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UV-induced Lactobacillus gasseri mutants resisting sodium chloride and sodium nitrite for meat fermentation
International Journal of Food Microbiology 56 (2000) 227–230 www.elsevier.nl / locate / ijfoodmicro
Short communication
UV-induced Lactobacillus gasseri mutants resisting sodium chloride and sodium nitrite for meat fermentation Keizo Arihara*, Makoto Itoh School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada-shi 034 -8628, Japan Received 22 November 1999; received in revised form 2 December 1999; accepted 4 December 1999
Abstract Lactobacillus gasseri, one of the predominant lactobacilli in human intestinal tracts, is utilized for probiotics and dairy starter cultures. However, since L. gasseri is relatively sensitive to sodium chloride and sodium nitrite (essential compounds for meat products), it is difficult to utilize this species for conventional fermented meat products. In this study, efforts were directed to generate mutants of L. gasseri resisting sodium chloride and sodium nitrite. UV irradiation of the strain of L. gasseri JCM1131 T generated several mutants resisting these compounds. A mutant strain 1131-M8 demonstrated satisfactory growth in meat containing 3.3% sodium chloride and 200 ppm sodium nitrite. Although proteins extracted from the cell surface of 1131-M8 were slightly different from those of the original strain, other biochemical characteristics of both strains were indistinguishable. These results suggest that the L. gasseri mutant obtained in this study could be utilized as a starter culture to develop probiotic meat products. 2000 Elsevier Science B.V. All rights reserved. Keywords: Lactobacillus gasseri; Meat fermentation; Sodium chloride; Sodium nitrite; UV irradiation
1. Introduction Probiotics are defined as cultures of live microorganisms that, applied to animals or humans, benefit the host by improving properties of indigenous microflora (Havenaar and Huis in’t Veld, 1992). Lactobacillus acidophilus group lactic acid bacteria, such as L. gasseri, have been widely utilized as probiotics (Klaenhammer and Kullen, 1999). Al*Corresponding author. Tel.: 1 81-176-23-4371; fax: 1 81176-23-8703. E-mail address: [email protected] (K. Arihara)
though these bacteria have been applied to various dairy products industrially (Holzapfel et al., 1998; Schillinger, 1999), they have not been applied to meat products. In our preceding study (Arihara et al., 1998), we reported that of the L. acidophilus group strains tested L. gasseri JCM1131 T exhibited the greatest fermentation performance in meats, and suggested that probiotic lactic acid bacteria could be effectively utilized for meat fermentation to produce healthy meat products. However, L. gasseri is relatively sensitive to sodium chloride and sodium nitrite (Arihara et al., 1996, 1998). In many countries, most
0168-1605 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00206-8
228
K. Arihara, M. Itoh / International Journal of Food Microbiology 56 (2000) 227 – 230
meat products must contain these compounds for preservation during storage. For example, regulations for the maintenance of meat products safety in Japan require the use of 3.3% sodium chloride and 200 ppm sodium nitrite (Sameshima et al., 1998). Therefore, effective starter cultures for fermented meat products are claimed to resist these compounds. In the present study, we carried out the generation of mutant strains of L. gasseri JCM1131 T resisting sodium chloride and sodium nitrite by UV irradiation. Such strains could be utilized for producing new probiotic meat products, and these products may develop new markets in the meat industry.
sodium chloride and sodium nitrite was varied to determine the resistance of lactobacilli to these compounds. The batter (50 g each) was stuffed into commercial high density polyethylene pouches (10 mm thickness) and incubated at 378C for 24 h.
2.4. SDS–polyacrylamide gel electrophoresis ( SDS–PAGE) Extraction of cell surface proteins of lactobacilli and their separation by SDS–PAGE was performed as previously described (Mukai and Arihara, 1994).
3. Results and discussion 2. Materials and methods
2.1. Bacterial strains Lactobacillus gasseri JCM1131 T , the type strain of L. gasseri isolated from the human intestine, was obtained from JCM (Japan Collection of Microorganisms, Wako, Japan). All Lactobacillus cultures including mutant strains obtained in this study were maintained as frozen stocks (kept at 2 558C) in MRS broth (Difco Laboratories, Detroit) plus 10% glycerol. Prior to use, they were transferred at least twice in MRS broth at 378C.
