Development and Survival Assessment of Microencapsulated Lactobacillus rhamnosus GG in Watermelon Juice -Sodium Alginate Beads at Different Storage Conditions

The present study adopted microencapsulation of bacteria to enhance its viability. Watermelon juice was found to be a suitable media for the growth of probiotic L.rhamnosus GG showing prebiotic effect. No significant difference in the viability of L.rhamnosus GG in MRS medium. The average viable count (log10cfu/ml) of L.rhamnosus GG in watermelon and MRS medium were 10.53±0.136 and 10.55±0.132 respectively. The survivability assessment of L.rhamnosus GG in microencapsulated beads at different storage conditions was studied. The probiotic level of 10 8 cfu/g of L.rhamnosus GG was maintained at both temperatures till 21 days of storage. The viability of L.rhamnosus GG was 8.404±0.019 and 8.027±0.008 log10cfu/ml in microencapsulated beads at refrigeration and ambient temperature respectively on 21 st day of storage after which there was a decline in the probiotic count in samples stored at ambient and refrigeration temperature.


INTRODUCTION
The name probiotic comes from the Greek word -pro bios‖ which means -for life‖. The history of probiotics began with the history of man; Cheese and fermented milk were well known to the Greeks and Romans, who recommended their consumption especially for children and convalescents. The concept of probiotics was introduced by Elie Metchnikoff in the early 20 th century (Metchnikoff, 1907). According to Food and Agriculture Organization (FAO) of the United States and World Health Organization (WHO), probiotics are ‗live microorganisms which when administered in adequate amounts confer health benefits to the host'.
Alternatively, probiotics have been defined as live microbial feed supplements that beneficially affect the host by improving its intestinal microbial balance (Fuller, 1989).
At present, several well-characterized strains of Lactic acid bacteria are available as potential probiotic organisms which are useful in improvising human health. The largest group of Lactic acid bacteria belongs to genus Lactobacillus that comprises of more than 50 different species. Lactobacillus species are found in the gut of humans and other animals, while their numbers may vary with species, age of host or their location within the gut. However, species of Lactobacillus like L.acidophilus, L.crispatus, L.plantarum, L.gasseri are involved in traditional and industrial food fermentations (de Vries et al., 2006).
The inclusion of probiotics in food matrices is a challenging area of research. Over the last few years, the probiotics industry experienced a remarkable market share increase. The development of functional food is associated to a large extent, with products containing probiotics and their contribution in supplementation to the bacterial flora in the intestines. Hence in the current study, the development of synbiotic watermelon beads. Reid (1999) elaborated the characteristics of Lactobacillus rhamnosus GG as a probiotic due to its ability to adhere to the cells, colonize the intestine, exclude or reduce the adherence of the pathogenic strains, persist and multiply, produce compounds that are antagonistic to pathogen growth, resist vaginal microbicides and form a normal, balanced flora. Land et al. (2005) in their review stated that Lactobacillus rhamnosus strain GG was originally isolated from the human intestinal flora and is the most widely used probiotic agent for adults and children. He highlighted its ability to prevent diarrhea and atopy among children.
Lactobacillus rhamnosus GG are large, white, creamy colonies. The colonies were reported to be gram positive, uniform rods in chains (Saxelin et al., 1993). Protection of probiotics has been proposed for various dairy fermentation, with microencapsulation in hydro colloidal beads for improving their viability in both the food products and in intestinal tract (Champagne et al., 1992). Viability of probiotic bacteria in a product at the point of consumption is an important consideration for their efficacy, as they have to survive during processing and shelf life of food and supplements, transit through high acidic conditions of the stomach and enzymes and bile salts in the small intestine (Kebary, 1996).
Microencapsulation is the process of encasing an active component in a shell and is defined as a technology of packaging solids, liquids or gaseous materials in miniature, sealed capsules that can release their contents at controlled rates under the influence of specific conditions (Kailasapathy & Masaondole, 2005).
According to Ding and Shah (2008), microencapsulation provides a favourable environment for sensitive probiotic bacteria as well as a physical barrier from the harsh environment. He also highlighted that food products containing microencapsulated probiotic bacteria were more stable than those containing free probiotic organisms.
Encapsulation is a process whereby cells are retained within a wall material to reduce cell injury. Encapsulation in hydrocolloid bead has been investigated as a means to protect and improve viability of probiotic microorganisms in food products and in the intestinal tract (Rao et al., 1989).
Microencapsulation by extrusion involves projecting an emulsion core and coating material through a nozzle at high pressure. Extrusion of polymer solutions through nozzles to produce capsules is mainly reported on a laboratory scale, where simple devices such as syringes are applied. If the droplet formation occurs in a controlled manner (contrary to spraying) the technique is known as prilling (Heinzen, 2002). The term synbiotic is used when a product contains both probiotic and prebiotic ingredients (Schrezenmeir & Vrese, 2001). Synbiotic are used not only for the improved survival of beneficial micro organisms added to food or feed but also for the stimulation of the proliferation of specific native bacterial strains present in the gastrointestinal tract (Gourbeyre et al., 2011).
According to Sivudu et al. (2014), fruits and vegetables are rich in nutrients and can be considered as a carrier to support probiotic and prebiotic delivery.
Watermelon is one of the underutilized fruits and is liked by consumers due to its flavour and attractive colour. The colour in the watermelon is due to the presence of lycopene that has potential to act as a biocolour and as an anticancerous agent (Huor et al., 1980). Wang et al. (1996) reported that the antioxidant activity, mineral and phenolics of pomegranate maintained the viability and stability of L. plantarum in microencapsulated beads.
Fruit juices represent a promising carrier for probiotic bacteria (Marianne et al., 2015).

