CHAPTER –I INTRODUCTION 1

CHAPTER –I
INTRODUCTION

1.Introduction
Dairy industry is primary sector involved in development of probiotic products, other food areas are now incorporating nuts, legumes or cereal based milk as vegetable milk (Neus Bernat, 2014). The demand for non-dairy product is increasing due to lactose intolerance, cholesterol content, allergenic milk caseins and desire for vegan alternatives. (Granato et.al.,2010). These are the major drawbacks related to the consumption of dairy products, which makes the development of new non-dairy probiotic essential foods (Heenan et al., 2005; Rivera-Espinoza and Gallardo-Navarro, 2010; Yoon et al., 2004).

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1.1 Functional food
A functional food is a food given an additional function by adding new ingredients or more of existing ingredients. The term may also apply to traits purposely bred into existing edible plants, such as purple or gold potatoes having enriched anthocyanin or carotenoid contents, respectively. Functional foods may be “designed to have physiological benefits and/or reduce the risk of chronic disease beyond basic nutritional functions, and may be similar in appearance to conventional food and consumed as part of a regular diet”. The functional food industry, consisting of food, beverage and supplement sectors, is one of the several areas of the food industry.

Functional beverage
A functional beverage is a drink typically intended to convey a health benefit. Some include ingredients like herbs, vitamins, minerals, nootropics, amino acids, or additional raw fruit or vegetables. Examples of functional beverages include sports and performance drinks, energy drinks, ready to drink (RTD) teas, smart drinks, enhanced fruit drinks, soy beverages, and enhanced water. Utilizing ingredients such as natural food/beverage colours, low-calorie natural sweeteners and kosher ingredients are in vogue. Manufacturers are constantly embracing open innovation to gain cost-leadership and fixed suppliers in early development stage. One of the major advantages of a functional beverage is that consumers are ready to pay a premium price for its various functional advantages. The market is largely fragmented as the categories differ significantly in the functional beverages market. Some of the key players in this industry are PepsiCo, Inc., Nestlé, Kraft Foods, General Mills, Campbell Soup Co., Monster Beverage Corporation, The Coca-Cola Company and Red Bull GmbH among others. A few major ingredient suppliers are Archer Daniels Midland Co., DuPont Nutrition & Health, LycoRed Ltd., Fortitech, Inc., BASF and Nutratech, Inc. Functional biscuits from soy flour and rice bran also been developed by Neha Mishra (2012).

1.2 Probiotics
The term ‘probiotic’ was coiled by Parker (1974) to describe “micro-organisms and substances which contribute to intestinal microbial balance”. Probiotics are live microbes that possess various beneficial effects to the host upon consumption and have been reported to inhibit the growth of pathogens, decreases cholesterol levels, and reduces the risks of cancer (Gomes, Xavier et.al., 1999.). Fermented milk has various advantages over non fermented milk. Fermentation may destroy pathogens, improve product flavour and reduce beany flavour, give textures. (Trindade et al., 2001). Probiotic food have been reported to improved digestibility of starch and protein (Saharan, 1994; Rani and Khetarpaul, 1999), reduced levels of antinutrients (Gupta et al, 1992) and enhanced bioavailability of minerals (Goyal and Khetarpaul, 1994). Besides nutrients, these fermented foods are known to contain different micro-organisms including probiotics, such as L. acidophilus.

1.2.1 History
The history recording the probiotic properties of live microbial food supplements is long. A good probiotic is that which is capable of exerting a beneficial effect on host animals and is non-pathogenic and non-toxic. The probiotics should be present as viable cells in large number and capable of surviving and metabolizing in the gut environment. Also, it should be stable and capable of remaining viable for long periods under storage and field conditions. The nutritional benefits of probiotics have been most extensively investigated with regard to the fermentation of food products with lactobacilli and biofidobacteria. Probiotic foods are nutritionally superior and acceptable products they have improved protein and starch digestibility, reduced levels of antinutrients and enhanced bioavailability of minerals (Khetarpaul, 1988). Lactic acid bacteria have also been shown to increase the content of the vitamin B-complex in fermented foods (Deeth and Tomine, 1981). At the beginning the development of probiotic food started with incorporation of vitamins or minerals such as vitamin C , Iron. Thereafter the development of product fortified with micronutrients such as omega-3-fatty acids, soluble fibre took place (Sloan, 2002). Now a days probiotic food is developing towards dietary supplementation which includes the area of probiotic, prebiotic and nutraceutical’s which improves the gut health.

1.2.2 Microorganisms used as probiotic:
The microbial culture is now been tested for their ability of preventing and curing diseases in humans and animals. Based on such applications the word ‘probiotics’ is used to describe the beneficial micro-organisms by Stillwell ; Lilly (1965). Fuller (1992) defined a probiotic as ‘live microbial feed supplement which beneficially affects the host animal by improving its microbial balance.

Probiotics come in two main forms i.e. foods and dietary supplements. Consumption of probiotics as part of foods such as dairy products has the advantage of having health benefits of foods (minerals, protein, vitamins, bioactive ingredients) increasing compliance (foods are regular part of our day) and improving the chances that the probiotics reach the intestine alive (Lee and Salminen, 1995; Sharma and Ghosh, 2006).Probiotic foods are claimed to have several health-specific advantages. In addition to their basic nutritive value, they contain a proper balance of ingredients which help in the prevention and treatment of illnesses and diseases. The products containing lactic acid bacteria or probiotics are increasingly gaining importance. The recognition of the beneficial effects of dairy products containing probiotics has been well established. The allergy to dairy products, lactose intolerance and cholesterol content are the major drawbacks related to the use of fermented dairy products for a large percentage of consumers. Modern consumers are increasingly interested in their personal health, and expect the food that they eat to be healthy or even capable of preventing illness. Because of this, probiotic food products made by the fermentation of cereals, fruits and vegetables are receiving attention from the scientific world as well as consumers.

1.2.3 Properties of probiotics
It must be viable
It must deliver a health benefits
Protection against certain types of cancer (Sharma & Ghosh, 2002)
It should not limit the mechanism of action but the living of metabolites/products by micro-organisms to the small intestine be consisted as a probiotic activity (Lee and Salminen, 1995; Conway, 1996; Sharma and Ghosh, 2006).

Improved digestibility
Decreasing the incidence of diarrhoea.

Reducing respiratory infections such as sinusitis, bronchitis and pneumonia in children.

Prevention of gastrointestinal disturbances.

Improving immune system, Vitamin production, availability of minerals and trace elements.

Cholesterol lowering effects.

Prevention and treatment of food allergy
Table 1.2.1 Microorganism commonly considered as probiotics
Lactobacillus spp. Bifidumbacterium spp.

Others
L. acidophilus B. adolescentis
Enterococcus faecalis
L.brevis B.animalis
Saccharomyces boulardii
L. casei B. bifidum
Streptococcus cremoris
L. delbrueckii subsp. Bulgaricus B. breve Streptococcus intermedius
L. gasseri B. infantis
Streptococcus salivarius
L. lactis B. lactis
Streptococcus thermophilus
L. paracasei B. longum
L. reuteri L. thermophilum
L. rhamnosus 1.2.4 Lactobacillus acidophilus as probiotic organism
Some of the fermented foods such as milk and whey, containing a micro-organism having probiotic property i.e. L. acidophilus, have been described to have nutritional as well as therapeutic advantages in the management of diarrhoea. Acidophilus yoghurts, acidophilus yeast milk, acidophilin, sweet acidophilus milk and acidophilus powders etc. have been developed and are consumed as more acceptable means of ingesting live L. acidophilus cells. L. acidophilus is an organism which is able to implant in intestine and can help the host by restoration of normal intestinal flora, resulting in prevention and cure of many intestinal disorders (Khedekar et al., 1990; Katelaris, 1996).

1.3 Lactose intolerance & non-dairy milk
Lactose intolerance is not an allergy, because it is not an immune response, but rather a sensitivity to dairy caused by lactase deficiency. Milk allergy, occurring in only 4% of the population, is a separate condition, with distinct symptoms that occur when the presence of milk proteins trigger an immune reaction.

