Journal of Food Engineering 98 (2010) 178–191
Contents lists available at ScienceDirect
Journal of Food Engineering
journal homepage: www.elsevier.com/locate/jfoodeng
Effects of gamma irradiation on bio-chemical and physico-chemical parameters
of fresh-cut red beet (Beta vulgaris L. var. conditiva) root
Maria Emilia Latorre a,1, Patricia Narvaiz b, Ana María Rojas a,2, Lía Noemí Gerschenson a,2,*
a
Departamento de Industrias, Facultad de Ciencias Exactas y Naturales (FCEN), Universidad de Buenos Aires (UBA) Ciudad Universitaria, Intendente Güiraldes 2620,
(1428) Ciudad Autónoma de Buenos Aires, Argentina
b
Food Irradiation Section, Radiation Technology and Applications, Ezeiza Atomic Center, National Atomic Energy Commission, Argentina
a r t i c l e
i n f o
Article history:
Received 8 September 2009
Received in revised form 17 December 2009
Accepted 20 December 2009
Available online 28 December 2009
Keywords:
Red beet
Gamma irradiation
Enzymes
Bio-chemical parameters
Mechanical parameters
a b s t r a c t
Red beet (Beta vulgaris L. var. conditiva) root is a popular item present in ready to eat salads and minimally processed foods. In this research, the effect of low doses of gamma radiation (1 and 2 kGy) on peroxidase (POX), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) activities, as well as on
the levels of compounds related to the response to the oxidative stress of plant metabolism, the changes
in colour and the mechanical behaviour of fresh-cut red beet root were analyzed, with the purpose of
understanding the influence of the processing on tissue characteristics. Cell wall modifications were also
studied through sequential extractions of polysaccharides from the alcohol-insoluble residues (AIR)
obtained from each tissue. Irradiation seemed to contribute to higher cell–cell adhesion through increasing of calcium-cross linking at the middle lamellae regions, in addition to an increment of cross-links of
polymers into the cell wall. Chemical modifications produced in the cell walls as a response to higher levels of H2O2 and subsequent POX mediated effects, were visualized structurally as a more elastic behaviour of irradiated tissues and rigidification of cell walls of treated roots, though puncture test did not
reveal significant differences. Microscopy showed a continuum of thick cell walls in beet root tissue,
which suffered slight modifications after irradiation, coherent with the bio-chemical results obtained.
It can be concluded that irradiation doses of 1 or 2 kGy produced bio-chemical changes in cellular contents as well as in the cell wall constitutive networks which not necessarily could be sensed by consumers as it was objectively evaluated through a puncture test. At the same time, the mentioned changes
involved an increase in the antioxidant capacity of red beet root tissue, showing that studied doses could
be interesting to be used in the frame of a combined technique for red beet processing.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Red beet (Beta vulgaris L. var. conditiva) is a traditional and popular vegetable in many parts of the world. It is especially rich in fibres as well as in sugars but with moderate caloric value. Content
of B-vitamins (B1, B2, B3 and B6) as well as folic acid is important.
Red beet roots are consumed either fresh or after thermal processing or fermentation. The soluble and cell wall associated phenolics
as well as betalains, the main pigments in red beet responsible for
its reddish-purple hue, are bioactive compounds (Schwartz et al.,
1980), being their antioxidant capacity beneficial for human health
(Kanner et al., 2001; Gliszczynska-Swiglo, 2006). Among other effects, betalains from beet root are important for cardiovascular
* Corresponding author. Tel.: +54 11 4576 3366/3397; fax: +54 11 4576 3366.
E-mail address: [email protected] (L.N. Gerschenson).
1
Fellow of the National Agency of Scientific and Technological Promotion of
Argentina (ANPCyT).
2
Research Member of the National Scientific and Technical Research Council of
Argentina (CONICET).
0260-8774/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2009.12.024
health contributing to reduce the concentration of homocysteine,
which can be harmful to blood vessels (Nagai et al., 2000; Olthof
et al., 2003; Detopoulou et al., 2008).
The increasing demand by consumers for minimally processed
(fresh-cut) fruits and vegetables has prompted the sale of pre-cut
red beet roots as well as other vegetables in trays commercialized
in markets or selled in bulk for institutions. Horticultural products
are susceptible to mechanical damage during harvesting, transport,
storage and minimal processing (Aquino-Bolaños and Mercado-Silva, 2004). Otherwise, minimally processed vegetables may have
high levels of microorganisms. Commercial processes for preparing
them usually can use chlorine in the wash water to control the
microbial population. However, chlorine cannot be relied onto
eliminate pathogenic microorganisms such as Listeria monocytogenes (Nguyen-the and Carlin, 1994). Non-thermal interventions
with minimal effects in quality deterioration of produce are one
of the alternatives currently available for disinfection of leafy vegetables (Gomes et al., 2008). Irradiation is a well-established nonthermal and non-chemical process with clearly documented safety
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
and efficacy as food preservation method (ICGFI, 1999). Its efficacy
is based on the fact that its activity is not limited to the surface; it
can penetrate the product and eliminate microorganisms that are
present in crevices and creases, cracks, and intercellular spaces of
the seeds and other parts of vegetables that harbor the pathogens
(Prakash et al., 2000a; Waje et al., 2009). The effect of irradiation
on food itself is usually minimal at doses up to 7.5 kGy. Shelf life
of treated foods is prolonged because organisms that cause spoilage are reduced along with pathogens. Low irradiation doses were
successfully applied early for shelf-life prolongation of foods and
have been thought and studied as a practical mean for treating
fruits for insect disinfestations (Noomhorm et al., 1998). Langerak
and Damen (1978) extended the shelf life of pre-packed
soup-greens stored at 10 °C by treatment with a low 1 kGy dose.
Cut romaine lettuce packaged under modified atmosphere (MAP)
and subjected to 0.15 and 0.35 kGy of c-irradiation, showed that
the highest dose was able to reduce aerobic bacteria as well as
yeast and molds, while maintaining attributes like colour, but
some tissue softening was found (Prakash et al., 2000a,b). Low dose
c-irradiation, used as a post-harvest and pre-shipment treatment,
and in combination with other processes, has shown to be a promising technique for extending the shelf life of fruits and for their
preservation. The D-values (the amount of radiation energy required to inactivate 90% of specific pathogens) of Escherichia coli
O157:H7 on fresh-cut vegetables were mostly between 0.12 and
0.20 kGy (Niemira et al., 2002; Foley et al., 2004; Niemira and
Fan, 2006). Therefore, a dose of 1 kGy can achieve at least a 5 log
reduction of Escherichia coli O157:H7 surface inoculated on fresh
produce, although the internalized pathogens may better be able
to survive irradiation than those on the surface (Niemira, 2007).
The cost of food irradiation depends on variety of food, applied
doses, required effects, packing densities, throughputs, special
handling, packaging, logistical requirements and local conditions
(Kunstadt, 2001). According to IGCFI (1999), irradiation costs range
from $10 to $15 per tonne for a low dose application.
In the recent proposal of labeling changes (FDA, 2007), FDA expressed interest in information on whether the control of foodborne pathogens by irradiation changes the characteristics of
food in a way that is outside of the normal variability of the food
and would therefore require additional labeling to inform the consumer of such change (Fan and Sokorai, 2008). Appearance and
texture changes are two fundamental characteristics determining
the acceptability of fresh-cut fruit and vegetables (Ilker and Szcsesniak, 1990; Toivonen and Brummell, 2008). Several studies have
shown that irradiation can cause changes in pectic and cellulosic
substances present in the cell walls of plant tissues, resulting in
softening and discolouration of soft tissues, as reported by Prakash
et al. (2000a). Moreover, damage to cellular membranes leads to
loss of turgor (Faust et al., 1967), contributing significantly to the
firmness loss. According to Lacroix and Ouattara (2000) most fruit
and vegetables tolerate irradiation treatments at a minimal dose of
0.25 kGy without undergoing a change in their quality. A dose of
2.25 kGy is usually the optimal one that fruits and vegetables
may tolerate to keep their quality intact (Lacroix and Ouattara,
2000). For that reason, combined techniques are usually applied
to enhance the safety of foods whilst maintaining a high level of
organoleptical quality (Thakur and Singh, 1995). According to Farkas (2006), irradiation results in reduced storage losses, shelf life
extension and/or improved microbiological and parasitological
safety. Thayer (1990) stated that minimal chemical changes are induced by irradiation of food. Besides preservation, irradiation has
been studied for other purposes. For example, Lee et al. (2001)
found the diminishing of milk protein allergenicity after irradiation, while Ahn et al. (2003) observed the enhancement of the antioxidant activity of phytic acid after irradiation of its aqueous
solutions. Jo et al. (2003) reported that irradiated green tea alco-
179
holic extracts showed colour change from dark brown to bright
yellow after irradiation, which allowed using these extracts in food
and cosmetic formulations without affecting the material colour,
whilst the antioxidant capacity was not impaired.
All biological pigments selectively absorb certain wavelengths
of light while reflecting others. The light absorbed may be used
by the plant to power chemical reactions, while the reflected wavelengths of light determine the colour the pigment will appear to
the eye. Betalains of red beet are water soluble nitrogen-containing
pigments derived from tyrosine that are found only in a limited
number of plants, specifically in plants belonging to the order
Caryophyllales and in the fungal genera Amanita and Hygrocybe
(Gandia-Herrero et al., 2005). Interest in betalains has grown after
finding their antiradical activity (Escribano et al., 1998; Kanner
et al., 2001; Pedreño and Escribano, 2001; Strack et al., 2003),
and they are widely used as additives in the food industry because
of their natural colourant properties and absence of toxicity, even
at high concentrations (Schwartz et al., 1980). All betalain pigments contain betalamic acid as the chromophore and act as visible signals to attract insects, birds and animals for pollination and
seed dispersal. They also protect plants from damage caused by UV
and visible light (Yoshikazu et al., 2008). Depending upon the nature of the betalamic acid addition residue, the betalains can be
classified as either betacyanins or betaxanthins. Betacyanins contain a cyclo-3,4-dihydroxyphenylalanine (cyclo-DOPA; usually glycosylated) residue and exhibit a red/violet colouration, while the
betaxanthins contain different amino acids or amine side chains
and exhibit a yellow/orange colouration. Betalains are located in
different parts of plants, in roots, fruits and flowers (Kujala et al.,
2000; Gandia-Herrero et al., 2005). According to Herbach et al.
(2004), about 1% (on dry basis) of the fresh roots consists of betalains, and 80–90% of the total pigment content of red beet are ascribed to betacyanins, mainly betanin and its isomer isobetanin,
being the rest betanidine, prebetanine and their C-15 isomers
(Knuthsen, 1981). Vulgaxanthin represents the predominant betaxanthin (Herbach et al., 2004).
Tyrosinase or polyphenol oxidase (sysname:monophenol Ldopa:oxygen oxidoreductase; EC 1.14.18.1; PPO) is the key enzyme
in melanin biosynthesis and in the enzymatic browning of fruits
and vegetables. The role of PPO in the secondary metabolism of
plants still remains unclear, but its implication in betalain biosynthesis has been proposed. PPO is a copper enzyme that catalyzes
two different reactions using molecular oxygen: the hydroxylation
of monophenols to o-diphenols (monophenolase activity) and the
oxidation of the o-diphenols to o-quinones (diphenolase activity;
Sánchez-Ferrer et al., 1995). This enzyme is widely distributed in
plants, microorganisms, and animals where tyrosinase is responsible for melanization.
