter was calibrated using an illuminant source C
(Average day light) on a standard white ceramic tile covered with the same film as the
ones used for meat samples to negate the color and light reflectance properties of the
packaging material. The color were expressed as CIE L*- (lightness), a*- (redness),
and b*- (yellowness) values (AMSA, 1991). The areas selected for color
measurement were free from obvious defects that may affect the uniform color
readings. An average from two random readings on each side (upper and lower) of
57
sample surface was used for statistical analysis.
4. Volatile compounds: Volatiles of samples were analyzed using a Solatek 72
Multimatrix-Vial Auto-sampler/Sample Concentrator 3100 (Tekmar-Dohrmann,
Cincinnati, OH, USA) connected to a GC/MS (Model 6890/5973; Hewlett-Packard
Co., Wilmington, DE, USA) according to the method of Ahn et al. (2001). Sample (3
g for raw meat and 2 g for cooked meat) was placed in a 40-mL sample vial, flushed
with helium gas (40 psi) for 3 s, and then capped airtight with a Teflon*fluorocarbon
resin/silicone septum (I-Chem Co., New Castle, DE, USA). Samples from different
treatment were randomly organized on the refrigerated (4°C) holding try to minimize
the variation of the oxidative changes in sample during analyses. The meat sample
was purged with helium (40 mL/min) for 14 min at 20 °C. Volatiles were trapped
using a Tenax/charcoal/silica column (Tekmar-Dohrmann) and desorbed for 2 min at
225 °C, focused in a cryofocusing module (-70 °C), and then thermally desorbed into
a capillary column for 2 min at 225 °C. An HP-624 column (7.5 m, 0.25 mm i.d., 1.4
m nominal), an HP-1 column (52.5 m, 0.25 mm i.d., 0.25m nominal), and an
HP-Wax column (7.5 m, 0.250 mm i.d., 0.25 m nominal) were connected using zero
dead-volume column connectors (J &W Scientific, Folsom, CA, USA). Ramped oven
temperature was used to improve volatile separation. The initial oven temperature of
25 °C was held for 5 min. After that, the oven temperature was increased to 85 °C at
40 °C per min, increased to 165 °C at 20 °C per min, and then increased to 230 °C at
5 °C per min and held for 2.5 min at that temperature. Constant column pressure at
22.5 psi was maintained. The ionization potential of MS was 70 eV, and the scan
range was 20.1 to 350 m/z. The identification of volatiles was achieved by the Wiley
Library (Hewlett-Packard Co.). The area of each peak was integrated using
ChemStationTM software (Hewlett-Packard Co.) and the total peak area (total ion
counts x 104) was reported as an indicator of volatiles generated from the samples.
5. Protein oxidation (total carbonyl): Protein oxidation was determined by the
58
method of Lund et al. (2008) with minor modifications. One gram of muscle sample
was homogenized with a Brinkman Polytron (Type PT 10/35), Brinkman Instrument
Inc., Westbury, NY, USA) in 10 mL of pyrophosphate buffer (2.0 mM Na4P2O7, 10
mM Trizma-maleate), 100 mM KCL, 2.0 mM MgCl2, and 2.0 mM ethylene glycol
tetraacetic acid, pH 7.4). Two equal amounts of meat homogenate (2 mL) were taken
from a sample, precipitated with 2 mL of 20 % trichloroacetic acid, and centrifuged at
12,000 × g for 5 min at room temperature. After centrifugation, the pellet from 1
sample was treated with 2 mL of 10 mM 2,4-dinitrophenylhydrazine dissolved in 2 M
HCL, and the pellet from the other was incubated with 2 M HCL as a blank. During
30 min of incubation in the dark, samples were vortex-mixed for 10 s every 3 min.
The protein was further precipitated with 2 mL of 20% trichloroacetic acid and
centrifuged at 12,000 x g for 5 min. The 2,4-dinitrophenylhydrazine was removed by
washing 3 times with 4 mL of 10 mM HCL in 1:1 (vol/vol) ethanol:ethyl acetate,
followed by centrifuging at 12,000 × g for 5 min. The pellets were finally solubilized
in 2 mL of 6.0 mM guanidine hydrochloride dissolved in 20 mM potassium
dihydrogen phosphate (pH = 2.3). The samples were kept at 5 °C overnight. In next
day, the samples were centrifuged to remove insoluble materials. The absorbance of
supernatant was read at 370 nm. The absorbance values for blank samples were
subtracted from their corresponding sample values. Briefly, Protein concentration was
measured using Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA)
following Microplate Assay protocol at 280 nm absorbance (BioTek-Gen5 Microplate
data collection & analysis software/BioTek Instruments, Inc., Model S4MLFPTA.,
Winooski, VT, USA). The carbonyl content was calculated as nmol/mg protein using
absorption coefficient of 22,000/M/cm as described by Levine et al. (1994).
59
Statistical analysis: Data were analyzed using the procedures of generalized linear
model (Proc. GLM, SAS program, version 9.3, 2012). Mean values and standard error of
the means (SEM) were reported. The significance was defined at P < 0.05 and Tukey test
or Tukey’s Multiple Range test were used to determine whether there is a significant
difference between the mean values.
Result and Discussion
Lipid Oxidation
Lipid oxidation is among the most critical quality parameters because it dose
not only interacts with protein oxidation (Hall, 1987) that causes meat discoloration
(Guillen-Sans and Guman-Chozas, 1998), but also is primarily responsible for
producing many off-odor and rancid flavor in the meat during storage (Vercellotti et
al., 1992; Min et al., 2005). The antioxidant activity of oregano essential oil in the
preliminary studies indicated that there were no significant differences (p > 0.05)
between treatments at day 0 in raw meat. TBARS values increased more in the
cooked meat compared to the raw meat during storage. In general adding oregano
essential oil for both raw and cooked ground chicken meat reduced the TBARS values
(Tables 1 and 4). This result agreed with other studies that added oregano oil directly
to the meat (Fasseas et al., 2007; Chouliara et al., 2007) or indirectly in the animal
diet (Botsoglou et al., 2002; Botsoglou et al., 2003a, b). Among the treatments,
oregano essential oil at 400 ppm showed the highest antioxidant effect. The TBARS
values of raw meat were very low compared to the cooked meat. Cooked meat is more
sensitive to oxidative changes than the raw meat because the antioxidant enzymes in
meat are denatured during cooking, iron ions were released from the intracellular to
extracellular compartment, membrane bi-layers became damaged and phospholipids
were open to catalysts and oxygen during cooking and storage (Gray et al., 1996).
However, the TBA values for the ground raw chicken meat showed very little changes
during storage regardless of the oregano essential oil treatments. The raw meat values
60
are in agreement with those of Chouliara et al. (2007) who reported TBA values of
0.1-0.28 in fresh breast chicken meat when 0.1 % oregano oil was added and stored
for 7 days. Kim et al. (2002) reported TBARS values of between 0.13-0.68 for turkey
meat were found after 7 day of storage. All the oregano essential oil levels (100, 300,
and 400 ppm) showed significant differences compared with the control for both raw
and cooked meat at day 7 of storage. The oregano essential oil at 400 ppm, however,
showed higher effect than BHA (5 ppm) especially on the cooked meat at day 7 (Table
4). The oregano essential oil at 100 and 200 ppm could be the minimum levels that
can be used to slow down the oxidative changes in ground chicken meat.
Protein oxidation
The functional properties of proteins such as protein solubility, gelation, and
emulsification capacity in the foods are highly dependent on their amino acid
composition and structure (Uchida and Kawakishi, 1992; Xiong, 2000). The
relationship between protein oxidation and overall food quality is also well
documented. Ground meat is usually more susceptible to oxidation than the whole
meat cuts due to their size and surface area that contact with the oxygen and the
presence of primary free radicals. Several other factors that may affect or increase
protein carbonyl formation in the meat system such as metal catalysts (iron, copper,
hem and non-heme iron, and myoglobin), pH, temperature, and presence of other
inhibitors (antioxidant phenolic compounds) should be considered (Xiong, 2000; Park
et al., 2007; Estevez., 2011; Promeyrat et al., 2011;). In this study oregano essential
oil significantly (P < 0.05) reduced the total carbonyl formation (nmol/mg of protein)
especially in the cooked meat (Table 8). There was no significant difference (P > 0.05)
in the total carbonyl values between the treatments in raw meat during the first 3 days
of storage (Table 2). The changes in the total carbonyl content were very low in raw
meat compared to the cooked meat during storage. This could have happened because
the production of free radicals was higher in the cooked meat than in the raw meat. In
addition, the total antioxidant capacity of the fresh chicken meat is usually higher than
61
that of the cooked meat. The reduction of antioxidant capacity in cooked meat is
caused by the denaturation of antioxidant enzymes, exposure of amino acid side
chains to the ROS, and loss of endogenous antioxidants (Fasseas et al., 2007; Serpen
et al., 2012) by heat treatment. Generally, the formation of total carbonyl agrees with
the TBA values in raw meat during storage. Oregano essential oil at 400 ppm showed
the strongest effect in reducing the total carbonyl values (Tables 2 and 5). Requena et
al. (2003) estimated that the total carbonyl content in various animal tissues is 1-2
nmol/mg protein. However, the result from the DNPH method from several studies
varied widely (0.5-4.8 nmol/mg protein) depending on the meat type, animal species,
experimental conditions, and analysis methods, which make it difficult to give a
general conclusion. However, total estimated carbonyl contents falls in the range of
1-3 nmol/mg protein for raw meat and up to 5 nmol/mg protein for cooked meat
products (Estevez et al., 2005; Sun et al., 2010; Estevea, 2011). The total carbonyl
values agreed well with the TBA results, and meats with the highest level of oregano
oil produced the lowest values. The result in this study agreed with Fasseas et al.,
(2007) who used oregano essential oil (3% w/w) in ground porcine and bovine raw or
cooked meat. They found that the oregano oil showed higher antioxidant activity than
sage and control treatments during the storage at 4 oC. There were no significant
differences between oregano oil treatments at 100 and 300 ppm, and BHA for both
raw and cooked meat at day 7 (Tables 2 and 5). The significant antioxidant effect of
oregano oil was more prominent when cooked meat was compared with the raw meat.
Color values
Color is one of the most important quality parameters that determine retail sales
and marketing strategies for raw ground chicken meat (Sandusky and Heath, 1998). In
addition, meat color for various products is very important for the consumer’s
decision for meat consumption (Hunt et al., 1993; Bekhit and Fasutman, 2005). The
lightness (L*-values) of ground chicken meat significantly decreased during storage.
Oregano essential oil added at level 400 ppm showed the highest stabilizing effects
62
for L*- and a*-values (Table 3). However, no significant difference (P > 0.05) among
the treatments on the redness or a*-values during storage was detected. All treatments
showed significant positive effects (P < 0.05) on L*-values compared to the control.
These results are very similar to those of Chouliara et al. (2007) and Mastromatteo et
al. (2009) who reported that oregano oil or their components had no significant effect
on a*- but significantly preserved the L*-values at the end of storage. In general,
b*-values decreased during storage time, but oregano oil maintained the b*-values of
the meat after 7 days of storage, which agreed with the results of Choulira et al.
(2007). There were no significant difference on a*-values between the treatments at
day 7 of storage. This may be due to the low myoglobin content in the chicken breast
meat (0.05 mg/g) that makes this change small. The same result was found by Ahn
and Lee (2004) who reported that a*-values did not change much during 15 days of
storage in both aerobic- and vacuum-packaged turkey breast meat. However, the
a*-values of meat with oregano oil were the highest at day 7, which showed the role
of oregano oil to stabilize the red color.
Volatiles production
Warmed-over flavor is the most critical problem affecting cooked meat
products (Jo et al., 2006). The primary volatiles produced in raw and cooked meat can
be further degraded into secondary products and cause rancid flavor. Adding oregano
essential oil reduced the amount of off-odor volatiles in cooked chicken meat (Tables
6-8). The research on adding oregano oil to ground meat is rare and nobody has
analyzed the effect of oregano oil on the volatiles (ketone, aldehydes, hydrocarbon
and sulfur compounds) formation in chicken meat. The volatiles produced by the raw
meat were very low and GC-MS did not detect any sulfur or aldehyde compounds
during storage time. No significant differences for most of volatiles between
treatments for both raw (data not shown) and cooked meat at day 0 of storage were
found. Heptanal, dimethyl disulfide, and 1-butanol were found only in the control
treatment at day 0 of storage time. This reflects lipid oxidation development in the
63
raw meat before cooking in the control treatment. However, oregano oil was able to
decrease aldehydes formation especially in the cooked meat during storage. Oregano
oil at 400 ppm showed the strongest effect in suppressing the volatile formation,
especially aldehydes such as hexanal and pentanal. Oregano oil levels at 100 ppm and
300 ppm also significantly (P < 0.05) reduced the amounts of most of the aldehydes
compared to the control in cooked meat. Lipid oxidation-dependent volatiles such as
aldehydes (e.g., propanal, hexanal, pentanal and heptanal) and hydrocarbons
(cyclohexane, hexane, heptane and octane) decreased by the addition of oregano oil
during storage. Sulfur volatiles decreased after 3 days of storage in the control
treatment. This agrees with several studies done using aerobically stored meat, from
which most of the sulfur volatile compounds escaped or disappeared after 5 days of
storage (Lee et al., 2003) due to their high volatility (Nam et al., 2002, 2003).
Oregano oil at 300 and 400 ppm reduced the amount of dimethyl disulfide at days 0
and 3 of cooked meat compared to other treatments (Tables 6 and 7). Hexanal and
pentanal were reported as the major indicators of lipid oxidation (Ahn et al., 1998;
Shadidi and Pegg, 1994). In addition, positive relationships between aldehydes and
TBARS values were reported (Du et al., 2003). Hexanal formation in the cooked meat
increased rapidly during storage time (Tables 6-8), which reflected the relationship
between total aldehydes and lipid oxidation status of ground meat. Volatile content of
aerobically-packaged meat increased more rapidly than the vacuum-packaged meat
during storage, indicating the role of oxygen in the formation of volatiles (Du et al.,
2000).
Minor compounds such as carbon disulfide, dimethyl sulfide, 2-butanone, ethyl
acetate, ethyl propionate that did not appear consistently in the samples within the
same treatment were not included in the tables. Samples from oregano oil treatment
showed some terpenoids such as sabinene, paracymen, limonene, camphene, gamma
terpinene, beta myrcene, and alpha terpinene in varying amounts. These compounds
have significant impact on the odor and flavor of meat products. Finally GC-MS
results did not show carvacrol and thymol probably due to their low volatility.
64
Conclusion
Oregano essential oil, at levels 300 and 400 ppm, showed the highest
antioxidant effect to preserve the ground chicken meat. Adding >100 ppm of oregano
essential oil improved color stability of raw meat and decreased off-odor volatile of
cooked meats, but their effects were much clearer in cooked than in raw meat. Over
all, oregano essential oil can be used in place of synthetic antioxidant (e.g., BHA) to
prevent quality deterioration in raw and cooked meat during storage.
65
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71
Table 1. TBARS value of raw ground chicken breast meat at different storage
time at 4 ℃.
Control
100 ppm
300 ppm
400 ppm
5 ppm
SEM
Time
without
Oregano
Oregano
Oregano
BHA
---------- TBARS (mg/Kg)meat ---------az
ay
ay
ax
ax
0.005
bcx
0.005
Day 0
0.13
Day 3
0.16
Day 7
0.23
0.15
0.13
0.12
0.14
0.005
0.008
0.006
0.006
0.009
SEM
a-c-
ay
ax
0.12
by
0.14
bx
0.11
bcxy
0.12
bx
0.11
cx
0.12
bx
0.13
0.13
bx
0.006
Value with different letters within a row are significantly different (P < 0.05).
n=4.
x-zValue with different letters within a column are significantly different (P < 0.05).
TBARS value in mg malonaldehyde/kg meat.
Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm
oregano oil, and 5 ppm BHA.
72
Table 2. Effect of adding different level of oregano oil on protein oxidation
of raw chicken breast patties during storage time.
Time
Control
100 ppm
300 ppm
400 ppm
5 ppm
Without
Oregano
Oregano
Oregano
BHA
-----------------ay
Day 0
0.7
Day 3
0.77
Day 7
0.94
SEM
a-c-
axy
ax
0.05
Carbonyl (nmol/ mg of protein)
ax
0.69
ax
0.75
abx
0.79
0.04
ax
0.69
ax
0.74
bx
0.77
0.04
SEM
---------------------ax
0.65
ax
0.65
bx
0.69
0.04
ax
0.69
ax
0.75
abx
0.80
0.04
0.05
0.03
0.06
Value with different letters within a row are significantly different (P < 0.05).
n=4.
x-zValue with different letters within a column are significantly different (P < 0.05).
Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano
oil, and 5 ppm BHA.
73
Table 3. CIE Color value of ground chicken patties with different level of
oregano oil during storage at 4℃.
Control 100 ppm 300 ppm 400 ppm
5 ppm
SEM
Time
without Oregano Oregano Oregano
BHA
L*
Day 0
64.47abx
64.40abx
64.35bx
64.82abx
64.91ax
0.12
Day 3
63.49ay
63.74axy
63.84ay
64.04ax
63.89ay
0.15
Day 7
62.71bz
63.64ay
63.74ay
63.95ax
63.67ay
0.18
SEM
a*
0.14
0.14
0.09
0.23
0.13
Day 0
8.65ax
8.62ax
8.55ax
8.62ax
8.59ax
0.10
Day 3
6.36
ay
6.35
ay
6.39
ay
6.50
ay
6.38
ay
0.09
Day 7
6.16ay
6.26ay
6.39ay
6.48ay
6.37ay
0.11
SEM
b*
0.07
0.16
0.1
0.08
0.06
Day 0
20.87cx
21.07cbx
21.27bx
21.85ax
21.70ax
0.09
Day 3
18.90cby
19.01cy
19.75cby
21.41ay
20.80bay
0.28
Day 7
18.62by
18.58by
18.68ay
19.29az
18.97abz
0.10
0.15
0.14
0.28
0.08
0.16
SEM
a-cValue with different letters within a row are significantly different (P < 0.05).
n=4.
x-zValue with different letters within a column are significantly different (P <
0.05).
CIE Color L*, a*, b*-value of ground chicken patties with different level of
oregano oil during storage at 4 ℃.
Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm
oregano oil, and 5 ppm BHA.
74
Table 4. TBA value of cooked ground chicken breast meat at different storage
time at 4℃.
Control
100 ppm
300 ppm
400 ppm
BHA
SEM
Time
without
Oregano
Oregano
Oregano
5 ppm
---------- TBARS (mg/Kg)meat ---------Day 0
0.26ay
0.14by
0.09by
0.08bz
0.11bz
0.01
Day 3
2.44ax
2.35ax
0.72cx
0.27dy
1.87by
0.06
Day 7
2.55ax
2.43bx
0.74dx
0.35ex
2.02cx
0.03
SEM
0.07
0.04
0.02
0.006
0.034
a-c
Value with different letters within a row are significantly different (P < 0.05). n=4.
value with different letters within a column are significantly different (P < 0.05).
TBA value in mg malonaldehyde/kg meat.
Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano
oil, and 5 ppm BHA.
x-z
Table 5. Effect of adding different level of Oregano oil on protein oxidation of
chicken breast patties during storage time/cooked meat.
