STRATEGIES FOR THE PREVENTION OF POTATO SPOILAGE DURING
STORAGE AND THE DISCOVERY OF THE ANTIMICROBIAL
ACTIVITY OF POTATOES
By
Amanda Rioux
B.S. Saint Joseph's College, 2003
A THESIS
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Master of Science
(in Food Science and Human Nutrition)
The Graduate School
The University of Maine
December, 2007
Advisory Committee:
Dr. Vivian C.H Wu, Assistant Professor of Food Science and Human Nutrition,
Advisor
Dr. Alfred Bushway, Professor of Food Science
Dr. Brian Perkins, Assistant Research Professor of Food Science and Human
Nutrition
STRATEGIES FOR THE PREVENTION OF POTATO SPOILAGE
DURING STORAGE AND THE DISCOVERY OF THE ANTIMICROBIAL
ACTIVITY OF POTATOES
By Amanda Rioux
Thesis Advisor: Dr. Vivian C.H Wu
An Abstract of the Thesis Presented
In Partial Fulfillment of the Requirements for the
Degree of Master of Science
(in Food Science and Human Nutrition)
December, 2007
Many potato farmers lose potatoes during storage due to the length of storage
time and microbial contamination. During harvesting, potatoes may become injured and
susceptible to microorganisms such as yeast and molds and bacteria. When in storage,
bacteria and fungi that were potentially introduced during harvesting may contaminate
some potatoes. As the potatoes are in such close contact during storage, the infection may
spread throughout the entire storage facility.
The first objective of this study was to develop a novel, simple gaseous chlorine
dioxide (CIO2) method that could effectively control natural flora, yeast and mold, and
Pseudomonas aeruginosa on potatoes during storage. Different treatments included the
combination of: 2 g of sodium chlorite and 2 g of acid into sachet to generate 16 mg/1
after 2.5 hrs and 20 mg/1 after 5 hrs (low treatment), 3g of sodium chlorite and 3 g of acid
to generate 24 mg/1 after 2.5 hrs and 30 mg/1 after 5 hrs (medium treatment), and 4 g of
sodium chlorite and 4 g of acid to generate 32 mg/1 after 2.5 hrs and 40 mg/1 after 5 hrs
(high treatment). Results were effective for yeast and mold, showing over a 5 log
CFU/potato reduction after 5 hr of treatment with a 4 g treatment of gaseous CIO2. The
natural flora study also showed over a 5 log CFU/potato reduction. For P. aeruginosa,
there was a slight increase in the reduction after 5 hr of the 4 g treatment with almost a 6
log CFU/potato reduction. The lowest treatment tested (2 g at 2.5 hrs) had reductions of
1.7, 1.9, and 2.3, log CFU/potato for natural flora, yeasts and molds, and P. aeruginosa,
respectively. Overall, gaseous CIO2 effectively killed natural flora, yeast and mold, as
well as P. aeruginosa on potatoes for each treatment.
As natural preservatives in food are desired, the second objective was to
determine the antimicrobial effects of peel and tubers on common foodborne pathogens.
Methods included using plain freeze dried potato juice that was tested at 50% (w/v) and
75% (w/v) concentrations with distilled water, as well as plain potato peel juice. These
compounds were tested utilizing the agar diffusion assay or the log reduction method.
Antioxidant levels were also studied to determine a correlation with the antimicrobial
study using the DPPH (l,l-Diphenyl-2 picrylhydrazyl) method.
The Russet potato peel treatment resulted in a complete 5 log CFU/ml reduction
over time (24 hr) against Escherichia coli 0157:H7. The freeze dried potato juice from
Russet potatoes at 75% (w/v) concentration, showed an inhibition zone of 13.3 mm
against Salmonella Typhurmium. The freeze dried tuber juice of Katahdin potatoes at a
50% (w/v) concentration had an average zone of inhibition of 13.2 mm (triplicate) against
S. Typhimurium. The antioxidant study showed that the potato peel resulted in slight
antioxidant activity, whereas the tuber did not.
Overall, potatoes showed antimicrobial activity, yet these studies may require
further research such as different extraction methods. Antioxidants in potatoes may be
further studied using quantitative methods.
ACKNOWLEDGEMENTS
As they were there for great guidance and inspiration, 1 would like to thank my
committee members; Dr. Vivian C.H Wu, Dr. Brian Perkins, and Dr. Alfred Bushway. I
would like to especially thank my advisor, Dr. Vivian C.H. Wu as she kept me strong and
positive.
I would also like to thank my family and friends especially my husband, Steve as
he supported me during both my times of disappointment and accomplishment, making
sure that love and comfort were provided throughout my entire journey. I am deeply
appreciative of Steve, Tom Christensen, Stig Callahan, Tom Lamontagne, and Scott
Nichols as well as the rest of The Advanced Manufacturing Center for their ideas and for
building the materials used in one of my studies. If it was not for their time and expertise,
I would not have been able to succeed. I would especially like to thank my close friend
Scott Nichols, as he not only provided emotional support, ensuring that I would succeed,
he provided technical support for my thesis which was of great appreciation. My parents,
Paul and Louise Henault are to be given special thanks as they brought me up with the
greatest inspiration that has given me the desire to succeed and never give up.
11
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
ii
LIST OF TABLES
viii
LIST OF FIGURES
ix
Chapter
1.
INTRODUCTION
1
2.
LITERATURE REVIEW
4
Potato Industry
4
Microorganisms Associated with Spoilage of Potatoes
5
Natural flora
6
Fungi
7
Pseudomonas aeruginosa
8
Sanitation Methods for Fresh Fruits and Vegetables
9
Ozone
10
Peracetic acid
12
Hydrogen peroxide
12
Chlorine
13
Ultra violet radiation
14
Curing
15
Other sanitation methods
15
Chlorine dioxide (C102)
16
Generation Methods for Aqueous and Gaseous Chlorine Dioxide
18
Applications of Chlorine Dioxide as a Sanitation Method
24
iii
3.
Antimicrobial Compounds in Potatoes
27
Aspartic Proteases in Potato Plant
28
MATERIALS AND METHODS: A SIMPLE GASEOUS CHLORINE
DIOXIDE TREATMENT FOR MICROBIAL DECONTAMINATION
ON POTATOES IN STORAGE
30
Cell Suspension and Media
30
Preparation of Desiccator to Duplicate Storage and Allow for
Measurement of Gaseous CIO2
4.
31
Sample Preparation and Inoculation
32
Activation of Gaseous C102and Treatment of Potatoes
33
Measurement of Gaseous CIO2
33
Microbial Enumeration
35
Effect of CIO2 Treatment on Visual Quality versus Control
36
Residual testing on the CIO2 treated potatoes
36
Statistical Analysis
37
MATERIALS AND METHODS: ANTIMICROBIAL ACTIVITY OF
POTATOES AGAINST FOODBORNE PATHOGENS
Bacteria and Media
38
38
Katahdin Potato Tuber Juice Treatment (50% (w/v)) Versus
Salmonella Typhimurium Using the Agar Diffusion Method
38
Preparation of potato samples
38
Agar diffusion method
39
iv
Russet Potato Tuber Juice Treatment (75% (w/v)) versus
Salmonella Typhimurium Using the Agar Diffusion Method
40
Preparation of materials and potato samples
40
Agar diffusion method
40
Potato Peel Juice Treatment on Escherichia coli 0157:H7 Using the
Log Reduction Method
41
Preparation of potato peel sample
41
Sample inoculation and antimicrobial study using log
reduction method
42
Antioxidant Study of Potatoes
5.
43
Preparation of food samples to be tested
43
Preparation of DPPH in test tubes
43
RESULTS: A SIMPLE GASEOUS CHLORINE DIOXIDE
TREATMENT FOR MICROBIAL DECONTAMINATION ON
POTATOES IN STORAGE
44
Natural Flora: Total Microorganisms
44
Natural Flora: Yeast and Mold
47
Pseudomonas aeruginosa
50
Visual Quality
53
Residue
54
v
6.
RESULTS: ANTIMICROBIAL ACTIVITY OF POTATOES AGAINST
FOODBORNE PATHOGENS
,
56
Katahdin Potato Tuber Juice Treatment (50% w/v) versus
Salmonella Typhimurium Using the Agar Diffusion Method
56
Russet Burbank Potato Tuber Juice Treatment (75% (w/v)) versus
Salmonella Typhimurium Using the Agar Diffusion Method
56
Potato Peel Juice Treatment on Escherichia coli 0157:H7 Using the
7.
Log Reduction Method
57
Antioxidant Study
57
DISCUSSION: A SIMPLE GASEOUS CHLORINE DIOXIDE
TREATMENT FOR MICROBIAL DECONTAMINATION ON
POTATOES IN STORAGE
8.
59
Natural Flora: Total Microorganisms
59
Natural Flora: Yeast and Mold
59
Pseudomonas aeruginosa
60
Visual Quality
62
Residue
63
DISCUSSION: ANTIMICROBIAL ACTIVITYOF POTATOES
AGAINST FOODBORNE PATHOGENS
64
Katahdin Potato Juice Treatment (50% (w/v)) versus Salmonella
Typhimurium Using Agar Diffusion Method
64
Russet Burbank Potato Tuber Juice Treatment (75%) (w/v)) versus
Salmonella Typhimurium Using the Agar Diffusion Method
vi
64
Potato Peel Juice Treatment on Escherichia coli 0157:H7 Using the
9.
Log Reduction Method
65
Antioxidant Study
65
CONCLUSION
67
Gaseous Chlorine Dioxide Study
67
Antimicrobial Activity of Potatoes
68
REFERENCES
69
APPENDIX: THE AMOUNT OF GASEOUS CHLORINE DIOXIDE RELEASE
OVERTIME
74
BIOGRAPHY OF THE AUTHOR
76
vn
LIST OF TABLES
Table 5.1: Visual quality of potatoes treated with gaseous chorine dioxide after 14
days using the hedonic scale
54
Table 5.2: Residue of chlorine dioxide on potatoes immediately following
treatment and after 14 days of treatment
55
Table 6.1: Results of agar diffusion assay for potato juice concentration against S.
Typhimurium
57
vm
LIST OF FIGURES
Figure 2.1: Diagram of the CUBE batch generator
20
Figure 2.2: Pictures of chlorine dioxide materials
23
Figure 3.1: Desiccator components
32
Figure 3.2: Flow chart of the methodology behind measuring gaseous chlorine
dioxide
35
Figure 4.1: Flow chart for preparation of 50% (w/v) and 75 % (w/v) potato juice
39
Figure 4.2: Flow chart for preparing potato peel juice
42
Figure 5.1: Microbial decontamination by gaseous chlorine dioxide on natural
flora-total microorganisms of potatoes at different times and
treatments
....46
Figure 5.2: Microbial decontamination by gaseous chlorine dioxide on natural
flora-yeast and molds of potatoes at different times and treatments
49
Figure 5.3: Microbial decontamination by gaseous chlorine dioxide on
inoculated Pseudomonas aeruginosa on potatoes at different times
and treatments
52
Figure 5.3: Microbial decontamination by gaseous chlorine dioxide on
inoculated Pseudomonas aeruginosa on potatoes at different times
and treatments
52
Figure A.l: Gaseous CIO2 release after 5 hrs
74
Figure A.2: Gaseous CIO2 release after 25 hrs
75
ix
Chapter 1
INTRODUCTION
Because potatoes are important to industry as well as in regard to human nutrition,
science must seek a way to ensure bulk contamination does not occur within storage
environments. Due to the long duration involved in the storage of potatoes, it is important
to prevent spoilage, which results in a great economic loss. Spoilage can come about
naturally or can be a result of rough handling or improper temperature control. During
processing; dirt, organic matter, and pathogens that may cause disease may be introduced
to the produce (Suslow, 1997). The disinfection process becomes important in
inactivating pathogenic and spoilage bacteria, fungi, viruses, cysts, and other
microorganisms (Suslow, 1997).
Tubers with an extreme infection may not only be useless, but may result in
ruining potatoes in storage. Mild and underdeveloped problems with yeast, mold, and
other infections will most likely worsen. As such storage diseases are not curable, finding
ways to limit pathogens from spreading to healthy tubers is important, but is challenging
due to their closeness during storage (Olsen et al, 2006).