Like many other natural pigments, betalains are very sensitive
to heat, light and oxidation, especially to that caused by the peroxidase (POX) activity, which is one of the major causes of pigment
discolouration. POX (sysname: donor hydrogen-peroxide oxidoreductase; EC 1.11.1.7) involves a group of enzymes known to play
a very crucial role in free radicals scavenging within plant systems
(Regalado et al., 2004), being also involved in various developmental and metabolic processes. The increase in hydrogen peroxide
(H2O2) like that occurred after c-irradiation can be controlled in
plants by POX through its hydroxylic and standard peroxidative cycles. Plants contain two classes of POX: the intracellular (ancient)
Class I, and the Class III, which is secreted into the cell wall or
the surrounding medium, being only present in land plants, as an
adaptation to the terrestrial life in presence of elevated oxygen
concentrations. The exchange of electrons and protons are produced through the Fe(III)-protoporphyrin IX (heme) group of the
Class III of POX. In its standard peroxidative cycle, the enzyme catalyzes the reduction of H2O2 by taking electrons from donor mole-
180
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
cules such as phenolic compounds, lignin precursors, auxin or secondary metabolites. It is considered that POX plays an important
role in processes such as lignification (Egley et al., 1983) as well
as insolubilization of pectin–extensin (HRGPs) complexes (Jackson
et al., 1999; Passardi et al., 2004). The diversity of substrates explains the implication of POX in a wide range of physiological processes like auxin catabolism, formation of lignin and suberin
(Oudgenoeg et al., 2002), cross linking of cell wall components, defense against pathogens, and cell elongation. By generating hydroxyl radicals (OH), POX could also play a crucial role in seed
protection as well as in the first days of germination by reducing
the pathogenic attack as soon as the radicle protrudes, event that
produces a ‘wounded’ region in the seeds. The production of OH
could further contribute to the breakage of the seed coat and to
the subsequent natural process of cell elongation by hydrolysis of
polysaccharides at specific sites of the cell wall network (Fry,
1998; Passardi et al., 2004).
Since phenolics are secondary metabolites implicated, as substrates of POX, in the defense of plant tissues from injury, the activity of the phenylalanine ammonia-lyase (PAL; sysname: Lphenylalanine ammonia-lyase; EC 4.3.1.24), the enzyme involved
in the biosynthesis of phenolic compounds, is also necessary to
be known after c-irradiation of red beet roots. In this sense, the effect of wounding or chilling injury on six minimally processed lettuce (Lactuca sativa L.) cultivars, derived from storage during
7 days at 5 °C, was evaluated through PPO, POX and PAL activities
(Cantos et al., 2001). Some enzymes are inactivated by c-irradiation at doses suitable for food processing, while others still remain
active (IAEA, 1998). Wang et al. (2006) observed that POX and PPO
were active in cantaloupe juice even after c-irradiation of 5 kGy.
The objective of the present work was to determine the effect of
low doses of c-radiation (1.0 and 2.0 kGy) on the characteristics of
red beet root tissue in order to evaluate the possibility of consider
irradiation as a hurdle barrier for shelf-life prolongation of
minimally processed (washed, peeled and cut) red beet roots. To
accomplish this, bio-chemical and physico-chemical parameters
involved in the prooxidant–antioxidant balance in vegetable cells
and their physical manifestation (mechanical performance) on tissue, were evaluated with the purpose of gaining knowledge on
optimization of vegetable processing and quality tending to contribute to healthier food development.
2. Materials and methods
2.1. Chemicals
They were of analytical grade unless stated.
2.2. Sample preparation
Red beet (Beta vulgaris L. var. conditiva) roots harvested in La
Plata (Argentina) were obtained at the local market. They were
carefully cleaned, peeled and cut into 10 mm-thick slices perpendicular to their longitudinal axis. Cylindrical samples of 15 mm
diameter were then obtained from each slice by using a cork borer.
This sample geometry was chosen by considering its adequacy for
mechanical assays.
2.3. Sample irradiation
Red beet root cylinders were divided into three lots (500 g each
one), being stored (from 6PM of one day to 8PM of the next day) in
a refrigerated chamber at 3.0 ± 0.5 °C (90–95% HR) before irradiation. One of them was not irradiated and used as a control
(0 kGy). The other two lots were exposed to ionizing radiation from
a 60Co-gamma source in a semi-industrial facility (Ezeiza Atomic
Center, National Atomic Energy Commission (CNEA) of Argentina;
activity: 600,000 Ci), at 20 °C, for approximately 8 min, time during
which the expected dose was absorbed. The absorbed doses were 1
or 2 kGy, with a dose rate of 7.7 kGy/h and dose uniformity of 1.04.
The non-irradiated cylinders (control) were also brought to the
irradiation facility with the aim of exposing them to similar transportation and environmental conditions as those of irradiated
samples.
2.4. Chemical and bio-chemical analyses
The samples (control and irradiated samples) were (a) directly
used to determine enzyme activities, colour, pigment and protein
contents, as well as to perform mechanical and turgor assays, or
(b) submitted to extraction of the alcohol-insoluble residue (AIR).
The AIRs were used (i) to determine the lignin, cellulose, total
(non-cellulosic) carbohydrates, uronic acids, protein and polyphenolic contents; (ii) to carried out a sequential extraction of cell wall
polymers. Unless stated, the analyses were performed in duplicate.
All chemical solutions used in the following analyses were prepared by using deionized water (Milli-Q, USA).
2.4.1. Enzyme assays
2.4.1.1. Acetonic extract. Following Walton and Sondheimer (1968),
raw tissue was homogenized using a Sorvall Omni Mixer (Sorvall,
Norwalk, Conn, USA), for 2 min, with 4 °C-pre-cooled acetone
(Merck Argentina S.A., Buenos Aires, Argentina) in a relation of
1 g to 4 ml. The homogenate was then filtered through a Büchner
funnel using filtering paper (Whatman GF/C, UK). The residue
was washed twice with acetone to eliminate colour and phenolic
interference and acetone was eliminated under labhood overnight
at room temperature. Powder was stored in a freezer (28 °C) till
its usage for enzyme activity determination.
2.4.1.2. Protein content. It was determined in the buffer extractive
solutions of enzymes according to Lowry et al. (1951) using bovine
serum albumin as standard (Sigma, St Louis, MO, USA).
2.4.1.3. Peroxidase (POX) activity. The powder (0.5 g) was suspended in 40 ml of 0.05 M-buffer phosphate (pH 7.0). Sodium chloride was added till reaching a 1 M-concentration in the suspension.
The system was maintained under stirring for 1 h at 5–7 °C and
then centrifuged (Model 5804R, Eppendorf AG, Hamburg, Germany) at 10,000 rpm for 15 min (4 °C). The supernatant was assayed for total POX activity, which was determined at 25 °C as
described by Marangoni et al. (1995), using guaiacol (o-methoxyphenol; Anedra, Buenos Aires, Argentina) as substrate and
expressing the activity as the change in absorbance at 470 nm
(Absorbance Units, AbU) per minute and milligram of protein
(AbU min1 mg1 protein), measured with an spectrophotometer
(Spectro SC model, LaboMed Inc., Culver City, CA, USA).
2.4.1.4. Polyphenol oxidase (PPO) activity. The powder was used for
evaluation of PPO activity according to Coseteng and Lee (1987)
and Xuan et al. (2008). The powder was suspended in 0.1 M-buffer
phosphate (pH 6.0) (1 g powder per 5 ml buffer) and 1 M-NaCl
solution during 45 min at 5–7 °C. The suspension was then centrifuged (Model 5804R, Eppendorf AG, Hamburg, Germany) at
9000 rpm for 10 min (7 °C). The supernatant was assayed for total
POX activity, at 25 °C by using 40 mM-pyrocatechol (Merck, Buenos Aires, Argentina) in 10 mM-phosphate buffer (pH 7.0). The
activity was evaluated following the change in absorbance at
420 nm with a spectrophotometer (SpectroSC model, LaboMed
Inc., Culver City, CA, USA). One unit of activity was defined as the
change in absorbance at 420 nm per minute and milligram of
protein.
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
2.4.1.5. Phenylalanine ammonia-lyase (PAL) activity. The powder
was suspended in 0.1 M-buffer phosphate (pH 8.8) (1 g of powder
per 20 ml buffer). The system was maintained under stirring for
30 min at 5–7 °C and, then, centrifuged (Model 5804R, Eppendorf
AG, Hamburg, Germany) at 9000 rpm for 10 min (7 °C). The supernatant was assayed for total PAL activity according to Duan et al.
(2007). Briefly, the supernatant was contacted with L-phenylalanine (20 mM) as substrate at 37 °C and the activity was evaluated
following the change in absorbance using a Shimadzu UV-Mini
1240 spectrophotometer (Shimadzu Corporation, Kyoto, Japon).
One unit of activity is defined as a 0.001-change in absorbance at
290 nm per hour and milligram of protein.
2.4.2. Amino acids
Amino acid composition was obtained after C-fraction treatment with 6 N HCl (Method 994.12, AOAC, 2000). For hydroxyproline analysis, sample was derivatized using 9-fluorenylmethylchloroformate and 3-mercaptopropionic acid (Herbert et al.,
2000; Henderson et al., 2000). Separation and analysis were performed with an HPLC Series 1100 (Agilent, California, USA)
equipped with a fluorescence detector. The chromatographic column was a 15 4.6 cm, 5 mm particle, C18 Hypersil BDS Gold
(Agilent, California, USA). A mixture of 40 mM-Na2HPO4, pH 7.8
(A) and methanol (B) with a linear gradient starting in a 50:50
(v/v) and ending, after 10 min, in a 10:90 (v/v) mixture of A and
B was used as mobile phase.
2.4.3. Alcohol Insoluble Fraction (AIR)
Either 350.00 g of each control or irradiated fresh tissue was
suspended in a 95% (v/v)-ethanol solution (1:4 ratio) for separation
of its insoluble residue (AIR) (Martin-Cabrejas et al., 1994), homogenized with a mechanical device and then boiled for 10 min under
stirring. The obtained residue was then extracted for 10 min with
65% (v/v)-ethanol boiling solution. The insoluble residue was separated and washed with 65% (v/v)-ethanol. Between each ethanol
treatment, the suspension was filtered (Whatman filter: GF/C,
UK) and the supernatant was discarded. The cell wall material
(AIR) obtained was left overnight under a lab hood to eliminate
the remaining ethanol, and then freeze-dried (Stokes freeze-drier,
Stokes Company, Philadelphia, MA, USA) after freezing with liquid
nitrogen. The product was then milled and the resulting powder
was distributed in plastic tubes which were packaged in Cryovac
(polyvinyl chloride-polyvinylidene chloride copolymer) bags under
vacuum and stored at 28 °C till usage. The AIR was used for
chemical analyses: (a) lignin, cellulose and non-cellulosic carbohydrates content, (b) quantification of uronic acids, degree of methylation (DM) and degree of acetylation (DA), (c) extraction and
quantification of total phenolics, (d) moisture content, (e) sequential extraction of cell wall polymers.
2.4.4. Lignin, cellulose, non-cellulosic carbohydrates, methanol and
acetyl contents
The AIR obtained was used to determinate uronic acid, total
(non-cellulosic) carbohydrate, cellulose and lignin contents.
Hydrolysis of cellulose and non-cellulosic polysaccharides of AIR
was performed according to Ng et al. (1998) by dispersion of
0.3000 g of sample product into 2080 ll of 72%-sulphuric acid
solution for 3 h at room temperature. This dispersion was made
1 M-sulphuric acid by addition of enough deionized water (until
25.00 ml-final volume) and each sample was heated at 100 °C for
2.5 h in a water-bath. After this, all dispersions were cooled, centrifuged at 12,000g for 10 min and the supernatant was separated,
carefully neutralized and total carbohydrate content was determined by phenol–sulphuric method (Dubois et al., 1956), using
D-galacturonic acid as standard curve. The residue was washed
three times with deionized water, centrifuged at 12,000g for
181
10 min and, finally, freeze-dried. The residue obtained was
weighed and reported as lignin.
