1.

INTRODUCTION

1
 INTRODUCTION
One of the main objectives of food research is to investigate and develop new food
products that promote health. During the last decade, consumers have progressively
believed that food contributes directly and efficiently to promote health. It has been
broadly suggested that high intake of fruits and vegetables, nutrient-rich plants and
antioxidant-rich beverages like tea and coffee may lessen the risk of degenerative
and oxidative-stress associated diseases. Fresh fruit and vegetables are, however,
quickly perishable; thus, the concern for processing which assures their safety for
consumption. Nevertheless, food processing can initiate undesirable chemical and
physical changes. For example, it has been testified that vitamins and natural
antioxidants are rapidly vitiated during thermal processing techniques like blanching,
cooking, pasteurisation or sterilisation, leading to a reduction in antioxidant activity
(Nicoli, M. et al., 1999). Post blanching, air drying, water immersion or boiling of
green leafy vegetables, celery and onion bulbs, significant loss in the vitamin C and
flavonoid contents was observed (Oboh, 2005).
Substantial epidemiological evidence has shown that intake of fruits and vegetables
is linked with reduced risk of chronic diseases of aging as well as cardiovascular and
numerous types of cancer (J. Yang, Y. Xiao, 2013). The phytochemicals existing in
plant foods, including fruits and vegetables, have been proven to be responsible for
health benefits of such foods. The compounds with antioxidant potentials such as
phenolics and polyphenolics appear to be important, while the mode of action of
phytochemicals present as well as their chemical composition is varied. Compared to
phenolics such as flavonoids which are dominant in fruits, the antioxidant activity of
vitamin C in many cases is less important (Nowak D. et al., 2018). Recent focus has
been on the identification of bioactives in an extensive range of foods, including
fruits, beverages and related commodities. The mechanism by which bioactives in
foods contribute their beneficial health effects has also been of much interest. These
developments have accelerated attention to nutraceuticals and functional foods. By
definition, nutraceutical is implied to components in food, both nutrient and non-
nutrient, that deliver beneficial health effect through disease prevention. These are
usually used in the medicinal form of pills, capsules, liquids and alike. On the other
hand, foods that comprise physiologically active ingredients with health benefits
above their basic nutrition are known as functional foods and these are in similar
appearance to traditional foods (Fereidoon Shahidi et al., 2003).
Beverages are regarded to be a brilliant medium for the supplementation of
nutraceutical components for amelioration such as soluble fiber or herbal extract (D.
T. Gordon et al., 2003). It is always more convenient to drink a beverage providing
health benefits rather than swallow vitamins or pills for the same health benefits.
Nutraceutical beverages are derived primarily from fruits and vegetables sources, but
also include those originated from other plants such as tea, coffee, cocoa, soybean as
well as animal products like milk and dairy-based and alcoholic drinks (F. Shahidi et
al., 2001).

2
The food industry has thus progressively considered new alternatives in order to
preserve the nutritional and antioxidant properties of foods during food processing.
Fermentation employing lactic acid bacteria is one of the oldest ways of food
processing and preservation, accountable to keep or increase the nutritional, safety,
shelf-life, and sensory properties of foods. Lactic acid bacteria (LAB)area group of
gram-positive bacteria which produce lactic acid as the main product of carbohydrate
fermentation. During fermentation, LAB produce substances such as bacteriocins,
exo-polysaccharides (EPS), enzymes (β-Galactosidase, β-D-phosphogalactoside
galactohydrolase), B vitamins (mainly folate, riboflavin, cobalamin), low-calorie
polyols (mannitol, sorbitol), or aroma compounds which enhance the antioxidant
activities, nutritional quality, safety, and sensory properties or of food (Beena Divya
et al., 2012).
Kimchi is a traditional fermented food containing mix of vegetables and Kombucha
which is a fermented tea beverage, were associated with antioxidative,
antimutagenic, antiaging, cholesterol-lowering activities and prevention against
cardiovascular diseases and cancer (Dufresne et al., 2000; Park, K.Y et al., 2014).
Several studies have also inspected the ability of lactic acid fermentation to enhance
the antioxidant properties of fruits and vegetables (Di Cagno, R. et al., 2011). An
improved preservation of ascorbic acid, phenolic compounds, glutathione and
antioxidant activity was observed following lactic acid fermentation of tomato juice
and smoothies with Lactobacillus plantarum, Pediococcus pentosaceus or Weissella
cibaria (Di Cagno, R. et al., 2009). Lactic acid fermentation started with L.
plantarum, Lb. delbrueckii subsp. lactis, Leuconostoc mesenteroides, P.
pentosaceus, Bifidobacterium breve and B. thermophilum increases Vitamin C
concentration, antioxidant activity, and phenolic content of carrots, black beans,
french beans, marrows, soy milk, pomegranate juice and cowpeas (Filannino et al.,
2013; Ng, C.C et al., 2011; Dueñas, M. et al., 2005; Marazza et l., 2012)

The enhancement of antioxidant property during fermentation is mostly attributed to

the release of bioactive compounds by Lactic acid bacteria (LAB) (Hur, S.J. et al.,
2014). It was testified that LAB was able to escalate the antioxidant activity of tea
extracts as modification of tea phenolics resulted (Zhao et al., 2016). Lactic acid
fermentation is thus a promising alternative to inflate the range of consumption of
processed foods, while simultaneously maintaining and/or boosting nutritional
benefits.
Fermented beverages play critical role in the diet of people not only as source of
nutrients but also in disease prevention and maintenance of health (D. Granato et al,
2010). The production of fermented beverages depends on the use of starter cultures
(F. Leroy et al., 2004). Microorganisms such as yeasts and LAB have been widely
employed to improve the functionality of foods (S.N. Liu et al., 2011). The use of the
latter is comparatively considered modest, energy efficient, cost-effective,
contributes to microbial safety and provide more organoleptic, technological,
nutritional as well as health benefits (N. Abu-Ghannam et al., 2015; M. Tripathi et
al., 2014; S. Borges et al., 2016).

