Journal of Pharmaceutical and Biological Sciences
ISSN: 2320-1924; CODEN: JPBSEV
Published by Atom and Cell Publishers © All Rights Reserved
Available online at: http://www.jpabs.org/
Review Article
Role of organic acids and hydrogen peroxide in fruit juice preservation: A review
P. Saranraj1* and M. Ramya2
1
Assistant Professor of Microbiology, Department of Biochemistry, Sacred Heart College
(Autonomous), Tirupattur – 635 601, Tamil Nadu, India.
2
Department of Biochemistry, Sacred Heart College (Autonomous), Tirupattur – 635 601,
Tamil Nadu, India.
Received: 12-01-2016 / Revised Accepted: 19-02-2016 / Published: 03-03-2016
ABSTRACT
Fruits contain high levels of sugars and other nutrients and they possess an ideal water activity for microbial
growth. Their low pH makes them particularly susceptible to fungal spoilage because a big part of the bacterial
competition was eliminated since most bacteria prefer near neutral pH. Some fungi are plant pathogens and can
start the spoilage from the field while others, although they could contaminate the fruits in the field, actually
proliferate and cause substantial spoilage only after harvest when the main plant defenses are reduced or
eliminated. In this present review, we clearly explained the role of organic acids and hydrogen peroxide in fruit
juice preservation. The topics covered in this present review are: Microbial spoilage of fruits and fruit products,
Sources of contamination of fruit juices, Fruit juice composition, Organic acids in fruit juice preservation,
Hydrogen peroxide in fruit juice preservation and Control of microbial spoilage in fruit juices.
Key words: Fruit juice, Microbial spoilage, Organic acids, Hydrogen peroxide and Preservation.
INTRODUCTION
Fresh, unpasteurized fruit juices hold a favorable
appeal too many consumers due to their distinct
flavor characteristics and perceived nutritional
superiority. Producers of unpasteurized juice have
traditionally relied upon a juices inherent acidity to
render their product microbiologically safe.
However, documented outbreaks of Salmonella and
Escherichia coli associated with unpasteurized
juices have dispelled this belief. As early as 1922
and 1944, outbreaks of typhoid fever have been
linked to sweet cider and orange juice
consumption. In 1980, before recognition of
Escherichia coli as a human pathogen, an outbreak
of hemolytic uremic syndrome, likely from
Escherichia coli was reported in apple cider [1].
Sixty six cases of Escherichia coli infection,
including the death of a child resulted from a 1996
outbreak of Escherichia coli in unpasteurized apple
cider [2].
Fruits are vital to our health and well being, as they
are furnished with essential vitamins, minerals,
fibres and other health-promoting phyotochemical.
The present health-conscious generation prefers a
diet exhibiting low calories and low fat/sodium
contents. A great importance of intake of fruits
everyday has been found to half the risk of
developing cancer and also reduce the risk of heart
disease, diabetes, stroke, obesity, birth defects,
cataract, osteoporosis and many more to count [3].
Contamination could arise from fecal contact, but
also other sources. Fecal contamination from the
use of dropped, unwashed apples has been
implicated as the source of Escherichia coli in
some apple cider outbreaks [4]. However, vectors
such as birds and insects could potentially deposit
this pathogen on tree-bound fruit [5]. In a 1995
outbreak of Salmonellosis from unpasterurized
orange juice, Salmonella spp. was isolated from
amphibians around the processing facility [6].
The fruits differ from vegetables in having
somewhat less water but more carbohydrate. The
protein, fat and ash content of fruits are
respectively, 0.9 % and 0.5 % somewhat lower than
vegetables except for ash content. Fruits contain
vitamins and other organic compounds, just as
vegetables do. On the basis of nutrient content,
*Corresponding Author Address: P. Saranraj, Assistant Professor of Microbiology, Department of Biochemistry, Sacred Heart College
(Autonomous), Tirupattur – 635 601, Tamil Nadu, India; E.mail: microsaranraj@gmail.com
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
these products would appear to be capable of
supporting the growth of bacteria, yeasts and
molds. However, the pH of fruits is below the level
that generally favors bacteria growth. This one fact
alone would seem to be sufficient to explain the
general absence of bacteria in the incipient spoilage
of fruits [7]. As natural components of fruits,
organic acids such as malic acid, citric acid and
tartaric acid lower the pH, and help maintain the
proper sugar/acid balance in fruit juices [8]. In
broth systems, their bacteriostatic and bactericidal
effects have been evaluated for both Escherichia
coli and Salmonella [9] with pH and degree of
dissociation being major factors in a particular
acids efficacy. Undissociated acids are more cell
permeable, and upon entering the cell can
dissociate and lower intracellular pH [10]. Thus,
organic acids can affect both the intercellular and
extracellular pH. As a natural method of lowering
juice pH, increasing the organic acid concentration
of juices may improve the antimicrobial efficacy of
hydrogen peroxide treatments.
have increased in worldwide. Changes in
agronomic, harvesting, distribution, processing and
consumption patterns, and practices have
undoubtedly contributed to this increase [17].
Microorganisms form part of the epiphytic flora of
fruits and vegetables and many will be present at
the time of consumption. The majority of bacteria
found on the surface of plants was usually Gram
negative and belong either to the Pseudomonas
group or to the bacteria belongs to the family
Enterobacteriaceae [18]. Many of these organisms
are normally non-pathogenic for humans. The
numbers of bacteria present will vary depending on
seasonal and climatic variation and may range from
104 to 108 per gram. The inner tissues of fruits are
usually regarded as sterile. However, bacteria can
be present in low numbers as a result of the uptake
of water through certain irrigation or washing
procedures. If these waters are contaminated with
human pathogens these may also be introduced.
About two thirds of the spoilage of fruits was
caused by fungal moulds [19]. Members of the
genera Penicillium, Aspergillus, Sclerotinia,
Botrytis and Rhizopus are well commonly involved
in this process. The spoilage was usually associated
with cellulolytic or pectinolytic activity which
causes softening and weakening of plant structures.
These structures are important barriers to prevent
growth in the products by contaminating microbes.
The survival or growth of contaminating
microorganisms was affected by intrinsic, extrinsic
and processing factors. Factors of importance are
nutrient composition, pH, presence of scales and
fibres, redox potential, temperature and gaseous
atmosphere. Mechanical shredding, cutting and
slicing of the produce open the plant surfaces to
microbial attack.
The bactericidal efficacy of hydrogen peroxide has
been demonstrated in both water and food systems
[11] with Gram negative organisms having the
most susceptibility [12]. It has been effective in
extending the shelf life of cantaloupe, mushrooms,
bell peppers, grapes and raisins [13]. This
antimicrobial action stems from its ability to form
reactive oxygen species such as the hydroxyl
radical and singlet oxygen, which can damage
DNA and membrane constituents [14]. Hydrogen
peroxide has GRAS (Generally Regarded as Safe)
status and is currently allowed as an antimicrobial
in starch processing and in milk for cheese
manufacturing.
Sensory changes as a result of hydrogen peroxide
and organic acid additions are an important
consideration. By contributing a sour or acidic
taste, organic acids are only practical in
concentrations that do not adversely upset the
sugar/acid ratio. Fruit juices are sensitive to oxygen
in terms of stability, appearance and flavor. In fact,
the color of apple juice is almost solely derived
from oxidative reactions with phenolic constituents
[15]. The use of hydrogen peroxide for extending
the shelf life of strawberries and raspberries was
negated due to anthocyanin bleaching [16]. Thus,
hydrogen peroxide, as both an oxidative molecule
and liberator of oxygen upon degradation, may
adversely affect sensory qualities of fruit juice.
Fruit juices has been identified as the vehicle of
transmission in at least 11 microbial disease
outbreaks since 1944, including Typhoid fever in
1944, Hepatitis A Virus in 1962, Viral
gastroenteritis in 1966, Typhoid fever in 1989,
Enterotoxigenic E. coli in 1992, Salmonella
enterica serotype and the largest Salmonella
outbreak with fresh orange juice, Salmonella
serotype [20]. In 2000, a Salmonella enteritidis
outbreak caused by unpasteurized orange juice
resulted in 88 illnesses in 6 of the western United
States [21]. Acidic fruit juices have also been
implicated in outbreaks of gastroenteritis.
Unpasteurized apple cider and apple juice were
associated with outbreaks of Salmonella
typhimurium, Escherichia coli [22], post diarrheal
haemolytic uremic syndrome and cryptosporidiosis.
The Food and Drug Administration (FDA) issued a
final rule to increase the safety of fruits and
vegetable juice and juice products [23]. According
MICROBIAL SPOILAGE OF FRUITS AND
FRUIT PRODUCTS
In the past decade, outbreaks of human illness
associated with the consumption of raw fruits or
unpasteurized fruit products produced from them
59
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
to this rule, juice processors must use Hazard
analysis and critical control point (HACCP)
principles for processing and utilize control
measures to achieve a 5 log (100000 fold)
reduction in the numbers of the most resistant
pathogen in their finished products compared to
levels that might be present in untreated juice. The
rule, effective January 22, 2002, states that
approved alternative technologies to pasteurization
can be used to achieve microbial reduction.
interface creates a slight vacuum due to the
decreased partial vapor pressure at the fruit surface,
thus potentially sucking bacteria beneath the skin,
calyx, or stem scar. Employing such a mechanism,
it is reasonable to assume that warm fruit, still on
the tree, could internalize pathogens from bird
feces, if subjected to a cool rain.