1.3.1 Lactose intolerance
Lactose intolerance is a condition in which people have symptoms due to the decreased ability to digest lactose, a sugar found in milk products. Those affected vary in the amount of lactose they can tolerate before symptoms develop. Symptoms may include abdominal pain, bloating, diarrhoea, gas, and nausea. These typically start between half and two hours after drinking milk or eating milk products. Severity depends on the amount a person eats or drinks. It does not cause damage to the gastrointestinal tract. Though dairy is the most popular matrix to deliver probiotics to the gastrointestinal tract, a trend of non-dairy probiotics is growing due to various issues with dairy probiotics. Dairy substrates may contain higher cholesterol content and potential allergens such as casein.

1.3.2 Non-dairy milk
In this work we have used soy milk, almond milk, Peanut milk and coconut milk as non-dairy milk. Non-dairy probiotic products have the advantage of being lactose- free and can be manufactured to sustain the growth of probiotics. Therefore, it could be used as a base product to develop new non- dairy fermented product with functional features, in which the nutritional and health benefits of soy, almond, and coconut milk.

1.3.2.1 Soy milk
Soybeans are composed of 40% high-quality protein and 20% oil. Soy contains high levels of a number of phytochemicals and is specially noted for the cholesterol-lowering effects of its protein (Liu, 2004). Among the numerous health-promoting compounds of soy, isoflavones play a key role, including the prevention and treatment of cardiovascular disease, cancer, osteoporosis, premenstrual and postmenstrual symptoms, and many more (Messina, 2003; Zubik & Meydani, 2003). Fermentation and heat treatment are methods to increase the digestibility of soy proteins (Liu, 2004; Han, Rombouts, & Nout, 2001). Consumers in western countries consume soymilk mainly as an important replacer of cow milk due to lactose intolerance or allergic reaction to cow?s milk, (Rosenthal et al., 2003; Lui, 1997; Kwok and Niranjan, 1995; Kanthamani et al., 1978).

1.3.2.2 Almond milk
Almonds have been found to be a valuable source of many nutrients. Chen, Lapsley, & Blumberg (2006) found that a 28 gram serving of almonds provides 36.4% of the Daily Value of vitamin E, 36.0% of manganese, 19.5% of magnesium, 16.0% of copper, 13.4% of phosphorus, 13.2% of fibre, 13.5% of riboflavin, and 12.1% of protein (Chen, Lapsley, & Blumberg, 2006). Almonds are naturally high in monounsaturated fatty acid content, which is inversely associated with cardiovascular disease when consumed (Chen, Lapsley, & Blumberg, 2006). Additionally, almond proteins are high in arginine content, making them highly digestible (Ahrens, Venkatachalam, Mistry, Lapsley, &Sathe, 2005).

1.3.2.3 Coconut milk
Coconut milk is another vegetable extract that may be used to replace cow milk in making probiotic drink. It is a popular substitute for cow milk in the tropics because it is simple to prepare, highly digestible and contains and abundance of nutrients (Wangcharoen, 2012). Coconut milk is rich in minerals (calcium, phosphors and potassium), vitamins (C, E and many B vitamins), proteins (rich in glutamic acid, aspartic acid and arginine), lipids and antioxidants (Yuliana, Rangga & Rakhmia, 2010). The fatty acids (high oleic and lauric acid) in coconut milk are instrumental in preventing arteriosclerosis (Belewu & Belewu, 2007).

1.3.2.4 Peanut milk
Peanut milk compares favourably well with cow’s milk and may be used as substitute. Peanut milk is white in colour, can be pasteurized or boiled without any sedimentation. The acceptability of this milk has been found to depend upon the colour, absence of undesirable flavour, taste, mouth feel and likeness (Chandrasekhara et al., 1971). It’s a good source of vitamin E, has at least double the amount of magnesium and vitamin B-6, and is packed with heart-healthy unsaturated fats.

1.4 Garcinia indica Choisy (Kokum) :
Garcinia indica Choisy belonging to the family Guttiferae is an indigenous tree of India (Chandran, 1996; Padhye, Ahmad et.al, 2009). Originally found only in the western coastal regions and the Western Ghats in the states of Maharashtra, Goa, Karnataka and Kerala, India as well as parts of Eastern India in the states of West Bengal, Assam and North Eastern Hill regions, but is today found growing in other parts of peninsular India (CHEMEXCIL, 1992; Chandran, 1996).Garcinia indica known by various names across country such as aamsul, bindin, biran, , bhinda, bhrinda, brinda, kokum, katambi, puanarpuli, kudam puli or ratamba (Anon., 1976). Kokum is a rich source of compounds like HCA (hydroxyl citric acid), garcinol, citric acid, malic acid, anthocyanin pigments and ascorbic acid (Mishra et al., 2006). The proximate composition of kokum rind contains moisture (30%), protein (1.92%), crude fiber (14.28%), pectin (5.71 %) , crude fat (10.0%), hydroxyl citric acid (22.80%) and carbohydrate (36.40%) (Krishanamurthy et al., 1982).

1.4.1 Culinary uses of kokum
Kokum is tropical fruit which ripens during the month of April-May. Kokum is an important culinary agent and is used as an acidulant for curries by people living in Maharashtra, costal Karnataka and Goa, India (Pritam Bafna, 2012). Addition of kokum is supposed to enhance the taste of coconut-based curries and to remove the unpleasant smell of mackerel and sardines (Menezes, 2000, 2002). Traditionally the fruit is sun-dried to preserve rinds after removing the pulp from fresh fruit to develop value added products. The rind and the pulp are often dried and then pulverized into a powder. The dried rind contains anthocyanin pigments which are of natural colourant for acid foods and kokum colourant are used in food applications in the area of food processing products such as juices and jams.

1.4.2 Medicinal uses of kokum
Kokum is used to treat illness related to obesity and multiple studies have shown that hydroxycitric acid (also known as garcinia acid) a component of kokum is reported to possess anti-obesity effects (Arseculeratne, S. N., Gunatilaka, A. A. L., & Panabokke, R. G ,1981). Studies have shown that consumption of hydroxycitric acid reduces appetite, inhibits fat synthesis, lipogenesis, decreases food intake and reduces body weight (Preussa et al., 2004; Jena, Jayaprakasha, Singh, & Sakariah, 2002). It also inhibits synthesis of fatty acid and lipogenesis from various precursors (Jena et al., 2002).

Kokum rind contains three important chemical constituents’ viz, Garcinol, Hydroxycitric acid and anthocyanin pigment. Garcinol is a fat soluble yellow pigment; Hydroxycitric acid is used as an acidulant and physiologically active compound has been shown to significantly reduce body weight. Kokum contains 2 to 3 % of red colour pigment. Anthocyanins of kokum are water soluble and possess antioxidant activity. Two major pigments characterized in kokum are cyanidin-3-glucoside and cyanidin-3-sambubioside which are usually present in the ratio of 4:1 (Nayak et al., 2010).

Hydroxycitric acid, both free acid and lactone forms, is present in fruit rind of Garcinia indica, which are commercially available in India. As an inhibitor of the synthesis of fat and cholesterol, hydroxycitric acid has been shown to significantly reduce the body weight and lower lipid accumulation. Extract containing HCA has proven its strength to reduce fat synthesis in the body from 40 to 70%. Garcinia fruit lowers blood lipids such as cholesterol and triglycerides by triggering fatty acid oxidation in the liver via thermogenesis (raising body temperature to speed up the body’s metabolism which increases burning of fats). It burns the fat slowly and gently without stimulating the central nervous system (Raju, 2001).

1.5 Indian traditional drink
With a climate as varied and extreme as India, the people require a myriad of options to keep their thirst appropriately quenched according to the weather conditions, varying from steaming hot beverages during winters to frosty cold drinks in summers. Different regions in the country serve drinks made with an eclectic assortment of ingredients including local spices, flavors and herbs. Available on the streets, as well as on the menus of posh hotels, these drinks add to the flavorful cuisine of India. These are some traditional indian drinks-Aam-panna, aamras,jal-jeera, kokum sarbat, thandai, solkadhi, rasam, neera, kala-khatta, kanji, liyo, kesar-kasturi etc.

