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REVIEW OF LITERATURE
2.1Fruit juice
The manufacture of juices from fruits and vegetables is as old (or older) than agriculture. In
simple words, juice is the extractable fluid contents of cells or tissues. It is defined as
fermentable but unfermented juice, intended for direct consumption, obtained by the
mechanical process from sound, ripe fruits, preserved exclusively by physical means. The
juice may be turbid or clear. The addition of sugars or acids can be permitted but must be
endorsed in the individual standard (Bates et al., 2001; ICMSF, 2005; Bevilacqua et al.,
2011).
Fruits and vegetables form a versatile and complex substance group category of
foods. The relevant substance groups are carbohydrates, acids, minerals, polyphenols
(tannins) including the colourful anthocyanins, water-soluble vitamins, amino acids, aroma
compounds, carotenoids, fibers and other bioactive substances. During processing, they are
essentially transferred into the pressed juice or into the puree (Bates et al., 2001).
2.2 Health benefits of fruit juices
Consumption of fruits and vegetables helps to prevent many degenerative diseases such as
cardiovascular problems and several cancers. Decades of research have found that fruits and
vegetables are crucial dietary components that can help to reduce the risk for numerous
chronic diseases which, in many cases, have been shown to be initiated by long term
inflammation. Fruit juices contain low sodium and high potassium which help in maintaining
normal blood pressure and absence of fat in fruit juices is beneficial for the cardiovascular
system. Many reports have revealed that fruit juices may play an important role in slowing
the progress of Alzheimer’s disease and development of cancer (Delichatsios and Welty,
2005; Matthews, 2006; Rico et al., 2007; Dai et al., 2006; Cutler et al., 2008; Kyle et al.,
2009; Holt et al., 2009).
2.3 pH of fruit juices
Fruit juices usually have low pH values that range between 2.0 and 4.5. Lime or lemons have
the lowest pH. The low pH of fruit juices is due to the presence of organic acids which varies
with the different type of juices (Table 2.1).
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Table 2.1. Typical pH values and the naturally occurring organic acids in fruit juices*
*Adapted from Lawlor et al., 2009
2.4 Spoilage
Food spoilage is defined as a change in the appearance, smell or taste of a food that makes it
unacceptable to the consumer (Aneja et al., 2008). Spoilage of fruit and vegetable juices is
primarily due to the proliferation of their natural acid tolerant and osmophillic microflora.
They contain high levels of sugar and possess ideal water activity for microbial growth; their
low pH (Table 2.1) makes them more susceptible to yeast and fungal spoilage because a big
part of bacterial contamination is eliminated due to the preference of bacteria to grow at
neutral pH (Worbo and Splittoesser, 2004; Patil et al., 2011; Bevilacqua et al., 2012).
2.5 Sources of contamination
Fruits and vegetables commonly used in juice processing are exposed to variety of potential
spoilage microorganisms during agricultural production, harvesting and transportation to fruit
sorting and juice extraction facilities. Most microorganisms that are initially observed on
Fruit juice pH range Major acid types
Apple 2.9-4.2 Malic , citric
Cherry 3.2-4.4 Malic , citric
Grape 2.9-4.5 Tartaric ,malic
Grapefruit 2.9-3.6 Citric
Kiwi 2.8-4.0 Citric, malic
Lemon 2.0-2.6 Citric
Lime 1.6-3.2 Citric
Mango 3.7-4.4 Citric , tartaric
Orange 3.0-4.3 Citric , malic
Pear 3.0-4.6 Malic, citric
Pineapple 3.1-4.0 Citric, malic
Raspberry 2.5-3.1 Citric
Strawberry 3.0-3.9 Citric
Tomato 3.9-4.5 Malic , citric
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whole fruit surfaces are soil inhabitants. Vectors for disseminating the microbes include soil
particles, airborne spores and irrigation water. Fruit and fruit juices are contaminated with
yeasts and moulds often from insect damage. Flavourings, water, processing machinery,
filling lines and other chemicals are all potential sources of microbial contamination
(Wareing and Davenport, 2005; Barth et al., 2009; Lawlor et al., 2009).
2.6 Spoilage microorganisms
Fresh fruit juices are more susceptible to spoilage because fluid contents are in touch with air
and microbes from the environment during the time of handling. Yeasts, heat sensitive
moulds and lactic acid bacteria are indicator for the quality of raw materials. Heat resistant
fungi and other spore forming bacteria such as Clostridium pasteurianum and Bacillus
coagulans are used as targets for fruit juice pasteurization processes (Tribst et al., 2009). The
various group of organisms involved in spoilage of various fruits, fruit products and fruit
juices are described here.
2.7 Yeasts
Yeasts predominate in the spoilage of acid fruit products because of high tolerance, frequent
ability to grow anaerobically and certain species are preservative resistance. More than 110
species of yeasts have been associated with foods; of which large proportion occur on fruits.
The presence of yeasts in fruit juices may result from failures in fruit juice pasteurization and
in sanitation practices. Pichia, Candida, Hansenula, Rhodotorula, Saccharomyces,
Torulopsis, Trichosporon and Zygossacharomyces, are some well known and important food
spoiling yeasts. Yeast species that cause spoilage in citrus fruits are Candida parapsilosis, C.
stellata, Saccharomyces cerevisiae, Torulaspora delbrueckii and Zygosaccharomyces rouxii
(Arias et al., 2002; ICMSF, 2005; Stratford, 2006; Tribst et al., 2009; Bevilacqua et al.,
2011; Vantarakis et al., 2011; Patil et al., 2011; Tyagi et al., 2013, 2014; Bukvicki et al.,
2014).
Tournas et al. (2006) also reported the presence of Rhodotorula rubra, C. sake,
Kloeckera apis and C. lambica being the most frequently encountered organism from apple,
carrot, grape, grapefruit and orange juices. Candida spp., Trichosporon mucoides, Kloeckera
sp., yeast-like fungus Cryptococcus neoformans were observed in freshly squeezed juices of
orange, lemon, grapefruit, and apple. Spoilage by yeasts in fruit juices is characterized by
formation of CO2 and alcohol. Yeasts may also produce turbidity, flocculation, pellicles, and
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clumping. Yeasts also produced pectinesterases which degrade pectin causing spoilage,
organic acids, and acetaldehyde, which contribute for a “fermented flavor,” may also be
formed (Lawlor et al., 2009).
Yeasts resistant to preservatives
Resistance to preservatives is a great threat to the stability of fruit juices. Examples of yeasts
resistant to preservatives include Zygosaccharomyces bailli, Candida krusei, Saccharomyces
bisporus, Schizosaccharomyces pombe and Pichia membranarfaciens. Resistance to
preservatives has been attributed to the ability of cells to tolerate chronic intracellular pH
drops by phosphofructokinase enzyme. P. membranifaciens is resistant to heat, moderate
amount of salt, SO2, sorbic, benzoic and acetic acid hence it is considered as target
microorganism for optimization of thermal pasteurization of fruit juices (ICMSF, 2005;
Lenovich et al., 2006; Stratford, 2006; Bevilacqua et al., 2011; Tyagi et al., 2013, 2014).
2.8 Moulds
Mould spoilage in fruits and fruit juices is divided into two categories:-
1) Growth of mould due to poor hygiene within factory or field conditions.
2) Growth of heat resistant moulds within heat processed juices.
The former type can cause tainting, discoloration and other problems associated with gross
mould growth. The latter type can result in slow growth of the mould within the processed
product. Juice cloud loss occurs through the activity of pectin esterases. The dominant
moulds recorded in fruit juices belong to Penicillium sp., Cladosporium sp., Aspergillus
niger, A. fumigatus, Botrytis sp., Aureobasidium pullulans. Rhizopus and Mucor are also
associated with spoilage of fresh fruits and vegetables (ICMSF, 2005; Wareing and
Davenport, 2005; Tournas et al., 2006; Moss, 2008; Lawlar et al., 2009).
Aspergillus and Peniciliium were the dominant mould genera isolated from orange,
guava and banana juices freshly prepared from the respective fruits collected from the local
markets of Zagazig city, Sharkia Govenmorate, Egypt (Helal et al., 2006). Penicillium,
Fusarium and Geotrichum were reported in pasteurized grapefruit juice (Tournas et al.,
2006). Among these, some moulds produce mycotoxins which are of great threat to human
health. Major mycotoxins associated with fruit juices are byssochlamic acid (Byssochlamys
fulva, B.nivea), patulin (B. fulva, B. nivea, P. expansum), ochratoxin (Aspergillus
carbonarius) and citrinin (Penicillium expansum, P. citrinum) (Delage et al., 2003; Wareing
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and Davenport, 2005). Presence of patulin in fruit juices is indicator of poor quality of fruits
used in processing of juices (Sylos et al., 1999).
