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Detection of Lactobacillus in Pork and Traditional Pork Products of Mizoram, India and Evaluation of its Probiotic Characteristics

K. Kalita1, D. Deka1,*, P. Hazarika2, E. Motina1, J. Lalmuanpuia1, S. Kumar3
1Department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences and Animal Husbandary, Central Agricultural University, Selesih, Aizawl-796 014, Mizoram, India.
2Department of Livestock Products Technology, College of Veterinary Sciences and Animal Husbandary, Central Agricultural University, Selesih, Aizawl-796 014, Mizoram, India.
3Department of Veterinary Microbiology, College of Veterinary Sciences and Animal Husbandary, Central Agricultural University, Selesih, Aizawl-796 014, Mizoram, India.

ABSTRACT

Background: Sa-Um, a traditional pork product of Mizoram could be an alternative and easily available source for Lactobacillus starter culture with probiotic properties.

Methods: Traditional pork product (Sa-Um) and pig faecal samples from Mizoram were collected and analysed for number of viable Lactobacillus bacteria by spread plate method. Samples above 106 cfu/ ml were considered for isolation of probiotics. Gram positive and catalase negative presumptive Lactobacillus isolates were confirmed by PCR analysis of 16S-rRNA gene. The Lactobacillus strains were studied for viability at different temperatures, species identification by fermentation of sugars and probiotic properties namely tolerance to acid, bile salt and digestive enzymes, antagonistic activity and antimicrobial sensitivity.

Result: Four Lactobacillus strains, namely L. plantarum SU 2, L. plantarum SU 11 and L. acidophilus SU 38 from Sa-Um and L. plantarum PF 11 from pig faeces were suggested as probiotic bacteria. Lactobacillus plantarum SU 2 was the most potential probiotic candidate based on the studied probiotic properties.

INTRODUCTION

Sa-Um is a traditional pork product with its ethnicity of Mizoram in North East India. Fat is collected from the inner abdominal portion and other body parts of pig, cooked, chopped into pieces and are placed in a special container called Sa-Um ‘Bur’. The container is then placed over the fireplace and after three days or a bit longer, the Sa-Um is ready for use in the preparation of other foods (Lalthanpuii et al., 2015).
       
Probiotics are defined as live microbes which transit the gastro-intestinal tract and in doing so; they benefit the health of consumers (Bhardwaj et al., 2012). Lactobacillus is one of the most important probiotic organisms found primarily in the small intestine capable of stimulating immune system and interactions with other microorganisms (Gupta et al., 2021). Lactobacillus is Gram positive, rod shaped and generally deficient in catalase and use fermentable carbohydrates as energy source (Duar et al., 2017; Muhammad et al., 2018). The evolution of the genus Lactobacillus has counted to nearly 200 species. These bacteria are also involved in the processes of fermentation and preservation of food products and they are generally regarded as safe.
       
Sa-Um could be an alternative and easily available source for Lactobacillus starter inoculums with probiotic potential. Therefore, the present study was aimed to isolate and identify the Lactobacillus from pork and Sa-Um and in vitro evaluation of its probiotic characteristics.
 

MATERIALS AND METHODS

The study was conducted on 50 Sa-Um and 50 pig faecal samples collected randomly from Aizawl district of Mizoram, India between April to September, 2021.
 
Enumeration, isolation and identification of Lactobacillus
 
One gram of each sample was diluted in 9 ml of 0.1% peptone water, mixed and then 10-fold serially diluted. A volume of 0.1 ml of appropriately diluted sample was spread in Lactobacillus selection agar (LSA) plate in duplicates and incubated at 37°C for 48 hours and bacterial colonies were enumerated. The bacterial colony forming unit was calculated by multiplying the number of colonies with the dilution factor and was expressed as mean log cfu/ml of the original sample. Morphologically distinct colonies were sub-cultured by using Lactobacillus selection broth (LSB) and LSA to isolate the pure Lactobacillus culture. Presumptive Lactobacillus isolates were identified by morphological characteristics, positive Gram’s reaction and negative catalase reaction (Mannan et al., 2017).
 
Molecular confirmation of Lactobacillus
The presumptive Lactobacillus isolates were confirmed by PCR analysis ofgenus specific 16S-rRNA gene (250bp) using R16-1 F (5'-CTT GTA  CAC ACC GCC CGT CA-3') and LbLMA1-rev R (5'-CTC AAA ACT AAA CAA AGT TTC-3') primers (Dubernet et al., 2002).
 
Growth of Lactobacillus strains in different temperature
 
The PCR confirmed Lactobacillus strains showing bacterial colony count of 106 cfu/ml and above in original samples were studied for growth at 20°C and 30°C in LSB for 48 hours.
 
