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Isolation, Identification and Analysis of Probiotic Properties of Lactobacillus Spp. from Selective Regional Yoghurts

M.Z. Hoque
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh.

F. Akter
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh.

K.M. Hossain
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh.

M.S.M. Rahman
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh.

M.M. Billah
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh.

K.M.D. Islam
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh.

Abstract/ Executive Summary:

Yoghurt is a potential source of probiotic lactobacilli. In the present study, Lactobacillus spp. were isolated from two regional yoghurts in Bangladesh, which were identified on the basis of their colony morphologies and some biochemical tests. It was observed that isolated Lactobacillus spp . were resistance to inhibitory substances like phenol (0.4%), NaCl (1-9%) and bile acid (0.05-0.3%). Additionally, good growths were observed in the presence of 1% NaCl and 0.3% bile acid. The isolated Lactobacillus spp. did show good survival abilities in acidic (pH 2.5) and alkaline (pH 8.5) conditions, while, their maximum growth was observed at pH 5.0 for lactobacilli isolated from Bogra yoghurt and at pH 6.5 for lactobacilli isolated from yoghurt of Khulna region of Bangladesh. Isolated lactobacilli were able to produce organic acid in skim milk which was determined by titrimetric method. The Lactobacillus spp. also did show good survival abilities in simulated gastric juice at pH 2.22 and pH 6.6 (Control). Their susceptibility to selected nine antibiotics was determined in terms of minimum inhibition concentration (MIC). The MICs results showed that, Lactobacillus spp. isolated from Bogra yoghurt were sensitive to amoxicillin, moderately sensitive to gentamycin, clindamycin, azithromicin and resistant to kanamycin, nalidixic acid, metronidazol, cefradine and tetracyclin. On the other hand, Lactobacillus spp. isolated from yoghurt of Khulna region were sensitive to gentamicin, clindamicin and resistant to amoxicillin, tetraciclin, kanamicin, nalidixic acid, metronidazol, azithromicin and cefradine. In conclusion, most of the results from the present experiments showed that, there were variations in probiotic properties of the isolated Lactobacillus spp. from different regions.

Keywords: Regional yoghurts, Inhibitory substances, Simulated gastric juice and MIC

Location: Grameen Dannone yoghurt, Bogra and Khulna Districts of Bangladesh.

Start Date:

End Date:

Program Area: Quality and Nutrition

Commodity: Yogurt

Objectives:

The experiment was carried out with the following objectives:

1. Isolation and identification of lactobacillus spp. from yoghurts of different regions in Bangladesh.

2. Determination of optimal growth and pH of isolated lactobacillus strains.

3. Quantification of lactic acid production in milk.

4. Determination of antibiotic and bile tolerance.

5. Determination of resistance to gastric juice.

Methodology:

Yoghurt samples were collected from Grameen Dannone yoghurt, Bogra and Khulna Districts of Bangladesh due to their wide acceptance among the consumers of Bangladesh. Immediately after collection, the samples were stored aseptically in low temperature (-4°C) refrigerator to protect from contamination and deterioration. In preliminary investigations the yoghurt sample of Grameen Dannone and Barisal region were discarded due to lack of sufficient probiotic properties. The bacteria Lactobacillus spp. were isolated from yoghurt samples by using modified MRS broth and MRS agar media. Additionally, 0.05% cysteine was added to MRS to improve the specificity of this medium for isolation of lactobacillus. The pH of the media was adjusted to 6.5. Lactobacillus was isolated from yoghurts by using MRS medium. One gram of each sample was dissolved into 100 ml of MRS broth at pH 6.5. After dissolving into MRS broth they were shaken homogeneously and were incubated at 37°C for 24 h in aerobic condition. The cultures were subjected to five subculture at 37°C under low pH (pH 4.5) and anaerobic condition in the presence of 10% CO₂ to remove unwanted bacteria. After seven subcultures, the bacterial culture was streak onto MRS agar media at pH 4.8. Finally, the single colony of lactobacillus was isolated by observing their colony morphology and some biochemical tests (Gram staining, catalase, endospore and motility test) and the culture were maintained in MRS broth at pH 5.5. The isolated bacteria were identified as Lactobacillus spp. by observing their morphological characteristics and by means Gram staining, motility test, catalase test, endospore test, milk coagulation activities, 0.4% bacteriostactic phenol tolerance test and 1-10% NaCl tolerance test. MRS broth containing inhibitory substances such as 0.4% phenol and 1-10% Nacl were inoculated with 1% (v/v) 24 h active culture of lactobacillus and incubated (anaerobically) for 24 h at 37°C in the presence of 10% CO₂. For the determination of optimal growth and pH of lactobacillus, 1% (v/v) fresh over night culture of lactobacillus were inoculated into MRS broth with varying pH ranging from 2.5-8.5. The pH were adjusted with concentrated acetic acid (99%) and 5 N NaOH. The inoculated broths were incubated in anaerobic condition 24 h at 37°C in the presence of 10% CO₂ . After 24 h of incubation growth of the bacteria were measured using a spectrophometer, reading the optical density at 560 nm (OD₅₆₀ ) against the uninoculated broth. Sensitivity of lactobacillus to different antibiotics was determined by the minimum inhibitory concentration (MIC) assay. The assay was done using the method. According to this method, stock solutions 128 µg/ml of 9 different selected antibiotics such as amoxicillin, gentamicin, tetracyclines, clindamicins, kanamycin, nalidixic acid, metronidazole, azithromicin and cefradine were prepared. All antibiotics except Kanamycin were obtained from Square Pharmaceuticals Ltd. Bangladesh Stock solutions of selected antibiotics were two-fold diluted in the range 0.25 µg/ml to 1024 µg/ml in MRS broths. For preparation of the test inoculums the 16 h active cultures of lactobacillus were adjusted to 0.5 McFarland standards (10⁷ to 10⁸ cfu/ml of bacteria) by adding sterile distilled water. 50 µl of test inoculums were inoculated into each dilution and incubation was done for 24 h at 37°C in the presence of 10% CO₂ in a CO₂ incubator.

Source of Information: World Journal of Dairy & Food Sciences 5 (1): 39-46, 2010, ISSN 1817-308X

Article Link (Online Journal): http://idosi.org/wjdfs/wjdfs5(1)/7.pdf

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, the experimental results showed that isolated Lactobacillus spp. are able to tolerate inhibitory substances such as 0.05-0.3% bile acid, 0.4% phenol, 1-8% NaCl and also able to survive in simulated gastric (pH 2.2) condition, as well as in alkaline (pH 8.5) condition. The isolated lactobacilli were able to produce organic acid in milk. From the MIC results, We have found that the Lactobacillus spp. are sensitive to amoxicillin, gentamycin, clindamycin, azithromycin resistant to kanamycin, nalidixic acid, metronidazol, cefradine and tetracyclin which are most frequently used antibiotics in Bangladesh regarding treatments of human and animals.

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