3
Lactic acid bacteria include of a group of beneficial bacteria with analogous
properties, degrade diverse carbohydrates and related compounds to produce mainly
lactic acid and energy. Strains of a few LAB species have been used to advance
health and wellbeing (A.A. Argyri et al., 2013). Lactic acid bacteria have been
described to hydrolyse complex polyphenols into simpler bioactive compounds
during the fermentation process (R.Y. Gan et al., 2016). Intrinsic characteristics such
as pH and brix as well as other extrinsic factors such as initial microbial load and
temperature affect the growth and product formation during fermentation. Therefore,
the final quality of the product considerably depends on the fermentation conditions.
The interest in the composition of berries has been increased because of the
improved awareness of their probable positive health effects. Berries are a rich
source of various bioactive compounds having fascinating biological activities. They
are rich in vitamins and minerals, fibre and especially wild berries are rich in various
phenolic compounds (Viberg and Sjöholm 1996; Törrönen et al. 1997; Häkkinen et
al. 1999, 2000; Vuorinen et al. 2000; Mullen et al. 2002) and organic acids
(Viljakainen 2003).
Phenolic compounds are those secondary metabolites which are omnipresent in all
higher plants. The role of these compounds in plants is not fully recognized.
Nevertheless, many of them act as defence compounds, for example, against plant
pathogens. Further they are often produced as a response to various stress conditions.
Phenolics are present in plant tissues as simple substituted phenols, majorly as
glycosides, or as complex polymerised molecules with high molecular weights.
Flavonoids, phenolic acids, complex phenolic polymers (polymeric tannins) and
lignans are characteristic for berries (Riitta Puupponen-Pimiä et al., 2004).
Berries are a rich source of flavonoids, such as flavonols. High flavonol contents are
found for example in cranberry, black currant and lingonberry (Häkkinen et al.
1999). In fact, wild berries and black currant contain high amount of flavonols than
many vegetables and fruits that are commonly used. Anthocyanins (anthocyanidin
glycosides) are the preponderating group of flavonoids present in berries (Määttä et
al. 2001). They absorb visible light, thus appear as coloured substances, thus are
responsible for the characteristic orange/ red/ blue colours of berries, such as
strawberries, bilberries, raspberries and red and black currants.
In strawberries, 44% of phenolic compounds are anthocyanins. Simple phenolic
acids, like, hydroxycinnamic acids and hydroxybenzoic acids, are common in many
berries (Herrmann et.al, 1989). Chlorogenic acid, an ester between caffeic acid and
quinic acid, is a commonly occurring compound in various berries. The building
blocks for polymeric tannins are flavonoids and phenolic acids. Polymeric tannins
can be classified into hydrolysable and condensed tannins. Hydrolysable tannins are
either gallotannins or ellagitannins. Upon hydrolysis gallotannins produce glucose
and gallic acid. Ellagitannins contain one or more hydroxydiphenoyl residues which
are linked to glucose as a diester along with gallic acid. The hydrodiphenoyl residue
undergoes lactonisation to produce ellagic acid upon hydrolysis ((Riitta et al., 2004).

4
Berries, belonging to family Rosaceae, genus Rubus (red raspberry, cloudberry, and
arctic bramble), are rich in ellagitannins (Häkkinen et al. 2000; Mullen et al. 2002).
Strawberry and these berries produce only ellagitannins based on stable glucose
conformation. These berries contain dimeric or polymeric ellagitannins with only
small amounts of monomers in addition to pentagalloylglucose, (Haslam 1989). All
berries of family Rubus contain a dimeric form called sanquin H6. Cranberry
Lingonberry, and strawberry are examples of berries rich in di- phenolic compounds
called lignans (Mazur et al. 2000). Cultivar, geographic and seasonal origin
contribute the individual characteristics of sugar and acid composition in berries. The
main acids of the northern region wild-berry juices are unvaryingly malic and citric
acids, even though their concentrations varied broadly from one berry to another
(2.9–16.2 g l−1 and 3.3–24.7 g l−1, respectively) according to Viljakainen et al.
(2002).
The pH of berry juices is low (2.4–3.5), which is beneficial in preventing microbial
contaminations. Consumption of fruits and vegetables is shown to lower the risk for
chronic diseases such as cancer, cardiovascular diseases, and stroke. The positive
health effects may be due to high contents of certain phenolic compounds in plant-
derived foods (Riboli, E. et al., 2003).
Strawberry (Fragaria sp.) is a native berry of temperate regions, but a number of
varieties are available which can be cultivated in subtropical climate. In India it is
commonly cultivated in the hills. The main bases of cultivation are Mahabaleshwar
(Maharashtra), Nainital (district) and Dehradun in Uttar Pradesh, Kashmir Valley,
Kalimpong (West Bengal) and Bangalore. In recent years, strawberry is being
cultivated successfully in plains of Maharashtra around Nashik, Pune and Sangali
towns. Strawberries are the most widely adapted among the small fruits. These fruits
are grown throughout Europe, in all states of the United States, in Canada and South
America. The extensive variation in climates within these regions and the widespread
adaptation of the strawberry plant enable harvesting and marketing of the fruit during
greater part of the year.
Strawberries (Fragaria×ananassa) are consumed in plentiful quantities and can thus
be a beneficial source of phenolic compounds. The phenolic compounds in
strawberries are anthocyanins which are responsible for the red color in strawberry
flesh, other than flavonols, flavanols, and derivatives of hydroxycinnamic and ellagic
acid (Seeram, N. P. et al., 2006). Strawberries are the chief dietary source of ellagic
acid-containing compounds, that is, ellagic acid, ellagitannins and ellagic acid
glycosides. About 40 phenolic compounds including glycosides of quercetin,
cyanidin, pelargonidin, kaempferol and ellagic acid, together with flavanols,
ellagitannins and derivatives of p-coumaric acid have been described. Ascorbic acid
was observed to be the only important contributor to electrochemical response in
strawberries (24%), whereas the ellagitannins and the anthocyanins showed the
highest contributions, 19 and 13% at 400 mV, respectively. (Kjersti, Ekeberg,
Skrede,2007)

5
There is a cumulative awareness that health benefits of dietary polyphenols may be
due to their role in modulating of cell signaling and gene expression, along with their
antioxidative activities (Williamson, G. et al., 2005).
Even though there are some reports on the fermentation effects on the phytochemical
and antioxidant properties of plant-based foods (Hur, Lee, Kim, Choi, & Kim, 2014).
There is paucity of information on the effect of LAB fermentation on strawberry
juice and its qualities essentially with regards to its health-related properties.
Therefore, in view of the value of this lactic acid fermentation and the desire to
increse the shelf-life of strawberry fruit juice, biotechnological applications of lactic
LAB could be beneficial in improving its functionality and availability. Therefore,
the study aimed to assess the influence of selected strains of LAB on the, polyphenol
profile and antioxidant activities of strawberry juice. Besides, the study sought to
develop a lactobacillus fermented strawberry drink.

