Introduction

Lactic acid bacteria are a group of related bacteria that produce lactic acid as a result of carbohydrate fermentation. This group includes representatives of the genus Lactobacillus, Lactococcus, Pediococcus, Leuconostoc and Streptococcus (Guessas et al, 2004) which are used in the production of fermented food products such as yogurt, cheese, sauerkraut and sausage (Kamaly et al, 1989, Nowroozi et al., 2004, Tserovska et al.,2002, Conter et al.,2005). Lactic acid bacteria are natural inhabitants of human gastrointestinal tract (GIT) and are also considered as a probiotic. These bacteria also support digestion and immune function enhancement in addition to controlling the pH of the large intestine through the liberation of lactic and acetic acid, which in turn restricts the growth of many potential pathogens (Nowroozi et al., 2004). In GIT these bacteria are capable of delivering enzymes and other substances into the intestine which possibly help to control intestinal microflora (Collins et. al., 1999). Lactic acid bacteria also produce biologically active peptides or protein complexes that display a bactericidal mode of action almost exclusively toward gram positive bacteria (Mojgani et al., 2006). Lactic acid bacteria naturally found in dairy products have been implicated in lipid degradation also (Kamaly et al.,1989; Marta et al.,2002; Kun et al.,2004; Andrews et al.,2006).

Since sedentary life style has given rise to the increased incidence of coronary heart problems such as atherosclerosis, high blood pressure etc which are consequences of increase in triglyceride content of blood. Isolated bacterial strains could be helpful to derive lipase preparation that could find therapeutic advantage in serum triglyceride reduction as well as for those peoples produce inadequate pancreatic lipase. Enzyme preparation could also have other applied benefits such as flavour and aroma enhancer in various dairy products and other fermented food products. It may also find application in development of probiotics which in today’s health- conscious scenario will not only help in natural reduction of serum triglycerides but also maintain gastrointestinal tract microflora that prevent infection and adhesion of pathogenic bacteria due to bacteriocin production and antagonistic activity.

Thus, the need of the hour is to identify such food and dairy products which can either be consumed directly or used to isolate these bacteria. Hence, in the present study samples of various dairy products were screened for the presence of and isolation of lactic acid bacteria.

Material and Methods

Sample collection

21 dairy samples viz. curd, cheese, milk (goat, buffalo, cow, camel) were used in this study. These samples were collected from local (domestic and commercial) sources of Udaipur (Rajasthan) in presterlized ampoules at room temperature and screened on same day of collection.

Isolation of lipolytic bacteria

Isolation of lipolytic strains was done by enrichment culture according to the modified method of Vargas et al, 2004. The (g/l) composition of enrichment medium was as follows: lipid substrate ( tributyrin and castor oil); 20 g, K₂HPO₄ ; 2.5 g, (NH₄)₂ SO4;1.3 g, MgSO₄; 0.5 g, yeast extract; 0.5 g. 6.5ml of filter sterilized urea (200 g/l stock) were added in media after autoclaving. pH was adjusted to 8.5 by addition of sterile sodium carbonate : bicarbonate mixture (3:1) to the medium. 10% (v/v) dilutions of samples were inoculated in enrichment medium and incubated in orbital shaker incubator at 220 rpm for 24h at 37 ⁰ c.

Lipolytic bacteria were isolated by streaking the enriched culture on tributyrin agar media containing fatty substrate. The components of media include 0.5% peptone, 0.3% yeast extract, 2% agar and1% fatty substrate. Lipolytic activity was observed as zone of clearance around bacterial colonies.

Isolation of lipolytic lactic acid bacteria

Isolation of lactic acid bacteria was done on selective medium i.e. MRS agar and M-17 agar (Tserovska et al.,2000). These isolates once again checked for lipolytic activity.

Identification of the bacterial strains

Identification of the strains as lactic acid bacteria was done on the basis of their morphological, cultural, physiological and biochemical characteristics as suggested in Bergey’s Manual (Bergey’s Manual, 1984).

Lipolytic activity

Lipolytic activity was determined by agar well diffusion assay (Blake et al,1996). 12 mm sized punched well in tributyrin agar plate were filled with 200 µl of cell free extract prepared from 4x 10⁶ CFU. These plates were incubated at 37 ⁰ C for 24h. A clear zone around the well indicated lipid hydrolysis. The size of clear zone was measured with the help of Hi- Antibiotic zone scale-C. Nutrient broth was used as negative control and pancreatic lipase as positive control.

Free fatty acids (FFAs) Titration

Titration method of Deeth et al.,1975 was used to measure the amount of FFA released  by the activity of crude lipase. Overnight culture of isolates, developed in basal medium containing tributyrin, were centrifuged at 5000 rpm and filtered to obtain cell free extract which was used as crude lipase preparation. The reaction mixture containing crude lipase,  tributyrin and phosphate buffer (0.2 M, pH 7.2) in 1:1:2 ratio respectively was placed in a 25 ml glass stoppered test tube and incubated for different time duration of 6h, 12h, 24h, 30h, 36h, 48h, 54h, 60h and 72h  at 37 ⁰ c temperature. After specified time of incubation, 2 ml of reaction mixture were mixed with 10 ml of extraction mixture (40:10:1, vol/vol/vol, isopropanol: petroleum ether: 4N H₂SO₄), 6 ml of petroleum ether and 4 ml of water. The mixture was shaken vigorously. The layers were allowed to separate for 10 min. at 25⁰ c. and 5 ml of the upper layer were then titrated with 0.02 N methanolic KOH using 50 µl of methanolic phenolphthalein as an indicator. Each time FFA content was determined using the formula (TN/PV)x10³ , where T is the net titration volume, N is the normality of the methanolic KOH , P is the proportion of the upper layer titrated, and V is the volume (in millilitres) of sample.

