Major Project #Draft 2

Reuterin production by lactobacilli isolated from pig faeces

and evaluation of probiotic traits

Rodríguez et al. (2003) conducted a study to determine the production of reuterin by lactobacilli isolated from pig faeces and to evaluate their potential as probiotic bacteria. Thirty-three samples of pig faeces were serially diluted and plated on de Man Rogosa and Sharp (MRS) agar. Approximately six colonies per sample were selected for further study. All isolates were screened for their reuterin production in the presence of glycerol by using colorimetric assay at 490 nm. Carbohydrate fermentation test kit (API 50 CHL) were used for the biochemical characterization of reuterin-producing isolates. A polymerase chain reaction (PCR) method based on amplification of 16S RNA coding sequences of L. reuteri and L. fermentum was used to identify selected isolates at species level, with primers pairs LOWLAC-REUT1 and LOWLAC-FERM1. Genomic DNA from L. reuteri CECT 925T and L. fermentum CECT 4007T were used as control in PCR reactions. Reuterin-producing isolates were exposed to HCl at pH 3 for 1 h and consecutively to 0.3% oxgall for 1 h to evaluate their survival under simulate gastric and intestinal conditions. Neutralized supernatants from cultures grown in MRS broth were used to determine the presence of bacteriocin-like antimicrobial activity by an agar diffusion assay using Enterococcus faecalis EF, L. buchneri St2A, Listeria monocytogenes Ohio, L. plantarum CECT 4645, Clostridium tyrobutyricum NZ8 and E. coli K12 as indicator strains. Antimicrobial activity was detected by the presence of inhibition zones. The results showed that 28 of 165 lactobacilli isolates produced reuterin in the presence of glycerol. Six isolates yielding high levels of reuterin (> 15 mg/mL) with respect to type strain Lactobacillus reuteri CECT 925T were identified as L. reuteri. They were able to survive at pH 3 and subsequent exposure to 0.3% oxgall and presented bacteriocin-like activities. The researchers suggested that high yields of reuterin may be obtained from selected isolates of L. reuteri. Probiotic characteristics of isolates studied could be applied in food and feed. However there are some limitations.

1) Resistance to low pH and bile salts in this study under simulate gastric and intestinal conditions in this study were not correlated with presented conditions in stomach and small intestine. According to Argyri et al. (2013), resistance to bile salts was assessed in terms of viable colony counts and enumerated after incubation culture in medium with 0.5% bile salts at 37 °C for 0, 1, 2, and 4 h, reflecting the time spent by food in the small intestine. For evaluation of resistance to low pH, the experiment was performed by incubation culture in medium with pH 2.5 at 37 °C for 0, 0.5, 1, 2, and 3 h, reflecting the time spent by food in the stomach.

2) Neutralized supernatant used for determination of bacteriocin-like antimicrobial activity were not exclude the inhibitory effect of other antimicrobial substances such as hydrogen peroxide and diacetyl which produced by lactobacilli. Therefore, inhibitory effect might not only be from bacteriocin.

3) The researchers used MRS agar without addition of CaCO₃ in the isolation of lactobacilli. Due to some microorganisms (such as yeast, mould, Bacillus, Staphylococcus) could grow in MRS medium, this isolation medium used might be difficult to select only lactobacilli. Thamacharoensuk et al. (2013) reported that lactic acid bacteria such as lactobacilli were isolated by an enrichment technique before plating medium on MRS-CaCO₃ agar. This procedure was easy to select acid-producing bacteria by occurrence of a clear zone around the colonies after incubated at room temperature for 48-72 hours.

The strength of this study is that biochemical characterization and PCR amplification of 16S RNA coding sequences used are widely accepted in many laboratories for bacterial identification in genus and species levels, respectively. Especially, 16S RNA coding sequences could differentiate bacterial species up to 99%. Another advantage of this study is that the results were clearly described with text, table, figures and conclusion which were made relation to results in this study.

References

Argyri, A. A., Zoumpopoulou, G., Karatzas, K. -A. G., Tsakalidou, E., Nychas, G. –J. E., Panagou, E. Z., & Tassou, C. C. (2013). Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests. Food Microbiology, 33: 282-291.

Rodríguez, E., Arqués, J. L., Rodríguez, R., Nuñez, M. & Medina, M. (2003). Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits. Letters in Applied Microbiology, 37: 259-263.

Thamacharoensuk, T., Thongchul, N., Taweechotipatr, M., Tolieng, V., Kodama, K., & Tanasupawat, S. (2013). Screening and characterization of lactic acid bacteria from animal faeces for probiotic properties. The Thai Journal of Veterinary Medicine, 43(4): 541-551.