Time
Control
100 ppm
300 ppm
400 ppm
5 ppm
without
Oregano
Oregano
Oregano
BHA
SEM
-----------Carbonyl (nmol/mg of protein)-------------Day 0
0.69ay
0.44by
0.43bx
0.40bx
0.47by
0.041
Day 3
1.25ay
0.50by
0.45bx
0.42bx
0.48by
0.038
Day 7
1.90ax
0.95bcx
0.56bcx
0.44cx
1.00bx
0.12
SEM
0.15
0.074
0.056
0.046
0.032
a-c
Value with different letters within a row are significantly different (P < 0.05). n=4.
Value with different letters within a column are significantly different (P < 0.05).
TBA value in mg malonaldehyde/kg meat.
Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm
oregano oil, and 5 ppm BHA
x-z
75
Table 6. Profile of volatiles cooked meat chicken patties with different level of
oregano oil at day 0.
Total Ion counts * 104
Compounds
2-propanone
1-butanol
2-propanol
Hexane
Heptane
1-propanol
Pentanal
Heptanal
Octane
Cyclohexane
Dimethy-DS
Hexanal
α-Pinene
Camphene
Limonene
β-Myrcene
γ-Terpinene
Sabinene
Control
without
2758b
548a
381bc
1775a
102a
2832a
215a
35a
86a
107a
507a
467a
0b
0b
0b
0c
0b
0c
100 ppm
Oregano
2552b
0b
319c
336b
59ab
2622a
270a
0b
235a
63abc
0b
167b
265a
256ab
72ab
162bc
285b
0c
300 ppm
Oregano
2466b
18b
1371a
244b
12b
1542b
125a
0b
406a
86ab
0b
93b
226a
457ab
173a
1056ab
2159ab
149b
400 ppm
Oregano
2733b
0b
959ab
243b
49ab
1027b
292a
0b
319a
30bc
0b
91b
362a
715a
165a
1891a
4637a
354a
5 ppm
BHA
6001a
0b
738bc
169b
0b
1720b
110a
0b
165a
0c
0b
551a
0b
0b
0b
0c
0b
0c
SEM
517
36
133
203
19
174
82
2
90
17
82
66
36
142
25
226
682
20
a-d
Different letters within a row are significantly different (P<0.05). n=4.
Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil,
and 5 ppm BHA.
76
Table 7. Profile of volatiles cooked meat chicken patties with different level of
oregano oil at day 3.
Total Ion counts * 104
Compounds
2-propanone
Pentane
2-propanol
Hexane
Heptane
1-propanol
Propanal
Pentanal
Heptanal
Octane
Cyclohexane
Dimethy-DS
Hexanal
α-Pinene
Camphene
Limonene
β-Myrcene
γ-Terpinene
Sabinene
a-d
Control
without
4106a
580a
620b
216a
86a
2279a
58b
1065a
0
147a
31a
23a
11371a
0b
0b
0b
0b
0b
0b
100 ppm
Oregano
4854a
0b
3324ab
20b
0b
1299b
15b
174bc
0
204a
0b
0b
3194b
0b
0b
0b
0b
0b
0b
300 ppm
Oregano
4530a
0b
3643a
0b
0b
2136a
0b
126bc
0
160a
0b
0b
1341b
475a
299a
381a
577ab
1609ab
180a
400 ppm
Oregano
4517a
0b
1337ab
0b
0b
1956a
0b
0c
0
175a
0b
0b
1448b
444a
328a
430a
740a
1984a
315a
5 ppm
BHA
3458a
0b
905ab
0b
0b
1703ab
144a
466b
0
179a
0b
0b
4167b
0b
0b
0b
0b
0b
0b
SEM
797
61
668
15
7
134
14
89
0
43
5
1
752
51
55
92
163
396
38
Different letters within a row are significantly different (P<0.05). n=4.
Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil,
and 5 ppm BHA.
77
Table 8. Profile of volatiles cooked meat chicken patties with different level of
oregano oil at day 7.
Total Ion counts * 104
Compounds
Control
without
100 ppm
Oregano
300 ppm
Oregano
400 ppm
Oregano
5 ppm
BHA
SEM
2-propanone
2307a
2031a
1966a
2065a
1224a
251
2-propanol
Hexane
Heptane
Acetaldehyde
Propanal
Pentanal
Heptanal
Octane
Cyclohexane
991a
47ab
160a
90a
297a
1153a
72a
322ab
127a
909a
172ab
0b
0b
0b
563b
0b
137b
45b
1008a
0b
0b
0b
0b
305bc
0b
397ab
45b
452a
0b
0b
0b
0b
297bc
0b
309ab
26b
951a
209a
132a
0b
0b
254c
0b
822a
184a
188
40
17
1
2
69
2
128
18
Hexanal
α-Pinene
Camphene
Limonene
β-Myrcene
γ-Terpinene
Sabinene
41262a
0c
0b
0c
0b
0b
0c
7887b
325bc
258b
114bc
335ab
491ab
73bc
1252c
762a
866a
349ab
640a
1326a
133b
1215c
378b
503ab
465a
412a
1020a
355a
2095bc
0c
0b
0c
0b
0b
0c
1422
85
133
62
92
200
23
a-d
Different letters within a row are significantly different (P<0.05), n=4.
Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil,
and 5 ppm BHA
78
Preliminary study data.
Table 9. TBA value of cooked ground chicken breast meat at different storage time at 4℃.
------------------- TBARS (mg/kg) ------------------
Time
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
SEM
78
Day 0
Day 3
Day 7
a-e
0.34a
0.20b
0.19bc
0.19bc
0.15c
0.15c
0.23b
0.38a
0.35a
0.35a
0.01
0.76ab
0.33cd
0.28dce
0.20de
0.16e
0.15e
0.37c
0.79a
0.71ab
0.70b
0.03
0.83a
0.37c
0.35cd
0.26ed
0.21e
0.18e
0.41c
0.81a
0.74a
0.64b
0.02
Value with different letter within a raw are significantly different (P<0.05).
Treatments : T1: Control; T2: 100 ppm Oregano;T3: 200 ppm oregano; T4:300 ppm oregano; T5:400 ppm oregano oil; T6:
500 ppm oregano; T7: 5 ppm BHA; T8: 2.5 ppm Tannic acid; T9:5 ppm Tannic acid; T10:10 ppm Tannic acid
79
Preliminary combination study
TBA Value
TBA Mg/kg meat
2.5
2
Day 0
Day 3
day 7
1.5
1
0.5
0
1
2
3
4
5
6
7
8
Treatment
9
10
11
12
13
Figure 1: TBA value of cooked meat at different levels of oregano and
tannic acid in different combinations.
The Antioxidant effect of tannic acid and oregano oil speratly or combined at
different level (12 combinations) using 0, 100, 200, and 400 of oregano oil with 5
and 10 ppm of tannic acid.
80
Table 10. TBA value of cooked meat using different level and combinations of
oregano oil and tannic acid at 4 °C.
Treatment
Day 0
Day 3
Day 7
SEM
1/control
0.27abz
1.78ay
2.19ax
0.022
2/ 5TA + 0 O
0.22bz
1.17by
1.87bx
0.005
3/ 10 TA + 0 O
0.23aby
0.82ex
0.93ex
0.047
4/0TA +100 O
0.23bz
1.04cdy
1.47cx
0.041
5/5TA + 100 O
0.25abz
0.98dy
1.38cx
0.028
6/10TA+ 100 O
0.26aby
0.35ghx
0.39fgx
0.018
7/0TA+ 200 O
0.26abz
0.54fy
1.17dx
0.019
8/5TA+ 200 O
0.35ay
0.41gxy
0.48fx
0.028
9/10TA+ 200 O
0.24aby
0.25hiy
0.29ghx
0.008
10/0TA+ 400O
0.29abx
0.32ghix
0.34fghx
0.015
11/5TA+ 400O
0.24aby
0.26hixy
0.29ghx
0.008
12/10TA+400O
0.21bx
0.22ix
0.24hx
0.005
13/BHA 5 ppm
0.29abz
1.14bcy
1.79bx
0.04
0.026
0.023
0.029
SEM
x-zValue with different letters within a row are significantly different (P < 0.05). n=4.
a-iValue with different letters within a column are significantly different (P < 0.05).
The Antioxidant effect of tannic acid and oregano oil speratly or combined at
different level (12 combinations) using 0, 100, 200, and 400 ppm of oregano oil
with 5 and 10 ppm of tannic acid.
81
CHAPTER 4
THE EFFECT OF ADDING DIFFRENT LEVEL OF TANNIC ACID ON THE
STORAGE STABILITY AND QUALITY CHARACTERISTICS OF GROUND
CHICKEN MEAT
Marwan Al-Hijazeen1, Aubrey Mendonca2,* and Dong Uk Ahn1,*
1
Department of Animal Science, Iowa State University, Ames, Iowa 50011, USA, and
2
Department of Food Science and Human Nutrition, and 2Department of Animal
Science, Iowa State University, Ames, IA 50011, USA.
*Corresponding author: D. U. Ahn
Phone: (515) 294-6595, Fax: (515) 294-6595
Email: [email protected]
82
Abstract
The objective of this study was to determine the effect of tannic acid (TA) on the
oxidative storage stability of ground chicken breast meat. Five different treatments
including 1) control (none added), 2) 2.5 ppm TA, 3) 5 ppm TA, 4) 10 ppm TA, and 5) 5
ppm butylated hydroxyanisole (BHA) were added to boneless, skinless ground chicken
breast meat, used for both raw and cooked meat studies. For the raw meat study, ground
chicken breast meat was packaged in oxygen-permeable bags and stored at 4 ℃ cooler for
up to 7 days. For the cooked study raw meat samples were packaged in
oxygen-impermeable vacuum bags and then cooked in-bag to the internal temperature
of 75 °C before transfer to the oxygen permeable bags for storage. Both raw and cooked
meats were analyzed for lipid and protein oxidation, color, and volatiles at 0, 3, and 7
days of storage. Both raw and cooked chicken breast meat added with 10 ppm of TA
had significantly (P < 0.05) lower lipid and protein oxidation than other treatments
during storage. In addition, TA at 10 ppm level maintained the highest color a*- and
L*-values during storage. In cooked chicken breast meat added with > 5 ppm TA had
significantly (p < 0.05) lower amounts of off-odor volatiles than other treatments.
Among the volatile compounds, the amount of hexanal increased rapidly during storage
for cooked meat. However, meats added with > 5 ppm TA showed the lowest amount of
hexanal and other aldehydes related to lipid oxidation, indicating a strong antioxidant
effect of TA in ground chicken breast meat. Furthermore, the differences among the
treatments were bigger in cooked meat than in raw meat, indicating that the antioxidant
efficiency of TA in cooked meat was high. Therefore, TA at > 5 ppm can be used as a
good natural preservative in ground chicken meat to improve its quality during storage.
Key Words: Tannic acid, BHA, Lipid and protein oxidation, Volatiles, Chicken patties.
83
Introduction
Recently, the poultry meat industry has been increasingly seeking natural
antioxidants to replace the synthetic antioxidants they currently use (Karre et al.,
2013). Something that is safe to add, does not cause human health problems, and
positively affect the consumers’ final purchase decisions (Solomakos et al., 2008;
Naveena et al., 2008; Gulcin et al., 2010). Several studies using natural antioxidants
from plant origins showed their positive effects on improving meat quality and
extending shelf life (Abdel-hamied et al., 2009). Meat color and odor are considered
the most important attributes affecting primary consumer evaluation for the meat
quality (Liu et al., 1995; Mancini and Hunt, 2005). Usually fresh meat is
characterized by cherry red color due to the formation of oxymyoglobin (OxyMb)
pigment. However, farther oxidation will convert the meat into brown color due to
metmyoglobin (MetMb) formation (Bekhit and Faustman, 2005; Bou et al., 2008).
Lipid oxidation is considered as the major problem affecting meat quality (Hui
et al., 2006; Ahn et al., 2007; Nam et al., 2007) because it changes color, generate
off-odor (Gray and Monahan, 1992; Gray et al., 1996; Min and Ahn, 2005), and
impair protein functionality (Hall, 1987). Protein functionality such as solubility,
emulsification, water binding capacity, and texture are also affected by lipid oxidation
secondary products and their interaction with protein oxidation products (Hall, 1987;
Gray et al., 1996). The progress of oxidation in fresh meat depends on many internal
factors such as catalyst (Iron Fe, Cu, etc), antioxidant capacity, pH, and free radical
formation. However, many external factors such as high storage temperature, oxygen
availability, meat processing method, and additives also can influence lipid and
protein oxidation in meat (Ahn et al., 2009).
Therefore, finding new natural antioxidants to resolve these problems in meat
and other foods is important (Wong et al., 2006; Descalzo and Sancho, 2008). Herbs
and plant extracts such as rosemary (Sebranek et al., 2005), oregano (Rojas and
Brewer, 2008), grape seed extract (Brannan, 2008), plum (Lee and Ahn, 2005),
84
bearberry (Carpenter et al., 2007) and several oleoresin plant extracts such as garlic
and onion (Nam et al., 2007) had been tested to prevent oxidation in meat because
they contained high levels of antioxidants (Karre et al., 2013). Tannins are one of the
secondary compounds produced by plants to protect themselves from external
herbivores and diseases (Salminen et al., 2011). These compounds have biological,
metal ion chelating and antioxidant (Hagerman, 2010), and protein precipitating
activities (Hagerman et al., 1998). In addition tannins showed positive effect on meat
color stability and extend their self life during refrigeration time (Luciano et al., 2009;
Maqsood and Benjakul, 2010c). The tannins are defined as water soluble phenolic
compounds with molecular weights between 500 and 3000 Da, and have special
properties such as precipitating alkaloids, gelatin, and other proteins (Bate-Smith and
Swain, 1962).
These properties of tannins are based on their chemical structures which have
two or three phenolic hydroxyl groups on phenyl ring. The antioxidant activity of
tannic acid was explained by several researchers through their ability to prevent
hydroxyl radical formation (Lopes et al., 1999), metal chelating activity (Okuda and
Ito, 2011), radical scavenging activity (Glucin et al., 2010) and through tannic acid
hydrolysis into gallic acid and a galloyl group (Kim et al., 2010).
Tannins are classified into two different main groups - hydrolysable and
condensed tannins. The hydrolysable type of tannins contains polyhydric alcohol and
hydroxyl groups, which are esterifies by gallic acid (gallotannins) or to hexahydroxy
diphenic acid (ellagitannins) (Okuda et al., 1985; Okuda, 2005; Haslam, 1989; Okuda
and Ito, 2011). Condensed tannins are more complex in their structure compared to
hydrolysable tannins. They are mainly produced by two main polymerized products of
flavan-3-ols and flavan-3,4-diols, or mixture of both (Chung et al., 1998).
Tannic acid is a specific commercial type of tannins that has a molecular
formula C76H52O46, and 10 ppm is the maximum level allowed in meat products (FDA,
2011). Tannic acid is a yellowish-white to light-brown powder, and is soluble in water
and alcohol. In addition, tannic acid is classified as an important gallotannins which
85
belong to the hydrolysable class (Miranda et al., 1996). These activities make the
tannic acid, especially the hydrolysable type, a possible replacement for the synthetic
antioxidants. Very little studies were done to determine the effect of tannic acid on the
quality of meat products (Luciano et al., 2009; Maqsood and Benjakul, 2010a; b; c).
Tannic acid is classified as generally recognized as safe (GRAS) by the Food and
Drug Administration (FDA) and can be used directly in foods. However, little work
has been done to determine their effect on the storage stability and quality
characteristics in ground chicken meat.
The objective of this study was to investigate the effect of adding tannic acid on
stability and meat quality of ground chicken meat (raw and cooked meat) during
storage.
Materials and Methods
Sample preparation: One hundred and twenty, 6-wk-old broilers raised on a
corn-soybean meal diet were slaughtered using the USDA guidelines (USDA, 1982).
The chicken carcass where chilled in ice water for 2 h and drained in a cold room, and
the breast muscles were separated from the carcasses at 24h after slaughter. Boneless
breast muscles were cleaned, skins removed, external fats trimmed off, and stored at
-20 ℃ freezer until used.
The frozen meats were thawed in a walk-in cooler (4 °C ), ground twice through a
10-mm and a 3-mm plates (Kitchen Aid, Inc., St. Joseph, MI, USA) before use. Five
different treatments including 1) (none added), 2) 2.5 ppm tannic acid, 3) 5 ppm
tannic acid, 4) 10 ppm tannic acid, and 5) 5 ppm butylatedhydroxy anisole (BHA)
were prepared. Tannic acid powder containing 90 % tannin was obtained from
Sigma-Aldrich (St. Louis, MO, USA). The tannic acid contained gallic acid,
monogalloyl glucose, digalloyl glucose, trigalloyl glucose, tetragalloyl glucose,
pentagalloyl glucose, ESA galloyl glucose, EPTA galloyl glucose, and octagalloyl
glucose. The product is hydrolyzable tannin obtained from oak gall nuts from Quercus
86
infectoria.
Tannic acid (0.1 g) was dissolved in 50 ml of de-ionized distilled water (DDW)
and stored in a dark area to prevent exposure to air and light. BHA powder (0.1 g) was
dissolved in 10 ml of 100% ethanol, and then mixed with 50 ml mineral oil to make
their stock solution. The ethanol added was removed using a rotary evaporator
(BUCH Rotavapor, Model R-200) at (70 °C, 175 mbar vacuum pressure) before
adding the stock solution to meat samples. Each additive treatment was added to the
ground breast meat and then mixed for 2 min in a bowl mixer (Model KSM 90;
Kitchen Aid Inc., St. Joseph, MI, USA). All treatments were added with the same
amounts of mineral oil to provide the same conditions.
For raw-meat study, the prepared meat samples (approximately 100 g each) were
individually packaged in oxygen-permeable bags (polyethylene, 4 x 6.2 mil, Associated
Bag Co., Milwaukee, WI), stored at 4 ℃ cooler for up to 7 days, and analyzed for lipid
and protein oxidation, and color at 0, 3, and 7 days of storage.
The same preparation method was used for cooked meat study, but the raw meat
samples were packaged in oxygen impermeable vacuum bags (O2 permeability, 9.3
mL O2/m2/24 h at 0 °C, Koch, Koch, Kansas City, MO, USA), and the meats were
cooked in-bag in a 90 °C water bath (Isotemp®, Fisher Scientific Inc., Pittsburgh, PA,
USA) until the internal temperature of the meat reached to 75 °C. After cooling to
room temperature, the cooked meat was transferred to a new oxygen-permeable bag
(polyethylene, 4 x 6.2 mil, Associated Bag Co., Milwaukee, WI, USA), and stored at
4 °C for up to 7 days, and analyzed for lipid and protein oxidation and volatiles at 0, 3,
and 7 days of storage.
2-Thiobarbituric acid-reactive substances (TBARS) measurement: Lipid oxidation
was determined using a TBARS method (Ahn et al., 1998). Five grams of ground
chicken meat were weighed into a 50-mL test tube and homogenized with 50μL BHT
(7.2%) and 15 mL of deionized distilled water (DDW) using a Polytron homogenizer
(Type PT 10/35, Brinkman Instruments Inc., Westbury, NY, USA) for 15s at high
87
speed. One milliliter of the meat homogenate was transferred to a disposable test tube
(13 × 100 mm), and thiobarbituric acid (15 mM TBA/15% TCA, 2 mL) was added.
The mixture was vortex mixed and incubated in a boiling water bath for 15 min to
develop color. Then samples were cooled in the ice-water for 10 min, mixed again,
and centrifuged for 15 min at 2,500 x g at 4 oC. The absorbance of the resulting
supernatant solution was determined at 532 nm against a blank containing 1 mL of
DDW and 2 mL of TBA/TCA solution. The amounts of TBARS were expressed as
mg of malondialdehyde (MDA) per kg of meat.