2.2. Isolation of mutants by UV irradiation UV irradiation was carried out by exposing L. gasseri JCM1131 T (ca. 10 8 2 10 9 cells per plate) spread on MRS agar plates containing 3.3% sodium chloride and 200 ppm sodium nitrite to UV light from a Toshiba (Tokyo, Japan) germicidal lamp (15 W) for 15 s at a distance of 60 cm. After UV irradiation, plates were incubated at 378C for 48 h under anaerobic conditions. The colonies formed on the plate were picked and purified on MRS agar plates containing sodium nitrite and sodium chloride.
2.3. Preparation of model sausage Fresh pork trim (ham) was obtained from a local packer. The meat was ground, mixed with glucose (1%), sodium chloride (0, 3.3%), sodium nitrite (0, 200 ppm), sodium ascorbate (550 ppm) and starter Lactobacillus (10 7 cfu / g meat). The concentration of
UV irradiation of Lactobacillus gasseri JCM1131 T generated several mutants resisting sodium chloride and sodium nitrite. The original strain of L. gasseri JCM1131 T did not grow on MRS plates containing 3.3% sodium chloride and 200 ppm sodium nitrite, but several mutants (e.g., 1131-M8) generated by UV irradiation grew on these plates (Fig. 1). Their resistance was well maintained after at least 20 transfers in MRS broth with or without sodium chloride and sodium nitrite. The pH changes of the model sausages fermented with L. gasseri strains were compared (Table 1). The pH drop of meats was utilized for estimating the growth of Lactobacillus strains. A mutant strain (1131-M8) demonstrated satisfactory pH decline caused by lactic acid production in meat containing 3.3% sodium chloride and 200 ppm sodium nitrite. Proteins extracted from the cell surface of 1131M8 strain were slightly different from those of the original strain (Fig. 2). However, other biochemical characteristics (e.g., carbohydrate fermentation ability, acid and bile-tolerance) of 1131-M8 were indistinguishable from those of the original strain (data not shown). Many functions of bacterial cell surface proteins have been reported. In lactobacilli these proteins are believed to control the transfer of nutrients and provide a protective barrier for the cell (Lotal, 1993; Moschl et al., 1993). Thus, one possibility is that mutants became resistant to sodium chloride and sodium nitrite due to changes in the surface structure (proteins) of cells. Intestinal lactobacilli, such as L. gasseri, have been reported to show beneficial health properties,
K. Arihara, M. Itoh / International Journal of Food Microbiology 56 (2000) 227 – 230
229
Fig. 1. Colonies of Lactobacillus gasseri JCM1131 T (A, B) and UV-induced strain 1131-M8 (C, D) formed on MRS agar plates with (B, D) and without (A, C) 3.3% sodium chloride and 200 ppm sodium nitrite. Table 1 Resistance of Lactobacillus gasseri strains (original strain of JCM1131 T and UV-induced strain 1131-M8) to sodium chloride and sodium nitrite in model sausages Strains of L. gasseri
JCM1131 T 1131-M8 a
pH of sausages after 24-h fermentation a Without NaCl and NaNO 2
With 3.3% NaCl and 200 ppm NaNO 2
4.3 4.4
5.8 4.6
The pH of sausages prior to fermentation was 6.0.
and have been utilized as probiotics and dairy starter cultures (Arihara and Luchansky, 1994). In recent years, several meat microbiologists have highlighted the possibility of developing healthy meat products
by using probiotic and / or intestinal lactic acid bacteria (Jessen, 1995; Hugas and Monfort, 1997; ¨ Hammes and Hertel, 1998; Incze, 1998; Lucke, 1999). However, to date, there are few published studies on this area (Arihara et al., 1996, 1998; Sameshima et al., 1998). Since meat products with such bacteria may develop new markets, research and development in this area is expected to increase. In our results, 1131-M8 strain generated from L. gasseri JCM1131 T exhibited satisfactory tolerance against sodium chloride and sodium nitrite for meat fermentation. Since L. gasseri has been reported to be one of the predominant lactic acid bacteria in human intestinal tracts (Benno et al., 1989), this mutant strain could be an appropriate probiotic starter culture for meat fermentation.
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K. Arihara, M. Itoh / International Journal of Food Microbiology 56 (2000) 227 – 230
Fig. 2. SDS–PAGE of cell surface proteins from Lactobacillus gasseri strains (original strain of JCM1131 T and UV-induced strain 1131-M8). Arrows indicate major difference in SDS–PAGE patterns.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (No. 11660272) from the Ministry of Education, Science and Culture of Japan, and the School of Veterinary Medicine and Animal Sciences, Kitasato University.
