DEVELOPMENT OF PROBIOTIC JUICE BY LACTIC ACID FERMENTATION OF FRUITS AND VEGETABLES

Most of the presently available probiotics foods are based on milk. But there are several problems associated with consumption of milk such as lactose intolerance and increase in the level of the cholesterol level of the consumers. Lactose intolerance and the cholesterol content are two drawbacks related to the consumption of milk and milk products. Currently, products are usually marketed in the form of fermented milk and yoghurt. It has also been suggested that fruit juice could serve as a good medium for cultivating probiotics (MattilaSandholm, 2002). The use of probiotics in the fruit and vegetable juice industry offer to consumers with special needs (vegetarian people with allergic reactions to milk proteins) the possibility to experience the positive effect of probiotic bacteria (Luckow and Delahunty, 2004). There is scanty of information on the lactic acid fermentation of fruits and vegetables juices especially with the respect to optimization of fermentation as probiotic juices and their evaluation as probiotic juices in the light of guidelines for such juices. Similarly, the storage behaviour and safety of such juices have not been documented. Recently, the antioxidant and antimicrobial phenomenon in fermented foods is being advocated, can these juices be designated having these characteristics, is not known (Stevans and Sheldon, 1992; Subrota et al., 2015).

DEVELOPMENT OF PROBIOTIC JUICE BY LACTIC ACID FERMENTATION OF FRUITS AND VEGETABLES

Thesis Thesis

COLLEGE OF HORTICULTURE

Dr YASHWANT SINGH PARMAR UNIVERSITY

OF HORTICULTURE AND FORESTRY, NAUNI

FOOD SCIENCE AND TECHNOLOGY

2015

Dr V K Joshi

Retd Professor & Head

Department of Food Science and Technology College of Horticulture

Dr Y S Parmar University of Horticulture and Forestry, Nauni, Solan – 173 230

CERTIFICATE - I

This is to certify that the thesis titled, “Development of probiotic juice by

lactic acid fermentation of fruits and vegetables”, submitted in partial fulfillment

of the requirements for the award of degree of MASTER OF SCIENCE (FOOD

TECHNOLOGY) to Dr Yashwant Singh Parmar University of Horticulture and

Forestry, (Nauni) Solan (HP) - 173230 is a bonafide research work carried out by

Ms Anuradha Thakur (H-13-21-M) daughter of Shri Madan Singh under my

supervision and that no part of this thesis has been submitted for any other degree or

diploma

The assistance and help received during the course of this investigation

have been fully acknowledged

Place: Nauni, Solan (Dr V K Joshi)

Dated: July, 2015 Chairman

Advisory Committee

CERTIFICATE - II

This is to certify that the thesis titled, “Development of probiotic juice

by lactic acid fermentation of fruits and vegetables” submitted by Ms Anuradha Thakur (H-13-21-M) daughter of Shri Madan Singh to Dr Yashwant Singh Parmar

University of Horticulture & Forestry, (Nauni), Solan (HP) - 173230 India, in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE

FOOD TECHNOLOGY has been approved by the Student’s Advisory

Committee after an oral examination of the same in collaboration with the external examiner.

(Retd Professor & Head)

Chairman, Advisory Committee

Members of Advisory Committee

Dr N S Thakur Dr (Mrs) Nivedita Sharma (Professor) (Professor)

Dr R K Gupta (Professor)

Dean College of Horticulture

Dr Y S Parmar University of Horticulture and Forestry

Nauni, Solan (HP)

At the onset of acknowledging the help of those who have contributed to the realization of this manuscript, I would like to express my profound gratitude and thanks to my esteemed teacher and chairman of

my Advisory Committee, Dr VK Joshi, retired Professor and Head of Department of Food Science and Technology for his inspiring vision, valuable suggestions, everlasting patience, logical and necessary criticism, advice and constant encouragement at every step of research work, and finalization of this manuscript It was great opportunity for me to work under his guidance.

It gives me adnascent pleasure in expressing my heartfelt gratitude to the worthy members of my Advisory Committee: Dr NS Thakur (Professor, Department of Food Science & Technology), Dr (Mrs) Nivedita Sharma (Professor, Microbiology, Department of Basic Science) and Dr RK Gupta(Professor, Statistics, Department of Basic Science) for their valuable suggestions and guidance with their scientific acumen during the investigation and thesis preparation

I earnestly acknowledge assiduous effort and sanguine gestures of Dr Anju Dhiman, Prof and Head Dept of Food Science and Technology, Mrs Surekha Attri, Dr KS Thakur, Dr Rakesh Sharma, Dr Devina Vaidya, and Dr Manisha Kaushal and all teachers of my department for their sincere help and valuable suggestions

I cordially acknowledge the assistance extended by Smt Hemlata Ji, Smt Shalini Parmar Ji, Sh Tapender Ji, Sh Manglesh Ji, Sh Khemchand Ji, Sh Hemchand Ji, Sh R L Junta Ji, Sh Lekh Ram Ji, Sh Raj Kumar Ji, Smt Kaushalya Ji, Sh Prakash Ji, Sh.Rohit Ji, Sh Narender Ji, Dipti di, Manju di and all staff members of Department of Food Science and Technology

Every effort is motivated by an ambition and all ambition has an inspiration behind I owe this place to

my ever loving family members, Papa (Sh Madan Singh), Mamma (Smt Mangla Devi), Archana (sister),Vicky (Brother) and Vandu (Bhabhi) for their selfless sacrifice, heartfull blessings, firm faith and supporting me psychologically, emotionally and morally which steered the life of this cipher to the present shape

I cant’ forget to mention the care and love showered upon me by Late Sh Kaliram Thakur (Dadaji), Late Smt Manti Devi, all tau ji’s and tai ji’ s, all chacha ji’ s and chachi ji’ s, all my cousins brother especially Devu bhai, Teshu bhai, cousins sisters, all bhabi ji’s, niece and nephew There are certain people in everyone’s life whose best wishes and blessings paved the way towards success and they are one of them

I cannot forget to express by the same occasions my deep and sincere thanks to my seniors Vikas sir, Navven sir, Abhimanyu sir, Sangeeta di, Priyanka di, Apoorva di, Nisha di, my classmates Jagriti, Pritika, Hamid, Dharmu sir, Babu, Niku, Abhishek, Aarti and my juniors Mona, Isha, Sheilja and Akriti for their continous support during entire course of my study I have special word of thanks for my friends Monika di, Shilpa, Neha, Rajni, Nitika, Cherry, Jagriti, Ankush, Deepak, Vikku and Late Ritu, who made my every moment living, enjoyable, stress free with their internal support and caring nature, helped me to feel better and encouraged me to carry out my work in time

I would like to thank DPT computers for their intense endeavour and kind cooperation in typesetting this manuscript

I solely claim all the responsibilities for the shortcomings and limitations in this work

Thanks to one and all and to those whose names could not appear but who at one stage or the other has helped me in some ways to achieve the goal

Place: Nauni

Date: (Anuradha Thakur)

CONTENTS

BRIEF BIO-DATA

LIST OF TABLES

2.3 Physico-chemical composition of orange 8 2.4 Physico-chemical composition of cabbage 8 2.5 Physico-chemical composition of cucumber 9 2.6 Physico-chemical composition of carrot 10 2.7 Characteristics of lactic acid bacteria 11 2.8 Key genera and species of microbes studied and used as

4.2 Physico-chemical characteristics of vegetable (cabbage,

cucumber and carrot) juices

43

4.3 Effect of lactic acid fermentation on TSS, pH and titratable

acidity of apple juice

46

4.4 Effect of lactic acid fermentation on reducing sugar, total

Sugar and total phenols of apple juice

48

4.5 Effect of lactic acid fermentation on antioxidant activity

and ascorbic acid of apple juice

50

4.6 Effect of lactic acid fermentation on viable cell counts,

LAB counts of apple juice

52

4.7 Effect of lactic acid fermentation on coliform counts, yeast

and mold counts of apple juice

53

4.8 Effect of lactic acid fermentation on antimicrobial activity

of apple juice

55

4.9 Comparison of quality of lactic acid bacteria of probiotic

apple juices on the basis of physico-chemical parameters

56

4.10 Comparison of quality of lactic acid bacteria of probiotic

apple juices on the basis of microbiological quality

57

4.11 Effect of lactic acid fermentation on TSS, pH and titratable

acidity of pear juice

59

4.12 Effect of lactic acid fermentation on reducing sugar and

total sugar of pear juice

61

4.13 Effect of lactic acid fermentation on antioxidant activity

and ascorbic acid of pear juice

63

4.14 Effect of lactic acid fermentation on viable cell counts and

LAB counts of pear juice

64

Table Title Page

4.15 Effect of lactic acid fermentation on coliform counts, yeast

and mold of pear juice

66

4.16 Effect of lactic acid fermentation on antimicrobial activity

of pear juice

68

4.17 Comparison of quality of lactic acid bacteria of probiotic

pear juices on the basis of physico-chemical parameters

69

4.18 Comparison of quality of lactic acid bacteria of probiotic

pear juices on the basis of microbiological quality

69

4.19 Effect of lactic acid fermentation on TSS, pH and titratable

acidity of orange juice

70

4.20 Effect of lactic acid fermentation on reducing sugar and

total sugar of orange juice

73

4.21 Effect of lactic acid fermentation on antioxidant activity

and ascorbic acid of orange juice

75

4.22 Effect of lactic acid fermentation on viable cell counts and

LAB counts of orange juice

76

4.23 Effect of lactic acid fermentation on coliform counts, yeast

and mold of orange juice

78

4.24 Effect of lactic acid fermentation on antimicrobial activity

of orange juice

80

4.25 Differentiation of quality of lactic acid bacteria of probiotic

orange juices on the basis of physico-chemical parameters

81

4.26 Differentiation of quality of lactic acid bacteria of probiotic

orange juices on the basis of microbiological quality

81

4.27 Evaluation of all probiotic fruits (apple, pear and orange )

juices on the basis of rate of fermentation (%/24hr) and

rate of acidification (°B/24hr) and lactic acid bacteria

counts(108cfu/ml)

