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Amylolytic lactic acid bacteria recovered and screened from plaa-som and its amylolytic enzyme
Abstracts / Journal of Biotechnology 136S (2008) S743–S750
References Chen, Y.S., Yanagida, F., Shinohara, T., 2005. Isolation and identification of lactic acid bacteria from soil using an enrichment procedure. Lett. Appl. Microbiol. 40, 195–200. Paludan-Müller, C., Madsen, M., Sophanodora, P., Gram, L., Moller, R.L., 2002. Fermentation and microflora of plaa-som, a Thai fermented fish product prepared with different salt concentrations. Int. J. Food Microbiol. 73, 61–70.
doi:10.1016/j.jbiotec.2008.07.1772 VIII2-Y-003 Amylolytic lactic acid bacteria recovered and screened from plaa-som and its amylolytic enzyme Narumon Phonyiam 1,2 , Sirinda Yunchalard 1,3,∗ 1
Department of Biotechnology, Khon Kaen University, Khon Kaen 40002, Thailand 2 Graduate school of Khon Kaen University, Khon Kaen 40002, Thailand 3 Centre of Fermentation for value added agricultural product (FerVAAP), Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand E-mail address: [email protected] (S. Yunchalard).
Plaa-som, a Thai fermented fish product, which production process consists of whole fish or fish fillets mainly from a freshwater fish, Barbodes gonionotus, raw or cooked rice, crushed fresh garlic, and salt. LAB was found to be the most dominant group of microorganisms from plaa-som. In this study, we screened LAB in plaa-som during its fermentation period. A total of 785 LAB isolates were confirmed to be LAB from 798 acid producing isolates recovered and recognized from plaa-som during its 7-day fermentation. These 785 LAB isolates were used as a source of screening for ALAB. Forty isolates from the LAB isolates were initially screened and primarily confirmed to be amylolytic lactic acid bacteria (ALAB). The ALAB were found at particular plaa-som processing steps; before mixing (sample A), at 6 hrs after mixing (sample B), and at overnight freezing at −18 ◦ C (sample C). The number of ALAB and LAB isolates for samples A, B, and C were 1 and 3, 13 and 105, and 26 and 106, respectively. LAB isolates only were found at other times during fermentation. The halo ratio from sample A was 5.5. The average halo ratios among the ALAB colonies isolated from samples B and C were found to be 4.61 and 4.81, respectively. Each high halo ratio of ALAB were further investigated for synthesis of amylolytic enzyme. The activity of amylolytic enzyme was carried out in MRS-ALAB broth without glucose, soluble starch and rice starch were added at 2.0% (w/v). The results showed that ALAB isolate number B62 gave the maximum of amylolytic activity at 1.15 and 1.26 units/ml of rice starch and soluble starch, respectively. Subsequently, all 40 ALAB isolates were initially identified to be Streptococcus sp. at 87.50% and Lactobacillus sp. at 12.50%.
S745
VIII2-Y-007 Characterization of virgibacillus sp. SK33 cell-bound proteinases and its application as a starter culture for fish sauce fermentation Sornchai Sinsuwan 1 , Siriwan Nawong 1 , Sureelak Nongnuch Raksakulthai 3 , Jirawat Yongsawatdigul 1,∗
Rodtong 2 ,
1
School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand 2 School of Microbiology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand 3 Department of Fishery Product, Faculty of Fisheries, Kasetsart University, Bangkok 10400, Thailand E-mail address: [email protected] (J. Yongsawatdigul). Fish sauce, a popular condiment in Asia, relies on natural fermentation of fish at 25–30% NaCl for 1–1.5 years (Beddows, 1998). The use of starter culture could be a potential means to accelerate fish sauce fermentation. Among 160 bacterial isolates obtained from 1–12 month-old Thai fish sauce mash, SK33 isolate showed the highest proteinase activity when cultivating in fish broth containing 25% NaCl. Based on morphological, biochemical characteristics, and 16S rRNA gene sequence analysis, the strain was belonged to Virgibacillus sp. SK33. Cell-bound proteinase of Virgibacillus sp. SK33 was also characterized. Cell-bound proteinase was extracted by incubating washed cells in 0.03 mg/mL lyzozyme in 0.05 M Tris–maleate (pH 7). Free cell-bound proteinases exhibited the dominant molecular weight (MW) of 32 and 17 kDa. Optimum proteolytic activity towards Suc–Ala–Ala–Pro–Phe–AMC was at 50 ◦ C and pH 8 and 11. Activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), indicating a characteristic of serine proteinase. All proteinases exhibited caseinolytic activity at 25% NaCl, suggesting their potential use at high salt concentration. Starter culture of Virgibacillus sp. SK33 was prepared using fish broth containing 25% NaCl for laboratory scale fish sauce fermentation. After 4-month fermentation, ␣-amino content of sample inoculated Virgibacillus sp. SK33 was higher than the control (no starter culture added) (p < 0.05). Histamine content of the inoculated sample was 50% lower than that of control. Glutamic acid content of sample inoculated Virgibacillus sp. SK33 was higher than the control (p < 0.05) and comparable to the sample fermented for 12 months (p > 0.05). These results demonstrated that Virgibacillus sp. SK33 can be a potential strain to reduce fish sauce fermentation time. Reference Beddows, C.G., 1998. Fermented fish and fish products. In: Wood, B.J.B. (Ed.), Microbiology of Fermented Foods, vol. 1. Blackie Academic & Professional, London, pp. 416–440.