Bacterial soft rot in fruit has also been associated
with increased levels of Salmonella contamination.
Wells and Butterfield [34] reported that Salmonella
contamination was present in at least 18 – 20 % of
soft rotted samples, compared to 9 -10 % in healthy
samples. In addition, fruits disks inoculated with
Erwinia carotovora (soft rot bacterium) and
Salmonella typhimurium supported 10 times the
Salmonella typhimurium levels of fruit disks
inoculated with Salmonella typhimurium alone.
SOURCES OF CONTAMINATION OF FRUIT
JUICES
Human pathogens are carried by an array of
animate vectors. Animals such as deer and cattle
are reservoirs for Escherichia coli [24]. Birds and
insects have also been implicated as carriers of this
pathogen [25]. Salmonella is harbored by a number
of domestic animals as well as humans [26].
Investigations into a 1995 outbreak of
Salmonellosis in orange juice isolated the pathogen
from frogs and toads around the processing facility
[27].
JUICE COMPOSITION
Fruit juice is mainly the liquid expressed from fruit
cell vacuoles, but also includes insoluble particles
and bits of fruit tissue. Though primarily water, this
organic medley contains sugars such as glucose,
fructose, and sucrose, organic acids (malic, citric,
and tartaric), fats, proteins, various volatile
compounds and vitamins [35]. Taste and flavor
qualities are formed by the sugars, organic acids
and aroma compounds present in juice. Sugars and
organic acids make up the bulk of the soluble solids
fraction, and a proper balance between the
concentrations of both are important in the
palatability of the juice. Thus, organic acid
additions are only reasonable within the scope of
maintaining an acceptable sugar/acid ratio. Aroma
arises from a number of volatile compounds whose
composition was essential to juice quality yet very
sensitive to processing techniques. Aroma profile
modification is of great importance when
considering processing methods for juice
manufacturing [36].
Pathogen contamination may occur via contact
with the feces of these vectors. Wind fallen or
dropped fruit, having a greater likelihood of fecal
contamination has been implicated as a pathogen
source in recent Escherichia coli outbreaks from
cider [28]. Under such circumstances, typical
brushing and washing techniques may remove
surface fecal contamination, but these techniques
become less effective if pathogens are internalized.
Though, the mechanisms by which pathogens enter
fruit are still questionable, internalization can
occur. External injuries such as cuts and abrasions
would offer easy attachment and access. Liao and
Sapers [29] found that the apple disks with no skin
retained 13 – 19 % more Salmonella Chester than
disks with skin, indicating easier attachment to
abraded or wounded fruit. Natural structures such
as the stem, stem scar and calyx are also potential
sites for internalization. Of those Salmonella
Chester attached to apples after artificial
inoculation, 94 % were located on the stem or
calyx region [30].
Enzymes released during juice expression cause a
host of chemical changes, some of which may
detract from the appearance and stability of a juice
product. In the case of apple juice under aerobic
conditions, polyphenol oxidase catalyzes the
polymerization of phenolic constituents which
leads to brown coloration (melanin) [37]. In
addition to being the sole production of color, these
oxidative polymerizations can change the flavor
and aroma of the juice. Thus, addition of an oxidant
such as hydrogen peroxide would likely have a
noted effect on the color, flavor, and aroma of
apple juice. In orange juice, pectin methyl esterase
converts pectin to pectic acid with the end result
being cloud loss and juice separation [38]. Such
changes are obvious in unpasteurized orange juice,
Wash water quality and temperature may also play
a role. Zhuang et al. [31] found that tomatoes at 25
°
C dipped in a 10 °C cell suspension of Salmonella
montevideo internalized a significant number of the
pathogen. Similar uptake of Escherichia coli was
witnessed in apples dipped in cold peptone water
[32]. Studies by the FDA using dyed water indicate
that microorganisms could potentially be
internalized simply through the skin of undamaged
fruit, when contacting aqueous suspensions of
lower temperature [33]. A warm fruit, cool water
60
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
which has had no heat treatment to inactivate
pectin methyl esterase.
acidified with between 0 and 3 % malic and citric
acid respectively, were analyzed for the survival of
Listeria monocytogenes. Untreated apple cider
reduced Listeria monocytogenes to undetectable
levels within 48 hours, but orange juice with 3 %
citric acid took at least 4 days to have the same
effect, and significant numbers survived at least 10
days in untreated orange juice. For Escherichia
coli, inactivation in acidified Tryptic Soy Broth
(TSB) and agar was demonstrated for citric, malic
and tartaric acids [47]. In one study, survival of
Escherichia coli was greater in acidified apple juice
compared to acidified TSB, suggesting a protective
effect of juice constituents. However, contrary to
other studies, acidified apple juice enhanced
survival compared to untreated apple juice,
suggesting a protective effect from the acid under
refrigeration [48].
As mentioned, the characteristic brown color of
apple juice is a result of oxidative reactions after
juice extraction. However, orange and grape juice
color is derived from pigments initially present in
the fruit. Orange juice derives its color from
carotenoids
such
as
β
–cryptoxanthin,
antheraxathin, α–carotene, β-carotene, and leutin
within the juice vesicles. These color components
are rather stable to processing, but vary in intensity
according to the fruit’s growing season [39]. In
purple grape juice, color comes from anthocyanins
and phenolic components mostly in the skin of the
grape, but must be extracted into the initially clear
juice. For wine production, fermentation aids in
color extraction, but in juice production, heat is
used to extract color components from the skins
[40].
Citric
acid
[HOOC-CH2-COH(COOH)-CH2COOH] is one of the more widely used food
acidulants. It is a common constituent of fruits,
namely citrus fruits and imparts a pleasant sour
taste. Citric acid was commonly employed as an
acidulant in canned vegetables and dairy products
[49]. In skim milk, citric acid was the most potent
inhibitor of Salmonella typhimurium compared to
lactic acid and HCl [50]. Fischer et al. [51]
reported a 0.75 % solution of citric acid to
sufficiently reduce Salmonella typhimurium,
Yersinia enterocolitica, Escherichia coli and
Staphylococcus aureus on hard-boiled eggs. Under
good manufacturing practices, citric acid was
approved as a GRAS substance.
ORGANIC ACIDS
Organic acids occur throughout nature and are used
extensively in food systems. In addition to their use
as microbial inhibitors, they can serve as
defoaming agents and emulsifiers, aid in setting of
pectin gels, and have a strong effect on the taste of
a food [41]. With a characteristically sour taste,
organic acids have an important role in the flavor
of fruits and their juices by balancing the
sugar/acid ratio [42].
The inhibitory effect of organic acids depends on
the undissociated form, as well as its ability to
donate hydrogen ions in an aqueous system [43].
The degree of dissociation for a particular acid was
related to its dissociation constant and the acidity
of the product. Dissociation constants indicate the
pH at which there is a 50/50 distribution of
undissociated and dissociated forms. At lower pH,
more undissociated acid was present [44]. In this
form, the cell membrane was more permeable to
the acid, allowing it to enter the cell. Upon entering
the cytoplasm, the acid dissociates, thus lowering
the internal pH of the cell and disrupting cellular
functions [45]. In addition to affecting enzymes,
excess protons in the cytoplasm upset the
membrane potential necessary for energy
production and transport across the cell membrane.
Thus, organic acids can act on a cell by affecting
both the external and internal pH.
Malic acid (HOOC-CHOH-CH2-COOH), along
with citric acid comprises the main organic acids in
fruits [52]. In apples, malic acid was the
predominant organic acid [53]. It was used for its
flavoring and acidification properties in beverages,
jams, jellies, and sherbets [54]. Malic acid has
GRAS status. Unlike most other fruits, the main
organic acid in grapes is tartaric acid
(HOOCCHOH-CHOH-COOH) [55]. Tartaric acid
is useful in supporting grape like flavors. As an
antimicrobial agent, tartaric acid was believed to
act only by lowering the pH of the product [56].
HYDROGEN PEROXIDE
Hydrogen peroxide (H2O2) is an antiseptic
(compared to a preservative) since it quickly acts to
kill microorganisms and has no long-term or
preserving effect [57]. This short-lived action was
due to hydrogen peroxide’s rapid decomposition to
oxygen and water upon contact with organic
material. The antimicrobial action of hydrogen
peroxide is not due to its oxidative properties as a
molecule, but primarily in the production of other
In both culture media and food system, the varying
bacteriostatic and bactericidal effects of organic
acids have been demonstrated. Chung and Goepfert
[46] showed that various organic acids are
bacteriostatic to Salmonella spp. at different pH
levels. In our lab, apple cider and orange juice
61
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
powerful oxidants such as singlet oxygen,
superoxide radicals and the hydroxyl radical [58].
These reactive oxygen species cause irreversible
damage to a host of cell components such as
enzymes, membrane constituents and DNA. In fact,
aqueous solutions of H2O2 alone will not cause
protein, lipid or nucleic acid modification without
the presence of radical formation catalysts [59].
The H2O2 is naturally produced by enzymatic
systems and is notably utilized by phagocytes in
the destruction of bacteria within the
phagolysozome [60].
and the vapor-phase [71]. As a water disinfectant,
hydrogen peroxide had only a moderate immediate
effect on Escherichia coli, but substantial
immediate effect on Salmonella typhi [72].
Hydrogen peroxide has been used as an
antimicrobial agent since the early 1800’s, and is
well known for its use as a topical skin application
in 3 % concentrations [73]. In foods, hydrogen
peroxide was used as a disinfectant in milk as early
as 1904 [74]. Hydrogen peroxide has GRAS status
and was approved by the FDA for packaging and
surface sterilization in the food industry.