82

4.28 Effect of lactic acid fermentation on TSS, pH and titratable

acidity of cabbage juice

85

4.29 Effect of lactic acid fermentation on reducing sugar, total

Sugar and total phenols of cabbage juice

87

4.30 Effect of lactic acid fermentation on antioxidant activity

and ascorbic acid of cabbage juice

88

4.31 Effect of lactic acid fermentation on viable cell counts,

LAB counts of cabbage juice

89

4.32 Effect of lactic acid fermentation on coliform counts, yeast

and mold of cabbage juice

92

4.33 Effect of lactic acid fermentation on antimicrobial activity

of cabbage juice

93

4.34 Differentiation of quality of lactic acid bacteria of probiotic

cabbage juices on the basis of physico-chemical parameters

94

4.35 Differentiation of quality of lactic acid bacteria of probiotic

cabbage juices on the basis of microbiological quality

94

4.36 Effect of lactic acid fermentation on TSS, pH and titratable

acidity of cucumber juice

97

Table Title Page

4.37 Effect of lactic acid fermentation on reducing sugar, total

sugar and total phenols of cucumber juice

99

4.38 Effect of lactic acid fermentation on antioxidant activity

and ascorbic acid of cucumber juice

100

4.39 Effect of lactic acid fermentation on viable cell counts and

lactic acid bacteria counts of cucumber juice

101

4.40 Effect of lactic acid fermentation on coliform counts ,yeast

and mold of cucumber juice

104

4.41 Effect of lactic acid fermentation on antimicrobial activity

of cucumber juice

105

4.42 Differentiation of quality of lactic acid bacteria of probiotic

cucumber juices on the basis of physico-chemical

parameters

105

4.43 Differentiation of quality of lactic acid bacteria of probiotic

cucumber juices on the basis of microbiological quality

106

4.44 Effect of lactic acid fermentation on TSS, pH and titratable

acidity of carrot juice

108

4.45 Effect of lactic acid fermentation on total Sugar, reducing

sugar and total phenols of carrot juice

110

4.46 Effect of lactic acid fermentation on antioxidant activity

and ascorbic acid during lactic acid fermentation of carrot

juice

112

4.47 Effect of lactic acid fermentation on viable cell counts and

lactic acid bacteria counts of carrot juice

113

4.48 Effect of lactic acid fermentation on coliform, yeast and

mold counts of carrot juice

115

4.49 Effect of lactic acid fermentation on antimicrobial activity

of carrot juice

116

4.50 Differentiation of quality of lactic acid bacteria of probiotic

carrot juices on the basis of physico-chemical parameters

117

4.51 Differentiation of quality of lactic acid bacteria of probiotic

carrot juices on the basis of microbiological quality

118

4.52 Evaluation of all probiotic vegetables (cabbage, cucumber

and carrot) juices on the basis of rate of fermentation

(°B/24hr) and rate of acidification (%/24hr) and lactic acid

bacteria counts(108cfu/ml)

119

4.53 Effect cold storage on physico-chemical parameters of

apple probiotic juice

4.57 Effect of cold storage on physico-chemical parameters of

pear probiotic juice

126

Table Title Page

4.58 Effect cold storage on microbiological analysis of pear

4.60 Acid tolerance and bile tolerance of lactic acid bacteria of

probiotic pear juice

130

4.61 Effect of cold storage on physico-chemical parameters of

orange probiotic juice

4.64 Acid tolerance and bile tolerance of lactic acid bacteria of

probiotic orange juice

135

4.65 Effect of cold storage on physico-chemical parameters of

cabbage probiotic juice

137

4.66 Effect of cold storage on microbiological quality of cabbage

probiotic juice

139

4.67 Effect cold storage on antimicrobial activity (mm) of

cabbage probiotic juice

140

4.68 Acid tolerance and bile tolerance of lactic acid bacteria of

probiotic cabbage juice

141

4.69 Effect of cold storage on physico-chemical parameters of

cucumber probiotic juice

142-143

4.70 Effect of cold storage on microbiological quality of

cucumber probiotic juice

144

4.71 Effect cold storage on antimicrobial activity (mm) of

cucumber probiotic juice

145

4.72 Acid tolerance and bile tolerance of lactic acid bacteria of

probiotic cucumber juice

145

4.73 Effect of cold storage on physico-chemical parameters of

carrot probiotic juice

4.76 Acid tolerance and bile tolerance of lactic acid bacteria of

probiotic carrot juice

152

4.77 Morphological, physiological and biochemical

characteristics of lactic acid bacteria of probiotic fruit and

LIST OF

Page(s)

2.1 Production growth trend for fruits and vegetables crops 5

3.1 Pictorial representation for preparation of probiotic apple 30-31 3.2 Pictorial representation for preparation of probiotic pear 30-31 3.3 Pictorial representation for preparation of probiotic orange 30-31 3.4 Pictorial representation for preparation of probiotic cabbage 30-31 3.5 Pictorial representation for preparation of probiotic cucumber 30-31 3.6 Pictorial representation for preparation of probiotic carrot 30-31 4.1 Effect of lactic acid fermentation rate of fermentation, rate of

acidification, retention of total phenols and vitamin C of apple

juice

44-45

4.2 Changes in TSS (°B) , pH , titratable acidity (%) , total sugar

(%), reducing sugar (%) and total phenols (mg/100g)(f)

during lactic acid fermentation of apple juice

46-47

4.3 Changes in antioxidant activity(%),vitamin C(mg/100g),lactic

acid bacteria counts(cfu/ml),antimicrobial activity against

S.aureus and E.coli during lactic acid fermentation of apple

juice

54-55

4.4 Effect of lactic acid fermentation rate of fermentation, rate of

acidification, retention of total phenols and vitamin C of pear

juice

58-59

4.5 Changes in TSS (°B) , pH, Titratable acidity (%) , Total sugar

(%) , Reducing sugar (%) and Total phenols (mg/100g)(f)

during lactic acid fermentation of pear juice

58-59

4.6 Changes in antioxidant activity(%),vitamin C(mg/100g),lactic

acid bacteria counts(cfu/ml),antimicrobial activity against

S.aureus and E.coli during lactic acid fermentation of pear

juice

64-65

4.7 Effect of lactic acid fermentation rate of fermentation, rate of

acidification, retention of total phenols and vitamin C of orange

juice

66-67

4.8 Changes in TSS (°B) ,pH, Titratable acidity (%),Total sugar

(%), Reducing sugar (%) and Total phenols (mg/100g)(f)

during lactic acid fermentation of orange juice

72-73

4.9 Changes in antioxidant activity(%),vitamin C(mg/100g),lactic

acid bacteria counts(cfu/ml),antimicrobial activity(mm) against

S.aureus and E.coli during lactic acid fermentation of orange

juice

76-77

Plate Title Between

Page(s)

