Isolation and identification of strains

Raw cow and buffalo milk and homemade yogurt were collected randomly from different dairy farms of Multan, Pakistan and were stored in sterile autoclaved containers at 4℃ until delivery to the laboratory for isolation of Lactobacillus strains.

For isolation Lactobacillus strains samples were diluted and were allowed to spread them on solidified MRS (de Man, Rogosa and Sharpe) agar (Oxoid). Plates were then incubated at 37°C for 24-48 h under anaerobic condition. Colonies having different morphological appearance were selected [18]. Gram staining and biochemical tests were done according to Bergey’s Manual of Bacteriology scheme to identify Lactobacillus strains [19]and strains were selected according to their biochemical tests. Bacterial strains that showed Gram positive reaction, catalase negative reaction, were fermenters of different carbohydrates like glucose, sucrose; maltose, lactose and dextrose were considered as Lactobacillus and were selected for antimicrobial activity [20].

Evaluation of probiotic properties

Acid and bile salt tolerance of isolated strains

Isolated strains were investigated for bile salt tolerance and for that purpose; MRS broth was prepared with varying concentrations of oxgall bile salt (0.2% and 2%) and pH (pH 2, 5). After preparation of MRS broth, each isolate was transferred into tubes (triplicates) containing broth and tubes were incubated at 37°Cfor 24 h. After incubation, optical density (O.D) was measured against the control. Isolates showing resistance more than 50% at pH 2 were considered acid tolerant strains. The percentage resistance in both cases (bile/acid pH values) was calculated as follows [18].

Resistance to stimulated gastric juice

Stimulated gastric juice was prepared and was adjusted to pH 3 by using HCl. Resistance to stimulated gastric juice was checked according to Feng et al., with slight modifications [21]. Lactobacillus strains were inoculated into MRS broth and were incubated at 37°C for 24 hours. Fresh cultures were harvested by centrifugation at 7000 x g for 10 min and suspended in saline solution. Dilutions were made to reach final dilution of 10⁹ CFU/ml. Thereafter, each cell suspension was mixed with stimulated gastric juice by vortex mixing and after that tubes were incubated at 37°C for 5 hours. Bacterial growth was estimated by taking optical density (O.D) at 560nm [21].

Physiological characterization

Physiological properties like ability of the strains to grow at different temperatures and different NaCl concentration were checked and the protocol is described briefly. MRS was inoculated with overnight cultures of selected bacterial strains and then these strains were grown at different temperatures i.e. 25°C, 37°C and 45°C and at three different concentrations of NaCl i.e. 1%, 3%, 6.5%. These different concentrations of NaCl were adjusted in MRS broth. After that the tubes were incubated for 24-48 hours [22].

Detection of antibacterial activity of selected strains

For antibacterial screening of isolated bacterial cultures, these cultures were inoculated in MRS broth and incubated at 37°C for 24-48 h on shaker to carry out fermentation process. 2 ml of the broth was taken in eppendorf and centrifuged at 14224 rcf for 10 min. After centrifugation, antibacterial activity of supernatant of strains was tested against test organisms by following the protocol of agar well diffusion method. Briefly, tested microorganisms were spread evenly on solidified agar plates and were allowed to dry. Wells (each 7mm in diameter) on agar plates were made with the help of sterile borer following with loading of wells with 60 – 70 µl of isolated bacterial cultures filtrate (supernatant). Plates were then incubated at 37°C for 24 hours [22].

Yogurt processing

Processing of fresh apple

Fresh apples were purchased from the market and were cut into wedges with the help of stainless-steel knife while precautions were taken to avoid cross contamination [23]. Apples were selected because of their prebiotic efficacy enriched with probiotic strains.

Production of Lactobacillus enriched apples

Lactobacillus enriched apple was prepared by mixing fresh apple pieces with biomass of Lactobacillus and was incubated at 37°C for 48 hours without agitation. Then liquid was drained off and enriched apple was washed twice with Ringer’s solution to remove free cells. The prepared enriched support was then freeze dried [23].

Yogurt production

For yogurt production, milk was dispensed equally in test tubes, pasteurized and was allowed to cool down at 45°C. The viability of yogurt starter culture was checked and was added in each part following the addition of Lactobacillus enriched apple pieces except the one which was used as a control [23]. For probiotic yogurt processing, pasteurized milk was cooled quickly to 45°C. After that yogurt starter cultures were added along with probiotic strains except in control part in which only yogurt cultures were added [24]. Tubes were then incubated at 37°C to 45°C until the pH was decreased to 4.6-4.8. The yogurt samples were then transferred to cold storage and stored at 4°C for 10 days. During that period different physiochemical properties were measured [24].

Determination of physiochemical properties

The pH value of yogurt and milk was measured by using the pH meter at 0 and 10 days of storage [12]. The total solid content of samples was determined by method described as follows; initial weight of sample was taken and then it is placed in hot dry oven at 110°C. After drying, samples were cooled at room temperature and final weight of sample was then measured. Final weight of sample after drying was considered as total solid contents. Total solid content was calculated by using following formula [23].

Total solid content = initial weight of sample – final weight

To determine syneresis, 3 tubes of 2ml of yogurt or fermented milk were weighed and placed in a centrifuge. Tubes were then centrifuged at 2,000 rcf for 5 min. After that separated serum was weighed. Syneresis was calculated using the following formula [24].