References Arihara, K., Luchansky, J.B., 1994. Dairy lactobacilli. In: Hui, Y.H., Khachatourians, G.G. (Eds.), Food Biotechnology: Microorganisms, VCH Publishers, New York, pp. 609–643. Arihara, K., Ota, H., Itoh, M., Kondo, Y., Sameshima, T., Yamanaka, H. et al., 1996. Utilization of probiotic lactic acid bacteria for meat products. Proc. Int. Cong. Meat Sci. Technol. 42, 501–502.
Arihara, K., Ota, H., Itoh, M., Kondo, Y., Sameshima, T., Yamanaka, H., 1998. Lactobacillus acidophilus group lactic acid bacteria applied to meat fermentation. J. Food Sci. 63, 544–547. Benno, Y., Endo, K., Mizutani, T., Namba, Y., Komori, T., Mitsuoka, T., 1989. Comparison of fecal microflora of elderly persons in rural and urban areas of Japan. Appl. Environ. Microbiol. 55, 1100–1105. Hammes, W.P., Hertel, C., 1998. New developments in meat starter cultures. Meat Sci. 49 (Suppl. 1), S125–S138. Havenaar, R., Huis in’t Veld, J.H.J., 1992. Probiotics: a general view. In: Wood, B.J.B. (Ed.), The Lactic Acid Bacteria in Health and Disease, The Lactic Acid Bacteria, Vol. 1, Elsevier Applied Science, London, pp. 151–170. Holzapfel, W.H., Haberer, P., Snel, J., Schillinger, U., Huis in’t Veld, J.H.J., 1998. Overview of gut flora and probiotics. Int. J. Food Microbiol. 41, 85–101. Hugas, M., Monfort, J.M., 1997. Bacterial starter cultures for meat fermentation. Food Chem. 59, 547–554. Incze, K., 1998. Dry fermented sausages. Meat Sci. 49 (Suppl. 1), S169–S177. Jessen, B., 1995. Starter cultures for meat fermentations. In: Campbell-Platt, G., Cook, P.E. (Eds.), Fermented Meats, Blackie Academic and Professional, Glasgow, pp. 130–159. Klaenhammer, T.R., Kullen, M.J., 1999. Selection and design of probiotics. Int. J. Food Microbiol. 50, 45–57. Lotal, S., 1993. Crystalline surface-layers of the genus Lactobacillus. In: Beveridge, T.J., Koval, S.F. (Eds.), Advances in Bacterial Paracrystalline Surface Layers, Plenum Press, New York, pp. 57–65. ¨ Lucke, F.-K., 1999. Utilization of microbes to process and to preserve meat. Proc. Int. Cong. Meat Sci. Technol. 45, 538– 547. Moschl, A., Schaffer, U., Sleytr, U.B., Messner, P., 1993. Characterization of the S-layer glycoproteins of two lactobacilli. In: Beveridge, T.J., Koval, S.F. (Eds.), Advances in Bacterial Paracrystalline Surface Layers, Plenum Press, New York, pp. 281–284. Mukai, T., Arihara, K., 1994. Presence of intestinal lectin-binding glycoproteins on the cell surface of Lactobacillus acidophilus. Biosci. Biotech. Biochem. 58, 1851–1854. Sameshima, T., Magome, C., Takeshita, K., Arihara, K., Itoh, M., Kondo, Y., 1998. Effect of intestinal Lactobacillus starter cultures on the behaviour of Staphylococcus aureus in fermented sausage. Int. J. Food Microbiol. 41, 1–7. Schillinger, U., 1999. Isolation and identification of lactobacilli from novel-type probiotic and mild yoghurts and their stability during refrigerated storage. Int. J. Food Microbiol. 47, 79–87.
