INTRODUCTION

1. 1 THE DEFINITION OF PROBIOTICS

Probiotics are just live bacteria and yeasts that are thought beneficial in several health
conditions. They are commonly ingested as supplements or yoghurts and are also known as
“good bacteria.” They restore the balance of gut bacteria when it has been disturbed
through long-term exposure to antibiotic or through gastrointestinal problems.

“Probiotics are “live microorganisms which, when administered in adequate amounts,

confer a health benefit on the host.” define by the World Health Organization (WHO) in
2001.

A wide range of microbes and their applications is included in this definition of probiotics.
By this one can easily distinguish between live microbes which are used as processing aids
or sources of useful compounds and those which are noticed primarily for their health
benefits. Also, one can recognise easily between commensal microorganisms and probiotics
by this definition. Although commensals present in the gut are mainly the source of
probiotic strains, but before their isolation, characterization and a convincing case proven
for health benefits, they can not be termed as ‘probiotics’. Even after 13 years of this known
definition, various researches have been done to challenge the limits of probiotic concept,
from faecal microbiota transplants (FMT) to live microbial culture present in fermented
foods. The term ‘probiotic’ has also been embezzled, for eg., on products such as shampoos,
disinfectants, mattresses and aftershaves for which effectiveness and maintenance of
viability of the microbes used are not established (Hill C et al.,2014).

The taxonomy of the probiotic candidate must be well defined. It has been proven that
efficacy of probiotics are strain specific and cannot be observed from other strain of same
genus or species. This introduces two terms which are genotype and phenotype used for the
precise identification of the isolated strains (Rijkers et al.,2010)
According to the 2002 FAO/WHO guidelines, the safety of the potential probiotic should be
assessed by the following minimum required tests:

 Antibiotic resistance patterns must be determined.

 Evaluation of some metabolic activities (e.g. bile salt deconjugation, D-lactate
production).

 Side effects evaluation during human studies.

 Epidemiological observation of unfortunate incidents in consumers (after market).

 Test for toxin production is required if the species of the strain is known to produce toxin
in mammals. One scheme for testing toxin production has been suggested by the EU
Scientific Committee on Animal Nutrition.

 Test for hemolytic activity is required if the strain under evaluation belong to a species
which have a hemolytic potential.

Most commonly used probiotics are from the species of lactobacillus and Bifidobacterium,
although some species of E. coli and Bacillus and the yeast (Saccharomyces cerevisiae) are
also used as probiotics.

1.2 ADVANTAGES OF PROBIOTICS

The balance or imbalance of bacteria in digestive system is directly related to overall health
and disease of the concerned organism. Probiotics increases the number of good bacteria of
gut and results in a range of health benefits (weight loss, digestive health, immune
function, etc).

1.2.1. Probiotics Help in balancing the friendly bacteria of your digestive system:

As already discussed probiotics include "good" bacteria. An imbalance means the number
of bad bacteria is more than that of the good ones which can be due to illness, medication
such as antibiotics, poor diet and more. Consequences can include allergies, mental health
problems, obesity, digestive issues, and more. Probiotic bacteria are generally found in
fermented foods or in supplements. . They have a tremendous ability to restore the natural
balance of gut bacteria by direct and indirect means(Brown AC and Valiere A, 2004).

1.2.2. Probiotics can help in preventing and treating diarrhea:

Diarrhea is a common side effect of taking antibiotics because antibiotics can negatively
affect the balance of good and bad bacteria in the gut. According to some studies, the use of
probiotic is linked with diminished risk of probiotic-associated-diarrhea. Probiotics can also
help in curing other forms of diarrhea which are not associated with antibiotics. In a large
review of 35 studies, it has been found that certain strains of probiotics can reduce the
duration of infectious diarrhea by an average of 25 hours. Efficacy of probiotics for
preventing diarrhea from other causes is 26% in adults and 57% in children. This efficiency
depends on the dose and type of the probiotic taken. Commonly known anti-diarrheal
probiotic strains are Lactobacillus casei, Lactobacillus rhamnosus and the yeast
Saccharomyces boulardii (Sazawal et al., 2006).

1.2.3. Probiotic supplements helps in improving some mental health conditions:

Gut health is directly linked with mood and mental health of an organism shown in several
studies. According to both animal and human studies probiotic supplements can improve
some mental health disorders. In a review of 15 human studies it is shown that
supplementing with Bifidobacterium and Lactobacillus strains for 1–2 months can reduce
anxiety, depression, autism, obsessive-compulsive disorder (OCD) and aids in memory. One
study followed 70 chemical workers for 6 weeks in which those who consumed 100 grams
of probiotic yogurt per day or took a daily probiotic capsule experienced benefits for general
health, depression, anxiety and stress. (Akkasheh et al., 2016)). Research shows probiotic
consumtion reduces symptoms of mental health disorders such as depression, anxiety, stress
and memory, among others.