A second procedure was carried out with other portion of
0.3000 g of each sample dispersed into 2080 ll of 72%-sulfuric acid
solution and water was immediately added up to 1 M-concentration followed by 2.5 h of heating at 100 °C. The final residue corresponded to cellulose + lignin, whilst the carbohydrate content of
the supernatant (Dubois et al., 1956) was constituted by non-cellulosic polysaccharides.
The third hydrolysis-procedure was performed with a new portion of each sample following the technique applied for the second
procedure, but 1 h of heating at 100 °C in a water-bath was applied
in this case. Only the supernatant was separated for quantification
as it was above indicated, and uronic acid content was determined
spectrophotometrically by the method reported by Filisetti-Cozzi
and Carpita (1991).
Methanol content was determined through saponification of
AIR with 0.5 N-NaOH for 1 h at room temperature, followed by
acidification with sulphuric acid solution needed for methanol
quantification through the spectrophotometric method of Wood
and Siddiqui (1971). Acetyl groups in the AIR of each fibre fraction
were determined according to the method of Naumenko and Phillipov (1992). Degree of methylation (DM) was calculated as the
percent ratio between moles of methanol and galacturonic acid
in a given sample. Degree of acetylation (DA) was calculated in
the same manner but with respect to the moles of total non-cellulosic carbohydrates in the sample (Fissore et al., 2007). Protein content was determined according to Lowry et al. (1951).
All determinations were performed in triplicate.
2.4.5. Cell wall phenolic contents
Total phenolics (ferulic and non-ferulic) determination was carried out on each AIR sample according to Bunzel et al. (2000). An
amount of 0.9000 g of AIR was mixed with 50.0 ml of a 1 MNaOH aqueous solution under vacuum and also protected from
light, at room temperature, during 18 h. The sample was acidified
with HCl (9.5 ml) to pH < 2 and then centrifuged for 15 min at
10,000 rpm (4 °C). The obtained supernatant was used to evaluate
total phenolics using the Folin–Ciocalteau technique reported by
Shui and Leong (2006). Galic acid (Anedra, Buenos Aires, Argentina)
was used as standard and the results were expressed as galic acid
equivalent (GAE), which means mg of galic acid per 100 g of AIR
sample (dried base).
Non-ferulic phenolic compounds were determined according to
Budini et al. (1980) and Parr et al. (1997). A sample of 0.9000 g of
AIR was mixed with 2 M-HCl solution in a 1:50 (w/v) ratio and the
system was heated for 30 min in a 95 °C water-bath. Centrifugation
was performed at 4 °C and 10,000 rpm for 15 min. The supernatant
obtained was used to evaluate phenolic (non-ferulic) content by
using the Folin–Ciocalteau technique above indicated.
2.4.6. FT-IR spectroscopy
Transmission spectra of the samples were recorded from KBr
(1% w/w) pellets in the range 4500–525 cm1 with a Nicolet
8700 FT-IR (Thermo Scientific Nicolet, MA, USA) spectrometer.
Each spectrum was obtained by recording transmittance (%)
through 64 scans performed with a resolution of 4 cm1. Spectra
were analyzed through the OMNIC software (version 7.3, Thermo
Electron Corp., USA).
2.4.7. Moisture
Moisture content was determined in a 1.0000 g sample of raw
or treated tissues as well as on their AIRs, using an IR moisture analyzer (OHAUS, Pine Brook, NJ, USA), working at 90 °C till constant
weight. This determination was performed in triplicate.
182
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
2.4.8. Sequential extraction of the cell wall polymers from AIR
It was made according to Ng and Waldron (1997) using deionized (Milli-Q™) water. AIR (0.5000 g) was suspended in deionized
water (50 ml) and stirred for 2 h at 18–20 °C. The water-insoluble
residue was further extracted in NaCl (50 ml, 0.136 M, pH 6.5) for
2 h at 18–20 °C. Residues were successively extracted in CDTA
(55 ml, 0.050 M, pH 6.8) for 5 h at 18–20 °C; Na2CO3 (20 ml,
0.050 M) for 16 h at 4 °C and Na2CO3 (20 ml, 0.050 M) for 3 h at
20 °C (Marry et al., 2006). The supernatants were centrifuged and
filtered through Whatman GF/C-filter paper. They were also dialyzed at 4 °C against 0.5 M-imidazol buffer (Mort et al., 1991) prior
to freeze-drying.
An extraction with sodium chlorite was also carried out separately from the AIRs.
The following analyses were performed in samples of the
extractive solutions obtained: uronic acids (Filisetti-Cozzi and
Carpita, 1991), total carbohydrates (Dubois et al., 1956) using
glucose for the standard curve, and methanol (Wood and Siddiqui,
1971).
2.5. Turgor
Cylindrical specimens of 20 mm-diameter of mesocarp tissue
were then respectively obtained from each slice using a cork borer.
Twelve 20 mm- and six 35 mm-diameter cylinders were equilibrated in different buffered (0.02 M KH2PO4 and 0.02 M K2HPO4)
polyethylene glycol 400 (PEG) solutions in the 0.000–1.200 M concentration range, except for one batch which was used as raw
(unsoaked) control tissue. This experience was performed in
duplicate.
2.5.1. Tissue volume measurement
Tissue volume (V) and weight (mf) of six 20 mm-diameter and
10 mm-thick cylinders were determined after 36 h of tissue equilibration in PEG-solutions at 12 °C. The volumes of the cylinders
were measured by liquid paraffin displacement in a calibratedmeasuring glass cylinder. The initial volumes (Vi) and weights
(mi) were determined on raw (unsoaked) pumpkin tissue cylinders.
The relative volume change [(V Vi)/Vi] and weight loss
[Dm = (mf mi)/mi] were then calculated considering the volume
(V) and mass (mf) corresponding to the equilibrium state with each
solution. The ratio between the weight of each PEG-solution and
the weight of tissue cylinders immersed was sufficiently high
(10:1 or more) as to maintain constant the initial value of solution
water activity along the experiment (Stadelmann, 1966). Turgor
pressure was calculated for each sample, from the change of volume experimented when equilibrated in the different PEG-solutions assayed as previously reported (Rojas et al., 2001). This
experience was performed in duplicate.
2.6. Pigment evaluation
A 20.00 g aliquot of each fresh tissue sample was crushed and
squeezed with a domestic juice extractor (Moulinex, Buenos Aires,
Argentina). The juice obtained was used for betalain spectrophotometric quantification according to Mobhammer et al. (2006) by
using UV-mini-1240UV–VIS spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Briefly, the evaluation of betacyanin (as betanin equivalents) or betaxanthin (as vulgaraxanthin equivalents)
content was performed by diluting the juice with a McIlvaine buffer (pH 6.3), until the absorbances measured at 536 and 476 nm
did not surpass a value of 1.00 (±0.05). Betacyanin content was calculated as:
BC ½mg L1 ¼
A F Mw 100
el
wherein A is the absorption value at betanin kmax (536 nm) corrected by the absorption at 600 nm, F is a dilution factor, Mw is
the betanin molecular weight (550 g mol1), e is the betanin molar
extinction coefficient (60,000 L mol1 cm1) and l is the pathlength
(1.0 cm) of the cuvette.
Betaxanthin content was calculated as:
BX ½mg L1 ¼
A F Mw 100
el
where A is the absorption value at vulgaraxanthin kmax (476 nm)
corrected by the absorption at 600 nm, Mw is the vulgaraxanthin
molecular weight (339 g mol1), e is the vulgaraxanthin molar
extinction coefficient (48,000 L mol1 cm1). Determinations were
performed in triplicate.
2.7. Colour measurement
The colour of red beet samples was assessed in triplicate by
using a colourimeter (Minolta Co. Ltd., Osaka, Japan) with illuminant D65 and 10° observer angle. Each sample was placed onto a
white tile and values of CIE colour space co-ordinates L* a* b* values were acquired, being L* the lightness, a* the grade of greenness/redness and b* the grade of blueness/yellowness. Chrome
(C*) and hue angle (h°) were calculated from a* and b* cartesian
co-ordinates by means of the following expressions:
2
C ¼ ða2 þ b Þ1=2
h ¼ arctan
b
a
C* is a measure of the chroma (saturation) and represents the
‘‘purity” of a colour, with lower chroma being less pure. The
parameter h° (0–360°) is a measure of Hue and takes values ranging from 0° to 90° for reds, oranges, and yellows; 90–180° for yellows, yellow–greens, greens; 180–270° for green–cyans (blue–
greens) and blues and from 270° to 360° for blues, purples, magentas, and returns again to reds (Barreiro et al., 1997).
2.8. Mechanical assays
Uniaxial (normal) compression and relaxation assays as well as
puncture test were performed using an Instron Testing Machine
model 3345 (Instron Corp, Norwood, Ma, USA) provided with a
5000 N-load cell and a 30 mm-diameter upper steel plate. In each
test, six 20 mm-diameter (A = area = p{diameter}2/4) and 10 mm in
height (HO) cylinders were assayed.
Puncture test was performed by using a puncher diameter of
2.5 mm and under a constant cross-head velocity of 10 mm/min.
Force–deformation curves were recorded and firmness was calculated as the ratio of force and the corresponding deformation at
failure.
In the compression assay, force–deformation curves were recorded up to 60% of sample deformation with a rate of 10 mm/
min. Force (Ff) and deformation (DH = Hf HO) at the first peak
of failure (f) were determined from the curves and the normal
stress (r, Pa = Ff/Area) and Henckýs strain (eH) were then
calculated.
eH ¼ ln
HO
H O DH
For relaxation test, specimens were compressed up to 10% of
deformation at a cross-head speed of 10 mm/min. This applied
deformation was the lowest one that allowed recording Instron
Machine response without macroscopic tissue failure, when raw
or treated red beet root cylinders were compressed. At this preset
deformation level, the crosshead was stopped and the relaxation
183
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
n
FðtÞ F 1 X
Fi
¼
þ
expðt=si Þ
FO
FO
FO
i¼1
A
800
700
600
mg / L
force [F(t)] was recorded for at least 10 min. Relaxation forces obtained were normalized dividing F(t) by the initial relaxation force
(FO). Curves of normalized forces (F(t)/FO) as a function of time (s)
were fitted to the generalized Maxwell model (Peleg and Calzada,
1976; Nussinovitch et al., 1989):
500
400
300
200
and the normalized parameters were obtained: the relative force at
infinite time of relaxation (F1/FO) which represents the free spring
of this mechanical model, the i-esim Maxwell relative-force component (F1/FO) associated to the corresponding characteristic relaxation time (si) of the i-esim Maxwell’s body.
100
0
0.0
1.0
Dose (kGy)
2.0
Betacyanins
Betaxanthins
2.9. Microstructural analyses
2.10. Statistical analysis
The results were reported as the average and standard deviation. Results were analyzed through ANOVA (a: 0.05) followed
by pair multiple comparisons evaluated by Tukeýs significant difference test, using the Statgraphic package (Statgraphic Plus for
Windows, version 5.0, 2001, Manugistic Inc., Rockville, MD, USA).
Regressions were performed by using the same software.