6
2.LITERATURE REVIEW

7
 LITERATURE REVIEW

2.1. Neutraseuticals and Functional foods

Human trials conducted over the last few decades have confirmed that the increased
consumption of carbohydrates, fats, or energy dense foods as stressors in meals may
lead to a low-grade inflammation as the common pathogenic denominator of
pathologies such as diabetes, obesity, atherosclerosis, and neurodegeneration
(Magrone and Jirillo 2010; Peluso et al. 2012). Polyphenol-rich foods might
counteract the inflammatory responses brought about by either stressor meals (Peluso
et al. 2012) or hypercaloric diet (Magrone et al. 2012; Marzulli et al. 2012).
There is a genuine interest in the development of fruit juice based functional
beverages, fortified with the probiotic and prebiotic ingredients despite potential
sensory challenges. The fruit juices have been recommended as an ideal medium for
the functional health ingredients because they innately contain beneficial nutrients,
they have taste profiles satisfying to all age groups, and because they are remarked as
being healthy and refreshing (Tuorila Cardello, 2002). The fruits and vegetables are
rich in the functional food components such as vitamins, minerals, dietary fibres,
antioxidants, and do not contain any dairy allergens that might restrain usage by
certain segments of the population (Luckow, Delahunty 2004).

2.2. Fruit and Vegetable beverages

Milk has been long considered as the only food containing almost all essential
substances for human nutrition. However, some researchers have recently focused
some constituents, such as , proteins, vitamin D, calcium, butyrate, CLA, saturated
fatty acids, and contaminants such as estrogen, pesticides, and insulin-like growth
factor I (IGF-I), which might be responsible for either a forthcoming or a harmful
association between dairy products and cancers (Davoodi and others 2013).
Moreover, cholesterol content and lactose intolerance are major disadvantages
related to functional dairy products (Pradoandothers2008)
Fruit juices could be ideal medium for probiotic due to their content of vital nutrients
(Granato and others 2010); some fruits used in commercial preparations include
pomegranate, apple, grapes, strawberries, cranberry, blueberry, guarana, mango,
blackcurrant, acai, acerola, cherries, kiwifruits, bilberries, feijoa, peach, and plums
(Sun-Waterhouse 2011). In addition, juices, food functionality can also be improved

8
using probiotics. The most significant traits for a promising probiotic rely upon (a)
adhesion to intestinal epithelium; (b) survival at low pH and in the presence of bile
salts; (c) immumodulation; (d) antimicrobial activity toward foodborne pathogens
and competitive adhesion to mucosa;(e) safety issues (production of harmful
metabolites, like biogenic amines, and transmission of genes encoding antibiotic
resistance; Sip and Grajek 2009; Nagpal et al., 2012). In addition to these classical
properties, some other features are required in functional beverages, like the
interaction with the starter cultures i.e. antagonistic interaction between probiotics
and starter cultures may bring about growth inhibition by acid, bacteriocins,
peroxide, and other metabolites. Therefore, the selection of compatible probiotics
and starter cultures could be crucial to prevent inhibition (Nagpal and others 2012;
Brajdes and Vizireanu 2013). Likewise, the ability of probiotics to grow well in
cereal, fruit or vegetable juices could depend, respectively, on their ability to exhibit
amylolytic activity or to resist preservatives (Yeo and others 2011).

2.3. Fermented beverages

Different studies have been carried out to explore the suitability of fruit juices such
as tomato, beetroot and cabbage juices as raw materials for the production of
probiotic drinks. L. plantarum, L. acidophilus and L. casei have been made use of as
probiotic bacteria cultures. Results have showed that all the strains are capable of
growth in the fruit juices mentioned and thus, the microbial population rises
significantly after 48 h of fermentation. Furthermore, L. plantarum, L. acidofilus and
L. delbruekii have exhibited to be resistant to the high acidic and low pH conditions
during storage periods at 4℃. However, results on L. casei have shown that this
strain loses its viability during cold storage (Yoon et al. 2004, 2005, 2006).

Thakur and V.K. Joshi (2017) studied fermentation of apple juice which was carried
out for 72 hrs by lactic acid bacteria (Lactobacillus plantarum and Streptococcus
thermophilus). During fermentation both vitamin C and total phenols deteriorated,
while, the antimicrobial activity, viable cell counts, antioxidant activity, and lactic
acid bacteria counts were increased. As the antimicrobial activity increased,
coliform, yeast and mold count declined.

Emmanuel, Yongkun, Tchabo et. al. (2018) examined the effect of lactic acid
bacteria (LAB) strains on phenolic profile, colour properties and antioxidant
activities of mulberry juice. Mulberry juice was individually fermented at 37 °C for
36 h using Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus
paracasei. The results exhibited that lactic acid fermentation affected the colour of
the juice. Furthermore, the study showed that LABs affected on the phenolic profile
of the juice. Cyanidin-3-O-rutinoside, syringic acid and quercetin were the
predominant anthocyanin, phenolic acid, and flavonol respectively in the lactic-acid-
fermented mulberry juice. The degree of radical scavenging activity was specific to
species with the juice fermented with L.plantarum having the highest radical
scavenging activity. The correlation analysis showed that flavonols and anthocyanins
were mainly responsible for the increase in 2,2′-azino-bis (3ethylbenzothiazoline-6-
sulfonic acid) scavenging activity while phenolic acids and flavonols were

9
accountable for 2,2-diphenyl-1-picrylhydrazyl scavenging activity and reducing
power capacity of the fermented juice.

Bao Toan, Bujna1, Fekete et.al.(2019) conducted a study in which fermentation of

pineapple juice with probiotic bacteria Lactobacillus and Bifidobacterium strains as
well as changes of some properties in the beverage during storage were investigated.
All tested strains displayed good growth properties on pineapple juice without
fortification of any nutrient compounds. Both total phenolic content and antioxidant
capacity improved slightly during fermentation and decreased during the storage
period.