1.2.4. Probiotic strains can maintain the health your heart:

As probiotics reduces the level of LDL("bad") cholesterol and blood pressure, so it will
definitely reduces the risk of artery or vein blockage and also the risk of heart stroke. Bile is
a fulid (made up of cholesterol) which occurs naturally in body, helps in digestion. There are
certain lactic acid-producing bacteria which break the bile in the gut and hence reduce
cholesterol so by breaking down bile, probiotics can prevent it from being reabsorbed in the
gut, where it can enter the blood as cholesterol (Kumar at al.,2012).Probiotic consumption
may also lower blood pressure.

1.2.5. Probiotics may decrease the severity of some allergies and eczema:

Mainly beneficial in children and infants. One study found that symptoms of eczema was
reduced for infants fed probiotic-supplemented milk, compared to infants fed milk without
probiotics (Isolauri et al.,2000). In another study, probiotics were given to women during
pregnancy as a result, children had an 83% lower risk of developing eczema in the first two
years of life (Rautava et al.,2012). Some probiotics are also known to reduce inflammatory
responses in people with milk or dairy allergies such as lactose intolerant people. However,
the evidence is weak and further studies are needed

1.2.6. Probiotics can help in reducing symptoms of certain digestive disorders:

In the US more than 1 million people suffer from inflammatory bowel disease. Many types
of probiotics from the Bifidobacterium and Lactobacillus strains have shown improved
symptoms in people with mild ulcerative colitis. Astoundingly, one study found that
supplementing with the probiotic E. coli either in the form of capsules or in the daily diet ,
it was enormously effective as drugs in maintaining revoation in people with ulcerative
colitis (Kruis et al.,2004). Early research suggests that they may help in reducing the
symptoms of irritable bowel syndrome (IBS). Probiotis have also been shown to diminish
the risk of severe necrotizing enterocolitis by 50%. So, one can easily conclude that
consumption of probiotics will have a recognisable effect on his/her digestive problems in a
better way which will further show a good impact on their mood as our food is directly
linked to our mood. If we consume good food there will be a good mood of us and a better
life further.
1.2.7. Probiotics helps in improving and maintain the Immune System:

Probiotics obviously help in giving your immune system a boost and retard the growth of
harmful or bad gut bacteria. Also, there are some probiotics which have been shown to
promote the synthesis of natural antibodies in the body. Immune cells like the IgA-
producing cells, T lymphocytes and natural killer cells are also increased and boosted by
probiotics. In a review it has been found that consuming probiotics reduced the occurrence
and duration of respiratory infections. In another study which including over 570 children, it
has been found that taking Lactobacillus GG reduced the frequency and seriousness of
respiratory infections by 17% (Hatakka et al.,2001). In women on consumption of probiotic
Lactobacillus crispatus, there is reduction in risk of urinary tract infections (UTIs) by 50%.
Probiotics may help boost your immune system and protect against infections.

1.2.8. Probiotics may help in loss of belly fat and weight loss:

There are number of different mechanisms by which probiotics helps in weight loss. For
example, some probiotics prevent the absorption of dietary fat in the intestine as a result the
fat is rather than being stored in the body gets excreted out through faeces. Probiotics may
also give you a feeling of satiety for longer, it helps in burning more calories and storing less
fat. This is partly caused by increasing levels of certain hormones, such as GLP-1 (a
hormone that stimulates insulin secretion in response to meals). In one study, women lost
her 50% weight when supplemented with Lactobacillus rhamnosus for 3 months than
women who didn't take a probiotic (Sanchez et al., 2014)

1.2.9. Probiotics are favourable to lactose intolerants:

Probiotic strains have also proved to solve the problem of lactose intolerance. There is an
enzyme called lactase or B-galactosidase which is responsible for breaking the milk sugar or
a disaccharide in milk i.e., lactose into glucose and galactose. So people lacking the ability
to produce lactase enzyme or B-galactosidase enzyme are known as lactose intolerant. These
individuals will not be able to digest milk and it mostly poses a problem in newborn infants.
People who are suffering with lactose intolerance problem express, cramps, flatulence,
abdominal discomfort, diarrhea, nausea, vomiting, etc. Lactobacillus produces the lactase
enzyme, among others. So, by consuming L. acidophilus and L. casei, we help our body
break up lactose and deliver it in a much more digestible form (in the form of glucose and
galactose).

