Screening of Probiotic Lactic Acid Bacteria Isolated from Fermented Pak-Sian and Its Application as a Starter Culture

Abstract

Fermented Pak-Sian is commonly consumed in the northeastern parts of Thailand and is rich in lactic acid bacteria (LAB). However, each region has a different fermentation recipe to ferment Pak-Sian which may affect the diversity of LAB. This research aimed to isolate and identify LAB from fermented Pak-Sian samples, to determine their probiotic properties and to use probiotic starter culture for fermentation of Pak-Sian. The samples were collected from local markets in Kalasin, Sakon Nakhon, Khon Kaen and Maha Sarakham Provinces. LAB were isolated on MRS agar supplemented with bromocresol purple (0.05%) as a pH indicator. The putative LAB were selected based on the yellow zone surrounding the colony, catalase-negative and gram-positive characters. A total of 234 presumptive LAB were selected. These presumptive LAB were grouped using whole-cell protein patterns by SDS-PAGE. The results presented 61 presumptive LAB from 8 local markets in this study. These LAB were confirm and identified by 16S rDNA analysis. LAB identified 17 isolates as follows Pediococcus pentosaceus (KS12, KS218, KS230, SK337, MK74), Pediococcus sp. KS215 Lactobacillus plantarum (SK321, KK53, KK518, MK711, MK724), Lactobacillus brevis (SK335), Lactobacillus fermentum (SK324, SK48, SK434), Weissella cibraria (SK415, SK432). The selected strains were investigated for probiotic properties namely bile salt tolerance (0.3% bile salt), pH tolerance (pH 2.5), survival in simulated gastric and intestinal tract, antimicrobial activity (Bacillus cereus, Staphylococcus aureus, Samonella typhymurium and Escherichia coli) antibiotic susceptibility (streptomycin, rifampicin, vancomycin, ampicillin, azithromycin and chloramphenicol), biogenic amine production and haemolytic activity. The results found that 14 strains showed resistance to the pH 2.5 and all LAB isolates showed resistance to bile salt. High survival rates of LAB in simulated gastric and intestinal tract indicated resistance to simulated gastric and intestinal tract. LAB demonstrated antimicrobial activity by inhibiting the pathogenic bacteria indicator. Moreover, most LAB strains were resistant to all antibiotics tested and some Lactobacillus strains were moderately susceptible to cholamphenical, rifampicin and ampicillin. All 14 strains did not produce biogenic amine., Only 8 out of the selected 17 strains showed no haemolysis activity (g-haemolysis) and therefore were selected for mucin adhesion capacity determination. Three strains (P. pentosaceus, Lb. fermentum and Lb. brevis) showed 0.03-2.39% adhesion. Lb. fermentum (SK324) and Lb. brevis (SK335) showed high percentage of adhesion capacity (2.39% and 2.34%, respectively). Therefore, it was chosen to be used as a starter culture for fermentation of fermented Pak-Sian. Fermented Pak-Sian was subjected to 4 treatments: Lb. fermentum (SK324), Lb. brevis (SK 335), mixed culture (Lb. fermentum (SK324); Lb. brevis (SK 335); 1:1) and compared with control (no add starter culture). In this study, 106 CFU/ml of initial starter culture was used and fermented for 3 days. The results showed that pH value of starter culture was lower than the control treatment The lactic acid bacteria (LAB) count and total plate count (TPC) showed an increase of up to 106 CFU/g during the fermentation of Pak-Sian of up to which is an added advantage to consumer health. The presence of short chain fatty acid indicated that all the treatments produced acetic acid while propionic acid and butyric acid were not found after fermentation. The sensory evaluation acceptance scores obtained during the utilization of starter culture and control treatment were not significant (P>0.05) in terms of colour, smell, taste, texture and overall acceptance. Therefore, these strains may be used as an alternative starter culture to produce fermented Pak-Sian thereby leading to an improvement in product development and overall increase in potential health benefits.

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