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Research Detail

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M. U. Habiba
Department of Dairy and Poultry Science, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

A. A. Jhinuk
Department of Dairy and Poultry Science, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

S. M. R. Sumon
Department of Medicine, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

M. Jahan
Department of Microbiology and Public Health, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

S. Ahmed
Department of Dairy and Poultry Science, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

M. D. Hossain
Department of Animal Science and Nutrition, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

A. S. M. Selim
Department of Animal Science and Nutrition, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

M. M. Rahman
Department of Dairy and Poultry Science, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

The present study explored the traditionally prepared fermented buffalo milk curd to isolate and characterize Bifidobacterium sp. towards developing future probiotics. Standard methods have been applied to identify Bifidobacterium sp. using bifidobacteria selective media, bifidobacteria specific mupirocin resistance test, phosphoketolase activity and commercial biochemical kit. The strains further confirmed by xfp gene PCR amplification. The growth dynamics of the identified isolates was also determined by monitoring the turbidity of the cultured media spectrophotometrically. A total of 20 isolates obtained from selective media were found gram positive, catalase negative, rod or v-shaped, non-motile and identified as presumptive bifidobacteria. All the isolates were resistant (zone diameter <10 mm) to the antibiotic mupirocin. However, the phosphoketolase activity of the isolates were varied significantly (p<0.05). A high, moderate and weak/no phosphoketolase activity was exhibited by 35%, 20% and 45% isolates, respectively. In the contrary, fourteen isolates (70%) amplified well with phosphoketolase activity encoded xfp gene and produced a PCR product of 704 bp. Combining the findings of these three tests, only six isolates (30%) displayed mupirocin resistance, high phosphoketolase activity and amplification with xfp gene. Of these six isolates biochemical test confirmed three isolates as Bifidobacterium mongoliense and two as Bifidobacterium pseudolongum subsp. globosum. The exponential phase growth rate of the six isolates also varied significantly (p<0.05) and ranged from 0.16±0.01/h to 0.34±0.02/h. Traditionally prepared buffalo milk curd from Charfassion upazila of Bhola district, Bangladesh could be a potent source of Bifidobacterium sp. However, species-specific identification and elucidation of probiotic potential are pivotal for future industrial use.

  Growth rate, Mupirocin, Phosphoketolase, xfp gene
  Char area of Ranga Bali, Monpura, Dhal Char, Char Montaj and Char Kukri-mukri, Charfassion upazila of Bhola district
  
  
  Postharvest and Agro-processing
  Curd, Milk

For the isolation and biochemical as well as genus specific identification of Bifidobacterium species from traditional buffalo milk curd.