3. Results and discussion
3.1. Pigments and colour
Exposure of living organisms to ionizing and non-ionizing irradiation constitutes a major exogenous source of reactive oxygen
species (ROS). Specifically, c-irradiation produces a whole range
of radical and non-radical species from ionization of intracellular
water (e.g. aqueous electron, OH, H2O2). Red beet accumulates
betacyanins, mainly betanin, in the store root (Sepúlveda-Jiménez
et al., 2004). Betalains (betacyanins and betaxanthins), the main
pigments of red beet roots, protect plants from damage caused
by UV and visible light, in addition to other pigment functions
(Yoshikazu et al., 2008). Betalains are located in the vacuole of
these Caryophyllales plant cells. Escribano et al. (1998) determined
that the antiradical activity of betacyanins was greater than that
observed for betaxanthins and increased with the pH of the reaction medium.
Betacyanin and betaxanthin contents determined in red beet
root tissue after c-irradiation showed a sharp decrease at 2 kGy
(Fig. 1A). In particular, betacyanin concentration decreased with
the irradiation dose and significantly (p 6 0.05), in a 35%, after
2.0 kGy of c-irradiation, whereas betaxanthin concentration increased after 1 kGy but decreased after 2 kGy, both significantly
(p 6 0.05) in an 11 and 19%-ratio with respect to control. These
B
70
60
Foo / FO (%)
Samples of about 4 mm3 were cut from raw or irradiated beet
root tissue cylinders. They were fixed in 2.5% w/w-glutaraldehyde
(Sigma, Saint Louis, Missouri) buffered by 0.2 M-phosphate (pH
7.2) solution and postfixed in 1.5% w/w OsO4 (Sigma, Saint Louis,
Missouri) into the same buffer solution, followed by fixation and
contrast in 5% w/w-uranyl acetate in distilled water. Dehydration
was performed in a graded acetone buffer series prior to embedding in Durcupan (epoxi) resin. Ultrathin sections of 0.5 microns
were first performed in a Reichert Jung Ultracut E (Leica, Germany)
microtome and stained with toluidine blue for observation in the
optical microscopy (Axioplan, Japan). After this work, ultrathin sections of 70–90 nm were obtained from the uranyl acetate stained
pieces and examined in an EM 10 C Zeiss transmission electron
microscope (Karl Zeiss, Berlin, Germany).
50
y = -1.069x + 91.951
R 2 = 0.999
40
30
20
A
10
0
20
30
40
50
F 1 / F O (%)
Fig. 1. (A) Betacyanin and betaxanthin concentrations, expressed as mg per litre of
red beet juice. (B) Correlation between the adimensional residual relaxation force
(F1/FO) and the Maxwell adimensionalized force component F1/FO associated to the
higher characteristic relaxation time (s1) determined at each irradiation condition
of red beet root tissue.
pigments seemed to overcome an irradiation dose of 1 kGy as
can be observed from considering the total pigment concentration.
Moreover, antioxidant cellular barriers led to a positive balance of
betaxanthin synthesis in this case, whereas ROS (e.g. H2O2, OH)
production as a consequence of 2 kGy-irradiation level consumed
significantly both betacyanin and betaxanthin, decreasing their ratio significantly from 1.4 (control system) to 1.1. Hence, betacyanin
seemed to be more sensible to c-irradiation, which may be associated to its higher capacity to withdraw one electron as well as from
the higher stability (and life-time) of the corresponding generated
radical, as indicated by Escribano et al. (1998).
Although pigments showed changes with irradiation dose, no
significant changes (p > 0.05) in colour could be observed between
samples, from comparison of all parameters evaluated (Table 1).
Hue angle (h°) always showed values in the red area of the
spectrum.
3.2. Mechanical behaviour and turgor characteristics
As can be observed in Table 2, irradiation increased the compressive stress (r) at the bioyield point but did not modify Hencky
strain (eH). Relaxation data obtained at constant compressive
deformation from non-fractured tissue samples showed the
change in the adimensional relaxation force (F(t)/FO) with time
for the different treatments; these data fitted to a mechanical model constituted by two Maxwell elements and a free spring (F1/FO)
(Table 3). The adimensional residual relaxation force (F1/FO) increased significantly after tissue irradiation, whereas the Maxwell
force component F1/FO associated to the higher characteristic
184
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
Table 1
Colour parametersA determined on red beet tissue samples.
Irradiation dose (kGy)
Storage time (d)
L*
a*
b*
C*
h°
0
1
2
0
0
0
10 ± 3a
8 ± 3a
9.9 ± 0.9a
40 ± 4a
38 ± 4a
40 ± 1a
17 ± 5a
15 ± 4a
17 ± 2a
43 ± 5a
41 ± 5a
43 ± 2a
22 ± 7a
21 ± 5a
23 ± 5a
L* a* b* CIE colour space parameters; C* chrome; h° hue angle.
A
Mean and standard deviation are shown (n = 10). Different letters in the same column indicate significant differences (p 6 0.05).
4
Doses (kGy)
Compressive stress 10
0
1
2
9.2 ± 0.5a
11 ± 1b
11 ± 1b
(r; Pa)
Hencky strain (eH)
0.23 ± 0.05a
0.22 ± 0.07a
0.24 ± 0.07a
A
The mean and standard deviation are shown (n = 6). Different letters in the
same row indicate significant differences (p 6 0.05).
Table 3
Maxwell parametersA determined from a relaxation test applied on untreated (0 kGy)
and irradiated (1 and 2 kGy) red beet root tissues. The change in the adimensional
relaxation forceB with time was calculated from the experimental data obtained at a
constant deformation (10%) selected from the elastic period of the initial compression
curves performed at a constant cross-head speed of 10 mm/min.
Doses (kGy)
F1/FO (%)
s1 (s)
F2/FO (%)
s2 (s)
F1/FO (%)
0
1
2
48 ± 6a
36 ± 11b
26 ± 9c
692 ± 182a
730 ± 247a
606 ± 224a
10 ± 2ab
9 ± 1a
9 ± 2a
22 ± 5ab
28 ± 4ac
28 ± 7ac
41 ± 8a
54 ± 11b
64 ± 11bc
si: characteristic relaxation time.
A
The mean and standard deviation are shown (n = 6). Different letters in the
same row indicate significant differences (p 6 0.05).
B
Adimensional relaxation force = ratio of the relaxation force recorded along
time [F(t)] to the initial force of relaxation (FO).
C
Adimensional residual relaxation force (F1/FO).
relaxation time (s1) significantly decreased as irradiation dose rose
(Table 3). This indicates the inverse relationship between the loss
of the mechanical energy by flow and the irradiation dose, fact that
is also coherent with the higher adimensional residual relaxation
force (F1/FO). The adimensional residual (F1/FO) and the first Maxwell F1/FO relaxation forces were inversely related and in a similar
ratio, and this can be observed in Fig. 1B. These relaxation results
can be associated to the rise in tissue elasticity with c-irradiation
dose probably due to an increase of cross-link density (Mc Evoy
et al., 1985; Grassi et al., 1996; Narine and Marangoni, 1999).
Since the rheology of the tissue is dependent upon the mechanics of the individual cell, and assuming that sample volume and cell
volume are proportional, the cell wall stress–strain relationship
derived from the change in tissue turgor pressure permitted us
to quantify the cell wall strength (Fig. 2), as indicated by Lin and
Pitt (1986). Control beet root tissue showed non-significant stiffness of cell wall at small stretch ratios, followed by a sharp increase from k 1.30 (30% strain) up to 1.33 (33% strain). A
decrease in strength was finally observed up tok 1.40 (40%
strain), which indicated the ability of the cell wall to deform under
hypotonic conditions, permitting the highest water uptake. This
event may be evaluating the performance of the pectin matrix in
allowing the viscous drag (deformability) of the cellulose–xyloglucan microfibrilar network, responsible for the elastic contribution
to the cell wall mechanical response (Rojas et al., 2002). Although
irradiated tissues also presented low stiffness of cell wall at small
stretch ratios, it was always higher than the values obtained from
0.600
0.500
Cell wall stress (MPa)
Table 2
ParametersA calculated from the force–deformation curves obtained by compression
of untreated (0) and irradiated (1 and 2 kGy) red beet root tissues up to 60% of
deformation, under a constant cross-head velocity of 10 mm/min.
Control tissue
0.400
0.300
TP =
0.1649 MPa
TP =
0.2286 MPa
0.200
Isotonicity
Isotonicity
0.100
1 kGy
TP =
0.1221 MPa
2 kGy
0.000
-0.100
1.00
B
1.10
1.20
1.30
1.40
Cell wall stretch ratio (λ)
1.50
Fig. 2. Relationship between the cell wall stress and cell wall stretch ratio
determined by osmotic stressing of each living red beet root tissue after irradiation
treatment. Turgor pressure calculated for each tissue is also shown, and the position
of each isotonic condition, indicated on the plot.
cell wall of control tissue (Fig. 2). Cell wall strength of irradiated
tissues increased from k 1.12 (12% strain) for 1 kGy and
k 1.14 (14% strain) for 2 kGy, with higher cell wall stiffness for
1 kGy-irradiated tissue up to k 1.19 (19% strain). After this, cell
wall stiffness of all irradiated tissues increased sharply since cell
wall stress went up to 0.46 MPa but accompanied by a non-significant change in the 19%-strain (k 1.19). Pitt and Chen (1983) observed that higher turgor pressures decreased the failure strain of
apple tissue but failure stress was unaffected.
All the above described mechanical assays allowed evaluating
the beet root tissue structurally. On the other hand, puncture test
was also performed with a punch whose diameter was lower than
that of the beet root tissue cylinder. Puncture test showed that 2kGy irradiated samples showed a lower ratio of puncture force to
deformation (firmness, resistance to puncture) with respect to control (Table 4). According to results obtained from puncture test,
probably, consumers will not notice differences between tissue
treatments, as puncture test, in general, did not reveal significant
differences (p > 0.05) for 1 kGy. It must be remembered that puncture force is not only evaluating the purely compressive (normal)
component but also the shear and cutting ones, as occurs in the
case of a bite (Bourne, 2002).
Table 4
Firmness at failure calculated from force–deformation curves obtained by puncture of
untreated (0 kGy) and irradiated (1 and 2 kGy) red beet root tissues with a punch
diameter of 2.5 mm and under a constant cross-head velocity of 10 mm/min.
Doses (kGy)
Firmness 103 (N/m)
0
1
2
6.9 ± 0.7ab
7 ± 1a
6 ± 1b
Different letters in the same row indicate significant differences (p 6 0.05).
Mean and standard deviation are shown (n = 6).
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
3.3. Microscopy study
As can be seen in Fig. 3A, optical microscopy images of living
red beet root cells cut at different levels show a continuum of thick
cell walls surrounding uniformly stained cellular contents, which
can then be ascribed to the tonoplast vacuole responsible for the
cell wall pressure and, hence, of the turgor pressure development.
The middle lamellae can be observed without separation along the
cell wall continuum. A similar microstructure can be seen in the
case of 1 kGy-irradiated tissue (Fig. 3B), with thickening of the
middle lamellae at the tricellular junction corners. The cell wall
continuum is also observed in 2 kGy-irradiated red beet root
(Fig. 3C). However, separation of the middle lamellae can be very
frequently seen at the corners, which may be associated to intercellular gas space opening (Roland, 1978), probably due to higher
metabolic activity. Cell separation forces are reduced by the introduction of intercellular spaces and decrease further as these expand (Jarvis, 1998). When adhesion breaks down between cells
in the living vegetables, it does so under strict developmental control or controlled enzymatic cell separation at specific tricellular
185
junctions. Where a tricellular junction has opened to make way
for an intercellular space, reinforcing zones with pectic polysaccharides structurally similar to those at the original tricellular junctions are present along the three cell–cell-space junctions where
the walls of adjacent cells diverge. In this way, higher plants have
evolved precisely defined and precisely localized polymer systems
to prevent cell separation as a consequence of the turgor pressure,
event which forces plant cells towards separation at the angles
from adjacent cells (Jarvis, 1998).