Due to the previously stated storage issues, it is important to work on determining
an efficient, safe, and cheaper way to control storage loss and spoilage in order to help
the potato farmers, packers, and processors survive; as well as to maintain one of
America's most beloved food sources. Therefore, the first objective of the study was to
develop a novel, simple, and inexpensive gaseous chlorine dioxide method to efficiently
decontaminate potatoes of natural flora, yeasts and molds, as well as Pseudomonas
1
aeruginosa. Various treatment times and concentrations of CIO2 treatment were
investigated using a constructed circulating chamber.
As previously discussed, keeping potatoes from spoiling is important from the
economical stand point of the farmer, as well as for the potato loving consumer. Aside
from being a delicacy to consumers across the nation, potatoes also might be of great
importance to science as they may contain antimicrobial properties that may help
preserve food in a natural way.
Chemical preservatives such as nitrites give food a longer shelf life and are used to
prevent spoilage and improve taste, appearance, texture, and nutritional value. Nitrites
may react with amino acids in order to form cancer-causing nitrosamines. Other common
chemical preservatives include sodium benzoate and potassium sorbate. Sodium benzoate
produces benzoic acid once it is dissolved in water. It may be used against yeasts, molds,
and foodborne pathogens, (but not spoilage bacteria) in food products such as: fruit
products, beverages, dressings, salads, pastry fillings, etc. Sodium benzoate must be used
at low levels as it is known to produce off flavors in some foods. By law, sodium
benzoate may only be used up to a concentration of 0.1%. Potassium sorbate has been
known to be effective against yeasts, molds, and selective bacteria in: cheeses, dips,
bread, cakes, beverages, fruit products, etc. Potassium sorbate is also restricted for use at
no greater than a concentration of 0.1%. The addition of sodium benzoate and/or
potassium sorbate to a food product will raise the pH by approximately 0.1 to 0.5 pH
units, depending on the original pH of the food, increasing the importance of monitoring
(New York State Agricultural Experiment Station, 1998).
2
Researchers continue to study natural compounds in various plants in order to
determine their potential utilization as natural preservatives. With a goal of using the
components as natural preservatives in foods, the second objective of this study was to
determine the antimicrobial effects of potato tubers and peel against common foodborne
pathogens such as: Escherichia coli 0157:H7, and Salmonella Typhimurium. It has been
shown in previous studies that fresh fruits and vegetables that exhibit antimicrobial
properties may contain antioxidants. As potato components were studied in search for
antimicrobial properties, antioxidant studies were also performed in order to determine
the correlation of these properties in the potato.
3
Chapter 2
LITERATURE REVIEW
Potato Industry
Potatoes comprise a major portion of the American diet. The USDA states that
Americans eat approximately 140.6 pounds of potatoes each year (USDA, 2001).
Potatoes are aptly nicknamed the "nutritional powerhouse"- they are an excellent source
of vitamin C (45% of daily recommended value), potassium (21% of recommended daily
value), and fiber (12% of recommended daily value). Potatoes are also low in sodium, a
good source of protein, and contain 10% of the daily recommended allowance of vitamin
B6 (Agricultural Marketing Resource, 2007). Dieticians have stated that those foods
which are high in potassium and low in sodium may help prevent heart disease, and that
foods high in fiber promote healthy digestive systems which in turn also prevent heart
disease as well as some types of cancer (Best Food Nation, 2006).
Potatoes are commercially grown in 36 of the U.S states. In 2004, 1108.8
thousand acres of potatoes were planted, while 1084.8 thousand acres were harvested
with an average yield of 389 hundred weight per acre (Agricultural Marketing Resource
Center, 2007). With over 5,500 potato growers, the annual U.S production is estimated at
45.6 billion pounds of potatoes valuing over $2.6 billion. The potato growers have began
to work even closer with the processors throughout the supply chain to ensure the
delivery of optimal quality potatoes to the consumer, thus increasing the total value of the
U.S economy (Best Food Nation, 2006).
Although important as table stock, as well as raw materials for many processed
foods such as potato chips, frozen fries, canned potatoes, starches, and flour, there are
4
challenges involved with the potatoes in storage. Unfortunately, according to the National
Potato Council 2004-2005 Potato Statistical Yearbook, U.S potatoes in storage have been
ruined at an average rate of 7.5% over the past several years (Olsen et al., 2006). Though
much potato loss is due to transpiration and respiration, the highest losses during storage
are due to disease (Olsen et al., 2006).
Potatoes are the number one agricultural commodity in Maine with sales of nearly
$105 million, or 24% of the state's total agricultural receipts (USDA, 2005). The Maine
potato industry is a major part of Maine's natural resources, supplying over $500 million
annually and contributes over 6,000 jobs to the state's economy (Maine Potato Board,
2006).
During storage, many pathogens may take over areas of the potatoes that have
been damaged. Such areas may contain nicks, cuts, abrasions, broken knobs, or even
shattered-bruised areas. Potatoes also have natural weak points, such as the area of stolen
attachment, lenticels, and eyes where yeasts, molds, and bacteria may fester (Olsen et al.,
2006). Because potatoes are kept in such close quarters, it becomes difficult to control the
infection because one potato can contaminate many surrounding potatoes, resulting in the
loss of all tubers in the storage facility.
Microorganisms Associated with Spoilage of Potatoes
Research has indicated that potato problems in storage may not always be due to
the storage conditions, but from conditions during harvesting. (Suslow, 1997). For
example, if a potato is harvested when soils are moist, the excess moisture on the surface
of the potato will result in optimal growing conditions for microorganisms during storage
5
(Suslow, 1997). The temperature is also a factor for proper harvesting as potatoes
harvested below 45°F are more likely to be injured than those harvested at higher
temperatures. Harvesting a potato when the temperature is above 60 °F can also ruin it
because there might be water loss as well as shrinkage (Yanta and Tong, 2007). If a
potato is mishandled, experiences adverse weather conditions, or has disease prior to
storage, the potatoes will not make it through storage and may spread disease to other
healthy tubers around them (Small and Pahl, 2003).
After being placed in storage, tubers injured during harvest may develop post
harvest diseases. Different types of flora that may become problematic to the injured
potato may live on the potato or in environment. For example, an oomycota issue
occurred in the Northwest by Pytophthora infestans (cause of late blight). This oomycota
species will not only eliminate potato plants within a specific field, it will also cause
immediate spoilage in stored tubers or cause a weakening of tubers which will then set up
the baseline for the bacteria such as Pseudomonas aeruginosa, a common soil bacterium
(Yanta and Tong, 2006). It is important to find methods to control and/or prevent this
natural flora from remaining on potatoes.
Natural flora
Natural flora on potatoes may include three soft rot coliforms: Erwinia
carotovora ssp. carotovora, Erwinia carotovora ssp. atroseptica, and Erwinia
Chrysanthemi. As gram negative, non spore forming, facultative anaerobes, these bacteria
rely on a large amount of extra cellular pectic enzymes along with other cell wall
degrading enzymes to cause disease. After the resistance of the host is impaired, these
6
bacteria become the main cause of tuber decay during storage and can also cause black
leg or stem rot while in the field (Perombelon, 2002). Bacteria commonly found on
potatoes include the saprophytic Pseudomonas spp., Bacillus spp., as well as Clostridium
spp. which will also cause potato rot if presented with the opportunity (Perombelon,
2002).
Fungi
The potato disease dry rot is caused by the Fusarium spp., fungi that are normally
dormant in soil as well as on the surface of the potato tuber. Fusarium spp. enter through
the growth cracks (or other sites of injury from harvesting) where spores are distributed.
Once the fungi begin to grow within the tuber tissue, dry rot lesions occur along the
points of injury. Symptoms of dry rot include a brown, firm, sunken flesh with wrinkled
surfaces and brown or white protuberances (Yanta and Tong, 2006).
Another organism, Pythium ultimum, the cause of "leak," invades tuber tissue
through wounds (generally from harvest) causing rot during transport or in storage.
Symptoms of leak include oozing tubers, pink then black flesh, and mushy rot. Further
loss of the potato after this infection may be due to secondary bacterial infections upon
the already damaged tuber tissue (Yanta and Tong, 2006).
A white mold commonly found on potatoes from the soil-borne fungus
Sclerontinia sclerotiorum (also called Sclerotinia), will result in stem rot and will form
hardened black surfaces on potato plants. The fungus produces lesions on areas of the
potato vine that will result in infection, killing the tissue of the vine above the lesion. If
the lesion forms near the soil, the whole vine will die. Cold temperatures and high
7
moisture are invitations for the germination of the sclerotia to form apothecia (mycelia).
When conditions become appropriate, the apothecia release ascospores, which when
airborne, will infect other plants that are in close quarters. When the host potato plant
dies, the white mold disease will repeat its process as soon as another susceptible host
arrives, since the sclerotia would have already returned to the soil. The white mold may
last for several years within the soil (Olsen, et al, 2003).
Pseudomonas aeruginosa
Tuber soft rot is caused by the bacteria naturally found on potatoes that are
located intercellular within the lenticels and wounds such as Pseudomonas aeruginosa
(pectolytic saprophytic bacteria). The bacteria are also likely to be located beyond the
phelloderm layer; the single layer of cork cells to the inside of the phelogen cork
cambium and also beyond the xylem (vascular system of the potato). The phelloderm
layer is an important part of the plant as it contains suberin, a waxy material that seals the
stem against a loss of water. This part of the plant is very susceptible to the invasion of
insects, as well as the infection by bacteria and fungal spores. After the absorption of
water, bacteria are in contact with the walls that are most hydrated. The amount of
bacteria depends upon the storage conditions as they increase with moisture within the
storage environment. Such bacteria may survive for several months which may continue
on from one growing season to the next (Perombelon, 2002).
Anaerobiosis (impairs oxygen-dependent host resistant systems) will not alone
cause rotting, but will increase cell membrane permeability which results in the leakage
of cell contents. With this leakage, bacteria such as Pseudomonas aeruginosa may grow
8
further into the tissue and cause degradation to the cell wall tissue as they multiply.
Anaerobiosis may increase the virulence of the bacteria (Perombelon, 2002). Even under
a low inoculum level with previously mentioned conditions, bacterial growth such as
Pseudomonas aeruginosa will result in rot. Although Erwinia spp. may grow quicker and
produce more pectic enzymes than proteolytic bacteria such as Pseudomonas spp., the
Erwinia spp. may then allow for the opportunity of the bacteria to produce a lesion that
will eventually lead to rot (Perombelon, 2002).
Sanitation Methods for Fresh Fruits and Vegetables
As potatoes are important to industry, as well as human nutrition, science must
seek ways to reduce possible microbial contamination within storage environments. The
importance of food safety to the food industry has increased due to foodborne illnesses
and crop losses. Our government, along with consumer groups and industries, have
become more aware of these issues as their food safety efforts have been forced to show
more of an improvement. In order to increase food safety, food processors and regulators
have upgraded regulations and standards, expanded research and monitoring, and have
also provided better detection and educational programs. In conjunction with these efforts
and because of a greater awareness of foodborne illnesses and environmental concerns,
the desire for a more effective and environmentally friendly sanitation method must be
recognized and applied to ensure that food is safe for consumers (Degremont
Technologies, 2007). As many "diseases" such as: silver scurf, pink rot, and late blight
have been identified during the bulk storage process of potatoes, a successful
decontamination method is important for the prevention/elimination of these diseases.
9
Although different methods are being utilized in industry for the reduction of
pathogens and spoilage organisms, new research has uncovered many problems in the
efficacy of the chemical decontaminants currently being utilized. As with all treatments,
the efficacy of the sanitation method of reducing pathogens depends upon many different
factors. For example: the produce condition and type are important such as if the produce
has cracks or striated surfaces, the texture, hydrophobic characteristics, the type and
characteristics of the microorganism to be treated, pH and temperature of the produce and
treatment, as well as the time and temperature of the treatment (FDA, 2001). Upon
acknowledging these individual factors, researchers must concentrate on creating new,
safe, and effective methods for reducing/eliminating pathogens and spoilage organisms
on fresh fruits and vegetables.
Ozone
As a newly studied decontamination method on fresh fruits and vegetables, ozone
is used in order to destroy pathogens in the air and on the surface of produce (Degremont
Technologies, 2007). It is effective because it oxidizes the cell membrane of the target
microorganism (Lenntech, 2007). Ozone was first utilized in France as a disinfecting
agent on potable water. The distinctive advantages include: it does not leave behind a
residual, it degrades into molecular oxygen after it has reacted, and it also degrades
naturally on its own. The food industry discovered that ozone may be of potential use as
it could be 52% stronger than chlorine and is effective against a broad spectrum of
microorganisms.