4.10 Effect of lactic acid fermentation rate of fermentation, rate of

acidification, retention of total phenols and vitamin C of

cabbage juice

82-83

4.11 Changes in TSS(°B), pH, titratable acidity (%),total

sugar(%),reducing sugar(%) and total phenols(mg/100g) during

lactic acid fermentation of cabbage juice

86-87

4.12 Changes in antioxidant activity (%), vitamin C(mg/100g),lactic

acid bacteria counts(cfu/ml),antimicrobial activity against

S.aureus and E.coli during lactic acid fermentation of cabbage

juice

92-93

4.13 Effect of lactic acid fermentation rate of fermentation, rate of

acidification, retention of total phenols and vitamin C of

cucumber juice

94-95

4.14 Changes in TSS(°B),pH, titratable acidity(%),total

sugar(%),reducing sugar(%)and total phenols(mg/100g) during

lactic acid fermentation of cucumber juice

98-99

4.15 Changes in antioxidant activity(%), vitamin C(mg/100g), lactic

acid bacteria counts(cfu/ml) and antimicrobial activity

(mm)against S aureus and E.coli of cucumber juice

104-105

4.16 Effect of lactic acid fermentation rate of fermentation, rate of

acidification, retention of total phenols and vitamin C of carrot

juice

106-107

4.17 Changes in TSS (°B), pH , titratable acidity (%) , total sugar

(%), reducing sugar (%) and total phenols (mg/100g)during

lactic acid fermentation of carrot juice

110-111

4.18 Changes in vitamin C (mg/100g),antioxidant activity(%), lactic

acid bacteria counts(cfu/ml) ,antimicrobial activity(mm)

against S.aureus and antimicrobial activity against E.coli

during lactic acid fermentation of carrot juice

116-117

4.19 Changes in TSS(°B) , pH, titratable acidity(%), total sugar(%) ,

reducing sugar(%), total phenols(mg/100g), vitamin

C(mg/100g) and antioxidant activity(%) during cold

storage(4°C) of apple probiotic juice

120-121

4.20 Changes in viable cell counts(cfu/ml), lactic acid bacteria

counts(cfu/ml) and antimicrobial activity against

Staphylococcus aureus and Escherichia coli during cold

storage (4°C) of apple probiotic juice

122-123

4.21 Changes in TSS(°B), pH, titratable acidity(%), total sugar(%) ,

reducing sugar(%), total phenols(mg/100g), vitamin

C(mg/100g) and antioxidant activity(%) during cold

storage(4°C) of pear probiotic juice

126-127

4.22 Changes in viable cell counts(cfu/ml), lactic acid bacteria

counts(cfu/ml) and antimicrobial activity (mm) against

Staphylococcus aureus and Escherichia coli during cold

storage (4°C) of pear probiotic juice

128-129

Plate Title Between

Page(s)

4.23 Changes in TSS(°B) , pH, titratable acidity(%), total sugar(%) ,

reducing sugar(%), total phenols (mg/100g), vitamin C

(mg/100g) and antioxidant activity(mm) during cold

storage(4°C) of orange probiotic juice

130-131

4.24 Changes in viable cell counts (cfu/ml), lactic acid bacteria

counts(cfu/ml) and antimicrobial activity (mm) against

Staphylococcus aureus and Escherichia coli during cold

storage (4°C) of orange probiotic juice

132-133

4.25 19 Changes in TSS(°B) , pH, titratable acidity(%), total

sugar(%) , reducing sugar(%), total phenols(mg/100g), vitamin

C(mg/100g and antioxidant activity(%) during cold

storage(4°C) of cabbage probiotic juice

136-137

4.26 Changes in viable cell counts(cfu/ml), lactic acid bacteria

counts(cfu/ml) and antimicrobial activity(mm) against

Staphylococcus aureus and Escherichia coli during cold

storage (4°C) of cabbage probiotic juice

138-139

4.27 Changes in TSS(°B) , pH, titratable acidity(%), total sugar(%) ,

reducing sugar(%), total phenols(mg/100g), vitamin

C(mg/100g and antioxidant activity(%) during cold

storage(4°C) of cucumber probiotic juice

142-143

4.28 Changes in viable cell counts (cfu/ml), lactic acid bacteria

counts (cfu/ml) and antimicrobial activity(mm) against

Staphylococcus aureus and Escherichia coli during cold

storage (4°C) of cucumber probiotic juice

144-145

4.29 Changes in TSS(°B) , pH, titratable acidity(%), total sugar(%) ,

reducing sugar(%), total phenols(mg/100g), vitamin

C(mg/100g and antioxidant activity(%) during cold

storage(4°C) of carrot probiotic juice

148-149

4.30 Changes in viable cell counts (cfu/ml), lactic acid bacteria

counts(cfu/ml) and antimicrobial activity(mm) against

Staphylococcus aureus and Escherichia coli during cold

storage (4°C) of carrot probiotic juice

LIST OF PLATES LIST OF PLATES

Page(s)

1 Effect of lactic acid fermentation on antimicrobial activity

(inhibition zone in mm) of all fruits and vegetable probiotic

juices against E.coli and S aureus

118-119

2 Probiotic juices of fruits(apple(f),pear (c)and orange(a)) and

vegetables(cabbage(e),cucumber(d) and carrot(b)) prepared by

lactic acid fermentation during storage

120-121

3 Colonies of lactic acid bacteria (Lactobacillus plantarum (a)

and Streptococcus thermophillus(b)

154-155

4 Lactic acid bacteria of selected treatment of fruits (apple (a)

pear (b) orange (c) and vegetables (cabbage (d) cucumber (e)

carrot (f) probiotic juice (100x 16X)

154-155

5 Catalase test of lactic acid bacteria of selected treatment of

probiotic fruits and vegetables (a) cucumber (b) carrot (c)

cabbage (d) pear (e)apple (f) orange

154-155

6 Fermentation of sugar by lactic acid bacteria of selected

treatment of probiotic fruits and vegetables (a) control (b)

apple (c) pear (d) orange (e) cabbage (f) cucumber (g) carrot

154-155

7 Amylase test of lactic acid bacteria of selected treatment of

probiotic fruits and vegetables (a) cucumber (b) carrot (c)

cabbage (d) pear (e)apple (f) orange

154-155

8 Casein hydrolysis test of lactic acid bacteria of selected

treatment of probiotic fruits and vegetables (a) cabbage(b)

carrot(c) cucumber (d) orange (e) apple (f) pear

154-155

9 Microbial reaction of lactic acid bacteria of selected treatment

of probiotic fruits and vegetables (a) control (b) apple (c) pear

(d) orange (e) cabbage (f) cucumber (g) carrot in litmus milk

154-155

LIST OF ABBREVATIONS

FAO : Food Agriculture Organization

LAB : Lactic acid bacteria

WHO : World Health Organization

GI : Gastrointestinal tract

NHB : National Horticulture Board

Mhac : Million hectares

GAE : Gallic acid equivalent

ATP : Adenosine trypsin phosphate

MRS : de Mann Rogousa Sharpe

Lb : Lactobacillus

CFU : Colony forming units

AOAC : Association of Official Analytical Chemists HPO3 : Metaphosphoric acid

Chapter-1

INTRODUCTION

Food is the most essential requirement of all the living systems and man is no exception With the advent of agriculture, the man cultivated all types of food crops resulting in a glut during the production season Consequently, a huge postharvest loss of perishables and economic loss to the farmers took place According to FAO, postharvest losses of fruit and vegetables are 20-40 per cent in 2013 (FAO, 2013) Preservation by fermentation is well known method and is also one of the oldest methods of food preservation in the world Fermentation of foodstuffs is a desirable process of biochemical modification of primary food products, with the major role in this respect played by microorganisms and their enzymes Fermentation improves flavour and taste, extends the shelf- life, and increases the nutritional value of fermented products (Nout and Ngoddy, 1997; Joshi, 2006) Fermented foods can generally be described as palatable and wholesome foods, prepared from raw or heated raw materials by microbial fermentation (Holzapfel, 1997)

Out of various fermentations, lactic acid fermentation is used for commercial bulk storage of seasonal fruits and vegetables to increase their availability and to obtain a desired sensory quality of the products (Frazier and Westhoff, 1998) Lactic acid fermentation using LAB (lactic acid bacteria) cultures is employed for the preparation of different products such

as fermented grape juice, fermented peanut milk, yoghurt, fermented corn meal (Kuhunzaki) and fermented beverages from wheat and maize (Joshi and Thakur, 2000) Kimchi and Sauerkraut are the well known products made by fermentation of vegetables Lactic acid

fermented foods such as ‘Dahi’ is considered as a probiotics Fermented foods like ‘dahi’ are more nutritious than unfermented ones (Joshi et al., 1999) and those foods are prepared

by using lactic acid bacteria (LAB) have better acceptability (Hang and Jackson, 1967)

In 1900, Metchnikoff discovered the use of fermented milk in the diet for prevention

of certain diseases of the gastro-intestinal tract and promotion of healthy day-to-day life Since then, a number of studies have shown that the fermented food products do have a positive effect on the health of the consumers (Sahlin, 1999) Moreover, LAB has GRAS (generally recognized as safe) status and lactic acid fermented foods constitute 25 per cent of

the European diet and 60 per cent of the diet in many developing countries (Stiles, 1996) Such foods are called as ‘probiotic’ foods The word ‘probiotics’ originates from the Greek word ‘for life’ and is currently used to describe the bacteria associated with beneficial affects for human and animals According to WHO, probiotics are defined as ‘live organism which when administered in adequate amount confer a health benefits on the host’ Probiotic bacteria were first studied by Metchinkoff, a Nobel laureate of 1908 in the field of medicine (FAO/WHO, 2003) Lactic acid bacteria are commercially used as a starter culture for the

manufacture of dairy based probiotic food products (Heenan et al., 2002) These bacteria

ferment sugar (e.g lactose) predominantly to lactic acid (Liu, 2003) and have strong inhibitory effects on the growth and toxin production by other bacteria Addition of probiotic bacteria to food provide several health benefits including reduction in the level of serum cholesterol, improvement of gastrointestinal function, enhancement of immune system and reduction in risk of colon cancer (Berner and Odonnell, 1998)