Blanca, Isabel Cerrill, et. al. 2013 fermented orange juice showing the consumption
of bioactive compounds and moderate alcohol reduces the risk of cardiovascular
diseases. These effects could be merged in a beverage formed by a controlled
alcoholic fermentation of orange juice. The effect of controlled alcoholic
fermentation on the bioactive compound profile of orange juice had not been
previously evaluated. The total and individual flavanones and carotenoids
significantly increased throughout the fermentation.

2.4. Lactic acid bacteria

Lactic acid bacteria (LAB) are a group of gram-positive bacteria including the genera
Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus. The
general description of the bacteria included in the group is gram-positive, non-spore
forming, cocci or rods, which produce lactic acid as the major end product during the
fermentation of carbohydrates. Lactic acid bacteria are nutritionally fastidious,
requiring carbohydrates, amino acids, peptides, nucleic acids and vitamins. Recent
taxonomic revisions of these genera suggest that the lactic acid bacteria comprise the
following: Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus,
Leuconostoc, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and
Weissella (M.E. Stiles, W.H. Holzapfel, 1997)

Lactic acid bacteria are industrially important organisms recognized for their
fermentative ability as well as their health and nutritional benefits (S.E. Gilliand,
1990). Species of Genera Lactococcus, Streptococcus, Pediococcus, Leuconostoc,
Lactobacillus, and the newly recognized Carnobacterium are employed for food
fermentations. These organisms have been isolated from green plants, grains, meat
and dairy products, fermenting vegetables, and the mucosal surfaces of animals.
These microbes were once used to restrict spoilage and preserve foods through
natural fermentations, now they have commercial applications as starter cultures in
the dairy, meat, vegetable, baking and alcoholic beverages industries. They produce
various compounds such as diacetyl, hydrogen peroxide, organic acids and
bacteriocins or bactericidal proteins during LAB fermentations (S.E. Lindgren, W.J.
Dobrogosz, 1990). These components are beneficial for their effects on food smell,
color, taste and texture, but they also inhibit undesirable microflora. Hence, lactic
acid bacteria and their products give fermented foods distinctive textures, flavors and

10
aromas while extending shelf-life, preventing spoilage and inhibiting pathogenic
organisms.

The genus Lactobacillus is a brilliant target for value addition of berry juice as it can
grow in the pH ranging from mild acidic to neutral values and temperature ranging
from 2 to 53°C. The optimum temperature range is of 30–40 °C and optimum pH
range is of 5.5-6.2 (Mayra, Fonteles, et. al. 2013). Lactobacillus plantarum, among
various species targets, is the species most frequently used to ferment food products
of plant origin (Rodriguez, Curiel, et al. 2009). In case of antioxidant enrichment
LAB with βglucosidase activity (including Lactobacillus casei, L. acidophilus,
Lactobacillus fermentum, Lactobacillus plantarum, and Bifidobacterium longum)
have potential based on the ability to upsurge isoflavone aglycone (Marazza
Nazareno et. al. 2013). Lactobacillus casei and Lactobacillus acidophilus could
produce active enzymes such as peptidases, amylase, lactate dehydrogenases and
proteinase that can transform primary food matrix into functional moieties.
Specifically L. plantarum is known to produce active enzymes such as amylase,
decarboxylase, β-glucosidase, lactate dehydrogenases, phenolic acid decarboxylases,
peptidase, proteinase, phenol reductase and tannase (Hur, Lee, Kim, Choi, 2014) and
these have significance for diverse food substrate biotransformations. On this basis,
several fruit juice substrates such as pomegranate juice (Mousavi, Razavi et. al.
2013) strawberry, blueberry, noni juice (Wang, Ng, Su et. al. 2009), and cashew
apple juice (Periera, Maciel, Rodriguez, 2011) were fermented using lactic acid
bacteria.

2.5. Strawberry: Phenolic profile

Strawberry is a fruit, rich in anthocyanin, ellagitannins, ellagic acid, gallotannins,
catechin, proanthocyanidins, quercetin, folic acid, ascorbic acid and minerals. This
fruit contains a significant number of beneficial molecules such as lignans,
sesquiterpenoids and flavonoids. (Basu, Nguyen, et.al. 2014), These molecules show
biological activity against many degenerative and chronic diseases.

Strawberries, in addition to the fresh or frozen forms, are also commercially

available as processed products, such as, jams, juices, puree and nectar, and are
extensively used in North American cuisine as a popular berry ingredient (Klopotek
et al., 2005; CSC, 2010). Keeping in view the widespread epidemic of obesity, and
related chronic diseases, especially, diabetes mellitus, cardiovascular disease
(CVD)and cancer in the United States (Jemal et al., 2010; Nguyen et al., 2010;
Towfighi et al., 2010), public health measures to improve lifestyle and diet factors
specifically highlight the role of fruits and vegetables in managing these conditions
(Bendinellietal.,2011).The strawberry fruit is now considered a functional food
contributing multiple health benefits apart from basic nutrition as proved by the
gathering evidence on its antioxidant, antihyperlipidemic, anti-inflammatory,
antiproliferative, or antihypertensive effects. These mechanisms of action are directly
linked to the modification of causes of chronic diseases.

11
Antioxidant properties of strawberries have been mostly accredited to their
polyphenol and vitamin content. Approximately 40 phenolic compounds have been
identified in strawberries, such as, glycosides of quercetin, cyanidin, kaempferol,
pelargonidin, ellagic acid, as well as ellagitannins. Ascorbic acid, anthocyanins and
ellagitannins were shown to be the most major contributors to the antioxidant
capacity of strawberries, as estimated by their electrochemical responses (Aaby
etal.,2007)

Strawberries were also ranked second in total soluble phenolic content among eight
horticultural crops, namely, papaya, nopal, black sapote, guava, avocado, mango, and
prickly pear (Corral-Aguayo et al., 2008). After comparing ORAC activity among
berries, Wang and Lin (2000) proved highest antioxidant activity of strawberries,
followed by black raspberries then black berries, and red raspberries.