1.2.10. Probiotics are active against certain vaginal infection and Candida ablicans:

An imbalance in the intestinal flora will definitely have effects on the whole body,
particularly the vaginal system, and include an extreme growth of pathogens which convert
into vaginal bacterial or yeast infections, such as C. albicans. One of the most important
bacteria found in the vaginal and intestinal flora is L. acidophilus. These friendly bacteria
maintain a low pH and produce bacteriocins in flora and hence makes the environment
hostile to pathogens. Moreover L.acidophilus produces hypothiocyanate and hydrogen
peroxide which retard the growth of C. albicans.

1.3. MODE OF ACTION OF PROBIOTICS

On ingestion in certain numbers, probiotics provides health benefits which are beyond
inherent general nutrition. Mechanism of action of probiotics is shown in figure 1 given
below. There are many good evidence that some specific strains
of Lactobacilli and Bifidobacteria can influence immune function through many different
pathways such as effects on enterocytes, antigen presenting cells (including both circulating
monocytes and local dendritic cells [DCs]), effector T and B cells, regulatory T cells. It is
not known to what extent a finding using a certain bacterial strain is related for other strains
even if they belongs to same species, because there are very few studies in which several
allegedly probiotic strains have been compared. There are only a few strains to date, which
are limited to Lactobacilli, which have been also reasonably well documented and defined
in clinical studies, mostly against infectious gastroenteritis and lactose intolerance. Locally
in the gut, there is affirmation that at least 1 probiotic strain has the potential to maintain the
nobility of the intestinal barrier, potentially minimizing systemic antigen load and
commensal gut bacteria help reduce local inflammation (Gaurav Tiwari et al.,2012).
Fig1. Mechanism of action of probiotic

1.4. HEALTH CLAIMS AND COMMERCE OF PROBIOTIC BAEL

DRINK

Market Potential

Products containing high-profile probiotics have been extremely successful in Europe, Asia,
and, more recently, in other regions of the world. This marketing success is promoting
consumption, product development, and research. Probiotics are mostly recommended by
nutritionists and sometimes by doctors, and a wide range of product types are available in
the market (Guarner et al., 2011).
 Food Meal Dietary
replacement supplement

Prescription Over-the Neutraceutical

drug counter drug

Fig 2 : Spectrum of interventions that can affect health and disease.

1.5. WOOD APPLE

Bael (Aegle marmelos Corr.) is an indigenous fruit of India belonging to family Rutaceae
and commonly known as Bael, Beli, Belgiri (Hindi); Bilva, Shivadruma, Shivaphala, Vilva
(Sanskrit); Holy fruit, Bengal quince, Indian quince, Golden apple, Bael (English); Bel, Bel
Kham (Urdu); Bel (Assamese and Marathi); Bilivaohal (Gujrati); Marredy (Malyalam); Belo
(Oriya); Vilvama, Vilva marum (Tamil); and Bilva, Bilva pandu(Telugu) in India (Ansary,
2005; Maity et al.,2009).

Bael is resident to Northern India but widely distributed throughout the Indian peninsula
and in Ceylon, Burma,Bangladesh, Thailand and Indo-China (Brijesh et al., 2009). Bael fruit
juice is more popular because of its exceptional functional, nutritional, and therapeutic
qualities (Bhardwaj and Pandey, 2011). Minerals are present in the form of electrolytes,
that’s why they are easily absorbabed by the human body(Bhardwaj et al., 2014). Bael juice
is rich in diverse sources of essential nutrients which comprise minerals like iron, boron,
molybdenum, iodine, zinc, selenium, copper, potassium iodine, sulfur, manganese and
magnesium; dietary fiber; antioxidants; amino acids and bioactive compounds(Bhardwaj,
2014) which are must for good nutrition, prevention of diseases and also provide a great
taste and health benefits (Nandal and Bhardwaj, 2012). Bael fruit juice is easily digestible; it
has no toxic effects on the body and gives a cleansing effect on the blood and digestive
tract; it also facilitates absorption of some nutrients (Nandal and Meena, 2012). The unripe
bael fruits have digestive, stomachic and astringent, properties and they are mostly
prescribed for treating diarrhea and dysentery. The ripe bael fruit is sweet, nutritious,
aromatic and very palatable and eaten by all classes of people (Singh et al., 2014a, 2014b).