Color measurement: The color of meat was measured on the surface of meat samples
using a Konica Minolta Color Meter (CR-410, Konioca Minolta, Osaka, Japan). The
colorimeter was calibrated using an illuminant source C (Average day light) on a
standard white ceramic tile covered with the same film as the ones used for meat
samples to negate the color and light reflectance properties of the packaging material.
The color were expressed as CIE L*- (lightness), a*- (redness), and b*- (yellowness)
values (AMSA, 1991). The areas selected for color measurement were free from
obvious defects that may affect the uniform color readings. An average from two
random readings on each side (upper and lower) of sample surface was used for
statistical analysis.
Volatile analysis: Volatiles of samples were analyzed using a Solatek 72
Multimatrix-Vial Auto-sampler/Sample Concentrator 3100 (Tekmar-Dohrmann,
Cincinnati, OH, USA) connected to a GC/MS (Model 6890/5973; Hewlett-Packard
Co., Wilmington, DE, USA) according to the method of Ahn et al. (2001). Each
sample (3 g for raw meat and 2 g for cooked meat) was placed in a 40-mL sample vial,
flushed with helium gas (40 psi) for 3 s, and then capped airtight with a
Teflon*fluorocarbon resin/silicone septum (I-Chem Co., New Castle, DE, USA).
Samples from different treatments were randomly organized on the refrigerated (4 ℃)
holding try to minimize the variation of the oxidative changes in sample during
88
analyses. The meat sample was purged with helium (40 mL/min) for 14 min at 20 °C.
Volatiles were trapped using a Tenax/charcoal/silica column (Tekmar-Dohrmann) and
desorbed for 2 min at 225 °C, focused in a cryofocusing module (-70 °C), and then
thermally desorbed into a capillary column for 2 min at 225 °C. An HP-624 column
(7.5 m, 0.25 mm i.d., 1.4 m nominal), an HP-1 column (52.5 m, 0.25 mm i.d.,
0.25m nominal), and an HP-Wax column (7.5 m, 0.250 mm i.d., 0.25 m nominal)
were connected using zero dead-volume column connectors (J &W Scientific, Folsom,
CA, USA). Ramped oven temperature was used to improve volatile separation. The
initial oven temperature of 25 °C was held for 5 min. After that, the oven temperature
was increased to 85 °C at 40 °C per min, increased to 165 °C at 20 °C per min, and
then increased to 230 °C at 5 °C per min and held for 2.5 min at that temperature.
Constant column pressure at 22.5 psi was maintained. The ionization potential of MS
was 70 eV, and the scan range was 20.1 to 350 m/z. The identification of volatiles was
achieved by the Wiley Library (Hewlett-Packard Co.). The area of each peak was
integrated using ChemStationTM software (Hewlett-Packard Co.) and the total peak
area (total ion counts x 104) was reported as an indicator of volatiles generated from
the samples.
Protein oxidation: Protein oxidation was determined by the method of Lund et al.
(2008) with minor modifications. One gram of muscle sample was homogenized with
a Brinkman Polytron (Type PT 10/35), Brinkman Instrument Inc., Westbury, NY,
USA) in 10 mL of pyrophosphate buffer (2.0 mM Na4P2O7, 10 mM Trizma-maleate),
100 mM KCL, 2.0 mM MgCl2, and 2.0 mM ethylene glycol tetraacetic acid, pH 7.4).
Two equal amounts of meat homogenate (2 mL) were taken from a sample,
precipitated with 2 mL of 20 % trichloroacetic acid, and centrifuged at 12,000 × g for
5 min at room temperature. After centrifugation, the pellet from 1 sample was treated
with 2 mL of 10 mM 2,4-dinitrophenylhydrazine dissolved in 2 M HCL, and the
pellet from the other was incubated with 2 M HCL as a blank. During 30 min of
incubation in the dark, samples were vortex-mixed for 10 s every 3 min. The protein
89
was further precipitated with 2 mL of 20% trichloroacetic acid and centrifuged at
12,000 x g for 5 min. The 2,4-dinitrophenylhydrazine was removed by washing 3
times with 4 mL of 10 mM HCL in 1:1 (vol/vol) ethanol:ethyl acetate, followed by
centrifuging at 12,000 × g for 5 min. The pellets were finally solubilized in 2 mL of
6.0 mM guanidine hydrochloride dissolved in 20 mM potassium dihydrogen
phosphate (pH = 2.3). The samples were kept at 5 oC overnight. In next day, the
samples were centrifuged to remove insoluble materials. The absorbance of
supernatant was read at 370 nm. The absorbance values for blank samples were
subtracted from their corresponding sample values. Briefly, Protein concentration was
measured using Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA)
following Microplate Assay protocol at 280 nm absorbance (BioTek-Gen5 Microplate
data collection & analysis software/BioTek Instruments, Inc., Model S4MLFPTA.,
Winooski, VT, USA). The carbonyl content was calculated as nmol/mg protein using
absorption coefficient of 22,000/M/cm as described by Levine et al. (1994).
Statistical analysis: Data were analyzed using the procedures of generalized linear
model (Proc. GLM, SAS program, version 9.3, 2012). Mean values and standard error of
the means (SEM) were reported. The significance was defined at P < 0.05 and Tukey test
or Tukey’s Multiple Range test were used to determine whether there is a significant
difference between the mean values.
Results and Discussion
Lipid Oxidation
In the raw chicken meat there was no significant difference (P > 0.05) in
TBARS between treatments at day 0. In addition tannic acid at 2.5 ppm did not show
any significant antioxidant effect on the TBARS values of chicken breast meat during
storage. However, 5 ppm BHA or tannic acid at 5 and 10 ppm showed significant
antioxidant effects after three days of storage (Table 1). Tannic acid at 10 ppm showed
90
the strongest antioxidant effects of all during storage. This result is in agreement with
that of Maqsood and Benjakul (2010a) who found that tannic acid at 100-200 ppm
effectively decreased both peroxide value and TBARS values in catfish slices. They
found that tannic acid exhibited the highest antioxidant activities (peroxide value,
conjugated diene, TBARS values) in both fish oil emulsion and fish mince among the
phenolic compounds (catechin, caffiec acid, ferulic acid, and tannic acid) they had
tested (Maqsood and Benjakul, 2010b). They explained that the high antioxidant
effect of tannic acid was due to its ability to chelate non-heme iron in the fish mince.
Iron is considered a strong pro-oxidant transitional metal that can increase lipid
oxidation in meat. Because of their chemical structure, polyphenol tannic acid inhibits
hydroxyl radical formation from the Fenton reaction by complexing ferrous ions
(Lopes et al., 1999). Lopes et al. (1999) found that the antioxidant activity of tannic
acid is mainly due to iron chelating rather than
·
OH scavenging activity. Maqsood
and Benjakul (2010c) also reported that when tannic acid (200 mg/kg) were added to
the refrigerated ground beef, its peroxide and TBARS values were significantly lower
than that of the control.
Research using tannic acid in cooked meat products has been very rare until
recently. The initial TBARS values in cooked meat showed that all treatments added
with tannic or BHA showed significantly antioxidant effects (Table 4). This could be
explained by the development of lipid oxidation from raw meat as reviewed by Ahn et
al. (2009). This was also in agreement with Rhee (1989) who reported that lipid
oxidation of cooked meat products and their effect on meat flavors depends on the
initial lipid oxidation in the raw meat. In addition, cooked meat is more susceptible to
lipid oxidation than raw meat because the denaturation of antioxidant enzymes and
the structural damages in membrane during cooking can expose phospholipids to
pro-oxidant environment (Ahn et al., 1992; Gray et al., 1996). Therefore, lipid
oxidation in cooked meat increased faster, and the variations between treatments were
clearer than the raw meat. The highest antioxidant effect was observed when 10 ppm
tannic acid was added to the meat. Maqsood and Benjaku (2010c) also found that
91
tannic acid can be a good additive that retards the initiation and propagation steps of
lipid oxidation reaction when added to the ground beef meat.
Antioxidant free radicals of tannic acid characterized as stable and have low energy or
low activity to initiate oxidation of unsaturated fatty acid usually formed (Maqsood
and Benjakul, 2010b). Tannic acid at 10 ppm had stronger antioxidant effect than 5
ppm tannic acid and 5 ppm BHA at day 7 of storage (Table 4). This strongly
suggested that tannic acid could be a good replacement for the synthetic antioxidant in
food containing fat. However, tannic acid at 10 ppm showed the strongest antioxidant
(p < 0.05) effect of all during storage
Protein oxidation
Protein and lipid oxidation are major concerns that affect meat quality during
storage (Nieto et al., 2013). It has been reported that protein oxidation can lead to
changes in overall properties of meat proteins such as gelation, viscosity, solubility,
and emulsification etc. (Xiong, 2000). Little information about the relations between
protein oxidation and their effect on some meat quality attributes are available. In this
study different levels of tannic acid were investigated to study their effect on the total
carbonyl formation (nmol/mg of protein). There were no significant differences (P >
0.05) among the tannic acid treatments in raw chicken breast meat during the first
three days of storage (Table 2). The changes of protein oxidation values in raw
chicken breast meat during the 7-day storage period were very low (0.54 at day zero
to 0.82 at day 7 for control samples). This was in agreement with Xiao et al. (2011)
who found that the total carbonyl content in raw chicken meat patties stored
aerobically at 4 ℃ increased from 0.46 to 0.81 nmol/mg of protein. Tannic acid at
level 10 ppm was the only treatment showed significant antioxidant effect (P < 0.05)
at day 7 of storage. Maqsood and Benjakul (2010c) found that tannic acid reduced the
degradation of myosin heavy chain and actin in meat by suppressing microbial growth
during storage. This indicated that the antimicrobial effect of tannic acid could have
contributed to the total carbonyl formation in meat with different levels of tannic acid.
92
On the other hand, the formation of secondary products (carbonyl and others) of
protein oxidation is also related to the degree lipid oxidation because lipid oxidation is
directly related to protein oxidation.
In contrast to raw meat, total carbonyl values in cooked meat were much higher
than those of raw meat and reached up to 2 nmol/mg protein after 7 days of storage
(Table 5). Other researchers reported that total estimated carbonyl contents were in
the range of 1-3 nmol/mg protein for raw meat and up to 5 nmol/mg protein for
cooked meat products (Estevez et al., 2005; Sun et al., 2010; Estevea, 2011). Adding
tannic acid at 10 ppm successfully delayed the total carbonyl formation in cooked
meat during storage. However, tannic acid at 5 and 10 ppm were more effective than
other treatments in inhibiting carbonyl formation during storage. These results are
well correlated with the TBARS values of cooked ground chicken meat during storage.
The mechanism of tannic acid in preventing protein oxidation still needs more
investigation, but the iron-chelating activity of tannic acid could be major reason for
delaying total carbonyl formation in meat (South and Miller, 1998; Lopes et al.,
1999).
Color values
Chicken breast meat contains much lower myoglobin content than meats from
other animal species such as beef, goat, pork and sheep. Meat color is an important
quality parameter that affects the consumer purchase decision (Bekhit and Faustman,
2005). However, very little research was done on the color of ground chicken meat
(Sanduskey and Heath, 1998). Table 3 showed that L* values of meat regardless of
treatments decreased significantly (P < 0.05) during storage. There were no significant
differences (P > 0.05) in L*-values- among the treatments at days 0 and 3 of storage.
Tannic acid showed the highest significant effect compared to the other treatments
using level of 10 ppm. The breast meat added with 10 ppm tannic acid had the highest
L*-value (lighter meat) and the control had the lowest value (darker meat). The meat
with high lightness value is considered more acceptable by the consumer than the
93
darker one because it can be considered old or spoilage in process (Hood and Riordan,
1973). Regardless of treatments, a*-values decreased during storage. However, no
significant difference was found (P > 0.05) between treatments at day 0. This agreed
with the results of Xiao et al. (2011) who reported that a*- and L*-values of ground
chicken meat decreased significantly after 7 day of refrigerated storage. Mancini and
Hunt (2005) reported that the decrease of a*-value during storage is due to
accumulation of metmyoglobin pigment. Tannic acid at 10 ppm showed the highest
effect in preventing changes in a*-value of all treatments. This is in agreement with
the results of Maqsood and Benjakul (2010c) who found that all ground beef samples
treated with tannic acid had higher oxymyoglobin and a*-value, and received higher
likeness score for color in sensory evaluation. However, no significant difference in
a*-value between tannic acid level at 5, 10 ppm tannic acid, and 5 ppm BHA during
storage. In comparison Luciano et al. (2009) found that dietary tannins improved lamb
minced-meat color stability and their shelf life. Changes in b*-values in chicken
breast meat during storage were not significant even though tannic acid -treated meat
showed higher values than control.
Volatiles production
No significant treatments effects on the volatile contents and profiles in raw
meat were found (data not shown). In cooked meat, tannic acid at 5 and 10 ppm
showed the highest significant effect on the formation of most of these volatiles after
3 days of storage (Tables 7 & 8). For example tannic acid at 10 ppm effectively
decreased pentane, octane, hexanl, and pentanal formation during storage. Hexanal,
which is considered as a good indicator for lipid oxidation (Shahidi et al., 1987; Ahn
et al., 2000), was significantly affected by 10 ppm of tannic acid at Day 7 of storage
(Table 8). Heptanal and nonanal were formed and increased only in control and 2.5
ppm tannic acid-added samples (Tables 7 & 8). The sulfur compounds formations
were found to be inconsistent and disappear from the samples. These sulfur
compounds such as dimethyl disulfide, dimethyl trisulfide were disappeared after the
94
first day of storage. Nam et al. (2002) and Lee et al. (2003) found that most of the
sulfur volatile compounds in turkey breast escaped or disappeared during storage
under aerobically packaging due to their high volatility.
Cooked meat showed higher volatiles formation and more diversity compared
with raw meat. At day 0, however, no significant differences in the amounts and
profiles of volatiles in cooked meat among the treatments were found. Hexanal
increased rapidly in cooked meat during storage due to high degree of lipid oxidation.
Many lipid oxidation-related aldehydes such as propanal, hexanal, and pentanal were
also detected during storage of cooked meat. Tannic acid at 10 ppm showed the
strongest effect in preventing aldehydes formation in cooked meat. This agreed and
reflected the positive relationships between the aldehydes and the degree of lipid
oxidation as reported by Du et al. (2000) and Ahn et al. (1998) (Tables 10 & 11).
Similar effects on the heptanal and nonanl formation were seen when 10 ppm of
tannic acid was added to the cooked meat. Overall, the profile of volatiles indicated
that tannic acid at 10 ppm effectively delayed the formation of lipid-oxidation-related
volatiles in cooked chicken breast meat during storage.
Conclusion
Tannic acid at 5 or 10 ppm could be effective in maintaining meat color and
retarding lipid and protein oxidation and off-odor-related volatiles in fresh ground
chicken breast during storage. Therefore, tannic acid could be a good candidate as a
natural antioxidant to prevent oxidative changes and color changes in raw and cooked
chicken breast meat during storage.
95
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Table 1. TBA value of raw ground chicken breast meat at different storage
time at 4 ℃.
Time
Control
2.5 ppm
5 ppm
10 ppm
5 ppm
SEM
without
Tannic
Tannic
Tannic
BHA
------ TBARS (mg/Kg) meat -----az
Day 0
0.14
Day 3
0.18
Day 7
0.34
SEM
0.001
a-c
ay
ax
az
0.13
ay
0.17
ax
0.33
0.009
ay
0.13
abx
0.15
bcx
0.17
0.007
ax
0.12
bx
0.13
cx
0.14
0.01
ay
0.007
ax
0.010
bx
0.008
0.13
0.18
0.19
0.016
Value with different letter within a row are significantly different (P<0.05).
Value with different letter within a column are significantly different (p<0.05).
N=4.
TBA value in a mg Malonaldehyde per Kg meat.
Treatment: Control, 2.5 ppm tanic acid, 5 ppm tannic acid, 7 ppm tannic
acid, and 5ppm BHA.
X-z
102
Table 2. Effect of adding different level of tannic acid on protein oxidation of
chicken breast patties during storage time.
Time
Control
without
2.5 ppm
Tannic
5 ppm
10 ppm
5 ppm
Tannic
Tannic
BHA
SEM
-------- Carbonyl (nmol/ mg of protein) -----ay
ay
ay
ax
ay
Day 0
0.54
Day 3
0.72
Day 7
0.82
0.81
0.77
0.66
0.77
SEM
0.040
0.040
0.034
0.040
0.033
a-c
ax
ax
0.54
ax
0.72
ax
0.52
ax
0.70
ax
0.52
ax
0.64
bx
0.53
ax
0.71
ax
0.025
0.055
0.020
Value with different letter within a row are significantly different (P<0.05).
Value with different letter within a column are significantly different (p<0.05).
N=4.
Treatment: Control, 2.5 ppm tannic acid, 5 ppm tannic acid, 7 ppm
tannic acid, and 5ppm BHA.
X-z
103
Table 3. CIE color value of ground chicken patties with different level of
tannic acid during storage at 4 ℃.
Time
Control
2.5 ppm
5 ppm
10 ppm
5 ppm
SEM
Without
Tannic
Tannic
Tannic
BHA
L*
ax
Day 0
64.52
Day 3
63.87
Day 7
61.44
SEM
a*
ax
by
0.29
ax
Day 0
8.53
Day 3
6.50
Day 7
6.09
SEM
b*
by
bz
0.09
bx
Day 0
20.09
Day 3
19.03
Day 7
20.34
SEM
cy
abx
0.19
ax
64.28
ax
63.74
aby
62.45
0.16
ax
8.45
by
6.42
by
6.07
0.2
ax
64.32
ay
63.63
az
bx
cy
19.02
abx
20.26
0.18
64.04
ax
ax
ay
62.58
63.27
0.14
0.18
ax
8.43
aby
6.71
aby
6.43
0.09
20.14
64.40
bx
19.63
cbx
19.75
bx
8.41
7.00
6.91
ax
ay
ay
0.12
21.33
21.41
ax
ax
ax
19.88
20.81
0.29
0.21
ax
0.17
ay
0.14
64.32
63.64
abz
62.44
0.24
0.08
ax
0.14
by
0.10
aby
0.15
8.43
6.40
6.35
0.13
bx
0.15
bax
0.27
abx
0.20
20.21
20.43
20.53
0.16
a-c
Value with different letter within a row are significantly different (P<0.05).
X-z
Value with different letter within a column are significantly different (p<0.05).
N=4.
CIE Color L*,a*,b*-value of ground chicken patties with different level
of oregano oil during storage at 4 C.
104
Table 4. TBA value of cooked ground chicken breast meat at different storage
time at 4℃.
Time
Control
2.5 ppm
5 ppm
10 ppm
5 ppm
SEM
Without
Tannic
Tannic
Tannic
BHA
------- TBARS (mg/km) meat ------az
Day 0
0.19
Day 3
1.42
Day 7
2.23
SEM
a-c
ay
ax
0.06
by
0.15
bx
1.08
bx
1.26
0.1
bz
0.13
cy
0.57
cx
0.79
0.03
0.11
bcz
0.28
0.34
dy
dx
0.01
by
0.01
bx
0.06
0.14
0.97
cx
0.99
0.05
0.03
Value with different letter within a row are significantly different (P<0.05).
Value with different letter within a column are significantly different (p<0.05).
X-z
N=4.