Hydroxyl radical (HO.) production likely plays the
largest role in the toxicity of hydrogen peroxide
[61]. When produced adjacent to DNA, hydroxyl
radicals are unique in that they can “both add to
DNA bases and abstract H-atoms from the DNA
helix” [62]. Hydroxyl radicals may also damage
cell membranes. In a study of model membrane
systems, Juven and Pierson [63] found that the
hydroxyl radicals (generated from hydrogen
peroxide) increased lipid peroxidation as well as
the ion permeability of model membrane systems,
though via independent mechanisms. Furthermore,
after 17 minutes of hydroxyl radical exposure,
complete membrane breakdown was observed.
Allowed uses of hydrogen peroxide as a direct
additive to foods are limited. For antimicrobial
purposes, H2O2 was allowed for treating milk used
in cheese manufacturing, thermopile free starch
production and the preparation of modified whey,
at levels of 0.05, 0.15 and 0.4 % respectively. It
was used as an oxidizing and reducing agent in
wine, dried eggs, and corn syrup, and as a
bleaching agent in tripe, beef feet, instant tea,
colored cheese whey and certain emulsifiers.
Residual peroxide must be removed by an
appropriate means, typically by addition of
catalase.
Production of HO from H2O2 has been reported to
occur in a number of ways. A commonly cited
example is the Fenton reaction whereby a reducing
agent such as the superoxide radical reduces Fe3+ to
Fe2+, which then reacts with H2O2 to produce
hydroxyl anions, hydroxyl radicals, and Fe3+ [64].
Accordingly, growing Staphylococcus aureus cells
in broths of increasing iron concentrations was
found to increase killing by H 2O2, whereas addition
of HO. scavengers had a protective effect against
such killing [65]. In a contrasting study, the ferryl
radical, not the hydroxyl radical was indicated as
the DNA damaging species in Escherichia coli
[66].
The use of hydrogen peroxide to extend the shelf
life of minimally processed fruits and vegetables
was reviewed by Sapers and Simmons [75].
Applied as a vapor, 60 minutes of hydrogen
peroxide exposure improved the shelf life of whole
cantaloupe and drastically reduced the mold count
on raisins. Similarly, in two varieties of grape, 10
minute applications of 40 ºC vapor phase H2O2 was
found to significantly reduce Botrytis cinera spores
and enhance shelf life without affecting grape color
[76].
Vapor phase treatments of mushrooms showed
excessive browning, but 30 second washes
treatment with 5 % H2O2 and subsequent
erythorbate dip (browning inhibitor) gave
acceptable bacterial control without compromising
color. On apple disks, 6 % hydrogen peroxide gave
a greater reduction of Salmonella Chester than
trisodium phosphate, calcium hypochlorite, or
sodium hypochlorite [77]. While products such as
zucchini and bell peppers show promise for use of
H2O2 in controlling soft rot, others like strawberries
and raspberries show great sensitivity to
anthocyanin bleaching at bactericidal peroxide
levels [78]. There is a substantiated correlation
between temperature and the antimicrobial efficacy
of hydrogen peroxide. Toledo et al. [79] found a
notable increase in the sporicidal activity of H2O2
as the temperature increased above room
temperature. In liquid whole egg, 1 % H2O2 was
A host of research related to the activity of
hydrogen peroxide on various bacteria, molds and
yeast has been performed. Its activity appears
greatest against anaerobic and Gram negative
bacteria [67]. Lillard and Thomson [68] found that
concentrations of 5,300 – 12,000 ppm in poultry
chiller water reduced Escherichia coli populations
97 to > 99.9 %. In addition, the Enterobacteriaceae
were found to be more sensitive than other
organisms tested. Escherichia coli showed a Dvalue of 0.57 minutes when exposed to 3 % H 2O2,
compared to 2.35 minutes for Staphylococcus
aureus, 8.55 for Aspergillus niger and 18.3 for
Candida parapsilosis [69]. The significant
sporicidal activity of H2O2 on Bacillus subtilis
spores was witnessed in both the liquid phase [70]
62
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
more effective (~1 log) at decreasing survival of
Salmonella typhimurium in 24 hours in comparison
to the same peroxide concentration [80]. Kuchma
[81] noted a synergistic effect (killing of
Escherichia coli and Pseudomonas aeruginosa)
between microwave heating and low H2O2
concentrations, with maximum lethality at 50 °C
and 0.08 % H2O2.
Smith and Rollin [86] showed that the sorbic acid
was an excellent fungistatic. It has also been placed
on the list of food additives generally recognized as
safe under conditions of intended use.
Ingram et al. [87] noted that the DHA has a low
dissociation constant, which makes it effective in
low-acid or neutral media, but that it has a low
activity against bacteria. They also stated that the
U. S. Food and Drug Administration have
evaluated the acute toxicity as approximately equal
to that of carbolic acid. Nevertheless, the frequent
appearance in the literature of DHA studies on
spoilage organisms made it desirable to include it
in their study. It was approved for bananas up to 10
ppm in the edible portion [88].
Though, little if any work related to H2O2 use in
juice has been done, an important observation is
that the efficacy of H2O2 appears to increase with
decreasing pH. Hydrogen peroxide was found to be
bacteriostatic towards Pseudomonas aeruginosa at
pH 5.0, yet 1.5 m mol were required at pH 8.0. In
addition, a 3 % solution of hydrogen peroxide was
sporicidal against Bacillus subtilis in 3 hours at pH
5.0, but needed 6 hours to achieve the same effect
at pH 6.5 and 8.0 [82]. Thus, hydrogen peroxide
might be more effective in combination with
acidulants such as organic acids.
Bell et al. [89] found that 0.1 % sorbic acid
inhibited lactic acid bacteria at pH 3.5, and yeasts
at pH 4.5. They found that the toxicity was directly
related to the concentration of undissociated acid
and therefore a function of pH, just as with sodium
benzoate. Robinson and Hills [90] reported that
sodium sorbate and mild heat increased the storage
life of apple cider, peach slices, and citrus fruit
salads. Deinhard et al. [91] reported the optimum
pH range for A. acidoterrestris growth of the
organism in BAM was 2.5 to 5.8 over a
temperature range of 35 to 55ºC with an optimum
at 42 to 53 ºC, and McIntyre et al. [92] reported
growth of A. acidoterrestris on PDA over a pH
range of 3.0 to 5.3 at 30 to 55ºC. Previdi et al. [93]
also stated that all A. acidoterrestris strains tested
were able to grow on TA (pH 4.91), OSA (pH
5.09), and MEA (pH 4.0).
CONTROL OF MICROBIAL SPOILAGE IN
FRUIT JUICES
Fruits contain high levels of sugars and other
nutrients and they possess an ideal water activity
for microbial growth. Their low pH makes them
particularly susceptible to fungal spoilage because
a big part of the bacterial competition was
eliminated since most bacteria prefer near neutral
pH. Some fungi are plant pathogens and can start
the spoilage from the field while others, although
they could contaminate the fruits in the field,
actually proliferate and cause substantial spoilage
only after harvest when the main plant defenses are
reduced or eliminated.
Walls and Chuyate [94] reported A. acidoterrestris
growth in Orange Serum Broth at pH 2.5 to 5.0
over a temperature range of 20 to 55 ºC.
According to Lewis [95], the Centre for Food
Safety and Applied Nutrition found in its
preliminary study that unpasteurized juices
accounted for 76 % of juice contamination cases
reported between 1993 and 1996. It is estimated
that 16000 to 48000 illnesses per y can be
attributed to juices [96]. Information compiled by
Beuchat [97] provides an overview of food borne
pathogens in different vegetable and fruit products.
The frequency of microorganisms such as
Salmonella, enterovirulent Escherichia coli,
Listeria monocytogenes and Campylobacter are
subject to wide variation from study to study. The
prevalence of Campylobacter was mostly at levels
< 3 %, whereas the prevalence of Salmonella is
higher. In a majority of reports the frequency of
Salmonella typhi
was between 4 and 8 %.
Escherichia coli and Listeria monocytogenes were
in general found in a higher frequency compared to
Salmonella. The presence of pathogenic
Phillips and Mundt [83] showed that the lactic acid
bacteria grew normally in pickle fermentations,
while the scum yeasts were completely inhibited by
0.1 % sorbic acid. Their observations had been
made without control of pH, which remained above
4.0 throughout their study. Wolf [84] reported that
the dehydro acetic acid (DHA) or its sodium salt
effectively inhibits undesirable microbial activity
when added to a waxed food wrapper, or when
used as a dip for dried fruits. He stated that it could
be added to foods if shown to have no harmful
effect on consumers.
Von Schelhorn [85] review indicates that the
effectiveness of preservatives has been frequently
observed to vary with the species of organism. This
study therefore designed to evaluate the above
preservatives against pure cultures of organisms
causing spoilage of citrus products under
conditions simulating citrus salads exposed to a
temperature favourable for microbial growth.
63
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
microorganisms on raw fruits and varies
considerably. Often no pathogens are detected. In
other surveys high percentages of samples
contaminated with pathogens was observed.
Surveys of the presence of parasites or viruses are
fewer because the lack of detection methods that
can be applied to fruits.
Saccharomyces cerevisiae ascospores in fruit juices
and a model juice buffer at pH 3.5 to 5.0.