The health benefits of certain foods had been investigated for several years Thus, development of foods that promote health and well being is one of the key research priorities

of food industry (Klaenhammer and Kullen, 1999) Fruit and vegetables are excellent source

of minerals, vitamins, sugars, acids besides phytochemicals and therefore, consumption of fresh or processed products would be healthful to the consumers In such products, if probiotic characteristics are also imparted it could add another feather in the already decorated cap of fruits and vegetables

Amongst the fruits, apple (Malus domestica) is a prominent fruit and is a member of

the rosaceae family, one of the most widely cultivated trees It can be processed into sauce, slices or juice, wine, cider and vinegar and is favoured for pastries, cakes, tarts and pies

(Downing, 1989) The pear (Pyrus communis) fruit, a member of family rosaceae is known for its texture and flavors (Hulme and Rhodes, 1971) Similarly, the sweet orange (Citrus sinensis L.osbeck) is a very good source of vitamins especially vitamin C and grown in many parts of world (Wardowski et al., 1986)

Vegetables are well known for their healthful benefits Amongst the vegetables,

cabbage (Brassica oleracea L var.Capitata) is a cruciferous vegetable which is rich in minerals, vitamins, dietary fibers and especially the phytochemicals (Chu et al., 2002)

Carrots are rich source of alpha-carotene and contain appreciable amount of thiamin and

riboflavin (Kotecha et al., 1998) Similarly, cucumber (Cucumis sativus) is widely cultivated

3

plant in the gourd family cucurbitaceae (McFeeters and Lovdal, 1987) It is one of the important summer vegetable, native to Asia and Africa, grown for its tender fruits for at least thousand years (Leppik, 1966)

Most of the presently available probiotics foods are based on milk But there are several problems associated with consumption of milk such as lactose intolerance and increase in the level of the cholesterol level of the consumers Lactose intolerance and the cholesterol content are two drawbacks related to the consumption of milk and milk products Currently, products are usually marketed in the form of fermented milk and yoghurt It has also been suggested that fruit juice could serve as a good medium for cultivating probiotics (Mattila-Sandholm, 2002) The use of probiotics in the fruit and vegetable juice industry offer

to consumers with special needs (vegetarian people with allergic reactions to milk proteins) the possibility to experience the positive effect of probiotic bacteria (Luckow and Delahunty, 2004) There is scanty of information on the lactic acid fermentation of fruits and vegetables juices especially with the respect to optimization of fermentation as probiotic juices and their evaluation as probiotic juices in the light of guidelines for such juices Similarly, the storage behaviour and safety of such juices have not been documented Recently, the antioxidant and antimicrobial phenomenon in fermented foods is being advocated, can these juices be

designated having these characteristics, is not known (Stevans and Sheldon, 1992; Subrota et

al., 2015) That is why, the present investigation has been carried out, keeping in view the following objectives:

OBJECTIVES:

i) To prepare apple, pear, orange, cabbage, cucumber and carrot probiotic juices by

lactic acid fermentation

ii) To determine the changes occurring during the lactic acid fermentation by LAB

(lactic acid bacteria)

iii) To study the cell viability and other physico-chemical characteristics of probiotic fruit

and vegetable juices during storage

Chapter-2

REVIEW OF LITERATURE

Food fermentation is the oldest and simple techniques of food preservation Lactic acid fermentation is one of the fermentations employed most extensively to preserve a variety of foods including milk Probiotics products like yakult, kefir etc are produced and consumed all over the world Recently, it has gained a lot of attention due to the probiotics effect contributing to the health of the consumers Probiotics are non-pathogenic microorganisms mostly of human origin which, when administered in adequate amounts, confer a health benefit on the host to prevent some diseases or improve health Probiotics may be a natural temporary constituent of the resident intestinal microflora, but their population is not always sufficient for therapeutic purposes The microbiota, the intestinal epithelium, and the mucosal immune system constitute the gastrointestinal ecosystem All the three components are essential for complete functional and developmental maturity of the system The use of antibiotics, immunosuppressive therapy, and irradiation, among other means of treatment, may cause alterations in the composition and have an effect on the gastrointestinal tract flora Therefore, the introduction of beneficial bacterial species to GI tract may be a very attractive

option to re-establish the microbial equilibrium and prevent diseases (Tiwari et al., 2012)

Various aspects connected with probiotics fruit and vegetable juices, their production, characteristic, mechanism of action, health benefits etc have been reviewed in this chapter under the following sub-heads:

2.1 FRUIT AND VEGETABLES

2.2 LACTIC ACID FERMENTATION

2.3 PROBIOTICS

2.4 PREPARATION OF PROBIOTIC JUICES

2.5 HEALTH BENEFITS OF PROBIOTICS

2.6 STORAGE OF PROBIOTIC JUICES

2.7 CURRENT STATUS OF PROBIOTIC JUICES IN WORLD AND INDIA

2.1 FRUIT AND VEGETABLES

Fruits and vegetables have been reported as important source of nutrients in many parts

of the world and offer many advantages over dietary supplements because of low cost and

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wide availability (Craig and Beck, 1999) Wargovich (2000) have reported that fruit and vegetables play a significant role in human nutrition, especially as sources of dietary fibre, minerals, vitamin C (ascorbic acid), vitamin A, thiamin (B1), niacin (B3), pyridoxine (B6), folic acid, vitamin E Thatis why fruits and vegetables are also called as protective foods

Some components of fruits and vegetables like fibers, mineral and vitamins, especially phytochemicals and antioxidants, are strongly associated with reduced risk of cancers, heart diseases, stroke and other chronic diseases (Hyson, 2002; Goldberg, 2003) USDA and HHS (2010) have reported that consumers are encouraged to eat up to 10 servings

of fruit and vegetables per day in some countries due to the highest antioxidant capacity of them

2.1.1 Fruits and vegetables: Production

NHB (2014) have recorded that production of fruits in India was 88,977 thousand MT over an area of 7216 thousand Mhac (Million hactares) during 2013-2014 Further, area and production of vegetables during 2013-2014 was 1, 62,897 thousand Mhac and 9,396 thousand

MT, respectively in India Year-wise (2000 onwards) area and production of fruits and vegetables in India have been reported by National Horticulture Board, New Delhi (Fig.2.1)

Fig 2.1 Production growth trend for fruits and vegetables crops

Source: NHB,2014

0 20000

2.1.2 Physico-chemical chemical composition of fruits (apple, pear and orange)

Physico-chemical characteristics of apple

Apple (Malus domestica) is commercially the most important temperate fruit and

ranks fourth among the most widely produced fruits in the world after banana, orange and grapes China is the largest apple producing country in the world with the production of 3700.16 thousand tonnes, while India is the fifth largest producer of apple with the production of 220.34 thousand tonnes (NHB, 2014) Apples are mostly consumed in fresh form but a small part of the production is processed into juices, jellies, canned slices and other items NHB (2014) have reported that production of apples was 2497.68 thousand MT from an area of 313.04 thousand hac, during 2013-2014

The physico-chemical characteristics of apples have investigated by many authors and

presented in Table 2.1 Kaushal and Sharma (1995); Chodak et al (2011); Jan and Rab

(2012) and Francini and Sebstiani (2013) have reported that apple is a good source of total soluble solids, reducing and total sugars, ascorbic acid, antioxidant activity and total phenolic content but have medium acid content as malic acid

Table 2.1 Physico-chemical characteristics of apple

GAE= Gallic acid equivalent

Source:- Kaushal BBL and Sharma PC, 1995; Savatovic et al., 2009; Chodak et al., 2011; Leahu et al., 2011; Jan and Rab, 2012; Francini and Sebstiani ,2013

Physico-chemical characteristics of pear

Pear (Pyrus communis) is one of the economically important fruit of temperate zone

Pear fruits are good source of pectin and contain phenolic compounds which have disease

Table 2.2 Physico-chemical characteristics of pear

GAE= Gallic acid equivalent

Source: - Kadam et al., 1995; Ozturk et al 2009; Hussain et al., 2013; Rocha et al., 2013

Physico-chemical characteristics of orange

Orange (C.sinensis L osbeck) is one of the important citrus fruit of the world occupies a prominent place in the horticultural field (Kale and Adsule, 1995) Nicolosi et al

(2000) have reported that orange was a crop of high economic importance and also a valuable source of vitamin C It can also be transformed into many processed products like juices, concentrated, marmalade, jams etc Production of orange was 3886.20 thousand MT over an area of 334.94 thousand hac in India as reported by NHB, 2014 during 2013-2014

The physico-chemical characteristics have been investigated and presented in Table

2.3 Hui (2006); Leahu et al (2011) have observed that orange was a good source of total

soluble solids, reducing and total sugars and pH value of juice was 3.23 It is a also good

source of antioxidants and total phenolic content (Stella et al., 2011)

Table 2.3 Physico-chemical characteristics of orange

GAE= Gallic acid equivalent

Source: - Kale and Adsule, 1995; Hui, 2006; Leahu et al., 2011; Stella et al., 2011

2.1.3 Physico-chemical chemical composition of vegetables (cabbage, cucumber and

carrot)