Detection of Lactobacillus species
 
The Lactobacillus strains showing growth in both 20°C and 30°C were identified for Lactobacillus species using a sugar fermentation panel of 17 sugars, namely arabinose, cellobiose, D- mannose, inositol, lactose, melezitose, melibiose, maltose, mannitol, raffinose, rhamnose, ribose, salicin, starch, sucrose, xylose and glucose (Ahirwar et al., 2017).
 
Evaluation of probiotic characteristics
 
Overnight cultured bacterial cells were harvested by centrifugation (10,000 × g, 5 minutes, 4°C), washed twice with PBS (pH 7.0) and then the cells were re-suspended applying required conditions for evaluation of following probiotic characteristics.
 
Acid resistance
 
The harvested bacterial cells were re-suspended in PBS with pH 2.0 and 3.0 and Lactobacillus strains were observed for the acid resistance based on viable colony counts on LSA in triplicates after incubation at 37°C for 0 and 3 hours (Maragkoudakis et al., 2006).
 
Bile salt tolerance
 
The harvested bacterial cells were re-suspended in PBS (pH 8.0) containing 0.5 and 1.0% bile salts and tolerance to the bile salt was evaluated based on viable colony counts in LSA in triplicates at 0 and 4 hours after incubation at 37°C (Argyri et al., 2013).
 
Digestive enzyme tolerance
 
The harvested bacterial cell suspension was prepared in PBS containing the enzymes, trypsin and pepsin, each at 2 and 8 gm/L concentration and incubated at 37°C for 2 hours and then viable colonies were counted in LSA plates in triplicate and incubated at 37°C for 36 hours (Veerapagu and Jeya, 2017).
 
Antagonistic activity
 
The antimicrobial activity of the Lactobacillus strains was determined against indicator bacterial pathogens, namely Salmonella Typhimurium, Staphylococcus aureus and Escherichia coli by using agar spot test and the average diameters (mm) of zones of inhibition were measured (Sharma et al., 2017).
 
Antimicrobial sensitivity
 
The Lactobacillus strains were subjected to antibiotic sensitivity assay by disk diffusion method against a panel of 12 antimicrobial agents used against Gram positive bacteria namelyAmpicillin (10 µg), Ceftriaxone (30 µg), Ciprofloxacin (5 µg), Chloramphenicol (30 µg), Clindamycin (2 µg), Erythromycin (15 µg), Gentamicin (10 µg), Kanamycin (30 µg), Penicillin(10 µg), Rifampicin (5 µg), Streptomycin (10 µg) and Tetracycline (30 µg) (Angmo et al., 2016).
 
 

RESULTS AND DISCUSSION

Enumeration, isolation and molecular identification of Lactobacillus
 
A total of 16 Sa-Um and 3 pig faecal samples have shown growth in LSA, out of which, 3 Sa-Um (SU 2, SU 11 and SU 38) and 1 pig faecal (PF 11) samples showed the viable colony count more than 106cfu/ ml (Table 1). Nineteen presumptive Lactobacillus strains were isolated contributing to 16 from Sa-Um and 3 from pig faeces and subsequently, 4 Lactobacillus strains from Sa-Um (SU 2, SU 11 and SU 38) and pig faeces (PF 11) were confirmed by PCR assay of 16S-rRNA gene (250 bp) (Fig 1). All the4 PCR positive strains have grown at both 20° and 30°C and were suggestive of probiotic Lactobacillus. The fermentation of 17 sugars by the 4 Lactobacillus strains identified that strains SU 2, SU 11 and PF 11 were L. plantarum and SU 38 was L. acidophilus (Table 2).

Table 1: Enumeration (log cfu/ml±SE) of Lactobacillus isolated from Sa-Um and pig faeces in Aizawl district, Mizoram.



Fig 1: Agarose gel electrophoresis plate showing PCR amplicons of 16S-rRNA gene (250 bp) of Lactobacillus M: 100 bp ladder; L1: Negative control; L2: Positive control; L3 to L6: Positive samples.



Table 2: Species detection of Lactobacillus strains isolatedfrom Sa-Um and pig faeces by sugar fermentation test.


 
Acid resistance
 
The viable colony counts of the 4 Lactobacillus strains ranged between 3.41±0.06 to 6.28±0.36 log cfu/ml after 3 hours of exposure to pH 3.0 and 2.0 (Table 3) indicating that all the strains were acid tolerant at pH 3.0 and 2.0. The L. plantarum SU2 strain showed the highest growth (5.17 ±0.29) and the growth was significantly (P<0.01) higher than the L. acidophilus SU 38 strain (3.41±0.06)at lower pH 2 after 3 hours. The ability to transit through the low pH of stomach (1.5-2) before reaching the intestine is a crucial characteristic of any probiotic bacteria (Gupta et al., 2021). The high survival rate of L. plantarum and L. acidophilus strains at gastric pH of 2.0 and 3.0 has been reported by Moreno et al. (2018); Betancur et al., (2020); Nath et al. (2020); Barzegar et al. (2021).