Approximately, 0.5 to 1.0 × 106 ascospores/mL
were pressurized at 300 to 500 MPa in juice or
buffer. D-values ranged from 8 sec to 10.8 min at
500 and 300 MPa, respectively. The range for zvalues was 115 to 121 MPa. No differences in D or
z-values among buffers or juices at any pH were
determined, indicating little influence of pH in this
range and absence of protective or detrimental
effects of juice constituents.
Beuchat [98] provides an overview of food borne
pathogens in different fruit products. The frequency
of microorganisms such as Salmonella, Escherichia
coli, Listeria monocytogenes, Campylobacter and
Cyclospora are subject to wide variation from
study to study. The prevalence of Campylobacter
was mostly at levels of 3 %, whereas the
prevalence of Salmonella was higher. In a majority
of reports, the frequency of Salmonella was
between 4 and 8 %. Escherichia coli and Listeria
monocytogenes were in general found in a higher
frequency compared to Salmonella. A conclusion
of the report was that the presence of pathogenic
microorganisms on raw fruits and vegetables varies
considerably. Often no pathogens were detected. In
other surveys high percentages of samples
contaminated with pathogens was observed.
Surveys of the presence of parasites or viruses are
fewer because the lack of detection methods that
can be applied to fruits.
According to Eguchi [101], K medium and OSA
are both suitable growth media for A.
acidoterrestris. Five strains of A. acidoterrestris
isolated from various juices and canned tomatoes
were streaked onto OSA, Tomato Juice Agar
Special (TJAS), PDA (each adjusted to pH 3.5, 4.0,
4.5 and 5.0), DTA (pH 7.4), and K medium (pH
3.7). All five isolates grew on OSA (pH 5.0) and K
medium (pH 3.7) at 35 ºC.
According to Pettipher [102], Alicyclobacillus
failed to grow on Nutrient agar and Tryptone Soy
agar at pH 7.3, but grew well on BAM medium,
PDA and OSA. Of these media, it was determined
that OSA allowed the highest recovery.
Narta Mari et al. [103] isolated Mucor puriformis
from orchard soils and from packing house dumptank waters. Pathogen propagates were not found in
fruit sample washing. The population of the
pathogen peopagules fluctuated in an annual cyclic
pattern declining in warm months and increasing
after harvest. The viability of sporangiospores was
markedly affected by rain. There was a good
correlation between the number of recovered
propagules in the soil and the amount of rainfall.
M. piriformis isolates caused decay on pear at 0 °C
after 14 days.
Wisse and Parrish [99] studied the occurrence of
spore-forming
thermo-acidophilic
bacteria,
including Alicyclobacillus and Sulfobacillus, in
citrus fruit growing and processing environments.
Isolates were obtained from seven of eighteen soil
samples taken from citrus orchards, surfaces of
unwashed fruit at eight of ten processing plants, on
surfaces of six of nine washed fruits and in
condensate water used to wash fruits in six of seven
facilities examined. Finding these bacteria in
condensate water generated in the processing of
citrus juice concentrates was significant because it
indicates that the microorganisms are likely to be
present in the water used to wash fruits. MPN based population estimates of washed and
unwashed fruits showed that approximately 46
spores/fruit of spore-forming, thermo-acidophilic
rods (STAR) were detected. Isolation of STAR
from fruit surfaces was expected due to cross
contamination with soil or other contaminated
fruits
during
fruit
growing
and
fruit
harvesting/handling
procedures.
However,
researchers were surprised that STAR was found
on washed fruit from six plants. Although, this may
be due to the fact that there were substantial
numbers of STAR spores in condensate water used
for fruit washing.
Mickee et al. [104] analyzed red rasp berry fruit
spreads sweetened with sugar or raspberry, red
grape and apple juice concentrates for chemical,
physical and sensory properties at 1.12 and 24
weeks. All pH values were between 3.0 and 3.5
while all aw were above 0.81 samples were dark red
but become duller over time. Mold was detected in
all samples at 24 weeks. Acceptable but inversion
processing was not recommended for fruit spread
preparation.
John Moore et al. [105] isolated a Gram negative
Bacillus from a batch of fruit flavored bottled
water, which had spoiled as a result of bacterial
overgrowth (>106 CFU/ml). The spoilage organism
was extremely difficult to identify phenotypically
and was poorly identified as Pasturella sp.
employing the identification scheme, which gave
Zook et al. [100] determined high pressure
inactivation kinetics (D and z values) of
64
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
the profile 5040000 base pairs. Molecular
identification through PCR amplification of a
partial region of the 16S rRNA gene followed by
direct automated sequencing of the PCR amplicon
allowed identification of the organism.
spoiled within 1 week. However, the addition of
2000 ppm vanillin resulted in suppression of the
growth of spoilage microbes. This level of vanillin
did not affect survival of acid-adapted Escherichia
coli. 1000 ppm vanillin was also effective in
controlling growth, but lower concentrations only
briefly delayed the onset of microbial spoilage.
Nisin was ineffective in preventing spoilage, and in
a test of yogurt containing fresh peaches, nisin
hastened growth of spoilage microbes.
Onimawo
et
al.
[106]
conducted
the
physicochemical and nutrient evaluations of
African bush mango seeds and pulp. The seeds
contained 3.36 %, 7.70 %, 65.46 %, 2.26 %, 10.23
% and 10.7 % of moisture, crude protein, crude fat
and mineral ash. The physicochemical analysis of
pulp showed that it contained 0.112 cm3 titrable
acidity, 0.21 %. Water soluble ash, 459.7 mg/100
ml reducing sugars, 49.1 % non reducing sugar,
10.0 % total solids, 1.2 × 103 lvsm-2 viscosity and
1.012 specific gravity. Ascorbic acid and calcium
contents were 66.7 mg/100 ml and 262.3 mg/100
ml, respectively. The pulp was slightly acidic (pH
5.8) which indicates that it may not be easily
spoiled by microorganisms.
Adisa [110] investigated the production of
amylolytic, cellulolytic and pectinolytic enzymes
by Aspergillus flavus and A. fumigatus. The two
fungi were cultured on wheat offal and liquid
crystalline carboxy methyl cellulose media. A.
flavus produced amylases on basal and starch
containing media while A. fumigatus could only
produce amylases on starch medium. The
cellulolytic activities of filtrates from culture or
infected fruits showed that A. flavus produced
lesser quantities of cellulolytic enzymes than A.
fumigatus. At 25 °C and at a pH range of 6 – 8, A.
flavus best produces amylases and cellulases, while
Aspergillus fumigatus showed highest activities of
the two enzymes at 35 - 40 °C and at pH 7.0. Two
pectinolytic enzymes polymethylgalacturonase and
pectinmethyltrans - eliminase were identified in
vivo with the two molds. An endo
polygalacturonase in addition to these two
pectinolytic enzymes was well associated with A.
fumigatus.
Dilnisi et al. [107] screened Lasiodiplodia
theobtomae,
Thielaviopsis
paradoxa,
Colletotrichum
musae,
Colletotrichum
gloeosporioides, Fusarium verticillioides and
Fusarium oxysporum for sensitivity to Na2CO3,
NaHCO3, CaCl2 and NaCl2. The spore germination
of all pathogens was completely inhibited by
Na2CO3 4 g/L, NaCl 5 g/L and NaHCO3, CaCl2,
and NaCl2 6 g/L each. Dipping the incidence of
crown rot 17 days after harvest in fruits treated
with NaCl2 by 67 % with NaHCO3 by 62 %, with
NaCl by 38 % and with CaCl2 by 33 % Na2CO3
treated fruits had the same incidence of crown rot
as untreated fruits.
Akpan and Kovo [111] examined the production
and preservation of Passion Fruit Juice to reduce
the spoilage and to increase the shelf life of the
juice. The preservation of the juice was carried out
using sugar, benzoic acid, citric and a combination
of citric and benzoic acid under room temperature.
The result revealed that the juice maintained its
colour, aroma and tastes for at least one month
when 30 % benzoic acid was used as preservative.
The juice under other preservation like 4 % sugar
went bad after three days, while that of 4 % citric
acid maintained its qualities for one week and some
days, but thereafter the aroma started to fade. The
combination of 3 % benzoic acid and 4 % citric
acid maintained the qualities of the juice fairly
between two to three weeks. The preservation used
also altered the pH so that it was impossible for
pathogens to exist at such a low pH environment.
Sulali Anthony et al. [108] collected the crown out
pathogen isolated from banana samples from 12
locations and the collected pathogens were
Lasiodiplodia theobromae, Fusarium, Proliferatum
and Colleotrichum musae. Fungal pathogens
isolated were able to cause crown rot disease alone
as in combination. Disease severity was highest
when combination of virulent pathogens were used
Cymbopogon nardus and Ocimum basilicum oils
displayed fungicidal activity against C. musae and
F. proliferatum between 0.2 - 0.6 % (v/v) in a
poisoned food bioassay.
Victoria Penney et al. [109] proposed that fruit
yogurt made with minimally processed ‘fresh’ fruit
has the potential to increase consumption rates of
yogurt. The efficacy of vanillin, nisin and fresh
cranberries to control microbial spoilage of a fresh
fruit yogurt containing wild blueberries was tested.
After introducing wild blueberries, yogurt
contained a large community of yeast and bacterial
cells. Yogurt with only wild blueberries was visibly
Vasantha Rupasinghe et al. [112] tested the
antimicrobial effect of vanillin against four
pathogenic or indicator organisms: Escherichia
coli, Pseudomonas aeruginosa, Enterobacter
aerogenes and Salmonella enterica subsp. enterica
serovar Newport and four spoilage organisms;
Candida albicans, Lactobacillus casei, Penicillium
65
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
expansum and Saccharomyces cerevisiae that could
be associated with contaminated fresh - cut produce
was
examined.