Collection of curd samples: The Charfassion upazila of Bhola district is one of the famous places to produce raw buffalo milk curd. The local curd manufacturers’ of the upazila collected raw milk from the buffalo farmers (called Bathan) of various Char areas like Ranga Bali, Monpura, Dhal Char, Char Montaj, Char Kukri-mukri. After preparation, curd samples from four different manufacturers’ were collected and transported to the laboratory of Dairy and Poultry Science department, Bangabandhu Sheikh Mujibur Rahman Agricultural University maintaining refrigerated temperature in ice box containing ice pack. Isolation of presumptive bifidobacteria: The presumptive bifidobacteria were isolated from the four buffalo milk curd samples using Bifidobacteria Selective Count Agar Base (HiMedia Laboratories Pvt. Ltd., India) supplemented with mupirocin (50 mg/L) and 1% glacial acetic acid (10 ml/L). After homogenization, each curd sample was serially diluted (from 10-1 to 10-6 ) with phosphate buffered saline (PBS; pH 7.2) and sample from each dilution was evenly spread onto the agar plates in duplicate. The inoculated plates were incubated under anaerobic condition (anaero-gas pack system includes anaero jar and gas pack, HiMedia Laboratories Pvt. Ltd., India) at 37°C for 48 hours. After incubation, at least five colonies from the high dilution plates for each sample were randomly selected. The selected colonies were re-purified by subsequent subcultures in the same media. The purified colonies were characterized using Gram stain, cell morphology, catalase reaction and motility tests. Gram-positive, rod shaped, non-motile and catalase-negative isolates were preserved at -86°C in Lactobacillus MRS broth (HiMedia Laboratories Pvt. Ltd., India) supplemented with 0.05% L-cysteine.HCl and 30% (v/v) glycerol for further analysis. Mupirocin resistance test: The antimicrobial susceptibility to mupirocin was performed by the agar disc diffusion method as described by Domingos-Lopes et al. (2017). After preparing, MRS agar plate supplemented with 0.05% L-cysteine. HCl was inoculated with the respective isolate by swab technique. Prior to swabbing, each isolate was grown in MRS broth containing 0.05% L-cysteine.HCl anaerobically at 37°C. Phosphoketolase assay: The phosphoketolase assay was carried out according to the protocol described by Orban and Patterson (2000). Each isolate was reactivated anaerobically by subculturing at least three times in MRS broth supplemented with 0.05% L-cysteine.HCl at 37°C. Cells were obtained from ten ml of overnight grown respective bacterial culture by washing twice (10,000×g, 4°C, 15 min) with phosphate buffer (0.05 M KH2PO4 and 0.05% L-cysteine. HCl mixed 1:1 (V/V), adjusted to pH 6.5) and resuspended in 1.0 ml of the same buffer. Washed bacterial cells were incubated with 0.4ml (450 mg/ml stock solution) of hexadecyltrimethylammonium bromide (cetrimonium bromide, CTAB) for 5 min prior to the assay. After pretreatment, 0.25 ml of sodium fluoride (NaF, 3 mg/ml) and sodium iodoacetate (5 mg/ml) solution in H2O was added followed by the addition of another 0.25 ml sodium fructose-6-phosphate (80 mg/ml in H2O). The solution was vortexed and then incubated at 37o C for 30 min. After incubation, 1.5 ml of hydroxylamine. HCl (13 g/100 ml) was added, vortexed and allowed to incubate at room temperature for 10 min. One milliliter of TCA (15%, W/V), 1.0 ml of 4N HCl and 1.0 ml of ferric chloride (FeCl3.6H2O, 5% W/V in 0.1 N HCl) were added to the tubes, followed by vortexing. The color formation was recorded either using a qualitative scale (visualization) or spectrophotometrically at 435 nm. For spectrophotometric determinations, the stopped reaction mixture was centrifuged (10,000×g, 4°C, 15 min) and the supernatant was measured using a spectrophotometer (Optizen POP, Mecasys Company, Korea). Test tubes containing all the reagents without cells were used as blank. All test tubes were prepared in triplicate. Genus specific identification of bifidobacteria isolates: The genus specific identification of the purified isolates was performed according to the protocol described by Kharchenko et al. (2015). The conventional PCR targeting the gene xfp was applied. After overnight reactivation at 37°C anaerobically, each presumptive pure bifidobacteria isolate was washed twice with PBS (pH 7.2) at 6,000×g at 4°C. According to the manufacturer guidelines, the genomic DNA from each washed isolate was extracted using E.Z.N.A. bacterial DNA Kit (Omega Bio-Tek, USA). Biochemical tests for identification of the isolates: The biochemical tests for identification of all the isolates were carried out using a bifidobacteria specific biochemical identification kit (HiMedia Laboratories Pvt. Ltd., India). Measurement of growth dynamics: The growth curve of the target bifidobacteria isolates was observed by monitoring absorbance and pH of the cultured media at different interval of incubation. After reactivating the frozen isolates for at least two times, the overnight culture of the respective isolates were inoculated at 1% (v/v) in a test tube containing 10 ml of MRS broth supplemented with 0.05% L-cysteine. HCl and incubated at 37°C for 24h under anaerobic condition. Test tubes without inoculum were used as control. At every 6 h interval, absorbance and pH of the cultured media was measured at 660 nm using a spectrophotometer and pH meter (Hanna Instruments, Inc. USA), respectively. Statistical analysis: The data generated from the study were inserted into excel sheet (Excel 2007, Microsoft, Redmond, Washington, USA) and were analyzed with the add-in software yStat 2008 (Shinya Yamazaki, Koriyama, Japan) as per the requirement of the study. One-way analysis of variance (ANOVA) with SNK multiple range tests among means was carried out. All tests of significance were two-tailed, and p<0.05 were considered as significant. 

  http://journal.safebd.org/index.php/jafe; Vol 2 No 1 March 2021 Pages 70-76 e-ISSN 2708-5694
  http://doi.org/10.47440/JAFE.2021.2112
Funding Source:
  

In the present study, a naturally fermented traditional buffalo milk curd obtained from Charfassion upazila of Bhola district, Bangladesh was explored to isolate and identify bifidobacteria, a potent probiotic candidate. A substantial number of isolates were confirmed as Bifidobacterium sp. based on their cultural, morphological, biochemical, phosphosketolase and genus specific characteristics, suggesting that buffalo milk curd is as a prospective niche of bifidobacteria. The source of bifidobacteria in the product could be mammalian secretion and/or environment during the entire production process. Since buffalo milk curd has already been using for human consumption, Bifidobacterium sp. obtained from this study could be considered as safe. However, identification at species level and evaluation of probiotic potential are crucial for future industrial application of these isolates.

  Journal
  


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