10
Oxygen (O2) is comprised of two bound oxygen atoms. If given the right
conditions, it will convert into ozone (O3). Ozone is naturally created in the atmosphere
by lightning as a result of electricity breaking apart the oxygen molecules, releasing two
single atoms of oxygen. These single oxygen atoms may then attach themselves to an
oxygen molecule, creating ozone (Olgear, 2007).
Lenntech et al. (2007) tested a treatment of ozone in wash water for carrots during
processing, resulting in a 3 log reduction of bacteria. The same researchers also showed
that there were positive results after ozone exposure for the prevention of decay due to
fungi on blackberries. After the fruit was stored for 12 days at 2 °C in 0.0, 0.1, and 0.3
ppm ozone, results showed that ozone delayed the fungal growth for 12 days, while up to
20% of the control decayed. It was also noted that the treated fruit did not show any
visual difference from the control (Lenntech, 2007). Lenntech et al. (2007) also tested
ozone against fungi, yeasts and bacteria on grapes at low and high doses. The low dose of
0.1 mg/g fruit for 20 min reduced the fungi, yeast, and bacteria. Unfortunately, the
medium and high doses ruined the quality of the fruit.
Disadvantages of ozone include the discoloration and bleaching of produce as
well as the possible formation of by-products such as bromate, in the presence of bromide.
Ozone is also easily removed by organic matter in water, and generates a high demand
for energy (Science Direct, 2005). In spit of these drawbacks, ozone has become popular
in the United States as an alternative method to eliminate pathogens in the cold storage
process on potatoes (Degremont Technologies, 2007).
11
Peracetic acid
Peracetic acid is typically created in concentrations of 5-15%. It can be created by
continuously feeding hydrogen peroxide and acetic acid to a reactive medium comprising
of an acid catalyst to form peracetic acid (Free Patents Online, 2004-07).
Today, peracetic acid is applied to medical supplies and is also used to prevent
bio-film formation. Peracetic works by oxidizing the outer cell membrane of the
microorganism and can be applied to deactivate a large number of microorganisms such
as viruses and spores (Lenntech, 2007).
A disadvantage of peracetic acid is that it is affected by pH and temperature
variations. Even at an optimal pH of 7 and at a temperature of 15 °C, 5 times more
peracetic acid is needed versus at a temperature of 35 °C at a pH of 7 (Lenntech, 2007).
Park et al. (1999) found that peracetic acid is a known decontaminant for Salmonella spp.
as well as Escherichia, coli 0157:H7 on melon surfaces at 40-80 mg/1. Unfortunately it
was found to cause a high corrosion rate and was extremely carcinogenic.
Hydrogen peroxide
Hydrogen peroxide is quick and reliable when applied to food contact surfaces in
industry, yet it is quickly diluted during transport and is a strong oxidant that generates
free radicals that can be cytotoxic (FDA, 2001). High concentrations are required for
optimal results and hydrogen peroxide will decompose with even the smallest elevation
of temperature as well as under the influence of other pollutants (Muller et al., 2003). In
a study by Park and Beuchat (1999), hydrogen peroxide showed bleaching of fresh fruit
at concentrations of 1 % and 5%.
12
Hydrogen peroxide vapor may be generated from hydrogen and oxygen within a
micro reactor, a novel means of an intensifying reactor system. The reaction includes:
H2 + 0 2 -> H 2 0 2 .
The traditional method for generating hydrogen peroxide was by using organic solvents
and alkylated anthraquinones (SITIS Archives, 2007).
Chlorine
As the most popular chemical sanitizer in industry, chlorine is used directly on
fresh produce as well as on facility surfaces. There are different forms of chlorine such as
aqueous chlorine and hypochlorite. These forms of chlorine are utilized between the 50200 mg/1 range of concentration for a contact time between 1-2 min (FDA, 2001).
For the decontamination on potatoes, the University of Idaho suggests minimizing
dry rot decay of fresh pack potatoes by applying a 500 ppm aqueous chlorine treatment
followed by a drying process before packing. This procedure is used to prevent a new
infection from occurring as well as to reduce any previous infection. The University of
Idaho also mentions that a post harvest chlorination treatment of chlorine disinfectants at
a level of 500 ppm when placed directly on potatoes and with the addition of air
circulation will reduce silver scurf infection throughout the storage pile (Shetty et al.,
1998).
Hypochlorous acid from the free form of chlorine has shown bactericidal effects
on many types of bacteria. In a study by Beuchat and Rye (1997), fresh-cut cantaloupe
given a treatment of 2000 mg/1 of chlorine showed less than a 90% reduction of
Salmonella spp. In another study by Beuchat (1998), a chlorine solution of 2000 mg/1
13
resulted in a 2.3 log CFU/cnr reduction of Salmonella spp. on tomatoes, lettuce leaves,
and apples that were dipped in the solution. Aqueous solutions such as sodium
hypochlorite or hypochlorous acid were previously being used to sanitize fresh fruits and
vegetables, yet pathogenic outbreaks are still problematic.
Factors such as light, temperature, air, and organic matter may hinder the
bactericidal effect of chlorine (FDA, 2001). Studies have also shown that chlorine
solutions may not be able to treat areas on produce that may contain crevices, cracks, or
deeply striated surfaces (FDA, 2001). Another disadvantage of chlorination is the toxic
by-products such as trihalomethane and chloramines (FDA, 2001). Also, chlorine is
effective in achieving acceptable microbial reduction rates after a treatment of up to 250
ppm which is very high. Chlorine is also corrosive, and when it was applied to equipment,
replacement costs have been shown to increase (PatentStorm, 2004-6). As aqueous
chlorination has its risks, chlorine gas is extremely toxic (PatentStorm, 2004-6). With
these drawbacks, other alternatives might be of great importance in order to effectively
and safely decontaminate fresh produce (FDA, 2001).
Ultra violet radiation
Ultra violet radiation (UV) has been utilized for years in industry for the
inactivation/destruction of microorganisms on produce and equipment. Short UV
radiation treatments ensure a 4 log reduction of microorganisms such as bacteria, mold,
yeast, and protozoa, as they are inactivated (Science Direct, 2005).
Advantages of UV use include no use of chemicals, cleanliness, ability to use with
ozone, and a broader spectrum of antimicrobial activity. Disadvantages include: its
14
effectiveness only in non-turbid water, inability to overcome particulates which will
protect microorganisms, contact times that may limit flow rates, and high maintenance
requirements (Science Direct, 2005).
Curing
Curing is another method commonly used to reduce the population of
microorganisms on potatoes. Curing helps to heal the skin of potatoes making it less
susceptible to disease. Curing may also reduce the chance of post harvest contamination
as this process includes exposing the potatoes to temperatures between 50-60 °F with a
relative humidity of 95% for 10-14 days (Yanta and Tong, 2007).
For potato farmers in Alaska, curing is an important process as the crops are
usually harvested while vines are green. This means that the skin on the tubers are very
susceptible to damage. At storage time, physical and chemical damage as well as bruising
due to separating vines and digging may not be noticed. Farmers will only see the
damage once it has gone too far, as bacteria and fungi penetrate and ruin the affected
potatoes. Such bacteria and fungi may then make their way throughout the entire storage
facility, costing farmers their entire crop (Vandre, 2005).
Other sanitation methods
A study by Somers and others (1994), found E. coli 0157: H7 to have a log
reduction of 5 - 6 CFU/g at 10 °C, as well as room temperature, after non-food surfaces
were given a treatment of 1% trisodium phosphate. Another study by Beuchat (1996),
with tomatoes, found a 5.2 CFU/cm2 reduction of S. Montevideo when treated with 15%
15
TSP after only 15 s. Unfortunately, phosphates are an environmental concern when
discharged in the environment. TSP might also be of concern due to its alkalinity (pH of
11-12), which may hinder its use for being a decontaminant for fruits and vegetables
(FDA, 2001).
Chlorine dioxide (ClOg)
Since the 1920's, chlorine dioxide has been acknowledged for its disinfection
properties. After first being recognized as a chemo sterilizing agent in 1984, it was then
acknowledged by the EPA in 1988 as a sterilant effective against bacteria, viruses and
protozoa, including oocysts of Chryptosporidium and Giardia cysts. Chlorine dioxide is
also recognized by the US Food and Drug Administration (FDA) for disinfecting
drinking water, THM (trihalomethane) and HAA (haloacetic acid) control in drinking
water, bacteria control in cooling water, bacterial control in paper processing, sanitizing
uncut fruits and vegetables, controlling bacteria in produce flume water, controlling
bacteria in poultry processing, controlling bacteria in beverage and brewing equipment,
as well as sanitizing food facilities and equipment (International Dioxcide, Inc., 2000).
The use of chlorine dioxide in an isolator (sealed chamber) has increased in popularity
within industries of the United States and in Europe. Pharmaceutical and medical device
industries have also adopted chlorine dioxide in gaseous form as it maintains the
properties of a true gas at ambient temperatures, is unaffected by temperature, and is
biocidal over a large range of pH. Gaseous chlorine dioxide is also known for its process
consistency as it can be accurately and precisely monitored and controlled
(Pharmaceutical Technology, 2005).
16
Research has shown that both aqueous and gaseous phases of chlorine dioxide are
effective sanitizing agents. They both treat a broad spectrum of microorganisms such as
bacteria, spores, viruses, and algae, however gaseous chlorine dioxide may be more
effective than aqueous when applied at equal concentrations and times as it may be able
to reach small spaces where its liquid form cannot (Pharmaceutical Technology, 2005).
Chlorine dioxide is an improvement over other chlorine based compounds as it
has 2.6 times the oxidizing capacity than that of chlorine (Lenntech, 2007). Upon
chlorination, hypochlorite, a very unstable compound degrades to a mixture of chloride
and chlorate, especially when not kept cold (Lenntech, 2007). Chlorine is also not
effective against many bacterial and fungal spores, resulting in the need for a longer
treatment time and higher concentration. Chlorine dioxide's major chemical reaction is
oxidation, thus preventing trihalomethane as well as haloacetic acid formation from
occurring. This allows for low phytotoxicity, making it safe and non-threatening to the
environment. Chlorine dioxide, unlike chlorine, will also not react with and not be
consumed by other impurities.
Upon treatment, chlorine dioxide reacts with the amino acids and RNA within the
cell, preventing the production of proteins and affecting the cell membrane by switching
membrane proteins and fats, also preventing inhalation (Lenntech, 2007). Numerous
studies have proven that gaseous CIO2 is extremely successful (greater than 5 log
reductions) in reducing food borne pathogens such as Escherichia coli 0157:H7, Listeria
monocytogenes, and Salmonella spp. on fruits and vegetables surfaces as well as on
spoilage organisms found on food-contact surfaces (Czarneski and Poisson, 2005).
17
As the consumption of fruits and vegetables have increased in the United States,
foodborne outbreaks have also risen, so consequently an effective way to eliminate
foodborne contamination is essential. Although chlorination has been used, gaseous CIO2
may be able to break down odor-causing phenolic compounds and will not react with
substances such as ammonia (S.Y Kaye et al., 2005).
Chlorine dioxide may be a positive sanitation method to treat potatoes in storage
since it is maintains the ability to kill microorganisms responsible for the late potato
blight as a result of Pytophthora infestans (Guevera et al., 2004). Another advantage of
gaseous CIO2 for the use on potatoes during storage is that it is also not sensitive to pH
and organic matter. This may help in decreasing the attraction to dirt that may be located
on the potato skin during storage (Guevera et al., 2004).
Generation Methods for Aqueous and Gaseous Chlorine Dioxide
There are different ways to generate both aqueous and gaseous chlorine dioxide.
The chosen method may be based on area of application (such as size and type of
surfaces and/or materials), desired concentration, as well as environmental factors. As
each of these concerns is important, scientists must determine the appropriate treatment
conditions before applying them in the food industry.
One common method of gaseous CIO2 generation takes place in an isolator and is
used in industry for the sterilization of supplies. This method of generation is similar to
those that use humidity or moisture along with a specific gas for sporicidal efficacy. This
chlorine dioxide method also uses either negative or positive pressure (2 KPa)
(Pharmaceutical Technology, 2005).