TBA value in a mg Malonaldehyde per Kg meat.
105
Table 5. Effect of adding different level of tannic acid on protein oxidation of
chicken breast patties during storage time.
Time
Control
2.5 ppm
5 ppm
10 ppm
5 ppm
SEM
Without
Tannic
Tannic
Tannic
BHA
------- Carbonyl(nmol/mg of protein)------az
Day 0
0.58
Day 3
1.21
Day 7
2.01
SEM
a-c
ay
ax
0.03
0.57
1.13
ay
axy
bx
1.38
0.14
ax
0.46
bx
0.62
cx
0.64
0.08
0.45
0.59
ay
bxy
0.60
cx
0.04
ay
0.07
bx
0.05
0.47
0.80
cx
0.82
0.10
0.03
Value with different letter within a row are significantly different (P<0.05).
Value with different letter within a column are significantly different (p<0.05).
N= 4.
X-z
106
Table 6. Profile of volatiles cooked meat chicken patties with different level of
Tannic acid at day 0.
4
-------- Total Ion counts * 10 ------Control
2.5 ppm
5 ppm
10 ppm 5 ppm SEM
Compounds
Without
Tannic
Tannic
Tannic
BHA
a
a
b
b
Pentane
238
240
0
0
0b
38
a
a
a
a
a
2-propanone
5267
3407
3325
4171
1923
1057
a
a
a
a
b
Ethanol
8757
8805
8302
8483
3557
830
a
a
a
a
a
2-propanol
810
551
564
494
779
142
b
b
bc
c
a
Hexane
331
277
144
24
830
50
ab
b
c
c
a
Heptane
56
50
0
0
85
7
a
b
b
b
b
Pentanal
566
69
0
0
0
51
Octane
274a
148ab
143ab
54b
162ab
31
a
a
b
b
b
Hexanal
1761
1447
78
0
162
174
a-c
Different letter (a-c) within a row are significantly different (P<0.05), n=4.
107
Table 7. Profile of volatiles cooked meat chicken patties with different
level of tannic acid at day 3.
4
-------- Total Ion counts * 10 ------Compounds
Control
2.5 ppm 5 ppm 10 ppm
5 ppm
SEM
Without
Tannic Tannic Tannic
BHA
a
a
b
b
Pentane
912
766
0
0
0b
50
Propanal
1459a
981a
0b
0b
0b
152
2-propanone
3792b
2283b
5796a
5638a
2674b
403
a
a
ab
a
b
Ethanol
5466
6959
4546
6540
2845
576
b
a
a
b
b
2-propanol
4826
8903
8552
4842
5671
461
a
a
b
b
b
Heptane
410
303
0
0
0
31
a
b
c
c
c
Pentanal
3916
1732
0
0
110
115
a
b
b
b
a
Octane
171
60
61
0
149
17
a
a
b
b
b
Hexanal
11755
9757
257
0
924
559
a
a
b
b
b
Heptanal
64
56
0
0
0
3
a
b
c
c
c
Nonanal
77
47
0
0
0
4
a-c
Different letter (a-c) within a row are significantly different (P<0.05), n=4.
108
Table 8. Profile of volatiles cooked meat chicken patties with different level of
tannic acid at day 7.
4
-------- Total Ion counts * 10 ------Compounds
Control
2.5 ppm
5 ppm
10 ppm 5 ppm SEM
Without
Tannic
Tannic
Tannic
BHA
b
a
c
c
Pentane
1195
2349
0
0
0c
101
a
a
b
b
b
Propanal
350
310
0
0
0
19
a
ab
ab
b
ab
2-propanone
6506
4420
3682
3348
3873
662
a
b
b
b
b
2-propanol
9122
1354
1435
1438
1364
249
a
b
b
b
b
1-Propanol
2625
1228
1377
1440
1322
172
a
ab
b
b
b
Heptane
302
144
0
0
0
42
a
a
b
b
b
Pentanal
3849
4148
0
0
126
225
Octane
776a
407b
56bc
0c
200bc
83
a
a
b
b
b
Hexanal
67351
51368
621
0
1328 5374
a
a
b
b
Heptanal
252
223
0
0
0b
8
a
ab
b
b
b
Nonanal
101
76
0
0
0
18
a-c
Different letter (a-c) within a row are significantly different (P<0.05), n=4.
109
CHAPTER 5
THE EFFECT OF OREGANO OIL AND TANNIC ACID COMBINATIONS ON
THE QUALITY AND SENSORY CHARACTERISTICS OF GROUND
CHICKEN MEAT
Marwan Al-Hijazeen1, Aubrey Mendonca2,* and Dong Uk Ahn1,*
1
Department of Animal Science, Iowa State University, Ames, Iowa 50011, USA, and
2
Department of Food Science and Human Nutrition, and 2Department of Animal
Science, Iowa State University, Ames, IA 50011, USA.
*Corresponding author: D. U. Ahn
Phone: (515) 294-6595, Fax: (515) 294-6595
Email: [email protected]
110
Abstract
The antioxidant effect of different combination levels of oregano essential oil and
tannic acid on both breast and thigh ground chicken meat were studied. Six different
treatments including 1) (none added), 2) 100 ppm Oregano essential oil + 5 Tannic
acid, 3) 100 Oregano essential oil + 10 Tannic acid, 4) 200 ppm Oregano essential oil
+ 5 Tannic acid, 5) 200 Oregano essential oil + 10 tannic acid and 6) 5 ppm butylated
hydroxy anisole (BHA) for breast or 14 ppm for thigh meat were prepared. For raw
meat study, samples were individually packaged in oxygen-permeable bags and stored at
4 ℃ cooler for up to 7 days. On the other hand cooked meat samples were packaged in
oxygen impermeable vacuum bags and then cooked in-bag to the internal temperature
of 75 ℃. After cooling to room temperature, the cooked meat was transferred to new
oxygen-permeable bags and stored at 4 ℃ for up to 7 days. Chicken meat patties were
analyzed for lipid and protein oxidation, color, and volatiles at 0, 3, and 7 days of
storage. The significant differences among the treatments were clearer in cooked meat
than in raw meat. Thigh meat patties showed higher TBARS, total carbonyl, and
volatiles values compared to the breast meat during storage. The combination of 200
ppm oregano oil and 10 ppm tannic acid showed the most significant effects (P < 0.05)
on TBARS, total carbonyl, and off odor volatile formation for both breast and thigh
meat. In addition, the combination of 200 ppm oregano oil and 10 ppm tannic acid
showed the highest effect in stabilizing color for both raw breast and thigh meat.
Oregano oil (200 ppm) and 10 ppm tannic acid combination also showed positive
effects on the sensory scores of chicken thigh meat. In conclusion, the combination of
200 ppm oregano oil and 10 ppm tannic acid could be a good natural replacement for
the synthetic antioxidants in the ground chicken meat.
Key Words: Oregano essential oil, butylated hydroxyanisole, ground meat, lipid
oxidation, thigh meat.
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Introduction
Recently it has become very interesting and highly recommended to use natural
antioxidants as food additives by the food industry (Solomakos et al., 2008; Karre et
al., 2013). This new approach considers a good replacement for the synthetic
antioxidant (butylated hydroxytoulene (BHT), butylated hydroxyanisole (BHA), and
tertiary butylhydroquinone (TBHQ)) which have a very limited usage (USDA, Food
additive regulation; 9 CFR 318.7, and 381.147) in a maximum 0.02 % based on the
fat content or meat preservative content (salt) in meat products such as dry sausage,
fresh pork, beef sausage, dried meat, and various poultry products. On the other hand
the natural antioxidant and particularly the plant sources have smaller limitation on
their use in food or meat products. Several herbs and plant extracts were used in
poultry meat to maintain their stability and extend shelf life (Fasseas et al., 2007;
Brannan, 2008; Abdel-hamied et al., 2009). Lipid and protein oxidations are the major
problems due to their effect on poultry meat quality and freshness. They are
responsible for quality deterioration, including color, flavor, and nutritive values of
meat (Lee et al., 2005a; Ahn et al., 2007; Nam et al., 2007). However, color and odor
are the two main quality parameters that influence consumer’s final decision on
purchasing meat (Liu et al., 1995; Mancini and Hunt, 2005). Natural antioxidants
from plant origins are a safe and healthy replacement for the synthetic ones that can
prevent oxidation in meat (Solomakos et al., 2008; Gulcin et al., 2010).
Oregano essential oil and tannins are extracted from plants and have strong
antioxidant properties (Goulas and Kontominas, 2007; Scramlin et al., 2010). The
antioxidant effect of oregano essential oil is due to its high polyphenol content such as
carvacrol, and thymol (Kosar et al., 2003; Capecka et al., 2005; Al-Bandak, 2007;).
However, very little work has been done to add oregano essential oil directly to the
meat to prevent oxidative changes in the meat (Fasseas et al., 2007; Chouliara et al.,
2007; Scramlin et al., 2010).
Tannins are secondary poly-phenol compounds produced by plants to protect
112
themselves from external herbivores and diseases (Salminen et al., 2011). These
compounds have biological, metal ion chelating, antioxidant (Hagerman, 2010; Okuda
and Ito, 2011), and protein precipitating activities (Hagerman et al., 1998). In addition,
tannins showed positive effect on meat color stability (Luciano et al., 2009; Maqsood
and Benjakul, 2010c).
These properties of tannins are due to their chemical structures which have two
or three phenolic hydroxyl groups on phenyl ring. Hydrolysable tannins are more
popular to use in the food preservation than the condensed type (Chung, 1998; Okuda
and Ito, 2011). The hydrolysable type of tannins contains polyhydric alcohol and
hydroxyl groups, which are esterified with gallic acid (Gallotannins) or to
hexahydroxydiphenic acid (Ellagitannins) (Okuda et al., 1985; Okuda, 2005; Haslam,
1989; Okuda and Ito, 2011).
Using one type of natural antioxidant to replace synthetic antioxidant is not
enough as many researchers have reported. Therefore, a combination of antioxidants
with different antioxidant mechanisms should be considered to increase their
effectiveness (Brewer, 2011). The term Synergism is widely used to explain this effect
between different combinations of antioxidants added to foods. For example, the
effect of adding dietary synthetic antioxidant combined with vitamin E were found to
be more effective in preventing lipid and protein oxidation in fresh frozen chicken
patties than using one natural antioxidant (Smet et al., 2008). The synergistic effect
can be explained by mixing the free radical acceptors for both antioxidants which will
work synergistically to prevent the oxidation process.
Aurand and Woods (1979) reported that the chemical associations between some
acid compounds (e.g. citric acid) and phenolic antioxidants decreased the
development of lipid oxidation. The antioxidant activity of phenolic compounds, the
ability of acid compounds to bind metal ions, and the complex structure between them
explained their synergistic effect. Furthermore, the combinations between the acid and
antioxidant suppressed the role of antioxidant radicals to break down lipid peroxides.
Lee et al. (2005b) found that the combination of chelators (soduim triphospahte or
113
sodium citrate) with reductants (e.g erythrobate, and rosemary extract) were more
effective in decreasing lipid oxidation and maintaining color of ground beef.
Several studies found positive effect of using oregano oil and tannic acid in meat
preservation (Chouliara et al., 2007; Maqsood and Benjakul, 2010c). Oregano oil were
added in animal diet (Botsoglou et al., 2002) or added directly to meat during
processing (Fasseas et al., 2007) to determine their effect on meat quality. Similar
applications for tannic acid or tannins extracts also had been tested (Luciano et al.,
2009; Lui et al., 2009; Maqsood and Benjakul, 2010a, b, c; Devatkal et al., 2010;
Maqsood and Benjakul, 2011; Brogna et al., 2014). The antioxidant effects of various
plants extracts such as green tea, fruit juices, and chestnut wood that contain high
levels of tannins (Liu et al., 2009; Staszewski et al., 2011) were also tested in various
foods and meat systems. Naveena et al. (2008) investigated the effect of adding
natural plant resources containing high levels of tannins in chicken patties and found
that 10 mg equivalent phenolics/100g meat would be sufficient to protect chicken
patties from oxidative rancidity for period longer than the most commonly used
synthetic antioxidant like BHT.
There is no study conducted to evaluate the combination effect of tannic acid and
oregano oil in meat quality and their storage stability. The objectives of this study
were: 1) to evaluate combination effect of tannic acid and oregano oil on the quality
and storage stability of raw and cooked ground chicken thigh or breast meat, and 2) to
select the best tannic acid-oregano oil combinations to control lipid oxidation, protein
oxidation, off-odor, and color in raw and cooked ground chicken meat.
Material and Methods
1. Sample preparation: One hundred and twenty, 6-wk-old broilers raised on a
corn-soybean meal diet were slaughtered using the USDA guidelines (USDA, 1982).
The chicken carcass where chilled in ice water for 2 h and drained in a cold room and
the breast and thigh muscles were separated from the carcasses at 24h after slaughter.
114
Boneless muscles (breast and thigh) were cleaned, skins removed, external fats
trimmed off, and stored at -20℃ freezer until being used.
The frozen meats were thawed in a walk-in cooler (4 ℃), ground twice a through
a 10-mm and a 3-mm plates (Kitchen Aid, Inc., St. Joseph, MI, USA) before use. Six
different treatments including 1) (none added), 2) 100 ppm Oregano essential oil + 5
Tannic acid, 3) 100 Oregano essential oil + 10 Tannic acid, 4) 200 ppm Oregano
essential oil + 5 Tannic acid, 5) 200 Oregano essential oil + 10 tannic acid and 6) 5
ppm butylatedhydroxy anisole (BHA) for breast or 14 ppm for thigh meat (based on
fat content) were prepared.
The oregano essential oil was obtained from a certified company in Turkey
(Healthy-Health, Oil of Oregano, Turkey). The GC/MS analysis of the oregano
essential oil indicated that 80.12% of the essential oil was carvacrol. BHA powder
(0.1 g) and oregano essential oil (1.25 g) were dissolved in 10 ml of 100% ethanol,
and then mixed with 50 ml mineral oil to make their stock solutions. The ethanol
added was removed using a rotary evaporator (BUCH Rotavapor, Model R-200) at
(70℃, 175 mbar vacuum pressure) before adding the stock solution to meat samples.
Tannic acid powder containing 90 % tannin was obtained from Sigma-Aldrich (St.
Louis, MO, USA). The tannic acid contained gallic acid, monogalloyl glucose,
digalloyl glucose, trigalloyl glucose, tetragalloyl glucose, pentagalloyl glucose, ESA
galloyl glucose, EPTA galloyl glucose, and octagalloyl glucose. The product’s name is
hydrolyzable tannin obtained from oak gall nuts from Quercus infectoria. Tannic acid
(0.1 g) was dissolved in 50 ml of de-ionized distilled water (DDW) and stored in a
dark area to prevent exposure to air and light.
Each additive treatment was added to the ground meat (breast or thigh) and then
mixed for 2 min in a bowl mixer (Model KSM 90; Kitchen Aid Inc., St. Joseph, MI,
USA). All treatments were added with the same amount of mineral oil to provide the
same conditions.
For raw-meat study, the prepared meat samples (approximately 100 g each) were
individually packaged in oxygen-permeable bags (polyethylene, 4 x 6.2 mil, Associated
115
Bag Co., Milwaukee, WI), stored at 4 ℃ cooler for up to 7 days, and analyzed for lipid
and protein oxidation, and color at 0, 3, and 7 days of storage.
The same preparation method was used for cooked meat study, but the raw meat
samples were packaged in oxygen impermeable vacuum bags (O2 permeability, 9.3
mL O2/m2/24 h at 0 °C, Koch, Koch, Kansas City, MO, USA), and the meats were
cooked in-bag in a 90 ℃ water bath (Isotemp®, Fisher Scientific Inc., Pittsburgh, PA,
USA) until the internal temperature of the meat reached to 75 ℃. After cooling to
room temperature, the cooked meat was transferred to a new oxygen-permeable bag
(polyethylene, 4 x 6.2 mil, Associated Bag Co., Milwaukee, WI, USA), and stored at
4 ℃ for up to 7 days, and analyzed for lipid and protein oxidation and volatiles at 0, 3,
and 7 days of storage.
2. 2-Thiobarbituric acid-reactive substances (TBARS) measurement: Lipid
oxidation was determined using a TBARS method (Ahn et al., 1998). Five grams of
ground chicken meat were weighed into a 50-mL test tube and homogenized with
50μL BHT (7.2%) and 15 mL of deionized distilled water (DDW) using a Polytron
homogenizer (Type PT 10/35, Brinkman Instruments Inc., Westbury, NY, USA) for
15s at high
speed. One milliliter of the meat homogenate was transferred to a
disposable test tube (13 × 100 mm), and thiobarbituric acid (15 mM TBA/15% TCA,
2 mL) was added. The mixture was vortex mixed and incubated in a boiling water
bath for 15 min to develop color. Then samples were cooled in the ice-water for 10
min, mixed again, and centrifuged for 15 min at 2,500 x g at 4 ℃. The absorbance of
the resulting supernatant solution was determined at 532 nm against a blank
containing 1 mL of DDW and 2 mL of TBA/TCA solution. The amounts of TBARS
were expressed as mg of malondialdehyde (MDA) per kg of meat.
3. Color measurement (Konioca Minolta): The color of meat was measured on the
surface of meat samples using a Konica Minolta Color Meter (CR-410, Konioca
Minolta, Osaka, Japan). The colorimeter was calibrated using an illuminant source C
(Average day light) on a standard white ceramic tile covered with the same film as the
116
ones used for meat samples to negate the color and light reflectance properties of the
packaging material. The color were expressed as CIE L*- (lightness), a*- (redness),
and b*- (yellowness) values (AMSA, 1991). The areas selected for color
measurement were free from obvious defects that may affect the uniform color
readings. An average from two random readings on each side (upper and lower) of
sample surface was used for statistical analysis.
4. Volatile compounds: Volatiles of samples were analyzed using a Solatek 72
Multimatrix-Vial Auto-sampler/Sample Concentrator 3100 (Tekmar-Dohrmann,
Cincinnati, OH, USA) connected to a GC/MS (Model 6890/5973; Hewlett-Packard
Co., Wilmington, DE, USA) according to the method of Ahn and others (2001).
Sample (3 g for raw meat and 2 g for cooked meat) was placed in a 40-mL sample
vial, flushed with helium gas (40 psi) for 3 s, and then capped airtight with a
Teflon*fluorocarbon resin/silicone septum (I-Chem Co., New Castle, DE, USA).
Samples from different treatment were randomly organized on the refrigerated (4 ℃)
holding try to minimize the variation of the oxidative changes in sample during
analyses. The meat sample was purged with helium (40 mL/min) for 14 min at 20 ℃.
Volatiles were trapped using a Tenax/charcoal/silica column (Tekmar-Dohrmann) and
desorbed for 2 min at 225 °C, focused in a cryofocusing module (-70 ℃), and then
thermally desorbed into a capillary column for 2 min at 225 °C. An HP-624 column
(7.5 m, 0.25 mm i.d., 1.4 m nominal), an HP-1 column (52.5 m, 0.25 mm i.d.,
0.25m nominal), and an HP-Wax column (7.5 m, 0.250 mm i.d., 0.25 m nominal)
were connected using zero dead-volume column connectors (J &W Scientific, Folsom,
CA, USA). Ramped oven temperature was used to improve volatile separation. The
initial oven temperature of 25℃ was held for 5 min. After that, the oven temperature
was increased to 85 ℃ at 40 ℃ per min, increased to 165 ℃ at 20 ℃ per min, and then
increased to 230 ℃ at 5 ℃ per min and held for 2.5 min at that temperature. Constant
column pressure at 22.5 psi was maintained. The ionization potential of MS was 70
eV, and the scan range was 20.1 to 350 m/z. The identification of volatiles was
117
achieved by the Wiley Library (Hewlett-Packard Co.). The area of each peak was
integrated using ChemStationTM software (Hewlett-Packard Co.) and the total peak
area (total ion counts x 104) was reported as an indicator of volatiles generated from
the samples.