Strawberries present a unique combination of several nutrients, fiber and

phytochemicals, which plays a synergistic role in its characterization as a functional
food. Strawberries are a significant source of B-vitamins, vitamin E, vitamin C,
potassium, carotenoids, folic acid, and specific flavonoids, such as, pelargonidin,
catechin and quercetin. Strawberries also contain significant amounts of ellagic acid,
phytosterols and tannins (Stoner et al., 2006; Aaby et al.,2007; Basu et al., 2010).

Anthocyanins are water-soluble plant secondary metabolites responsible for the deep
colors of berry fruits, such as, blueberries, strawberries and blackberries.
Anthocyanins are among the principal bioactives in strawberries, hence food
scientists have carried out comprehensive analysis for their quantification and
characterization (Aaby et al., 2007; Buend´ıa et al., 2010). The major forms of
anthocyanidins identified were cyanidin-3-glucoside, 3-rutinoside, pelargonidin3-
glucoside and 3-malonyl glucoside.

Ellagic acid and ellagitannins have been demonstrated as important contributors to

the antioxidant and anticarcinogenic effects of strawberries. Ellagic acid is a widely
distributed phenolic acid in foods, such as, strawberries, grapes, raspberries, and
walnuts, and has shown to exert strong free radical scavenging, as well as
antiproliferative effects. Ellagitannins are hydrolysable tannins that usually consist of
glucose and a phenolic acid i.e. gallic acid or hexahydroxydiphenic acid. Strawberry
cultivars that were grown in Spain showed variation in ellagitannin and ellagic acid
content (10–23 mg/100 g and 1–2 mg/100 g fresh weight, respectively), although
these were lesser than total anthocyanin content in strawberries (Buend´ıa et al.,
2010). Ellagitannins have also been shown to be the second potential antioxidant
factor in strawberries, apart from vitamin C (Aaby et al., 2007).

Flavonoids, such as, catechin, kaempferol, naringenin, quercetin, and hesperidin have
a common chemical structure and exist in plant foods as aglycones or flavonoid
glycosides (Crespy etal.,2002). Free radical scavenging activity has been shown to be
the most effective biological mode of action of flavonoids, followed by anti-
inflammatory, antiproliferative and vasodilatory functions.

12
Bioavailability of dietary polyphenols is a complex process and several pathways
involving factors affecting their absorption and metabolism, have been proposed in
pig, rat or human model.Some anthocyanins have been shown to be absorbed intact
as glycosides (Cao et al., 2001; Wu et al., 2002). It has been suggested that
bilitranslocase, an organic anion membrane carrier, may play a role in the
bioavailability of anthocyanins though the mechanism of absorption is not clear,
(Vanzo et al., 2008).

3. MATERIALS AND METHODS

13
 MATERIALS AND METHODS

3.1. Materials
3.1.1. Raw material
Strawberry fruit, Nabila variety from Mahabaleshwar (Fragaria×ananassa Duch)
were purchased from the APMC fruit market, Vashi in February, 2019. The ripened
fruits were manually sorted and the damaged ones were discarded. The berries were
dipped in 0.02% Sodium hypochlorite and then washed with sterile distilled water to
eradicate the surface microbial load. The fruits were subsequently stored at -20℃
before the beverage formulation.

3.1.2. Bacterial culture

The lactobacillus cultures of the species namely, Lactobacillus plantarum, L.casei,
L.halvaticus, Leuconostoc mesenteroids, Pediococcus pentosis were procured from
Institute of Chemical Technology, Matunga in glycerol stock. The cultured were
stored and preserved at -20℃. The LAB strains were activated before being used.
(Kwaw, E., Ma, Y., Tchabo,2018).

3.2. Beverage formulation

3.2.1. Activation of starter culture
0.01g of the culture was added to 200mL DeMan, Rogosa and Sharpe (MRS) broth
(MV369, Rush Chem) and activated at 37℃ for 18 hours. Thereafter, the strains
were sub-cultured twice in MRS broth at 37℃ for 24hours.
Each culture was centrifuged at 3500rpm for 10minutes at 4℃. The microbial cells
were collected and washed with 0.1% sterile Sodium Chloride solution.
The cell concentration (108 cfu/ml) was determined roughly by dilution to makeup to
OD 0.2-0.4 at 600nm under UV Spectrophotometer.

3.2.2. Preparation of fruit extract

14
The frozen strawberry fruits were thawed for 8 hours at 4℃ and crushed using a
Food Processor in slow speed. The pulp obtained was treated with Pectinase enzyme
(NS33613, Novozymes Bangalore) with concentration of 0.1mL per Kg of pulp.
The juice was filtered using 3-4 layers of muslin cloth to remove the achenes.
The juice was centrifuged for 15minutes at 8000rpm at 4℃. The pellet was discarded
and the supernatant clear juice was sterilized by ultrasonication for 10minutes. These
conditions ensure microbial safety without thermal treatment.

3.2.3. Beverage fermentation

The juice was aseptically contained to six 250mL Erlenmeyer flasks containing
100ml of the clarified Strawberry juice. A 1% (v/v) of the individual LAB inoculants
(L.plantarum, L.casei, L.halvaticus, Leuconostoc mesenteroids, Pediococcus
pentosis) were allotted into 5 separate flasks and labelled (Table 1). One of the juice
flasks was allotted as control (CON) and was not inoculated and stored in the
refrigerator at 4℃. The five inoculated sample flasks were incubated at 37℃ for 36
hours in a rotary incubator at 100rpm. Thereafter, the lactic acid fermented
strawberry juice (LFSJ) was sterilized by ultrasonication for 10minutes. After this
sterilization process, the LFSJ were refrigerated at 4℃.