The
minimal
inhibitory
concentration (MIC) of vanillin was dependent
upon the microorganism and this ranged between 6
and 18 mM. When incorporated with a commercial
anti-browning dipping solution calcium ascorbate,
Nature Seal, 12 mM vanillin inhibited the total
aerobic microbial growth by 37 % and 66 % in
fresh-cut ‘Empire’ and ‘Crispin’ apples,
respectively, during storage at 4 °C for 19 days.
Vanillin (12 mM) did not influence the control of
enzymatic browning and softening by Nature Seal.
These results provide a new insight for vanillin as a
potential antimicrobial agent for refrigerated freshcut fruits.
propolis showed greater antifungal activity than
sodium benzoate.
Izuagie and Izuagie [115] determined the ascorbic
acid content of the juices of four different citrus
fruits – orange, tangerine, grapefruit and lime in
order to know which fruit would best supply the
ascorbic acid need for the body. The results of their
research showed that the orange had the highest
value of ascorbic acid, 600 μg/ml followed by
grape, 446 μg/ml and then tangerine, 415 μg/ml.
Lime had the least value, 306 μg/ml. It follows that
orange would supply more ascorbic acid per
millilitre for body need compare to the other three
fruits. In fact, the value of ascorbic acid in orange
was about twice that of lime.
Magashi Abdulkadir and Bukar Aminu [116]
determined the antibacterial and antifungal effects
of high pH (9, 10) and paraffin wax. Determination
of antibacterial and antifungal activity of the
combined treatments was achieved by aerobic
mesophilic count of bacteria and fungi on the
surface of the tomatoes, peppers and oranges using
serial dilution and pour plate techniques and
compared prior to and after 4 days of treatment
with buffer (pH 9, 10) and wax for 3 min using
dipping method. Reduction in bacterial and fungal
count indicates antifungal and antibacterial activity.
A bacterial count reduction of 84.3 (control), 63.4
(pH 9) and 78.2 % (pH 10) and fungal count
reduction of 53.6 (control), 43.4 (pH 9) and 73.5
(pH 10) were achieved after 4 days of treatment
respectively. Their study showed that the control
(unwaxed) had similar antibacterial and antifungal
effect as waxed fruits at pH 9 and 10, except for pH
10 that had higher reduction of fungal counts than
the control, showing prospect of higher activity
with wax at higher pH than 10 [117].
Wissanee Supraditareporn and Renu Pinthong
[113] conducted experiments to study the physical,
chemical and microbiological properties of fresh
orange juices immediately after harvest and
different storage periods and storage temperatures.
Some of the parameters could be used as indicator
of quality loss of the juices such as colour and, total
soluble solid, titratable acidity, ascorbic acid and
total plate counts varied with storage time and
temperature. The shelf life of orange juices had
only 1 day shelf life at 25 ºC, 6 days at 4 ºC and
more than 21 days at -18 ºC. The orange juice at 18 ºC still has a good quality throughout the
storage time; however, ascorbic acid contents were
reduced.
Ayse Nedret Koc et al. [114] examined the
antifungal effect of ethanol extract of Turkish
propolis (EETP) treatments in four non-pasteurized
fruit juices including apple, orange, white grape
and mandarin against 6 different yeasts isolated
from the corresponding spoiled juices. These
isolated yeasts include: Candida famata, C.
glabrata, C. kefyr, C. pelliculosa, C. Parapsilosis
and Pichia ohmeri. Minimum Inhibitory
Concentration (MIC) ranges were determined
responding to the National Committee for Clinical
Laboratory Standards (NCCLS) M27-A that were
slightly modified with broth microdilution method.
In their study, the presence of propolis in apple (pH
= 3.9), orange (pH = 3.7), white grape (pH = 3.8)
and mandarin (pH = 3.4) juices ranging from 0.01
to 0.375 mg/ml inhibited the growth of all spoilage
yeasts at 25 °C. The MIC ranges of propolis were
0.02 – 0.375, 0.04 – 0.375, 0.01 – 0.185, 0.02 –
0.185 and 0.04 – 0.375 mg/mL in mandarin, apple,
orange, white grape juices and RPMI medium.
MIC ranges of sodium benzoate which was used as
positive control, were 80 – 320, 80 – 320, 40 – 640,
40 – 80 and 320 – 1280 mg/ml in mandarin, apple,
orange, white grape and RPMI medium as blank
control, respectively. In terms of MIC ranges,
Rosalia Trias et al. [118] evaluated the efficacy of
lactic acid bacteria (LAB) isolated from fresh fruits
as biocontrol agents against the phytopathogenic
and spoilage bacteria and fungi, Xanthomonas
campestris, Erwinia carotovora, Penicillium
expansum, Monilinia laxa and Botrytis cinerea.
The antagonistic activity of 496 LAB strains was
tested in vitro and all tested microorganisms except
Penicillium expansum were inhibited by at least
one isolate. The 496 isolates were also analyzed for
the inhibition of Penicillium expansum infection in
wounds of Golden Delicious apples. Four strains
reduced the fungal rot diameter of the apples by 20
%; only Weissella cibaria strain TM128 decreased
infection levels by 50 %.
Ermi Sukasih and Setyadjit [119] determined the
heat resistance and heat adequacy value of pure
citrus juices. The method used was heating the
tubes containing pure citrus juices with
66
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
combination of time and temperature of
pasteurization at 55 ºC, 60 ºC, 65 ºC, 70 ºC, 75 ºC
and 80 ºC during 5, 10, 15 and 20 minutes. Their
results showed that bacterium population with z
value equal to 30 ºC had higher heat resistance
value than yeast/mold population with z value
equal to 17.24 ºC. P value for 3D pasteurization of
pure citrus juices was 11.26 minutes for acid foods,
which meant that it will achieve heat adequacy
treatment if it was pasteurized at time and
temperature having P value equal to 11.26 minutes.
Perni et al. [120] described the inactivation by cold
atmospheric plasmas of one pathogenic and three
spoilage organisms on the pericarps of mangoes
and melons. The operating voltage necessary for
efficient microbial decontamination of fruit
pericarps was first established using Escherichia
coli at a concentration of 107 CFU/cm2 on the
surface of mango. It was found that, when the
plasma was sustained slightly above its breakdown
voltage of 12 kV, no inactivation was detected
when cells were plated onto Tryptone soya extract
agar (TSA). However, when plated onto Eosin
methylene
blue
agar,
sublethal
injury
corresponding to approximately 1 log reduction
was achieved, whereas on TSA supplemented with
4 % NaCl a greater reduction of 1.5 log was
revealed. When the voltage was increased by 33 %
to 16 kV, a reduction in cell counts of 3 log was
achieved on all three plating media. Further
investigations at these new operating conditions
were conducted using a range of spoilage
microorganisms all at a surface concentration of
106 CFU/cm2 on the pericarps of mango and melon.
Pantoea agglomerans and Gluconacetobacter
liquefaciens were reduced below the detection limit
after only 2.5 s on both fruits, whereas Escherichia
coli required 5 s to reach the same level of
inactivation. Saccharomyces cerevisiae was the
most resistant organism studied and was reduced in
numbers below the detection limit after 10 s on
mango and 30 s on melon. The optical emission
spectra generated by the cold atmospheric plasma
at both high and low operating voltages were
compared in order to identify putative lethal
species.
ambient temperature were also determined. After
10 days of storage, the total viable counts increased
from 0.6 × 103 cfu/g to 5.3 × 103 cfu/g and to 5.5 ×
103 cfu/g in the HDP- and LDP-packaged
watermelon samples, respectively. The total fungal
counts increased from 0.5 × 103 cfu/g to 6.7 × 103
cfu/g and to 7.2 × 103 cfu/g in the HDP- and LDPpackaged watermelon samples.
Reddy et al. [122] estimated the losses caused by
post-harvest fungal diseases in sweet orange and
acid lime at field, wholesale, retail and consumer
levels. The extent of loss due to the post-harvest
fungal spoilage was varied at different stages of
marketing. The post-harvest fungal spoilage was
mostly due to green mold (Penicillium digitatum),
black mold (Aspergillus niger) and sour rot
(Geotrichum candidum) while the other diseases
were only to limited extent. Generally, the fungal
spoilage was more in sweet orange compared to
acid lime. The extent of damage was high at retail
level which was 43.8 % in sweet orange and 36.8
% in acid lime respectively.
Tamaliza et al. [123] evaluated Bacillus
licheniformis for the control of gray mold of apple
caused by Botrytis mali. Dual culture cell free
metabolite and volatile tests showed that Bacillus
licheniformis inhibited growth of the pathogen.
Bacillus lichniformis appeared to be a good
antagonist of gray mold on apple 20 °C and 4 °C. It
reduced Bacillus lesion diameter to 9 - 11 mm
compared with to 32 - 41 mm in the control at 4 °C.
At 20 °C, the lesion diameter was reduced to 3.5 8.4 mm for the antagonistic treatment and to 24.8 38.2 mm for the control treatment after 14 days.