18
The generation of gas within the isolator is performed by using solid sodium
chlorite from small cartridges. After the addition of a chlorine-nitrogen (2:98%) gas
mixture, pure chlorine dioxide will result in the generation of nitrogen without any form
of by product as there might have been upon the generation of the aqueous form of
chlorine dioxide (Pharmaceutical Technology, 2005). The reaction is as follows:
Cl2 (g) + 2NaC102 (s) -> 2C102 (g) + 2NaCl (s)*
*The letter (g) stands for gas as (s) stands for solid
Sieman's Water Technologies has put out the Wallace and Tieman Millenium
III™ CUBE batch generator (Fig. 2.1) which is made for smaller scale chlorine dioxide
decontamination. This system may decontaminate using two methods. The first method
generates gas by the reaction of molecular chlorine gas and a 25% aqueous sodium
chlorite solution. The formula is as follows:
2NaC102 + Cl2 -»• 2C102 +2NaCl.
The second method of generation is first produced by the reaction between a 12.5%o
solution of sodium hypochlorite with a 15% solution of hydrochloric acid. In the second
step of this process, the chlorine gas that is produced reacts with 25% sodium chlorite
solution to produce chlorine dioxide. The reactions are as follows:
Step #1- NaOCl + 2HC1 -> Cl2 + 2NaCl + H 2 0; and
Step #2- 2NaC102 + Cl2 -»> 2C102 + 2NaCl
19
^
Figure 2.1: Diagram of the CUBE batch generator
Another method for generating gaseous CIO2 is a tablet called Aseptrol®,
manufactured by Engelhard. Aseptrol® allows for the delivery of chlorine dioxide in a
stable form. The Aseptrol® tablet consists of proprietary activators that react with
chlorite salt within the tablet to generate chlorine dioxide when in contact with water or
moisture from the air. Aseptrol® is available in forms of loose powder, dissolvable
tablets, hot melts and sachets for the application of deodorizing and disinfecting
(Cochrane, 2005).
Many methods have been used to generate aqueous chlorine dioxide.
For
example, Purogene® and Purogene Professional® are premium blends of chlorine
dioxide produced by the company Bio-cide International. Purogene® is scientifically
proven to be a broad spectrum antimicrobial agent used to eliminate both gram negative
and gram positive bacteria, yeasts, mold, and fungi (Bio-cide International, 2007).
Purogene® (2% chlorine dioxide solution) is used pre-storage on potatoes as a
mist at treatments between 200-400 ppm. Purogene® is shipped as sodium chlorite and
activated by a small amount of food grade acid to produce chlorine dioxide. Purogene
20
Professional® offers a 5% chlorine dioxide solution that uses citric acid and sodium
chlorite to produce chlorine dioxide. One method uses 45 g/L of citric acid dissolved in a
5% concentrated sodium chlorite solution. This product is then diluted to 2000 ppm and
further diluted to get the desired working solution concentration (Bio-cide International,
2007).
The product Anthium AGP® (5% chlorine dioxide solution), manufactured by
International Dioxcide, generates aqueous chlorine dioxide similarly to Purogene
Professional® and is also used on potatoes during storage in order to prevent spoilage
organisms from contaminating the storage facility.
A form of germicide produced by Agranco Corp. U.S.A, called Vibrex®, is an
advanced formulation of chlorine dioxide and oxygen in an aqueous solution. Vibrex®
includes many of the advantages of chlorine, but with better performance. It is 160%
more effective than chlorine, 10 times more stable, and will not dissipate. As a colorless
odorless liquid, Vibrex® is nontoxic, non corrosive, and nonflammable. Vibrex® is a
wide spectrum bactericide, fungicide, virucide, and algaecide. It can be used as a
disinfectant in many applications, especially post harvest on fresh fruits and vegetables
and has been approved by the Food and Drug Administration (FDA) (Agranco Corp.,
2007).
In the potato industry, Vibrex® at 50 ppm, is part of a humidification system for
storage and a germicide for common potato bacteria such as Pseudomonas aeruginosa, as
well as many fungi, and viruses. Vibrex® applied to Pseudomonas spp. reduces 100%) of
the bacteria after 10 min with a concentration of 50 ppm. For treatments on Erwinia
21
carotovora, a common potato pathogen, Vibrex® killed 99.9% of the bacteria after 60
seconds at a concentration of 50 ppm (Agranco Corp., 2007).
As previously mentioned, gaseous CIO? may be generated by utilizing many
different methods. A simple, inexpensive, fast release Z-Series
product manufactured
by ICA TriNova, LCC was (Fig. 2.2) used in this study. Gaseous chlorine dioxide is
activated on site in an easy to use sachet. Different concentrations of desired gas may be
studied without the need for expensive equipment is safe, reliable, and affordable. With
this fast gas release method, 30 times less chlorine dioxide will be needed compared to a
chlorine treatment. The Z-Series fast gas release works by combining an equal amount of
sodium chlorite activating acids in a sachet without adding any solution. After activation,
the sachet is then placed in the application area.
22
Figure 2.2: Pictures of chlorine dioxide materials. A.) Impregnates of sodium chlorite and
activating acid compounds and B.) Sachet where compounds are added to activate
gaseous chlorine dioxide by mixing and shaking (supported by ICA TriNova, LCC).
A.)
B.)
The effect of chlorine dioxide has been analyzed through many procedures
(summarized below) such as direct spraying on the potatoes in storage, manual
application, and even by using a humidifier. These procedures failed due to the formation
of excess water among the pile (invitation for fungi and bacteria) and/or by inconsistent
chlorine dioxide administration (Kleinkopf, 2001).
It was found that manual spraying resulted in poor coverage. When gaseous
chlorine dioxide was applied in an enclosed room (without potatoes), it was shown to be
extremely effective on all surfaces of the entire environment, including beneath
equipment. It was also noted that gaseous chlorine dioxide does not leave a residual
behind, which is important when dealing with the quality of food (Kleinkopf, 2001).
Based on previous studies, and the desire to control pathogens during potato
storage, it would be optimal to actually construct a simple gaseous CIO2 unit that may be
used directly on potatoes in storage. Gaseous CIO2 is efficient in covering the entire
environmental perimeter, and does not involve excess water and inconsistent treatments.
There has been a desire to continue this research in hope to accomplish a truly cost
23
efficient, effective, quick, and safe methods that farmers may use in their very own
facilities.
Applications of Chiorine Dioxide as a Sanitation Method
Han et al. (2001) tested the reduction of L. monocytogenes after water washing,
aqueous chlorine dioxide, and gaseous chlorine dioxide treatments on injured and noninjured green pepper surfaces, as chlorination and water washing have limiting effects on
the decontamination of whole fruits and vegetables on a commercial scale (Han et al.,
2001). Gaseous chlorine dioxide was generated by a laboratory generator using 4%
chlorine in nitrogen gas. The gas was then collected in sampling bags. A sampling
syringe was used to add a certain amount of gas into the Plexiglas cylinder where the
samples were placed.
Results were positive for gaseous chlorine dioxide as it showed a higher log
reduction which was also significantly different than aqueous chlorine dioxide and water
treatments for both injured and uninjured surfaces at 0.3 mg/1 concentration as well as the
3.0 mg/1 concentration (Han et al., 2001).
As the previous study indicated, gaseous chlorine dioxide is effective in
decontaminating both injured and non-injured surfaces on peppers. Potatoes contain
many "injured" areas where weak spots develop. These injured surfaces are sites for
bacteria and fungi growth. Due to the quick spread of fungi and bacteria during potato
storage, gaseous chlorine dioxide may help in controlling potential infestations as it maypenetrate throughout the pile and decontaminate a significant portion of the total surface
area.
24
Gaseous chlorine dioxide was then studied by use of a dry chemical sachet, and
tested against food borne pathogens (Listeria monocytogenes, Escherichia coli 0157:H7,
and Salmonella Typhimurium) on lettuce leaves (Lee et al., 2004). A model gas cabinet
was used (20 L gas bucket). Lettuce leaves were treated with chlorine dioxide after
inoculation; using 4.3, 6.7, and 8.7 mg for time periods of 30 mins, 1 hr, and 3 hrs. The
gaseous chlorine dioxide not only reduced the amount of each bacterium tested from 3 - 6
log reductions, it did not cause any type of distortion to the leaf itself (Lee et al., 2004).
Therefore, as opposed to aqueous chlorine dioxide that might leave a liquid residue
behind, it will be interesting to test the gas on potatoes with hope that it will also not
affect their sensory quality as well.
Gaseous chlorine dioxide was also studied on strawberries using batch and
continuous chlorine dioxide generating treatments (Han et al. 2004).
Both L.
monocytogenes and E. coli 0157:H7 decreased over time and treatment at 15 mins, and
30 mins using 0.2 mg/1 and 0.6 mg/1 of gaseous chlorine dioxide. After 15 mins with 0.2
mg/1, E. coli 0157:H7 showed a 1.2 CFU/strawberry reduction and a 2.4 CFU/strawberry
log reduction after 30 min. L. monocytogenes showed a 1.8 CFU/strawberry reduction
after 15 mins and a 2.8 CFU/strawberry reduction after 30 mins. At the 0.6 mg/1
treatment, E. coli 0157:H7 showed a 1.9 CFU/strawberry reduction after 15 mins, and a
3.0 CFU/strawberry reduction after 30 mins. L. monocytogenes showed a 2.6
CFU/strawberry reduction after 15 min and a 3.6 CFU/strawberry log reduction after 30
min. L. monocytogenes had a greater log reduction for each treatment than E. coli 0157:
H7 (Han et al., 2004). The results also indicated that the continuous gas treatment was
much more effective as it showed more than a 3 log reduction after 10 min at a 0.6 mg/1
25
treatment, whereas the batch gas treatment showed over a 1.5 log reduction with the 0.6
mg/1 treatment after 15 mins (Han et al., 2004).
In a study by Kaye et al. (2005), gaseous chlorine dioxide was generated using a
Plexiglas desiccator to determine its effectiveness as a sanitizcr for killing Salmonella,
yeasts, and molds on blueberries, strawberries, and raspberries as small fruits have been
associated with foodborne illnesses. Their results showed that the 8.0 mg/1 treatment (for
120 min) significantly reduced the Salmonella population on blueberries (a reduction of
2.4 to 3.7 log CFU/g), strawberries (a reduction of 3.8 to 4.4 log /CFU/g) and raspberries
(a reduction of 1.5 log CFU/g). Treatment results from 4.1 to 8.0 mg/1 (30 min to 120 min)
for yeasts and molds on blueberries, strawberries, and raspberries showed 1.4 to 2.5, 1.4
to 4.2, and 2.6 to 3.0 log CFU/g reductions, respectively (Kaye et al., 2005).
Reducing yeast and molds on potatoes becomes very important especiall> during
storage. As potatoes maintain a natural flora very high in yeast and molds, it is best that
these microorganisms are controlled as potatoes may contain weakened or damaged
points which become sites for microbial infestation. As the previous study by Kaye et al.,
(2005) indicated that gaseous chlorine dioxide was very effective in killing yeasts and
molds on fresh vegetables, potatoes might also be a treated with gaseous CIO2 as they are
being stored to prevent the major diseases such as; pink rot, Pythium leak, late blight, and
soft rot, etc. It will also be interesting to study the potato and to determine different times
and treatments of gas in order to determine the optimal time periods and treatments for
the potato itself as all fruits and vegetables vary in their compositions.
Another gaseous chlorine dioxide effectiveness study was done on the growth of
the fungi; Stachybotrys chartum, Chaetomium globosum, Penicillium chrysogenum, and
26
Cladosporium cladosporioides in attempt to control sick building syndrome due to poor
indoor air quality (Wilson et al., 2006). Gaseous chlorine dioxide was prepared by using
a modified pressure cooker as a gas chamber with 6g of Aseptrol S10- Tab tablets
dissolved in water at 500 ppm and 1000 ppm concentrations. Results showed that gaseous
chlorine dioxide seems to be quite effective on inactivating some fungi, yet S. chartarum,
even though it was inactivated by the fumigation treatment still remained toxic after all
the dosages (Wilson et al., 2005).
Kleinkpf et al. (2001), used a humidifier as a method of chlorine dioxide
application against late blight in potato storage. The chlorine dioxide was added to the
humidification water at 50-200 ppm. The experiment showed an unacceptable 30%
reduction on the surface infection. The same researchers speculated that many active
ingredients might be lost in the gas from solution into the environment.
Based on the results from these studies, scientists have been working to continue
this study in order to determine if the chlorine dioxide itself is effective enough to
actually penetrate within the potato pile during storage. Researchers are also interested in
developing methods that will allow chlorine dioxide to be effective for the entire potato
population during storage by determining appropriate times and concentrations
appropriate for reducing yeast, mold, and bacteria on the surface of potatoes.