TABLE 1:SAMPLE ABBREVIATIONS

Sample Name Sample Abbreviations
L.plantarum fermented strawberry juice PLFSJ
L.mesenteroides fermented strawberry
MEFSJ
juice
L.pediococcus fermented strawberry juice PEFSJ
L.halvaticus fermented strawberry juice HAFSJ
L.casei fermented strawberry juice CAFSJ
Control CON

15
 Figure 1 Fresh strawberries

Figure 2 Frozen strawberries

Figure 3 Clear ultrasonicated strawberry juice

Figure 4 MRS Broth containing lactobacillus cultures

16
 Figure 5 Centrifuged pellets of LB cells

Figure 6 Lactobacillus fermented strawberry juice

3.3. Phytochemical Concentration assay

3.3.1. Total Phenolic Concentration

The Folin-Ciocalteu method was adopted in the determination of the total Phenolic
concentration (TPC) of LFSJ.
The Folin–Ciocalteu reagent which is a mixture of tungstates and molybdate. It
works on the mechanism of oxidation–reduction reaction i.e. based on the reduction
of the mixture heteropolyphosphotungsates–molybdates by the phenolic compound
which forms a blue coloured chromogen. The phenolic compounds react with Folin–
Ciocalteu reagent only under basic conditions provided by sodium carbonate
solution. Under basic conditions it has been observed that the phenolic compound
undergoes dissociation to form a phenolate anion which reduces the Folin–Ciocalteu
rendering a blue coloured solution. The colour intensity of the formed blue
chromogen can be measured by the absorbance readings using a UV
Spectrophotometer.
To 500µl LFSJ (1:100), 2.5mL freshly prepared Folin-Ciocalteu reagent (1:10v/v)
was added in a test tube after which 2mL sodium carbonate (75g/L) was added and

17
vortexed for 1min.The mixture was henceforth incubated for 40min at 25℃ and its
absorbance at 760nm using UV Spectrophotometer. Standards were also prepared
taking 25mM Gallic acid made in 95% methanol. TPC was expressed as milligram of
Gallic acid equivalent per millilitre of LFSJ.

3.3.2. Total Flavonoid Concentration

Total flavonoid content was estimated by using Aluminium chloride colorimetric
method.
The principle behind in Aluminium chloride (AlCl3) colorimetric method is that
AlCl3 forms acid stable complexes with the C-4 keto groups and either the C-3 or C-
5 hydroxyl group of flavones and flavonols. It also forms acid labile complexes with
the orthodihydroxyl groups in the A- or B-ring of flavonoids.
Studies have stated quercetin to be suitable reference for estimation of total flavonoid
content in plant sample extract. Therefore, quercetin solutions of various
concentrations were used to make the standard calibration curve. (Bag, Grihanjali
and Th. Bhaigyabati,2015)
10mg of quercetin was dissolved in 100ml methanol and then diluted to 6.25, 12.5,
25, 50, 80, and 100µg/ml using methanol. Stock solution of LFSJ in 1:100 ratio was
prepared. 10% aluminium chloride and 1M potassium acetate were prepared using
distilled water. The assay was determined using 0.5ml of each extract stock solution
and each dilution of standard quercetin taken separately in test tubes. To each test
tube 1.5mL methanol, 0.1mL aluminium chloride solution, 0.1mL potassium acetate
solution and 2.8mL distilled water were added and mixed well. Sample blank for all
the dilution of standard quercetin was prepared in similar manner by replacing
aluminium chloride solution with distilled water. Absorbance was taken at 415 nm
against the suitable blank. The TFC was expressed as milligram of quercetin per
millilitre LFSJ.

3.3.3. Total Anthocyanin concentration

pH Differential method was used to estimate Anthocyanin content. (Cheng and
Breen 1991)

Monomeric anthocyanins undergo

a reversible structural
transformation as a function of pH
(colored oxonium form at
pH 1.0 and colorless hemiketal
form at pH 4.5; Figure 2). Thus,

18
the difference in absorbance at the l
vis-max
(ca 520 nm) of the
pigment is proportional to the
concentration of pigment
Monomeric anthocyanins undergo
a reversible structural
transformation as a function of pH
(colored oxonium form at
pH 1.0 and colorless hemiketal
form at pH 4.5; Figure 2). Thus,
the difference in absorbance at the l
vis-max
(ca 520 nm) of the
pigment is proportional to the
concentration of pigment
Monomeric anthocyanins undergo a reversible structural transformation as a function
of pH (colored oxonium form at pH 1.0 and colorless hemiketal form at pH 4.5).
Thus, the difference in absorbance at the λ vis-max (ca 520 nm) of the pigment is
proportional to the concentration of pigment. Results were expressed as equivalents
of cyanidin-3-glucoside, which is the most common anthocyanin pigment found in
nature. (Barnes; Eisele; Giusti,2005)
3 ml of extracts were diluted in 5 ml of two different buffers; 0.025 M potassium
chloride pH = 1.0 and 0.4 M sodium acetate pH = 4.5, respectively. After 30 minutes
of incubation at room temperature, absorption (A) was measured at Ȝ = 510 and Ȝ =
700 nm. All extracts were analysed in duplicate.

19
For calculation of total anthocyanins as C3g, the molar absorptivity coefficient (İ)
values 26900 M-1cm-1 (Meyers et al. 2003) and 29600 M-1cm-1 (Cao et al., 2011) and
molecular weight 449 was used. The results were calculated similarly to
Giusti and Wrolstad [2001] as follows:
Asp = (A510 – A700) pH1.0 – (A510 – A700) pH 4.5
The content of total anthocyanins (TA) were calculated as follows:
TA = (Asp × M ×DF × 1000) / (İ × Ȝ × m),
where DF is the dilution factor, Ȝ is the cuvette optical pathlength (1 cm) and m is
the weight of the sample (g). The total anthocyanin content was expressed as mg
anthocyanin per 1ml LFSJ.

3.4. Antioxidant activity assay

The radical scavenging activity of LFSJ was determined by using DPPH assay
according to Chang et al. (2001). The decrease in the absorption of the DPPH
solution after the addition of an antioxidant was measured at 517nm. Ascorbic acid
was used as reference.
1, 1 Diphenyl 2- Picryl Hydrazyl is a stable (in powder form) free radical with red
color which turns yellow when scavenged. The DPPH assay uses this character to
show free radical scavenging activity. The scavenging reaction between (DPPH) and
an antioxidant (HA) can be written as,
(DPPH) + (H-A) → DPPH-H + (A)
Antioxidants react with DPPH and reduce it to DPPH-H and as consequence the
absorbance decreases. The degree of discoloration indicates the scavenging potential
of the antioxidant compounds or extracts in terms of hydrogen donating ability.
0.1mM DPPH solution was prepared by dissolving 2.45mg of DPPH in 100ml of
ethanol. The standards of ascorbic acid(1500µM) were prepared in different
concentrations (30,60,..150µM).In essence 100µL of LFSJ (1:100) and in the
different stock solutions, 900µL of 62.13µM solution of DPPH reagent was added.
The reaction mixture was incubated in dark condition at room temperature i.e. 25℃
for 30 minutes. After 30 minutes, the absorbance of the mixture was read at 517 nm.
3ml of DPPH was taken as control.
The % radical scavenging activity of the plant extracts was calculated using the
following formula, Where, RSA is the Radical Scavenging Activity; Abs control is
the absorbance of DPPH radical + ethanol; Abs sample is the absorbance of DPPH
radical +LFSJ.
|control|−|sample|
% RSA = × 100
|control|

3.5. pH and Titrable Acidity

The pH value of prepared fermented juice was evaluated by a pH meter. Total

acidity, expressed as percent lactic acid, was conducted by titrating with 0.1 N NaOH
to pH 8.