Protein Determination

The protein content of cell free extract was determined according to Bradford et al., 1976 with bovine serum albumin as a standard.

Statistical analysis

The statistical analysis of data was done by using software origin 6.1. The data were analysed by one way ANOVA and also by determining linearity of lipase activity with time.

Results and discussion

Enrichment culture allows the selective growth of strains with desired characteristics hence in the present study enrichment culture technique was used to isolate lipolytic bacteria. Five lipolytic strains of lactic acid bacteria were isolated from enriched samples. Identification of strains was done by studying morphological and biochemical characteristics as indicated in table. no. 1 . These strains were identified as Streptococcus (MRLa) , Streptococcus (MRLb), Lactobacillus (MRLc), Lactobacillus. (MRLd) and Lactobacillus (MRLe).

Lipolytic activity of crude lipase, obtained from isolated lactic acid bacteria, was determined by agar well diffusion method (Blake et al,1996). The basic principle behind this method is the hydrolysis of lipid substrate present in the media by the microbial enzyme which can be observed as a clear zone around the bacterial colonies or wells containing crude lipase preparation. The size of zone of clearance indicates the magnitude of activity. Results indicate (Table no 2) that maximum zone of clearance (14mm) was produced by crude lipase from Lactobacillus (MRL d) while minimum (3mm) was produced by Streptococcus (MRLa). Lipolytic activity has been reported in lactic acid bacteria by several workers (Kamaly et al, 1990, Woo et al, 1984). Lipase enzyme responsible for this activity may be extracellular, intacellular or it may be cell membrane bound (Mourey., 1981, Lee and Lee., 1989 and Large et al., 1999). Presence of extracellular lipase in cell free extract is therefore possibly responsible for the lipolytic activity of the isolates (Kalogridou-Vassiliadou,1984).

Further quantitative assay of lipase activity was done by titrimetric method.  In this method lipase activity was measured as a release of free fatty acids during specific time duration by using phenolphthalein as end point indicator. This enzyme hydrolyzes the lipid into fatty acids and glycerol. Hydrolysis of tributyrin results in increase in acidity of reaction mixture which can be measured titrimetrically (Deeth et.al.,1975).

Lipase assays measure fatty acid released in periods less than an hour (Chen et al., 2003; Deeth & Touch, 2000). However, some authors, in attempting to detect trace levels of lipase have performed longer incubations (Blake et al., 1996; Deeth & Touch, 2000). Hence, for each strain release of free fatty acids was determined over 72 hours and was found to be linear with time (Figure 1,2,3,4 and 5). After 72 h of incubation of reaction mixture, maximum lipase activity was observed for strain MRLd followed by MRL e, MRL c, MRL b and MRL a respectively.

Factors like environmental conditions at the time of sample collection, type of sample, chemical and physical nature of sample , substrate specificity etc. might be responsible for variations in lipase activity among lipolytic lactic acid bacterial strains. Either single or cumulative effect of above mentioned factors may affect the expression of gene coding for lipase degrading principle and subsequently in the amount of FFAs release by each strain. On the basis of this results obtained, it can be suggested that physical and chemical nature as well as environmental condition may be responsible for better enzymatic activity in the strain isolated from camel milk as compare to cow milk .

The value of correlation coefficient (R) for each strain was found to be significant (Table 3). In addition to these, values of coefficient (P<0.0001) were also found to be more significant for each strain. Analysis of data by one -way ANOVA at 0.05 level of significance also indicates that this work is significant.

Of the various dairy samples screened, strains isolated from camel milk show best results. Therefore it can be suggested that camel milk can be a potential source for isolation of lipolytic lactic acid bacteria as compared to cow milk.

Apart from this it can also be suggested that consumption of camel milk can be used for natural enhancement of inherent gastrointestinal lipolytic flora and therefore it can be a part of natural therapy for hypertriglyceridemia.

Since lipolytic activity of crude lipase preparation of MRLd is comparable with that of pure lipase preparation (pancreatin) and hence there is a possibility that pure lipase prepration might have greater activity than pancreatin.

Table 1: Morphological ,culture and biochemical characteristics of isolates used for study.

A* Catalase test, B* Nitrate Reduction test, C* Vogus Proskar test, D* Gelatinase test, E* T.S.I. Reduction test, F* Litmus test, G* Methyl Red test

Table 2: Zone of hydrolysis produced by LAB isolates on Tributyrin agar media.

*values are mean of three replicates N.D. = Not Determined

Table 3: Statistical analysis of data given in figure1, 2, 3, 4 and 5.

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