1.6 Solkadhi
Kokum solkadhi is a refreshing appetizer drink originated in Konkan region, Maharashtra. Traditionally, kokum are soaking in water for overnight. After soaking, kokum extract is collected by squeezing. Coarse crush of coriander, cumin, green chilly, garlic and salt is added. All the ingredients are mixed with fresh coconut milk, kokum extract, crushed spices with salt and mix well (Shrikant Swami, 2014).

1.6.1 Solkadhi as funcctional beverage
Garcinia indica, or Kokum, owing to the presence of high concentration of anthocyanins, is a great antioxidant that works like anti carcinogenic and anti-ageing agent. It is known for its digestive, refreshing, cleansing and cooling properties. As the kokum is having cooling effect, people in coastal region enjoy drinking this delicious, refreshing kokum solkadhi especially during hot summers as it has a cooling effect on body.

1.6.1.1 Appetizer
Solkadhi can improve digestion too. It relieves all kinds of digestive problems and cleanses your system entirely. Solkadhi helps in regulating absorption of food; prevent your body from heartburn, indigestion, gas, cramps, constipation, diarrhea and abdominal pain. It also allows perfect bowel movement over time.

1.6.1.2 Antioxidative property
Solkadhi is rich in antioxidants that impart several health benefits. It protects your heart from diseases, heart attacks and lowers the level of cholesterol. It improves the immune system, protects your DNA and lowers the chances of developing cancer. It protects your body from diabetes and defends against dementia too.

1.6.1.3 Other properties
Solkadhi can also prove to be great for your eyes because of the high level of antioxidants contained in it. Solkadhi nourishes and detoxifies your body effectively. It prevents cold, flu, migraines, nausea, constipation and other health conditions such as diarrhea. It also helps you shed unwanted weight and ensures your metabolism is accelerated. Solkadhi is great for your skin. Consuming it will not only improve the texture and quality of your skin but also fights rashes. Drinking solkadhi will help in reducing the appearance of rash and its symptoms over time (Shrikant & Thakor, 2014).

1.7 Objectives
Using L. acidophilus, fermented drinks based on milk or whey have been prepared but no such work has been conducted till today on the development of non-dairy probiotic drink based on solkadhi or Garcinia indica flavoured drink after fermentation with L. acidophilus. The effect of fermentation with probiotic micro-organism i.e. L. acidophilus can have added advantages. They may not only improve the nutritional quality of products but also add therapeutic benefits. Further, addition of non-dairy milk with spice and kokum extract will add more nutrients, flavour and it will also suitable for people with lactose intolerance. With this perspective, the present study was planned with the following objectives:
To develop non-dairy probiotic solkadhi using soy, almond, peanut and coconut milk and
Evaluation of its physical, organoleptic and functional analysis
CHAPTER –II
LITERATURE REVIEW
Probiotics are preparation of live micro-organisms which beneficially affect the host by improving the properties of the indigenous microbes. Probiotics are used to improve intestinal health and stimulate the immune system. The microbes commonly used as probiotics for humans are the lactic acid bacteria. Consuming fermented products containing these organisms allows the continuous passage of these organisms through the gut (Modler et al., 1990).

The functional food is an expanding sector of the food industry. A major development in functional food pertains to foods containing probiotics and prebiotics that enhance health promoting microbial flora in the intestine. A healthy gut microflora may provide protection against gastrointestinal disorders (Sharma and Ghosh, 2006). Probiotic fermentation can also play an excellent role in the production of wholesome foods ranging from fermented fresh vegetables such as cabbage (Sauerkraut) and cucumber (pickles), fermented cereal buttermilk mixture (rabadi), fermented cereal-legume mixture (dhokla, idli, wadi), fermented cereal yoghurt (Nigerian ogi/Kenyan uji), sour dough bread (puto), fermented milks (dahi/yoghurt/cheese) and fermented meats (European salami). These products are either fermented naturally or with the help of pure cultures of micro-organisms (Soni and Sandhu, 1990; Binita, 1995; Chahal, 1999).

Foods fermented with Lactobacilli have been found to be more nutritious than their unfermented counterparts. This improvement can come about in at least three different ways (Potter and Hotchkiss, 1996). Micro-organisms not only are catabolic, breaking down more complex compounds, but they also are anabolic and synthesize several B-complex vitamins and other growth factors. The second important way in which fermented foods can be improved nutritionally has to do with the liberation of nutrients locked into plant structures and cells by indigestible material. A third mechanism by which fermentation can enhance nutritional value, especially of plant materials, involves enzymatic splitting of cellulose, hemicellulose and related polymers that are not digestible by humans into simpler sugars and sugar derivatives. Other workers also reported that the fermentation with probiotics had been improved the starch and protein digestibility (Antony and Chandra,, 1998; Binita and Khetarpaul, 1999). Improvement in starch digestibility during fermentation can be related to enzymatic properties of fermenting microflora that brings about the breakdown of starch to oligosaccharides. The presence of ?-amylase in the fermenting microflora was indicated by Bernfeld (1962). These enzymes bring about the cleavage of amylase and amylo-pectin to maltose and glucose. Reduction in amylase inhibitor activity may also be responsible for increased starch digestibility (Sharma and Kapoor, 1996).

Similarly, improved protein digestibility is mainly associated with proteolytic activity of fermenting microflora. This increase in digestibility may also be due to reduced antinutrient content of the fermented food as antinutrients are known to inhibit amylolysis and proteolytic activity. Reduction in antinutrients may also explain the increased bio-availability of various minerals as indicated by a number of studies (Jood and Khetarpaul, 2005). In plant foods, minerals like Ca, Fe and Zn are bound with phytic acid and a protein-phytate mineral complex is formed (Prattley et al., 1982). Such a complex inhibits the availability of minerals and therefore, extractability of minerals is low in raw foods. Decrease in level of phytic acid after fermentation, which is possibly through hydrolysis by inherent phytate in fermenting microflora may release these metallic ions in free form and therefore, may account for increased bioavailability of these in fermented products.

Kokum solkadhi is a traditional coastal region drink in India, having numerous health benefits on body. Shrikant swami, 2014 have worked or preparation of solkadhi mixture powder to increase its shelf life and to preserve its functional properties. Till date no author have done study on ‘probiotic solkadhi’ . Hence the objective of the work is to design solkadhi with different non-dairy milk and its probiotic product.

CHAPTER –III
MATERIALS AND
METHODS

3. MATERIAL AND METHODS
The present study was carried out at D.Y Patil School of biotechnology and Bioinformatics, CBD Belapur, Navi Mumbai to study the formulation, development, utilization and nutritional evaluation of probiotic drink. This chapter contains relevant information pertaining to the research design and methodological steps used for the present investigation.

3.1 Procurement of raw materials
Kokum rinds, Garlic, Cumin, Green chilly, Salt, Soybean, Coconut, Peanut and Almond were purchased from the APMC market Vashi, Navi mumbai in a single lot. The lyophilized culture of probiotic microorganism Lactobacillus acidophilus was purchased from Jevan Chemicals and Pharmaceuticals, Turbhe, Navi Mumbai. Lactobacillus MRS broth was purchased from Hi-media, Mumbai.

3.2 MICROBIAL STOCK CULTURE
3.2.1 Isolation of Probiotic Organism
Around 0.5g powder of lyophilized powder of Lactobacillus acidophilus was transferred to sterile saline under aseptic condition to make a suspension. The inoculum from saline was inoculated on sterile MRS agar plates and was incubated at 37 ? for 24hr in an incubator. After 24 hrs inoculation well isolated colonies were obtained. The culture was confirmed before by use of Gram’s staining and catalase test and motility test.