doi:10.1016/j.jbiotec.2008.07.1774
References VIII2-P-004 Diaz-Ruiz, G., Guyot, J.P., Ruiz-Teran, F., Morlon-Guyot, J., Wacher, C., 2003. Microbial and physiological characterization of weakly amylolytic but fast-growing lactic acid bacteria: a functional role in supporting microbial diversity in pozol, a Mexican fermented maize beverage. Appl. Environ. Microbiol. 69, 4367–4374. Olympia, M., Fukuda, H., Ono, H., Kaneko, Y., Takano, M., 1995. Characterization of starch-hydrolyzing lactic acid bacteria isolated from a fermented fish and rice food, “Burong Isda,” and its amylolytic enzyme. J. Ferment. Bioeng. 80, 124–130. Paludan-Müller, C., Huss, H.H., Gram, L., 1999. Characterization of lactic acid bacteria isolate from a Thai low-salt fermented fish product and the role of garlic as substrate for fermentation. Int. J. Food Microbiol. 46, 219–229.
doi:10.1016/j.jbiotec.2008.07.1773
Application of HACCP in the production of lactobacillus acidophilus functional koumiss Ruixiang Zhao ∗ , Junliang Sun, Shengyang Niu, Lili Zhao, Han Gao School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China E-mail address: [email protected] (R. Zhao). HACCP system was applied into the food safety control of Lactobacillus acidophilus functional koumiss in this study. According to the seven HACCP Principles, the characteristics of Lactobacil-
References Chen, Y.S., Yanagida, F., Shinohara, T., 2005. Isolation and identification of lactic acid bacteria from soil using an enrichment procedure. Lett. Appl. Microbiol. 40, 195–200. Paludan-Müller, C., Madsen, M., Sophanodora, P., Gram, L., Moller, R.L., 2002. Fermentation and microflora of plaa-som, a Thai fermented fish product prepared with different salt concentrations. Int. J. Food Microbiol. 73, 61–70.
doi:10.1016/j.jbiotec.2008.07.1772 VIII2-Y-003 Amylolytic lactic acid bacteria recovered and screened from plaa-som and its amylolytic enzyme Narumon Phonyiam 1,2 , Sirinda Yunchalard 1,3,∗ 1
Department of Biotechnology, Khon Kaen University, Khon Kaen 40002, Thailand 2 Graduate school of Khon Kaen University, Khon Kaen 40002, Thailand 3 Centre of Fermentation for value added agricultural product (FerVAAP), Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand E-mail address: [email protected] (S. Yunchalard).
Plaa-som, a Thai fermented fish product, which production process consists of whole fish or fish fillets mainly from a freshwater fish, Barbodes gonionotus, raw or cooked rice, crushed fresh garlic, and salt. LAB was found to be the most dominant group of microorganisms from plaa-som. In this study, we screened LAB in plaa-som during its fermentation period. A total of 785 LAB isolates were confirmed to be LAB from 798 acid producing isolates recovered and recognized from plaa-som during its 7-day fermentation. These 785 LAB isolates were used as a source of screening for ALAB. Forty isolates from the LAB isolates were initially screened and primarily confirmed to be amylolytic lactic acid bacteria (ALAB). The ALAB were found at particular plaa-som processing steps; before mixing (sample A), at 6 hrs after mixing (sample B), and at overnight freezing at −18 ◦ C (sample C). The number of ALAB and LAB isolates for samples A, B, and C were 1 and 3, 13 and 105, and 26 and 106, respectively. LAB isolates only were found at other times during fermentation. The halo ratio from sample A was 5.5. The average halo ratios among the ALAB colonies isolated from samples B and C were found to be 4.61 and 4.81, respectively. Each high halo ratio of ALAB were further investigated for synthesis of amylolytic enzyme. The activity of amylolytic enzyme was carried out in MRS-ALAB broth without glucose, soluble starch and rice starch were added at 2.0% (w/v). The results showed that ALAB isolate number B62 gave the maximum of amylolytic activity at 1.15 and 1.26 units/ml of rice starch and soluble starch, respectively. Subsequently, all 40 ALAB isolates were initially identified to be Streptococcus sp. at 87.50% and Lactobacillus sp. at 12.50%.