TEM analysis revealed the integrity of the plasmalemma on the
edges of the cell walls (Figs. 3D–F), where some organelles were
pushed along by the intracellular tonoplast. Cell walls of raw tissue
(Fig. 3D) as well as of 1 kGy- (Fig. 3E) and 2 kGy- (Fig. 3F) irradiated
root show slight electronic density of longitudinal and parallel fibres at both sides of the darker middle lamellae. Plasmodesmata
areas were frequently observed in 2 kGy-irradiated tissues
(Fig. 3G) with the stain intensity that characterized cytoplasmic
bridges and surrounded by the intracellular smooth endoplasmic
reticulum. These areas constitute reinforcement points of cell–cell
adhesion (Hallett et al., 1992).
Fig. 3. Optical images taken from unstored control (0 kGy) (A) and irradiated (B: 1 kGy, C: 2 kGy) red beet root living tissues. Arrows indicate either middle lamellae (ML) or
gas spaces (GS). TEM images got from unstored control (0 kGy) (D) and irradiated (E: 1 kGy; F–G: 2 kGy) red beet root living tissues. (E) image-arrows indicate the cell content
pushed against the cell membrane along the edges of the cell wall (CW) of each neighbor cell. M: mitochondria; SER: smooth endoplasmic reticulum. (G) Image shows a
plasmodesmata area between contiguous cells and arrows indicate some cytoplasmic bridges. Bar = 10 lm (A–C); bar = 1 lm (D–G).
186
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
3.4. FT-IR
3.5. Cell wall composition
The FTIR analysis of AIRs obtained from control and irradiated
tissues (Fig. 4A) did not reveal differences coming from appearance
of any different cross-link beyond the presence of feruloylated pectins into the cell wall materials, as determined through the band
observed at 1520 cm1, which is absent in citrus pectin also
showed for comparison in Fig. 4B (Synytsya et al., 2003). Among
1020–1170 cm1, cellulose masked the typical pectin bands, like
the characteristic peaks of the polygalacturonic backbone in the
fingerprint zone (1200 to 900–850 cm1) of pectins at 1020 and
1105 cm1 (McCann et al., 1992). Typical cellulose peaks at 1062
and 1110 cm1 corresponding to the asymmetric stretching of
the glycosidic linkages, as well as the shoulder at 985 cm1 can
be seen in its fingerprint zone. The cellulose contribution can also
be observed at 1390 cm1, which differed from citrus pectin
(Fig. 4B). The water adsorbed in the cell wall pectins led to a sharp
peak at 1650 cm1 (Wilson et al., 2000), which masked the broad
band usually manifested at 1615–1650 cm1 by the carbonyl
stretching of the carboxylic (non-esterified) group of galacturonic
and glucuronic, in the case of red beet (Strasser and Amado,
2001) residues of pectins. The band in the zone attributed to the
characteristic C@O stretching of the esterified carboxyl group
was observed at 1740 cm1, as a single major band. The broad
OH-stretch band at a frequency of 3430 cm1, reported as characteristic of polysaccharides from cell walls (Coimbra et al., 1999),
was seen as well as the broad shorter band at 2940 cm1, which
corresponds to the OH-stretching in the carboxylic group, both also
present in pure citrus pectin (Fig. 4B).
The lignin content showed a higher value for the AIR (cell wall
polymers) of 1 kGy tissue (Table 5). This trend was also observed
for total cell wall phenolic content (Fig. 5A) but in the case of lignin, differences between treatments were non-significant
(p > 0.05). In Pinus taeda cell suspension cultures, lignin synthesis
was correlated with the generation of the oxidizing co-substrate
H2O2, as reported by Oudgenoeg et al. (2002). It is suggested in
our work that monolignol and lignin radicals can undergo b-O-4
coupling to form quinone methide intermediates, which are stabilized by the addition of nucleophiles such as water, uronic acids or
neutral sugars, to form structures substituted with a-hydroxyl
groups or cross-linked to the cell wall polysaccharides and proteins by benzyl a-ester linkages, as indicated by Grabber (2005).
Water addition is coherent with initial stages of lignification,
where a hydrophilic medium exists in the cell wall polysaccharide
network, as occurs in the present work with irradiated red beet
root tissue (Table 5). Radical scavenging efficiency of lignins has
also been recognized and some structure–activity relationships
proposed (Pouteau et al., 2003; Dizhbite et al., 2004). The connection of the antibacterial effect of kraft lignin with radical scavenging activity of its soluble fraction was also mentioned by Dizhbite
et al. (2004).
Cellulose tended to rise after irradiation (Table 5) but the difference resulted non-significant (p > 0.05). De Lara et al. (2006) determined that structural/morphological changes such as cross-linking
of polymer chains, seemed to justify the effect of c-irradiation on
cellulose membrane permeability to electrolytes like NaCl.
A
1062
1020
985
3430
1110
1650
co ntro l A IR
Absorbance
991
1520
2940
1740
835
1kGy-A IR
co ntro l A IR
550
800
1050
1300
1550
1800
2050
2300
2550
2800
3050
3300
3550
3800
-1
wavelength (cm )
1012
B
998
1050
1102
Absorbance
967
835
1740
3330
1520
1650
2945
pectin
550
800
1050 1300
1550
1800
2050 2300
2550
2800
3050 3300
3550
3800
-1
wavelength (cm )
Fig. 4. (A) FTIR spectra obtained from the alcohol-insoluble residues (AIR) or cell wall materials extracted from not irradiated (control) and 1 kGy or 2 kGy-irradiated red beet
root tissue. (B) The spectrum of citrus pectin is also shown for comparison purposes.
187
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
Table 5
Chemical compositionA of the alcohol-insoluble residue (AIR) or cell wall material extracted from non-irradiated (control) and 1 or 2 kGy c-irradiated red beet root tissues.
Yield of AIR (g dry AIR/100 g red beet tissue)
Moisture content of AIR (% db)
Lignin of AIR (% db)
Cellulose of AIR (% db)
Non-cellulosic carbohydrates of AIR (% db)
Uronic acid of AIR (% db)
Degree of methylation (DM) of AIR pectins
Degree of acetylation (DA) of AIR pectins
Protein3 (g BSA / 100 g AIR, db)
Hydroxyproline (mg/100 g protein of AIR, db)
Tyrosine (mg/100 g protein of AIR, db)
Carbohydrate content of WSF (% db)
UA content of WSF (% db)
DM of pectins in the WSF (% db)
Carbohydrate content of SSF (% db)
UA content of SSF (% db)
0.0 kGy (control sample)
1.0 kGy
2.0 kGy
3.9 ± 0.8
3.5 ± 0.3a
5.9 ± 0.9a
26 ± 2a
52 ± 4a
14 ± 1a
79 ± 2a
74 ± 3a
9.0 ± 0.6a,b
5.467a
4.449a
100.0 ± 0.9a
11.3 ± 0.6a
63 ± 2ab
102 ± 2a
16.5 ± 0.1a
3.5 ± 0.9
3.4 ± 0.2a
7.6 ± 1.5a
31 ± 6a
53 ± 2a
14.1 ± 0.1a
86.2 ± 0.6b
71 ± 1a
9.8 ± 0.3b
4.325b
4.014a
105 ± 3a
13.6 ± 0.2b
67 ± 3a
98 ± 3a
19.7 ± 0.3b
4.0 ± 0.8
2.5 ± 0.1b
6.5 ± 1.5a
32 ± 7a
56 ± 1a
13 ± 1a
80.4 ± 0.9a
72 ± 1a
9.6 ± 0.4a
4.621c
4.878b
103 ± 5a
12.8 ± 0.6 ab
60 ± 1b
101 ± 7a
27.6 ± 0.7c
WSF: water soluble fraction of AIR; SSF: salt soluble fraction of AIR.
UA: uronic acid content.
db: dry bases.
A
Mean and standard deviation are shown (n = 3). Different letters in the same row indicate significant differences (p 6 0.05).
g GA / 100 g AIR (db)
A
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Phen
Phen
Non
Fer
Non
Fer
Non
Fer
Fer
Fer
0.0
Phen
Fer
1.0
2.0
Dose (kGy)
18
1.70
16
1.60
POX
14
1.50
12
10
1.40
PPO
8
1.30
6
1.20
4
PAL
2
0
0.0
1.0
B
1.10
ΔAbs (PPO) / min mg protein
Abs(POX) / min mg protein
[ Abs(PAL) / min mg protein]x10 3
B
1.00
2.0
Dose (kGy)
Fig. 5. (A) Total phenolics (Phen), non-ferulic and ferulic acid contents, expressed
as grams of galic acid (GA) per 100 g of alcohol-insoluble residue (AIR) or cell wall,
both of them on dry bases. (B) peroxidase (POX), poliphenol oxidase or tyrosinase
(PPO) and phenyl-alanine ammonia-lyase (PAL) activity against gamma irradiation
absorbed doses for red beet root tissue.
The uronic acid content of AIRs as well as the high degree of
pectin acetylation (DA 73%), typical of beet root (Dongowsky,
2001), did not change after irradiation, but the high degree of
methylation (DM) increased significantly (p 6 0.05) for 1 kGy-cell
wall pectins (Table 5). The moisture content of the AIR showed a
significant decrease (p 6 0.05) for an absorbed dose of 2 kGy. Rastogi (2005) observed that water effective diffusivity along dehydration was higher for pre-irradiated carrots, explaining this based on
rupture of cellular walls. In our case, it is not coherent to associate
the AIR-moisture diminishing observed at 2 kGy with rupture of
cell walls but with lower water retention or polysaccharide swelling derived from crosslinking of the cell wall polymers.
Protein content in the cell wall or AIR fraction showed non-significant differences (p > 0.05) between control and irradiated tissues (Table 5) but varied significantly between doses of 1 and
2 kGy, being higher for 1 kGy. Hydroxyl radical generated by cradiolysis of cell water is considered the most reactive radical in
biological systems. By the OH attack on proteins most closely surrounding it, a series of compounds can be formed, including
hydroxyproline, glutamyl semialdehyde, and others. However, a
trend to lower hydroxyproline content was found in the cell wall
proteins isolated from irradiated beet root tissues, though it was
higher for 2 kGy than for 1 kGy-tissue and the highest tyrosine
content was found in the AIR-proteins of 2 kGy-irradiated tissue
(Table 5). The extensins, the most studied hydroxyproline-rich glycoprotein (HRGPs) group, are secreted into the apoplast as soluble
monomers where the positively charged lysine and protonated histidine residues are thought to interact ionically with the negatively
charged uronic acids of pectins (Fry, 1986). Ribeiro et al. (2006)
confirmed that the H2O2-mediated increase in wall resistance to
digestion was accompanied by the formation of an insoluble extensin-like network in grapevine callus cell walls. Bradley et al. (1992)
reported that extensin cross-linking begins a few minutes after
vegetable stress. A sequential extraction of cell wall polymers
was carried out from the AIR residues obtained from control and
irradiated red beet tissues, beginning with water at 18 °C (Brett
and Waldron, 1996). The proportion of water soluble fractions extracted from AIRs did not show variations with radiation dose
(Fig. 6); they were essentially constituted by carbohydrates
(100%), having a lower proportion (13%) of highly methylated
(63%) uronic acids (Table 5). It is known that water extracts pectins loosely attached to the cell walls (Fry, 1986). The proportion of
ionically related extensin-pectin polymers extracted by the subsequent 0.136 M-NaCl aqueous solution (Fry, 1986; Brett and Waldron, 1996) significantly (p 6 0.05) decreased as radiation dose
increased, mainly in 2 kGy-AIR (Fig. 6). Cell wall polymers extracted by the 0.136 M-NaCl aqueous solution were essentially
constituted by carbohydrates (100%) with a lower proportion of
uronic acids, where the latter increased significantly with the radiation doses (Table 5).