3.2.2 Preparation of stock culture
Stock culture of the L. acidophilus was maintained on MRS agar by stab inoculation. After incubating at 370C for 24 h, the stock culture tubes were stored in a refrigerator for further use. For maintenance, the cultures were sub-cultured regularly at 2 to 4 weeks’ intervals. The culture was further used as inoculum.

3.2.3 Identification of bacteria
Identification of bacteria is a crucial step in probiotic product development to insure food safety.

3.2.3.1 Gram staining – Methods (Hucker’s Modification)
Procedure – Preparation of smear
Bacterial suspensions in broth: With a sterile cooled loop, place a loopful of the broth culture on the slide. Spread by means of circular motion of the inoculating loop to about one centimetre in diameter. Excessive spreading may result in disruption of cellular arrangement. A satisfactory smear will allow examination of the typical cellular arrangement and isolated cells.

Place slide with heat fixed smear on staining tray.

Gently flood smear with crystal violet and let stand for 1 minute.

Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

Gently flood the smear with Gram?s iodine and let stand for 1 minute.

Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The smear will appear as a purple circle on the slide.

Decolorize using 95% ethyl alcohol. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize.

Immediately rinse with water.

Gently flood with safranin to counter-stain and let stand for 45 seconds.

Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

Blots dry the slide with bibulous paper.

View the smear using a light-microscope under oil-immersion.

3.2.2.2 Catalase test
1. 2 drops of H2O2 solution were dropped on test slide.

2. The effervescences for the oxygen were observed.

3.2.2.3 Motility test- Wet mount microscopic method by (ASHABIL AYGAN, 2007)
Wet Mount Preparation:
Take a clean, scratch free glass slide.

Label the slide with the name of the organism
Place 15 – 20 uL of the culture in the middle of the slide
Lower a clean cover slip over the drop as though it were hinged at one side avoiding bubbles.

Examine the preparation under microscope first under 4 x followed by 40 x and 100x magnification.

First focus with the 4x objective on the edge of the coverslip.  It is easier to find and focus on than the nearly transparent suspension.

Find a bubble in the liquid suspension, and adjust the fine focus on the edge of the bubble.

Switch to the 10x objective, repeat the careful focusing.

Switch to the 40x objective, repeat the careful focusing.

Apply oil and examine with the 100x oil immersion lens, again using the edge of the bubble as a focusing point.

Identify the motile organisms.

Clean up the slide with alcohol first (because it had live bacteria on it), followed by soap and water.

Discard the cover slip.

3.2.3 Preparation of inoculum
A single colony of Lactobacillus acidophilus was transferred to 100 ml of sterile MRS broth under aseptic condition by nicrome wire loop. The inoculated sterilized MRS agar broth was incubated at 37°C for 48 hours in an incubator. Further cells were washed with sterile saline by centrifugation at 8000 rpm for 10 minutes at 4°C until the colourless pellet will get. Then adjust the optical density (OD) by UV spectrophotometer at absorbance 660 nm to get 104 cells/ml of saline. Washed cells were used within 12 hrs.

100 ml sterile MRS broth
?
Autoclave at 1210 C, 1.5 kg/cm2for 15 min
?
Cooled (40°C)
?
Inoculate L. acidophilus
?
Incubate at 370 C for 24 hrs.

?
Centrifuge MRS Broth at 8000 rpm for 10 min
?
Discard supernatant and wash the pellet with equal amount of saline water until colourless pellet will get.

?
Adjust the OD at absorbance 660 nm
Fig. 3.1 preparation of inoculum
For cell count 106 cells ml optical density should be in between ~ 0.1- 0.2 at absorbance 660 nm. For inoculation the require cell count is 10-4 cells/ml therefore by formula,
N1 V1= N2 V2
106 cells/ml × X = 104 cells/ml × 10
X = 104 cells/ml × 10
106 cells/ml
X = 0.1 ml
Therefore, 0.1 ml of 106 cells/ml in 9.9 ml of saline = 104 cells/ml
3.3 Development of probiotic solkadhi
3.3.1 Preparation of non-milk
200 g of soybean,250 g of peanut, 250 g of almond and 250 g of coconut were soaked overnight in water. Seed cover of peanut, soybean and almond were removed manually then grind soaked seeds with the help of electrical grinder and strained thought double layered cheese cloth. A seed to water ration is 1:6 (w/v) for soybean and peanut, 1:4.2 (w/v) for almond and coconut. Freshly prepared milk was used for preparation of probiotic drink.

(a)Fresh coconut slices(b) 12 Hr soaked soybean
(c)12 hr soaked almonds(d)12 hr soaked peanuts
282102342161000right6978650028294066484500right5517245Fig.3.4 (D)
00Fig.3.4 (D)
right3441781005308605627370Fig. 3. 3 (C)
00Fig. 3. 3 (C)
right2681605Fig. 3.2 (B)
00Fig. 3.2 (B)
4508502673350Fig. 3.1 (A)
00Fig. 3.1 (A)

3.3.2 Preparation of spice mixture
For preparation of spice mixture different concentrations of spices were took in 100 ml of non-dairy milk to evaluate taste and texture of drink
Table 3.3.2.1 Concentration of spice g/100 ml of non-dairy milk
Sr. No. Cumin Seeds Garlic Green chilly Salt
1 2 2 1 2
2 2 1 2 2
3 2.5 1.25 1.25 2
4 2.5 1.25 1 2
5 2.5 2 1 2
Among all the concentrations of, ratio of cumin: garlic: green chilly ie. 2.5 :1.25:1.25 (w/w/w) was good in sensory therefore, this ratio was selected for further analysis using kokum extract of selected ratio ie. 0 ml, 2 ml, 4ml, 6ml and 8ml per 100 ml of soy, peanut, almond and coconut milk.

3.3.3 Preparation of kokum extract
The dried kokum rinds were soaked overnight in water at room temperature in 1:2 (w/v) ratio. After overnight soaking the solution is boiled for 5 mins and strained through double layer muslin cloth. This extract was used for preparation of probiotic solkadhi drink.

3.3.4 Preparation of probiotic drink
The prepared milk was boiled for 1 min at 100? and cooled upto 40?. The developed spice mixture was mixed with almond milk (100ml), soymilk (100ml), peanut milk (100ml) and coconut milk (100ml) with different concentrations of kokum (Garcinia indica choisy) extract. Prepared inoculum (3.2.4) at a level of 1ml/100ml was added to the liquid portion containing the formulation mixture. Hold on 5 min followed by incubation at 37? for 6 hrs. All the samples were prepared in triplicate in a batch of 300 ml.

Table 3.3.4.1 Preparation of probiotic solkadhi drink
Sr. No. Concentration of kokum extract (ml/100ml) Milk
(ml) Inoculum
(ml/ml)
1 0 100 1
2 2 100 1
3 4 100 1
4 6 100 1
5 8 100 1
Methods
All analysis were carried out after fermentation of 6 hrs.

3.4 pH and Titrable acidity
The pH was measured by a pH meter – Accumax chemical labouratory, India. against a standard buffer of 4.0 pH.

Titratable acidity was determined as lactic acid per 100 ml by the method of Amerine et al. (1967).

1. Pipette out 5 ml fresh sample in 250 ml conical flask.

2. Add about 75 ml boiling water and 5 drops of phenolphthalein indicator.

3. Read the level of 0.1 N NaOH in the burette.

4. Added NaOH slowly to the sample until finally one drop gave a pink colour lasting for a minute or longer.

5. The amount of alkali in ml used times 0.150 equalled the total acidity expressed as g lactic acid per 100 ml.

Titrable acidity as lactic acid (g/100ml)=(V)×(N)×75×100
(V’)×1000
Where,
V = ml of NaoH used for titration
N = Normality of NaoH ie.0.1N
V’= Volume of sample
3.5 ENUMERATION OF CELLS COUNT IN PROBIOTIC DRINK
0.5 ml of probiotic drink was added to 4.5 ml sterile saline solution with a sterile one ml delivering glass pipette. Further dilutions upto 10-10 were made using a fresh pipette for each. 0.1 ml of each dilution was poured plated on sterile MRS agar plates. Spread it by using glass spreader in circular movements in different directions for 10 seconds. These petri plates were incubated at 370 C for 24 hrs. The colonies were counted and multiplied by the dilution factor to obtain the viable count per ml in probiotic drink.