Syneresis % = (Wsx 100)/Wy

Where Ws was the supernatant weight after centrifugation and Wy was the weight of the yogurt or fermented milk in the tube.

Statistical analysis

Ninteen (19) strains were isolated from different dairy products out of which 10 strains were considered as Lactobacillus strains on the basis of colony morphology and biochemical characterization done according to Bergey’s manual of Bacteriology scheme [19]. According to morphological characterization, all of the strains were round in shape and all strains have entire edges. Antimicrobial activity of 10 selected Lactobacillus strains was checked against different pathogens. For a strain to be of a good probiotic character, it is necessary that they should inhibit the growth of harmful microorganisms. Strains such as S4BM2, S1BM4, S4Y2, S2Y1, and S2Y2 showed viable zone of inhibition against different pathogens including Enterobacter spp., E. coli (9473), E. coli (ETCC), MRSA 6, Klebsiella spp., Salmonella typhi and P. aeruginosa. Maximum zone of inhibition was shown by S4BM2 (15mm) against Salmonella and same strain showed zone of inhibition (14mm) against E. coli. Study of Kumar and Kumar (2015) showed that only one strain (LBS2) inhibited the growth of E. coli, however, in our study it was shown that strains S4BM2, S1BM4, S4Y2, S2Y2, S2Y1 inhibited the growth of E. coli [18]. Five strains with best zones were selected for the evaluation of further probiotic properties. The growth of isolates at different temperatures (25°C, 37°C and 45°C) and different NaCl concentration (1%, 3% and 6.5%) confirms that isolated strains are Lactobacillus. Similar results were present in the study of Pundir et al., [22]; they isolated strains from different food samples. In their study, all of the strains were also able to grow in the presence of different NaCl concentrations and at different temperatures. Main reason to select such temperature range (25°C, 37°C, and 45°C) was to find out whether the isolated strains were able to grow within normal body temperature range or not. NaCl was considered as an inhibitory substance so, it was necessary to check whether the strains were able to grow in the presence of different concentrations of NaCl concentrations or not [22]. Activity of probiotics was influenced by several gastrointestinal tract factors including low pH, pepsin and bile salt. For the strains to 

be selected as suitable probiotic, they should be able to grow at low pH and in the presence of 0.2% of bile salt. Isolates showing ≥50% resistance at pH 2 are considered as pH tolerant. At pH 2, 100% of the selected strains were able to resist this low pH and showed a percentage resistance of more than 50% and the greatest resistance was shown by S4BM2 (74%). However, in the study of Kumar and Kumar, highest resistance was shown by LBS2 at pH 3 which was 60.52% [18]. Isolated strains were also tested for bile salt (0.2%) tolerance. Strains showing percentage resistance ≥50% were considered to be tolerant to bile salt (0.2%). In the screening study all of the strains were able to grow at 0.2% bile salt after 24 hours of incubation. In an earlier study, researchers had observed highest resistance of strain LBS2 with bile salt (0.3% w/v) 72.36% [18]. In our study, the maximum growth was being shown by S4BM2 which was 81%. Pepsin is an enzyme of stomach which helps in the breakdown of protein which affects the activity of these bacterial strains in acidic condition of stomach. Tested strains maintain constant viability in the presence of pepsin enzyme. Our work was correlated with Feng et al., and the isolated strains were also able to grow at stimulated gastric juice [21].

Strains with good probiotic properties were selected for the production of probiotic yogurt and yogurt formed with Lactobacillus enriched apple pieces. Initial pH of buffalo milk was 6.5 and was decreased due to production of lactic acid by yogurt starter cultures. Yogurt sample without any strain (control) showed more decrease in pH as compared to the yogurt sample with Lactobacillus and the yogurt with Lactobacillus enriched apples at 0 day of storage. After 10th day of storage, decrease in pH of control showed more decrease in pH due to conversion of lactose into lactic acid. However, in case of yogurt produced with incorporation of enriched apple pieces, there was a delay in lactic acid production and hence less decrease in pH during the 10 day of storage [23]. The analysis of syneresis is of particular importance especially during storage. In our research, the value of syneresis at 10 day of storage varied from 23.5% to 14.9% and showed significant results as compared to yogurt processed by Lactobacillus strains only. Least syneresis was shown by yogurt sample processed by the addition of apple pieces enriched with S1BM4 strain. Our work was correlated with Bosnea et al., [23]. In their research, less syneresis was observed in yogurt produced with enriched apple pieces as compared to control and yogurt processed with probiotic strains [23].

Total solid content of yogurt being processed by combination of Lactobacillus strains and apple pieces showed significant result as compared to yogurt processed by Lactobacillus strains and starter cultures only and the best result was shown by S2Y2 and apple pieces.

In our study we use apple pieces because they have pectin that account for total of 50% and have prebiotic benefit and when they are combined with probiotic Lactobacillus, they allow these strains to survive better under storage conditions. It was concluded that yogurt formed with synbiotic effect (probiotics combined prebiotics) showed better results as compared to other yogurt with only probiotic effect. This combination of apple pieces and probiotic strains can further be used in different dairy industries for yogurt processing to improve the quality of yogurt.