MATERIALS AND METHODS Materials
Lactobacillus rhamnosus GG (ATCC 53103) was obtained from National Dairy Research Institute, Karnal, Haryana. De Man Rogosa and Sharpe (MRS) broth (Himedia GM369) and De Man Rogosa and Sharpe (MRS) Agar (Himedia M641) were used for the propagation and enumeration of the freeze dried culture respectively. Food grade Sodium alginate and Calcium chloride was purchased from Loba Chemie Pvt Ltd. Watermelon was purchased from from local market, Chennai, TamilNadu.

Propagation and enumeration of freeze dried Lactobacillusrhamnosus GG
The freeze dried culture of L.rhamnosus GG (ATCC 53103) was inoculated in MRS broth and incubated overnight at 37°C. The propagated culture was then enumerated in MRS agar. Stock cultures were maintained by sub-culturing once in 15 days, whereas the working cultures were freshly prepared in skim milk as and when needed. Preparation of microencapsulated L.rhamnosus GG beads Assessing the prebiotic property of watermelon on the viability of L.rhamnosus GG The prebiotic property of watermelon was assessed as per the modified procedure of Saranyambiga et al. (2017).

Preparation of microencapsulated L.rhamnosus GG beads
Microencapsulation technique in the present study was adopted from Krasaekoopt et al. (2003). Lactobacillus rhamnosus GG was inoculated in skim milk and incubated at 37°C overnight. The overnight culture was suspended in the carrier solution containing 2 per cent sodium alginate in sterilized watermelon juice. This was then filled in a pre sterilized syringe and dropped slowly into 0.1M Calcium chloride solution from a height of 20cm get pink coloured L.rhamnosus GG microencapsulated beads. The flow diagram for the preparation of L.rhamnosus GG beads is given below.
Sterilized skim milk  Inoculation of L.rhamnosus GG (1 per cent)  Incubation at 37°C overnight  Cell suspension  Sodium alginate (2 per cent) in 100ml of watermelon juice  Dropping in 0.1M Calcium chloride solution  Microencapsulated beads Assessment of survivability of Lactobacillus rhamnosus GG in microencapsulated beads during the storage period at different temperatures L.rhamnosus GG was enumerated in microencapsulated beads stored at ambient and refrigeration temperatures as per the procedure followed by Savedboworn et al. (2015) for a period of 28 days at 7 days interval.