2.9 Heat resistant fungi
Spoilage of pasteurized fruit juices is caused by heat resistant fungi. Principal heat resistant
moulds belong to Byssochlamys nivea, B. fulva, Neosartorya fischeri, Eupenicillium
brefeldianum and Talaromyces macrospores. These moulds survive commercial heat
pasteurization treatment, usually applied to fruits and fruit products, due to the presence of
heat resistant ascospores. Byssochlamys spp. are historically most widely encountered
moulds causing spoilage of heat processed fruits (ICMSF, 2005; Salomao et al., 2007;
Lawlor et al., 2009). Kutama et al. (2010) reported the presence of heat resistant moulds such
as Byssochlamys, Neosartorya and Talaromyces in orange, mango, tomato and pineapple
juices. The presence of heat resistant fungi such as Paecilomyces variotii, Aspergillus tamari,
A. flavus and A. ochraceous has been reported in sixty packaged Nigerian fruit juices
consisting of mango, pineapple, orange and tomato (Obeta and Ugwuyani, 2007).
Chlamydospores, sclerotia and aleurospores are the resistant structures/spores produced by
these moulds (Voldrich et al., 2004; Salomao et al., 2007).
2.10 Bacteria
Bacteria are usually present in low numbers on fresh fruits and vegetables. Some bacteria
such as heterofermentative lactic acid bacteria (LAB), acetic acid bacteria, Erwinia sp.,
Enterobacter sp., Clostridium, Alicyclobacillus acidoterristeris, Propionibactreium
cyclohexanicum, Pseudomonas sp. and Bacillus have been reported as deteriorative in cut
fruits and juices (ICMSF, 2005; Lawlor et al., 2009; Raybaudi-Massilia et al., 2009b; Tribst
et al., 2009; Bevilacqua et al., 2011).
Lactic Acid Bacteria
Heterofermentative LAB was reported as the most important group of spoilage
microorganisms in fruit juices. Lactobacillus and Leuconostoc are the two taxa frequently
isolated from fruits and spoiled fruit juices. They produce lactic acids in fruit juices along
with lesser amount of acetic and gluconic acids, ethanol and CO2, but some species of LAB
such as Leuconostoc mesenteroides ssp. cremoris, Leuconostoc paramesenteroides and
Leuconostoc dextranicum are more prominent as they produce diacetyl and acetoin as
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metabolites in spoiled fruit juices, contributing to buttery or butter milk off flavor to citrus
juices (ICMSF, 2005; Lawlor et al., 2009; Steyn et al., 2011).
Acetic Acid Bacteria
Acetic acid bacteria belong to three taxa, namely, Acetobacter, Gluconobacter, and
Gluconacetobacter are involved in the spoilage of juices. Production of sour and vinegar like
flavours in fruit juices is due to the formation of acetic acid by these bacteria (Worbo and
Splistosser, 2004; ICMSF, 2005; Lawlor et al., 2009).
Alicyclobacilli
In recent years, Alicyclobacillus a thermoacidohile, endospore producing bacterium has
emerged as major concern to the beverage industry worldwide as many high concentrated
fruit products which are valuable semi prepared food components to the bakery, dairy,
canning, baby foods, distilling and beverage industries have been found to be contaminated
with these spoilage microbes. The thermoacidophile nature and presence of highly resistant
endospores is responsible for their survival during the production of concentrated fruit
products. Soil is considered to be the main source of contamination of fresh fruits during
harvesting (Walls and Chuyate, 2000; Parish and Goodrich, 2005; Bahceci et al., 2007;
Groenewald et al., 2008, 2009; Steyn et al., 2011).
Of the over 20 species of Alicyclobacillus isolated from different environments. A.
acidocaldarious, A. hesperidium, A. acidophilus, A. cyclohaptanicus, A. fastidious and A.
pomorum have been implicated in spoilage incidents in high acid fruit and vegetable products
(Goto et al., 2007). Alicyclobacillus acidoterrestris has emerged as new spoilage bacterium
for commercialized fruit juices that can survive pasteurization at 95oC for 2 minutes and can
spoil heat treated fruit juices by the formation of taint chemicals (guaiacol and halophenolic)
(Witthuhn et al., 2007; Steyn et al., 2011). Alicyclobacillus contains ω- alicyclic fatty acids
(ω-cyclohexane and ω- cycloheptane fatty acids) in their cell membrane that are responsible
for heat resistance of by forming a protective coating with strong hydrophobic bonds. These
hydrophobic bonds stabilize reduced membrane permeability in extreme and high
temperature environments. Another factors contributing to the heat stability of
Alicyclobacillus is its endospores along with presence of heat stable proteins and
mineralization by divalent cations especially calcium- dipicolinate complex (Wisotzsky et
al., 1992; Chang and Kang, 2004; Jay et al., 2005; Smit et al., 2011).
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Contamination of Alicyclobacillus in fruit juices results from sources like soil,
water and processing facilities. Spoilage of fruit juices by Alicyclobacillius is difficult to
detect because it does not produce any visible changes such as gas during growth and
incipient swelling of containers does not occur so that spoilage in retail products cannot be
noticed. It produces a smoky, medicinal and antiseptic off odour associated with guaiacol.
Other compounds such as 2,6- dibromophenol and 2,6- dichlorophenol have also been
detected (Silva and Gibbs, 2004; Witthuhn et al., 2007; Durak et al., 2010; Danyluk et al.,
2011; Smit et al., 2011; Witthuhn et al., 2013). Endospores of Alicyclobacillus have D
values in the range of 16-23 minutes at 900C, greater than the pasteurization treatments
applied in fruit juice processing (Walker and Phillips, 2008b). Hence, Silva and Gibbs (2004)
suggested that Alicyclobacillus be designated as the target microbe in the design of
pasteurization processes for acidic foods and beverages.
Propionibacterium cyclohexanicum
Propionibacterium cyclohexanicum was first isolated from spoiled orange juice in 1993. It
possesses ω-cyclohexyl undecanoic acid in cell membrane as Alicyclobacillus genus but
lacks the production of endospores (Kusano et al., 1997; Walker and Phillips, 2008a).
Walker and Phillips (2007) reported that P. cyclohexanicum survives at 950C for 10 minutes
in orange juice and hence would survive treatments commonly used in pasteurization process
used in fruit juice industry.
Bacillus
Bacillus coagulans, B. marcesens and B. polymyxa spoil several fruit juices (Stratford et al.,
2000). B. coagulans spoils canned tomato juice and vegetable products. It causes flat sour
spoilage in juice (ICMSF, 2005; Silva and Gibbs, 2004; Steyn et al., 2011; Daryaei and
Balasubramanium, 2013).
Clostridium
Two species of Clostridium mainly C. pasteurianum and C. butyricum have been isolated at
low pH of fruit juices (Stratford et al., 2000).
Members of Enterobacteriaceae
Psychrotrophic bacteria such as Klebsiella sp., Serratia sp., Citrobacter sp., and Cedecea sp.,
are capable of multiplying in citrus juices with pH values below 4.3. These strains cause a
mixed-acid fermentation resulting in citrate, acetate, and CO2 production, along with
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“unclean” flavor and aroma defects. In certain cases, enteric bacteria may produce a sulfur-
like off-aroma in spoiled citrus juices (Lawlor et al., 2009). Suguna et al. (2011) observed
the presence of Klebsiella pneumoniae in dragon-fruit (pitaya) juices in Penang city of
Malaysia.
2.11 Pathogenic microorganisms
In tropical countries consumer preference is for fresh-cut fruits and juices rather than their
processed counterparts. Fruit juices sold by the street vendors are consumed regularly by the
local owing to their fresh look, original nutritional and sensory attributes (Brackett, 2001;
Thunberg et al., 2002; Suguna et al., 2011). In India, a large population of all income and age
groups consume freshly squeezed fruit and vegetable juice, but the presence of pathogenic
microorganisms in street vended fruit juices have been reported in various parts of India such
as Vishakhapatnam (Lewis et al., 2006), Mumbai (Mahale et al., 2008), Amravati (Tambaker
et al., 2009), Nagpur (Titarmare et al., 2009), Kolkata (Mukhopadhyay et al., 2011), Mysore
(Divyashree et al., 2013) and Tirumula (Suneetha et al., 2013). Other researchers have also
carried out study on the microbiological quality of street vended fruit juices in other parts of
the world as summarized in table 2.2. Food borne pathogens such as Escherichia coli and
Salmonella survive in acidic environment of fruit juices due to acid stress response
(Ghenghesh et al., 2005; Tribst et al., 2009; Ray-Baudi Massilia et al, 2009). Some strains of
E. coli, Shigella and Salmonella may survive for several days and even weeks in acidic
environment by regulating their internal pH that maintained at neutral pH by combination of
passive and active mechanisms (Vantarakis et al., 2011).
Shigella flexneri and S. sonnei survive in apple (pH 3.3) and tomato juices (pH
4.0) at 70C for at least 14 days (Opstal et al., 2005). Sospedra et al. (2012) reported the
presence of Salmonella sp. and Staphylococcus aureus in orange juice extracted by squeezing
machine used in restaurants. Because of the presence of pathogens in fruit juices, the food
borne outbreaks associated with consumption of fruit juices have been increased (CDC,
2007; Van Opstal et al., 2006; Raybaudi-Massilia et al., 2009b; Vantakratis et al., 2011;
Sospedra et al., 2012). Fruit juice borne outbreaks of last two decades from 1991-2010 are
summarized in table 2.3. Several outbreaks associated with consumption of fruit juices have
been reported maximum in year 1999 (5) (CDC, 1999; Krause et al., 2001; Mahale et al.,
2008; CDC, 2011) and 1996 (4) (CDC, 1997;Cody et al., 1999; FDA, 2001).