Antimicrobial Compounds in Potatoes
Developing a safer and more effective way of microbial decontamination of the
potato becomes important not only for the farmer and consumer, but also for scientists.
As society continues to demand the "natural" way of life, scientists (especially food
27
scientists) are focusing on plant research in order to utilize nature's natural compounds.
With that in mind, keeping our potatoes healthy by using a decontamination method such
as gaseous CIO2, especially during storage, becomes important for the study of the
antimicrobial agents found in potato leaves/tubers, What if potatoes could be used for
more reasons than human consumption?
Potato tubers and peels have been known to contain phenolic compounds that
may show antimicrobial activity against many gram positive and negative bacteria
(Guevera et al., 2004). Potatoes have also been shown to contain antioxidants within their
peel which may also be coixelated with their antimicrobial properties. Such isolated
component(s) could become an alternative to artificial preservatives such as sorbates,
benzoates, and nitrites that may have harmful effects to the consumer over time. Though
chemical preservatives such as nitrites give food a longer shelf life and are used to
prevent spoilage and improve taste, appearance, texture, and nutritional value, they may
react with amino acids in order to form nitrosamines which are cancer causing agents.
Utilizing the potato for its antimicrobial properties may be very important and readily
applied to food industry.
Aspartic Proteases in the Potato Plant
Aspartic proteases, a large class of highly distributed proteases in animals,
microbes, and plants have been shown to play a major role in plant defense against
abiotic stress (Guevera et al., 2004). Guevera et al. (2004) observed the aspartic proteases
in plants that are related to abiotic stress responses and introduced an aspartic protease in
potato leaves (StAP3) and an aspartic protease in potato tubers (StAPI). They found that
28
both StAP3 and StAF\ were effective antimicrobial agents against P. infestans as well as
Fusarium solani. With the induction of the StAFs on tubers and leaves, after wounding
the potato, it was observed that the StAFs were a major part of the plant defense
mechanisms. Though both of these enzymes contain a similar cleavage position, the
StAF\ had two cleavage places, whereas the StAP3 cleaves the peptide bond Phe24Phe25 which is very similar to the aspartic proteases of other plants. The differences in
the specificity of the substrate between StAF3 and StAPI, is related to the different
antimicrobial activities within each of the StAFs (Guevera et al., 2004). Final results
showed that the aspartic protease containing potato leaf (SlAF3) is activated after the
infection from P. infestans. The differences within the StAFs that may be linked to the
protein and activity level could be related to a lower or higher plant defense. The
differences between StAF3 and StAPI could be related to the variety of antimicrobial
activities which are justified by the specifications of the substrates as well as the
activation of StAFs. These results indicate that the antimicrobial activities of the enzymes
are controlled by the presence of particular aspartic protease inhibitors (Guevera et al.,
2004).
This study is very informative, and continuing research is important, especially
when the effect of these compounds on common foodborne pathogens are acknowledged.
As defense mechanisms, these compounds work against P. infestans as well as Fusarium
solani. Further research is needed in order to determine antimicrobial effectiveness,
which is important for developing effecti ve natural compounds that may be active against
foodborne pathogens, thus used as natural preservatives.
29
Chapter 3
MATERIALS AND METHODS: A SIMPLE GASEOUS CHLORINE DIOXIDE
TREATMENT FOR MICROBIAL DECONTAMINATION ON POTATOES IN
STORAGE
Cell Suspension and Media
Inoculum suspensions were prepared from pure cultures of Pseudomonas
aeruginosa cultures (ATCC #10145 and #27853) obtained from the Pathogenic
Microbiology Laboratory in the Department of Food Science and Human Nutrition at
University of Maine (Orono, ME).
Each strain of Pseudomonas aeruginosa was grown in Brain Heart Infusion broth
(BHI, Difco, Becton Dickinson, Sparks, MD) at 37 °C for 24 hrs. Cultures were kept
under refrigeration (4 °C) as stock cultures and transferred weekly to maintain viability.
A cocktail mixture of two strains was utilized in this study. The culture cocktail was
made by combining 75 ul of both fresh cultures (a total of 150 ul) and adjusted to
approximately 1 x 108 CFU/ml using 0.1% peptone water (Difco), prior to the inoculation
onto the potato surfaces. The natural flora (including total microorganisms as well as
yeasts and molds) originally from potatoes were also enumerated.
Tryptic soy agar (TSA, Difco) and Dichloran Rose Bengal Chloramphenicol agar
(DRBC, Difco) were used to enumerate the total microbial counts and yeast and molds
respectively. Pseudomonas isolation agar (PIA agar; Difco) was used for inoculated
Pseudomonas aeruginosa on potatoes.
30
Preparation of Desiccator to Duplicate Storage and Allow for Measurement of
Gaseous CIO2
A 5.5 L vacuum tight desiccator (Scienceware, Pequannock, New Jersey) was
used as the housing chamber (Fig, 3.1). A plastic cover was constructed so that it could
screw onto the vacuum port (threads on both port and cover). A silicon based septa used
in gas chromatography (11 mm Agilent Technologies, Richardson, TX) was placed over
the newly threaded pon opening and screwed in between the new plastic threaded cover
as to "sandwich" the septa tightly between to create a seal. A small fan was wired (UC
Fan, Copal Co., LTD, Malaysia) through the top of the desiccator and connected to a
control panel at the top which consisted of an on/off switch, as well as speed control.
31
Figure 3.1: Desiccator components. A.) assembled desiccator, B.) sandwiched port where
needle is inserted for gas removal, C.) small installed fan used for circulation of gas
within the desiccator, and D.) control panel used to control the speed of the fan
A.)
B.)
D.)
C.)
Sample Preparation and Inoculation
For the natural flora study, six unwashed potatoes were weighed. Four of the
potatoes were placed in the dessicator: 2 of similar size to be used for testing, 1 used for a
14 day visual check and the other to be used as a residue check. For the P. aeruginosa
32
study, 3 potatoes were separately weighed, washed with tap water, then with 95% ethanol,
and finally sterile distilled water. Cleaned potatoes were then placed under a bio-safety
hood and propped erectly in a small beaker under the germicidal light for 2 hrs,
continuously rotating for drying. After drying, 150 ul of P. aeruginosa cocktail was then
inoculated onto 15 locations on the surface of the potato using a micropipette. Potatoes
were then left to dry under the hood for another 2-3 hr. The inoculated dried potatoes
were then ready for the gaseous CIO? study (1 for the control and 2 for CIO2 treatment).
Activation of Gaseous ClQ^and Treatment of Potatoes
Gaseous CIO2 was generated by combining an equal amount of impregnates
sodium chloride and activating acids in a sachet (ICA TriNova, LLC, Forest Park; GA),
mixing and shaking for activation. Different treatments included the combination of: 2 g
of sodium chlorite and 2 g of acid into sachet to generate 16 mg/1 after 2.5 hrs and 20
mg/1 after 5 hrs (low treatment), 3g of sodium chlorite and 3 g of acid to generate 24 mg/1
after 2.5 hrs and 30 mg/1 after 5 hrs (medium treatment), and 4 g of sodium chlorite and 4
g of acid to generate 32 mg/1 after 2.5 hrs and 40 mg/1 after 5 hrs (high treatment). The
sachet was sealed and placed on the bottom of the desiccator where potatoes were stored
for, 2.5 and 5 hrs treatment time, for each treatment.
Measurement of Gaseous CIO2
After 2.5 hrs and 5hrs, headspace treatments of gaseous CIO2 were determined byinsertion of the gas tight syringe (50 ml, Hamilton Co., Reno, Nevada)/needle complex
(Fisher Scientific, Hampton, NH), into the unit containing the silicon-based septa to
33
remove 10 ml of gas from the chamber (Fig 3.2). The needle was then placed (with
syringe containing 10 ml of gas) in 10 ml of distilled water contained in a test tube. Gas
was slowly released (water was drawn back into the syringe and released to completely
remove gas into the sample tube)
The mixture was vortexed and measured
colorimetrically using the DPD (A^.A'-diethyl-p-phenylenediamine) method for aqueous
chlorine dioxide. This method is used to determine chlorine dioxide concentrations using
indicators that change color when oxidized by chlorine dioxide. This method will also
measure gaseous CIO2. The spectrophotometric analyzers work by reading the treatment
of chlorine dioxide by measuring the optical absorbance of the indicator in the liquid
sample. The absorbance is proportional to the treatment of the chlorine dioxide in the
water.
34
Figure 3.2: Flow chart of the methodology behind measuring gaseous chlorine dioxide
•
Concentration will then be analyzed
using the DPD Method
Microbial Enumeration
In the natural flora study, the control potato sample (without CIO2 treatment) was
placed in a sterilized sample bag (Whirl Pak: Nasco, Ft. Atkinson, WI) along with 50 ml
of peptone water. The treated potato samples from the desiccator (after CIO2 sanitation)
were individually wrapped in sterilized sample bags (Whirl Pak: Nasco, Ft. Atkinson, WI)
along with 25 ml of peptone water. The potato samples were massaged thoroughly by
hand for 2 min. A series of dilutions were prepared for plating. Dilutions of 0.1 ml were
spread plated in duplicate onto TSA (Difco) for total microbial counts. The plates were
inverted and incubated at 37 °C for 24 hrs. From the same dilutions, 0.1 ml of sample was
35
spread-plated in duplicate onto DRBC agar (Difco) for yeast and mold counts. The
DPvBC plates were inverted and kept at room temperature (21 °C) for 5 days.
In the Pseudomonas study, the inoculated control and treated samples were
diluted using the same priced ares as the natural flora stud}' described above. Samples
were spread-plated in duplicate on PIA agar (Difco). Plates were inverted and incubated
at 37 °C for 24 hrs.
Effect of CKVTreatment on Visual Quality versus Control
One ClOo treated potato from the desiccator after each trial (for visual) was placed
in a sterilized sample bag (Whirl Pak, Nasco, USA) and stored at 4°C for 14 days. One
potato (visual control) without CIO2 treatment was also placed in a Whirl Pack bag after
each trial and stored at 4°C for 14 days. Evaluation of potato visual quality was
performed daily. In the 9 point hedonic test (1 = dislike extremely, 2 = dislike very much,
3 = dislike moderately, 4 = dislike slightly, 5 = neither like nor dislike, 6 = like slightly, 7
= like moderately, 8 = like very much, 9 = like extremely) was used for the evaluation of
visual quality. Between 1 and 3 persons evaluated the samples. Pictures were taken for
the evaluation of any possible differences between control and treated samples.
Residual testing on the CIO2 treated potatoes
For each treatment, the CIO2 residue was checked immediately following the
treatment (2.5 and 5 hrs) on day 0 and on day 14 by adding 50 ml of distilled water to
bagged samples and massaging the bag by hand for 5 min. Ten milliliters of liquid
sample was then removed from each bag and tested using the DPD method.
36
Statistical Analysis
All experiments were repeated three times with duplicate samples. Data was
analyzed by analysis of variance (ANOVA) using the SAS General Linear Models
procedure with SAS software (SAS Institute, Cary, NC). Mean values were analyzed to
determine statistically significant differences (a = 0.05) by the Least Square Difference
(LSD) test.
37
Chapter 4
MATERIALS AND METHODS: ANTIMICROBIAL ACTIVITY OF POTATOES
AGAINST FOODBORNE PATHOGENS
Bacteria and Media
Escherichia coli 0157:H7 (ATCC 129000 and 35150), and Salmonella
Typhimurium (ATCC 14028) obtained from the Pathogenic Microbiology Laboratory in
the Department of Food Science and Human Nutrition at University of Maine (Orono,
ME) were used in this study.
MacConkey Sorbitol Agar (MSA, Difco) was used to enumerate E. coli 0157:H7
and tryptic soy agar (TSA, Difco) was used to enumerate S. Typhimurium.
Katahdin Potato Tuber Juice Treatment (50% (w/v)) Versus Salmonella
Typhimurium Using the Agar Diffusion Method
Preparation of potato samples
Figure 4.1 illustrates the preparation of potato tuber juice. Materials to be used
were autoclaved (the plastic materials were placed under the hood). Katahdin potatoes
were purchased at a local grocery store (Orono, ME). One potato was peeled and weighed.
