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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,
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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*

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
*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
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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

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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). 8
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
9 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 10 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
11 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 12
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
13 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 Orange, Pomegranate, Mango, Faecal coliforms and faecal Lewis et al., 2004
(India) Pine apple, Grape streptococci
2 Mumbai (India) Sugarcane,Lime, Carrot Vibrio cholerae, E. coli, Mahale et al., 2008
S.aureus
3 Jimma town Avocado, Papaya, Pine apple Klebsiella, Enterobacter, Ketema et al., 2008
(South west Serratia
Ethopia)
4 Nagpur (India) Pine apple, Sweet Lime, Carrot Salmonella, coliforms, Titarmare et al., 2009
juice S.aureus
5 Amravati (India) Apple, Orange, Pineapple, Salmonella, coliforms, Tambaker et al., 2009
Pomegranate, Sweet lemon , S.aureus,Pseudomonas,
Mixed fruit Proteus
6 Kolkata Mango , Pineapple, Sweet Vibrio and Salmonella Mukhopadhyay et al., 2011
lime, lemon, Pomegranate,
Sugarcane
7 Mysore Orange, Sweet lime and Micrococcus spp., Bacillus, Divyashree et al., 2013
Pineapple Streptococcus,
Staphylococcus
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 Pathogens Year Country Venue Number Reference


juice of cases
(deaths)

Unpasteurized Escheichia coli 1991 USA Small cider 23(0) Besser et al.,
apple juice O157:H7 mill 1993

Unpasteurized Enterotoxigenic 1992 India Roadside 6 (0) Singh et al.,


orange juice E. coli vendor 1995

Unpasteurized Cryptosporidium 1993 USA School 213(0) Millard et


apple juice al., 1994

Carrot Clostridium 1993 USA Home 1 (0) Buzby and


homemade botulinum Crutchfield,
juice 1999

Unpasteurized Salmonella 1995 USA Retail 63 (0) CDC, 1995;


orange juice gaminara, Cook et al.,
S.hartford and S. 1998;
rubislaw Parish, 2000

Unpasteurized Shigella flexneri 1995 South Restaurant 14(0) Thurston et


orange juice Africa al., 1998

Unpasteurized C. parvum 1996 USA Small cider 31 (0) CDC, 1997


apple juice mill

Unpasteurized E. coli O157:H7 1996 USA Small cider 14 (0) CDC, 1997
apple juice mill

Unpasteurized E. coli O157:H7 1996 USA Small cider 6 (0) FDA, 2001
apple juice mill
15
Unpasteurized E. coli O157:H7 1998 Canada Farm/Home 14 (0) Tamblyn et
apple juice al., 1999

Unpasteurized E. coli O157:H7 1999 USA Not 25 (0) CDC, 2011


apple juice reported

Unpasteurized S. muenchen 1999 Canada, Restaurant 423 (1) CDC, 1999


orange juice USA

15
Unpasteurized S. anatum 1999 USA Roadside 6 (0) Krause et
orange juice stand al., 2001

Unpasteurized S. typhimurium 1999 Australia Retail 405 (0) Mahale et


orange juice al., 2008

Unpasteurized Vibrio cholerae 1999 India Not ------------ Mahale et


sugar cane reported al., 2008
juice

Unpasteurized S. enteritidis 2000 USA Retail and 88(0) Butler, 2000


orange juice food
service

Juice Cryptosporidium 2002 Colombia Not 56 Botero-


cayetanensis reported Graces et
al., 2006

Raspberry C. cayetanensis 2003 Guatemala Not 7 Puente et


juice reported al., 2006

Unpasteurized C. parvum 2003 USA Farm/Retail 144 (0) Vojdani et


apple juice al., 2008

Unpasteurized E. coli O111 and 2004 USA Farm/Home 212 (0) Vojdani et
apple juice C. parvum al., 2008

Unpasteurized S. typhimurium 2005 USA Retail and 152 (0) Jain et al.,
orange juice and S. saintpaul food 2009
service

Unpasteurized Trypanosoma 2005 Brazil Roadside 25 (3) Pereira,


sugar cane cruzi kiosk 16 2009
juice
Pasteurized C. botulinum 2006 USA Retail 4(0) CDC, 2006
carrot juice

Unpasteurized E. coli O157:H7 2007 USA Not 9(0) CDC, 2011


apple juice reported

Unpasteurized E. coli O157:H7 2008 USA Retail 7 CDC, 2011

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apple juice

Unpasteurized S. panama 2008 Netherlands Not 33(0) Bevilacqua


orange juice reported et al., 2011

Unpasteurized E. coli O157:H7 2010 USA Fair 7(0) FDA, 2010


apple juice

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 17 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.