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Table 2.2. Street vended fruit juices in different locations and associated pathogenic microorganisms
Sr.no. Place Fruit juice Pathogens Reference
1 Vishakhapatnam
(India)
Orange, Pomegranate, Mango,
Pine apple, Grape
Faecal coliforms and faecal
streptococci
Lewis et al., 2004
2 Mumbai (India) Sugarcane,Lime, Carrot Vibrio cholerae, E. coli,
S.aureus
Mahale et al., 2008
3 Jimma town
(South west
Ethopia)
Avocado, Papaya, Pine apple Klebsiella, Enterobacter,
Serratia
Ketema et al., 2008
4 Nagpur (India) Pine apple, Sweet Lime, Carrot
juice
Salmonella, coliforms,
S.aureus
Titarmare et al., 2009
5 Amravati (India) Apple, Orange, Pineapple,
Pomegranate, Sweet lemon ,
Mixed fruit
Salmonella, coliforms,
S.aureus,Pseudomonas,
Proteus
Tambaker et al., 2009
6 Kolkata Mango , Pineapple, Sweet
lime, lemon, Pomegranate,
Sugarcane
Vibrio and Salmonella Mukhopadhyay et al., 2011
7 Mysore Orange, Sweet lime and
Pineapple
Micrococcus spp., Bacillus,
Streptococcus,
Staphylococcus
Divyashree et al., 2013
8 Tirumula Orange and Pineapple B. cereus, S. aureus Suneetha et al., 2013
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Table 2.3. Fruit juice borne outbreaks caused by pathogenic bacteria
Type of fruit
juice
Pathogens Year Country Venue Number
of cases
(deaths)
Reference
Unpasteurized
apple juice
Escheichia coli
O157:H7
1991 USA Small cider
mill
23(0) Besser et al.,
1993
Unpasteurized
orange juice
Enterotoxigenic
E. coli
1992 India Roadside
vendor
6 (0) Singh et al.,
1995
Unpasteurized
apple juice
Cryptosporidium 1993 USA School 213(0) Millard et
al., 1994
Carrot
homemade
juice
Clostridium
botulinum
1993 USA Home 1 (0) Buzby and
Crutchfield,
1999
Unpasteurized
orange juice
Salmonella
gaminara,
S.hartford and S.
rubislaw
1995
USA
Retail
63 (0)
CDC, 1995;
Cook et al.,
1998;
Parish, 2000
Unpasteurized
orange juice
Shigella flexneri 1995 South
Africa
Restaurant 14(0) Thurston et
al., 1998
Unpasteurized
apple juice
C. parvum 1996 USA Small cider
mill
31 (0) CDC, 1997
Unpasteurized
apple juice
E. coli O157:H7 1996 USA Small cider
mill
14 (0) CDC, 1997
Unpasteurized
apple juice
E. coli O157:H7 1996 USA Small cider
mill
6 (0) FDA, 2001
Unpasteurized
apple juice
E. coli O157:H7 1998 Canada Farm/Home 14 (0) Tamblyn et
al., 1999
Unpasteurized
apple juice
E. coli O157:H7 1999 USA Not
reported
25 (0) CDC, 2011
Unpasteurized
orange juice
S. muenchen 1999 Canada,
USA
Restaurant 423 (1) CDC, 1999
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Unpasteurized
orange juice
S. anatum 1999 USA Roadside
stand
6 (0) Krause et
al., 2001
Unpasteurized
orange juice
S. typhimurium
1999
Australia
Retail
405 (0) Mahale et
al., 2008
Unpasteurized
sugar cane
juice
Vibrio cholerae 1999 India Not
reported
------------ Mahale et
al., 2008
Unpasteurized
orange juice
S. enteritidis 2000 USA Retail and
food
service
88(0) Butler, 2000
Juice Cryptosporidium
cayetanensis
2002 Colombia Not
reported
56 Botero-
Graces et
al., 2006
Raspberry
juice
C. cayetanensis 2003 Guatemala Not
reported
7 Puente et
al., 2006
Unpasteurized
apple juice
C. parvum 2003 USA Farm/Retail 144 (0) Vojdani et
al., 2008
Unpasteurized
apple juice
E. coli O111 and
C. parvum
2004 USA Farm/Home 212 (0) Vojdani et
al., 2008
Unpasteurized
orange juice
S. typhimurium
and S. saintpaul
2005
USA
Retail and
food
service
152 (0) Jain et al.,
2009
Unpasteurized
sugar cane
juice
Trypanosoma
cruzi
2005 Brazil Roadside
kiosk
25 (3) Pereira,
2009
Pasteurized
carrot juice
C. botulinum 2006 USA Retail 4(0) CDC, 2006
Unpasteurized
apple juice
E. coli O157:H7 2007 USA Not
reported
9(0) CDC, 2011
Unpasteurized E. coli O157:H7 2008 USA Retail 7 CDC, 2011
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E. coli O157:H7 is associated several outbreaks attributable to consumption of unpasteurized
apple juice. Salmonella is main causal organism for outbreaks related to unpasteurized orange
juice. Clostridium botulinum was reported from homemade as well as pasteurized carrot juice.
Vibrio cholorae had been reported for outbreak in India by the consumption of unpasteurized
sugarcane juice (table 2.3). In year 1999, 423 people in the USA and Canada and 405 people in
Australia were affected by consuming unpasteurized orange juice (Mahale et al., 2008).
2.12 Factors affecting shelf life of juices
The shelf life of a food can be defined as the time period within which the food is safe to
consume and/or has an acceptable quality to consumers or shelf life is also defined as the time to
reach a microbial population of 6 log cfu/mL which determined experimentally (Andres et al.
2001).The shelf life of juices is affected by both the intrinsic and extrinsic factors. Intrinsic
factors include pH, oxidation–reduction potential, water activity, availability of nutrients, the
presence of antimicrobial compounds, and competing microflora. Extrinsic factors encompass
storage temperatures and times, relative humidity conditions during storage and packaging
material characteristics. Among intrinsic factors pH and water activity are the most influential
factors affecting spoilage rates. Bacteria prefer to grow at pH 6.5-7.5 but tolerate a pH range of 4
to 9. Yeasts are more tolerant than bacteria to low pH values. However, moulds can grow in the
widest range of pH conditions. Therefore, one way to control the microbial growth in foods by
increasing the acidity of food. In the past, fruit juices were considered as safe foods because of
their low pH caused by naturally occurring organic acids. These acids are different in different
fruit juices as described in table 2.1. Organic acids affect a number of systems in the target
organism. Organic acids has direct influence on pH of the substrate or growth medium due to an
increase in proton concentration, reduction in internal cellular pH by ionization of undesociated
acid molecule, or disruption of substrate transport by alternation of cell membrane permeability.
apple juice
Unpasteurized
orange juice
S. panama 2008 Netherlands Not
reported
33(0) Bevilacqua
et al., 2011
Unpasteurized
apple juice
E. coli O157:H7 2010 USA Fair 7(0) FDA, 2010
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In addition to inhibit substrate transport, organic acids may also inhibit NADH oxidation, thus
eliminating supplies of reducing agents to electron transport systems (Davidson, 2001;
Mosqueda-Melgar et al., 2008a; Lawlor et al., 2009).
Water activity of juices is associated with ºBrix. In juices, ºBrix is used to indicate the
percentage of soluble solids and is one of the most important factors for grading the quality of a
juice. Microorganisms cause fruit juice spoilage by fermentation of sugars, and can therefore
increase the ºBrix value owing to the conversion of complex sugars into monosaccharides (Rivas
et al., 2006; Lawlor et al., 2009).
Extrinsic factors such as temperature also influence the shelf life of juices. The shelf life
of freshly squeezed, un-pasteurized orange juice is less than 20 days at 1oC. Low temperature is
necessary during manufacturing and storage of juice. The primary purpose of low temperature
storage is to increase the shelf life by slowing down degradatory reactions and limiting microbial
growth. Therefore the combination of reduction in chemical, biochemical and microbial kinetics,
can increase the shelf life of fresh and processed foods (Hartel and Heldman, 1997; Bates et al.,
2001; Sandhu and Minhas, 2006; Raccach and Mellatdoust, 2007).