20
3.6. Brix
Degree brix which is a measure of the number of dissolved solids in a liquid via its
specific gravity, used especially to measure dissolved sugar. One-degree Brix is 1g
sucrose in 100g of solution. It was measured using a Refractometer.

21
 4. RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

22
The total phenolic concertation (TPC), total anthocyanin concentration (TAC) and
the total flavonoid concentration (TFC) of the fermented strawberry juices as shown
in the tables, compared to the CON, the LFSJ had higher phytochemical
concentrations which demonstrates that LABs improved on the phytochemical
concentrations of the juice during the fermentation (Gan, Shah, Wang, et al, 2016;
Kachouri, Ksontini, Kraiem, et al, 2015; Kaltsa, Papaliaga, et al, 2015). The higher
phytochemical concentrations of the LFMJ could be attributed to the ability of LABs
to produce hydrolytic enzymes which hydrolyzed the complex phytochemicals into
simpler forms (Gan, Shah, et al, 2016; Kaltsa, Papaliaga, et al, 2015). The
differences in the phytochemical concentrations among the LABs strains could be
due to their individual adaptability as well as their ability to produce more of the
hydrolytic enzymes.
The ability of L. plantarum to enhance the TPC, TFC, TAC, and antioxidant activity
more than the other LABs may be suggested to be to its ability to deglycosylate more
glycosylated phenolics of the strawberry juice during the fermentation thereby
releasing soluble conjugated or insoluble bounded phenolic compounds from plant
cell wall (Benincasa, Muccilli, et al, 2015; Landete, Curiel, et al, 2014).

23
4.1. EFFECT OF LACTOBACILLUS FERMENTATION ON TOTAL
PHENOLIC CONTENT
The result (Table 2, Figure 7) depicted an upsurge in concentration of the individual
phenolic compounds after the fermentation. It was observed that the total
concentration of the phenolic compounds quantified in the fermented sample
increased from 0.776±0.065 mg /mL (CON) to the following: 1.114± 0.013,
0.852±0.036, 1.060±0.089, 0.920±0.087, 0.824±0.009, 0.776±0.065 mg/ml after
fermenting it with L. plantarum (PLFSJ), L. mesenteroids (MEFSJ), L. pediococcus
(PEFSJ), L. halvaticus (HAFSJ), L. casei (CAFSJ) respectively. This could be due to
the conversion of complex phenolics into free forms and the depolymerization of
high molecular weight phenolic compounds by (poly) phenoloxidases in LABs (Hur,
Lee, Kim, Choi, & Kim, 2014). Furthermore, the stability of phenols is pH-
dependent (Kwaw, et al., 2017) hence, the decrease in pH of the LFSJ might have
stabilized the phenolics hence the high phenolic content of the LFSJs compared to
the control.
Table 2: TOTAL PHENOLIC CONTENT

LFSJ SAMPLES Gallic acid equivalence mg/ml

PLFSJ 1.1141± 0.013151
MEFSJ 0.8520±0.036316
PEFSJ 1.06006±0.08039
HAFSJ 0.92047±0.087082
CAFSJ 0.82430±0.009453
CON 0.77649±0.065115

PLFSJ and PEFSJ shows a high upsurge in the phenolic content compared to the
other LFSJs, among which PLFSJ shows the highest phenolic content. MEFSJ,
HAFSJ and CAFSJ samples did not show much difference in the total phenolic
content with respect to the CON sample.

Total Phenolic Content

1200
Conceentration of Gallic acid µg/ml

1000

800

600

400

200

0
PLFSJ MEFSJ PEFSJ HAFSJ CAFSJ CON

24
 Figure 7: TOTAL PHENOLIC CONTENT

4.2. EFFECT OF LACTOBACILLUS FERMENTATION ON TOTAL

FLAVONOID CONTENT
The TFC values (Table 3, Figure 8) increased from 4.9384±0.1771mg/ml (CON) to
13.248±0.0696, 12.216±0.0167, 5.253±0.0542, 5.453±0.2902, 4.2324±0.5023 mg/ml
in PLFSJ, MEFSJ, PEFSJ, HAFSJ and CAFSJ respectively.

Table 3: TOTAL FLAVONOID CONTENT

LFSJ SAMPLES Quercetin equivalence mg/ml

PLFSJ 13.248±0.0696
MEFSJ 12.216±0.0167
PEFSJ 5.253±0.0542
HAFSJ 5.453±0.2902
CAFSJ 4.2324±0.5023
CON 4.9384±0.1771

PLFSJ and MEFSJ showed the most increment in flavonoid content compared to the
other LFSJs of which PLFSJ showed highest flavonoid content. PEFSJ, HAFSJ, and
CAFSJ did not show much change in flavonoid content compared to the control.

Total Flavonoid Content

14000
Concentration of Quercetin µg/ml

12000

10000

8000

6000

4000

2000

0
PLFSJ MEFSJ PEFSJ HAFSJ CAFSJ CON
LFSJ Samples

Figure 7: TOTAL FLAVNOID CONTENT

25
4.3. EFFECT OF LACTOBACILLUS FERMENTATION ON TOTAL
ANTHOCYANIN CONTENT
The TAC values (Table 4, Figure 9) increased from 23.451 mg/ml (CON) to 32.214,
23.546, 23.785 in PLFSJ, MEFSJ, HAFSJ mg/ml respectively, but decreased
to19.145 and 22.883 mg/ml in PEFSJ and CAFSJ respectively.