0.5 ml of probiotic drink were added in 4.5 ml of sterile saline solution
?
0.5 ml of 10-1 diluted NSS to 4.5 ml NSS (10-2) and so on till 10-10 dilution was achieved
?
10-1 10-2 10-3 10-4 …………………………………1-10
?0.1 ml ?0.1 ml ?0.1 ml ?0.1 ml ?0.1 ml
Spread plating was done
?
Incubated (370C, 24 h)
?
Colonies were counted on each plate
Fig. 3.1: Flow diagram showing the method used for enumeration of bacteria
Calculation:
No. of colonies counted on plate x Dilution factor = No. of bacteria per ml
3.6 LEVEL OF ANTIOXIDANT PRESENT IN THE PROBIOTIC DRINK BY ‘DPPH ASSAY’
1, 1-Diphenyl-2-picryl-hydrazyl (DPPH) is a free radical but stable. DPPH solution is initially violet in colour which fades when antioxidants donate hydrogen. The change in colour is monitored by spectrophotometer. Antioxidant capacity based on DPPH radical for extracts of products was analysed following the method given by (Molyneux, 2004) with some modification.

1. Working solation was prepared freshly prior to analysis by taking 62.13 ?M of DPPH in 50 ml amber colored reagent bottle and dissolving in 50 ml methanol.

2. Different concentrations of ascorbic acid were prepare (0, 3, 6, 9, 12, 15, 18) by diluting 1500 ?M stock of ascorbic acid solution.

3. All samples of probiotic drink were diluted in a ratio of 1:100 (1 ?l + 99 ?l of D/W = 100 ?l)
4. 100 ?l of sample and 900 ?l of DPPH were added.

5. Measurements in the measuring cuvette were performed 30 min after addition of DPPH in order to give enough time for the reaction of the cellular antioxidants with DPPH.

6. For blank determination 1000 ?l methanol was measured immediately against methanol. Absorbance of the measuring cuvette was measured at 517 nm.

7. The assay was performed in triplicate. The results of scavenging activity were expressed as
%DPPH scavenging activity = (A515 nm blank -A515nm sample) x 100
515nm blank
3.7 ESTIMATION OF PHENOLIC CONTENT PRESENT IN THE PROBIOTIC DRINK
Determination of polyphenols was determined using Folin–Ciocalteu reagent (Singleton and Rossi, 1965).

1. All samples of probiotic drink were diluted in a ratio of 1:100 (1 ?l + 99 ?l of D/W = 100 ?l)
2. Take 100 ?l of sample to this add 200 ?l of 10 % F. C. reagent and leave for 1 minute.

3. To this, 800 ?l of 0.7 mM sodium carbonate (Na2CO3) were added and incubated for 30 minutes.

4. The absorbance of the solution was measured at 765 nm. Gallic acid was used as a standard. Each sample was analyzed trice with triplicates.

5. Results were expressed as milligram Gallic acid equivalent per 100 g dry weight.

3.8 ORGANOLEPTIC EVALUATION OF PROBIOTIC DRINK
Sensory characteristics of probiotic drink samples were evaluated three times different by a group of nine panelists (Vos, 2010). The judges were drawn from the Department of Food Science and Technology, Dr. D. Y. Patil University, CBD Belapur, Navi Mumbai. 100 ml of probiotic drink sample in the glass containers, duly coated with three digit code was given to the panelist for evaluating the sensory attributes like appearance, body, odor, flavor and taste and over all acceptability of the probiotic drink samples using 9-Point Hedonic Scale. Hedonic Scale was in following sequence; like
9. Like Extremely
8. Like very much
7. Like Moderately
6. Like Slightly
5. Neither like nor dis like
4. Dislike slightly
3. Dislike Moderately
2. Dislike very much
1. Dislike Extremely
CHAPTER –IV
RESULT AND
DISCUSSION

4. Result
In the present study, efforts were made to develop probiotic drink using various processing treatments like autoclaving, extraction and probiotic fermentation. The results pertaining to this study have been presented and discussed under the following heads and subheads:
4.1 ISOLATION OF PROBIOTIC ORGANISAM
Probiotic bacteria were isolated by streak plate method as shown in following figure.

center1524000

Fig. 4.1.1 Pure culture of L. acidophilus by streak plate method
4.2 IDENTIFICATION OF BACTERIA
Isolated bacteria was identified by Gram’s staining and Catalase test
4.2.1 Gram’s staining – Methods (Hucker’s Modification)
Gram?s staining was done and stained bacteria were observed under microscope (Fig. 4.2.1.1)
center3335100
Fig. 4.2.1 Microscopic Observation of L. acidophilus
4.2.2 Catalase test
Catalase test was done (Fig. 4.2.2.1). As a result of catalase test the bacterial was catalase Positive because after drops adding of H2O2 bubbles were observed on the glass slide. Catalase is an enzyme that splits up hydrogen peroxide into oxygen and water. Chemically catalase is a hemoprotein, similar in structure to hemoglobin. Catalase is present, often in high concentrations in the majority of aerobic organisms but is absent from most obligate anaerobes. Thus when H2O2 is added externally in the medium, catalase activity results in the production of molecular gaseous oxygen.

10217151016000
Fig. 4.2.2.1 Catalase test
4.2.3 Motility test
Motility test was done using wet mount method (Fig.4.2.3). The organism didn’t shown any kind of movement or vibration; hence test organism is ‘non-motile’. Motility is interpreted using high dry magnification to locate the bacteria within the drop of water. If they swim randomly and “against the current” of water streaming across the slide surface, they are positive for motility. If they seem to be buffeted around, all moving the same direction and at the same speed, there is no motility.

156972022098000
Fig.4.2.3.1 Motility test
4.3 Preparation of Kokum extract
The kokum extract was prepared and used for development of non-dairy probiotic solkadhi.

1678305444500
Fig. 4.3.1 Kokum extract
4.4 Titrable acidity
right75438000As shown in table acidity increases with increasing concentrations of kokum extract 0, 2, 4, 6 and 8 after 6 h of incubation. In figure 4.4.1 Probiotic drink containing highest amount of kokum extract have highest percentage acidity.

169862532575542418002997200019989805943600469455560325000Fig. 4.4.1 Effect of level of different concentration of kokum extract in Coconut milk probiotic drink, Almond probiotic drink, Peanut milk probiotic drink and Soy milk probiotic drink on titrable acidity
Table 4.4.1 Effect of level of different concentration of kokum extract in soymilk, Coconut milk, Almond milk and Peanut milk on acidity
Samples Concentration Acidity
Coconut milk probiotic solkadhi 0 0.20 ± 0.007
2 0.42 ± 0.02
4 0.59 ± 0.01
6 0.83 ± 0.02
8 1.21 ± 0.01
Almond milk probiotic solkadhi 0 1.62 ± 0.03
2 2.16 ± 0.08
4 2.42 ± 0.03
6 2.87 ± 0.03
8 3.36 ± 0.04
Peanut milk probiotic solkadhi 0 0.55 ± 0.07
2 0.67 ± 0.03
4 1.22 ± 0.03
6 1.41 ± 0.01
8 1.69 ± 0.007
Soy milk probiotic solkadhi 0 1.05 ± 0.003
2 1.31 ± 0.02
4 1.89 ± 0.007
6 2.41 ± 0.02
8 2.69 ± 0.01
In table 4.4.1 Titrable acidity of coconut milk probiotic drink increases from 0.20 ± 0.007 to 1.21 ± 0.01, for soymilk probiotic drink it increases from 1.05 ± 0.03 to 2.69 ± 0.01, for almond milk probiotic drink it increases from 1.62 ± 0.03 to 3.36 ± 0.04 and in peanut milk probiotic drink it increases from 0.55 ± 0.007 to 1.69 ± 0.007
4.5 pH
165107410450As shown in figure 4.5.1 pH of control probiotic drink is high as and pH of probiotic drink containing highest amount of kokum extract is low as compare to other probiotic drinks due to acidic nature of kokum.