The bael fruit juice has lots of pharmacological activity like anti-dyspepsia, anti-diarrhea
and anti dysentery properties. It is largely known to cure antiproliferative activity,
intermittent fever, mental disease and has hypoglycemic, anti-inflammatory, antifungal and
antimicrobial, analgesic, antipyretic, immune-modulatory activity, wound-healing activity,
anti-fertility, insecticidal activity, etc.(Lambole et al., 2010). It has inhibitory effects against
the HIV virus and promotes metabolic functions also it reduces unwanted fat, cures ulcers
and maintain proper digestion (Sanchez-Moreno et al., 2009). Bael fruit pulp extracts also
showed good antioxidant property (Rajan et al., 2011). It is also beneficial in curing typhoid,
anaemia, fracture,pregnancy troubles, swollen joints(Paricha, 2004), colitis (Agrawal,
1990), bleeding sores and cramps (Agarwal, 1997). Bael juice extracts have shown
activities, including antiulcer, antidiabetic,anti-hyperlipidemic, antioxidant, anticancer,
antimicrobial, radio-protective, anti-inflammatory, antipyretic, anti-spermatogenic and
analesic effects on various animal models (Karakaya, 2004; Jagetia et al., 2005). As dietary
supplement in daily diet for an adult about 60 g of the bael fruit juice/pulp is sufficient (Roy
and Singh, 1980).

1.5.1. BIOACTIVE COMPOUNDS OF BAEL FRUIT JUICE

Nutrients:
The relevant nutritional groups are vitamins, carbohydrates, minerals (K,Ca,P), organic
acids, amino acids, fibers and polyphenols like colorful carotenoids, aromatic compounds
and other bioactive substances (Nandal, 2013). Bael fruit juice is also an important source of
certain vitamins, especially vitamins A, B(mainly riboflavin) and C. Bael fruit juice, is a rich
source of glucose, sugar and fiber (Nandal, 2013). The average nutritional composition of
100g of bael fruit juice is water 55.0-61.5 g, energy 137 calories, protein 1.8-2.62 g, fat 0.2-
0.39 g, fiber 2.9 g, carbohydrates 28.11-31.80 g, minerals 1.7 g, carotene 55 mg, thiamine
0.13 mg, riboflavin 1.19 mg, niacin 1.1 mg, vitamin C 8.0-60.0 mg, tartaric acid 2.11 mg,
calcium 85 mg, phosphorus 50 mg and potassium 600 mg.
Flavonoids and polyphenols:
Bael fruit pulp contains steroids, flavonoids, terpenoids, phenolic compounds, lignin, fat and
oil, proteins, carbohydrates, alkaloids and cardiac glycosides (Bhaskara Rao et al., 2011).
Fruit juice also contains phenolic constituents such as chlorogenic, arbutin, caffeic, p-
coumaroyl quinic and p-coumaric acids and many procyanidins(Ma,2005). The unripe bael
fruit juice has tannins (9%), alkaloids, coumarins, steroids, polysaccharides, carotenoids, 2-
furocoumarinpsoralen, riboflavin and marmelosin (Singh et al., 2009), flavan-3-ols, rutin
and flavones glycosides (Sivraj and Balakrishana, 2011).
Alkaloids:
The bael fruit juice contains a number of alkaloids, among which aegeline, marmin and
marmelosin are the major ones(Johnson, 2010). In unripe bael fruit juice number of
alkaloids like aegeline and marmelline is more(Patel et al., 2012). Bael fruit juice contains
several alkaloids like y-sitosterol, rutacine, aegelemine, marmeline, fragrine, aegeline,
dictamine and cinnamides (Sharma et al., 2011; Malviya et al., 2012).
Polysaccharides:
Galactose, arabinose and L-rahaminose are the major polysaccharides resent in bael fruit
juice (Patel et al., 2012; Malviya et al., 2012). It is rich in reducing sugars, like uronic acid
galactose, arabinose, and L-rhamanose,(Basak et al., [1982] and Laphookhieo [2011]).
Tannins and phenols:
Bael fruit juice provides a number of health benefiting organic aids phenolic compounds and
tannins. In the month of January when the fruit is in unripe stage, the maimum number of
phenolic compounds and tannin are observed.. There is about 9% of phenolic compounds
and tannins in the pulp of wild bael fruit juice (Sharma et al., 2007). Oxalic acid , tartaric,
malic and ascorbic acids, are mainly found in bael fruit juice (Bansal and Bansal, 2011).

1.6. OBJECTIVES

1. Isolation of native probiotic strains and

2. Production of novel probiotic bael juice/drink by incorporating the isolated probiotic
strains.