Table 3: Acid tolerance of the Lactobacillus strains isolated from Sa- Um and pig faeces.


 
Bile salt tolerance
 
All the 4 Lactobacillus strains tolerated 0.5% and 1%bile salt up to 4 hours of incubation although their growth rate decreased at higher bile salt concentration. The highest growth was visible in L. plantarum SU 2 strain (3.25±0.24) after an exposure of 4 hours in 1% bile salt. However, L. plantarum SU 11 showed significantly (P<0.05) higher growth (4.90±0.30) after an exposure to 4 hours at 0.5% bile salt than L. plantarum PF 11 strain (3.46±0.59) (Table 4). It is necessary to evaluate the bile salt resistance of probiotic bacteria for the ability to resist the unfavourable conditions of the intestinal tract for their colonization and metabolic activity. Tolerance to the bile salt among the probiotic Lactobacillus strains were also reported by Huang et al. (2021).

Table 4: Bile salt tolerance of Lactobacillus strains isolated from Sa- Um and pig faeces.


 
Digestive enzymes tolerance
 
The 4 Lactobacillus strains were found to be tolerant to the digestive enzymes, trypsin and pepsin at 2 and 8 g concentration up to 2 hours although the viability decreased at higher concentration (8 g) of both the enzymes. Among the strains, L. plantarum SU 2 strain showed the highest growth at 8g concentration of trypsin (4.30±0.08) and pepsin (4.26± 0.03) (Table 5). Similarly, Nath et al. (2020); Barzegar et al., (2021); Huang et al. (2021) had reported that Lactobacillus could survive different concentrations of trypsin and pepsin.

Table 5: Digestive enzymes tolerance of Lactobacillus strains isolated from Sa- Um and pig faeces.


 
Antagonistic activity
 
The antibacterial activity of Lactobacillus strains showed weak to moderate inhibition against the three indicator organisms (Table 6). Lactobacillus acidophilus SU 38 and L. plantarum PF 11 showed weak inhibition of E. coli and L. plantarum SU 2 and SU 11 showed moderate inhibition against the organism. However, L. plantarum SU 2, L. acidophilus SU 38 and L. plantarum PF 11 showed weak inhibition against S. aureus and L. plantarum SU 11 showed moderate inhibition against it. All the 4 strains showed weak inhibition against S. typhimurium Antibacterial activity is a functional requirement for probiotic bacteria as they could be used as bio-preservatives. The antibacterial effect exerted by Lactobacillus is due to the production of antibacterial metabolites like different organic acids, hydrogen peroxide, bacteriocins and other antibacterial substances (Kabir, 2009). The variable antibacterial activity against common pathogens was also observed by Wang et al. (2020); Erdogmus et al. (2021).

Table 6: Antagonistic activity of Lactobacillus strains from Sa- Um and pig faeces.

  
 
Antimicrobial sensitivity
 
All the 4 Lactobacillus strains were sensitive to most of the antibiotics namely, Chloramphenicol, Clindamycin, Erythromycin, Penicillin, Ceftriaxone and Amipicillin followed by 3 strains to Tetracycline, Rifampicin and Gentamicin and 2 strainsto Ciprofloxacin and Streptomycin indicating considerable food safety characteristic. However, all the strains were resistant to Kanamycin. Lactobacilli possess intrinsic resistance to Kanamycin, Gentamicin and Streptomycin (Fraqueza, 2015). The L. plantarum SU2 strain was sensitive to 11 antibiotics except Kanamycin. Variable resistance pattern of Lactobacilli to different antibiotics were also reported by Handa and Sharma (2016); Bindu and Devi (2020).
       
Thus, in vitro evaluation of probiotic characteristics of 4 Lactobacillus strains has suggested their probiotic potential and indicated that L. plantarum SU 2 was the most potential probiotic candidate. Similarly, Moreno et al. (2018) and Betancur et al., (2020) reported L. plantarum with probiotic properties from meat and meat products.
 

CONCLUSION

The present study primarily confirmed4 Lactobacillus with promising probiotic properties from the ethnic pork product (Sa-Um) and pork faeces of Mizoram. Based on the studied probiotic properties, 3Sa-Um Lactobacilli (L. plantarum SU2, L. plantarum SU 11 and L. acidophilus SU 38) and 1 faecal Lactobacillus (L. plantarum PF 11) were suggested as probiotic strains. The susceptibility of the strains to most of the studied antibiotics is promising from food safety point of view. Lactobacillus plantarum SU 2 was the most promising probiotic candidate and Sa-Um may be explored as a potential traditional food source of probiotics. However, to characterize as ideal probiotic microbes, in depth in vitro and in vivo studies for safe application of these Lactobacilli in food are necessary.
 

CONFLICT OF INTEREST

None.
 

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