The potato was then sliced into pieces and were fed (227 g) into a juicer (Model #6001,
AcmeJuicer MFG. Co., Sierra Madre, CA). Approximately 25 g of retained juice was
distributed into 4 centrifuge tubes (Nalgene, Lima, OH). The tubes were then centrifuged
(Avanti J-E Centrifuge, Fullerton, CA) at 6,000 rpm for 20 min at 4 °C. After
centrifugation, samples were placed in a freezer at -80 °C for 24 hrs. After 24 hrs,
38
samples were freeze dried (Freeze Dry System/Freeze Zone 4.5, Labconco, Kansas City,
KS) at -40 °C for 24 hrs,
Figure 4.1: Flow chart for preparation of 50% (w/v) and 75 % (w/v) potato juice
Agar diffusion method
The freeze dried sample was weighed (subtracting container). A (w/v) amount of
sterile distilled water was added to the lyophilized material to make a 50% (w/v) dilution.
The mixture was then vortexed. A "lawn of bacteria" was created on 4 TSA plates by
spread plating (2 for tests, 2 for controls) using 100 ul of the chosen pathogen Salmonella
Typhimurium (a mixture of 2 strains at lxlO 8 CFU/ml). After 10 min of drying, 3 sterile
discs (12.5 mm in diameter) were placed on each of the four plates in uniform: 2 plates (6
discs, 100 ul/disc) were inoculated with sterile distilled water (control) while the other 2
plates (6 discs, 100 ul/disc) were each inoculated with potato extract. After 10 min, plates
were inverted and incubated for 24 hrs at 37 °C. The zones of inhibition were measured
with a ruler in millimeters after incubation.
39
Russet Potato Tuber Juice Treatment (75% (w/v)) versus Salmonella Typhimurium
Using the Agar Diffusion Method
Preparation of materials and potato samples
Russet Burbank potatoes were obtained from a local grocery store in Orono ME.
Potato samples were prepared, centrifuged and freeze-dried as described above. Fifty ml
of potato juice was centrifuged. Fifteen ml of resulting liquid was freeze-dried.
Agar diffusion method
The freeze-dried sample was weighed (0.8 g). Fifty percent (w/v) of sterile
distilled water (0.4 g) was added to freeze dried material to make a 75% (w/v) dilution. A
"lawn of bacteria" was created on 3 plates of TSA agar using 100 ul of the chosen
pathogen Salmonella Typimurium (a mixture of 2 strains at 1x10
8
CFU/ml). After 10
min of drying, 3 sterile discs (12.5 mm in diameter) were placed on each of the 3 plates in
uniform: On the first plate, 100 ul of ethanol was placed on each of the 3 discs, on the
second plate, 100 ul of potato extract was placed on each of the discs, and on the third
plate, 100 ul of distilled water was placed on each of the discs. Plates were inverted and
incubated for 24 hrs at 37 °C. The zones of inhibition were measured with a ruler in
millimeters after incubation.
40
Potato Peel Juice Treatment on Escherichia coli 0157:H7 Using the Log Reduction
Method
Preparation of potato peel sample
Figure 4.2 illustrates the preparation of potato peel juice. Russet Burbank potatoes
were obtained from a local grocery store in Orono ME. All materials, including the knife,
food processor and beaker were sterilized. Potatoes were washed (2 medium sized
Russets) with sterile distilled water while wearing sterile gloves. The potatoes were
peeled under a sterilized hood and the germicide hood was turned on for 20 min. The
peelings were flipped halfway through to assure sterilization on both sides. The peel was
weighed and then cut into fine pieces. Pieces were then ground using a small food
processor (General Electric, Fairfield, CT). The same amount of sterile distilled water
was added gradually throughout the grinding process until frothy (approximate 20 min) to
make 50% (w/v) potato peel extract. Four milliliters of liquid extract was removed and
used as sample treatment in the log reduction study
4!
Figure 4.2: Flow chart for preparing potato peel juice
Sterilize all materials
Remove 4 ml for treatment
A
Wash, peel potatoes
Under hood
t
I
u
Add w/v sterile distilled water
Continue grinding ~20 min.
Grind in food processor
t
Sample inoculation and antimicrobial study using log reduction method
To make the treatment sample, 4ml of previously prepared potato peel extract was
added to 1 ml of E. coli 0157:H7 cocktail (5 log CFU/ml). To make the control sample, 4
ml of sterile distilled water was added to 1ml of E. coli 0157:H7 cocktail (5 log CFU/ml).
After 24 hrs, a series of dilutions were performed. One hundred ul of samples from each
dilution for both the control and potato treatment are spread-plated on duplicate MSA
plates. Plates were inverted and incubated at 37 °C for 24 hrs for the comparison of the
viable cell count between the treatment and control samples.
42
Antioxidant Study of Potatoes
The DPPH method (l,l-Diphenyl-2 picrylhydrazyl) (Sigma-Aldrich; St. Louis.
State) was used to examine antioxidant activity of different components of the potato
such as the peel and tubers and a few small fruits The DPPH radical is taken up by
antioxidants (if present) through the donation of a hydrogen, forming a reduced DPPH-H.
As a qualitative method, the addition of the DPPH to the food containing antioxidants
will change the color of the solution from deep purple to light yellow (Miliauskas et al.,
2003).
Preparation of food samples to be tested
Samples that were used included: 0.45 ml of cranberry concentrate, 0.45 ml of
Cornus fruit concentrate, 1 macerated blueberry (discarding skin), 1 tea bag (soaked in
water and 0.45 ml was used), a clipping of an orange peel, a small piece of potato tuber
(mortared), and a small clipping of potato peel (mortared). All samples were placed in
test tubes. The samples in each of the test tubes were mixed well occasionally for 15 min
Preparation of DPPH in test tubes
One ml of DPPH (l,l-Diphenyl-2 picrylhydrazyl) was added to each of 7 test
tubes containing food samples, and then 1 ml of 95% EtOH was added to the DPPH
solution within each of the 8 tubes. One test tube was kept free of a sample to use as a
reagent control (containing DPPH and EtOH with a deep purple color). The components
were thoroughly mixed. The amount of antioxidant levels in the samples were measured
by the color change from purple to the light yellow if antioxidants were present in foods.
43
Chapter 5
RESULTS: A SIMPLE GASEOUS CHLORINE DIOXIDE TREATMENT FOR
MICROBIAL DECONTAMINATION ON POTATOES IN STORAGE
Natural Flora: Total Microorganisms
As the total microbial trials for both treatment and time were performed in
triplicate, results were determined based on the log reduction by bacterial enumeration
seen on the TSA plates. In studying the results of the total microorganisms CFU/potato
(Fig. 5.1A), there was no significant difference (P > 0.05) between the 4 g and 3 g
treatments after 2.5 hrs as the 4 g treatment showed a 2.7 log CFU/potato log reduction
and the 3 g treatment showed a 2.4 mg/1 log reduction. There was a significant difference
{P < 0.05) between the 2 g and the 3 g and also the 2 g and 4 g after 2.5 hrs as the 2 g
treatment gave a log reduction of only 1.7 log CFU/potato. After 5 hrs, there was a
significant difference {P < 0.05) between each treatment as the 4 g treatment reached a
log reduction of 5.4 log CFU/potato, the 3 g gave a log reduction of 3.2 log CFU/potato,
and the 2 g resulted in 2.6 log CFU/potato.
In studying the treatment time (Fig. 5.1 A), there was a significant difference (P <
0.05) between each treatment after 2.5 and 5 hrs as the 2 g increased from 1.7 log
CFU/potato after 2.5 hrs to 2.6 log CFU/potato after 5 hrs of treatment. The 3 g treatment
increased from 2.4 log CFU/potato after 2.5 hrs to 3.2 log CFU/potato after 5 hrs, and the
4 g treatment showed a large reduction in the total microorganisms as it went from 2.7
log CFU/potato after 2.5 hrs to 5.4 log CFU/potato after 5 hrs.
44
With the CFU/g analysis (Fig. 5.IB), results were slightly different. For example,
after 2.5 hrs of treatment there was no significant difference between each treatment as
the 4 g and 3 g treatments both reached 1.8 log CFU/g and the 2 g had a 1.3 log CFU/g
reduction. After 5 hrs for the CFU/g there was a significant difference (P < 0.05) among
each of the treatments due to the 4 g giving a 3.4 log CFU/g reduction, and the 3 g at 2.4
log CFU/g reduction, while the 2 g only reached 1.9 log CFU/g reduction.
When looking at the effect of treatment time from CFU/g analysis (Fig. 5.IB),
they differ from the CFU/potato in that there is no significant difference for the 2 g
treatment after 2.5 and 5 hrs as the reduction went from 1.3 log CFU/g after 2.5 hrs to 1.9
log CFU/g after 5 hours. After treatments of 3 g and 4 g for CFU/g, results show that just
as with the CFU/potato, there was a significant difference (P < 0.05) between the 2.5 and
5 hr as the 3 g went from 1.2 log CFU/g after 2.5 hrs to 2.5 log CFU/g after 5 hrs. Results
from the CFU/potato and the CFU/g after 2.5 hrs with the 4 g treatment were similar due
to a large increase in the log reduction as compared to the 2 g and 3 g treatments after 2.5
hrs.
45
Figure 5.1: Microbial decontamination by gaseous chlorine dioxide on natural flora-total
microorganisms of potatoes at different times and treatments. A.) Log reduction
CFU/potato and B.) Log reduction CFU/g. Different capitalized letters vertically indicate
a significant difference (P < 0 05) between different treatments over the some period.
Different lower case letters horizontally indicate a significant difference (P < 0.05)
between different times at the same treatment.
A.)
•4g
•3g
2g
0
2
3
Treatment (hr)
46
4
6
B.)
c
o
6.0 5.5
5.0 4.5 -
*•>
o3
0)
4.0
3.5 -
4g
3g
2g
l_
O)
3
UL
oO)
O
3.0 2.5
20 i
1.5
1.0 J
0.50.0 '
2
3
4
Treatment (hr)
Natural Flora: Yeast and Mold
As the yeast and mold trials for both treatment and time were performed in
triplicate, results were determined based on the log reduction by bacterial enumeration
seen on the DRBC plates. For results of yeast and mold based on CFU/potato analysis
(Fig. 5.2A), there was not a significant difference between the 3 g treatment and the 2 g
treatment after 2.5 hrs as the 3 g had a reduction of 2.0 log CFU/potato and the 2 g had a
reduction of 1.9 log CFU/potato. There was however, a significant difference (P < 0.05)
between the 2 g and 4 g, and between the 3 g and 4 g, treatments after 2.5 hrs. The 4 g
treatment showed a much higher log reduction of 3.0 log CFU/potato after 2.5 hrs. The 5
hr treatment showed similar results, with a significant difference (P < 0.05) between the 4
g treatment with a 5.2 log CFU/potato reduction and the 3 g treatment with a 3.3 log
47
CFU/potato reduction. There was also a significant difference (P < 0.05) between the 4 g
treatment and the 2 g treatment which only showed a reduction of 2.6 log CFU/potato
reduction (similar to the 3 g treatment).
The CFU/g analysis (Fig. 5..7B) is similar to that of the CFU/potato. The 4 g
treatment was significantly different (P < 0.05) from both the 3 g and 2 g treatments after
2.5 hrs. There was not a significant difference, however, between the 3 g and the 2 g
treatment. The 4 g reduction reached 2.5 log CFU/g, yet the 3 g and 2 g treatments only
showed a reduction of 1.2 log CFU/g and 1.1 log CFU/g respectively. The 5 hr treatment
CFU/g varied slightly from the CFU/potato analysis. The 4 g treatment (3.2 log CFU/g)
was significantly different from the 2 g treatment (2.1 log CFU/g), yet there was no
significant difference between the 3 g (2.5 log CFU/g) and 2 g (2.1 log CFU/g) as well as
the 3 g (2.5 log CFU/g) and the 4 g (3.2 log CFU/g) treatment after 5 hrs.
Results from the effect of various treatment times for CFU/g data showed a
significant difference (P < 0.05) after 2.5 and 5 hour treatments. The 2 g treatment
increased from a log reduction of 1.1 log CFU/g after 2.5 hrs to a reduction of 2.1 log
CFU/g after 5 hrs; the 3 g from 1.2 log CFU/g to 2.5 log CFU/g, and the 4 g from 2.5 log
CFU/g to 3.2 log CFU/g respectively. These results were similar to the CFU/potato data
with the exception of the 2 g treatment, which rose only slightly from a reduction of 1.9
log CFU/potato to 2.6 log CFU/potato. The 3 g went from a reduction of 2.0 log
CFU/potato after 2.5 hrs to 3.3 CFU/potato after 5 hrs. The 4 g treatment increased the
reduction significantly, rising from 3.0 log CFU/potato after 2.5 his to 5.2 log
CFU/potato after 5 hrs of treatment time.