2.13 Preservation of fruit juices
Food preservation is defined as to control the growth of spoilage and pathogenic organisms
(Aneja et al., 2008). Preservation of fruit juice depends on the low pH, pasteurization,
refrigeration and on the addition of preservatives. Pasteurization of fruit juices is often done by
applying temperature of 85-95ο
C for 2 minutes. However, some problems associated with this
technique, as pasteurization temperature is only effective against pathogens such as E. coli and
Salmonella but are not effective against ascospores of heat resistant fungi and heat resistant
bacteria. In addition the thermal treatment also affects the sensory and nutritional quality of fruit
juices (Salomao et al., 2007; Kutama et al., 2010; Smit et al., 2011; Steyn et al., 2011;
Mosqueda-Melgar et al., 2012). Several non-thermal technologies have been developed that
include high hydrostatic pressure (HHP), high pressure homogenization (HPH), pulsed electric
field (PEF), ultrasound and irradiations proving to be beneficial to inactivate microorganisms,
decrease the activity of enzymes and increase the shelf life of foods (Rico et al., 2007; Tribst et
al., 2009; Rupasinghe and Yu, 2012). Among these High Hydrostatic Pressure (HHP) is the
best one to be applied for juice treatment. In this process, fruit juices are subjected to 400MPa
pressure for a few minutes at 20oC or below which is sufficient to reduce the numbers of
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19
spoilage microorganisms such as yeasts, moulds and lactic acid bacteria. The lethal effect of
HHP treatment on microorganisms by affecting their cell membrane along with inactivation of
some key enzymes which are involved in DNA replication and transcription processes
(Kuldiloke and Eshtiaghi, 2008; Mckay et al., 2011; Vercammen et al., 2012). The efficacy of
HHP against pathogens in juices have been evaluated and according to Bayindrill et al. (2006),
five log reductions or greater of S. aureus, E. coli and S. enteritidis were observed in apricot,
orange, sour cherry and apple juices by using a pressure treatment of 350MPa/5
minutes/30oC.Other authors (Linton et al., 1999; Slifko et al., 2000; Ramaswamy et al., 2003;
Guerrero-Beltran et al., 2011) also achieved the same level of inactivation for Listeria innocua,
E. coli 29055, Cryptosporidium parvum. HHP is effective against vegetative cells of A.
acidoterresteris (Alpes et al., 2003; Buzrul et al., 2005) and heat resistant moulds (Voldrich et
al., 2004) but not effective against bacterial and mold spores (Tribst et al., 2009). Another factor
which limits the effect of HHP in juice is the soluble solid content of juice. Lee et al. (2006)
observed that pressure (207 MPa) and temperature (45oC) were sufficient to inactivate 2 log
cycles of A. acidoterresteris in juice at 17.5o Brix, however a temperature of 71
oC was required
to achieve the same inactivation in juice at 30o Brix.
Pulsed Electric Field (PEF) has also shown potential against in the inactivation of
pathogens, with 5 log reduction cycles of S. enteritidis, L. innocua, E. coli and S. aureus in
orange and apple juices (Evrendilek et al., 1999; Jin and Zhang, 1999; Evrendilek et al., 2000;
McDonald et al., 2000; Iu et al., 2001; Liang et al., 2002; Heinz et al., 2003; Elez-Martinez et
al., 2005; Sampedro et al., 2007; Evrendilek et al., 2008; Mosqueda- Melgar et al., 2008 a;
Gurtler et al., 2011). This process inactivates microorganisms and enzymes with only small
increase in temperature affects the cell membrane of microorganisms by electroporation which
leads to leakage of cytoplasmic content from cells (Cserhalmi et al., 2006; Charles-Rodriguez et
al., 2007; Gurtler et al., 2011). PEF showed synergistic effect with other antimicrobials such as
lysozyme, nisin, clove oil (Liang et al., 2006), cinnamon bark oil and citric acid (Mosqueda-
Melgar et al., 2008 a, b). However, PEF was not effective against heat resistant microorganisms
(Tribst et al., 2009).
High Pressure Homogenization (HPH) is an alternative to eliminate pathogens from
unpasteurized fruit juices. Initially, HPH was purposed as a suitable method for the stabilization
of dairy products but in last decades it has been suggested for its use for prolongation of the shelf
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20
life of fruit juices. The inactivation of 2 to 5 log cycles of E. coli, Saccharomyces cerevisiae,
Lactobacillus plantarum, Penicillium, Aureobasidium, Aspergillus, and Z. bailli in apple, apricot,
carrot and orange juices was achieved with different levels of pressure treatment (100-300 MPa).
HPH inactivates microorganisms by damaging their structural integrity coupled with sudden rise
in temperature produced in this process. But the possibility of homogenization generating strange
odour and colour in juices, reduce their acceptability in treatment of juices (Brinez et al., 2006;
Campos and Cristianini, 2007; Mckay, 2009; Patrignani et al., 2009, 2010; Mckay et al., 2011;
Bevilacqua et al., 2012).
Many authors have observed the effect of UV radiation against pathogens inoculated in
fruit juices. Ultraviolet radiation involves the use of radiation from electromagnetic spectrum
from 100-400nm. It is classified as: UV-A (320-400nm), UV-B (280-320nm), UV-C (200-
280nm) Keyser. UV-C is effective against bacteria and viruses. UV treatment is performed at
low temperature. 254nm wavelength of UV light is widely used in juice and beverage industry.
Five log reductions were achieved for E.coli K-12 using UV radiation at 2.34 kJ/m2 in apple
juice; for E.coli treated at 450 kJ/m2 for 30 minutes in apple nectar and for C. parvum in apple
cider by applying 0.14 kJ/m2 for <2 seconds (Hanes et al., 2002; Guerrero- Beltrin and Barbosa-
Canovas, 2006; Keyser et al., 2008; Rupasinghe and Yu, 2012). No data is reported about the
activity of UV against heat resistant spores.
Ultrasound has already been tested as potential technology against pathogens in juices
but it seems to have a limited effect to inactivate 1-2 log cycles of E.coli O157:47 and L.
monocytogenes in cider treated for 3 minutes at 44-48 kHz (Rodgers and Ryser, 2004).
2.14 Antimicrobial agents
Antimicrobials are chemical compounds or substances that may delay microbial growth or cause
microbial death in a food matrix. The major targets for such antimicrobials are food poisoning
microorganisms (infective agents and toxin producers) and spoilage microorganisms whose
metabolic end products or enzymes cause off-odors, off-flavors, texture problems, and
discoloration. The food antimicrobials are usually classified into traditional or natural and
synthetic substances depending on their origin. Antimicrobials are called traditional when they
have been used for many years and many countries approve them for inclusion in foods.
However, many synthetic antimicrobials are found naturally (benzoic acid in cranberries, sorbic
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acid in rowanberries, citric acid in lemons, malic acid in apples, tartaric acid in grapes, the
perception of natural has become important for many consumers (Davidson, 2001; Negi, 2012).
The efficacy of antimicrobials depends on the type, genus, species and strain of the
target microorganism. Factors such as pH, water activity (aw), temperature, atmosphere
composition, initial microbial load and acidity of the food substrate also influence the activity of
antimicrobials. The antimicrobial nature of phytochemical depends on its chemical properties,
such as pKa value, hydrophobicity/ lipophilicity ratios, solubility, and volatility. The pH and
polarity are the most prominent factors influencing the effectiveness of a food antimicrobial.
Polarity is related to both the ionization of the molecule and the contribution of any alkyl side
groups or hydrophobic parent molecules (Davidson, 2001). Therefore, specific characteristics of
the food system that needs to be preserved must be known because high proportion of lipids
could render the effectiveness of some antimicrobial agents. Further, hydrophobic properties of
some antimicrobial substances can make their dissolution difficult in water limiting their use in
foods (Beuchat, 2001; Davidson, 2001; Stratford and Eklund, 2003; Owen and Palombo 2007).
2.15 Chemical preservatives
Benzoic acids, sorbic acids, sulphur dioxide and p-hydroxy benzoic acids are permitted as
preservatives in fruit based products (Bates et al., 2001; ICMSF, 2005). The permitted level of
sodium benzoate and potassium sorbate in foods is 0.1% (Chipley, 2005). Different studies have
showed the effect of addition of organic acids in fruit juices to inhibit and reduce population of
spoilage and pathogenic microorganisms as shown in table 2.4. Addition of chemical
preservative in fruit juices increases the shelf life of fruit juices. There is strong consumer
demand to avoid the use of artificial preservatives. There was report of formation of benzene
from benzoic acids in foods. S. cerevisiae and P. anomola are able to decarboxylate sorbic acids
to 1, 3- pentadiene causing a kerosene like off odour and S.pombe may produce off flavours.
Such problems with chemical synthesized preservatives, growing demand of consumer for
natural food preservatives. A variety of substances have been investigated in an effort to replace
benzoate and sorbate such as bacteriocins, lysozyme, chitosan, essential oil, vanillin (Burt, 2004;
Shi et al., 2010; Tajkarimi et al., 2010; Tserennadmid et al., 2011; Tyagi et al., 2013, 2014).