5. Protein oxidation (total carbonyl): Protein oxidation was determined by the
method of Lund et al. (2008) with minor modifications. One gram of muscle sample
was homogenized with a Brinkman Polytron (Type PT 10/35), Brinkman Instrument
Inc., Westbury, NY, USA) in 10 mL of pyrophosphate buffer (2.0 mM Na4P2O7, 10
mM Trizma-maleate), 100 mM KCL, 2.0 mM MgCl2, and 2.0 mM ethylene glycol
tetraacetic acid, pH 7.4). Two equal amounts of meat homogenate (2 mL) were taken
from a sample, precipitated with 2 mL of 20 % trichloroacetic acid, and centrifuged at
12,000 × g for 5 min at room temperature. After centrifugation, the pellet from 1
sample was treated with 2 mL of 10 mM 2,4-dinitrophenylhydrazine dissolved in 2 M
HCL, and the pellet from the other was incubated with 2 M HCL as a blank. During
30 min of incubation in the dark, samples were vortex-mixed for 10 s every 3 min.
The protein were further precipitated with 2 mL of 20% trichloroacetic acid and
centrifuged at 12,000 x g for 5 min. The 2,4-dinitrophenylhydrazine was removed by
washing 3 times with 4 mL of 10 mM HCL in 1:1 (vol/vol) ethanol:ethyl acetate,
followed by centrifuging at 12,000 × g for 5 min. The pellets were finally solubilized
in 2 mL of 6.0 mM guanidine hydrochloride dissolved in 20 mM potassium
dihydrogen phosphate (pH = 2.3). The samples were kept at 5℃ overnight. In next day,
the samples were centrifuged to remove insoluble materials. The absorbance of
supernatant was read at 370 nm. The absorbance values for blank samples were
subtracted from their corresponding sample values. Briefly, Protein concentration was
measured using Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA)
following Microplate Assay protocol at 280 nm absorbance (BioTek-Gen5 Microplate
data collection & analysis software/BioTek Instruments, Inc., Model S4MLFPTA.,
Winooski, VT, USA). The carbonyl content was calculated as nmol/mg protein using
118
absorption coefficient of 22,000/M/cm as described by Levine et al. (1994).
6. Sensory evaluation: Trained sensory panels were used to evaluate the sensory
characteristics of the ground chicken thigh meat. Sensory panels were evaluated the
color, aroma, and over all acceptability of both raw and cooked meat. Six treatments
were prepared with the same method described in the quality analysis to evaluate the
effect of adding different level of oregano oil and tannic acid combinations: control,
combinations of 5 ppm tannic acid (TA) and 100 ppm oregano oil (Org), 10 ppm TA
and 100 ppm Org, 5 ppm TA and 200 ppm Org, 10 ppm TA and 200 ppm Org, and 14
ppm BHA. The meat was refrigerated at 4 ℃ for three day before each evaluation
session. Ten trained panelists (Iowa State University. Graduate Student and staff),
were participated for each session. The evaluations were done twice after three day of
storage time at 4 ℃. For training, 3 one-hour sessions were held using commercial
and experimental products to develop descriptive terms for the desired attributes. Raw
ground chicken meat was evaluated for color (redness), oregano aroma or spice odor,
oxidative odor, and over all acceptability. Cooked meats were evaluated for cooked
chicken, oxidative odor, spice odor or oregano odor, and over all acceptability.
All attributes were measured using a line scale without numbers (numerical
value 9 units) with graduation from 0 to 9. For example overall acceptability; where 9
represented extremely desirable, and 0 represent extremely undesirable (9-extremely
desirable, 8 very, 7 moderate, 6-slightly, 5-neithtly nor, 4 slightly undesirable,
moderately, very, and extremely). Similar terminology (e.g detectable or undetectable)
underline was used for the other attributes. Evaluation sessions for cooked and raw
meat were done in a separate days to decrease any variability.
Refrigerated (4 °C) samples were evaluated by the panelists for each treatment.
The panelist was served 1 glass vial, 20 ml volume, of each treatment to evaluate
color and odor of raw thigh meat. All ground meat samples were packaged in oxygen
119
permeable bags before evaluation session. The panelists were asked to evaluate the
ground meat patties (color) and open the vials to evaluate odor. Raw meat samples of
10g each were placed in small vials (20-mL) capped with a septum (I-Chem CO.) in
order to evaluate odor attributes. All samples vials were labeled by a three digit
number selected randomly. For cooked meat, samples (4g) at 0 day of cooking were
placed in a three digit coded 20-mL sample vial and capped with a septum (I-Chem
Co.). Panelists were asked to smell samples in random order and record the intensity
of odor or over all acceptability on the scale line.
Statistical analysis: Data were analyzed using the procedures of Generalized Linear
Model (Proc. GLM, SAS program, version 9.3, 2012). Mean values and standard error of
the means (SEM) were reported. The significance was defined at P < 0.05 and Tukey test
or Tukey’s Multiple Range test were used to determine whether there is a significant
difference between the mean values. Pearson correlation coefficient (CORR Procedure)
was used to determine the relation between TBARS, total carbonyl, and hexanal.
Results and Discussion
Lipid Oxidation
The TBARS values of raw breast and thigh meat among the treatments at day 0
did not differ significantly (P > 0.05) (Table 1). However, the TBARS values of all
treatments were significantly lower than that of the control (p < 0.05) at day 3 and 7.
The highest combination effect was found when 200 ppm oregano oil and 10 ppm of
tannic acid was used. Similar trends were found in thigh meat, but the TBARS values
in thigh meat were higher than the breast meat (Tables 1). Several studies showed that
oregano oil (Botsoglou et al., 2003a, b; Chouliara et al., 2007) and tannic acid
(Luciano et al., 2009; Maqsood and Benjakul, 2010b; Maqsood and Benjakul, 2010c)
have antioxidant effect when added directly or indirectly (animal diet) to the foods. It
was also reported that a combination of antioxidants shows stronger antioxidant
effects if the antioxidants work synergistically (Lee et al. 2005b; Brewer, 2011).
120
Cooked chicken breast and thigh meats showed a clearer difference (p < 0.05)
between treatments than the raw meat (Tables 5). Cooked meat is more susceptible to
lipid oxidation than raw meat because of the denaturation of antioxidant enzymes and
the structural damages in membrane, which can expose phospholipids to pro-oxidant
environment in cooked meat (Ahn et al., 1992; Gray et al., 1996). The TBARS values
of cooked ground thigh meat were higher than the cooked breast meat but the
antioxidant effects were very similar in both breast and thigh muscle tissues. The
higher TBARS values of thigh meat than breast are due to the higher fat and
polyunsaturated fatty acid content, which increases their oxidation susceptibility to
free radicals.
Protein Oxidation
Total carbonyl (nmol/mg of protein) content of raw meat breast was lower than
the thigh meat during storage (Table 2). Both ground chicken meat (raw breast and
thigh meat) had no significant difference (P > 0.05) in total carbonyl values at day 0
of storage, and the formation of carbonyl was very slow in the raw meat during
storage. This was in agreement with Xiao et al. (2011) who found that the total
carbonyl content in raw chicken meat patties stored aerobically at 4 ℃ increased from
0.46 to 0.81 nmol/mg of protein. In thigh chicken meat, however, all treatment had
significantly (P < 0.05) lower carbonyl content than the control at Day 3. Both breast
and thigh meat treated with antioxidants (oregano, tannic acid, and BHA) had
significantly (P > 0.05) lower carbonyl content than the control at day 7 of storage.
However, combination of 200 ppm oregano oil and 10 ppm of tannic acid showed the
highest effect in preventing carbonyl formation among the treatments.
Both cooked breast and thigh meat showed higher total carbonyl values than the
raw meat (Table 6) probably due to higher free radical formation in cooked meat than
the raw meat. The highest total carbonyl value was found in cooked thigh meat
(control), which reached to 4.58 nmol/mg protein. The results of total carbonyl
formation were similar to those of other researchers who found that total estimated
121
carbonyl contents were in the range of 1-3 nmol/mg protein for raw meat and up to 5
nmol/mg protein for cooked meat products (Estevez et al., 2005; Sun et al., 2010;
Estevea, 2011). Again, the combination of 200 ppm oregano oil and 10 ppm tannic
acid had the highest effect in preventing carbonyl formation in cooked meat.
Generally a positive relation between TBARS values and total carbonyl formation
were found during storage in both breast and thigh chicken meats. It is known that the
antioxidant activity of oregano oil and tannic acid was due to their polyphenols, which
works to prevent oxidation in the foods (Chung et al., 1998; Goulas and Kontominas,
2007; Okuda and Ito, 2011).
Color values
There were no significant differences (P > 0.05) in color L*- and a*-values of
breast meat among treatments at day zero (Table 3). However, significant differences
in color L*- and a*-values were found after day 3 of storage. Both L*- and a*-values
decreased, but the changes in b*-values were inconsistent during storage. Mancini and
Hunt (2005) reported the decrease of a*-value during storage was due to the
accumulation of metmyoglobin in meat. A strong relationship between Metmyoglobin
(MetMb) accumulation and fresh meat discoloration were reported. However, several
factors that can affect the MetMb reducing activity in fresh meat such as pH, storage
temperature, storage time, lipid peroxidation, oxygen, ions and chemicals may also
have been involved (Bekhit and Faustman, 2005). Since oregano oil and tannic acid
slowed the oxidation in meat, they might have increased the MethMb reducing
activity and stabilized meat color. The combination of 200 ppm oregano oil and 10
ppm TA acid showed the highest effect in stabilizing color values. This treatment had
the highest L*- and a*-values at the end of storage time. This is in agreement with the
results of others (Chouliara et al., 2007; Mastromatteo et al., 2009; Maqsood and
Benjakul, 2010c) who found that oregano oil and tannic acid improved meat color
stability. Thigh meat had lower L*-values, but higher a*-value than the breast meat
(Tables 3 & 4). This agrees with the results of Sandusky and Heath (1998) who
studied the differences between sensory and instrument-measured ground chicken
122
meat color. The differences in a*- and L*-values between breast and thigh meat was
due to their myoglobin content, water holding capacity, fatty acid content, and their
freshness (Froning, 1995; Fletcher, 1999; Berri et al., 2005). The meat with high
lightness value is considered more acceptable by the consumer than the darker ones
because it is considered fresher (Hood and Riordan, 1973). The combination of 200
ppm of oregano oil and 10 ppm of tannic acid showed the highest effect in stabilizing
color values. However, the differences between treatments and control were clearer in
thigh meat than breast meat due to their higher myoglobin content than breast meat.
Volatiles production
Generally, the amounts and number of volatiles compounds increased during
storage time in both breast and thigh cooked meat patties. Raw meat patties showed
very low volatiles production even after 7 days of storage (Data not shown). Among
the carbonyl compounds, hexanal increased by five- to six-fold in untreated patties
during the 7 day storage time. This was inagreement with Du et al. (2000) who found
that the amount of total volatiles in aerobically-packaged chicken meat increased by
five to six folds during 7 day of refrigerated storage conditions. However, in this
study cooked thigh meat had higher volatiles values than the breast cooked meat
(Tables 7-9 & Tables 10-12). The higher volatile values in thigh meat were due to
their different chemical composition such as fat, water holding capacity, and
polyunsaturated fatty acid. Control samples showed the highest aldehyde values
among the treatments. Combination of 200 ppm oregano oil and 10 ppm of tannic
acid showed the highest effects in inhibiting the volatile formation in both breast and
thigh cooked meat during storage. Other treatments (oregano + tannic acid, and BHA)
also decreased the volatile formation during storage. In addition, it showed significant
effect (P < 0.05) on aldehydes, and other volatiles which are responsible for the
off-odor and rancidity of ground chicken meat during storage time. Over all, this
agrees with the TBARS values of ground cooked meat compared to the raw meat due
to the higher oxidation susceptibility of cooked meat compared to the raw meat (Ahn
123
et al., 1992; Gray et al., 1996). Lee et al. (2003) also found that the volatile
aldehyde-reducing activity of antioxidants were consistent with the result of TBARS
values in raw and cooked turkey breast. Similar relation was found between TBARS
values and aldehydes formation in both breast and thigh meat. In addition, very high
correlation coefficient between TBARS, total carbonyl, and hexanl values were found
during storage time (Table 13). This explains the positive relationships between these
three variables during storage time. Among aldehydes, hexanal and pentanal were the
most predominant compounds affected by the combination of 200 ppm oregano oil
and 10 ppm tannic acid. Jo et al. (2006) found that hexanal contents of control cooked
chicken breast meat increased by about 2.7 times at day 1 compared with 0 day.
Hexanal and pentanal are considered good indicators for lipid oxidation (Shahidi et al.,
1987; Ahn et al., 2000) during storage time. The volatile profile also showed some
terpenoids such as camphene and limonene when ground chicken meats mixed with
oregano oil, and their amounts increased as the amount of oregano oil increased.
These compounds are responsible for the spice odor of meat added with oregano oil.
Combination of 200 ppm oregano oil and 10 ppm tannic acid also showed significant
effect (P < 0.05) deteriorate most of aldhydes related-lipid oxidation such as propanal,
hexanal, and pentanal in cooked thigh meat. Similar results were observed on the
content of sulfur compounds (dimethyl trisulfide and dimethyl disulfide) and other
volatiles which found in-consistant and in a very low concentration in the treated
samples. Overall, very low amounts of sulfur volatiles were detected because of their
high volatility, and susceptibility to escape from the oxygen permeable bags (Nam et
al., 2002; and Lee et al., 2003). The combination (200 ppm oregano oil and 10 ppm
tannic acid) treatment showed lower volatile formation than the BHA treatment in
both breast and thigh cooked meat patties during storage.
124
Sensory evaluation
Panelists could not detect any significant treatment difference in color attribute of
raw thigh chicken meat (Table 14). On the other hand, panelists easily distinguished
oregano oil odor in raw thigh meat. However, sensory panels could not distinguish the
treatment differences in cooked meat due to cooking effect on volatiles related to
oregano odor (Table 15). Combination of 10 ppm of tannic acid and 200 ppm oregano
oil treatment reduced oxidation odor score significantly ( P < 0.05). In addition,
combination of 10 ppm of tannic acid and 200 ppm oregano oil treatment showed the
highest score on the overall acceptability for both raw and cooked meat patties.
Conclusion
Tannic acid and oregano essential oil at level 200 ppm and 10 ppm showed
positive effect on the decreasing TBARS values, total carbonyl, improve color
stability, and deteriorate off-odor volatile formation. Data of lipid oxidation, protein
oxidation, and color, volatile agreed with the sensory evaluation results. In conclusion
the combination of 200 ppm oregano oil and 10 ppm tannic acid could be a good
replacement for the synthetic antioxidant (BHA) in the ground chicken meat.
125
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Table 1. TBARS value of raw ground chicken breast and thigh meat added with
different levels of oregano oil and tannic acid combinations during refrigerated
storage at 4℃.
Storage
Control
Time
without
CM1
CM2
CM3
CM4
BHA
SEM
5 ppm
---------------------- TBARS (mg/Kg) ---------------------Breast meat
Day 0
Day 3
Day 7
SEM
0.12az
0.10az
ay
aby
0.19
ax
0.34
0.01
0.16
bcx
0.26
0.01
0.11ay
abcy
0.14
cdx
0.22
0.01
0.09ay
0.10ax
0.10az
0.01
cy
cx
aby
0.01
bx
0.01
0.13
dex
0.17
0.02
0.12
ex
0.14
0.01
Thigh meat
0.17
0.28
0.01
14 ppm
Day 0
0.25az
0.22ay
0.24ay
0.24ay
0.22ay
0.24ay
0.017
Day 3
0.90ay
0.30bcy
0.27bcy
0.25bcy
0.23cy
0.33by
0.022
Day 7
2.21
ax
1.54
bcx
1.16
cdx
0.98
dex
0.60
ex
1.73
bx
0.102
SEM
0.077
0.091
0.036
0.029
0.034
0.069
a-e
Values with different letter within a row are significantly different (P < 0.05).
x-z
Values with different letter within a column are significantly different (P < 0.05).
N= 4.
Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm
oregano oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic
acid + 200 ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil;
BHA.
133
Table 2. Effect of adding different level of oregano oil and tannic acid on protein
oxidation of raw chicken breast and thigh patties during storage time.
Storage Control
CM1
CM2
CM3
CM4
BHA
SEM
Time
without
5 ppm
------- Carbonyl (nmol/ mg of protein)------Breast meat
Day 0
0.76ay
Day 3
0.84ay
Day 7
1.04ax
SEM
0.04
0.75ax
0.80ax
0.87abx
0.04
0.74ax
0.77ax
0.81bx
0.02
0.74ax
0.75ax
0.77bx
0.04
0.74ax
0.74ax
0.75bx
0.03
Thigh meat
Day 0
Day 3
Day 7
SEM
a-c
0.86az
1.27ay
1.68ax
0.28
0.75ax
0.82ax
0.88abx
0.05
0.03
0.04
0.05
14 ppm
0.84ax
0.94bx
1.11bcx
0.071
0.83ax
0.87bx
0.92bcx
0.056
0.83ax
0.83bx
0.86bcx
0.079
0.81ax
0.83bx
0.83cx
0.041
0.83ay
0.95by
1.13bx
0.038
0.035
0.061
0.065
Value with different letter within a row are significantly different (P<0.05).
Value with different letter within a column are significantly different (p<0.05).
N = 4.
Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm oregano
oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic acid +
200 ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil; BHA.
x-z
134
Table 3. *CIE color values of ground chicken breast patties with
of oregano oil and tannic acid stored at 4℃.
Time
Control
CM1
CM2
CM3
CM4
without
L*
Day 0
63.0ax
62.7ax
63.2ax
62.9ax
62.9ax
Day 3
61.1by
61.5aby
61.7aby
61.7aby
62.1ay
Day 7
60.6by
61.2aby
61.3aby
61.5ay
60.9abz
SEM
0.2
0.19
0.17
0.15
0.14
a*
Day 0
8.28ax
8.24ax
8.31ax
7.84ax
8.24ax
Day 3
6.52by
6.39by
6.86aby
6.69by
7.32ay
cy
cby
cbz
aby
Day 7
5.89
6.12
6.24
6.49
6.76az
SEM
0.20
0.20
0.10
0.09
0.13
b*
Day 0
19.4by
18.9by
20.4ax
20.2ax
20.1ax
Day 3
19.9ay
19.4cx
19.6abcy
19.8aby
19.5bcy
Day 7
20.3ax
19.3bx
19.06bz
19.97ay
19.49by
SEM
0.14
0.08
0.06
0.14
0.11
a-c
different level
5 ppm
BHA
SEM
62.8ax
61.1by
61.05aby
0.21
0.18
0.18
0.20
8.40ax
6.64by
6.43aby
0.18
0.21
0.13
0.12
20.6ax
19.9ay
20.32ax
0.11
0.12
0.09
0.10
Value with different letter within a row are significantly different (P<0.05).
Value with different letter within a column are significantly different (p<0.05).
N = 4.
Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm oregano
oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic acid + 200
ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil; BHA.
x-z
135
Table 4. *CIE color value of ground chicken thigh patties with different level of
oregano oil and tannic acid during storage at 4 ℃.
Time
Control
CM1
CM2
CM3
CM4
14 ppm SEM
without
BHA
L*
Day 0
57.12ax 56.15bx
56.09bx
56.25abx
56.24abx
56.70abx 0.22
Day 3
55.02ay 54.78ay
54.85ax
55.09axy
54.56ay
55.02ay
0.25
bz
abz
aby
aby
ay
bz
Day 7
52.26
53.47
53.50
53.74
54.10
52.39
0.34
SEM
0.085
0.18
0.314
0.431
0.216
0.271
a*
Day 0
10.03ax
9.85ax
9.78ax
9.62ax
9.63ax
10.0ax
0.10
by
ax
ay
ax
ax
abx
Day 3
8.88
9.43
9.48
9.46
9.85
9.52
0.08
cz
aby
abz
aby
ay
bcy
Day 7
7.29
8.53
8.64
8.78
9.02
7.99
0.22
SEM
0.138
0.199
0.076
0.124
0.064
0.221
b*
Day 0
16.51ay 16.74axy
16.96ay
16.95ay
16.82ay
17.00ax
0.15
ax
ay
az
ay
ay
ax
Day 3
17.16
16.26
16.32
16.70
16.69
17.31
0.23
ax
ax
ax
ax
ax
ax
Day 7
17.33
17.54
17.36
17.42
17.65
17.57
0.08
SEM
0.151
0.284
0.097
0.147
0.108
0.144
a-c
Value with different letter within a row are significantly different (P<0.05).
value with different letter within a column are significantly different (p<0.05).
N=4.
Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm oregano
oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic acid +
200 ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil; BHA.
X-z
136
Table 5. *TBARS values of cooked ground chicken meat with different
combination level of oregano oil and tannic acid at 4℃.
Time
Control
CM1
CM2
CM3
CM4
Without
BHA
5 ppm
SEM
------- TBARS (mg/kg) meat ------Breast meat
Day 0
0.21az
0.18az
0.19ay
0.21az
0.19ay
0.21az
0.015
Day 3
1.36ay
0.87by
0.77bcx
0.74bcy
0.57cx
0.96by
0.066
Day 7
2.03ax
1.32bcx
0.97cdx
0.88dx
0.75dx
1.50bx
0.079
SEM
0.084
0.038
0.076
0.014
0.064
0.056
Thigh meat
14 ppm
Day 0
1.23az
1.10az
1.09az
0.97az
0.94ay
1.27az
0.074
Day 3
3.40ay
1.64cy
1.36dy
1.11ey
1.01ey
1.88by
0.036
Day 7
3.48ax
2.58bcx
2.32cdx
2.07dx
1.75ex
2.82bx
0.062
SEM
0.056
0.04
0.052
0.026
0.071
0.082
a-e
Values with different letter within a row are significantly different (P < 0.05). n = 4.
x-z
Values with different letter within a column are significantly different (P < 0.05).
*TBARS value in mg Malonaldehyde per Kg meat.
Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm oregano
oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic acid + 200
ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil; BHA.
137
Table 6. Effect of adding different level of oregano oil and tannic acid on
protein oxidation of cooked chicken patties during storage time.
Time
Control
CM1
CM2
CM3
CM4
Without
Breast
meat
BHA
SEM
5 ppm
------- Carbonyl (nmol/ mg of protein)-------
Day 0
1.00az
1.05ay
0.99ay
0.98ay
0.98az
1.01az
0.06
Day 3
1.69ay
1.45abxy
1.31bx
1.26bxy
1.15by
1.42aby
0.07
Day 7
2.15ax
1.75abx
1.41bx
1.34bx
1.30bx
1.76abx
0.11
SEM
0.04
0.16
0.04
0.08
0.03
0.07
Thigh
Meat
14 ppm
Day 0
1.55az
1.45ay
1.42az
1.42az
1.37ay
1.48az
0.049
Day 3
3.10ay
2.63bx
2.16cy
1.75dy
1.51dxy
2.67by
0.088
Day 7
4.58ax
3.03bx
2.52cx
2.06dx
1.70dx
3.21bx
0.086
SEM
0.083
0.118
0.071
0.076
0.053
0.029
a-d
Value with different letter within a row are significantly different (P<0.05).
Value with different letter within a column are significantly different (p<0.05).
N = 4.
Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm
oregano oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic
acid + 200 ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil;
BHA.
X-z
138
Table 7. Profile of volatiles cooked breast meat chicken patties with different
level of oregano oil and tannic acid at day 0.
Total Ion counts*104
Compound
Control CM1
CM2
CM3
CM4
without
5 ppm SEM
BHA
Propanal
165a
0b
0b
0b
0b
0b
10
2-propanone
4388a
2677a
3127a
4125a
2627a
2100a
514
2-propanol
592a
611a
561a
615a
713a
848a
75
Hexane
Heptane
Pentanal
a
177
a
b
b
0
0
b
76
1043
a
148
b
0
b
0
b
b
0
b
0
a
b
0
b
0
0
b
9
b
66
b
0
b
0
b
5
b
0
b
0
b
b
0
b
Heptanal
41
0
0
0
0
0
1
Octane
333a
118b
82b
130b
94b
90b
17
Hexanal
3401a
561b
329b
335b
314b
316b
277
α-Pinene
0c
93bc
160ab
272a
229ab
0c
31
c
bc
b
a
a
c
25
c
12
b
18
c
21
Camphene
Limonene
β-Myrcene
γ-Terpinene
0
98
c
78
b
b
0
0
0
c
b
168
bc
51
b
0
152
a
167
a
0
b
296
199
128
b
311
a
284
a
157
a
176
286
a
0
0
0
0
Sabinene
0b
0b
0b
46a
52a
0b
4
Different letter (a-d) within a row are significantly different (P<
0.05), n=4.
Treatments: Control, Combination of 100 ppm oregano oil and 5 ppm Tannic acid,
100 ppm Oregano oil + 5 ppm Tannic acid, 200 oregano oil + 5 Tannic acid, 200 ppm
oregano oil + 10 Tannic acid, and 5 ppm BHA.
139
Table 8. Profile of volatiles cooked breast meat chicken patties with different level
of oregano oil and tannic acid at day 3.
Total Ion counts*104
Compound
Control
CM1
CM2
CM3
CM4
5 ppm SEM
without
BHA
a
b
b
b
b
Pentane
536
112
0
0
0
0b
43
a
b
b
b
b
b
Propanal
1736
0
0
0
0
0
153
ab
bc
c
c
c
a
2-propanone 4119
2921
2657
2427
2046
4370
304
a
a
a
a
a
a
2-propanol
278
336
466
392
385
396
48
a
b
c
c
c
c
Heptane
133
58
0
0
0
0
3
a
b
b
b
b
b
Pentanal
4385
168
62
71
91
76
158
a
a
a
a
a
a
Octane
259
249
93
160
109
168
39
a
b
b
b
b
b
Hexanal
39357
2086
701
925
870
1136 1778
a
b
b
b
b
Heptanal
132
0
0
0
0
0b
15
b
b
b
a
a
b
α-Pinene
0
126
124
372
337
0
30
c
b
b
a
a
c
Camphene
0
136
123
334
330
0
20
c
ab
bc
ab
a
c
Limonene
0
94
34
107
136
0
16
c
bc
c
a
ab
c
β-Myrcene
0
32
0
188
151
0
27
c
b
b
a
a
c
γ-Terpinene
0
77
122
337
401
0
14
c
b
c
c
a
c
Sabinene
0
20
0
0
41
0
2
Different letter (a-d) within a row are significantly different (P<
0.05), n=4.
Treatments: Control, Combination of 100 ppm oregano oil and 5 ppm Tannic acid,
100 ppm Oregano oil + 5 ppm Tannic acid, 200 oregano oil + 5 Tannic acid, 200 ppm
oregano oil + 10 Tannic acid, and 5 ppm BHA.
140
Table 9. Profile of volatiles cooked breast meat chicken patties with different level of
oregano oil and tannic acid at day 7.
Total Ion counts*104
Compound
Control
without
CM1
CM2
CM3
CM4
5 ppm
BHA
SEM
Pentane
Propanal
2-propanone
2-propanol
Heptane
Butanal
697a
2156a
1899a
274ab
137a
198a
0c
127b
2412a
172b
0c
0b
0c
0b
2766a
149b
0c
0b
0c
0b
2672a
394a
0c
0b
0c
0b
2676a
426a
0c
0b
195b
0b
2441a
152b
52b
0b
25
38
198
38
10
9
Pentanal
Octane
Hexanal
Heptanal
Nonanal
α-Pinene
Camphene
Limonene
β-Myrcene
γ-Terpinene
Sabinene
10241a
405a
84538a
449a
63a
0b
0d
0b
0b
0b
0b
244bc
348a
3741bc
0b
0b
241a
243bc
52b
70b
203b
0b
81c
101c
1269c
0b
0b
216a
165c
55b
57b
208b
0b
91c
98c
1165c
0b
0b
278a
280ab
146a
517a
1362a
140a
81c
92c
1014c
0b
0b
282a
369a
196a
433a
1156a
154a
994b
227b
9363b
0b
0b
0b
0d
0b
0b
0b
0b
171
24
1451
17
2
29
20
14
35
101
7
Different letter (a-d) within a row are significantly different (P< 0.05), n=4.
Treatments: Control, Combination of 100 ppm oregano oil and 5 ppm Tannic acid,
100 ppm Oregano oil + 5 ppm Tannic acid, 200 oregano oil + 5 Tannic acid, 200 ppm
oregano oil + 10 Tannic acid, and 5 ppm BHA.
141
Table 10. Profile of volatiles cooked thigh meat chicken patties with different level
of oregano oil and tannic acid at day 0.
Total Ion counts*104
Compound
Control
CM1
CM2
CM3
CM4
14 ppm SEM
without
BHA
a
ab
ab
b
ab
2-propanone 4994
4762
4183
3098
3752
4336ab
416
a
a
a
a
a
a
2-propanol
819
754
693
643
816
897
89
a
b
b
b
b
a
Cyclohexane
106
0
0
0
0
91
8
a
c
c
c
c
b
Pentanal
691
0
0
0
0
161
9
a
ab
b
b
b
b
Octane
306
179
146
163
153
212
24
a
c
c
c
c
b
Hexanal
3395
311
427
463
213
1360
184
c
b
ab
ab
a
c
α-Pinene
0
274
383
282
416
0
30
c
ab
b
a
ab
c
Camphene
0
284
278
406
358
0
27
b
a
a
a
a
b
Limonene
0
72
69
105
107
0
15
c
b
b
a
a
c
β-Myrcene
0
137
98
305
329
0
21
c
b
bc
a
a
c
γ-Terpinene
0
486
408
1081
1416
0
100
c
c
c
a
b
c
Sabinene
0
0
0
105
83
0
5
Different letter (a-d) within a row are significantly different (P< 0.05), n=4.
Treatments: Control, Combination of 100 ppm oregano oil and 5 ppm Tannic acid,
100 ppm Oregano oil + 5 ppm Tannic acid, 200 oregano oil + 5 Tannic acid, 200 ppm
oregano oil + 10 Tannic acid, and 5 ppm BHA.
142
Table 11. Profile of volatiles cooked thigh meat chicken patties with different level
of oregano oil and tannic acid at day 3.
Total Ion counts*104
Compound
Control CM1
CM2
CM3
CM4
14 ppm SEM
without
BHA
a
c
c
c
c
Pentane
989
0
0
0
0
307b
18
a
b
c
c
c
b
Propanal
1258
0
0
0
0
598
35
b
b
a
a
a
b
2-propanone
2069
2114
4777
4392
3827
2140
318
a
a
a
a
a
a
2-propanol
1649
939
1358
1240
1125
1081
188
a
a
a
b
b
a
Hexane
649
403
438
0
0
572
62
a
b
c
c
c
a
Cyclohexane
133
98
0
0
0
149
7
a
c
c
c
c
b
Pentanal
4358
229
285
180
0
875
88
a
b
b
b
b
b
Heptane
125
0
0
0
0
0
5
Octane
691a
438c
513abc
456bc
423c
672ab
51
Hexanal
54364a 2708c
1845c
1623c
565c
11797b 995
Heptanal
216a
0b
0b
0b
0b
0b
14
c
b
b
b
a
c
α-Pinene
0
319
382
458
687
0
50
d
c
c
b
a
d
Camphene
0
169
210
411
514
0
21
c
c
b
bc
a
c
Limonene
0
0
43
98
192
0
13
b
b
b
a
a
b
β-Myrcene
0
0
74
199
244
0
26
c
b
bc
a
a
c
γ-Terpinene
0
364
250
1058
874
0
72
b
b
b
a
a
b
Sabinene
0
0
0
82
86
0
7
Different letter (a-d) within a row are significantly different (P< 0.05), n=4.
Treatments: Control, Combination of 100 ppm oregano oil and 5 ppm Tannic acid,
100 ppm Oregano oil + 5 ppm Tannic acid, 200 oregano oil + 5 Tannic acid, 200
ppm oregano oil + 10 Tannic acid, and 5 ppm BHA.
143
Table 12. Profile of volatiles cooked thigh meat chicken patties with different level
of oregano oil and tannic acid at day 7.
Total Ion counts*104
14
Compound
Control CM1
CM2
CM3
CM4
SEM
ppm
without
BHA
a
c
c
c
c
Pentane
474
0
0
0
0
176b
26
ab
a
bc
bc
c
abc
2-propanone 4064
4622
2824
2807
2465
3714
301
a
b
b
b
b
b
2-propanol
2983
1620
938
809
742
629
222
a
ab
bc
d
d
c
Hexane
243
173
139
0
0
83
17
a
b
c
c
c
a
Cyclohexane
152
106
0
0
0
143
7
a
c
c
c
c
b
Pentanal
2477
375
150
44
76
1154
89
a
b
b
b
b
b
Heptane
347
0
0
0
0
57
18
a
bc
c
c
c
b
Octane
1337
627
494
478
458
850
56
a
c
c
c
c
b
Hexanal
88685
5573
1871
1652
832
25172 1175
a
c
c
c
c
Heptanal
395
0
0
0
0
135b
14
a
b
b
b
b
b
Octanal
70
0
0
0
0
0
4
d
bc
c
ab
a
d
α-Pinene
0
434
333
517
669
0
37
d
c
bc
b
a
d
Camphene
0
246
283
449
638
0
38
c
b
b
a
a
c
Limonene
0
43
53
140
144
0
5
c
b
b
a
a
c
β-Myrcene
0
129
100
245
280
0
9
c
b
b
a
a
c
γ-Terpinene
0
319
309
638
792
0
58
c
c
c
b
a
c
Sabinene
0
0
0
41
59
0
4
Different letter (a-d) within a row are significantly different (P< 0.05), n=4.
Treatments: Control, Combination of 100 ppm oregano oil and 5 ppm Tannic acid,
100 ppm Oregano oil + 10 ppm Tannic acid, 200 oregano oil + 5 Tannic acid, 200
ppm oregano oil + 10 Tannic acid, and 5 ppm BHA.
144
Table 13. Correlation Coefficients of TBARS, total carbonyl, hexanal values during
storage time of control treatment.
_____________________________________________________________________
Pearson Correlation Coefficients, N = 12, Prob > | r | under HO: Rho = 0
Cooked Breast
Cooked Thigh
Var
TBA
CAR
TBA
1
CAR
0.98262*
Var
TBA
0.98262* 0.94888*
TBA
1
0.97103*
CAR
0.98085*
HEX
0.94888* 0.97103*
1
HEX
0.96005* 0.99116*
1
CAR
HEX
HEX
HEX
0.98085* 0.96005*
1
1
TBA
MEAN
1.1999
1.6147
42431.75 MEAN
3.8995
3.0758
48814.5
STD
0.8042
0.4976
35156.5
2.517
1.3
36767.26
STD
* P < 0.01 : Highly significant
** 0.01 < P < 0.05 : Strong significant difference
*** 0.05 < P < 0.1 : Low significant difference
**** P > 0.1 : No significant difference
CAR
0.99116*
TYPE
TYPE
TBA
CAR
HEX
145
Table 14. Sensory attributes means of raw ground thigh chicken meat patties.
Sensory attributesb
TRTa
Color
redness
Oregano
Odor
Oxidation
Odor
Over All
Acceptability
Control
CM1
CM2
CM3
CM4
BHA
SEMc
6.9a
7.0a
7.4a
6.9a
8.0a
7.1a
0.34
0.9c
2.3bc
3.2b
5.6a
6.1a
1.2c
0.37
3.8a
3.6ab
2.8ab
1.7ab
1.1b
3.2ab
0.6
4.9b
5.3b
5.8ab
6.3ab
7.6a
6.4ab
0.47
a
Treatments: Control, Combination (CM) of 100 ppm oregano oil and 5 ppm
Tannic acid; 100 ppm Oregano oil + 10 ppm Tannic acid; 200 oregano oil + 5
Tannic acid; 200 ppm oregano oil + 10 Tannic acid; and 5 ppm BHA.
b
Sensory attributes: samples were evaluated on day 3.
SEMc: Standard error of the means.
d-f
Mean within same column with different superscripts are different (P < 0.05)
n = 10.
146
Table 15. Sensory attributes means of ground cooked thigh chicken meat patties.
Sensory attributesb
Cooked
Oregano
Oxidation
Over All
TRTa
chicken
Odor
Odor
Acceptability
b
d
a
Control
4.6
0.7
5.3
4.1c
CM1
5.9ab
2.1cd
4.7ab
4.6c
CM2
6.4ab
2.9bc
3.5ab
5.7bc
CM3
6.7ab
4.7a
2.3b
7.5ab
CM4
7.9a
4.3ab
2.6b
7.9a
BHA
6.6ab
1.1d
3.1ab
7.3ab
0.51
0.37
0.6
0.44
SEMc
a
Treatments: Control, Combination (CM) of 100 ppm oregano oil and 5 ppm
Tannic acid; 100 ppm Oregano oil + 10 ppm Tannic acid; 200 oregano oil + 5
Tannic acid; 200 ppm oregano oil + 10 Tannic acid; and 5 ppm BHA.
b
Sensory attributes: samples were evaluated on day 0.
c
SEM: Standard error of the means.
d-f
Mean within same column with different superscripts are different (P < 0.05)
n = 10.
147
CHAPTER 6
SURVIVAL AND GROWTH OF NATURAL BACTERIAL FLORA AND
HUMAN ENTERIC PATHOGENS IN GROUND CHICKEN MEAT WITH
ADDED OREGANO ESSENTIAL OIL OR TANNIC ACID, ALONE OR IN
COMBINATION
Marwan Al-Hijazeen1, Aubrey Mendonca2,* and Dong Uk Ahn1,*
1
Department of Animal Science, Iowa State University, Ames, Iowa 50011, USA, and
2
Department of Food Science and Human Nutrition, and 2Department of Animal
Science, Iowa State University, Ames, IA 50011, USA.
*Corresponding authors: Aubrey F. Mendonça and D. U. Ahn
Phone: (515) 294-9078. Fax: (515) 294-8181.