Physico-chemical characteristics of cabbage

Cabbage (Brassica oleracea L.var capitata) is a good source of protein (1.3%) which

contains all essential amino acid, particularly sulphur containing amino acids (Delahaut and Newenhouse, 1997) NHB (2014) reported that India produced 9039.22 thousand MT cabbages over an area of 400.14 thousand hac, during 2013-2014

The physico-chemical characteristics of cabbage have been investigated (Lee et al., 1970; Champa et al., 2007; Ferreira et al., 2015) and are presented in Table 2.4 It has been reported

that cabbage was a good source of total soluble solids, reducing and total sugar, but had low acid content (0.11%) It has also a good quantity of antioxidants, ascorbic acids and total phenolic content

Table 2.4 Physico-chemical characteristics of cabbage

GAE= Gallic acid equivalent

Source:- Lee et al., 1970; Yoon et al., 2006; Champa et al., 2007; Ferreira et al.,2015

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Physico-chemical characteristics of cucumber

The cucumber (Cucumis sativus) is one of the most important market vegetable in the

tropics and it is also the basis of an extensive pickling industry It is used for a culinary purpose or salad It contains 95% water, 0.7% protein, 0.1% fat, 3.4% carbohydrates, 0.4%

fiber and 0.4% ash (Renner et al., 2007) Production and area of cucumber in India was

678.15 thousand MT and 43.28 thousand hac, respectively (NHB, 2014)

Lee et al (1970); Kaur et al (2014) have observed the physico-chemical characteristics and

are presented in Table 2.5 Accordingly, cucumber has been found as good source of total soluble solids, reducing and total sugar, total phenols, antioxidants and ascorbic acids but have low acid content (0.029%)

Table 2.5 Physico-chemical characteristics of cucumber

GAE= Gallic acid equivalent

Source: - Lee et al., 1970; Kinh et al 2001; USFDA, 2007; Kaur et al., 2014,

Physico-chemical characteristics of carrot

Carrot (Daucus carota) is an ancient cool season root vegetables and its roots are used

for preparation of soups, stews, curries, pies, pickles and as salad It is a rich source of α and

β carotene (1890µg/100g) and contains sucrose 10 times that of glucose or fructose (Hedren

et al., 2002) Production and area of carrot in India was 1073.71 thousand MT and 62.41

thousand hac during 2013-2014 (NHB, 2014) Sharma et al (2006); Leahu et al (2013); Ferreira et al (2015) have determined the physico-chemical characteristics where are

presented in Table 2.6 They reported carrot was a good source of total soluble solids (8°B), reducing and total sugar (1.40% and 6.28% respectively), total phenol content (65.2mg/100g), antioxidant activity (20.39%) and ascorbic acid (5.54mg/100g) but has low acid (0.10-0.24%) content and huge pH 5.98

Table 2.6 Physico-chemical composition of carrot

GAE= Gallic acid equivalent

Source: - Carpenter, 1940; Sharma et al 2006; Leahu et al., 2013; Ferreira et al., 2015

2.2 LACTIC ACID FERMENTATION

Food fermentation is one of the oldest methods of food preservation in the world

There are four types of fermentation: alcoholic, lactic acid, acetic acid and alkaline fermentation Out of these, lactic acid fermentation is used for commercial bulk storage of seasonal fruits and vegetables to increase their availability and to obtain a desired sensory quality of products Fermented foods, are the food substrates that are invaded or overgrown

by edible microorganisms whose enzymes, particularly amylases, proteases and lipases, hydrolyse polysaccharides, proteins and lipids into different products with flavours, aromas and textures, are pleasant and attractive to the human consumer (Steinkraus, 1997)

Caplice and Fitzerald (1999) have reported that there are 21 different commercial vegetable fermentations in Europe along with a large number of fermented vegetable juices and blends, the most economically relevant of them are the fermentations of olives, cucumber

(pickle) and cabbage (Sauerkraut, Korean kimchi) Further, Joshi and Thakur (2000) have

reported that lactic acid fermentation using LAB cultures is employed for the preparation of different products such as fermented grape juice, fermented peanut milk, sogurt, fermented

corn meal Kuhunzaki and fermented beverages from wheat and maize

2.2.1 Historical

Lactic acid fermentation using LAB cultures is used throughout the world The Chinese were the first to ferment the fruit and vegetables and there is evidence to show that they had prepared such fruits and vegetables at the time of building the Great Wall of China

(Pederson, 1971) Etchells et al (1961) and Mundt (1970) have observed that spontaneous

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lactic acid fermentation occurs when vegetables are brined because lactic acid bacteria are naturally present on most of the living plants The lactic acid fermentation results in products with a longer shelf-life compared to fresh vegetables

2.2.2 Lactic acid bacteria

LAB have taditionally been associated with food and feed fermentations, and are

generally considerd beneficial micro-organisms, including some strains even as a health

promoting (probiotic) bacteria such as Lactobacillus, Streptococcus etc (Von Wright and

Axelsson, 2011) Gibbs (1987) reported that lactic acid bacteria (LAB) are one of the important microorganisms in food fermentation Lactic acid bacteria are defined as bacteria which produce lactic acid as their major fermentation product This large group includes

Lactobacillus, Streptococcus , Enterococcus, Lactococcus, Bifidobacteria and Leuconostoc (Prescott et al., 2002; Azizpour, 2009) Hoque et al (2010) and Sobrun et al (2012) have

found that LAB are Gram positive usually non-motile, non-spore forming rod and cocci, catalase negative (Table 2.7) LAB can only get ATP by fermentation, usually of sugar Since they do not use oxygen in their energy production, lactic acid bacteria grow under anaerobic condition, but they can also grow in presence of oxygen They are protected from oxygen by by-products (H2O2) because they have peroxidase That is why these organisms are

aerotolerant anaerobes (Yien et al., 2012; Guetouache and Guessas, 2015)

Table 2.7: Characteristics of lactic acid bacteria

Lactic acid bacteria (genus) Characteristics

Lactobacillus Gram positive, rod shaped, non-motile, catalase negative,

milk coagulation positive, NaCl tolerance positive, sugar fermentation positive, non-spore forming

Streptococcus Gram positive, spherical or coccus, catalase negative, non

-motile, NaCl tolerance positive, sugar fermentation positive, non- spore forming

Lactococcus Gram positive, coccus shaped, facultative anaerobe, catalase

negative, fermentation of carbohydrates positive, non motile and non- spore forming

-Leuconostoc Gram positive, coccus shaped, catalase negative, sugar

fermentation positive, arginine negative, citrate utilization negative, non -motile and non -spore forming

Source:- Azadnia and Khan, 2009

Among this group of lactic acid bacteria, Lactobacillus is the largest genus which

contains almost 80 species and are used in preparation of different fermented products such

as pickle, sauerkraut, beer, wine, juices, yogurt, fermented grape juice, fermented peanut milk

,sausage sogurt, fermented corn meal Kuhunzaki and fermented beverages from wheat and

maize (Caplice and Fitzerald, 1999; Joshi and Thakur, 2000)

2.2.3 Lactic acid fermentation

Lactic acid fermentation has been described as an anaerobic process in which lactic acid bacteria transform sugar into lactic acid (Fig 2.2) (Blake and Donald, 1995a) Lactic acid is a natural, low pH and effective preservative The production of lactic acid creates acidification, which decreases the pH of product from 6.5-4.5 within 48 hrs under optimum conditions (Morrow and Ferket, 2002)

Lactic acid fermentation has been reported to decrease in anti-nutritional factors such

as phytic acid, trypsin inhibitors, ionositol phosphates etc Badau et al (2005) have reported

that fermentation of processed pearl millet for 24 hr reduced the phytic acid content He reported that it was due to the action of enzyme phytase released by micro-organism during fermentation It also improves the protein content, vitamins such as thiamine, riboflavin, niacin or folic acid which have direct effect on the health of consumers of such foods

Pyruvate Fermentation

Lactic acid

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(Steinkraus, 1997) Lactic acid fermentation increased the utilization of iron from food by breakaway of inorganic iron from complex substances under the influence of vitamin C

(Siegenberg, 1991; Venkatesh, 1998) Not only this, Nout and Motarjemi (1997) have

reported that vitamin C is better preserved in lactic acid fermentation of vegetable product,

compared with those which are processed by alternative method

2.3 PROBIOTICS

The term ‘probiotic; is a relatively new word meaning “for life” and it is currently used to name the bacteria associated with beneficial effects for humans and animals Probiotics are viable microbial dietary supplements that, when introduced in sufficient quantities, positively influence the health of consumers mainly by improving the composition

of intestinal microbiota For this reason, they are called probiotics (Sharma et al., 2012)

2.3.1 Historical development of probiotics

Tissier (1906) observed that children with diarrhea had in their stools a low number of bacteria characterized by a peculiar, Y shaped morphology These “bifid.” bacteria were abundant in healthy children It was suggested that these bacteria could be administered to patients with diarrhoea to help restore a healthy gut flora In 1965, the term ‘probiotics’ was first coined by Lilly and Stillwell in a different context to represent ‘substances secreted by one organism which stimulate the growth of another’ (Lilly and Stillwell, 1965)