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Table 2.4:- Organic acids used as preservatives in fruit juices to control pathogenic and spoilage microorganisms
Sr.no Organic acid Fruit juice Target microorganisms Reference
1 Benzoic acid Apple cider E.coliO157:H7 Zhao et al., 1993
2 Benzoic acid Grape juice Yeast populations Pederson et al., 1961
3 Citric acid Apple cider E.coliO157:H7 Pederson et al., 1961
4 Citric acid Tomato juice Salmonella enteriditis Mosqueda–Melgar et al.,
2008a
5 Citric acid Orange juice, apple juice,
pear juice
E.coliO157:H, Salmonella enteriditis Mosqueda–Melgar et al.,
2008b
6 Fumaric acid Apple cider E.coliO157:H7 Comes and Beelman, 2002
7 Lactic acid Apple cider E.coliO157:H7, Salmonella enteriditis, yeast
and molds
Uljas and Ingahm ,1999
8 Malic acid Apple juice, pear juice,
melon juice
E.coliO157:H7, Salmonella enteriditis,
Listeria monocytogenes
Raybaudi- Massilia et al.,
2009a
9 Potassium sorbate Apple juice Alicyclobacillus acidoterrestris Walker and Phillip, 2008a
10 Potassium sorbate Orange juice Propionobacterium cyclohexanicum Walker and Phillips, 2008a
11 Potassium sorbate Apple juice E.coliO157:H7 Ceylon et al., 2004
12 Potassium sorbate Apple juice Byssochlamys nivea Roland and Beuchat ,1984
13 Sodium benzoate Apple cider E.coliO157:H7 Fisher and Golden,1998
14 Sodium benzoate Apple juice Alicyclobacillus acidoterrestris Walker and Phillips, 2008a
15 Sodium benzoate Orange juice Propionobacterium cyclohexanicum Walker and Phillips, 2008b
16 Sodium benzoate Apple juice E.coliO157:H7 Ceylon et al., 2004
17 Sorbic acid Apple cider E.coliO157:H7 Uljas and Ingham, 1999
18 Sorbic acid Grape juice Yeast Uljas and Ingham, 1999
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2.16 Natural Antimicrobials
Use of natural antimicrobial compounds is one of the oldest and most traditional food
preservation techniques. Consumers demand for food without chemical preservatives has
promoted the search of preservatives from natural sources such as animals, plants and
microorganisms (Figure 2.1) (Vigil et al., 2005; Tiwari et al., 2009; Raybaudi- Massilia et al.,
2009b). Enzymes from animal origin have also shown potential as preservative in food.
Lysozyme is a protein present in milk and eggs that catalyzes the hydrolysis of the β-1,4
linkages betweenN-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer of
the bacterial cell wall. The FAO/WHO joint and several countries including Austria, Australia,
Belgium, Denmark, Finland, France, Germany, Italy, Japan, Spain, and United Kingdom have
approved its use in some foods when used in accordance with good manufacturing practices
(GMP). Lysozyme exhibits antibacterial activity against Gram positive bacteria. It is used as
antimicrobial agent in casing for frankfurters, on cooked meat and poultry products, and to
prevent the blowing caused by Clostridium tyrobutyricun in semi hard cheeses (Losso et al.,
2000; Raybaudi- Massilia et al., 2009b; Lucera et al., 2012).
. Fig. 2.1. Sources of natural antimicrobials
Lactoperoxidase, a hemoprotein present in milk and other secretions, which catalyzes
the oxidation of thiocyanate (SCN−) and iodide ions to generate highly reactive oxidizing agents.
These products possess broad spectrum antimicrobial activity against bacteria, fungi, and viruses
(Naidu, 2000).The lactoperoxidase system exerts its antimicrobial action through short-life
oxidation products, mainly hypothiocyanate (OSCN−) and hypothiocyanous acid (HOSCN),
which produce microbiocidal or microbiostatic effects by the oxidation of thiol groups (-SH) of
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24
cytoplasmic enzymes and damage to the outer membrane, cell wall or cytoplasmic membrane,
transport systems, glycolytic enzymes, and nucleic acids. The regulatory organization, Food
Standards Australia New Zealand (FSANZ), has permitted the use of the lactoperoxidase system
for the treatment of meat (including poultry), fish and milk products as an antimicrobial at
maximum levels of 20 mg/kg meat or 30 mg/L milk (Naidu, 2000; Raybaudi- Massilia et al.,
2009b).
Chitosan, a heteropolysaccharide composed of β−1, 4-linked 2-amino-2-deoxy-β-D-
glucose obtained commercially by deacetylation of chitin, which is an abundant constituent of
crustacean shells and fungi. Chitosan got GRAS (Generally Recognized as Safe) status in 2005
by USFDA and is marketed as food additive or supplement in Japan, Korea, England, Italy,
Portugal, and today in the United States. It is more active against yeast and moulds but has also
been shown potential against Gram negative bacteria may be owing to polycationic structure at
pH 6.3; interact with anionic components such as lipopolysaccharide and proteins of the
membrane cell surface responsible for the disruption of the integrity of the outer membrane
resulting in loss of barrier function but lacking direct bactericidal activity (Rhoades and Roller,
2000; Novack et al., 2003; Sebti et al., 2005; No et al., 2007; Raybaudi- Massilia et al., 2009b).
One of the most common forms of food preservation is fermentation, a process based
on the growth of microorganisms in foods, whether natural or added. These organisms mainly
comprise lactic acid bacteria (LAB), which produce organic acids and other compounds that, in
addition to antimicrobial properties, also confer unique flavours and textures to food products.
Traditionally, a great number of foods have been protected against spoiling by natural processes
of fermentation. Lactic acid and other end products of LAB metabolism, including hydrogen
peroxide, diacetyl, acetoin, reutericyclin, antifungal peptides, and bacteriocins and other organic
acids, act as bio preservatives by altering the intrinsic properties of the food to such an extent as
to actually inhibit spoilage microorganisms. Bacteriocins are the antimicrobial proteins or
peptides produced by bacteria. They are ribosomally synthesized and kill closely related bacteria.
Nisin and pediocin are two bacteriocins which received a great deal of attention because of their
beneficial effects to human health and to food production as well as the replacement of chemical
preservatives that are being continuously questioned with regard of safety (Cleveland et al.,
2001; Deegan et al., 2006; Gálvez et al., 2007; Tiwari et al., 2009).
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25
Edible, medicinal, herbal plants and their derived essential oil and isolated compounds
contain a large number of compounds that possessed the antimicrobial activity. A variety of plant
and spice based antimicrobials is used for reducing or eliminating pathogenic bacteria and
increasing the overall quality of food products (Tajkarimi et al., 2010).
2.17 Plant antimicrobials
Plant products have also been used since ancient time for flavoring foods and beverages, and for
medicinal purposes with varying success to cure and prevent diseases. It is estimated that there
are 250,000 to 500,000 plant species on the earth and only one-tenth of these have been exploited
till date (Cowan, 1999; Tajkarimi et al., 2010; Negi, 2012). In the last few years, a number of
studies have been conducted in different countries to prove potency of plant products and
thousands of compounds have been isolated from these plants, which exhibit antimicrobial or
medicinal properties. The use of plant extracts with known antimicrobial properties can be of
great connotation in food preservation (Kubo et al., 1993; Silva et al., 1996; Nimri et al., 1999;
Rauha et al., 2000; Ahmad and Beg, 2001; Negi and Jayaprakasha, 2001, 2004; Chauhan et al.,
2007; Negi et al., 1999, 2003a, 2003b, 2005, 2008, 2010; Shan et al., 2007, 2009; Butkhup et al.,
2010; Tornuk et al., 2011; Bhatt and Negi, 2012; Zeng et al., 2012, Tyagi et al., 2013,2014;
Bukvicki et al., 2014).
2.18 Natural antimicrobials of plant origin
As far as the use of natural antimicrobials contains great diversity of the compounds, however
their use in foods is concerned; the lack of reproducibility of their activity is one of the major
restrictions. There is variation in qualitative and quantitative analysis of bioactive
phytochemicals in plant extracts result in their variable effectiveness. Further, the extrapolation
of results obtained from in-vitro experiments with laboratory media to food products is not
straightforward as foods are complex, multicomponent systems consisting of different
interconnecting microenvironments. Herbs, spices and essential oils are used by the food
industry as natural agents for extending the shelf life of juices. There are more than 1340 plants
with defined antimicrobial compounds and over 30,000 components have been isolated from
phenol group containing plant oil compounds and used in food industry (Tiwari et al., 2009;
Tajkarimi et al., 2010; Negi, 2012; Lucera et al., 2012).
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2.19 Essential oils
Essential oils are aromatic and volatile liquids extracted from plant parts (flowers, roots, bark,
leaves, seeds, peel, fruits and whole plant. These oils are secondary metabolites enriched with
isoprene structure. They are called terpenes. When compounds contain additional element such
as oxygen, they are called terpenoids. Essential oils have been used in medicine, perfumery,
cosmetic, and have been added to foods as part of spices or herbs. Their initial application was in
medicine, but in the nineteenth century their use as aroma and flavor ingredients increased and
became their major employment. Essential oils are considered to be secondary metabolites which
play an important role in plant defense as they often possess antimicrobial properties. They also
possess antiviral, antifungal, antiparasitic, antioxidant and insecticidal properties. However,
majority of essential oils are classified as GRAS substance, but their application in food as
preservative is limited due to flavour considerations. Therefore, application of essential oils as
food preservatives requires detailed knowledge about their properties, i.e., the minimum
inhibitory concentration (MIC), the range of target organisms, the mode of action, and the effect
of food matrix components on their antimicrobial properties (Cowan 1999; Tajakarimi et al.,
2010; Bassole and Juliani, 2012; Hyldgaard et al. 2012).