444690561277500188468058420004229735299720001508125316230Fig. 4.5.1 Effect of level of different concentration of kokum extract in Coconut milk probiotic drink, Almond probiotic drink, Peanut milk probiotic drink and Soy milk probiotic drink on pH
In table 4.5.1 pH is decreasing as increasing the concentration of kokum extract. pH of probiotic drink was slightly changed after fermentation of 6 h as shown in table 4.5.1 The reduction in pH may be due to natural acidity of kokum extract. Acidity and pH found dependent on concentration of kokum extract per 100 ml of milk.

4.5.1 Effect of level of different concentration of kokum extract in soymilk, Coconut milk, Almond milk and Peanut milk on pH
Samples Concentration pH
Coconut milk probiotic solkadhi 0 5.23 ± 0.007
2 4.66 ±0.01
4 4.28 ±0.07
6 3.26± 0.01
8 3.06 ±0.01
Almond milk probiotic solkadhi 0 5.25 ± 0.007
2 4.93 ±0.007
4 4.33 ± 0.007
6 3.88 ± 0.007
8 3.62 ± 0.03
Peanut milk probiotic solkadhi 0 5.33 ± 0.007
2 4.86 ± 0.03
4 4.25 ± 0.01
6 3.74 ± 0.007
8 3.55 ± 0.01
Soy milk probiotic solkadhi 0 5.34 ± 0.01
2 4.74 ± 0.01
4 4.17 ± 0.007
6 3.76 ± 0.01
8 3.55 ± 0.007
4.6 BACTERIAL COUNT OF PROBIOTIC DRINKS
Bacterial count was done by using spread plate method in triplicates. In table 4.6.1 The probiotic count of peanut probiotic drink is decreasing from 13×108 to 4×105 cfu/ml. In soybean milk probiotic drink bacterial count is decreasing from 12×109 to 2×105 cfu/ml and in almond milk probiotic drink bacterial count is decreasing from 14×108 to 2×106 cfu/ml. As compared to all probiotic drinks coconut milk probiotic drink has more probiotic count i.e. 12×108 to 3×106 cfu/ml and soy milk has less probiotic bacteria as compared to coconut milk probiotic drink. As concentration of kokum extract increases bacterial count is decreasing. As showed in following figure 4.6.1
Table 4.6.1 Effect of level of different concentration of kokum extract in soymilk, Coconut milk, Almond milk and Peanut milk on probiotic count
Sample Conc. Count
Cfu/ml Sample Conc. Count
Cfu/ml
Coconut milk probiotic solkadhi 0 12×108 Peanut milk probiotic solkadhi 0 13×108
2 9×108 2 10×108
4 7×107 4 8×107
6 5×107 6 6×106
8 3×106 8 4×105
Almond milk probiotic solkadhi 0 14×108 Soy milk probiotic solkadhi 0 12×109
2 11×108 2 8×108
4 8×107 4 7×108
6 5×106 6 5×106
8 2×106 8 2×105
right10351
101282531623036487103473450012179306032500370395560261500Fig. 4.6.1 Effect of level of different concentration of kokum extract in Coconut milk probiotic drink, Almond probiotic drink, Peanut milk probiotic drink and Soy milk probiotic drink on probiotic count
4.7 DETERMINATION OF ANTIOXIDANT LEVELES IN PROBIOTIC DRINKS
Total antioxidative potential of all 20 probiotic drinks were by two method DPPH assay and Total phenolic content present in probiotic drink. Any substance that when present at lower concentration compared with those of the oxidizable substrate significantly delay or inhibit the oxidation of that substrate (H. Subrota, 2013). Table 4.7.1 is observation table for standard i.e. ascorbic acid, its optical density at 515 nm and percentage scavenging activity.

Table 4.7.1 Observation table of ascorbic acid as standard
Concentration of Ascorbic acid (mg/ml) O.D. at 517 nm Percent scavenging activity
0 0.985 ± 0.005 0
3 0.887 ± 0.008 9.94 ± 0.3 %
6 0.806 ± 0.006 18.11 ± 1.11 %
9 0.750 ± 0.008 23.85 ± 0.42 %
12 0.608 ± 0.001 38.57 ± 0.06 %
15 0.560 ± 0.007 43.145 ± 0.38 %
18 0.512 ± 0.007 48.06 ± 0.037 %
IC50 is a free stable radical quenching method to determine IC50 concentration to decrease concentration of the test free radical by 50%.

From the standard graph IC50 was calculated as fallows
From Graph, solved equation
y= mx+c
y= -0.0271x + 0.9733
y= Highest OD point = 0.985 = 0.492
2 2
y = mx+c
0.492=0.0271x + 0.9733
x= 17.76
Therefore, IC50 = 17.76 mM/ml
IC50 of Ascorbic acid is 17.76 mM/ml
right23295000
Fig. 4.7.1 Absorbance Vs concentration of Ascorbic acid
In table 4.7.2 the optical density of the probiotic drink decreases as the concentration of kokum extract decreases.

Table 4.7.2 Effect of level of different concentrations of kokum extract in Peanut milk, soymilk, almond milk and coconut milk probiotic drink on antioxidants
Sample Conc. O.D. at 517 nm P.S.A A.A.E
Coconut milk probiotic solkadhi 0 0.956 ± 0.002 1.94 ± 0.5 % 0.73 ± 0.02 A.A.E.

2 0.952 ± 0.003 3.10 ± 0.08 % 0.73 ± 0.01 A.A.E.

4 0.946 ± 0.002 3.70 ± 0.06 % 1.35 ± 0.21 A.A.E.

6 0.931 ± 0.007 5.23 ± 0.20 % 1.9 ± 0.14 A.A.E.

8 0.923 ± 0.002 9.10 ± 0.04 % 1.9 ± 0.14 A.A.E.

Almond milk probiotic solkadhi 0 0.956 ± 0.001 1.42 ± 0.14 % 0.77 ± 0.03 A.A.E.

2 0.949 ± 0.007 2.58 ± 0.62 % 1.65 ± 0.2 A.A.E.

4 0.936 ± 0.004 5.35 ± 0.43 % 1.9 ± 0.14 A.A.E.

6 0.914 ± 0.001 7.20 ± 0.38 % 2.1 ± 0.14 A.A.E.

8 0.902 ± 0.007 8.37 ± 0.45 % 2.9 ± 0.14 A.A.E.

Peanut milk probiotic solkadhi 0 0.954 ± 0.004 0.70 ± 0.13 % 0.82 ± 0.10 A.A.E.

2 0.949 ± 0.001 0.60 ± 0.71 % 1.65 ± 0.21 A.A.E.

4 0.944 ± 0.007 1.06 ± 0.63 % 1.55 ± 0.07 A.A.E.

6 0.937 ± 0.007 1.77 ± 0.49 % 1.65 ± 0.21 A.A.E.

8 0.934 ± 0.002 2.12 ± 0.85 % 1.7 ± 0.14 A.A.E.

Soy milk probiotic solkadhi 0 0.957 ± 0.007 0.75 ± 0.50 % 0.9 ± 0.14 A.A.E.

2 0.952 ± 0.001 1.31 ± 0.42 % 0.87 ± 0.17 A.A.E.

4 0.946 ± 0.001 1.92 ± 0.41 % 1.55 ± 0.07 A.A.E.

6 0.943 ± 0.002 3.19 ± 0.33 % 1.4 ± 0.14 A.A.E.

8 0.937 ± 0.007 5.32 ± 0.47 % 1.9 ± 0.14 A.A.E.

(OD- Optical density, PSA- Present scavenging activity, AAE- Ascorbic Acid Equivalence)
The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was increased by increasing the concentration of the kokum extract.