After nine years, Parker (1974) described probiotics as the “organisms and substances which contribute to intestinal microbial balance” Fifteen years later, Fuller (Fuller, 1989) proposed that probiotics were ‘live microbial supplements which beneficially affects the host

animal by improving its microbial balance Further, Saliminen et al (1998) have reported that

probiotics have a long history of human use and are traditionally consumed in several parts of

the world

2.3.2 Definition and characteristics of probiotics

The probiotic word was discovered many years ago, but it was defined a few years ago Fuller (1989) defined as probiotics “live microbial feed supplement that beneficially

affects the host by improving its intestinal balance” while Salminen et al., 1998 characterized

that probiotics are live microbial food ingredients that have a beneficial effect on human

health Almost on the similar time, FAO/WHO (2001) defines probiotics as ‘Live

microorganisms which when administered in adequate amounts confer a health benefit on the

host’ Ouwehand et al (1999) reported that probiotics must be shown to exert a beneficial

effect on the consumer, should be non-pathogenic, non-toxic, and free of any significant

adverse side effects, retain stability during the intended shelf- life of the product, contain an

adequate number of viable cells to confer the health benefit, compatible with product format

to maintain desired sensory properties and labeled in a truthful and informative manner to the

consumer

Certain physiological characteristics are considered important for probiotics targeted

toward particular applications, for example, resistance to stomach acid and pancreatic

secretions such as bile and digestive enzymes that would be important for probiotics needing

to survive in high numbers through the human small intestine (CAST, 2007)

2.3.3 Probiotic bacteria and their action

In 1907, Metchnikoff postulated that the bacteria involved in yoghurt fermentation,

Lactobacillus bulgaricus and Streptococcus thermophilus, suppress the putrefactive-type

fermentations of the intestinal flora and that the consumption of yoghurts played an

important role in maintaining the human health (Metchnikoff, 2004) Sharma et al (2012)

reported that lactic acid bacteria (LAB) and bifidobacteria are the most common types of

microbes used as probiotics; but certain yeasts and bacilli may also fit into the definition

Probiotics are also called "friendly bacteria" or "good bacteria"

Table 2.8 Key genera and species of microbes studied and used as probiotics

Bacteria Lactobacillus L acidophilus, L brevis, L reuteri, L casei, L

rhamnosum, L bulgaricus, L cellobiosus, L

delbrueckii, L fermentum

Bifidobacterium B thermophilus, B infantis, B longum, B

bifidum, B animalis

Pediococcus P acidilactici Leuconostoc

Enterobacter

L mesenteroides

E faecium, E faecalis

Escherichia coli Bacillus coagulansc , clausii

Yeast Saccharomyces S boulardii, S cerevisiae, S carlsbergensis

Source: - Council for Agricultural Science and Technology (CAST), 2007

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Sartor (2004) have reported the mechanisms explaining the health benefits of probiotics which includes many steps Firstly, probiotic bacteria adhere and colonize the gut then, suppress the growth or epithelial binding or invasion by pathogenic bacteria and finally, production of antimicrobial substances Lactic acid bacteria produce several metabolites (Fig 2.3) like fatty free acids, hydrogen peroxide, bacteriocins, etc., which prevent the growth of food-borne pathogens in dairy products (Lindgren and Dobrogosz, 1990) After production of antimicrobial substances, there is improvement of intestinal barrier functions that ultimately control the transfer of dietary antigens and stimulate the mucosal and systemic host immunity

Fig 2.3 Metabolites of lactic acid bacteria

(Source: - Modified from Tiwari et al., 2012)

2.4 PREPARATION OF PROBIOTIC JUICES

2.4.1 Fruits

2.4.1.1 Red and yellow watermelon

Red and yellow watermelons have carotenoids pigments, lycopen and lutein, respectively They have antioxidant properties and fundamental contribution to human health

(Tadmor, 2005) Alavi et al (2012) found that to conduct the fermentation, inoculum (Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus plantarum) was prepared

by growing the culture at 30°C for 24 h in MRS broth Fermentation were conducted in 250

mL flask, each containing 100 mL of sterile red and yellow watermelon juices that were pasteurized for 10 min at 85°C All samples were inoculated with a 12-h old culture (105

Bacteriocins

Co-aggregation Hydrogen molecules peroxide

Biosurfactants Lactic acid

Adhesion inhibitors

Lactic acid bacteria

CFU/mL) and incubated at 37°C for 48 h Both fruit juices were proved to be a suitable media for production of a fermented probiotic drink

2.4.1.2 Peach probiotic juice

Peaches (Prunus persica) are rich in minerals, vitamin C and contain a lot of sugar (Yoon et al., 2004) They are high in phytochemicals, dietary fibers and polyphenols that

could lead to improvement in health of consumers (Magerramov, 2006) Peach juice was

fermented with probiotic lactic acid bacteria (Lactobacillus plantarum DSMZ 20179, L delbrueckii DSMZ 15996, L casei DSMZ 20011) Fermentation was conducted in test tubes

(25 × 200 mm) and each tube was filled separately with 40 mL of sterile peach juice All the samples were incubated with a 24-hour-old probiotic culture (> 105 CFU/mL) at 30°C for 72 hours Peach juice could prove to a healthy beverage for vegetarians and lactose-allergic

consumers

2.4.1.3 Roselle Juice

Morton, (1987) reported that roselle (Hibiscus sabdariffa L.) is widely cultivated in Thailand where it is known as krachiap daeng Roselle calyces contain 9% moisture, 1.14%

protein, 2.61% fat, 12% fibre and 6.9% ash It is high in calcium, niacin, riboflavin and iron

It is used as an antibacterial, antifungal, hypocholesterolemic, diuretic, mild laxative and

antihypertensive substance (Hirunpanich et al., 2006)

To conduct the fermentation, probiotic lactic acid bacteria (Lactobacillus plantarum TISTR 863 and Lactobacillus casei TISTR 390) were obtained from culture collection centre and the cultures were grown at 37°C for 24 h in MRS broth and were used as an inoculum

Fermentations were conducted in 250-ml Erlenmeyer flasks, each containing 100 ml of sterile roselle juice All the samples were inoculated with a 24-h culture (>105cfu/ml) and incubated

at 30 or 37°C for 72 h (Tantipaibulvut et al., 2008) Lb plantarum and Lb casei were found suitable for use as probiotic cultures for production of a healthy beverage from roselle calyces

for vegetarians or consumers who are allergic to lactose present in probiotic dairy products

2.4.1.4 Cashew Apple

Cashew apple has high ascorbic acid , phenols content, reducing sugars (fructose and

glucose), minerals, amino acids and also a good source of antioxidant compounds ( Rabelo et

al , 2009) Pereira et al., (2010) reported that fermentation was conducted in Erlenmeyers

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flasks, each containing 100 mL of clarified and pasteurized cashew apple juice All the

samples of cashew apple juice were inoculated with 7.00 log cfu/mL of L casei This

concentration was chosen based on the recommendation for probiotic foods: minimal counts

of 7.00 Log cfu/mL for gave better efficacy in regulating beneficial effects (Assuncao and Mercadante, 2003) Cashew apple juice showed to be as efficient as dairy products for

Lactobacillus casei growth and considered that fermented juice with Lactobacillus casei is a

good and healthy alternative functional food containing probiotics

2.4.2 Vegetables

2.4.2.1 Tomato

Tomato juice contains 93.1% moisture, 4.89% carbohydrate, vitamins, and minerals,

and is low in protein and fat (Abdel-Rahman and Abdel-Hamd, 1982) So, Suzuki et al

(2002) recognized that tomato juice was one of the healthy beverages

Yoon et al (2004) procured that probiotic lactic acid bacteria (Lactobacillus acidophilus LA39, Lactobacillus casei A4, Lactobacillus delbrueckii D7, and Lactobacillus plantarum C3) from culture collection centre and conducted the fermentation for preparation

of tomato probiotic juices Cultures were grown at 30ºC for 24 h in MRS broth and were used

as inocula in tomato juice, which was conducted in test tubes (25×200 mm), each containing

40 ml of pasteurized tomato juice All the samples were inoculated with a 24 h culture (>105 CFU/ml) and incubated at 30°C for 72 h After completion of fermentation, viable cell counts reached nearly 1.0 to 9.0x109 cfu/ml after 72h of fermentation Similarly, Babu et al (1992) reported that addition of tomato juice to skimmed milk stimulated the growth of L acidophilus and resulted in higher viable counts of fermented tomato juice Probiotic tomato

juice could serve as a health beverage for vegetarians or consumers who are allergic to dairy products

All that samples were inoculated with a 24-h-old culture and incubated at 30°C (Yoon et al.,

2005) On completion of the fermentation, all the lactic acid cultures were found capable of

rapidly utilizing beet juice for cell synthesis and lactic acid production (Yoon et al., 2005),

who reported that all the lactic acid cultures were found capable of rapidly utilizing beet juice for cell synthesis and lactic acid production