Many researchers have conducted study on the efficacy of essential oil and their active
compounds to control or inhibit the growth of pathogenic and spoilage microorganisms in fruit
juices (Table 2.5). The effectiveness of essential oil depends on the pH of the fruit
product, kind and concentration of used EOs or active compound, and microorganism
type. In this way, Mosqueda-melgar et al (2008a) reports higher reduction in S.
entritidis and E.coli in strawbarry and orange juices containing 0.1% (v/v)
cinnamomum bark oil than in apple and pear juices under same condition. Similar
studies have been observed by Mosqueda-melgar et al (2008c) and Raybaudi-Massilia
et al (2006) in melon and watermelon juices with added cinnamom bark oil and
among apple and pear juices in comparison with melon juice containing cinnamom
oil, lemongrass oil and geranoil. With decrease in pH, the effectiveness of essential
oil increases owing to increase in hydrophobicity of essential oil enabling them to
more easily penetrate in the lipids of cell membrane of the target bacteria (Burt
2004).
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Table 2.5. Antimicrobial effect of essential oil on pathogenic and spoilage microflora of fruit juices
Essential oil or
active
compound
Fruit juice (pH) Storage conditions Target
microorganisms
Effect Reference
Temper
ature
Time
Cinnamon oil
Apple (4.20), pear
(3.97), melon
(5.91)
35 24 h Listreia innocua,
Salmonella enteritidis,
Escherichia coli
Reduced > 5log
CFU/ml
Raybaudi-Massilia et
al., 2006
Strawberry (3.16),
orange (3.44),
apple (4.46), pear
(4.40) and tomato
(4.30)
22 1h S. enteritidis, E. coli
O157:H7
Reduced > 5log
CFU/ml
Mosqueda-Melgar et
al., 2008a,b
Melon (6.11) and
watermelon (5.73)
22 1h S. enteritidis, E. coli
O157:H7, L.
monocytogenes
Reduced 3.1 to
3.9, 1.4 to 1.9 and
3.4 to 4.4 log
CFU/ml of S.
enteritidis, E. coli
O157:H7 and L.
monocytogenes
respectively
Mosqueda-Melgar et
al., 2008c
Apple (3.7) 37 1h S. enteritidis, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al.,2004
Clove oil Tomato (4.2) 50 0.5h Native microbiota Reduced 3.9 log
CFU/ml
Nguyen and Mittal,
2007
Apple (3.7) 37 1h S. hadar, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al., 2004
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Lemon oil Apple (3.7) 37 1h S. hadar, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al., 2004
Lemongrass oil
Apple (4.20), pear
(3.97), melon
(5.91)
35 24 h Listreia innocua,
Salmonella enteritidis,
Escherichia coli
Reduced > 5log
CFU/ml
Raybaudi-Massilia et
al., 2006
Apple (3.7) 37 1h S. hadar, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al., 2004
Apple orange
mixture
28 4h Saccharomyces
cerevisiae,
Zygosacsharomyces
bailli, Pichia
fermentans
Reduced to 3, 2.3
and 2.1 log
CFU/ml of S.
cerevisiae, Z.
bailli, P.
fermentans
Tyagi et al., 2014
Lime oil Apple (3.7) 37 1h S. hadar, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al., 2004
Oregano oil Apple (3.7) 37 1h S. hadar, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al., 2004
Cavacarol Apple (3.7) 37 1h S. hadar, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al., 2004
Cinnamaldehyde Apple (3.7) 37 1h S. hadar, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al., 2004
Citral Orange (3.5) 45 0.5h L. monocytogenes Reduces 1.1 to
1.3 log CFU/ml
Ferrante et al., 2007
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29
Apple (3.7) 37 1h S. hadar, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al., 2004
Eugenol Apple (3.7) 37 1h S. hadar, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al., 2004
Menthol Apple-orange
mixture
37 2 h Saccharomyces
cerevisiae,
Reduced 2 log
cycles
Tyagi et al., 2013
Geraniol Apple (4.20), pear
(3.97), melon
(5.91)
35 24 h Listreia innocua,
Salmonella enteritidis,
Escherichia coli
Reduced > 5log
CFU/ml
Raybaudi-Massilia et
al., 2006
Apple (3.7) 37 1h S. hadar, E. coli
O157:H7
Reduced 50% of
bacterial
population
Friedman et al., 2004
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2.20 Plant extracts
Plant extracts have also shown great potential in the food industry and approved by various
regulatory agencies such as US Food and Drug Act (USFDA), the European Union standards and
Codex Alimentarius and Food Standard Safety of India (FSSAI) (Raju and Bawa 2006; Negi,
2012). The extracts of several plant species contain many bioactive molecules which gain
momentum for pharmaceutical and food processing sectors. The antimicrobial activity of plant
forms the basis for many applications including raw and processed food preservation,
pharmaceuticals, alternative medicines and natural therapies. The first scientific evidence of the
preservation potential of spices, describing antimicrobial activity of cinnamon oil against spores
of anthrax bacilli were reported in 1830. A variety of plant and spice based antimicrobials are
used for reducing or eliminating pathogenic microorganisms and increasing the shelf life of food.
In India, natural herbs and spices are consumed either in food or used as medicine in order to
maintain proper sanitation, health and hygiene and to increase longevity of life. Several spices
such as ajowan, clove, ginger, black pepper, cumin and asafetida are commonly used in the
Indian diet. Herbs and spices are used as one of the safest and effective remedies in curing
various diseases and long term consumption is not known to produce any side effects. They do
not exhibit toxicity (Arora and Kaur, 1999; Shan et al., 2007; Sofia et al., 2007; Sunilson et al.,
2009; Tajkarimi et al., 2010).
Numerous studies have been conducted to prove efficacy of plant extracts as
antimicrobial agents (Beuchat, 2002; Friedman et al., 2002, 2004; Burt, 2004; Raybaudi-
Massilia et al., 2009b; Tajkarimi et al., 2010), very few studies are available for food products
owing to use of crude extracts in most studies which did not produce marked inhibition as many
of the pure compounds in foods. The low activity of extracts is attributed to presence of
flavonoids in glycosidic form where sugar present in them decreases effectiveness against some
bacteria (Kapoor et al., 2007; Parvathy et al., 2009). Despite of the antimicrobial activity of
essential oil in fruit juices, literature search reveals that the antimicrobial activity of plant
extracts in different solvents have not been reported against microbes associated with fruit juices.
There have been many studies published on the activities of plant extracts and essential
oils against different microbes, including food-borne pathogens. The results of these studies are
difficult to compare directly because different methodologies including solvents concentrations,
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31
microbial strains and antimicrobial test methods were used (Thongson et al., 2004; Shan et al.,
2007).
Arora and Kaur (1999) studied the antimicrobial activity of common Indian spices
aqueous extracts; Garlic, ginger, clove, black pepper and green chillies against Bacillus
sphaericus MTCC 511, Staphylococcus aureus MTCC 87, Staphylococcus epidermidis MTCC
435, Enterobacter aerogenes MTCC 111, Escherichia coli MTCC 118, Pseudomonas
aeruginosa MTCC1034, Salmonella typhi MTCC531, Shigella flexneri MTCC 1457 and yeasts
such as Candida albicans MTCC 183, 227, C. apicola MTCC 1445, C. acutus MTCC 536, C.
catenulata MTCC 535, C. inconspicua MTCC 1074, C. tropicalis MTCC 184, Rhodotorula
rubra MTCC 248, Trignopsis variabilis MTCC 256. Of the five plant aqueous extracts studied,
clove and ginger exhibited antiyeast and antibacterial activity.
In vitro antimicrobial activity of six Indian spice extracts of Syzygium aromaticum (bud),
Cinnamomum zeylanicum (bark), Brassica jancea (seeds), Allium sativum (bulb), Zingiber
officinale (rhizome) and Mentha piperita (leaf) was evaluated against E. coli, S. aureus and B.
cereus. S. aromaticum, C. zeylanicum, B. jancea showed inhibitory effect against all the tested
pathogens while extracts of A. sativum and M. piperita possessed negligible inhibitory effect
(Sofia et al., 2007).
Shan et al. (2007) studied the in vitro antibacterial activities of a total of 46 methanolic
extracts from dietary spices and medicinal herbs against five foodborne bacteria (Bacillus cereus,
Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Salmonella anatum). A
total of 12 spices and herbs e.g. Punica granatum, Myrica nagi, Salvia officinalis, Areca catechu,
Eugenia caryophylata, Polygonum cuspidatum, Rhus succedanea, Matteuccia struthiopteris,
Origanum vulgare, Cinnamomum burmannii, Terminalia bellirica and Cassia auriculata showed
relatively high inhibitory activities against the five foodborne pathogenic bacteria tested.
Weerakkody et al. (2010) compared the antimicrobial activities of extracts from four
under-utilized spices and herbs including Garcinia quaesita, Alpinia galanga, Eucalyptus
staigerana and Tasmannia lanceolata to the three common spices and herbs Piper nigrum,
Rosmarinus officinalis and Oreganum vulgare in water, ethanol and hexane extraction solvents.
These extracts were tested against four food-borne bacteria such as E. coli, S. typhimurium, L.
monocytogenes and Staphylococcus aureus using agar disc diffusion and broth dilution assays.
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32
They observed that antimicrobial effect of spices and herbs tested was more effective against
Gram positive bacteria than Gram negative bacteria.