In table 4.7.2 percent scavenging activity increases from 1.94% ± 0.5 to 9.10% ± 0.04 for Coconut milk solkadhi, 0.75% ± 0.50 to 5.32% ± 0.47 for Soymilk probiotic solkadhi, 1.42% ± 0.14 to 8.37% ± 0.45 for Almond milk probiotic solkadhi and 0.70% ± 0.13 to 2.12% ± 0.85 for peanut milk probiotic solkadhi. Ascorbic acid equivalence is increased as the concentration of kokum extract were increased in the milk per 100 ml. scavenging activity of Almond milk is high and in soymilk it is low as compare to other probiotic solkadhi drinks.

In the above table Ascorbic acid equivalence increased from 0.73 ± 0.02 A.A.E. to 1.9 ± 0.14 A.A.E. for Coconut milk probiotic solkadhi, 00.9 ± 0.14 A.A.E. to1.9 ± 0.14 A.A.E. for soymilk probiotic solkadhi, 0.77 ± 0.03 A.A.E. to 2.9 ± 0.14 A.A.E. for almond milk probiotic solkadhi which is high as compare to other probiotic drinks and 0.82 ± 0.10 A.A.E. to 1.7 ± 0.14 A.A.E. for peanut milk probiotic solkadhi respectively. Ascorbic acid equivalence of probiotic drink was calculated from graph by using the straight line equation.

4.8 DETERMINATION OF PHENOLIC CONTENT IN PROBIOTIC DRINK
The amount of polyphenols and Gallic acid equivalence in probiotic drink increases as concentration of kokum extract increases.

Table 4.8.1 Observation table of Gallic acid as standard
Concentration of Galic acid (mg/ml) O.D. at 765 nm
20 0.276 ± 0.001
40 0.384 ± 0.001
60 0.504 ± 0.007
80 0.658 ± 0.002
100 0.677 ± 0.001
center14584700
Fig. 4.8.1 Absorbance Vs Concentration of ascorbic acid
Table 4.8.2 Effect of level of different concentrations of Kokum extract in Peanut milk, soymilk, almond milk and coconut milk probiotic drink on total phenolic content
Sample Concentration O.D. at 765 nm G.A.E.

Coconut milk probiotic solkadhi 0 0.283 ± 0.007 22.85 ± 0.21 G.A.E
2 0.286 ± 0.001 23.25 ± 0.35 G.A.E
4 0.287 ± 0.007 23.35 ± 0.21 G.A.E
6 0.289 ± 0.001 23.9 ± 0.14 G.A.E
8 0.292 ± 0.003 26.1 ± 0.14 G.A.E
Almond milk probiotic solkadhi 0 0.282 ± 0.003 22.9 ± 0.07 G.A.E
2 0.285 ± 0.001 23.65 ± 0.21 G.A.E
4 0.286 ± 0.001 23.75 ± 0.35 G.A.E
6 0.287 ± 0.003 23.95 ± 0.07 G.A.E
8 0.290 ± 0.007 26.1 ± 0.14 G.A.E
Peanut milk probiotic solkadhi 0 0.285 ± 0.001 23.35 ± 0.21 G.A.E
2 0.287 ± 0.003 23.65 ± 0.21 G.A.E
4 0.288 ± 0.007 24.1 ± 0.14 G.A.E
6 0.290 ± 0.003 25.9 ± 0.14 G.A.E
8 0.292 ± 0.001 26.1 ± 0.14 G.A.E
Soy milk probiotic solkadhi 0 0.285 ± 0.007 23.35 ± 0.21 G.A.E
2 0.287 ± 0.002 23.95 ± 0.07 G.A.E
4 0.290 ± 0.001 26.1 ± 0.14 G.A.E
6 0.293 ± 0.003 26.3 ± 0.14 G.A.E
8 0.295 ± 0.002 26.65 ± 0.21 G.A.E
(OD- Optical density, G.A.E. – Gallic Acid Equivalence)
4.9 ORGANOLEPTIC EVALUATION OF PROBIOTIC SOLKADHI
organoleptic evaluation of final probiotic drink was done on the measures of organoleptic evaluation using nine point hedonic scale.

Mean scores for probiotic solkadhi containing 6% kokum extract was the highest scores of appearance, consistency, flavour, texture and overall acceptability in each probiotic drink. Though there is variation in scores in all probiotic drinks. It is as shown in table 4.9.1 and in figures.

center0
23801825839970047144153418190255029531877000468116652975Fig. 4.9.1 Effect of level of different concentration of kokum extract in Coconut milk probiotic drink, Almond probiotic drink, Peanut milk probiotic drink and Soy milk probiotic drink on appearance
In above figure 4.9.1 Appearance of coconut milk probiotic drink containing 6% of kokum extract was good i.e. 8.55 ± 0.11 as compare to other probiotic drinks.

-486-1702
17570045740670043932003308760237925032809200492765452489Fig. 4.9.2 Effect of level of different concentration of kokum extract in Coconut milk probiotic drink, Almond probiotic drink, Peanut milk probiotic drink and Soy milk probiotic drink on consistency
In figure 4.9.2 consistency of almond milk probiotic drink was good i.e.6.3 ± 0.23 as compare to other probiotic drink.

Table 4.9.1.1: Organoleptic evaluation of final probiotic solkadhi drink
Samples Conc. Appearn. Consistency Flavour Taste Overall Acceptability
Coconut milk probiotic solkadhi 0 3.65 ±0.21 4.7± 0.1 4.4 ± 0.19 4.4± 0.14 5.65 ± 0.21
2 4.85± 0.21 5.2 ± 0.1 5.6 ± 0.25 5.4± 0.14 6.75 ± 0.35
4 5.4 ± 0.14 5.4 ± 0.3 6.4 ± 0.14 5.4± 0.14 6.9 ± 0.14
6 8.7 ± 0.28 5.7 ± 0.1 7.3 ± 0.28 6.2± 0.21 7.9 ± 0.14
8 8.1 ± 0.14 5.3 ± 0.3 6.9 ± 0.04 5.8± 0.07 7.25 ± 0.35
Almond milk probiotic solkadhi 0 3.2 ± 0.14 4.2 ± 0.2 4.3 ± 0.21 4.6±0.14 5.4 ± 0.14
2 5.0 ± 0.28 5.2 ± 0.1 5.3 ± 0.14 5.1± 0.21 6.35 ± 0.21
4 5.4 ± 0.14 4.8 ± 0.1 6.0 ± 0.21 5.3± 0.21 6.55 ± 0.35
6 8.3 ± 0.21 5.6 ± 0.3 7.1 ± 0.21 6.3± 0.14 7.4 ± 0.14
8 7.7 ± 0.14 5.1 ± 0.1 6.6 ± 0.21 5.7± 0.14 6.8 ± 0.21
Peanut milk probiotic solkadhi 0 3.4 ± 0.07 4.2 ± 0.2 4.6 ± 0.21 4.3± 0.21 5.15 ± 0.21
2 4.6 ± 0.14 4.2 ± 0.1 5.4 ± 0.28 5.1± 0.24 5.9 ± 0.14
4 5.7 ± 0.28 4.8 ± 0.2 6.5 ± 0.21 5.4± 0.30 6.1 ± 0.14
6 8.1 ± 0.28 5.6 ± 0.3 6.9 ± 0.04 6.2± 0.09 6.6 ± 0.21
8 7.8 ± 0.14 5.1 ± 0.1 6.7 ± 0.14 5.7±0.19 7.15 ± 0.35
Soy milk probiotic solkadhi 0 3.1 ± 0.35 3.8 ± 0.1 4.3 ± 0.44 4.7± 0.07 4.9 ± 0.14
2 4.2 ± 0.42 4.4 ± 0.7 5.4 ± 0.14 5.2± 0.28 5.3 ± 0.14
4 5.3 ± 0.21 4.8 ± 0.2 6.3 ± 0.14 5.2± 0.19 6.2 ± 0.14
6 7.9 ± 0.14 5.3 ± 0.1 6.8 ± 0.19 6.2 ±0.12 6.6 ± 0.21
8 7.5 ± 0.07 5.2 ± 0.2 6.5 ± 0.35 5.7± 0.21 6.4 ± 0.21

23994356227050046853413172970254360727013100475108439397Fig. 4.9.3 Effect of level of different concentration of kokum extract in Coconut milk probiotic drink, Almond probiotic drink, Peanut milk probiotic drink and Soy milk probiotic drink on flavour
262647600102In figure 4.9.3 flavour of coconut milk probiotic drink was good i.e. 7.3 ± 0.2 as compare to other probiotic drinks.