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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
18 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

18
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 71oC 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
19 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

19
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
20 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

20
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
21 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).

21
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, E.coliO157:H, Salmonella enteriditis Mosqueda–Melgar et al.,
pear juice 2008b
6 Fumaric acid Apple cider E.coliO157:H7 Comes and Beelman, 2002
7 Lactic acid Apple cider E.coliO157:H7, Salmonella enteriditis, yeast Uljas and Ingahm ,1999
and molds
8 Malic acid Apple juice, pear juice, E.coliO157:H7, Salmonella enteriditis, Raybaudi- Massilia et al.,
melon juice Listeria monocytogenes 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

22
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 antimicrobials23


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

23
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 are24the 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).

24
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
25 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).

25
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
26 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).

26
Table 2.5. Antimicrobial effect of essential oil on pathogenic and spoilage microflora of fruit juices

Essential oil or Fruit juice (pH) Storage conditions Target Effect Reference
active Temper Time microorganisms
compound
ature
Apple (4.20), pear 35 24 h Listreia innocua, Reduced > 5log Raybaudi-Massilia et
(3.97), melon Salmonella enteritidis, CFU/ml al., 2006
(5.91) Escherichia coli
Strawberry (3.16), 22 1h S. enteritidis, E. coli Reduced > 5log Mosqueda-Melgar et
orange (3.44), O157:H7 CFU/ml al., 2008a,b
apple (4.46), pear
(4.40) and tomato
(4.30)
Cinnamon oil Melon (6.11) and 22 1h S. enteritidis, E. coli Reduced 3.1 to Mosqueda-Melgar et
watermelon (5.73) O157:H7, L. 3.9, 1.4 to 1.9 and al., 2008c
monocytogenes 3.4 to 4.4 log
CFU/ml of S.
enteritidis, E. coli
O157:H7 and L.
monocytogenes
respectively
Apple (3.7) 37 1h S. enteritidis, E. coli Reduced 50% of Friedman et al.,2004
O157:H7 bacterial
population
Clove oil Tomato (4.2) 50 0.5h Native microbiota Reduced 3.9 log Nguyen and Mittal,
CFU/ml 2007
Apple (3.7) 37 1h S. hadar, E. coli Reduced 50% of Friedman et al., 2004
O157:H7 bacterial
population

27
Lemon oil Apple (3.7) 37 1h S. hadar, E. coli Reduced 50% of Friedman et al., 2004
O157:H7 bacterial
population
Apple (4.20), pear 35 24 h Listreia innocua, Reduced > 5log Raybaudi-Massilia et
(3.97), melon Salmonella enteritidis, CFU/ml al., 2006
(5.91) Escherichia coli
Apple (3.7) 37 1h S. hadar, E. coli Reduced 50% of Friedman et al., 2004
O157:H7 bacterial
population
Lemongrass oil Apple orange 28 4h Saccharomyces Reduced to 3, 2.3 Tyagi et al., 2014
mixture cerevisiae, and 2.1 log
Zygosacsharomyces CFU/ml of S.
bailli, Pichia cerevisiae, Z.
fermentans bailli, P.
fermentans
Lime oil Apple (3.7) 37 1h S. hadar, E. coli Reduced 50% of Friedman et al., 2004
O157:H7 bacterial
population
Oregano oil Apple (3.7) 37 1h S. hadar, E. coli Reduced 50% of Friedman et al., 2004
O157:H7 bacterial
population
Cavacarol Apple (3.7) 37 1h S. hadar, E. coli Reduced 50% of Friedman et al., 2004
O157:H7 bacterial
population
Cinnamaldehyde Apple (3.7) 37 1h S. hadar, E. coli Reduced 50% of Friedman et al., 2004
O157:H7 bacterial
population
Citral Orange (3.5) 45 0.5h L. monocytogenes Reduces 1.1 to Ferrante et al., 2007
1.3 log CFU/ml

28
Apple (3.7) 37 1h S. hadar, E. coli Reduced 50% of Friedman et al., 2004
O157:H7 bacterial
population
Eugenol Apple (3.7) 37 1h S. hadar, E. coli Reduced 50% of Friedman et al., 2004
O157:H7 bacterial
population
Menthol Apple-orange 37 2h Saccharomyces Reduced 2 log Tyagi et al., 2013
mixture cerevisiae, cycles

Geraniol Apple (4.20), pear 35 24 h Listreia innocua, Reduced > 5log Raybaudi-Massilia et
(3.97), melon Salmonella enteritidis, CFU/ml al., 2006
(5.91) Escherichia coli
Apple (3.7) 37 1h S. hadar, E. coli Reduced 50% of Friedman et al., 2004
O157:H7 bacterial
population

29
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 30 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,

30
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 31
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.