2.4.2.3 Cabbage juice

Cabbage is a cruciferous vegetable, which is rich in minerals, vitamin C, dietary

fibers, and especially phytochemicals (Chu et al., 2002) Yoon et al (2006) reported that to conduct the cabbage juice fermentation, cultures (Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7) were grown at 30°C for 24 h in MRS (de Mann

Rogousa Sharpe) broth Fermentations were conducted in test tubes (25-200 mm), each containing 40 mL of sterile cabbage juice and juice was inoculated with a 24-h-old lactic

culture and incubated at 30°C They reported that L casei, L delbrueckii, and L plantarum

grew well on cabbage juice and reached nearly 10x108 CFU/mL after 48 h of fermentation at 30°C.Fermented cabbage juice could serve as a healthy beverage for vegetarians and lactose-allergic consumers

2.4.2.4 Radish

Joshi and Sharma (2009) have found that lactic acid fermentation of radish as one of

the alternatives to preserve this vegetable besides providing consumer healthful product The physico-chemical characteristics of radish (TSS 6°B, total sugars 2.04% and ascorbic acid 12.8 mg/ 100g) showed its suitability for lactic acid fermentation The grated radish was fermented after addition of 2.5% salt at a temperature of 25±1°C With natural microflora, the fermentation was completed in 16-18 days, giving a titratable acidity of 1.80% as lactic acid

It was observed that the fermented radish could be stored up for a period of 15 days under

refrigerated conditions without any spoilage Similarly, Joshi et al (2008) reported that lactic

acid fermentation of carrot and radish with 2.5% salt and with sequential culturing of

Streptococcus lactis and Lactobacillus plantarum at 26°C had the best physico-chemical

characteristics

2.4.3 Cereals

2.4.3.1Cereal-Based Probiotic Beverages

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Cereal products often ferment spontaneously, resulting in improved shelf-life and nutritional properties compared with the raw material (Blandino, 1996) Hassan et al (2012)

observed that rice and millet grains were fermented for 16hr at 37°C with 5% ABT-2 starter

culture (S thermophillus, L acidophilus and Bifidobacterium BB-12) Fermentation with ABT-2 starter culture (S thermophillus, L acidophilus and Bifidobacterium BB-12)

improved the color, flavor, texture and overall acceptability of the beverages

2.4.4 Effect of lactic acid fermentation on physico-chemical characteristics and

microbial quality of probiotic juices

pH

Yoon et al (2004) have reported that lactic acid cultures (Latobacillus acidophilus LA39, Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii

D7) reduced the pH value to 4.1 or below of tomato juice after 72 h fermentation Similarly,

in another study, Yoon et al (2005) reported that Lactobacillus acidophilus, Lactobacillus casei , Lactobacillus delbrueckii, Lactobacillus plantarum reduced the pH value of beet juice

from 6.3 to 3.7, 4.1, 5.0 and 5.0 after 72 hr of fermentation, respectively

Yoon et al (2006) found that the pH values at the end of cabbage juice fermentation were 3.5, 3.6 and 3.6 mg/ml for L casei, L delbrueckii, and L plantarum, respectively Similarly, L plantarum and L casei reduced the pH value of roselle juice from 4.74 to 3.91 and 3.90 after 72 hr fermentation at 30°C, respectively (Tantipaibulvut et al., 2008) In another study, Guo et al (2009) documented that during fermentation L.casei was able to

reduce the pH value of milk to 5.59 after 24 h and after 28 days the pH further declined 4.60

Similarly, Hassan et al., (2012) reported that pH value of fermented rice and millet beverages

decreased markedly to 4 at 8h until 12 h and further decreased afterward The time required

to reach pH 4.5 for fermented plain rice beverage samples was recorded as 16 h while it was reduced to 10h for fermented plain millet beverage

Titratable acidity

Shah and Jelen, (1990) reported that lactic acid (LA) was the major end product of the

fermented cabbage juice, attaining the concentrations of 9.69, 12.2 and 6.97 g/l LA for L casei , L delbrueckii, and L plantarum, respectively Further, probiotic bacteria could tolerate

high acid medium and survived during fermentation process (Holzapfel and Schillinger, 2002)

Yoon et al (2004) found that lactic acid cultures (Latobacillus acidophilus LA39, Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7)

increased the acidity to 0.65% or higher of tomato juice after 72 hr of fermentation Similarly,

Yoon et al (2005) found that Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus plantarum increased the titratable acidity in term of lactic acid

of beet juice from 0.13 to 0.98, 0.56, 0.25 and 0.23% after 72 hr of fermentation,

respectively L plantarum and L casei increased the acidity in term of lactic acid of roselle

juice from 0.25 to 0.44 and 0.52% after 72 hr fermentation at 30°C, respectively as observed

by Tantipaibulvut et al (2008) Joshi and Sharma (2009) reported that fermentation of

reddish with natural microflora was completed in 16-18 days and gave a titratable acidity of 1.80% as lactic acid

Sugar

It was observed that the lactic acid cultures rapidly fermented tomato juice and

reduced the level of sugar (Yoon et al., 2004) L plantarum consumed the sugar at a much faster rate than L acidophilus, L casei and L delbrueckii Such as L plantarum and reduced

the sugar level from an initial value of 32.4 mg/mL to 25.2, 21.0, and 19.3 mg/mL after 24,

48, and 72 h fermentation, respectively Yoon et al (2006) reported that concentrations of sugar at the end of cabbage juice fermentation were 36.5, 19.33 and 28.35 mg/ml for L casei,

L delbrueckii , and L plantarum, respectively which were further decreased after fermentation from the initial value Similarly, Tantipaibulvut et al (2008) found that L plantarum and L casei reduced the sugar level of roselle juice from 9.66 to 1.82 and

4.07mg/ml after 72 hr fermentation at 30°C, respectively

Total phenols and antioxidant activity

There are several reports on the total phenols and antioxidant activity in the fermentation by lactic acid bacteria Whiting and Coggins (1974) and Whiting (1975) have described that L plantarum in anaerobic conditions reduced quinate to

dihydroxycyclohexane carboxylate and acetic acid Further, L plantarum not only reduced

quinate but at the same time, even under anaerobic conditions, oxidized a proportion to

catechol However, Serraino et al (1985) reported that these antinutrients interfere with

mineral bioavilability and digestibility of proteins and carbohydrates Reduction in polyphenolic content through fermentation may imply improved digestibility of proteins and

ABTS method, DPPH method and FRAP method (89.09, 50.09 and 801.25% inhibition, respectively) and proteolytic activity as well as reduced polyphenolic content (29.10 mg/100

ml to 11.98 mg/ 100 ml), which were incubated for 24 h at 37°C (Subrota et al., 2015) The

diminishing effect of fermentation on polyphenols may be due to the activity of polyphenols oxidase present in the food grain or microflora This reduction in polyphenolic content by

fermentation results in less astringency L plantarum was able to degrade some food

phenolic compounds giving compounds which influence the food aroma as well as increased

antioxidant activity (Subrota et al., 2015)

Ascorbic acid

L-ascorbic acid is an indicator of the sparingness of the technological process, the concentration of this acid was also measured in the end products It was concluded that about

20 to 70% of the initial content of L-ascorbic acid remains preserved in the end-products,

depending on the used method of processing (Kopec, 2000) Kohajdova et al (2006) reported

that 43% and 56% of the original content of L-ascorbic acid was preserved at the end of

fermentation of tomato juice and cabbage juice, respectively

Viable cell counts

Shah (2000) reported that the viability of probiotic organisms was dependent on the level of oxygen in products, oxygen permeation of the package, fermentation time, and

storage temperature.Yoon et al (2004) found that lactic acid cultures (Latobacillus acidophilus LA39, Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7 increased the viable cell counts of tomato juice (CFU) from 1.0 to 9.0×109/ml

after 72 h fermentation Similarly, Yoon et al (2006) in another study reported that L casei,

L delbrueckii , and L plantarum grew well on cabbage juice and reached nearly 10x108CFU/mL after 48 h of fermentation at 30°C The cell number of L plantarum in roselle juice

increased from 3.3x107 to 4.6x108 cfu/ml and cell number of L casei from 8.5x107 to 2.7x108

reported by Tantipaibulvut et al (2008) in another study

Pereira et al (2010) observed that cashew apple juice inoculated with 7.30 and 7.48 Log cfu/mL of L casei, presented a fast growth At 6 hours of fermentation viable cell counts

of 8.04±0.00 and 8.08±0.00 Log CFU/mL, respectively, was observed Similarly, Pakbin et

al (2014) studied that probiotic lactic acid bacteria grew well in peach juice, reached nearly

10 × 109 CFU/mL, after 48 hours of fermentation at 30 °C and was capable of consuming more sugar decreasing pH decrease and production of lactic acid during fermentation

Antimicrobial activity

Lactic acid bacteria produced various compounds such as organic acids, diacetyl, hydrogen peroxide, and bacteriocins or bactericidal proteins during lactic fermentations

(Lindgren and Dobrogosz, 1990) Tagg et al (1976) found that bacteriocins produced by

lactic acid bacteria, such as nisin, inhibited not only closely related species but were also effective against food-borne pathogens and many other Gram-positive spoilage microorganisms Similarly, Schillinger and Lucke (1989) revealed that the LAB inhibited all the pathogenic bacteria and the inhibition was scored positive in the study when the width of the clear zone around the colonies of the producer strain was 0.5 mm or larger