Juan Calvo et al. [124] assessed the antagonistic
activity of the mixtures Rahnella aquatitis,
Rhodotorula
glutinis
and
R.
aquatitis,
Cryptococcus laurentii against Penicillium
expansum (cause of blue rot) and Botrytis cinerea
(cause of grey rot) on apple fruit at 4 °C and 9.5 %
relative humidity (RH). Under these cold storage
conditions, the mixture R. aquatitis - R. glutinis
inhibited the development of B. cinerea and
Penicillium expansum in apples stored for 40 days
and reduced the incidence of disease produced by
these moulds to nearly zero.
Nwachukwu et al. [121] collected freshly sliced
watermelon from different street vendors to
determine their microbiological quality. Eight
different microbial isolates were obtained from the
sliced watermelon samples, namely Escherichia
coli, Klebsiella aerogenes, Proteus mirabilis,
Staphylococcus aureus, Lactobacillus spp.,
Saccharomyces cerevisiae, Rhizopus stolonifer and
Mucor spp. The effects of high density
polyethylene (HDP) and low density polyethylene
(LDP) packaging bags on the microbiological
quality of freshly sliced watermelon, stored at
Behnas Solaimani et al. [125] investigated the
antifungal effects of the herbal essential oil of
Shiraz thyme against the producing agent of
Penicillium digitatum and Penicillium italicum on
the Washington Navel Orange fruit. In their study,
treatments were three level of Shiraz thyme
essential oil (0 µl, 200 µl and 400 µl) in the forms
of spray and dipping for 10 and 20 min in lab (in
vivo) condition. This investigation was arranged by
67
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
the Completely Randomized Design (CRD) with
three replications that each containing four fruit
and the contamination of fruits was recorded for 4
week. The chemical composition of essential oils
isolated by hydrodistillation from the aerial parts of
Shiraz thyme was analyzed by GC and GC–MS.
Carvacrol (63.17 %), thymol (15.1 %), p-cymene
(7.87 %), linalool (3.88 %), á-pinene (3.19 %) and
Carvacrol methyl ether (1.92 %) were found to be
the main constituents in Zataria multiflora essential
oil. The results indicated that the essence of Shiraz
thyme was not effective on the green fungus and
the highest preventing effect of the essence was in
the joint from related to the mixture of the three
essences treatment.
paper and incubated at 25 °C to allow the growth of
fungi for a period of 1 week. Fungal species
developed on seeds and fruit pieces were isolated
on Potato dextrose agar for identification. Twenty
species from 14 genera of fungi have been isolated
from 13 different varieties of date-palm as seedborne fungi while 39 species of 16 genera of fungi
were isolated as fruit spoilage fungi. Alternaria
alternata, Aspergillus flavus, Aspergillus niger,
Fusarium oxysporum and Fusarium solani were the
predominant species in both seed-borne and fruit
spoilage fungi.
Galgozy et al. [129] evaluated the antibacterial
effect of fruit juices and pomace extracts from 13
wild and cultivated fruits (Prunus avium, P.
cerasus, P. armeniaca, Crataegus monogyna,
Morus alba, M. nigra, Ribes nigrum, R. rubrum, R.
uvacrispa, R. nidigrolaria, Rubus idaeus and R.
fruticosus) against two foodborne enteric pathogens
(Salmonella ser. Typhimurium and Campylobacter
jejuni) by Broth micro dilution assays. Juices and
extracts of sour cherry, apricot, raspberry,
blackcurrant, redcurrant, gooseberry and jostaberry
efficiently inhibited the growth of both bacteria
(growth ≤ 25 %). Juices and extracts from cherry
(red and yellow cultivars), hawthorn, blackberry
and pomace extracts from black and white
mulberry had a similar strong inhibitory effect on
the growth of C. jejuni, but had weak or no effect
on Salmonella typhimurium. Sour cherry, josta
berry and raspberry pomace extracts revealed a
substantial antibacterial effect at both acidic and
neutral pH.
Zamani et al. [126] used the antagonistic
bacterium, Pantoea agglomerans for controlling
citrus green mould caused by Penicillium digitatum
at 20 °C and 4 °C. This isolate was also assessed in
combination with dipping in 3 % sodium
bicarbonate solution at 24 °C and 45 °C on
artificially inoculated Thomson navel oranges.
Application of the antagonist alone reduced green
mould by more than 75 % at both temperatures, but
was not as effective as Imazalil. The antagonistic
bacterium was completely tolerant to sodium
bicarbonate upto a concentration of 3 %. In
addition, its efficacy for controlling green mould
was improved at least by 5 % and 11 % when
combined with 3 % sodium bicarbonate at 24 °C
and 45 °C.
Abhinaba Gosh [127] found out the organisms
which make tomato more susceptible to spoilage.
Out of the 30 Rose Bengal agar plates which were
inoculated and incubated vigorous growth of fungi
was observed in 26 plates with moderate growth on
other 4 plates. Also 30 other plates of Nutrient agar
were inoculated with the sample and very scanty
growth of bacterial colonies was observed in 3 - 4
plates and mostly occupied by fungal colonies.
Thus, it was found that fungi were the source of
spoilage in most of the samples rather than
bacteria. Further morphological studies were done
to know the fungal member responsible for the
spoilage. Among the fungi, it was found that
Aspergillus niger and Fusarium were found in most
of the spoiled samples with a few samples
containing Penicillium too with Aspergillus niger
dominating all the plates.
Akpan and Kovo [130] examined the production
and preservation of Passion Fruit Juice to reduce
the spoilage and to increase the shelf life of the
juice using chemical
preservatives. The
preservation of the juice was carried out using
sugar, benzoic acid, citric and a combination of
citric and benzoic acid under room temperature.
The result revealed that the juice maintained its
color, aroma and tastes for at least one month when
30 % benzoic acid was used as preservative. This
happens to be the best among all. The juice under
other preservation like 4 % sugar went bad after
three days, while that of 4 % citric acid maintained
its qualities for one week and some days, but
thereafter the aroma started to fade. The
combination of 3 % benzoic acid and 4 % citric
acid maintained the qualities of the juice fairly
between two to three weeks.
Hashiem Al Sheik [128] collected the seeds and
fruits of different date palm varieties from local
market, where further experiments for isolation of
fruit spoilage and seed-borne fungi were conducted
by using common technique of wet blotter method.
A total number of 100 seeds and 100 cubes
obtained from the fruits were put on wet filter
Gobbi et al. [131] described the application of an
electronic nose equipped with a Metal Oxide
Semiconductor sensor array for the detection of
Alicyclobacillus
acidoterrestris
and
A.
acidocaldarius artificially inoculated in peach,
68
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
orange and apple fruit juices. Overall the system
was able to detect the presence of Alicyclobacillus
spp. in all the tested fruit juices at 24 hrs from
inoculation. The electronic nose could detect
bacterial concentration as low as <102 colony
forming unit/ml and it was also able to classify
bacterial contamination independently of the
Alicyclobacillus species. The Gas Chromatography
– Mass Spectrometry (GC-MS) characterization of
the volatile profile of orange juices confirmed the
existence of quantitatively different patterns
between contaminated and uncontaminated
samples.
Isabel Alegre et al. [134] proposed that Escherichia
coli, Salmonella and Listeria innocua increased by
more than 2 log10 units over a 24 hrs period on
fresh cut Golden Delicious apple pings stored at 25
°C and 20 °C. Listeria innoua reached the same
final population level at 10 °C meanwhile
Escherichia coli and Salmonella only increased 1.3
log10 units after 6 days. Only Listeria innoua was
able to grow at 5 °C. No significant differences
were observed between the growths of food born
pathogen on fresh-cut Golden Delicious. Granny
Smith and Shampion apples stored at 25 °C and 5
°C. These results highlight the importance of
avoiding contamination of fresh cut fruit with food
borne pathogens and the maintenance of the cold
chain during storage until consumption.
Ethiraj and Suresh [132] studied the nature and
distribution of microorganisms associated during
processing of mango. Bacteria outnumbered yeasts
in both unwashed and washed fruits. Washing the
fruits in running water reduced the surface flora
considerably. Because of low pH and high sugar
content, mango products are highly susceptible to
spoilage by yeasts. Therefore, the yeast flora
isolated during different stages of processing was
identified. Species of Kloeckera and Hyphopichia
in unwashed fruit and Kloeckera and Pichia in
washed fruits were the predominant yeasts.
However, flesh from both unwashed and washed
fruits contained species of Kloeckera, Hyphopichia
and Candida as the major yeasts. Species of
Candida, Kloeckera and Kluyveromyces were the
predominant yeasts in the unheated raw mango
pulp whereas heated pulp did not show the
presence of any yeast. Effect of sodium benzoate,
potassium sorbate and potassium metabisulphite on
growth of some predominant yeast was studied. It
was found that the sodium benzoate at 500 ppm
level inhibited all the yeasts except Saccharomyces
ludwigii, while potassium sorbate and potassium
metabisulphite at the same concentration inhibited
all the yeasts.
Jageethadevi et al. [135] investigated the inhibitory
effect of chemical preservatives and organic acids
on the growth of bacterial pathogens. Decrease in
the growth of all the four bacteria were observed
with increase in the concentration of acetic acid and
citric acids. The growth of all the bacterial culture
were effectively inhibited at 1000 µg ml-1 and
lower inhibition zone was found at 200 µg ml-1.
The inhibitory effect on the bacterial culture was
more in acetic acid compared to citric acids. The
effect of preservatives (potassium sorbate and
calcium propionate) on the inhibition of growth of
bacteria was studied and for all the cultures, the
inhibition zone area increased with increase in the
concentration of the preservatives. Vibrio
parahaemolyticus, Shigella sonnei, Staphylococcus
aureus and Salmonella typhimurium were
effectively inhibited at 1400 µg ml-1. The inhibitory
effect for all the bacteria was more in potassium
sorbate compared to calcium propionate.