31
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), 32
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

32
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,33prevention 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

33
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
34 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

34
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.

35

35
Table 2.6. Ethanobotanical description, phytochemical composition, regulatory status and part of plants used in
antimicrobial study
Scientific Common Family Plant Phytoconstitu Traditional uses Regulatory References
name name part ents of part status
tested used
Amomum Badi Zingiberaceae Fruit/ Carbohydrates, Curative for throat trouble, FSSAI Madhusoodan
subulatum elachi seeds flavonoids, Congestion of lungs, 2.9.9.4 an and Rao,
Roxb. amino acids, inflammation of eyelids, 2001; Bisht et
(Fig. 2.2) steroids, digestive disorders and in the al., 2011
triterpenoids, treatment of pulmonary
glycosides, tuberculosis, flavouring agent
tannins, in confectionery, hot or sweet
alkaloids, 1,8- pickles and in beverages
cineole,
limonene
Cinnamomum Tejpatta Lauraceae Leaves Phellandrene, Use in the treatment of ----------- Shah and
tamala Nees eugenol, rheumatism, colic, diarrhoea, Panchal,
(Fig. 2.3) linalool and nausea 2010; Panday
some traces of et al., 2012
α-pinene,
pcymene,
ß-pinene and
limonene,
phenylpropano
ids
Cinnamomum Dalchini Lauraceae Bark Cinnamaldehy Used in the treatment of FSSAI Ranasinghe et
zeylanicum de, tannins diarrhea, flatulent dyspepsia, 2.9.4, al., 2012
Breyn (5,7,3,,4,- poor appetite, low vitality, GRAS, 21
(Fig. 2.4) tetrahydroxy kidney weakness and CFR182.10

36
flavan-3,4– rheumatism, influenza, cough,
diol) 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 Dhania, Apiaceae Fruits Flavonoids, Used for indigestion, against FSSAI Asgarpanah
sativum Linn. Coriander isocoumarins, worms, rheumatism,pain in 2.9.7, and
(Fig. 2.5) fatty acids, the joints, against intestinal GRAS, 21 Kazemivash,
sterols and parasites, seeds in sweet CFR182.10 2012
coriandrones, vodka, ingredient of pickles
coumarins,
catechins,
polyphenolic
compounds
Cumin Jeera Apiaceae Fruits Diverse Used in the treatment of mild FSSAI Amin, 2001;
cyminum flavonoids, digestive disorders, diarrhea, 2.9.8, Johri, 2011
Linn. isoflavonoids, dyspepsia, flatulence, GRAS, 21
(Fig. 2.6) flavonoid morning sickness, colic, CFR182.10
glycosides, dyspeptic headache and
monoterpenoid bloating, flavouring agent in
glucosides, confectionery, meat, sausage
lignins and and bread manufacturing and
alkaloids and as a preservative in food

37
other phenolic processing
compounds
Curcuma Haldi Zingiberaceae Rhizome Curcumin Used to treat gastrointestinal FSSAI Chattopadhya
longa Linn. (diferuloylmet upsets, arthritis pain, tonic for 2.9.18, y et al., 2004
(Fig. 2.7) hane), a- the digestive system GRAS, 21
phellandrene, CFR182.10
sabinene,
borneol,
zingiberene,
sesquiterpines
Elettaria Chhoti Zingiberaceae Fruits/ α-terpineol, Used in aromatherapy to FSSAI Korikanthimat
cardamomum elachi seeds 1,8-cineole, stimulate energy, aphrodisiac 2.9.2.1, h, 2001;
Maton with smaller and remedy in case of GRAS, 21 Kaushik et al.,
(Fig. 2.8) amounts of digestive problems, asthma, CFR182.10 2010
borneol, bronchitis, and urinary
camphor, complaints and several other
limonene, α- human ailments, in flavouring
terpenyl pickles, meat and canned
acetate and α- soups.
pinene
Emblica Amla Euphorbiaceae Leaves Gallic caid, Source of Vitamin C, ------ Aneja et al.,
officinalis ethyl gallate, enhances food absorption, 2010
(Fig. 2.9) 1,2,3,4,6- balances stomach acids,
penta-O- fortifies the liver, supports the
galloylglucose heart, promotes healthier hair
and luteolin -
4’-
Oneohesperiod
oside