Table 4: TOTAL ANTHOCYANIN CONTENT

LFSJ SAMPLES Cyanidin-3-glucoside mg/ml

PLFSJ 32.214
MEFSJ 23.546
PEFSJ 19.145
HAFSJ 23.785
CAFSJ 22.883
CON 23.451

PLFSJ showed highest anthocyanin content compared to the other LFSJ samples.
MEFSJ and HAFSJ did not show much change in anthocyanin with respect to the
CON sample. Whereas PEFSJ and CAFSJ showed a decrease in anthocyanin content.
This may be due to the instability of anthocyanin after fermentation.

Total Anthocyanin Content

Concentration of cyanidin-3-glucoside mg/ml

35
30
25
20
15
10
5
0
PLFSJ MEFSJ PEFSJ HAFSJ CAFSJ CON
LFSJ Samples

Figure 8:TOTAL ANTHOCYANIN CONTENT

26
4.4. EFFECT OF LACTOBACILLUS FERMENTATION ON ANTIOXIDANT
ACTIVITY
The results on the antioxidant activities -DPPH•-SA, are presented in Table 6,
Figure10, and Figure 11. The results illustrate the considerably positive effects that
lactic acid fermentation had on the scavenging activities of DPPH radical as revealed
by the increase in the inhibition of DPPH•-SA (ranged from 15.045%, 14.630%,
9.642%, 14.131%, 10.473%, to 8.312% for PLFSJ, MEFSJ, PEFSJ, HAFSJ, CAFSJ
and CON respectively).
During DPPH•-SA, the anomalous electron of nitrogen atom in DPPH is reduced by
accepting hydrogen atom from antioxidants to the corresponding hydrazine (Kedare
& Singh, 2011). The increase in DPPH•-SA stipulates that, lactic acid fermentation
might have certainly increased the availability of compounds with proton-donating
properties.
It has been stated that phenolics antioxidant activities are affected by the molecular
structure of phenolic compounds and can be influenced depending on the group
linked to their basic aglycon (Pérez-Gregorio, Regueiro, Alonso González, Pastrana-
Castro, & Simal-Gándara, 2011). The association between the phenolic compounds
particularly the flavonoids and the antioxidant activities could be due the number of
acidic and phenolic hydroxyl groups. The resonance between their aromatic benzene
rings and free electron pair on the phenolic oxygen induce an increase in electron
delocalization, thereby increasing their antioxidant activities towards free radicals
(Cheynier, 2006; Granato, Katayama, & de Castro, 2011). Thus, antioxidant
activities may differ, based on the disparities in the chemical composition of the
product (Aydın & Mammadov, 2017).

Table 5: TOTAL ANTIOXIDANT CONTENT

LFSJ SAMPLES Ascorbic acid equivalence µg/ml

PLFSJ 769.8743±0.03924
MEFSJ 744.4988±0.9987
PEFSJ 492.7937±0.7854
HAFSJ 715.8547±0.6547
CAFSJ 506.5087±0.8966
CON 383.3236±0.4678

27
 Total Antioxidant Content
900

Concentration of Ascorbic acid µg/ml

800
700
600
500
400
300
200
100
0
PLFSJ MEFSJ PEFSJ HAFSJ CAFSJ CON
LFSJ Samples

Figure 9: ANTIOXIDANT CONTENT

DPPH SCAVENGING ACTIVITY

16
14
12
10
DPPH-SA %

8
6
4
2
0
PLFSJ MEFSJ PEFSJ HAFSJ CAFSJ CON
LFSJ Samples

FIGURE 10:DPPH -SA

4.5. EFFECT OF LACTOBACILLUS FERMENTATION ON pH

The pH values of LFSJs were lower than the CON sample due to the fermentation
product i.e. lactic acid. pH reduced from 3.64 to 3.11. The FJs were more acidic
compared to the CON, this may be due to the production of lactic acid.

4.6. BRIX
The brix of the CON and LFSJ samples were studied to be 2.5⸰Brix. Thus no
changes in Brix or sugar content.

28
5. CONCLUSION

29
 CONCLUSION
The LABs strains used certainly had effects on the phenolic constituents of the
strawberry juice. The fermentation process significantly improved on the total
anthocyanin, phenolic and flavonoid concentration of the strawberry juice. There was
a potent increase in free radical scavenging activities of DPPH•-SA of the strawberry
juice after the LAB fermentation. Fermentation using Lactobacillus plantarum
however, exhibited a higher improvement in total phenolic concentration and
antioxidant activities than L.casei, L.halvaticus, Leuconostoc mesenteroids and
Pediococcus pentosis. Moreover, the antioxidant activities of the juice were related
to the phenolic profile after the LAB fermentation. Nevertheless, further research is
needed to optimize the fermentation conditions to ascertain how they will impact on
the bioactive constituent and bioactivity of the fermented strawberry juice.

30
6. FUTURE ASPECTS

31
 FUTURE ASPECTS
Functional foods have a huge and growing global market and are reckoned as one of
the most actively researched areas of food science. The progression in popularity of
functional foods is credited to the phenomena of consumer health care, cumulative
health awareness and knowledge in the many benefits of functional foods. Common
advantages of functional foods include health maintenance by reduction of disease
and prevention of nutrition related diseases.
Taking the present study forward, GC-MS could be employed to identify and
quantify the different phenolics present in the fermented strawberry juices to
specifically characterize as functional drink.
Recently, beverages based on fruits, vegetables, soybeans and cereals have been
proposed as novel products containing probiotic strains; mainly, fruit juices have
been reported as a new and appropriate medium for probiotic because they contain
essential nutrients. Hence strawberry can be used as an excelled media for carrying
probiotics.
The juice could be fortified with prebiotics further serving the gut health and for the
growth and stability of probiotic strains.
Antimicrobial studies can be carried out against various pathogenic microbe, adding
up to the functional profile of the drink.
Due to its highly risky, complex and expensive nature, wide-ranging research is
essential in the development of a marketable new functional food product. Thus,
functional foods have a great marketing potential due to the fact that it is a
differentiated version of a pre-existing food product which has a supplementary
health value.

32
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33
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