CONCLUSION
The present review concludes that the organic acids
and hydrogen peroxide are of predominant efficacy
in preservation of fresh fruit juices from the
bacterial, fungal and other microbial isolates.
Majority of the previous research findings
proposed that the organic acids are highly effective
in the biological control of fruit juice spoilage
causing bacteria when compared to hydrogen
peroxide.
Foley et al. [133] determined the effects of
different doses of gamma irradiation on reducing
the microbial pathogens Listeria monocytogenes
and Salmonella enterica in fresh orange juice, and
to determine whether significant reduction could be
achieved without compromising sensory qualities.
While irradiation was effective in destroying
pathogens, the development of off flavours
precludes its use as an alternative processing
technology.
69
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
Parish, M. E. Critical Reviews in Microbiology 1997; 23 (2): 109 – 119.
CDC (Centers for Disease Control and Prevention). MMWR 1997; 46 (1): 4 – 8.
Besser, R. E., S. M. Lett, J. T. Weber, M. P. Doyle, T. J. Barrett, J. G. Wells and P. M. Griffin. JAMA
1993; 269 (17): 2217 - 2220.
Conner, D. E and J. S. Kotrola. Applied Environmental Microbiology 1995; 61(1): 382 – 385.
Wallace, J. S., T. Cheasty and K. Jones. Journal of Applied Microbiology 1997; 82: 399 - 404.
Parish, M. E. Critical Reviews in Microbiology 1997; 23(2): 109 – 119.
Conner, D. E and J. S. Kotrola. Applied Environmental Microbiology 1995; 61 (1): 382 – 385.
Nagy, S., C. S. Chen and P. E. Shaw. Fruit Juice Processing Technology 1993; Agri. science, Inc.,
Auburndale, Florida.
Chung, K. C and J. M. Goepfert. Journal of Food Science 1970; 35: 326 - 328.
Luck, E and M. Jager. Antimicrobial Food Additives: Characteristics, Uses, Effects 1997; 2nd edition
Springer - Verlag, Germany.
Liao, C and G. M. Sapers. Journal of Food Protection 2000; 63 (7): 876 - 883.
Davidson, M. P and A. L. Branen. Antimicrobials in Foods 1993; 2nd ed. Marcel Dekker, New York.
Sapers, G. M and G. F. Simmons. Food Technology 1998; 52 (2): 48 – 52.
Juven, B. J and M. D. Pierson. Journal of Food Protection 1996; 59 (11): 1233 - 1241.
Nagy, S., C. S. Chen and P. E. Shaw. Fruit Juice Processing Technology 1993; Agri. science, Inc.,
Auburndale, Florida.
Ryu, J. H., Y. Deng and L. R. Beuchat. Journal of Food Protection 1999; 62 (5): 451 - 455.
Beuchat, L.R and J. Ryy. Emerging infectious diseases 1997; 8 (4): 459 - 465.
Lund, B. M. Journal of Applied Bacteriology 1992; 73 (21): 115 - 135.
ICMSF. 1998. Microbial Ecology of Food Commodities. Microorganisms in Foods. Blackie Academic
& Professional.
Murphy, J. Food Chemical News 1999; 41 (2): 24.
FDA. Food and Drug Administration: U.S. Dept. of Health and Human Services. 2001. FDA publishes
final rule to increase safety of fruit and vegetable juices.
Parish, M. Fruit Processing 2000; 7: 246 - 250.
CFR. 2001. January 19 Hazard analysis and critical control point (HACCP); Procedures for the safe
and sanitary processing and importing of juice.
Jay, J. M. 1996. Modern Food Microbiology. 5th ed. Chapman and Hall, New York.
Wallace, J. S., T. Cheasty and K. Jones. Journal of Applied Microbiology 1997; 82: 399 - 404.
Jay, J. M. 1996. Modern Food Microbiology. 5th ed. Chapman and Hall, New York.
Parish, M. E. Critical Reviews in Microbiology 1997; 23(2): 109 – 119.
Besser, R. E., S. M. Lett, J. T. Weber, M. P. Doyle, T. J. Barrett, J. G. Wells and P. M. Griffin. JAMA
1993; 269 (17): 2217 - 2220.
Liao, C., G. M. Sapers. Journal of Food Protection 2000; 63 (7): 876 - 883.
Nagy, S., C. S. Chen and P. E. Shaw. Fruit Juice Processing Technology 1993; Agri. science, Inc.,
Auburndale, Florida.
Zhuang, R. Y., L. R. Beuchat and F. J. Angulo. Applied Environmental Microbiology 1995; 61 (6):
2127 - 2131.
Buchanan, R. L., S. G. Edelson, R. L. Miller and G. M. Sapers. Journal of Food Protection 1999; 62
(5): 444 - 450.
Merker, R., S. Edelson Mammel, V. Davis and R. L. Buchanan. 1999. Preliminary experiments on the
effects of temperature difference on dye uptake by oranges and grapefruits. U.S. Food and Drug
Administration.
Wells, J. M and J. E. Butterfield. Plant Disease 1997; 81: 867 - 872.
Nagy, S., C. S. Chen and P. E. Shaw. Fruit Juice Processing Technology 1993; Agri. science, Inc.,
Auburndale, Florida.
Fennema, O. R. Food Chemistry 1996; 3rd ed. Marcel Dekker, Inc., New York.
Zhuang, R. Y., L. R. Beuchat and F. J. Angulo. Applied Environmental Microbiology 1995; 61 (6):
2127 - 2131.
Saranraj, P., M. Geetha. International Journal of Pharmaceutical and Biological Archives 2012; 3 (1):
204 - 214.
Baldy, M. W. 1998. The University Wine Course. The Wine Appreciation Guild, San Francisco, CA.
Fennema, O. R. Food Chemistry 1996; 3rd ed. Marcel Dekker, Inc., New York.
Arthey, D and P. R. Ashurst. Fruit Processing 1996; Chapman and Hall, Great Britain.
70
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
Uljas, H. E and S. C. Ingham. Journal of Food Protection 1998; 61(8): 939 - 947.
Bruice, P.Y. Journal of Food Science 1995; 35: 326 - 328.
Luck, E and M. Jager. Antimicrobial food additives: characteristics, uses, effects 1997; 2nd edition
Springer - Verlag, Germany.
Chung, K. C and J. M. Goepfert. Journal of Food Science 1970; 35: 326 - 328.
Conner, D. E and J. S. Kotrola. Applied Environmental Microbiology 1995; 61(1): 382 – 385.
Uljas, H. E and S. C. Ingham. Journal of Food Protection 1998; 61 (8): 939 - 947
Davidson, M. P and A. L. Branen. Antimicrobials in Foods 1993; 2nd ed. Marcel Dekker, Inc., New
York.
Subramanian, C. S and E. H. Marth. Journal of Milk and Food Technology 1968; 31: 323.
Fischer, J. R., D. L. Fletcher, N. A. Cox, and J. S. Bailey. Journal of Food Protection 1985; 4: 252.
Arthey, D and P. R. Ashurst. Fruit Processing 1996; Chapman and Hall, Great Britain.
Darwina, M., D. Kanchana, P. Saranraj. Novus International Journal of Biotechnology and Biosciences
2012; 1 (1): 1 - 13.
Davidson, M. P and A. L. Branen. Antimicrobials in Foods 1993; 2nd ed. Marcel Dekker, Inc., New
York.
Ryu, J. H., Y. Deng and L. R. Beuchat. Journal of Food Protection 1999; 62 (5): 451 - 455.
Davidson, M. P and A. L. Branen. Antimicrobials in Foods 1993; 2nd ed. Marcel Dekker, Inc., New
York.
Luck, E and M. Jager. Antimicrobial food additives: characteristics, uses, effects 1997; 2nd edition
Springer - Verlag, Germany.
Saranraj, P., D. Stella and D. Reetha. International Journal of Natural Product Science 2012; 2 (2): 1 12.
Juven, B. J and M. D. Pierson. Journal of Food Protection 1996; 59 (11): 1233 – 1241.
Brock, T. D., M. T. Madigan, J. M. Martinko and J. Parker. Biology of Microorganisms 1997; Prentice
Hall, Upper Saddle River, NJ.
Imlay, J. A and S. Linn. Journal of Bacteriology 1987; 169 (7): 2967 - 2976.
Cadenas, E. Annual Reviews in Biochemistry 1989; 58: 79 – 110.
Juven, B. J and M. D. Pierson. Journal of Food Protection 1996; 59 (11): 1233 – 1241.
Kanchana, D., R. Kavitha, P. Saranraj. Advances in Biological Research 2015; 9 (4): 253 – 256.
Repine, J. E., R. B. Fox and E. M. Berger. Journal of Biological Chemistry 1981; 256 (14): 7094 7096.
Imlay, J. A., S. M. Chin and S. Linn. Science 1988; 240: 640 - 642.
Saranraj, P., P. Sivasakthivelan, S. Kavi Karunya and N. K. Shanmugavel. Science Park Research
Journal 2015; 3 (25): 1 – 8.
Lillard, H. S and J. E. Thomson. Journal of Food Science 1983; 48: 125 - 126.
Turner, F. J. 1983. Hydrogen peroxide and other oxidant disinfectants. In Disinfection, Sterilization and
Preservation. 3rd ed. edited by S.S. Block, Lea, and Febiger, Philadelphia. 240 -250.