48
Figure 5.2: Microbial decontamination by gaseous chlorine dioxide on natural flora-yeast
and molds of potatoes at different times and treatments. A) Log reduction CFU/potato
and B) Log reduction CFU/g. Different capitalized letters vertically indicate a significant
difference (P < 0.05) between different treatments over the same period. Different lower
case letters horizontally indicate a significant difference (P < 0.05)
times at the same treatment.
A.)
0
1
2
3
Treatment (hr)
49
4
between different
B.)
6.0
•4g
5.5
•3g
2q
5.0
c
o
o
3
•o
0)
4.5 4.0 3.5 -
l_
«!? 3.0
li.
O
O)
o
_l
2.5
2.0
1.5 1.0 0.5 0.0 '
0
2
3
4
Treatment (hr)
Pseudomonas aeruginosa
As the Pseudomonas trials for both treatment and time were performed in
triplicate, results were determined based on the log reduction by bacterial enumeration
seen on the PIA plates. Results for CFU/potato analysis (Fig. 5.3A) showed a significant
difference (P < 0.05) between each treatment after 2.5 and 5 hrs. The 4 g treatment
resulted in a reduction of 3.9 log CFU/potato after 2.5 hrs and 5.8 CFU/potato after 5 hrs.
The 3 g treatment was also significantly different (P < 0.05) between the 2.5 hrs and 5 hrs
as it increased from 3.27 log CFU/potato to 4.3 log CFU/potato. The 2 g treatment rose
from 2.2 log CFU/potato after 2.5 hrs of treatment time to 2.7 log CFU/potato after 5 hrs.
The results also showed a significant difference (P < 0.05) between the 2.5 hrs and 5 hrs
with each of the three treatments. After 2.5 hrs, the 4 g treatment showed a 3.9 log
50
CFU/potato reduction, the 3 g showed a reduction of 3.27 log CFU/potato, and the 2 g a
2.2 log CFU/potato reduction. After 5 hrs, the treatments were also significantly different
(P < 0.05) from one another as the 4 g showed a 5.8 log CFU/potato reduction, the 3 g
showed a 4.3 log CFU/potato reduction, and the 2 g showed a ? 7 !og CFU/potato
reduction.
Results for the CFU/g analysis (Fig. 5.3B) showed a slightly different result than
the CFU/potato. Just as the CFU/potato results showed significant difference (P < 0.05)
between each treatment (2 g, 3 g and 4 g) after 2.5 and 5 hrs the CFU/g did as well. The 4
g increased from 2.87 log CFU/g after 2.5 hrs to 4.3 leg CFU/g after 5 hrs, the 3 g
increased from 2.5 log CFU/g to 3.5 log CFU/g, and the 2 g increased from 1.2 log
CFU/g after 2.5 hrs to 2.0 log CFU/g. Results differed with the CFU/g analysis as there
was not a significant difference between the 4 g treatment and the 3 g treatment after 2.5
hrs. The 4 g showed approximately a 2.8 log CFU/g reduction and the 3 g showed a 2.5
log CFU/g reduction. There was a significant difference (P < 0.05) between the 4 g
treatment (2.8 CFU/g) and 2 g treatment (1.2 log CFU/g) after 2.5 hrs as well as a
significant difference (P < 0.05) between the 3 g (2.5 log CFU/g) and the 2 g (1.2 log
CFU/g) treatment. Similar to the CFU/potato analysis, there was a significant difference
(P < 0.05) between the all 3 treatments after 5 hrs of treatment time, as the 4 g showed a
reduction of 4.3 log CFU/g, the 3 g showed a reduction of 3.5 log CFU/g, and the 2 g
showed
a
reduction
of
51
»'
2
log
CFU/g.
Figure 5.3: Microbial decontamination by gaseous chlorine dioxide on inoculated
Pseudomonas aeruginosa on potatoes at different times and treatments.
A) Log
reduction CFU/potato and B) Log reduction CFU/g. Different capitalized letters
vertically indicate a significant difference (P < 0.05) between different treatments over
the same period. Different lower case letters horizontally indicate a significant difference
(P < 0.05) between different times at the same treatment.
A.)
Log CFU/pcrtato educti
r
6.00
5.50
c 5.00
o
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
1.00
2.00
3.00
4.00
Treatment (hr)
52
5.00
6.00
B.)
6.00
5.50
5.00
c
>g CFU/g nsductii
ft
J3
•4g
'3g
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
1.00
2.00
3.00
4.00
Treatment (hr)
5.00
6.00
Visual Quality
After each trial, one treated potato was taken from the desiccator to use as the
sample for visual quality against one un treated potato. The treated and control potatoes
were stored in whirlpak bags and both were observed over a 14 day period for signs of a
decrease in visual quality. A decrease in visual quality may manifest as wrinkling and/or
lightening of the skin. Upon observing the visual quality of the control potato in
comparison to the treated sample, the results did not indicate a difference in appearance
when observing the 2 g and 4 g trials (Table 5.1). Both of the 3 g, 2.5 hr, and 3 g 5 hr trial
treated potatoes showed slightly lighter brown skin coloration than the control. This
result was not unacceptable, as it differed only slightly from the others.
53
Table 5.1: Visual quality of potatoes treated with gaseous chorine dioxide after 14 days
using the hedonic scale.
Treatment
Average
2 g 2.5 hr.
2 g 5 hr.
9.00 ± 0.00*
9.00 ± 0.00
2g2.5hr.
3g5hr.
8.67 ±0.58
8.33 ±0.58
4 g 2.5 hr.
9.00 ± 0.00
4g5hr.
9.00 ±0.00
*Data are averages of triplicate ± the standard deviation. The hedonic scale ranges from
0-9; 9 being rnosi acceptable.
Residue
After each trial, one treated potato was tested immediately following treatment to
determine the residual CIO2 at day 0. After the visual check of the 1 treated potato in
comparison to the 1 control potato after 14 days, each potato (control and treated potatoes)
was tested for the amount of remaining residue after the 14 day period. Even though the
control received no treatment, it was checked as it may have had residue left behind from
other outside sources. Results look uniform as the residue decreased after the 14 days
consistently for each treatment, indicating that CIO2 may dissipate naturally following the
treatments over time (Table 5.2).
54
Table 5.2: Residue of chlorine dioxide on potatoes immediately following treatment and
after 14 days of treatment.
Treatment
„, .
, -'
Treatment ^g)
Residue After 14 Days (g)
2g2.5hr.
2g5hr.
0.09 ±0.02*
0.11 ±0.08
0.02 ± 0.03
0.01 ± 0.01
3g2.5hr.
3g5hr.
0.16 ±0.01
0.17 ±0.08
0.02 ± 0.01
0.05 ± 0.06
4g2.5hr.
4g5hr.
1.19 ±0.57
0.66 ±0.13
0.86 ±1.01
0.35 ±1.53
J
* Results are an average of the individual trials in triplicate ± the standard deviation.
55
H
Chapter 6
RESULTS: ANTIMICROBIAL ACTIVITY OF POTATOES AGAINST
FOODBORNE PATHOGENS
Katahdin Potato Tuber Juice Treatment (50% (w/v)) versus Salmonella
Typhimurium Using the Agar Diffusion Method
Duplicate plates containing discs of the 50% (w/v) potato tuber juice treatment
along with duplicate plates containing the control (sterile distilled water) were observed
after 24 hours of activity for each of the three trials based on zones of inhibition. The
control showed a 0 mm zone of inhibition (or 12.5 mm when including the disc). Results
of the freeze dried 50% (w/v) Katahdin potato tuber juice showed a slight inhibition of
Salmonella Typhimurium. As the experiment was performed in triplicate, the average
zone of inhibition was 13.2 mm (Table 6.1). Although these zones did not completely
remove all bacteria from the surrounding area, the juice did show an indentation in the
lawn of bacteria surrounding the disc.
Russet Burbank Potato Tuber Juice Treatment (75% (w/v)) versus Salmonella
Typhimurium Using the Agar Diffusion Method
Duplicate plates containing discs of the 75% (w/v) potato tuber juice treatment
along with duplicate plates containing the control (sterile distilled water) were observed
after 24 hours for activity based on the zones of inhibition. The control showed a 0 mm
zone of inhibition (or 12.5 mm when including the disc). The 75% (w/v) Russet Burbank
juice resulted in a 13.3 mm zone of inhibition, similar to the 50% Katahdin juice (Table
56
6.1). Again, there was not a completely clear zone of inhibition, as it was more of an
indentation in the bacteria surrounding the treated disc.
Table 6.1: Results of agar diffusion assay for potato juice concentration against S
Typhimurium.
Concentration (%)*
Zone of Inhibition (mm)
0%
12.5 mm
50%
13.2 mm
75%
13.3 mm
*The 50% (w/v) concentration is an average of 3 trials with Katahdin potatoes. The 75%
(w/v) shows results from 1 trial with Russet Burbank potatoes.
Potato Peel Juice Treatment on Escherichia coli 0157:H7 Using the Log Reduction
Method
The potato peel treatment and control containing 5 log CFU/ml of E. coli 0157:
H7 were observed on MSA for each dilution in duplicate after 24 hrs. After observing the
MSA plates, results showed positive effects. There was a complete 5 log CFU/ml
reduction ofE. coli 0157:H7 after the 24 hr treatment of the potato peel as not one colony
of E. coli 0157:H7 was observed on any of the inoculated plates containing the treatment.
Antioxidant Study
After the addition of samples to the DPPH solution, it took approximately five
min for the samples to eventually change from purple to yellow. The samples that
57
changed include: cranberry concentrate, Cornus fruit concentrate, macerated blueberries
(discarding skin), tea, and orange peel. The potato psel changed color as well but only
resulted in a light greenish color. The potato tuber showed no coloration change from the
original deep purple color of the DPPK mixture,
58
Chapter 7
DISCUSSION: A SIMPLE GASEOUS CHLORINE DIOXIDE TREATMENT FOR
MICROBIAL DECONTAMINATION ON POTATOES IN STORAGE
Natural Flora: Total Microorganisms
The results from the 2.5 hour trials for CFU/g showed no significant difference
between the 2 g, 3 g, and 4 g treatment. However, the CFU/potato results did show a
difference between both the 4 g and 3 g and the 3 g and 2 g treatments. Based on these
results, the 3 g treatment is recommended as it differs slightly from the 4 g, which allows
for a less toxic, more economic, and more efficient use of the product.
When looking at the 5 hr trials for both the CFU/potato and CFU/g there are
significant differences (P < 0.05) between all three treatments. The results show that
using the 4 g treatment during this time is optimal because it achieves the industry
standard 5 log CFU/potato reduction.
Natural Flora: Yeast and Mold
Results for yeast and mold reduction at 2.5 hrs are similar between the 2 g and 3 g
treatments. The 4 g treatment proved to be much more effective than the others, giving an
increased reduction of greater than 1 log. The 5 hr trials as also showed the 4 g treatment
to be much more effective than the others. The 5 hr showed better results than the 2.5
hour trials for each treatment, with the slight exception of the 2 g treatment foi the
CFU/potato which showed no significant difference after the 2.5 and 5 hr treatment times.
59
For optimal log reduction , the 4 g treatment for 5 hrs would be best as there as it shows a
much greater reduction than the two other concentrations.
Pseiidomcnas aeruginosa
Though there was no significant difference between the 4 g and 3 g treatment
after 2.5 hr treatment with the CFU/g analysis, the recommendation would be to use the 4
g to ensure the optimal log reduction. After the 5 hr treatment time, the results still
indicate that it would be best to use the 4 g treatment, as there is an approximate 1 log
CFU/g reduction difference between the 3 g and 4 g treatments, and a much higher
reduction (almost 2 log) for CFU/potato.
As gaseous CICh reduced P. aeruginosa by nearly 6 log after 5 hrs of the 4 g
treatment, this time and treatment is ideal since a 5 log reduction is optimal in industry.
Results in this research show that P. aeruginosa is more affected by gaseous CIO2
than yeast and mold. Other studies showed similar results after testing the effect of
gaseous CIO2 against bacteria and yeast and mold on produce. Gaseous CIO2 was tested
against Salmonella and yeast and mold on tomatoes, onions, apples, and peaches by S.Y
(2005) showing a greater reduction of Salmonella than yeast and mold. S.Y (2005) also
tested gaseous CIO? against Salmonella and yeast and mold on blueberries, strawberries,
and raspberries; again Salmonella was reduced more on blueberries and strawberries, yet
raspberries were an exception as yeast and mold was reduced more than Salmonella.
