Asian Journal of Microbiology, Biotechnology & Environmental Sciences Paper

LAB ISOLATED, SCREENED AND SELECTED FROM LOCAL INDIGENOUS FERMENTED BEEF SAUSAGE (NAEM-NEUA) PRODUCTS FOR PROBIOTIC STARTER CULTURES IMPLEMENTATIONS

TITINAN BUABAN AND BORWONSAK LEENANON

Abstract

The high potential lactic acid bacteria (LAB) were isolated from local indigenous fermented beef sausage (Naem-Neua) products collected from three different small-scale food fermentation industries which are famous for their good quality beef in Sakon Nakhon province located in the Northeastern region of Thailand. Naem-Neua products were collected after they had been produced for 48-72 h and subsequently maintained under refrigerated condition (below 4 °C). The sample location codes were SN1, SN2 and SN3. Naem-Neua products were investigated for their physicochemical characteristics including pH, Aw, % titratable acidity, surface color and texture profile analysis (TPA). It was found that pH of SN3 was the highest value (pH 5.05) whereas Aw values of SN2 and SN3 were the highest ones (0.97) (p≤0.05). Also, % titratable acidity of SN1 was the highest value (0.45) (p≤0.05). However, only the pH of SN1 was complied with Thai Community Product Standard; Fermented Beef Sausage, Naem-Neua; TCP 470-2555 (2012). In addition, L* of SN1 was the highest value (41.32) while a* and b* of SN2 and SN3 were the highest ones (a* = 4.63 and 5.33, respectively, b* = 20.80 and 20.23, respectively) (p≤0.05). Moreover, hardness, gumminess and chewiness values of SN2 were the highest ones as they were 22.05 N, 12.45N and 7.35N.mm, respectively whereas springiness values of SN2 and SN3 were the highest ones as they were 0.59 mm and 0.63 mm, respectively (p≤0.05). Besides, cohesiveness value of SN1 was the highest one (0.69) (p0.05). For microbiological analysis, total aerobic plate count, lactic acid bacteria, yeast and mold were reported for all samples. In addition, Staphylococcus aureus, Bacillus cereus, Salmonella were not found in any samples which were complied with Thai Community Product Standard; Fermented Beef Sausage, Naem-Neua; TCP 470- 2555 (2012) regardless of Clostridium perfringens in SN2 (3.43 Log CFU/g). For enumeration, isolation and screening of LAB as starter cultures from Naem-Neua products, colonies were randomly selected from MRS agar plates of Naem-Neua samples (SN1, SN2, SN3) and streaked again on MRS agar for further purification of the isolates. Primary screening of 45 LAB isolates were conducted using agar well diffusion assay and clear zones were recorded. The results showed that only 16 out of 45 LAB isolates could inhibit the growth of the tested pathogens. After that, secondary screening of 16 LAB isolates was then explored for their tolerances to high temperatures, acidic pHs, bile, high concentrations of lactic acid and sodium chloride. It was revealed that LAB isolates could not grow at 4 °C, 15 °C and 50 °C whereas most LAB isolates could grow at 30 °C, 37 °C and 45 °C. However, some LAB isolates including ITP01, ITP07, ITP12, ITP14 and ITP15 could not grow at 45 °C. In addition, for tolerances of LAB isolates to acidic pHs and bile salt, it was discovered that ITS14 could survive the most in MRS broth at pH 3.0 (adjusted with 1N HCl), however, all of LAB isolates did not appear in MRS broth at pH 2.0. Also, all of LAB isolates could survive in MRS broth supplemented with bile salt and it was found that ITP01 and ITP09 could survive the most in MRS broth added with 1% bile salt (p≤0.05). Furthermore, all of LAB isolates were not tolerant to lactic acid whereas most of them could tolerate to NaCl from 1.5% up to 5% as well as ITY11 and ITS14. Finally, two high potential LAB isolates including ITY11 and ITS14 were selected for probiotic starter cultures implementations and they were subsequently identified by 16s rDNA sequencing as Lactobacillus plantarum for ITY11 and Lactobacillus pentosus for ITS14 with 99.86 and 100% similarity, respectively.