Email: [email protected] or [email protected]
148
Abstract
The aim of this study was: 1) to evaluate the effect of oregano essential oil (EO)
and tannic acid (TA) on the survival and growth of Salmonella enterica, Listeria
monocytogenes, Escherichia coli O157:H7, Enterobacteriaceae, and total aerobic
count in raw ground chicken meat, 2) to evaluate the effect of adding EO and TA on
the survival and growth of Stapylococcus aureus, and aerobic plate count (APC) in
cooked meat during storage at different temperature conditions. Five different
treatments including 1) control (no antimicrobial agent), 2) 200 ppm EO, 3) 10 ppm
TA, 4) 10 ppm TA + 200 ppm EO, and 5) 5 ppm butylated hydroxyanisole (BHA)
were prepared. Oregano oil, tannic acid, BHA treatments showed significant (P < 0.05)
growth inhibitory effects on the APC when used separately. However, the
combination of EO + TA showed the strongest (P < 0.05) effect in reducing the APC
of raw chicken breast meat during storage at 4 and 10 oC. Of all the treatments tested,
the combination treatment also showed the strongest antimicrobial effects against
Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7, and
Enterobacteriaceae (ENT) in raw meat during storage. In cooked chicken breast meat,
EO and EO + TA combination were efficient (P < 0.05) in suppressing the growth of
aerobic bacteria in meat at all temperatures (10, 25, and 43 oC) tested. The
antimicrobial effect of TA was weaker than that of the OE, and TA did not show
significant inhibitory effect at the end of storage period compared to BHA. Oregano
essential oil treatment was more effective than the BHA and tannic acid in slowing
down the growth of Stapylococcus aureus in cooked meat during storage at 10, 25,
and 43 oC, but the OE + TA treatment was more effective than the EO alone. Based on
these results it is concluded that EO has strong antimicrobial activities but its effect
can be further improved when it is combined with TA. This study demonstrates that
EO + TA combination could be a good natural antimicrobial treatment to suppress
natural bacterial flora and food borne pathogens in ground chicken meat.
Keywords: Oregano essential oil, tannic acid, aerobic plate count, pathogens, chicken
meat.
149
Introduction
Several foods including ground meats serve as a vehicle for transmission of
enteric pathogenic bacteria to humans. The World Health Organization (WHO)
reported that foodborne diseases are still highly responsible for the high levels of
morbidity
and
mortality
in
the
general
population
and
especially
in
immunocompromised groups of people including infants and young children, the
elderly, pregnant women, organ transplant patients, and patients undergoing
chemotherapy (WHO, 2014). The Center for Disease Control and Prevention (CDC,
2011) estimated that approximately 1 in 6 Americans (or 48 million people) became
ill, 128,000 were hospitalized, and 3,000 died of foodborne diseases in the U.S.
annually (caused by known and unknown agents), and the recent cost estimation for
the foodborne illness is around $ 51.0 billion to $ 77.7 billion each year (Scallan et al.,
2011a; 2011b; Scharff, 2012). Although the 2011 estimation showed improvements
from that of the 1999 (Mead et al., 1999), more efforts are needed to control the top
known pathogens and determine the causes of foodborne illness (due to unknown
agents), death without known causes, and economic cost estimation (Buzby and
Roberts, 2009; Scallan et al., 2011a; 2011b; Scharff, 2012). In addition, the CDC
indicated that reducing foodborne illness by 10% would keep about 5 million
Americans from getting sick each year (CDC, 2014). The CDC considers that
Salmonella and Staphylococcus aureus among the top five pathogens causing
domestically acquired foodborne illnesses, Salmonella and E. coli O157 among the
top five pathogens causing domestically acquired foodborne illnesses resulting in
hospitalization, and Salmonella and Listeria monocytogenes among the top five
pathogens causing domestically acquired foodborne illnesses resulting in death (CDC,
2011).
Meat is an important part of our food and is an excellent medium in which
many pathogens can grow. The contamination of meat by pathogens can occur before
150
or after processing of the meat products (Jay, 2000), and through cross contamination
between carcasses, handling, processing equipment, water, air, and food contact
surface in the kitchen (Perez-Rodriguez et al., 2007). However the development of
foodborne illness depends on the ability of pathogens to survive or grow in raw or
cooked meat. For example, compared to cooked meat products, raw meat has a higher
probability of being contaminated with Salmonella enterica, Listeria monocytogenes,
and Escherichia coli and causing foodborne illness. Although each of these pathogens
has their optimal conditions for survival, all can survive in fresh or ready-to-eat (RTE)
poultry meat products. On the other hand, Staphylococcus aureus has higher ability to
survive in cooked meat than in raw meat because their ability to compete with other
microorganisms is very poor in raw meat products (Casman et al., 1963; Normanno et
al., 2007).
Poultry meat is the most popular meat consumed but can be easily contaminated
by pathogens that cause food borne illness (Chouliara et al., 2007). Storage
temperature and hygienic food handling are among the most important factors that can
prevent contamination or suppress the growth of pathogens (Vernozy-Rozand et al.,
2002; Solomakos et al., 2008). Most of the foodborne illnesses occur due to
ineffective preservation methods, food contamination during processing, improper
cooling of foods, and unhygienic practices of food handlers. Some pathogens such as
Salmonella and Escherichia coli O157:H7 can be easily killed if proper cooking
methods for the meat are used (Ingham et al., 2005) while others are resistant to heat
treatment or produce toxins that cannot be destroyed by cooking. Many synthetic
antimicrobial agents such as sodium lactate, acetate, benzoic acid etc. have been used
to prevent or slow down the growth of pathogens in meat. Recently, the food industry
has developed a greater interest in using natural antimicrobial agents in place of the
synthetic ones to satisfy consumers who are concerned about the health effects of
synthetic antimicrobial agents (Solomakos et al., 2008; Zhang et al., 2009; Davidson
et al., 2013).
Many spices, herbs and plant extracts have been studied in the food safety area
151
because of their antimicrobial activity (Chung et al., 1998a; Horosova et al., 2006;
Chouliara and Kontominas, 2006; Venkitanarayanan et al., 2013). Tannic acid and
oregano oil exhibit antimicrobial activity because of their chemical structure and
their properties (Scalbert, 1991; Botsoglou et al., 2003; Burt, 2004). Oregano
essential oil contains high level of phenolic compounds such as carvacrol and thymol
that are responsible for their antimicrobial activity (Beuchat, 1994; Sivropoulou et
al., 1996). Tannic acid has been well studied and proven for its activity against
several types of pathogens (Kim et al., 2010).
The antimicrobial mechanisms of tannic acid are through their astringent effect,
enzyme and substrate complex formation, and metal chelating activity (Chang, 1993,
1998b). However, no study has been conducted to determine the combined effects of
these two natural antimicrobial agents on the growth of pathogens and the storage
stability of ground chicken meat. A limitation of using these two natural
antimicrobial agents together in meat is due to their effects on the flavor, odors, and
their interactions with other food ingredients (Zucker, 1983; Chang 1998a; Burt,
2004). Thus, finding the optimal concentrations of these two natural antimicrobial
agents to avoid changes in eating quality while maintaining the microbial safety of
the meat are an important issue.
The objectives of the present study were: 1) to evaluate the effect of oregano
essential oil and tannic acid on the survival and growth of Salmonella enterica,
Listeria monocytogenes, Escherichia coli O157:H7, aerobic mesophilic bacteria and
Enterobacteriaceae in raw ground chicken meat, and 2) to evaluate the effect of
adding oregano oil and tannic acid on the survival and growth of Stapylococcus
aureus, and aerobic mesophilic bacteria in cooked meat during storage at different
temperatures.
152
Materials and Methods
Sample preparation: Chicken breast meat was obtained from a local grocery store
and ground twice a through a 10-mm plate then a 3-mm plate (Kitchen Aid, Inc., St.
Joseph, MI). Five different treatments were prepared: 1) control (no antimicrobial
agent), 2) 200 ppm oregano essential oil, 3) 10 ppm tannic acid, 4) 10 ppm tannic acid
+ 200 ppm oregano essential oil, and 5) 5 ppm butylatedhydroxyanisole (BHA). The
amounts of oregano essential oil and tannic acid used were selected from the chemical
studies to achieve the maximum antioxidant benefits of the additives. The oregano
essential oil used was obtained from a certified company in Turkey (Healthy-Health
Oil of Oregano, Turkey). The GC/MS analysis of the oregano essential oil indicated
that 80.12% of the essential oil was carvacrol. BHA powder (0.1 g) and oregano
essential oil (1.25 g) were dissolved in 10 ml of 100% ethanol, and then mixed with
50 ml mineral oil to make their stock solutions. The ethanol added was removed using
a rotary evaporator (Model BUCH Rotavapor R-200) at (70 °C, 175 mbar vacuum
pressure) before adding the stock to meat samples. Tannic acid powder (Sigma
Chemical Co., St Louis, MO) was dissolved in de-ionized sterilized water before adding
it to meat. Each treatment was added to the ground chicken meat and then mixed for 2
min in a bowl mixer (Model KSM 90; Kitchen Aid Inc., St. Joseph, MI, USA). All
treatments were added with the same amounts of water and mineral oil to provide the
same moisture and oil conditions. Samples of ground chicken meat (approximately 25
g each) were individually packaged in separate oxygen-permeable bags (polyethylene, 4 x
6.2 mil, Associated Bag Co., Milwaukee, WI), and used for the raw-meat study
performed in the Microbial Food Safety Laboratory (MFSL) in the Department of
Food Science and Human Nutrition. The same preparation method was used for
cooked meat study, but the samples were packaged in oxygen impermeable vacuum
bags (O2 permeability, 9.3 mL O2/m2/24 h at 0 °C, Koch, Koch, Kansas City, MO,
USA). The patties were cooked in bag in a 90 °C water bath (Isotemp®, Fisher
Scientific Inc., Pittsburgh, PA, USA) until the internal temperature of the meat
153
reached 75 °C. After cooling to room temperature, the cooked meats were packed in
insulated coolers and carried to MFSL for the cooked meat study. All meat samples
were held in a walk-in cooler (4.4 ºC) and used in experiments within two hours after
preparation.
Water activity and pH: The initial water activity (wa) of raw meat was measured
using water activity meter (AQUA-LAB 4-TEV, Pullman, WA, USA). A 2-g portion
of each non-inoculated raw ground meat sample was spread on the surface of a white
plastic container. After the calibration of the instrument, the sample was inserted
inside the meter and the water activity value was obtained and recorded. The water
activity meter was calibrated and checked using an empty container, and AQUA-Lab
Standard solution before use. The pH values of the ground chicken raw meat samples
were determined using a pH meter (Accumet 925: Fisher Scientific, Fair Lawn, N.J.,
U.S.A) after homogenizing the 1.0-g samples with 9 ml de-ionzed distilled water
(Sebranek et al., 2001).
Preparation of bacterial cultures: Five-strains each of Salmonella enterica, E. coli
O157:H7, and Listeria monocytogenes, and four strains of Staphylococcus aureus
were obtained from the Microbial Food Safety Laboratory at Iowa State University.
Frozen stocks (in 10 % glycerol at -80 ºC) were thawed and separately transferred
twice to 10 ml sterile tubes of tryptic soy broth (TSB; Difco, BD, Sparks, MD) before
making the working cultures. Before the inoculation process each working culture
(parent cells) was individually grown in 10 mL of tryptic soy broth (Difco laboratory,
Detroit, MI) supplemented with 0.6 % yeast extract (TSBYE) at 35 ºC for 24h. These
strains were adapted gradually in the TSBYE with different concentrations of added
Naldixic acid (NA; antibiotic; M.W 232.24, 10 ug/ml, 30 ug/ml, and 50 ug/lm,
Sigma-Aldrich, St. Louis, Missouri). Two consecutive transfers 24-h for each
154
Naldixic acid resistant strain (NAR) cultures were prepared. For each pathogen, at the
beginning of each study, 6 ml of each individual NAR adapted culture in TSBYE was
aseptically transferred to a sterilized centrifuge tube to give a total 30 ml of strain
mixture culture (NARC).
Bacterial cells from each mixture of strains were harvested by centrifugation at
10,000 x g for 10 min at 4 ºC using a Sorvall Super T21 centrifuge (American
Laboratory Trading, Inc., East Lyme, CT). The pelleted cells were re-suspended in a
30-ml sterilized saline (0.85 % NaCl), washed by vortexing, and then centrifuged
again at the same speed and temperature conditions. After harvesting the cells from
the second centrifugation, the harvested cells were suspended in fresh saline (0.85%
w/v NaCl) and diluted (10-fold) using tubes of saline to obtain 106 CFU/ml in a
suspension of washed cells used for inoculating the ground chicken meat.
Preparation and inoculation of meat samples: Samples of ground chicken meat
were transported to the Microbial Food Safety Laboratory (Dept. of Food Science &
Human Nutrition) for inoculation and microbial analysis. Each sample was
inoculated with a mixture of five nalidixic-acid resistant serotypes of Salmonella
enterica (S. Typhimurium, S. Newport, S. Kentucky, S. Oranienburg, and S.
Montevideo), 5-strain mixture of Escherichia coli O157:H7 (ATCC 35150, ATCC
43894, ATCC 43895, WS 3062 and WS 3331) and 5-strain mixture of Listeria
monocytogenes (Scott A, H7969, H7596, H7762 and H7962; all serotype 4b) to give
an initial cell concentration of ~104 colony forming units (CFU)/g for each pathogen
indiviually. All inoculated packages of ground chicken meat were loosely closed,
manually massaged for 30 s from outside of the bag, and held at different storage
times and temperature. At set intervals during storage, samples of raw meat were
analyzed for Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes
survivors. In addition, separate bags of non-inoculated ground meat were held at 4
and 10 °C and analyzed for aerobic plate counts and numbers of viable
Enterobacteriaceae.
155
The same inoculation preparation procedure was performed for the cooked meat
using a 4-strain mixture of Staphylococcus aureus (ATCC 6538, ATCC 25923, ATCC
10390, and ATCC 29213) obtained from American Type Culture Collection. In
addition, separate bags of non-inoculated ground cooked meat were held at different
temperatures (10, 25, and 43 °C) and analyzed for aerobic plate counts.
Microbial analysis: Packages of ground chicken meat were aseptically opened,
and two-25 g portions of meat were aseptically transferred into separate sterile
filter-lined stomacher bags. Sterile 0.1% (w/v) peptone water (225 ml) was added to
each bag. The resulting mixtures were homogenized for one minute in a laboratory
stomacher blender operating at medium speed. Further 10-fold serial dilutions of meat
samples were prepared in tubes of 0.1% peptone and 0.1 ml aliquots of diluted
samples were surface plated on appropriate selective agar media to enumerate
pathogenic bacteria.
Sorbitol MacConkey agar (SMA), Modified Oxford (MOX) agar, Xylose lysine
Deoxycholate XLD agar, and Baird-Parker Agar base were used to enumerate
Escherichia coli O157:H7,
Listeria monocytogenes, Salmonella enterica, and
Staphylococcus aureus, respectively. Inoculated agar plates were incubated at 35 °C
and bacterial colonies were counted after 48 hours. The aerobic plate count (APC) was
determined by surface plating aliquots (0.1 ml) of meat homogenate on tryptic soy agar
(TSA), incubating the inoculated TSA plates at 35 °C, and counting bacterial colonies
at 48 hours. The numbers of viable Enterobacteriaceae were determined using the agar
overlay technique. Aliquots (0.1 ml or 1.0 ml) of meat homogenate were placed in a
petri dish and molten TSA (48°C) was poured onto the dish to form the first layer. The
TSA plate was held for 60 minutes at ambient temperature before pouring a layer of
violet red bile agar (VRBA) over the TSA. The solidified TSA/VRBA plates were
incubated at 35 °C and bacterial colonies were counted at 24 hours.
Statistical analysis: The experiment was a completely randomized design (CRD).
156
Two separate samples per treatment per replication were analyzed over 2 replications
of each independent experiment. The statistical analysis was performed using the
SPSS (Chicago, IL, USA) package for Windows (Ver. 20.0). The mean values were
compared using the one-way analysis of variance (ANOVA) followed by Duncan’s
multiple range tests (p < 0.05). Mean values and standard error of the means (SEM)
were reported at (P<0.05).
Results and Discussion
The aerobic plate count (APC) provides an estimate of the level of numbers of
viable bacteria that can grow aerobically in a product. Obtaining these data may help
evaluate good manufacturing practices for foods as well as determine potential
sources of contamination (American public Health Association, 1984). In addition
Enterobacteriaceae is a large family of rod-shaped Gram-negative, facultative
bacteria, which includes some pathogens such as Salmonella, Yersinia, Escherichia
coli, Klebsiella and Shigella. Most members of this family are a normal part of the gut
flora and are found in the intestines of human and other animals. However, some are
also found in water, soil, or parasites of a variety of different animals and plants.
Enterobacter spp and Escherchia coli (members of the coliform group) are commonly
used to indicate the level of sanitary quality of foods and water. Salmonella enterica,
Listeria monocytogenes, and Staphylococcus aureus are considered important
pathogens related to the microbial safety of meats (FSIS, 2014) in the US.
The initial pH value of the ground chicken breast meat without additive was
approximately 6.2 (Table 1), and was similar to that of meat samples containing the
other treatments. Adding tannic acid or oregano oil to the meat did not change the pH
values. In addition, the pH values for all treatments remained unchanged during
storage time (data not shown). The initial water activity (aW) values (0.98, Table 1)
were similar among all treatments, indicating that all the treated samples of meat have
almost
similar intrinsic conditions (with respect to pH and water activity) for
157
microbial growth at the beginning of the study.
For the raw meat samples, the storage temperatures of 4 ºC and 10 ºC were
selected to represent a typical refrigeration temperature (4 ºC) and temperature abuse
(10 ºC) of the meat in a faulty refrigeration unit. The total aerobic count of raw meat
patties significantly (p < 0.05) increased during storage time in control group
incubated at both 4 ºC and 10 ºC (Table 2). The initial APC (day 0) for the fresh
ground chicken meat used for all treatments was almost similar (around 4.15 to 4.37
log10 cfu/g) indicating that the microbial quality of chicken meat was
acceptable
(Dawson et al., 1995). The differences between the treatments started to appear after 2
days of storage. The combination of oregano oil and tannic acid treatments resulted in
lower total plate counts compared to the control and other treatments after two days.
Meat samples with BHA also had lower APC than the control group over the storage
time. Oregano essential oil treatment reduced the APC by 1.8 log compared with the
control at Day 8; however, the combination of oregano oil and tannic acid showed the
highest antimicrobial activity against the APC in raw meat samples (Table 2). The
antimicrobial effect of combination treatment reduced the APC by 2.07 log unit
compared with the control during storage at 10 oC (at Day 4). The ICMSF (1986)
considered 7.0 log10 cfu/g the microbiological limit for microbial spoilage of fresh
poultry meat. Therefore, the combination treatment was able to extend the shelf-life of
the raw chicken meat by 2 days compared to the control (Tables 2). Chouliara et al.
(2007) reported that oregano oil (0.1 %) extended the shelf-life of fresh chicken breast
meat when stored at 4 oC. Skandamis and Nychas (2001) found that oregano essential
oil (0.5% and 1%) delayed microbial growth and suppressed the final count of the
spoilage micro-organisms in minced beef meat stored at 5 oC.
The antimicrobial effect caused by adding oregano oil can be explained by the
high level of carvacrol, thymol and the other polyphenolic structures (Sivropoulou et
al., 1996). Tannic acid has high binding efficiency to iron which makes it like a
siderophore to complex iron from the entire media affecting the iron availability to the
microorganisms. The microorganisms living under aerobic conditions need iron for
158
many biological functions, such as reducing ribonucleotide precursor of DNA,
making heme compounds for the respiratory chain, and many essential processes for
survival (Chung et al., 1998b).
