Introduction

‘Functional’ dairy products are ideal vehicles for delivering a defined number of live, beneficial bacteria to the human gut. Since times immemorial, they have been central in the manufacture of various fermented foods like yogurt, cheeses, fermented milk, vegetables, meat, wine and therefore, are heavily used as starters in the food industry [1-5]. Therefore, lactic acid bacteria (LAB), producers of lactic acid as their primary metabolic product, are diversely applied organisms in the food industry. Their extended and safe use as starter cultures in fermented products qualifies them as generally recognized as safe (GRAS) microorganisms [6]. LAB is a heterogeneous group of spherical (cocci) or rod-shaped (bacilli), non-sporulating, non-motile gram-positive, catalase negative, anaerobic, and aero-tolerant bacteria [7]. They are non-pathogenic [8], and can be homo-fermentative, facultative homo-fermentative or hetero-fermentative. They can metabolize mono- and disaccharides like glucose and lactose to harness energy. Using beta-galactosidase enzyme, homo-fermentative LABs hydrolyze lactose in the milk and produce two moles of lactate and ATP by Entner-Meyerhof-Parnas (EMP) pathway [9]. Besides lactic acid, these bacteria produce a variety of compounds like acetic acid, ethanol, aroma compounds, enzymes, exopolysaccharides (EPS) [2]. Low pH, the non-dissociated acid, and production of antimicrobial metabolites called bacteriocins also contribute to their antagonistic property [9,10]. They are acid tolerant and bile tolerant[8], making them suitable for the food industry; although presence of the 'inhibitory' secondary metabolites[11], and additional stresses based on the technique used for their preservation, such as spray drying [12], freeze drying [13], and fluidized bed drying [14] may restrict their applicability.

Recently, LAB derived EPS have gained popularity due to various reasons. Their ability to improve rheology (viscosity and elasticity) makes them excellent candidates as natural gelling agents, biothickeners, emulsifiers, and physical stabilizers to bind water and limit syneresis [15]. Although EPS are themselves tasteless, they prolong the time that the milk product spends in the mouth, enhancing its taste through improved volatilization of the intrinsic flavor in the product [16] and contribute to the organoleptic quality of the fermented foods, in texture and taste perception, mouth-feel, and stability [15,17]. Also, due to increased viscosity, the foods may remain in the gastrointestinal tract for a longer duration and therefore be beneficial to the transient colonization by probiotic bacteria [18]. Various factors such as bacterial growth, phase, medium composition (carbon and nitrogen source), pH, and temperature influence their production [15]. Also, LAB synthesized EPS appear to have anti-tumor, anti-ulcer, immunomodulating, and cholesterol-lowering activity [14]. In Maharashtra and Gujarat (India), the fermentation of milk into curd and straining and separation of the whey to get the concentrated curd, called 'Chakka' is a routine in households and dairies. Chakka is used as a base for making Shrikhand, a popular culinary concept. The preparation of Shrikhand involves mixing Chakka with the required amounts of sugar, color and flavor to get the product. This thick curd marinade has an exciting, sour-sweet taste and holds great economic potential. Therefore, this study aimed to isolate, identify, and characterize the naturally occurring LABs from Chakka, to assess their potential as starters. The study also included the optimization of culture media and other parameters and evaluation of lactic acid and exopolysaccharides produced.

Materials and Methods

Chemicals

All chemicals were purchased from Qualigens, India and HiMedia, India and were of analytical grade and were used directly without further purification. M17 Agar and De Man Rogosa Sharpe (MRS) agar were obtained from HiMedia, India and Accumix, India, respectively.

Isolation and purification of cultures

All the strains were isolated from the Chakka prepared using pasteurized full cream milk, obtained from M/s. B.G. Chitale Dairy at Bhilawadi in Sangli District, Maharashtra, India. The LAB isolates were purified according to the method adopted [19]. The plates were incubated in micro-aerobic conditions (5% CO2 ) [20], at 37°C for mesophilic and at 42°C for the thermophilic LAB for 48 hours. All experiments were performed in triplicate. The colonies were randomly selected from the agar surface and were purified by streaking twice on fresh MRS [21] and M17 [22] agar. Colonies were selected according to their morphology. The purity of isolates was checked by four streak method on the respective media followed by macroscopic and microscopic examination. The strains displaying the general characteristics of lactic acid bacteria were chosen from each plate for further studies. The strains were stored without appreciable loss of properties in reconstituted sterile skimmed milk at -20°C. Working cultures were also kept on MRS or M17 agar slants at 4°C.

Phenotypic characterization of isolates

The cultures were characterized based on differentiation scheme for genera level identification of lactic acid bacteria and profile matching method based on Bergey’s manual of systematic bacteriology [7]. The individual colonies were subjected to preliminary screening by examination for cellular morphology, the colony appearance, were Gram stained, observed for motility, and tested for cytochrome oxidase and catalase [23]. Hugh and Leifson’s test, which allows determination of ability of an organism to act as oxidizer or fermenter under aerobic and anaerobic conditions was performed in Oxidizer/Fermenter (O/F) medium [24]. The isolates were also tested for acid production from carbohydrates 1% (w/v) with glucose, sucrose, lactose, arabinose, maltose and sorbitol.