Statistical analysis
The Statistical analysis was done for the data obtained using VETSTAT as per the standard procedure of Snedecor and Cochran (1980). Results were expressed as Mean ± Standard Error.

Comparative study on viability of Lactobacillus rhamnosus GG in MRS broth and Watermelon juice. Table shows the viability of L.rhamnosus GG in MRS broth and watermelon juice
The Mean±SE values for the growth (log 10 cfu/ml) of L.rhamnosus GG in MRS broth was 10.55±0.132 and in watermelon juice was 10.53±0.136.
Statistical analysis revealed no significant difference in the counts of L.rhamnosus GG in MRS broth and watermelon juice.  Table 1 presents the viability of L.rhamnosus GG in pasteurized water melon juice. The log values of 10.53±0.136 log 10 cfu/ml showed no significant difference with MRS medium and suggest that water melon can be considered as a suitable medium for the growth of L.rhamnosus GG. The results concur with the observations of Tuorila and Gardello (2002) that fruit and vegetable juices may be considered as an alternate vehicle for the delivery and incorporation of probiotics into human intestine. The medium can be

Microencapsulated beads
Single microencapsulated bead Statistical analysis revealed a highly significant difference (P≤ 0.01) in the survivability of L.rhamnosus GG between 0, 7, 14, 21 and 28 days of storage at refrigerated temperature. There was a one log reduction in the viable count of L.rhamnosus GG on the 28 th day of enumeration.
Statistical analysis revealed a highly significant difference (P≤ 0.01) in the survivability of L.rhamnosus GG between 7 th day and other days of storage at ambient temperature. The maximum viable count was enumerated on the 7 th day followed by 0 and 14 th day. There was a gradual decline of viability on the 21 st day of storage and a one log reduction in the viable count of L.rhamnosus GG on the 28 th day of enumeration.
The survival rate and the probiotic level of 10 8 cfu/g of L.rhamnosus GG were maintained at both temperatures till 21 days of storage. The viability of L.rhamnosus GG was maintained up to 8log 10 cfu/g for 21 days at refrigerated temperature and ambient temperature after which there was a decrease in the viable count. This may be due to the reasoning of Charteris et al. (2002) that stress associated with encapsulation and other adverse conditions such as refrigerated storage may cause culturable cells to enter a growth phase that does not produce colonies on media that noraly support their growth.

CONCLUSION
Probiotic foods are becoming increasingly popular due to their contribution to good health. Lactobacillus rhamnosus species has a long history of being used in probiotic foods and possess a genome that allows it to adapt to a range of environments including the human gastrointestinal and urogenital tracts. In order to exert health promoting probiotic effects, it is important for the bacteria to survive the inhospitable environment of the human gastrointestinal tract. The present study adopted microencapsulation of bacteria to enhance its viability. Watermelon juice was found to be a suitable media for the growth of probiotic L.rhamnosus GG showing prebiotic effect. No significant difference in the viability of L.rhamnosus GG in MRS medium. The average viable count (log 10 cfu/ml) of L.rhamnosus GG in watermelon and MRS medium were 10.53±0.136 and 10.55±0.132 respectively. The survivability assessment of L.rhamnosus GG in microencapsulated beads at different storage conditions was studied. The probiotic level of 10 8 cfu/g of L.rhamnosus GG was maintained at both temperatures till 21 days of storage. The viability of L.rhamnosus GG was 8.404±0.019 and 8.027±0.008 log 10 cfu/ml in microencapsulated beads at refrigeration and ambient temperature respectively on 21 st day of storage after which there was a decline in the probiotic count in samples stored at ambient and refrigeration temperature.