Though a non-significant difference (p > 0.05) was observed in
water-extractability of the cell wall polymers from AIRs of control
188
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
Solubilized AIR (%, w/w)
40
Water
NaCl
CDTA
CO3 (4 ºC)
Na Chlorite
CO3 (20 ºC)
30
20
10
0
0 1 2
0 1 2
0 1 2
0 1 2
0 1 2
0 1 2
Doses (kGy)
Fig. 6. Percent of alcohol-insoluble residue (AIR) or cell wall material solubilized
alter each sequential extraction with water, 0.136 M-NaCl, CDTA and Na2CO3 (CO3)
against gamma irradiation dose absorbed by red beet root tissue. Results for
treatment with NaClO (Na Chlorite) are also shown.
or irradiated tissues, solubilization of Ca-cross-linked pectins in
CDTA increased significantly (p 6 0.05) with the irradiation dose
(Fig. 6). Hence, irradiation seemed also to contribute to higher
cell–cell adhesion through increasing calcium-cross linking at the
middle lamellae region as well (Vincken et al., 2003). Marry et al.
(2006) observed that not only calcium-chelated homogalacturonan-pectin regions were important in cell–cell adhesion of sugar
beet tissue but also feruloyl ester (covalent) bridges in the arabinogalactan side chains, which are usually removed through successive Na2CO3 treatments at 4° and 20 °C. It is recognized that the
special mechanical strength that characterizes beet root tissue
which survives to cooking in boiling water, is associated to the diferuloylated pectins above mentioned, occurring in the middle
lamellae and cell corners between neighboring cells (Waldron
et al., 1997a,b; Marry et al., 2006).The following extractions performed with Na2CO3 aqueous solution at 4° and 20 °C, only showed
a significant lower percent of polysaccharide extractability from
2 kGy-irradiated residue of cell walls (Fig. 6). The cell wall polysaccharides sequentially solubilized in CDTA and Na2CO3 (4 °C – 16 h;
20 °C – 2 h) were only constituted by uronic acids, as chemically
determined.
On the other hand, a parallel extraction of AIR samples with sodium chlorite produced a significant lower percent of extraction
from the AIR obtained from 1 kGy-irradiated tissue (Fig. 6).
Total cell wall phenolics as well as the non-ferulic component of
them increased in concentration after an absorbed radiation dose
of 1 kGy whereas decreased after 2 kGy with respect to control system, being the latter change only significant (p 6 0.05) in the case
of total cell wall phenolics (Fig. 5A). Ferulic acid concentration,
which was calculated as the mathematical difference of the previously mentioned phenolic fractions, showed a similar trend but the
difference was only significant (p 6 0.05) between samples irradiated at 1 and 2 kGy.
3.6. Enzyme activity
3.6.1. POX
The increase in H2O2 like that occurred after c-irradiation can
be controlled in plants by the peroxidases (POX), through its
hydroxylic and standard peroxidative cycles. POX can be considered as bifunctional enzymes that can oxidize various substrates
in the presence of H2O2 but also produce ROS like OH, which is
implicated in the scission of polysaccharides such as pectin and
xyloglucans of the cell wall, to accomplish the natural process of
cell elongation (Fry, 1998).
In our study, POX activity increased significantly (p 6 0.05) with
the c-irradiation dose as can be observed in Fig. 5B. This trend can
be associated to the higher stiffness and to the rise in tissue elasticity after irradiation. This rise is probably due to higher cross-link
density which can be, in part, ascribed to the insolubilization of the
extensin-pectin network in the cell wall as well as to the higher
calcium cross-linked pectins observed through sequential extraction (Fig. 6) in the cell wall-middle lamella, as a consequence of
higher POX-activity in response to increasing H2O2 production.
All this gave origin to a matrix which did not permit an extreme
deformability for maximal water uptake at hypotonic condition
and/or to higher turgor pressures in irradiated tissues. The formation of a less transient structure which, probably, involves covalent
bonds derived from oxidative processes mediated by H2O2-POX
activity (Fry, 1986) determined the higher elasticity observed for
irradiated tissues (Table 2).
Also, the increase in POX might have influenced the changes in
cell wall phenolics.
This higher POX activity could have also promoted the polymerization of lignin precursors (phenolics) in the cell wall of the 1 kGyirradiated beet tissues. Class III POX can oxidize a variety of physiological substrates (e.g. ferulic acid) demonstrating little substrate
specificity (Price et al., 2003). Ferulic acid is often found esterified
to plant cell wall polysaccharides and it is a good substrate for
most plant POX. These POX are hence the major players in the formation of diferulic bonds, which occurs after oxidation leading to
the free ferulic radical. Dehydrodimers of ferulic acid stemming
from radical combination have been identified in plant cell walls
(Oudgenoeg et al., 2002), particularly, in sugar beet roots (Levigne
et al., 2004) and red beet (Strasser and Amado, 2001). Baydoun
et al. (2004) observed a net loss of around 50% of the phenolic
groups (monomers plus dimers) during in vitro dimerisation,
though this ratio can vary in vivo due to surrounding macromolecules into the cell wall network. Actually, phenolic synthesis can
be increased in vivo as a consequence of the enzymatic pathway
stimulation as a response to the increased requirements for
cross-linking of cell wall biopolymers. However, in the present research, the low PAL activity detected did not change significantly
(p > 0.05) with the absorbed dose (Fig. 5B). The variation in the
ferulic content (Fig. 5A) of red beet root with the absorbed dose,
may be ascribed to the fact that each dose probably determined
variable H2O2 level, controlled in different ways by the increasing
POX activity.
3.6.2. PPO
Concerning PPO, it can be observed in Fig. 5B that there was no
change in PPO activity with 1 kGy irradiation while it increased
significantly (p 6 0.05) for 2 kGy.
Formation of betanidin (violet) from dopaxanthin-quinone (yellow) would make PPO (tyrosinase) the decisive enzyme responsible for the change of colour from yellow to violet; PPO also
catalyzes reactions involved in tyrosine-betaxanthin to betanidin
conversion (Gandia-Herrero et al., 2005).Therefore, betaxanthin
content rise while betacyanin content felt after 1 kGy of c-irradiation, can be ascribed to the absence of an increment in the PPO
activity at this irradiation dose (Fig. 5B).
The betacyanin decrease after 2 kGy-irradiation coincided with
the significant increment (p 6 0.05) in the PPO activity observed
for this absorbed dose as well as with the highest POX activity
(Fig. 5B). It is suggested that higher PPO levels may be, in part, ascribed to the increased necessity for tyrosinase activity in order to
compensate with higher synthesis the increased consumption of
betacyanin and betaxanthin, by the generated OH and other ROS
compounds. The non-significant (p > 0.05) change in colour parameters observed for this tissue due to irradiation (Table 1) may also
confirm that the increment in PPO activity developed at 2 kGy
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
(Fig. 5B) was not mainly deviated to melanin biosynthesis and
enzymatic browning in red beet tissue herein assayed, but to betacyanin–betaxanthin synthesis. It has also to be mentioned that the
proposed detention of the oxidation process (Gandia-Herrero et al.,
2005) mediated by tyrosinase in the biosynthetic scheme of betalamic acid formation, implies the natural existence of a reducing
agent in the raw (living) tissue, like L-(+)-ascorbic acid.
4. Conclusions
Betacyanin and betaxanthin contents determined in red beet
root tissue after c-irradiation showed a sharp decrease at 2 kGy.
These pigments seemed to overcome an irradiation dose of 1 kGy
as can be concluded from the null change of total pigment concentration. In general, a non-significant change was observed with reference to colour parameters for the studied tissue and values were
always in the red area of the spectrum.
POX activity increased significantly with the c-irradiation dose
and PPO activity increased only for a radiation dose of 2 kGy. Total
cell wall phenolics as well as the non-ferulic component of them
increased in concentration after an absorbed radiation dose of
1 kGy whereas decreased after 2 kGy with respect to control system, being the latter change only significant in the case of total cell
wall phenolics.
Uronic acid content as well as its high DA (73%), typical of pectins from beet root, did not change after irradiation. However, the
DM (80%) increased slightly though significantly (86%) for
1 kGy-cell wall pectins, whereas lower moisture content was found
in the AIR of 2 kGy-irradiated tissue and this can be attributed to
higher polysaccharide cross-linking determining a change in swelling capacity of pectin network after irradiation, associated with
lower water retention.
Sequential extraction of cell wall polysaccharides showed that
irradiation seemed to contribute to higher cell–cell adhesion
through increasing of calcium-cross linking at the middle lamellae regions, in addition to an increment of cross-link of polymers
into the cell wall. Chemical modifications produced in the cell
walls as a response to higher levels of H2O2 and subsequent
POX mediated effects, were visualized structurally as a more
elastic behaviour of irradiated tissues and rigidification of cell
walls of treated roots. Anyhow, puncture test did not reveal, in
general, significant differences for 1 kGy. Microscopy showed
that irradiation at 1 kGy seemed to develop some thickening of
the middle lamellae at the tricellular junction corners, whereas
intercellular gas space opening was observed after irradiation
at 2 kGy, as a bio-chemical response to this level of irradiation
stress.
It can be concluded that irradiation doses of 1 or 2 kGy produced bio-chemical changes in cellular contents as well as in the
cell wall constitutive networks which not necessarily could be
sensed by consumers as objectively evaluated through puncture
and colour tests. At the same time, the mentioned changes involved an increase in the antioxidant capacity of red beet root tissue, showing that studied doses could be interesting to be used in
the frame of a combined technique for red beet processing with the
purpose of shelf-life prolongation. Anyhow, for the implementation of irradiation as a hurdle barrier, more studies must be performed in order to evaluate the influence of sample shape on
results obtained as well as the incidence of this technique on final
product cost.
Acknowledgements
The authors acknowledge the financial support from University
of Buenos Aires, National Agency of Scientific and Technological
189
Promotion of Argentina (ANPCyT) and National Research Council
of Argentina (CONICET).
References
Ahn, H., Kim, J., Yook, H., Byun, M., 2003. Irradiation effects on free radical
scavenging and antioxidative activity of phytic acid. J. Food Sci. 68, 2221–2224.
AC, A.O., 2000. Official methods of analysis, (17th ed.). Method 994.12. Association of
Official Analytical Chemists, MD.
Aquino-Bolaños, E.N., Mercado-Silva, E., 2004. Effects of polyphenol oxidase and
peroxidase activity on browing of cut jicama. Postharvest Biology and
Technology 33, 275–283.
Barreiro, J.A., Milano, M., Sandoval, A.J., 1997. Kinetics of colour change of double
concentrated tomato paste during thermal treatment. J. Food Eng. 33, 359–371.
Baydoun, E.A.H., Pavlencheva, N., Cumming, C.M., Waldron, K.W., Brett, C.T., 2004.
Control of dehydrodiferulate cross-linking in pectins from sugar-beet tissues.
Phytochemistry 65 (8), 1107–1115.
Bourne, M., 2002. Food texture and viscosity. Concept and Measurement. Second
Edition, Chapter 4. Food Science and Technology. International Series. Academic
Press Publisher, New York. pp. 113–126.
Bradley, D.J., Kjellbom, P., Lamb, C.J., 1992. Elicitor- and wound-induced oxidative
cross-linking of a proline-rich plant cell wall protein: A novel, rapid defense
response. Cell 70, 21–30.