Antimicrobial activity of Zingiber officinalis, Curcuma longa and Alpinia galanga was
assessed in four solvents against five bacteria E.coli, S. enteriditis, S. aureus, Campylobacter
jejuni, B. cereus and four fungi e.g. Saccharomuces cerevisiae, Hensenula anomola, Mucor
mucedo and Candida albicans. It was found that Z. officinalis and C. longa possessed greater
antimicrobial activity than Alpinia galangal (Sunilson et al., 2009).
2.21 Major groups of plant phytochemical compounds (Secondary metabolites)
The value of plants lies in some chemical substances that produce a definite action on the
microbiological, chemical and sensory quality of foods, and these phytochemicals have been
grouped in several categories including polyphenols, flavonoids, tannins, alkaloids, terpenoids,
isothiocyanates, lectins, polypeptides or their oxygen substituted derivatives. These substances
are naturally produced in plants as defense mechanisms against pathogenic microorganisms and
insect pests. These secondary metabolites are the major sources of pharmaceuticals, food
additives, fragrances and pesticides (Cowan, 1999; Edeoga et al., 2005).
Alkaloids
They are low-molecular-weight, nitrogen-containing compounds found in about 20% of plant
species. The term ‘alkaloid’ meaning ‘alkali like’, was coined by W. Meibner, a German
pharmacist. Later it was demonstrated that the alkalinity was due to the presence of a basic
nitrogen atom. Alkaloids occur in more than 150 families of plants. The important ones are
Apocynaceae, Papaveraceae, Fabaceae, Ranunculaceae, Rubiaceae, Rutaceae, Solanaceae, and
less common lower plants and fungi (ergot alkaloids). In plants, alkaloids generally exist as salts
of organic acids like acetic, oxalic, citric, malic, lactic, tartaric, tannic and other acids. A few
alkaloids also occur as glycosides of sugar such as glucose, rhamnose and galactose, e.g.
alkaloids of the solanum group (solanine), as amides (piperine), and as esters (atropine, cocaine)
of organic acids. The mechanism of action of alkaloids is owing to their ability to intercalate
with DNA (Cowan, 1999; Ramawat, 2007).
Colchicine from Colchicum autumnale, piperine from black pepper (Piper nigrum),
indicine-n-oxide (Heliotropium indicum), di-n-oxide trilupine (Lupinus barbiger, L. laxus),
betaines e.g. stachydrine (Medicago sativa) and trigonelline (in fenugreek, garden peas, oats,
potatoes, coffee, hemp) are some examples of neutral alkaloids. Barberine and sanguinarine are
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33
33
two important alkaloids which possess antidiarrhetic and anticaries activities respectively
(Ramawat, 2007; Ramawat et al., 2009).
Phenolics
Phenolics are the compounds which have at least one hydroxyl group attached to an aromatic
ring such as catechol. Catechol and pyrogallol are hydroxylated phenols with antimicrobial
potential. The increase in number of hydroxyl groups in phenolics rings is attributed to increase
in relative toxicity. The mechanism of antimicrobial activity of phenolic compounds involves
enzyme inhibition by the oxidized compounds by reaction with sulfhydral or through more
nonspecific interactions with proteins. The phenolic group includes metabolites derived from the
condensation of acetate units (e.g. terpenoids), those produced by the modification of aromatic
amino acids (e.g. phenylpropanoids, cinnamic acids, lignin precursors, hydroxybenzoic acids,
catechols and coumarins), flavonoids, isoflavonoids and tannins . A phenyl group having three
carbon side chains is known as a phenylpropanoid, such as hydroxycoumarins, phenylpropenes
and lignans. The phenylpropenes are important components of many essential oils, e.g. eugenol
in clove oil (Syzygium aromaticum) and anethole and myristicin in nutmeg (Myristica fragrans)
(Cowan, 1999; Dewick, 2003; Ramawat et al., 2009).
Flavones, flavonoids, and flavonols
Flavones are phenolic structures consisting one carbonyl group. The addition of a 3-hydroxyl
group yields a flavonol. Flavonoids are derivatives of flavones composed of two benzene rings
attached by propane unit. Flavonoids are generally produced in plants in response to microbial
infection and their activity is owing to their ability to form complex with extracellular and
soluble proteins and form complexes with bacterial cell wall. The reduced forms of flavonoid
catechins exert antimicrobial activity in oolong green teas. Isoflavones are rearranged flavonoids
and occurred in pulses particularly in soybeans and chickpeas. Isoflavones possess other health-
promoting activities, such as chemoprevention of osteoporosis, prevention of postmenopausal
disorders and cardiovascular diseases and reduced the risk of prostate and breast cancer (Cowan,
1999; Uesugi, 2001).
Tannins
Tannins are group of polymeric phenolic substances found in almost every plant part: bark,
wood, leaves, fruits and roots. They are formed by condensations of flavan derivatives which
have been transported to woody tissues of plants. Consumption of tannin containing beverages
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34
especially green teas and red wines help in preventing a variety of diseases. Tannins help in the
stimulation of phagocytic cells, host mediated tumor activity, and a wide range of anti-infective
actions. The mode of antimicrobial action of tannins is related to their ability to inactivate
microbial adehsins, enzymes, and cell envelope transport proteins (Cowan 1999; Raskin et al.,
2002).
Quinones
Quinines are aromatic rings with two or more ketone substitutions. The natural quinone pigments
range in colour from pale yellow to almost black and there are over 450 known structures. These
compounds are responsible for the browning reactions in cut or damaged fruits and vegetable and
are an intermediate in the melanin synthesis pathway in human skin. Hypercin, an
anthroquinone, an example of quinine, is obtained from St. John’s Wort (Hypericum perforatum)
and has received much attention as an antidepressant, antiviral, and also for several other
antimicrobial properties. Anthroquinone from Cassia italica has been found to be bacteriostatic
for Bacillus antracis, Corynebacterium pseudodiphtherium and Pseudomonas aeruqinosa and
bactericidal for P. pseudomonilliae (Kazmi et al., 1994).
Saponins
Saponins are glycosides of both triterpenes and steroids that are characterized by their bitter or
astringent taste, foaming property, haemolytic effect on red blood cells and cholesterol binding
properties. Saponins are divided into two groups: Sterodal and terpenoids saponin. Terpenoids
saponins are found in many legumes such as soybean, peas and also in tea, spinach, sugarbeet
and sunflower while the steroidal saponins have been reported in oats, peppers, capsicum,
fenugreek and tomato (Okwu, 2005).
2.22 Taxonomical details of the plants evaluated for their bioactivity
On the basis of medicinal property, FSSAI standard and GRAS status, twenty plants belonging to
ten families were selected to find out their antimicrobial potential against locally isolated
microbes associated with juices. The plants used in the present study were identified by
consulting various books, monographs and manuals: Indian Materia Medica (Nandkarni, 2009);
Indian Medicinal Plants (Kirtikar and Basu, 1988) ; Encyclopedia of Indian Medicinal plants
(Khare, 2004); Indian Medicinal Plants- An illustrated Dictionary (Khare, 2007); Handbook of
Herbs and Spices (Peter, 2001); Chemistry of Spices (Parthasarathy et al., 2008) and A
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35
35
Handbook of Medicinal Plants: A Complete Source Book (Prajapati et al., 2003). Regulatory
status and brief descriptions of these plants are summarized in table 2.6.
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36
Table 2.6. Ethanobotanical description, phytochemical composition, regulatory status and part of plants used in
antimicrobial study
Scientific
name
Common
name
Family Plant
part
tested
Phytoconstitu
ents of part
used
Traditional uses Regulatory
status
References
Amomum
subulatum
Roxb.
(Fig. 2.2)
Badi
elachi
Zingiberaceae Fruit/
seeds
Carbohydrates,
flavonoids,
amino acids,
steroids,
triterpenoids,
glycosides,
tannins,
alkaloids, 1,8-
cineole,
limonene
Curative for throat trouble,
Congestion of lungs,
inflammation of eyelids,
digestive disorders and in the
treatment of pulmonary
tuberculosis, flavouring agent
in confectionery, hot or sweet
pickles and in beverages
FSSAI
2.9.9.4
Madhusoodan
an and Rao,
2001; Bisht et
al., 2011
Cinnamomum
tamala Nees
(Fig. 2.3)
Tejpatta Lauraceae Leaves Phellandrene,
eugenol,
linalool and
some traces of
α-pinene,
pcymene,
ß-pinene and
limonene,
phenylpropano
ids
Use in the treatment of
rheumatism, colic, diarrhoea,
nausea
----------- Shah and
Panchal,
2010; Panday
et al., 2012
Cinnamomum
zeylanicum
Breyn
(Fig. 2.4)
Dalchini Lauraceae Bark Cinnamaldehy
de, tannins
(5,7,3,,4,-
tetrahydroxy
Used in the treatment of
diarrhea, flatulent dyspepsia,
poor appetite, low vitality,
kidney weakness and
FSSAI
2.9.4,
GRAS, 21
CFR182.10
Ranasinghe et
al., 2012
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37
flavan-3,4–
diol)
rheumatism, influenza, cough,
bronchitis, fever, arthritic
angina, palpitations,
hypertension and nervous
disorders, stimulating the
circulatory system and
capillary circulation, spasms,
vomiting and controlling
infections, reducing blood
sugar levels in diabetics and
as a skin antiseptic
Coriandrum
sativum Linn.