24278085651500023879653161090045802553365500479425049530Fig. 4.9.4 Effect of level of different concentration of kokum extract in Coconut milk probiotic drink, Almond probiotic drink, Peanut milk probiotic drink and Soy milk probiotic drink on taste
In figure 4.9.4 taste of almond milk probiotic solkadhi was good i.e. 6.3 ± 0.14as compare to other probiotic drinks.

-4860
32945366036220054518946865253614133272760301053531877000Fig. 4.9.5 Effect of level of different concentration of kokum extract in Coconut milk probiotic drink, Almond probiotic drink, Peanut milk probiotic drink and Soy milk probiotic drink on overall acceptability
In figure 4.9.5 overall acceptability of of coconut milk probiotic drink was good i.e. 7.9 ± 14 as compare to other probiotic drinks.

CHAPTER-V
CONCLUSION
Non-dairy milk like coconut milk, almond milk, peanut milk and soy milk were used to prepare probiotic solkadhi drink. Solkadhi is traditional appetizer drink consume widely in Indian coastal area. Components in non-dairy milk are suitable for the growth of probiotic organism ie. Lactobacillus acidophilus. From the analysis it can conclude that acidity of probiotic solkadhi drink was increased with increase in concentration of kokum extract whereas pH of probiotic solkadhi drink was dropped as the concentration of kokum extract increased in non-dairy milk. Probiotic count was decreased as concentration of kokum extract increased.

Antioxidants and total phenolic content of probiotic solkadhi was done. Antioxidant level and phenolic content was increased in the probiotic drink as the concentration increases. All the parameters of analysis were increased as the concentration of kokum extract increased in the non-dairy milk.

In the organoleptic evaluation probiotic drink containing 6 ml of kokum extract was good in all aspects of evaluation like flavor and color of coconut milk was good, appearance and colour as well was of soy milk, coconut milk, peanut milk almond milk probiotic drink was good in texture and consistency.

CHAPTER – VI
SUMMARY
6.0 summery
In the present study, the probiotic organism i.e. L. acidophilus was isolated from lyophilized powder for development of non- dairy probiotic solkadhi drink. Probiotic drinks were prepared by mixing prepared kokum extract and spice mixture of ratio of cumin: garlic: green chilly ie. 2.5:1.25:1.25 (w/w/w) in non- dairy milk of peanut, soybean, almond and coconut milk. Salt was added for taste. The kokum extract was prepared from overnight soaked kokum extract in 1:2 ratios. Soybean, peanut and almond were soaked overnight and freshly cut coconut was used for preparation of respective milk. L. acidophilus inoculum containing 104cells per ml of sterilized saline solution were prepared. 1ml of inoculum was inoculated in 100 ml of milk containing kokum extract, spice mixture and salt.

On the basis of their organoleptic characteristics and acceptability, spice mixture with the ratio of cumin: garlic: green chilly ie. 2.5:1.25:1.25 (w/w/w)) was selected for the fermentation of developed probiotic drink. Twenty types of probiotic drinks were prepared using five different concentration of kokum extract i.e. 0ml, 2ml, 4ml, 6ml and 8ml per 100 ml of non-dairy milk by inoculating probiotic bacteria and adding. Milk was boiled for 5min before inoculation. Inoculated drink was incubated at 370 C for 6 hr. The probiotic counts were increased in all the probiotic drinks. Microbial count was decreased as concentration of kokum extract increased in milk. More bacterial count was observed in coconut milk probiotic drink as compare to other three probiotic drinks.

There were decrease in pH and increase in titratable acidity was observed after 6 hr of incubation as the concentration of kokum extract was increased. In coconut milk probiotic drink pH decreased from 5.23±0.007 to 3.06±0.01, in almond milk 5.25±0.007 to 3.62±0.03, in peanut milk 5.33±0.007 to 3.55±0.01, in soy milk 5.34±0.01 to 3.55±0.007.

For detection of antioxidants in probiotic drink DPPH assay and total phenolic content for detection of phenols in probiotic drink was carried out. In DPPH assay absorbance was inversely proportional to the concentration of kokum extract in milk. Non-dairy milk like soymilk, peanut milk and almond milk is rich in isoflavones and bio-isoflavones, which are reported to beneficial estrogenic effects with potential bioactive antioxidant properties. Due production of ?-glucosidase activity by probiotic lactobacilli, isoflavoneaglycones increases antioxidant activity therefore peanut milk probiotic drink has highest absorbance followed by soy milk probiotic as compared to Almond milk, Coconut milk, which an indication of higher amount of antioxidants in it. Percentage scavenging activity was also inversely proportional to the concentration of kokum extract per 100 ml of milk. Percentage scavenging activity was also decreasing as concentration increasing because any substance that when present at lower concentration compared with those of the oxidizable substrate significantly delay or inhibit the oxidation of that substrate. i.e. lower the concentration less the free radicals. Scavenging activity of soymilk probiotic drink was 0.10% to 2.19%, 0.20% to 5.84 for Almond milk probiotic drink; 0.41% to 3.65% for Coconut milk probiotic drink and 0.41% to 2.50% for Peanut milk probiotic drink.

Gallic acid equivalence increase from 22.85±0.21 G.A.E. to 26.1±0.14 G.A.E. for coconut milk, 22.95±0.07 G.A.E. to 26.1±0.14 G.A.E. for almond milk, 23.35±0.21 G.A.E. to 26.1±0.14 G.A.E. for peanut milk and 23.35±0.21 G.A.E. to 26.65±0.21 G.A.E. for Soy milk. IC50 was calculated from the graph by using straight line equation by taking higher OD point as y the equation.

Phenols are constituting of the most numerous and ubiquitous plant metabolites. They are a large group of compounds with closely related classed that consists bioisoflavonoids, coumesstance and stilbenes. Coconut milk probiotic drink has highest contents of polyphenols in it that is ranges from 0.73±0.021 A.A.E. to 1.9±0.14 A.A.E. and for Coconut milk, 0.77±0.03 A.A.E. to 2.9±0.14 A.A.E. for Almond milk, 0.82±0.10 A.A.E. to 1.7±0.1 A.A.E. for Peanut milk, and 0.9 A.A.E. to 1.9 A.A.E. for soy milk probiotic drinks. Content of polyphenols was higher as increased concentration of kokum extract. That is content of polyphenols was directly proportional to the concentration of kokum extract per 100 ml of milk.

Form this study it can be conclude that spice mixture ratios and concentrations of kokum extract in non-dairy milk was suitable for growth of probiotic organism Lactobacillus acidophilu. And have good amount of antioxidants in it which insoluble isoflaovnes soluble after 6 hours fermentation with probiotic bacteria.

CHAPTER – VII
FUTURE ASPECTS
Presents study we have developed non- dairy probiotic solkadhi drink for the lactose intolerance people who cannot digest lactose. Addition to this to increase its nutritional value kokum extract is added which is extremely nutritious underutilized Indian tropical fruit; packed with lots of minerals, vitamins, HCA like anti-obesity components and a potent antioxidant of red coloured pigment anthocyanin. Besides nutritional benefits, such developed functional foods containing probiotic fermenting organism may have therapeutic value too.

In future we can study shelf life of these products by adding various preservatives or by appropriate packaging of the probiotic drink, storage at controlled temperature and relative humidity. By extending its shelf life such product can be exported which will ultimately lead to employment in rural area and economic benefits to nation.

Instead of kokum extract we can add germinated barley, ragi and moth bean coarse grains and legumes or other concentration of drink mixture like jowar, brown rice and green gram.

CHAPTER – VIII
BIBLIOGRAPHY
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