Many researchers believe that using combinations of natural antimicrobial plant
extracts may increase their effectiveness through the synergistic effects (Pol and Smid,
1999; Lin et al., 2004; Brewer., 2011; Abd El-Kalek and Mohamed, 2012). Although,
the combination of oregano oil and tannic acid showed better antimicrobial effects
than using them alone, no synergistic antimicrobial effect of tannic acid and oregano
oil was found in this study (Table 2).
Similar results were found when tannic acid + oregano oil was added to the raw
chicken meat that was later cooked. The initial number of APC was very low
compared to the raw meat with around two log differences. Oregano oil, tannic acid +
oregano oil combination, and BHA had the lowest initial APC numbers among the
treatments and those numbers were significantly lower than that of the control and
tannic acid treatments (Tables 5 & 6). These results strongly suggested that these
preservatives, including the combination treatment, reduced the initial aerobic count
in the meat during cooking. The APC for cooked meat incubated at 10 oC showed the
highest bacterial count in the control group (around 8.31 Log10 CFU/g) at day 5 of
storage. Tannic acid + oregano oil combination and oregano oil showed the strongest
effect in suppressing the microbial growth at 10 oC. Tannic acid alone did not show
significant effect at days 3 and 5 (Table 5). Also, tannic acid showed no antimicrobial
effect at 0, 2, and 8 hour in cooked chicken meat held at 25 oC. These results indicate
that tannic acid’s antimicrobial activity on the APC of the cooked meat patties was
very low. All additive treatments showed significant antimicrobial effect in
suppressing the growth of aerobic microorganisms at 43 oC after 6 hours of storage
(Table 6).
The same antimicrobial trend was found with oregano oil + tannic acid
treatment on the total viable count of Enterobacteriaceae (ENT) in raw meat. The
control group showed the highest ENT cell counts (8.74 CFU/g) after 8 days of
159
storage at 4 oC, and reached to 7.60 CFU/g after storage at 10 oC for 4 days. The
growth rate of Enterobacteriaceae was suppressed further when the oregano oil +
tannic acid combination was used (Table 3). These results indicate the good potential
of the combination treatment to suppress growth of Gram-negative, facultative
bacteria, including pathogens in this group, in raw chicken during temperature abuse
at 10 ºC.
Among the pathogenic microorganisms, Escherichia coli O157:H7 was the
most susceptible to oregano oil + tannic acid combination compared to the other
treatments. After 6 days of incubation at 10 oC, E. coli O157:H7 with the oregano oil
+ tannic acid treatment showed the lowest log10 number (around 6.99 CFU/g) while
the control had 8.51 CFU/g. No significant difference (P > 0.05) between tannic acid
and BHA treatment during storage time was observed (Table 4).
Oregano oil at 200 ppm showed higher effect in suppressing the growth of E.
coli O157:H7 than the tannic acid and BHA treatments. The log10 difference between
the oregano oil + tannic acid combination and the control was around 1.52 units at
day 6. Oregano oil + tannic acid combination and oregano oil treatments showed the
strongest antimicrobial effect against Salmonella. Adding a combination of oregano
oil and tannic acid to raw ground chicken meat reduced the Log value by 1.5 unit
compare to the control at day 6 of storage time (Table 4).
Listeria monocytogenes was more affected by the oregano oil + tannic acid
combination treatment than other treatments. The Listeria monocytogenes data
showed that oregano essential oil has stronger antilisterial effect than tannic acid or
BHA treatment. These results agree with those of Lin et al. (2004) who found that
oregano suppressed the growth of Listeria monocytogenes in fish and other meats.
Oregano oil + tannic acid combination treatment showed 1.63 log10 lower than the
control at Day 4, and 1.95 log10 lower than control at Day 8 of storage (Table 4).
The oregano oil + tannic acid combination treatment did not show any
synergistic effect against Enterobacteriaceae, Escherichia coli O157:H7, Salmonella
enterica, and Listeria monocytogenes although the combination showed the highest
160
antimicrobial effects among the treatments. The antimicrobial effect of oregano oil in
suppressing the growth of E. coli O157:H7 is due to the high carvacrol content in the
essential oil (Xu et al., 2008). Other researchers (Ultee et al., 1998; 2002; Di Pasqua
et al., 2010) reported that oregano essential oil and its major component carvacrol
changed the membrane potential of bacterial cell wall and disturbed the biological
activities inside the cell.
For the cooked meat, the temperatures of 10, 25, and 43 oC were all selected to
represent temperature abuse conditions that allow the growth of Staphylococcus
aureus in a faulty refrigeration unit (10 ºC), during inadvertent holding of cooked
meat at ambient temperature (25 ºC) and during unsafe (43 ºC) hot-holding
temperature that may often occur in foodservice operations. At all the temperature
conditions used in the present study (10, 25, and 43 oC), Staphylococcus aureus in
cooked meat was more sensitive to oregano oil + tannic acid combination compared
to other treatments. The initial number of Staphylococcus aureus was the same for all
treatments at the beginning of all studies. The oregano oil + tannic acid combination
treatment in cooked meat samples resulted in numbers of viable Staphylococcus
aureus
that were 1.42 log10 lower than those of the control following 8 hours of
exposure of the meat at 43 oC (Tables 7 & 8). The antimicrobial activity of oregano oil
and tannic acid against Staphylococcus aureus growth in various foods is also
reported by many research studies (Beuchat and Heaton, 1975; Akiyama et al., 2001;
Ozcan, 2001).
Conclusion
Oregano oil at level 200 ppm showed a good antimicrobial activity in both raw
and cooked meat. However, if oregano essential oil (200 ppm) and tannic acid (10
ppm) were combined, their antimicrobial effect could be better than using them singly.
In terms of general food safety, the combination exhibited strong antimicrobial effects,
but the effects were additive not synergistic. This combination could be a good
antimicrobial treatment that can suppress most of the food borne pathogens as well as
microorganisms that can grow aerobically in raw and cooked chicken breast meat.
161
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166
Table 1. Water activity and pH of raw chicken breast meat at 0 day.
Treatment
Water activity (wa)
pH value
Control
0.986a
6.22a
Tannic acid
0.987a
6.21a
Oregano
0.991b
6.23a
Combination
0.989ab
6.20a
BHA
0.987a
6.24a
SEM
0.001
0.061
a-f
Statistically significant differences (p<0.05) between treatment. n = 4 for water
activity and n = 3 for pH value
Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil;
Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.
167
Table 2. Total Plate Counts of raw chicken breast meat during storage at 4 °C
and 10 °C.
Storage time (days)
Treatments
0 day
2 days
4 days
6 days
8 days
SEM
----------------------------------- Log CFU/g meat ------------------------Storage at 4°C
Control
4.37aw
6.35bx
8.34dy
9.36cz
9.82dz
0.16
Tannic acid
4.51aw
5.31ax
7.42cy
7.99abz
8.37bz
0.13
Oregano
4.59av
5.46aw
7.20bx
8.04aby
8.34bz
0.05
Combination
4.58av
5.47aw
6.84ax
7.65ay
8.02az
0.05
BHA
4.39av
5.57aw
7.23bx
8.28by
8.89cz
0.08
SEM
0.09
0.15
0.02
0.14
0.07
4.15abx
7.20dy
9.35cz
-1
-
0.04
bx
cy
bz
-
-
0.03
8.37
bz
-
-
0.08
Storage at 10 °C
Control
Tannic acid
1
4.26
abx
6.63
by
8.27
Oregano
4.15
6.42
Combination
4.07ax
5.44ay
7.28az
-
-
0.11
BHA
4.18abx
6.66cy
8.24bz
-
-
0.01
SEM
0.04
0.06
0.09
Not determined. n=4.
a-e
Different superscripts within a column (for a specified storage temperature) differ
significantly (p < 0.05).
x-z
Different superscripts within a row (for a specified storage temperature) differ
significantly (p < 0.05).
Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil;
Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.
168
Table 3. Numbers of viable Enterobacteriaceae in ground raw chicken breast
meat stored at 4 °C and 10 °C.
Treatments
Storage time (days)
0 day
2 days
4 days
6 days
8 days
----------------------- Log CFU/g meat ------------------------
Storage at 4 °C
Control
Tannic acid
Oregano
5.20bw
6.35cx
8.13cy
8.74dz
0.05
av
4.75
aw
bx
by
cz
0.07
4.70
aw
bz
0.06
aw
az
4.14
av
4.17
av
4.23
4.59
BHA
SEM
4.15av
0.05
Storage at 10 °C
Control
Oregano
1
4.21av
Combination
Tannic acid
SEM
5.42
bx
5.49
ax
6.89
by
6.72
ay
7.79
7.53
4.86
6.20
7.10
0.07
4.71aw
0.05
5.50bx
0.06
6.73by
0.08
7.75cz
0.06
0.05
4.15ax
6.27cy
7.60dz
-1
-
0.06
bx
5.34
by
cz
-
-
0.09
5.08
by
bz
-
-
0.03
5.69
az
-
-
0.05
6.13bz
0.02
-
-
0.04
4.33
abx
4.22
ax
ay
Combination
4.16
4.80
BHA
SEM
4.15ax
0.05
5.24by
0.08
6.71
6.08
Not determined. n=4.
a-e
Different superscripts within a column (for a specified storage temperature) differ
significantly (p < 0.05).
x-z
Different superscripts within a row (for a specified storage temperature) differ
significantly (p < 0.05).
Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil;
Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.
169
Table 4. Number of viable Escherichia coli O157:H7, and Salmonella enterica
in ground raw chicken breast meat during storage at 10 °C.
Treatments
0 day
8 days
SEM
------------------------ Log CFU/g meat -----------------------------Escherichia coli
Control
4.19aw
5.34cx
6.78cy
8.51dz
-1
0.03
Tannic acid
aw
cx
4.15
5.26
ax
abx
by
6.43
aby
6 days
cz
-
0.12
bz
-
0.12
7.91
Oregano
4.23
4.60
6.13
7.29
Combination
4.04ax
4.21ax
6.00ay
6.99az
-
0.06
aw
bcx
by
cz
0.18
BHA
SEM
5.01
0.18
6.40
0.11
7.93
0.09
-
Listeria monocytogenes
Control
4.21av
5.14cw
6.35dx
8.13dy
8.71dz
0.05
av
bw
cx
cy
cz
0.08
az
0.15
az
0.09
bz
Tannic acid
Oregano
Combination
4.09
0.05
4.15
aw
4.17
aw
4.23
av
4.79
abw
4.59
aw
4.45
abw
5.63
bx
5.12
ax
4.72
cx
7.69
ay
6.14
ay
6.06
by
8.51
6.86
6.76
BHA
SEM
4.15
0.06
4.63
0.07
5.50
0.12
6.73
0.12
7.68
0.04
0.05
Salmonella enterica
Control
4.07aw
5.37cx
6.78cy
8.34dz
-
0.06
aw
cx
bcy
cz
Tannic acid
4.06
5.30
6.61
7.67
-
0.05
Oregano
4.18ax
4.60abx
5.90 ay
7.14bz
-
0.17
aw
ax
ay
az
-
0.04
cz
-
0.17
Combination
BHA
SEM
1
Storage (days)
2 days
4 days
4.03
aw
4.09
0.06
4.21
bcx
5.01
0.17
5.75
by
6.30
0.12
6.84
7.74
0.07
Not determined. n = 4.
a-e
Different superscripts within a column of the same meat differ significantly (p <
0.05).
x-z
Different superscripts within a row differ significantly (p < 0.05).
Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil;
Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.
170
Table 5. Aerobic plate count of cooked chicken meat patties during storage at
10 °C.
Storage (days)
Treatments
0 day
1 day
3 days
5 days
-------------------------- Log CFU/g meat -------------------------Control
Tannic acid
Oregano
Combination
BHA
SEM
1
2.58dw
2.52dw
2.01bw
1.79aw
2.30cw
0.04
4.29cx
3.93abx
4.16cx
3.73ax
4.12bcx
0.07
7.54cy
7.28cy
5.70ay
5.71ay
5.86by
0.03
8.31cz
8.28cz
6.33az
6.25az
6.71bz
0.05
SEM
0.04
0.08
0.04
0.03
0.03
Not determined. n = 4.
a-e
Different superscripts within a column differ significantly (p < 0.05).
x-z
Different superscripts within a row differ significantly (p < 0.05).
Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil;
Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.
171
Table 6. Aerobic plate counts of cooked meat patties held at 25 or 43 °C during
storage.
Storage (hrs)
Treatments
0 hr
2 hr
4 hr
6 hr
8 hr
------------------- Log CFU/g meat ----------------------
SEM
Storage at 25 °C
Control
2.60cv
3.46acw
4.51dx
5.49ey
6.88cz
0.04
Tannic acid
2.54cv
3.49cw
4.30cx
5.31dy
6.68cz
0.04
bv
bw
ax
by
bz
0.04
5.26
az
0.05
0.09
Oregano
2.32
av
2.89
Combination
2.16
2.62
BHA
2.11av
SEM
aw
3.62
ax
4.32
ay
5.70
3.59
4.07
2.61aw
3.77bx
4.94cy
5.58bz
0.03
0.05
0.04
0.04
0.09
Control
2.60cv
3.88dw
5.11ex
6.16dy
8.32dz
0.03
Tannic acid
2.54cv
3.69cw
4.85dx
5.82cy
7.79cz
0.03
Oregano
2.31bv
2.89bw
3.67bx
4.65by
6.69bz
0.03
av
aw
ax
ay
az
0.01
bz
0.03
Storage at 43 °C
Combination
BHA
SEM
2.14
av
2.06
0.02
2.68
2.86
bw
0.04
3.48
cx
3.79
0.02
4.31
4.67
by
0.02
6.46
6.75
0.03
a-e
Different superscripts within a column (for a specified storage temperature) differ
significantly (p < 0.05).
x-z
Different superscripts within a row (for a specified storage temperature) differ
significantly (p < 0.05). n = 4.
Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil;
Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.
172
Table 7. Numbers of viable Staphylococcus aureus in cooked ground chicken
meat during storage at 10 °C.
Storage (days)
Treatments
0 day
1 day
3 day
5 day
7 day
SEM
-------------------------------- Log CFU/g meat--------------------------------Control
4.32av
5.05dw
6.77dx
7.48dy
8.39dz
0.06
Tannic acid
Oregano
4.27av
4.64bw
5.45bx
6.70cy
7.30bz
0.02
aw
4.33
aw
bx
by
bz
0.07
aw
6.63
az
0.06
7.33bz
0.08
0.04
4.31
aw
Combination
4.24
4.28
BHA
SEM
4.19av
0.07
4.93cw
0.02
5.44
ax
6.50
ay
5.30
6.00
5.69cx
0.02
6.68cy
0.05
7.13
a-e
Different superscripts within a column of the same meat are differ significantly (p <
0.05).
x-z
Different superscripts within a row are differ significantly (p < 0.05). n = 4.
Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil;
Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.
173
Table 8. Numbers of viable Staphylococcus aureus in cooked ground meat
patties held at 25 or 43 °C.
Storage (hrs)
Treatments
0 hr
2 hr
4 hr
6 hr
8 hr
SEM
---------------------------- Log CFU/g meat ----------------------------Storage at 25 °C
Control
4.32av 4.65cw
5.37dx
5.65dy
6.32cz
0.01
Tannic acid
4.33av
4.45abw
4.77bcx
5.15cy
5.79bz
0.03
Oregano
4.31aw
4.44abw
4.76bx
4.91by
5.77bz
0.04
ax
ay
az
0.07
bz
Combination
aw
4.24
aw
4.43
ax
bx
4.60
cy
4.78
by
5.31
BHA
SEM
4.32
0.07
4.47
0.01
4.86
0.03
4.92
0.01
5.81
0.04
0.02
Storage at 43 °C
Control
Tannic acid
4.30av
5.33bw
6.45dx
7.65cy
8.72dz
0.01
av
4.67
aw
bx
by
bz
0.02
4.62
aw
abz
0.05
aw
az
Oregano
4.27
av
4.31
av
Combination
4.24
4.62
BHA
4.32av
SEM
0.06
5.61
bx
5.60
ax
6.82
by
6.77
ay
7.46
7.39
5.39
6.48
7.30
0.07
4.68aw
5.75cx
6.81by
7.63cz
0.02
0.02
0.04
0.02
0.03
a-e
Different superscripts within a column (for a specified storage time) are differ
significantly (p < 0.05).
x-z
Different superscripts within a row are differ significantly (p < 0.05). n = 4.
Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil;
Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.
174
CHAPTER 7
SUMMARY AND CONCLUISONS
Finding good combinations of natural antioxidants and antimicrobials from
plant origins has been a hot topic in recent years. Researchers and food industry all
share this approach because it reflects consumer preferences. Replacing synthetic
antioxidants with natural ones is an excellent idea but requires further investigation.
Several factors can affect the antioxidant and antimicrobial activity of these natural
plant extracts in the meat system. For example the concentration and interaction of
natural antioxidant or antimicrobial agents with other compounds, pro-oxidant, free
radical, and oxygen availability in the meat system will affect their activity. So the
objectives of the present study were: 1) to evaluate the antioxidant effect of oregano
essential oil, tannic acid, and/or their combinations in ground chicken meat, 2) to
evaluate the antimicrobial effect of oregano essential oil, tannic acid, and/or their
combinations in ground chicken meat, and 3) to evaluate the sensory characteristics of
combined oregano oil and tannic acid in ground chicken meat.
Oregano essential oil, at levels 300 and 400 ppm, showed the highest
antioxidant effect in ground chicken meat. Adding >100 ppm of oregano essential oil
improved color stability of raw meat and decreased off-odor volatile of cooked meat,
but their effects were much clearer in cooked than in raw meat. Over all, oregano
essential oil could be used in place of synthetic antioxidant (e.g., BHA) to prevent
quality deterioration in raw and cooked meat during storage.
Tannic acid at 5 or 10 ppm was effective in maintaining meat color and
retarding lipid and protein oxidation in raw ground meat. In addition, tannic acid at >
5 ppm effectively reduced lipid oxidation, protein oxidation, and off-odor-related
volatiles in cooked ground breast during storage. Therefore, tannic acid could be a
good candidate as a natural antioxidant to prevent oxidative changes and color
changes in raw and cooked chicken breast meat during storage.
Combination of tannic acid and oregano essential oil at level 200 ppm and 10
175
ppm, respectively, showed positive effects on the TBARS values, total carbonyl, color
stability, and off-odor volatile formation. The combination of 200 ppm oregano oil
and 10 ppm tannic acid was a good replacement for the synthetic antioxidant in the
ground chicken meat. In addition, combinations of tannic acid and oregano essential
oil showed positive effects on the most sensory attributes of raw and cooked chicken
meat. This combination has the highest over all acceptability score of both raw and
cooked meat compared to the other treatments.
Oregano oil at level 200 ppm showed a good antimicrobial activity for both raw
and cooked meat. However, when oregano essential oil (200 ppm) and tannic acid (10
ppm) were combined, their antimicrobial effects were better than using them singly.
In term of general food safety, the combination exhibited strong antimicrobial effects,
but the effects were additive not synergistic. This combination showed a good
antimicrobial treatment and suppressed most of the food borne pathogens as well as
aerobic microorganisms in raw and cooked chicken breast meat. In addition, this
combination showed higher score than other treatments for most of the sensory
attributes used in this study.
Over all the combination of tannic acid (10 ppm) and oregano essential oil (200
ppm) could be a good replacement for the synthetic antimicrobials and antioxidants
currently used by the meat industry.