(Fig. 2.5)
Dhania,
Coriander
Apiaceae Fruits Flavonoids,
isocoumarins,
fatty acids,
sterols and
coriandrones,
coumarins,
catechins,
polyphenolic
compounds
Used for indigestion, against
worms, rheumatism,pain in
the joints, against intestinal
parasites, seeds in sweet
vodka, ingredient of pickles
FSSAI
2.9.7,
GRAS, 21
CFR182.10
Asgarpanah
and
Kazemivash,
2012
Cumin
cyminum
Linn.
(Fig. 2.6)
Jeera Apiaceae Fruits Diverse
flavonoids,
isoflavonoids,
flavonoid
glycosides,
monoterpenoid
glucosides,
lignins and
alkaloids and
Used in the treatment of mild
digestive disorders, diarrhea,
dyspepsia, flatulence,
morning sickness, colic,
dyspeptic headache and
bloating, flavouring agent in
confectionery, meat, sausage
and bread manufacturing and
as a preservative in food
FSSAI
2.9.8,
GRAS, 21
CFR182.10
Amin, 2001;
Johri, 2011
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38
other phenolic
compounds
processing
Curcuma
longa Linn.
(Fig. 2.7)
Haldi Zingiberaceae Rhizome Curcumin
(diferuloylmet
hane), a-
phellandrene,
sabinene,
borneol,
zingiberene,
sesquiterpines
Used to treat gastrointestinal
upsets, arthritis pain, tonic for
the digestive system
FSSAI
2.9.18,
GRAS, 21
CFR182.10
Chattopadhya
y et al., 2004
Elettaria
cardamomum
Maton
(Fig. 2.8)
Chhoti
elachi
Zingiberaceae Fruits/
seeds
α-terpineol,
1,8-cineole,
with smaller
amounts of
borneol,
camphor,
limonene, α-
terpenyl
acetate and α-
pinene
Used in aromatherapy to
stimulate energy, aphrodisiac
and remedy in case of
digestive problems, asthma,
bronchitis, and urinary
complaints and several other
human ailments, in flavouring
pickles, meat and canned
soups.
FSSAI
2.9.2.1,
GRAS, 21
CFR182.10
Korikanthimat
h, 2001;
Kaushik et al.,
2010
Emblica
officinalis
(Fig. 2.9)
Amla Euphorbiaceae Leaves Gallic caid,
ethyl gallate,
1,2,3,4,6-
penta-O-
galloylglucose
and luteolin -
4’-
Oneohesperiod
oside
Source of Vitamin C,
enhances food absorption,
balances stomach acids,
fortifies the liver, supports the
heart, promotes healthier hair
------ Aneja et al.,
2010
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39
Ferula
asafoetida
Linn.
(Fig. 2.10)
Hing Apiaceae Gum
resin
Sesquiterpene
coumarins, 2-
butyl 1-
propenyl
disulfide, 1-
(methylthio)pr
opyl 1-
propenyl
disulfide and
2-butyl 3-
(methylthio)-2-
propenyl
disulfide
Used for Flatulence, hysteria
and nervous disorders,
asthma, flavoring spice in a
variety of foods
FSSAI
2.9.29
Iranshahy and
Iranshahi,
2011
Foeniculum
vulgare
Gaertn
(Fig. 2.11)
Fennel
/Saunf
Apiaceae Fruit Anethole,
fenchone
Essence in cosmetics and
perfumes industry
FSSAI
2.9.9.2,
GRAS 21
CFR182.10
Oktay et al.,
2003
Illicium
verum Hook.
(Fig. 2.12)
Chinese
star anise
Illiciaceae Fruits Seco-
prezizaane-
type
sesquiterpenes,
phenylpropano
ids, lignans,
flavonoids
Used to treat infant colic GRAS 21
CFR182.10
Wang et al.,
2011
Mentha
arvensis
Linn.
(Fig. 2.13)
Pudina Lamiaceae Leaves Tannins,
phenols,
steroids,
flavonoids
and volatile
Used to treat liver and spleen
diseases, asthma and jaundice.
------- Kumbalwar et
al., 2014
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40
oils,
Myristica
fragrans
Houtt.
(Fig. 2.14)
Jaiphal Myristicaceae Fruits Myristicin,
Lignans,
monoterpene
hydrocarbons
pinene and
sabinene
Used for flatulence,nausea
and vomiting, for
convalescents, as an ointment
for piles, for leucorrhoea and
as a local stimulant to the
gastro-intestinal tract,
flavouring agent for food
products and liquors
FSSAI
2.9.14,
GRAS 21
CFR182.10
Krishnamoort
hy and Rema,
2001;
Chatterjee et
al., 2007
Ocimum
sanctum Linn.
(Fig. 2.15)
Tulsi Lamiaceae Leaves β-bisabolene ,
methyl
chavicol, 1,8-
cineole ,
eugenol, (E)-a-
bisabolene and
a-terpineol
Antimicrobial,
immunomodulatory, anti-
stress, anti-inflammatory,
antipyretic, anti-asthmatic,
hypoglycemic, hypotensive
and analgesic activities
GRAS 21
CFR182.10
Singh et al.,
2011
Piper nigrum
Linn.
(Fig. 2.17)
Black
pepper
Piperaceae Leaves Piperine Stimulating the digestive
enzymes of pancreas,
enhances the digestive
capacity and significantly
reduces the gastrointestinal
food transit
Time
FSSAI
2.9.15,
GRAS 21
CFR182.10
Srinivasan,
2007
Syzygium
aromaticum
Linn.
(Fig. 2.16)
Clove,
Laung
Myrtaceae Dry
flower
buds
Eugenol,
eugeniin,
acetyl eugenol,
quercetic acid,
gallic acid,
vanillin
Used in toothache,
particularly to aid digestion,
cure stomach disorders and in
pain relief, antiseptic, for
topical anesthesia in dentistry
FSSAI 2.9.6 Arora and
Kaur, 1999;
Nurdjannah
and
Bermawie,
2001; Negi,
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41
2012
Terminalia
arjuna Wight
& Arn.
(Fig. 2.18)
Arjun Combretaceae Leaves Flavonoid Used as a remedy for the
treatment of ear ache
---------- Aneja et al.,
2012
Terminalia
chebula Retz.
(Fig. 2.19)
Harad,
black
myroblans
Combretaceae Fruits Hydrolysable
tannins, gallic
acid,
chebulagic
acid,
punicalagin,
chebulanin,
corilagin,
neochebulinic
acid, ellagic
acid,
chebulinic acid
Household remedy against
asthma, sore throat, vomiting,
hiccough, diarrhea, bleeding
piles, gout, and heart and
bladder disease
---------- Sharma et al.,
2012;
Rathinamoort
hy and
Thilagavathi,
2014
Trachyspermu
m copticum
Linn.
(Fig. 2.20)
Ajowan Apiaceae Fruits Thymol ,
terpinene, p-
cymene,
pinene
Used as a digestive stimulant
or to treat liver disorders
FSSAI
2.9.22
Nagalakshmi
et al., 2000;
Murthy et al.,
2009
Zingiber
officinale
Roscoe
(Fig. 2.21)
Saunth,
dried
ginger
Zingiberaceae Rhizome Gingerol (5-
hydroxy-1-(4
hydroxy-3-
methoxy
phenyl) decan-
3-one)
Commonly used in food
products and beverages,
carminative, antispasmodic,
digestive, stomachic,
vasodilator, appetizer,
expectorant, bronchodilator,
topical and local stimulant,
analgesic, antiflatulent,
FSSAI
2.9.11,
GRAS 21
CFR182.10
Sunilson et
al., 2009
Page 37
42
aphrodisiac, digestive,
antitussive, antiflatulent,
arthritis, rheumatism, sprains,
muscular aches, pains and
laxative
FSSAI- Food Safety and Standards Authority of India; GRAS-Generally Recognized As Safe; CFR- Title 21 of the U.S. Code of
Federal Regulations
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43
Fig.2.2. Amomum subulatum- plant, fruits and Fig.2.3.Cinnamomum tamala- plant with
seeds (inset) leaves
Fig.2.4. Cinnamomum zeylanicum-trunk and Fig. 2.5. Coriandrum sativum plant and fruits
(inset)bark (inset)
Fig.2.6. Cumin cyminum seeds
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44
Fig.2.7. Curcuma longa- rhizome Fig.2.8. Elettaria cardamomum- plant and fruits
(inset)
Fig.2.9. Emblica officinalis- plant Fig.2.10. Ferula asafoetida- gumresins
Fig.2.11. Foeniculum vulgare- branches with inflorescence and seeds (inset)
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45
Fig. 2.12. Illicium verum- fruits Fig. 2.13. Mentha arvensis- plant
Fig. 2.14. Myristica fragrans- fruits Fig. 2.15. Ocimum sanctum- plant
Fig 2.16. Syzygium aromaticum- dry flower buds
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46
Fig 2.17. Piper nigrum- fruits Fig 2.18. Terminalia arjuna-tree with leaves
Fig. 2.19. Terminalia chebula- plant with leaves Fig 2.20. Trachyspermum copticum- fruits
and fruits (inset)
Fig. 2.21. Zingiber officinale- plant with rhizome (inset)