Stevans and Sheldon (1992) reported that inhibition of Escherichia coli and Salmonella under conditions that disrupt the outer membrane, including truncated lipopolysaccharides (LPS), low pH and high salt concentrations However, Caplice and Fitzerald (1999) found that antimicrobial metabolites had a very broad mode of action and inhibit both Gram-positive and Gram-negative bacteria as well as yeast and moulds Kalalou

et al (2004) studied the activity of LAB on some Gram positive and negative pathogenic

bacteria such as E.coli, Pseudomonas aeroginosa, Klebsiella pneumonia, Staphylococcus aureus and Bacillus cereus and the inhibition zones were in the range of 1.4 to 2.8cm

Similarly, cell free supernatant of the Lactic acid bacteria inhibited the growth of all

organisms (E coli, Klebsiella, Pseudomonas, Streptococcus, Proteus) (Saranya and

Hemashenpagam, 2011) The antimicrobial effect of lactic acid was found to be due to

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undissociated form of acid that penetrated the membrane and liberated hydrogen ion in the neutral cytoplasm thus, leading to inhibition of vital cell functions

2.4 HEALTH BENEFITS OF PROBIOTICS

Probiotics, especially Lactobacillus and Bifidobacterium have been suggested to be associated with alleviation of lactose intolerance (Ouwehand et al.,1999); prevention and cure of viral, bacterial and antibiotic or radiotherapy induced diarrhoeas (Parvez et al., 2006); immunomodulation (Forsythe and Bienenstock, 2010); antimutagenic (Chalova et al., 2008)

and anticarcinogenic effects (Liong, 2008); and even blood cholesterol reduction (Ooi and

Liong, 2010) Ouwehand et al (1999); Zubillaga et al (2001); Holzapfel and Schillinger

(2002) reported that these health benefits related to probiotic bacteria are due to the reduction

of gut pH, production of some digestive enzymes and vitamins, production of antibacterial substances, e.g., organic acids, bacteriocins, hydrogen peroxide, diacetyl , acetaldehyde and lactones by lactic acid bacteria The beneficial effects of lactic acid fermented products were revealed by a Russian Scientist E Metchnikoff (1845–1919) long ago who proposed that

ingesting fermented milk products extended longevity of people (Hove et al., 1999) (Table

2.9) LAB is useful for human beings and animals in many aspects as discussed here:

2.5.1 Hypercholesterolemia and Cardiovascular Diseases

Survarna and Boby (2005) studied that elevated blood cholesterol levels can be reduced by consumption of probiotic-containing dairy foods Further, the dietary cholesterol absorption was reduced in three ways: assimilating, binding, or degradation Probiotic strains assimilate the cholesterol for their own metabolism Probiotic strains can get to the

cholesterol molecule, and can degrade cholesterol to its catabolic products (Parvez et al

2005)

2.5.2 Diarrhea

Parvez et al (2006) reported that diarrhea is a major world health problem,

responsible for several million deaths each year Probiotics can potentially provide an important means to reduce these problems Moreover, probiotics might prevent infection because they compete with pathogenic viruses or bacteria for binding sites on epithelial cells

Similarly, Lactobacillus GG strain has been able to be very effective against viral and

idiopathic diarrhea, as identified by Harish and Vargese in their studies (Harish and Vargese, 2006) on this aspect

2.5.3 Pancreatitis

Pezzilli and Fantini (2006) studied that patients with acute pancreatitis received L plantarum 299v showed a decrease in occurrence of pancreatic infection/abscess and a shorter hospital stay

2.5.4 Hypertension

Sanders (2007) reported that consumption of certain lactobacilli, or products made from fermented milk may reduce blood pressure in mildly hypertensive people It was found that systolic blood pressure was decreased on the order of 10-20 mm Hg due to consumption

of fermented milk

2.5.5 Cancer

Sanders (2007) have hypothesized that probiotic cultures might decrease the exposure to chemical carcinogens by detoxifying ingested carcinogens, altering the environment of the intestine and thereby, decreasing the populations or metabolic activities of bacteria that may generate carcinogenic compounds, producing metabolic products (e.g., butyrate) which improve the ability of cell to die when it should die (a process known as apoptosis or programmed cell death), producing compounds that inhibit the growth of tumor cell and at last stimulating the immune system to better defend against cancer cell

proliferation Similarly, Kim et al (2006) have found that LAB such as Lactobacillus rhamnosus ATCC 9595 was also useful in preventing colon cancer in human beings

2.5.6 Lactose intolerance

Yogurt is one of the milk product to aid digestion of lactose because the lactic acid

bacteria viz L acidophilus, Bifidobacteria, Lactobacillus bulgaricus and Streptococcus thermophillus used as starter culture to make yogurt, produce lactase and digest the lactose

before it reaches the colon reported by Sanders (2007) and Marteau et al (1990)

2.5.7 HIV and Immune System Stimulation

Sanders (2007) studied that effect of yogurt or lactic acid bacteria on enhancing levels

of certain immunoreactive cells (e.g macrophages, lymphocytes) or factors (cytokines,

25

immunoglobulin’s, and interferon Results accumulated so far have suggested that probiotics may provide an additional tool to help your body protect itself

2.5.8 Kidney Stones

Sanders (2007) found that a probiotic preparation that contained bacteria able to

degrade oxalate in vitro was shown to reduce oxalate fecal excretion in patients This study suggested that manipulation of the gut flora with the right probiotic bacteria may improve gastrointestinal tract oxalate levels and may decrease oxalate absorption

2.5.9 Antibiotic Therapy Disease

The basic purpose of antibiotics is to kill the harmful bacteria Unfortunately, they frequently kill the normal bacteria as well, often resulting in disruption of the bacterial flora, leading to diarrhea and other intestinal disturbances Replenishing the flora with normal bacteria during and after antibiotic therapy seems to minimize disruptive effects of antibiotic

use Sanders (2007) found that probiotics can prevent antibiotic associated diarrhea

Table: 2.9 Effect of lactic acid fermentation on several diseases

bacteria on epithelial cells)

Parvez et al., 2006; Harish

and Vargese, 2006

pancreatic infection)

Pezzilli and Fantini, 2006

Positive (Minimize the disruptive effect

of antibiotics to normal bacterial flora)

Sanders, 2007

2.6 STORAGE OF PROBIOTIC JUICES

Storage study of probiotic juice has been done by many researchers to study the effect

of cold storage on viable cell counts of lactic acid bacteria of fermented fruit juices But, most

of literature of the effect has been on cold storage on physico-chemical properties of yogurt and fermented milk than that of probiotic juices Therefore, studies on the storage of probiotic fermented milk or yogurt have been cited

Damin et al (2006) studied the chemical changes occurred during cold storage of

fermented milk which was prepared by using probiotic bacteria It was found that fermented milk showed a fall in pH during the refrigerated storage, that was due to lactose consumption

by lactic acid production, also called post acidification while the viability of probiotic

bacteria were decreased during storage Karsheva et al (2013) have also observed the

changes in physico-chemical parameters of yoghurt during the storage and found the greatest decrease of pH values or increase in titratable acidity of yoghurt during first 24 hrs of the storage, known as “secondary acidification” or main factor for the viability loss of the probiotic bacteria took place

Lourens-Hattingh and Viljoen, (2001) have reported that the probiotic culture should

be able to multiply to reach high cell counts in the fermented product and possessed a high acid tolerance to ensure high viable cell numbers during storage Further, Shah (2001) found that for the maximum health benefits, the minimum number of probiotic organisms in a food product should be 106 CFU/g Therefore, the viability of the lactic cultures is the most important factor during refrigerated or frozen storage

The viable cell counts of the four lactic acid bacteria in the fermented tomato juice ranged from 106 to 108 CFU/ml after 4 weeks of cold storage at 4°C (Yoon et al., 2004) Similarly, Yoon et al (2005) found that the viable cell counts of Lactobacillus acidophilus, Lactobacillus casei , Lactobacillus delbrueckii, Lactobacillus plantarum in the fermented beet

juice still remained at 106–108 CFU/ml except L acidophilus after 4 weeks of cold storage at 4°C Yoon et al (2006) also studied that after 4 weeks of cold storage of fermented cabbage juice at 4°C, the viable cell counts of L plantarum and L delbrueckii were still 4.1 x 107 and 4.5 x 105 mL -1, respectively L.casei, however, did not survive the low pH and high acidic

conditions in fermented cabbage juice and lost cell viability completely after 2 weeks of cold storage at 4°C The cell number of fermented roselle juice was also reduced to approximately104 cfu/ml after 3 weeks of cold storage at 7°C (Tantipaibulvut et al., 2008)

Kohajdova et al (2006) have reported that colour, turbidity, sediment and overall

appearance are the most important parameters to evaluate the appearance of lactic acid fermented fruit and vegetable juices Sensory characteristics of probiotic blackcurrant juice