Baldry, M.G.C. The Journal of Applied Bacteriology 1983; 54: 417 - 423.
Klapes, N. A, D. Vesley. Applied Environmental Microbiology 1990; 56 (2): 503 - 506.
Yoshpe Purer, M. S and E. Eylan. Health Laboratory Science 1968; 5 (4): 233 - 238.
Kanchana, D., P. Saranraj, R. Kavitha. World Journal of Fish and Marine Sciences 2015; 7 (4): 243 –
246.
Prem Kumar, D., M. Jayanthi, P. Saranraj, S. Kavi Karunya. Life Science Archives 2015; 1 (4): 217 –
222.
Sapers, G. M and G. F. Simmons. Food Technology 1998; 52 (2): 48 – 52.
Forney,
C.
F.,
R.
E.
Rij,
R.
Dennis
Arrue
and
J.
L.
Smilanick.
Horticulture Science 1991; 26 (12): 1512 - 1514.
Liao, C and G. M. Sapers. Journal of Food Protection 2000; 63 (7): 876 - 883.
Sapers, G. M and G. F. Simmons. Food Technology 1998; 52 (2): 48 – 52.
Toledo, R. T., F. E. Escher and J. C. Ayers. Applied Microbiology 1973; 26: 592 - 567.
Forney, C. F., R. E. Rij, R. Dennis Arrue and J. L. Smilanick. Horticulture Science 1991; 26 (12): 1512
- 1514.
Kuchma, T. Journal Microwave Power and Electromagnetic Energy 1998; 33 (2): 77 – 87.
Baldy, M. W. 1998. The University Wine Course. The Wine Appreciation Guild, San Francisco, CA.
Phillips, G. F and J. O. Mundt. Food Technology 1950; 4: 291 - 293.
Wolf, P. A. Food Technology 1950; 4: 294 - 297.
Von Schelhorn, M. Food Technology 1953; 7: 97 - 101.
Smith, D. P., and N. J. Rollin. Food Research 1954; 19: 59 - 65.
71
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
Ingram, M., F. J. H. Ottaway, and J. B. M. Coppock. Chemistry and Industry 1956: 1154 - 1163.
Anonymous. 1961. Food regulations. Food Processing, 22 (9): 29.
Saranraj, P., P. Sivasakthivelan, S. Sivasakthi and D. Sujitha. Weekly Science Research Journal 2015;
3 (24): 1 – 8.
Bell, T. A., J. L. Etchells and A. F. Borg. Journal of Bacteriology 1959; 77: 573 – 580
Robinson, J. F and C. H. Hills. Food Technology 1959; 13: 251 - 253.
Deinhard, P., K. Blanz, K. Poralla, and E. Altan. System and Applied Microbiology 1987; 10: 47-53.
McIntyre, U., Rice, S., L. Land and L. R. Beuhat. Applied and Environmental Microbiology 1995: 791
- 796.
Previdi, M. P., S. Quintavalla, C. Lusardi, and E. Vicini. Industrial Conserve 1997; 72: 353 - 358.
Walls, I. and R. Chuyate. Food Australia 1998; 52 (7): 286 - 288.
Lewis, C. Critical controls for juice safety. FDA Consumer 1998, 32(5): 16 - 20.
FDA. Food and Drug Administration: U.S. Dept. of Health and Human Services. 2001. FDA publishes
final rule to increase safety of fruit and vegetable juices.
Beuchat, L. R. 1998. Surface decontamination of fruits and vegetables eaten raw: A review. Food
Safety Unit, World Health Organization WHO/FSF/FOS/98.2.
Prem Kumar, D., M. Jayanthi, P. Saranraj and S. Kavi Karunya. Indo-Asian Journal of
Multidisciplinary Research 2015; 1 (3): 273 – 279.
Wisse, C. C. and M. Parrish. Dairy, Food, Environment and Sanitation 1998; 18 (8): 504 - 509.
Zook, C. D., M. E. Parish, R. J. Braddock, and M. O. Balaban. Journal of Food Science 1999; 64 (3): 1
- 3.
Eguchi, S.Y., G.P. Manfio, M.E. Pinhatti, E. Azuma, and S.F. Variane. 1999. Acidothermophilic spore
forming bacteria (ATSB) in orange juices: detection methods, ecology, and involvement in the
deterioration of fruit juices.
Pettipher, G. L and M. E. Osmundson. Food Australia 2000; 52(7): 293 - 295.
Narta Mari, L., Mohan, M. S and K. S. Manja. 2000. Evaluation of coliform presence in food samples
by a simple qualitative test. AMI abstract, D.F.R.L., Mysore - 38.
Mickee, L. H., Matook Saif Mokbel and Toshihiko Suganuma. Plant foods for Human Nutrition 2000;
57: 343 - 352.
John Moore, L., Khan, S. H and Faqir Muhammad. Journal of Agriculture and Social Science 2002; 14
-15.
Onimawo, I.A, Miguel, A and Come-Lim. Plant Foods for Human Nutrition 2003; 58: 1 - 6.
Dilnisi, G, Eckert, J. W., Ogawa, J. M. Journal of Genetics And Plant Pathology 2004; 70: 61 - 65.
Sulali Anthony, John Tournas and Eugenia Katsoudes. Mycopathologia 2004; 157: 91 - 97.
Victoria Penney, Gemma Henderson, Carolyn Blum and Perry Johnson Green. Innovative Food
Science and Emerging Technologies 2004; 5(3): 369 - 375.
Adisa, V. A. Biomedical and Life Sciences 2004; 91 (2): 101 -108.
Akpan, P. E. and D. K. Kovo. 2005. Fruit and Vegetable Juice Processing Technology, Ail Publishing
Co. Inc., New York, p. 342 - 370.
Vasantha Rupasinghe, H.P., Jeanine Boulter - Bitzer, Taehyun Ahn and Joseph A. Odumeru. Food
Research International 2006; 39(5): 575 – 580.
Wissanee Supraditareporn and Renu Pinthong. International journal of Agriculture and Biology 2007; 9
(5): 726 – 730.
Ayse Nedret Koc, Sibel Silici, Fatma Mutlu-Sariguzel1 and Osman Sagdic. Food Technology and
Biotechnology 2007; 45 (1): 57 – 61.
Izuagie, A. A and F. O. Izuagie. Research Journal of Agriculture and Biological Sciences 2007; 3 (5):
367 - 369.
Magashi Abdulkadir, M and N. Bukar Aminu. African Journal of Biotechnology 2007; 6 (6): 720 - 722.
Ayse Nedret Koc, Sibel Silici, Fatma Mutlu-Sariguzel1 and Osman Sagdic. Food Technology and
Biotechnology 2007; 45 (1): 57 – 61.
Rosalia Trias, Lluis Baneras, Emilio Montesinos, Esther Badosa. International Microbiology 2008; 11:
231 - 236.
Ermi Sukasih and Setyadjit. Indonesia Journal of Agriculture 2008; 1(1): 22 – 27.
Perni, S., D. W. Liu, G. Shama and M. G. Kong. Journal of Food Protection 2008; 71 (2): 302 - 308.
Nwachukwu. E., C. F. Ezeama and B. N. Ezeanya. African Journal of Microbiology Research 2008; 2:
192 - 195.
Reddy, C., Raghubeer, J and A. Matches. Annual. Reviews in Microbiology 2008; 101 - 120.
Tamaliza, H., Thomas, C. J and U. Masters. Journal of Applied Bacteriology 2008; 23: 163 - 171.
Juan Calvo, S. L. Lafourcade and P. R. Gayon. Food Bioprocess Technology 2008; 4: 667 – 674.
72
Saranraj and Ramya, J Pharm Biol Sci 2016; 4(2): 58-73
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
Behnaz Solaimani, Sadrollah Ramezani, Majid Rahemi and Mohammad Jamal Saharkhiz. Advances in
Environmental Biology 2009; 3 (3): 249 - 254.
Zamani. M, A. Sharifi Tehrani, M. Ahmadzadeh, V. Hosseininaveh and Y. Mostofy. Journal of Plant
Pathology 2009; 91 (2): 437 - 442.
Abhinaba Gosh, M., Anand, J. C. M and D. S. Johar. Journal of Science and Technology Industrial
Research 2009; 16 (A): 270 - 272.
Hashiem Al Sheik, N. Journal of Science and Industry Research 2009; 118: 480 - 481.
Galgoczy, L., T. Hevér, L. Orosz, J. Krisch, C. Vagvolgyi, M. Tolgyesi and T. Papp. The Internet
Journal of Microbiology 2009; 7 (1): 13 - 17.
Akpan, P. E. and D. K. Kovo. Journal of Food Science 2010; 46: 677 - 680.
Gobbi, M., M. Falasconi, I. Concina, G. Mantero, F. Bianchi, M. Mattarozzi, M. Musci and G.
Sberveglieri. Food Control 2010; 21 (10): 1374 - 1382.
Ethiraj, U. G and T. Suresh. J. Sci. Food Agric., 2010; 79: 1197 – 1202.
Foley, M., Q.H. Zhang and D.B. Min. Journal of Agricultural and Food Chemistry 2010; 46: 2744 2747.
Isabel Alegre, D., Inukai, K., R. Shankar and P. Laxminarayana. Indian Journal of Dairy Science 2010;
38(3): 208 - 216.
Jageethadevi, A., P. Saranraj and N. Ramya. Asian Journal of Biochemical and Pharmaceutical
Research 2012; 1 (2): 1 – 9.
73