S745
VIII2-Y-007 Characterization of virgibacillus sp. SK33 cell-bound proteinases and its application as a starter culture for fish sauce fermentation Sornchai Sinsuwan 1 , Siriwan Nawong 1 , Sureelak Nongnuch Raksakulthai 3 , Jirawat Yongsawatdigul 1,∗
Rodtong 2 ,
1
School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand 2 School of Microbiology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand 3 Department of Fishery Product, Faculty of Fisheries, Kasetsart University, Bangkok 10400, Thailand E-mail address: [email protected] (J. Yongsawatdigul). Fish sauce, a popular condiment in Asia, relies on natural fermentation of fish at 25–30% NaCl for 1–1.5 years (Beddows, 1998). The use of starter culture could be a potential means to accelerate fish sauce fermentation. Among 160 bacterial isolates obtained from 1–12 month-old Thai fish sauce mash, SK33 isolate showed the highest proteinase activity when cultivating in fish broth containing 25% NaCl. Based on morphological, biochemical characteristics, and 16S rRNA gene sequence analysis, the strain was belonged to Virgibacillus sp. SK33. Cell-bound proteinase of Virgibacillus sp. SK33 was also characterized. Cell-bound proteinase was extracted by incubating washed cells in 0.03 mg/mL lyzozyme in 0.05 M Tris–maleate (pH 7). Free cell-bound proteinases exhibited the dominant molecular weight (MW) of 32 and 17 kDa. Optimum proteolytic activity towards Suc–Ala–Ala–Pro–Phe–AMC was at 50 ◦ C and pH 8 and 11. Activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), indicating a characteristic of serine proteinase. All proteinases exhibited caseinolytic activity at 25% NaCl, suggesting their potential use at high salt concentration. Starter culture of Virgibacillus sp. SK33 was prepared using fish broth containing 25% NaCl for laboratory scale fish sauce fermentation. After 4-month fermentation, ␣-amino content of sample inoculated Virgibacillus sp. SK33 was higher than the control (no starter culture added) (p < 0.05). Histamine content of the inoculated sample was 50% lower than that of control. Glutamic acid content of sample inoculated Virgibacillus sp. SK33 was higher than the control (p < 0.05) and comparable to the sample fermented for 12 months (p > 0.05). These results demonstrated that Virgibacillus sp. SK33 can be a potential strain to reduce fish sauce fermentation time. Reference Beddows, C.G., 1998. Fermented fish and fish products. In: Wood, B.J.B. (Ed.), Microbiology of Fermented Foods, vol. 1. Blackie Academic & Professional, London, pp. 416–440.
doi:10.1016/j.jbiotec.2008.07.1774
References VIII2-P-004 Diaz-Ruiz, G., Guyot, J.P., Ruiz-Teran, F., Morlon-Guyot, J., Wacher, C., 2003. Microbial and physiological characterization of weakly amylolytic but fast-growing lactic acid bacteria: a functional role in supporting microbial diversity in pozol, a Mexican fermented maize beverage. Appl. Environ. Microbiol. 69, 4367–4374. Olympia, M., Fukuda, H., Ono, H., Kaneko, Y., Takano, M., 1995. Characterization of starch-hydrolyzing lactic acid bacteria isolated from a fermented fish and rice food, “Burong Isda,” and its amylolytic enzyme. J. Ferment. Bioeng. 80, 124–130. Paludan-Müller, C., Huss, H.H., Gram, L., 1999. Characterization of lactic acid bacteria isolate from a Thai low-salt fermented fish product and the role of garlic as substrate for fermentation. Int. J. Food Microbiol. 46, 219–229.
doi:10.1016/j.jbiotec.2008.07.1773
Application of HACCP in the production of lactobacillus acidophilus functional koumiss Ruixiang Zhao ∗ , Junliang Sun, Shengyang Niu, Lili Zhao, Han Gao School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China E-mail address: [email protected] (R. Zhao). HACCP system was applied into the food safety control of Lactobacillus acidophilus functional koumiss in this study. According to the seven HACCP Principles, the characteristics of Lactobacil-