Brett, C., Waldron, K., 1996. Physiology and Biochemistry of Plant Cell Wall.
Chapman and Hall.
Budini, R., Tonelli, D., Girotti, S., 1236. Analysis of total phenols using Prussian blue
method. J. Agric. Food Chem. 26 (6), 1236–1238.
Bunzel, M., Ralph, J., Marita, J., Stainhart, H., 2000. Identification of 4-O-50 coupled
diferulic acid from insoluble cereal fiber. J. Agric. Food Chem. 48, 3166–3169.
Cantos, E., Espin, J.C., Tomás-Barberán, F.A., 2001. Effect of wounding of phenolic
enzymes in six minimally processed lettuce cultivars upon storage. J. Agric.
Food Chem. 49, 322–330.
Coimbra, M.A., Barros, A., Barros, M., Rutledge, D.N., Delgadillo, I., 1999. FTIR
spectroscopy as a tool for the analysis of olive pulp cell-wall polysaccharide
extracts. Carbohydr. Res. 317, 145–154.
Coseteng, M.Y., Lee, C.Y., 1987. Changes in apple polyphenol concentrations in
relation to degree of browning. J. Food Sci. 52 (4), 985–989.
de Lara, R., Vazquez, M.I., Galan, P., Benavente, J., 2006. Modification of cellulose
based membranes by gamma-radiation: effect of cellulose content. J. Membr.
Sci. 273 (1–2), 25–30.
Detopoulou, P., Panagiotakos, D.B., Antonopoulou, S., Pitsavos, C., Stefanadis, C.,
2008. Dietary choline and betaine intakes in relation to concentrations of
inflammatory markers in healthy adults: the ATTICA study. Am. J. Clin. Nutr. 87
(2), 424–430.
Dizhbite, T., Telysheva, G., Jurkjane, V., Viesturs, U., 2004. Characterization of the
radical scavenging activity of lignins-natural antioxidants. Bioresour. Technol.
95 (3), 309–317.
Dongowski, G., 2001. Enzymatic degradation studies of pectin and cellulose from
red beets. Mol. Nutrit. Food Res 45 (5), 324–331.
Duan, X., Su, X., You, Y., Qu, H., Li, Y., Jiang, Y., 2007. Effect of nitric oxide on pericarp
browning of harvested longan fruit in relation to phenolic metabolism. Food
Chem. 104 (2), 571–576.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric
method for determination of sugars and related substances. Anal. Chem. 28,
350–356.
Egley, E., Paul Jr., R.N., Vaughn, K.C., Duke, S.O., 1983. Role of peroxidase in the
development of water-impermeable seed coats in Sida spinosa L.. Plant 157 (3),
157–224.
Escribano, J., Pedreño, M.A., García-Carmona, F., Muñoz, R., 1998. Characterization
of the antiradical activity of betalains from Beta vulgaris L.. Roots. Phytochem.
Anal. 9 (3), 124–127.
Fan, X., Sokorai, K.J.B., 2008. Retention of quality and nutritional value of 13 freshcut vegetables treated with low-dose radiation. J. Food Sci. 73 (7), S367–S372.
Farkas, J., 2006. Irradiation for better foods. Trends Food Sci. Technol. 17, 148–152.
Faust, M., Chase, B.R., Massey Jr., L.M., 1967. The effect of ionizing radiation and
diphenylamine treatment on glucose metabolism and membrane permeability
of ‘‘Cortland’ apples. Proc. Am. Hort. Sci. 90, 25–32.
FDA, 2007. Irradiation in the production, processing and handling of food. Proposed
rules. Fed. Reg. 72 (64), 16291–16306. <http://www.cfsan.fda.gov/lrd/
fr070404.html>.
Filisetti-Cozzi, T.M.C.C., Carpita, N.C., 1991. Measurement of uronic acids without
interference from neutral sugars. Anal. Biochem. 197, 157–162.
Fissore, E.N., Ponce, N.M.A., Stortz, C.A., Rojas, A.M., Gerschenson, L.N., 2007.
Characterization of fiber obtained from pumpkin (Cucumis moschata, Duch.)
mesocarp through enzymatic treatment. Food Sci. Technol. Int. 16 (1), 1–7.
Foley, D., Euper, M., Caporaso, F., Prakash, A., 2004. Irradiation and chlorination
effectively reduces Escherichia coli O157:H7 inoculated on cilantro (Coriandrum
sativum) without negatively affecting quality. J. Food Prot. 67 (10), 2092–2098.
Fry, S.C., 1986. Cross-linking of matrix polymers in the growing cell walls of
angiosperms. Annu. Rev. Plant Physiol. 37, 165–186.
Fry, S.C., 1998. Oxidative scission of plant cell wall polysaccharides by ascorbateinduced hydroxyl radicals. Biochem. J. 332, 507–515.
Gandia-Herrero, F., Escribano, J., Garcia-Carmona, F., 2005. Betaxanthins as
substrates for tyrosinase. An approach to the role of tyrosinase in the
biosynthetic pathway of betalains. Plant Physiol. 138, 421–432.
190
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
Gliszczyńska-Świgło, A., 2006. Antioxidant activity of water soluble vitamins in the
TEAC (trolox equivalent antioxidant capacity) and the FRAP (ferric reducing
antioxidant power) assays. Food Chem. 99 (1), 131–136.
Gomes, C., Moreira, R.G., Castell-Perez, M.E., Kim, J., Da Silva, P., Castillo, A., 2008. Ebeam irradiation of bagged, ready-to-eat spinach leaves (Spinacea oleracea): an
engineering approach. J. Food Sci. 73 (2), E95–E102.
Grabber, J.H., 2005. How do lignin composition, structure, and cross-linking affect
degradability? A review of cell wall model studies. Crop. Sci. 45, 820–831.
Grassi, M., Lapasin, R., Pricl, S., 1996. A study of the rheological behaviour of
scleroglucan weak gel systems. Carbohydr. Polym. 29, 169–181.
Hallett, I.C., MacRae, E.A., Wegrzyn, T.F., 1992. Changes in kiwifruit cell wall
ultrastructure and cell packing during postharvest ripening. Int. J.Plant Sci. 153,
49–60.
Henderson, J., Ricker, R., Bidlingmeyer, B., Woodward, C., 2000. Rapid, Accurate,
Sensitive, and Reproducible HPLC Analysis of Amino Acids. Technical Report
from Agilent 8 Technologies.
Herbach, K., Stintzing, F., Carle, R., 2004. Impact of thermal treatment on colour and
pigment pattern of red beet (Beta vulgaris L.) preparations. J. Food Sci. 69 (6),
491–498.
Herbert, P., Barros, P., Ratola, N., Alves, A., 2000. HPLC Determination of amino acids
in 4 musts and port wine using OPA/FMOC derivatives. J. Food Sci. 65 (7), 1130–
1133.
IAEA, International Atomic Energy Agency, 1998. Combination processes for food
irradiation. In: Proceedings of the Final Research Co-ordination Meeting,
Pretoria, South Africa, 27 February–3 March 1995 (Panel Proceeding), Vienna.
ICGFI, International Consultative Group on food, 1999. Facts about Food Irradiation.
IAEA, Vienna, Austria.
Ilker, R., Szcsesniak, A.S., 1990. Structural and chemical bases for texture of plant
foodstuffs. J. Texture Stud. 21, 1–36.
Jackson, P., Paulo, S., Brownleader, M.D., Freire, P., Ricardo, C.P.P., 1999. An extensin
peroxidase is associated with white-light inhibition of lupin (Lupinus albus)
hypocotyl growth. Aust. J. Plant Physiol. 26, 313–326.
Jarvis, M.C., 1998. Intercellular separation forces generated by intracellular
pressure. Plant Cell Environ. 21 (12), 1307–1310.
Jo, C., Son, J., Lee, H., Buym, M., 2003. Irradiation application for colour removal
and purification of green tea leaves extract. Radiat. Phys. Chem. 66, 179–
184.
Kanner, J., Harel, S., Granit, R., 2001. Betalains. A new class of dietary cationized
antioxidants. J. Agric. Food Chem. 49, 5178–5185.
Knuthsen, P., 1981. Investigations on beetroot colours for the purpose of regulation.
Z. Lebensm. Unters. Forsch. 172, 195–200.
Kujala, T., Loponen, J., Klika, K., Pihlaja, K., 2000. Phenolics and betacyanins in red
beetroot (Beta vulgaris) root: distribution and effect of cold storage on the
content of total phenolics and three individual compounds. J. Agric. Food Chem.
48, 5338–5342.
Kunstadt, P., 2001. Economic and technical considerations in food irradiation. Peter
Chapter 16. In: Molins, Ricardo (Ed.), Food Irradiation: Principles, Applications.
John Wiley and Sons Inc., NY, USA, pp. 415–442.
Lacroix, M., Ouattara, B., 2000. Combined industrial processes with irradiation to
assure innocuity and preservation of food products. A review. Food Res. Int. 33,
719–724.
Langerak, D.I., Damen, G.A.A., 1978. Influence of irradiation on the keeping quality
of pre-packed soup greens at 10 °C. Food Preservation by Irradiation, vol. 1.
International Atomic Energy Agency, Vienna, Austria. pp. 275–282.
Lee, J., Kim, J., Yook, H., Kang, K., Lee, W., Hwang, H., Buyn, M., 2001. Effects of
gamma radiation on the allergenicity and antigenicity properties of milk
proteins. J. Food Prot. 64, 272–276.
Levigne, S., Ralet, M.C., Quéméner, B., Thibault, J.F., 2004. Isolation of diferulic
bridges ester-linked to arabinan in sugar beet cell walls. Carbohydr. Res. 339
(13), 2315–2319.
Lin, T.-T., Pitt, R.E., 1986. Rheology of apple and potato tissue as affected by cell
turgor pressure. J. Texture Stud. 17, 291–313.
Lowry, O.H., Rosebrough, N.J., Randall, R.J., 1951. Protein measurement with the
Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Marangoni, A.G., Jackman, R.L., Stanley, D.W., 1277. Chilling associated softening of
tomato fruit is related to increased pectinmethylesterase activity. J. Food Sci. 60
(6), 1277–1281.
Marry, M., Roberts, K., Jopson, S.J., Huxham, I.M., Jarvis, M.C., Corsar, J., Robertson, E.,
McCann, M.C., 2006. Cell–cell adhesion in fresh sugar-beet root parenchyma
requires both pectin esters and calcium cross-links. Physiol. Plant. 126 (2), 243–
256.
Martin-Cabrejas, M.A.M., Waldron, K.W., Selvendran, R.R., 1994. Cell wall changes in
Spanish pear during ripening. J. Plant Physiol. 144, 541–548.
Mc Evoy, H., Ross-Murphy, S.B., Clark, A.H., 1985. Large deformation and ultimate
properties of biopolymer gels: 1. Single biopolymer component systems.
Polymer 26, 1483–1492.
McCann, M.C., Hammouri, M., Wilson, R., Belton, P., Roberts, K., 1992. Fourier
Transform Infrared Microspectroscopy is a new way to look at plant cell walls.
Plant Physiol. 100, 1940–1947.
Mobhammer, M.R., Maier, C., Stintzing, F.C., Carle, R., 2006. Impact of thermal
treatment and storage on colour of yellow-orange cactus pear (Opuntia ficusindica L. Mill. cv. ‘Gialla’) juices. J. Food Sci. 71 (7), 400–406.
Mort, A.J., Moerschbacher, B.M., Pierce, M.L., Maness, N.O., 1991. Problems
encountered during the extraction, purification, and chromatography of pectic
fragments, and some solutions to them. Carbohydr. Res. 215, 219–227.