38
Ferula Hing Apiaceae Gum Sesquiterpene Used for Flatulence, hysteria FSSAI Iranshahy and
asafoetida resin coumarins, 2- and nervous disorders, 2.9.29 Iranshahi,
Linn. butyl 1- asthma, flavoring spice in a 2011
(Fig. 2.10) propenyl variety of foods
disulfide, 1-
(methylthio)pr
opyl 1-
propenyl
disulfide and
2-butyl 3-
(methylthio)-2-
propenyl
disulfide
Foeniculum Fennel Apiaceae Fruit Anethole, Essence in cosmetics and FSSAI Oktay et al.,
vulgare /Saunf fenchone perfumes industry 2.9.9.2, 2003
Gaertn GRAS 21
(Fig. 2.11) CFR182.10
Illicium Chinese Illiciaceae Fruits Seco- Used to treat infant colic GRAS 21 Wang et al.,
verum Hook. star anise prezizaane- CFR182.10 2011
(Fig. 2.12) type
sesquiterpenes,
phenylpropano
ids, lignans,
flavonoids
Mentha Pudina Lamiaceae Leaves Tannins, Used to treat liver and spleen ------- Kumbalwar et
arvensis phenols, diseases, asthma and jaundice. al., 2014
Linn. steroids,
(Fig. 2.13) flavonoids
and volatile

39
oils,
Myristica Jaiphal Myristicaceae Fruits Myristicin, Used for flatulence,nausea FSSAI Krishnamoort
fragrans Lignans, and vomiting, for 2.9.14, hy and Rema,
Houtt. monoterpene convalescents, as an ointment GRAS 21 2001;
(Fig. 2.14) hydrocarbons for piles, for leucorrhoea and CFR182.10 Chatterjee et
pinene and as a local stimulant to the al., 2007
sabinene gastro-intestinal tract,
flavouring agent for food
products and liquors
Ocimum Tulsi Lamiaceae Leaves β-bisabolene , Antimicrobial, GRAS 21 Singh et al.,
sanctum Linn. methyl immunomodulatory, anti- CFR182.10 2011
(Fig. 2.15) chavicol, 1,8- stress, anti-inflammatory,
cineole , antipyretic, anti-asthmatic,
eugenol, (E)-a- hypoglycemic, hypotensive
bisabolene and and analgesic activities
a-terpineol
Piper nigrum Black Piperaceae Leaves Piperine Stimulating the digestive FSSAI Srinivasan,
Linn. pepper enzymes of pancreas, 2.9.15, 2007
(Fig. 2.17) enhances the digestive GRAS 21
capacity and significantly CFR182.10
reduces the gastrointestinal
food transit
Time
Syzygium Clove, Myrtaceae Dry Eugenol, Used in toothache, FSSAI 2.9.6 Arora and
aromaticum Laung flower eugeniin, particularly to aid digestion, Kaur, 1999;
Linn. buds acetyl eugenol, cure stomach disorders and in Nurdjannah
(Fig. 2.16) quercetic acid, pain relief, antiseptic, for and
gallic acid, topical anesthesia in dentistry Bermawie,
vanillin 2001; Negi,

40
2012
Terminalia Arjun Combretaceae Leaves Flavonoid Used as a remedy for the ---------- Aneja et al.,
arjuna Wight treatment of ear ache 2012
& Arn.
(Fig. 2.18)
Terminalia Harad, Combretaceae Fruits Hydrolysable Household remedy against ---------- Sharma et al.,
chebula Retz. black tannins, gallic asthma, sore throat, vomiting, 2012;
(Fig. 2.19) myroblans acid, hiccough, diarrhea, bleeding Rathinamoort
chebulagic piles, gout, and heart and hy and
acid, bladder disease Thilagavathi,
punicalagin, 2014
chebulanin,
corilagin,
neochebulinic
acid, ellagic
acid,
chebulinic acid
Trachyspermu Ajowan Apiaceae Fruits Thymol , Used as a digestive stimulant FSSAI Nagalakshmi
m copticum terpinene, p- or to treat liver disorders 2.9.22 et al., 2000;
Linn. cymene, Murthy et al.,
(Fig. 2.20) pinene 2009
Zingiber Saunth, Zingiberaceae Rhizome Gingerol (5- Commonly used in food FSSAI Sunilson et
officinale dried hydroxy-1-(4 products and beverages, 2.9.11, al., 2009
Roscoe ginger hydroxy-3- carminative, antispasmodic, GRAS 21
(Fig. 2.21) methoxy digestive, stomachic, CFR182.10
phenyl) decan- vasodilator, appetizer,
3-one) expectorant, bronchodilator,
topical and local stimulant,
analgesic, antiflatulent,

41
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

42
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

43
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)

44
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

45
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)

46