Could gaseous C102be more effective in reducing bacteria than yeast and mold? It would
be interesting to understand and compare the mechanism of how the gas kills the bacteria
and fungi cells in order to determine the answers to these questions.
60
Due to gaseous CIO2 ability to be utilized at ambient temperatures, the gas may
penetrate hard to reach areas throughout the entire storage area. Gaseous CIO2 gas will
not condense within cold environments, nor will it become reduced in treatment in
warmer areas such as those including the vapors of hydrogen peroxide (see literature
review for example). This is a positive aspect of the treatment as condensation and
uneven treatment occurs within many vapor systems (Czarneski and Lorcheim, 2005). As
visual quality is important, chlorine dioxide is superior to other decontamination methods,
such as ozone and sodium hypochlorite, as it does not significantly decrease the overall
appearance of the potato.
Purogene® and Anthium AGP® products are buffered solutions of sodium
chlorite, and have been tested on potatoes. The reaction that is required to produce this
chlorine dioxide from a buffered solution needs the addition of food grade acid to be
added to the Purogene® and Anthium AGP® products. These methods are time
consuming and require a risky and lengthy activation process that may not generate
adequate treatments of chlorine dioxide if not properly activated. With these two products,
the potatoes must be washed before further processing and consumption (Olsen, 2007).
The simple gaseous CIO2 generation presented in this thesis may not only require less of
an activation process, but the gas may be easily studied using the DPD method
(previously mentioned) in order to make sure that the activation is done correctly,
producing the appropriate amount of gas for each treatment after a certain amount of time.
Kleinkopf (2001) reported a method of using chlorine dioxide created by adding
food grade acid to buffered solutions and then dissolved it into an aqueous solution,
which was applied to potatoes by way of misting. Unfortunately, during misting, much of
61
the gas dissipated into the air instead of being applied to the potatoes themselves. After
attempting to apply more liquid to the potatoes to resolve this issue, it was noted that the
potatoes were too moist. The addition of moisture will make them more susceptible to
microbial invasion. The same stud*' then tested chlorine dioxide efficacy through a
humidification system. Unfortunately it was found that much of the chlorine dioxide was
again lost as a gas from solution into the surrounding atmosphere (Kleinkopf, 2001). The
gaseous CIO2 used in this research will not add moisture to the potatoes during
application, preventing the optimal environment that many pathogens desire.
Visual Quality
Gaseous CIO2 did not affect the overall visual quality of the potato, with a slight
exception for the 3 g treatment. Two of these trials resulted in potatoes that were noted to
have a bit lighter, and drier, looking skin. Conditions such as the relative humidity may
influence the visual quality of the potato during treatment. Might a lower relative
humidity be the cause?
In industry, visual quality is a very important criterion as consumers generally
purchase fresh produce based on its appearance. As previously mentioned, different
forms of decontamination treatments may affect the visual quality of produce. Gaseous
CIO2 may be a better disinfectant to utilize in storage as the visual quality of the potato is
appropriately maintained. Just as potatoes showed no severe loss of visual quality after
treatments, Lee et al, (200) studied gaseous CI02on lettuce leaves and also leported that.
the quality of the lettuce leaves were not compromised after treatments.
62
Residue
Though the residue increased with both time and treatment during the CIO2 trials,
results showed an obvious reduction of chlorine dioxide residue on the potato after 2
weeks. With such a great reduction, if might be appropriate to havr high treatments of
CIO2 as it naturally dissipates over time. For further research, it might be interesting to
see what the residue would be at the time of purchase by the consumer. For example,
potatoes require time for shipping and also for sitting on display in the grocery store or
wherever they may be sold. It may be several months before the potato is released from
storage, giving it plenty of time to rid itself of any lingering residual. Gaseous CICbmay
leave less of a residue behind than any other liquid decontamination such as aqueous
chlorine dioxide as it is directly applied onto the produce as a liquid and may
permanently adhere to its surface.
63
Chapter 8
DISCUSSION: ANTIMICROBIAL ACTIVITYOF POTATOES AGAINST
FOODBORNE PATHOGENS
Katahdin Potato Juice Treatment (50% (w/v)) versus Salmonella Typhimurium
Using Agar Diffusion Method
As this method using of Katahdin potato juice (50% w/v) was performed three
times against Salmonella Typhimurium, and showed positive and consistent results, it
may be safe to assume that tuber juice may contain antimicrobial properties. These
properties may be the result of proteases or other phenolic compounds such as: (+) catechin, chlorogenic acid, caffeic acid, p - coumaric acid, and ferulic acid (Mendez,
2004), in the 50% (w/v) concentration of tuber juice. Aspartic proteases in plants have
been known to play a major role in plant defense against abiotic stress, as the induction of
the StAPs in rubers is seen after wounding the potato. If the antimicrobial activities of the
enzymes are controlled by the presence of these particular aspartic protease inhibitors,
antimicrobial compounds might not readily be active if the inhibitors are not activated.
Russet Burbank Potato Tuber Juice Treatment (75% (w/v)) versus Salmonella
Typhimurium Using the Agar Diffusion Method
As the 50% (w/v) freeze dried potato concentration showed a slight inhibition
when applied to Salmonella Typhimurium, the 75% (w/v) concentration showed similar
results. The 75% (w/v) juice trial was only successfully performed one time, and should
not be readily compared to the 50% (w/v) treatment because the potatoes were of
64
different variety and treatment concentrations. Along with the differing variety of
potatoes used in the 50% (w/v) and 75% (w/v) trials, the quality of the potato should be
determined. Were the potatoes used in the 50% (w/v) trials perhaps more injured, hence
releasing the antimicrobial compounds more readily9 Tf R^ssot potato juice a' 75% (w/v)
was to contain as many compounds as Katahdin at only 50% (w/v) concentration could
that mean that the Katahdin variety may contain more antimicrobial compounds? These
are questions to be investigated in future studies.
Potato Peel Juice Treatment on Escherichia coli 0157:H7 Using the Log Reduction
Method
Results indicate that potato peel juice may be effective against E. coli. 0157:H7,
as it completely eliminated the bacterial load of 5 log CFU/ml in the treated sample.
However, since this method was only successfully performed one time, it cannot be
stated that this method truly is effective against E. coli. 0157:H7. As antimicrobial
compounds may only be activated under certain circumstances, it may be possible that
the potatoes were not similar in their quality. These initial results look promising, but
need to be replicated and then used as a preliminary study for future research. Upon
repeating this study, it might be interesting to see if the peel will reduce other bacteria
using the same log reduction method.
Antioxidant Study
Although the results indicate that there is a slight antioxidant level in the potato
peel, they also show that the potato tuber does not contain any significant antioxidant
65
compounds. Even though both treatments (peel and tuber) were tested and showed
antimicrobial activity against bacteria, they were tested using different methodologies
and bacteria, so it may not be possible to draw strong conclusions concerning their lack
of antioxidant compounds. If the tuber antioxidant compounds w very 'light they might
not have been readily detected by this particular method, therefore other procedures may
need to be utilized for further determination. Quantitative methods for measuring
antioxidants in food may also be utilized for determining antioxidants in potatoes. One of
these methods is the oxygen radical absorbance capacity (ORAC), which measures the
oxidative degradation of fluorescein after being mixed with peroxyl radical. The reaction
is then compared to a standard antioxidant like Tolox (EAC), a vitamin E analogue
(Wikipedia, 2007).
Another quantitative method that may be utilized in determining antioxidants in
potatoes is the FRAP (ferric reducing ability of plasma) assay. This assay is measured by
the ferric to ferrous ion reduction at low pH, and will result in a colored ferroustripyridyltriazine complex to occur. When comparing the absorbance change at the 593
nm wavelength in test reaction mixtures with those containing ferrous ions in known
concentration, FRAP values are determined. With antioxidant mixtures, absorbance
changes are linear over a wide concentration range (Benzie, 1996).
The DPPH study might be a good preliminary for further research in determining
what the compounds may be, how strong they are, and where they may be most
prominently located within the tuber.
66
Chapter 9
CONCLUSION
Ga?eou* Chlorine Dioxide Study
In conclusion, gaseous CIO2 showed positive results as a sanitization method on
potatoes against yeast and mold, total microorganisms, and P. aeruginosa as it effectively
reduced each microorganism by approximately 5 log CFU/potato after 5 hrs of 4 g
treatments. These results were consistent within each time and treatment of trials without
leaving behind much residue and not compromising the visual quality of the potato.
Interestingly enough, when comparing the reduction among the different organisms, it
became obvious that both fungi (yeast and mold) and total microorganisms showed very
similar reduction results. This is likely due to the fact that most of the "natural flora" on
potatoes consist of fungi (yeast and mold).
There are many positive attributes of gaseous CIO2, such as safe, effective, and
convenient generation, as well as having a wide range of opportunities for further
research. For example, many other organisms commonly associated with potatoes in
storage could be studied. It would also be interesting to see by what mechanisms the gas
affects the fungi and bacteria (ex: reaction with the cell membrane, inactivation of
essential enzymes, or the destruction of genetic materials).
Results may be more promising if gaseous CIO2 is applied under such more
realistic conditions, becoming one of the most safe and effective ways to control and/or
limit microbial contamination on potatoes during storage. The application of gaseous
CIO2 on a larger scale could initially be tested in a small storage room similar to a real
67
storage environment at the facility of Aroostook Farm in Presque Isle, Maine. The
appropriate treatment and time of gas application may then be studied for use in the
larger scale facility. This study would definitely help Maine potato farmers to grow and
prosper without having te de»l with the loss of their crop? in storage due to microbial
contamination.
Antimicrobial Activity of Potatoes
Potatoes contain antimicrobial properties as seen in the tuber juice and peel study,
however further research may be performed as many factors must be taken into
consideration. Such factors may include: repeats, type of potato, methodology, and
especially the potato health as injured potatoes may include more aspartic proteases
(antimicrobial compounds) than healthy tubers. Also, different methods to extract the
antimicrobial compounds may be developed in order to determine what concentrations
may be effective for use in the food industry as possible natural preservatives.
The antioxidant test was performed on the peel and tuber to determine the linear
relationship between antioxidant levels and antimicrobial compounds. It was interesting
to discover that the peel had slight antioxidant levels, but the tuber did not, yet both had
shown antimicrobial properties. This could have been due to the physical state of the
potato, or possibly the tuber contained such slight amounts that the compounds were not
detected by this method. This study might be further tested using different methods and
on different potatoes during different stages of growth (fresh vs old).
68
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73
APPENDIX: THE AMOUNT OF GASEOUS CHLORINE DIOXIDE RELEASE
OVER TIME
Appropriate treatments and times for the fast gas release were determined based
on the chart below.
Figure A.l: Gaseous CIO2 release after 5 hrs
ICA TriNova
Research Materials
10
8
4
0
0
2
3
Time, hr
74
4
Figure A.2: Gaseous C102 release after 25 hrs
ICA TriNova
Research Materials
14
12
10
8
6
4
0
10
15
Time, hr
75
20
25
BIOGRAPHY OF THE AUTHOR
Amanda Rioux was born in Lewiston Maine on January 6, 1981 and grew up in
the small town of Greene Maine until she moved away to college in 1999 where she
attended Saint Joseph's College in Standish Maine. At Saint Joseph's College she
received her Bachelor's degree with a major in Biology and a minor in Chemistry,
Immediately following graduation from Saint Joseph's College in 2003, Amanda
married her former fiancee Steven Rioux from Lewiston. Amanda and Steve then moved
to the small town of Lisbon where Amanda worked at Poland Spring Bottling taking on
the appropriate positions of being the sensory coordinator for the factory as well the
position of being one of the microbiologists.
After two years of great experience in industry, Amanda and Steve decided to
move to Plymouth, Maine due to Steve receiving a job opportunity at the University of
Maine. As Steve was working at the university, Amanda then decided that this would be
the optimal time to continue on with her education in microbiology. Amanda then entered
the biochemistry molecular microbiology program. Upon the realization that food science
was her calling (as she worked in sensory and microbiology at Poland Spring and loved
it), she then entered into the Food Science and Human Nutrition program under Dr.
Vivian C. H. Wu who gladly accepted her with open arms. Amanda is a candidate for the
Master of Science degree in Food Science and Human Nutrition from The University of
Maine in December, 2007.
76