Nagai, T., Ishizuka, S., Hara, H., Aoyama, Y., 2000. Dietary sugar beet fiber prevents
the increase in aberrant crypt foci induced by gamma-irradiation in the
colourectum of rats treated with an immunosuppressant. J. Nutr. 130 (7), 1682–
1687.
Narine, S.S., Marangoni, A.G., 1999. Fractal nature of fat crystal networks. Phys. Rev.
E 59, 1908–1920.
Naumenko, I.V., Phillipov, M.P., 1992. Colourimetric method for determination of
acetyl groups in pectic substances. Akademi Nauk Republi Moldova Biologi i
Khimie Nauki*** 1, 56–59.
Ng, A., Waldron, K.W., 1997. Effect of cooking and pre-cooking on cell wall
chemistry in relation to firmness of carrot tissues. J. Sci. Food Agric. 73, 503–
512.
Ng, A., Parr, A.J., Ingham, M.L., Rigby, N.M., Waldron, K.W., 1998. Cell wall chemistry
of carrots (Daucus carota L.; cv. Amstrong) during maturation and storage. J.
Agric. Food Chem. 46, 2933–2939.
Nguyen-the, C., Carlin, F., 1994. The microbiology of minimally processed fresh
fruits and vegetables. Crit. Rev. Food Sci. Nutr. 34, 371–401.
Niemira, B.A., Sommers, C.H., Fan, X., 2002. Suspending lettuce type influences
recoverability and radiation sensitivity of Escherichia coli O157:H7. J. Food Prot.
65 (9), 1388–1393.
Niemira, B.A., Fan, X., 2006. Low dose irradiation of fresh and fresh-cut produce.
safety, sensory, and shelf life. Chapter 10. In: Sommers, C.A., Fan, X. (Eds.), Food
Irradiation. Research and Technology. Blackwell Publishing and IFT Press,
Oxford, UK, pp. 169–184.
Niemira, B.A., 2007. Relative efficacy of sodium hypochlorite wash versus
irradiation to inactivate Escherichia coli O157:H7 internalized in leaves of
Romaine lettuce and baby spinach. J. Food Prot. 71 (11), 2526–2532.
Noomhorm, A., Ilangantileke, S.G., Upadhyay, I.P., Karki, D.B., Apintanapong, M.,
1998. Use of irradiation in combination with preservation techniques to extend
the shelf life of tropical fruits and their products. In: International Atomic
Energy Agency (Ed.), Combination processes for food irradiation. International
Atomic Energy Agency, Vienna, Austria, pp. 53–75.
Nussinovitch, A., Peleg, M., Normand, M.D., 1989. A modified Maxwell and a nonexponential model for the characterization of the stress-relaxation of agar and
alginate gels. J. Food Sci. 54, 1013–1016.
Olthof, M.R., van Vliet, T., Boelsma, E., Verhoef, P., 2003. Low dose betaine
supplementation leads to immediate and long term lowering of
plasma homocysteine in healthy men and women. J. Nutr. 133 (12), 4135–
4138.
Oudgenoeg, G., Dirksen, E., Ingemann, S., Hilhorst, R., Gruppen, H., Boeriu, C.G.,
Piersma, S.R., van Berkel, W.J., Laane, C., Voragen, A.G.J., 2002. Horse radish
peroxidase-catalyzed oligomerization of ferulic acid on a template of a tyrosinecontaining tripeptide. J. Biol. Chem. 277, 21332–21334.
Parr, A.J., Ng, A., Waldron, K.W., 1997. Ester-linked phenolic components of carrot
cell walls. J. Agric. Food Chem. 45 (7), 2468–2471.
Passardi, F., Longet, D., Penel, C., Dunand, C., 2004. The class III peroxidase
multigenic family in rice and its evolution in land plants. Phytochemistry 65,
1879–1893.
Pedreño, M.A., Escribano, J., 2001. Correlation between antiradical activity and
stability of betanine from Beta vulgaris L. roots under different pH, temperature
and light conditions. J. Sci. Food Agric. 81, 627–631.
Peleg, M., Calzada, J.F., 1976. Stress relaxation of deformed fruits and vegetables. J.
Food Sci. 41, 1325–1329.
Pitt, R.E., Chen, H.L., 1983. Time-dependent aspects of the strength and rheology of
vegetative tissue. Trans. ASAE 26, 1275–1280.
Pouteau, C., Dole, P., Cathala, B., Averous, L., Boquillon, N., 2003. Antioxidant
properties of lignin in polypropylene. Polym. Degrad. Stab. 81, 9–18.
Prakash, A., Guner, A.R., Caporaso, F., Foley, D.M., 2000a. Effects of low-dose gamma
irradiation on the shelf life and quality characteristics of cut romaine lettuce
packaged under modified atmosphere. J. Food Sci. 65 (3), 549–553.
Prakash, A., Inthajak, P., Huibregtse, H., Caporaso, F., Foley, D.M., 2000b. Effect of low
dose gamma irradiation and conventional treatments on shelf life and quality
characteristics of dicey celery. J. Food Sci. 65, 1070–1075.
Price, N.J., Pinheiro, C., Soares, C.M., Ashford, D.A., Ricardo, C.P., Jackson, P.A., 2003. A
Biochemical and Molecular Characterization of LEP1, an Extensin Peroxidase
from Lupin. J. Biol. Chem. 278 (42), 41389–41399.
Rastogi, N.K., 2005. Impact of gamma-irradiation on some mass transfer driven
operations in food processing. Radiat. Phys. Chem. 73, 355–361.
Regalado, C., Garcia-Almendarez, B.E., Duarte-Vanquez, M.A., 2004. Biotechnological
applications of peroxidases. Phytochem. Rev. 3 (1–2), 243–256.
Ribeiro, J.M., Silva Pereira, C., Soares, N.C., Vieira, A.M., Feijó, J.A., Jackson, P.A., 2006.
The contribution of extensin network formation to rapid, hydrogen peroxidemediated increases in grapevine callus wall resistance to fungal lytic enzymes.
J. Exp. Bot. 57 (9), 2025–2035.
Rojas, A.M., Gerschenson, L.N., Marangoni, A.G., 2001. Contributions of cellular
components to the rheological behaviour of kiwifruit. Food Res. Int. 34, 189–
195.
Rojas, A.M., Delbon, M., Marangoni, A.G., Gerschenson, L.N., 2002. Contribution of
cellular structure to the large and small deformation rheological behaviour of
kiwifruit. J. Food Sci. 67 (6), 2143–2148.
Roland, J.C., 1978. Cell wall differentiation and stages involved with intercellular gas
space opening. J. Cell Sci. 32 (1), 325–336.
Sánchez-Ferrer, A., Rodríguez-López, J.N., García-Cánovas, F., García-Carmona, F.,
1995. Tyrosinase: a comprehensive review of its mechanism. Biochim. Biophys.
Acta 1247, 1–11.
M.E. Latorre et al. / Journal of Food Engineering 98 (2010) 178–191
Schwartz, S.J., von Elbe, J.H., Jackman, R.L., Smith, J.L., 1980. Quantitative
determination of individual betacyanin pigments by high performance liquid
chromatography. J. Agric. Food Chem. 28, 540–543.
Sepúlveda-Jiménez, G., Rueda-Benítez, P., Porta, H., Rocha-Sosa, M., 2004.
Betacyanin synthesis in red beet (Beta vulgaris) leaves induced by wounding
and bacterial infiltration is preceded by an oxidative burst. Phys. Mol. Plant
Pathol. 64, 125–133.
Shui, G., Leong, L.P., 2006. Residue from star fruit as valuable source for functional
food ingredients and antioxidant nutraceuticals. Food Chem. 97, 277–284.
Stadelmann, E., 1966. Evaluation of turgidity, plasmolysis and deplasmolysis of
plant cells. In: Prescott, D.M. (Ed.), Methods in Cell Physiology, vol. II. Academic
Press, New York, pp. 143–216.
Strack, D., Vogt, T., Schliemann, W., 2003. Recent advances in betalain research.
Phytochemistry 62 (3), 247–269.
Strasser, G.R., Amado, R., 2001. Pectic substances from red beet (Beta vulgaris
conditiva). Part I. Structural analysis of rhamnogalacturonan I using enzymic
degradation and methylation analysis. Carbohydr. Polym. 44, 63–70.
Synytsya, A., Copíková, J., Matějka, P., Machovič, V., 2003. Fourier transform Raman
and infrared spectroscopy of pectins. Carbohydr. Polym. 54, 97–106.
Thakur, B., Singh, R., 1995. Combination processes in food irradiation. Trends Food
Sci.Techn. 6, 7–11.
Thayer, D.W., 1990. Food Irradiation: Benefits and concerns. J. Food Qual. 13, 147–
169.
Toivonen, P.M.A., Brummell, D.A., 2008. Biochemical bases of appearance and
texture changes in fresh-cut fruit and vegetables. Postharvest. Biol. Technol. 48
(1), 1–14.
Vincken, J.P., Schols, H.A., Oomen, R.J.F.J., Beldman, G., Visser, R.G.F., Voragen, A.G.J.,
2003. Pectin: the hairy thing. In: Advances in pectin and pectinase research.
191
Kluwer Academic Publishers, Voragen A. G. J., Schols H. A., Visser R. eds.,
Dordrecht. pp. 47–59.
Waje, C.K., Jun, S.Y., Lee, Y.K., Kim, B.N., Han, D.H., Jo, C., Kwon, J.H., 2009. Microbial
quality assessment and pathogen inactivation by electron beam and gamma
irradiation of commercial seed sprouts. Food Control 20, 200–204.
Waldron, K.W., Smith, K.C., Parr, A.J., Ng, A., Parker, M.L., 1997. New approaches to
understanding and controlling cell separation in relation to fruit and vegetable
texture. Trends Food Sci Technol. 8, 213–221.
Waldron, K.W., Ng, A., Parker, M.L., Parr, A.J., 1997b. Ferulic acid dehydrodimers in
the cell walls of Beta vulgaris and their possible role in texture. J. Sci. Food Agric.
74, 221–228.
Walton, D.C., Sondheimer, E., 1968. Effects of Abscisin II on Phenylalanine
Ammonia-Lyase Activity in Excised Bean Axes. Plant Physiol. 43 (3), 467–469.
Wang, Z., Ma, Y., Zhao, G., Liao, X., Chen, F., Wu, J., Chen, J., Hu, X., 2006. Influence of
gamma irradiation on enzyme, microorganism and flavor of cantaloupe
(Cucumis melo L.) juice. J. Food Sci. 71 (6), M215–M220.
Wilson, R.H., Smith, A.C., Kacurakova, M., Saunders, P.K., Wellner, N., Waldron, K.W.,
2000. The mechanical properties and molecular dynamics of plant cell wall
polysaccharides studied by Fourier-transform infrared spectroscopy. Plant
Physiol. 124, 397–405.
Wood, P.J., Siddiqui, I.R., 1971. Determination of methanol and its application for
measurement of pectin ester content and pectin methyl esterase activity. Anal.
Biochem. 39, 418–428.
Xuan, L., Yanxiang, G., Xiaoting, P., Bin, Y., Honggao, X., Jian, Z., 2008. Inactivation of
peroxidase and polyphenol osidase in red beet (Beta vulgaris L.) extract with
high pressure carbon dioxide. Innovative Food Sci. Emerg. Technol. 9, 24–31.
Yoshikazu, T., Nobuhiro, S., Akemi, O., 2008. Biosynthesis of plant pigments:
anthocyanins, betalains and carotenoids. The Plant J. 54 (4), 733–749.