Short communication
UV-induced Lactobacillus gasseri mutants resisting sodium chloride and sodium nitrite for meat fermentation Keizo Arihara*, Makoto Itoh School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada-shi 034 -8628, Japan Received 22 November 1999; received in revised form 2 December 1999; accepted 4 December 1999
Abstract Lactobacillus gasseri, one of the predominant lactobacilli in human intestinal tracts, is utilized for probiotics and dairy starter cultures. However, since L. gasseri is relatively sensitive to sodium chloride and sodium nitrite (essential compounds for meat products), it is difficult to utilize this species for conventional fermented meat products. In this study, efforts were directed to generate mutants of L. gasseri resisting sodium chloride and sodium nitrite. UV irradiation of the strain of L. gasseri JCM1131 T generated several mutants resisting these compounds. A mutant strain 1131-M8 demonstrated satisfactory growth in meat containing 3.3% sodium chloride and 200 ppm sodium nitrite. Although proteins extracted from the cell surface of 1131-M8 were slightly different from those of the original strain, other biochemical characteristics of both strains were indistinguishable. These results suggest that the L. gasseri mutant obtained in this study could be utilized as a starter culture to develop probiotic meat products. 2000 Elsevier Science B.V. All rights reserved. Keywords: Lactobacillus gasseri; Meat fermentation; Sodium chloride; Sodium nitrite; UV irradiation
1. Introduction Probiotics are defined as cultures of live microorganisms that, applied to animals or humans, benefit the host by improving properties of indigenous microflora (Havenaar and Huis in’t Veld, 1992). Lactobacillus acidophilus group lactic acid bacteria, such as L. gasseri, have been widely utilized as probiotics (Klaenhammer and Kullen, 1999). Al*Corresponding author. Tel.: 1 81-176-23-4371; fax: 1 81176-23-8703. E-mail address: [email protected] (K. Arihara)
though these bacteria have been applied to various dairy products industrially (Holzapfel et al., 1998; Schillinger, 1999), they have not been applied to meat products. In our preceding study (Arihara et al., 1998), we reported that of the L. acidophilus group strains tested L. gasseri JCM1131 T exhibited the greatest fermentation performance in meats, and suggested that probiotic lactic acid bacteria could be effectively utilized for meat fermentation to produce healthy meat products. However, L. gasseri is relatively sensitive to sodium chloride and sodium nitrite (Arihara et al., 1996, 1998). In many countries, most
0168-1605 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00206-8
228
K. Arihara, M. Itoh / International Journal of Food Microbiology 56 (2000) 227 – 230
meat products must contain these compounds for preservation during storage. For example, regulations for the maintenance of meat products safety in Japan require the use of 3.3% sodium chloride and 200 ppm sodium nitrite (Sameshima et al., 1998). Therefore, effective starter cultures for fermented meat products are claimed to resist these compounds. In the present study, we carried out the generation of mutant strains of L. gasseri JCM1131 T resisting sodium chloride and sodium nitrite by UV irradiation. Such strains could be utilized for producing new probiotic meat products, and these products may develop new markets in the meat industry.
sodium chloride and sodium nitrite was varied to determine the resistance of lactobacilli to these compounds. The batter (50 g each) was stuffed into commercial high density polyethylene pouches (10 mm thickness) and incubated at 378C for 24 h.
2.4. SDS–polyacrylamide gel electrophoresis ( SDS–PAGE) Extraction of cell surface proteins of lactobacilli and their separation by SDS–PAGE was performed as previously described (Mukai and Arihara, 1994).
3. Results and discussion 2. Materials and methods
2.1. Bacterial strains Lactobacillus gasseri JCM1131 T , the type strain of L. gasseri isolated from the human intestine, was obtained from JCM (Japan Collection of Microorganisms, Wako, Japan). All Lactobacillus cultures including mutant strains obtained in this study were maintained as frozen stocks (kept at 2 558C) in MRS broth (Difco Laboratories, Detroit) plus 10% glycerol. Prior to use, they were transferred at least twice in MRS broth at 378C.
2.2. Isolation of mutants by UV irradiation UV irradiation was carried out by exposing L. gasseri JCM1131 T (ca. 10 8 2 10 9 cells per plate) spread on MRS agar plates containing 3.3% sodium chloride and 200 ppm sodium nitrite to UV light from a Toshiba (Tokyo, Japan) germicidal lamp (15 W) for 15 s at a distance of 60 cm. After UV irradiation, plates were incubated at 378C for 48 h under anaerobic conditions. The colonies formed on the plate were picked and purified on MRS agar plates containing sodium nitrite and sodium chloride.
2.3. Preparation of model sausage Fresh pork trim (ham) was obtained from a local packer. The meat was ground, mixed with glucose (1%), sodium chloride (0, 3.3%), sodium nitrite (0, 200 ppm), sodium ascorbate (550 ppm) and starter Lactobacillus (10 7 cfu / g meat). The concentration of
UV irradiation of Lactobacillus gasseri JCM1131 T generated several mutants resisting sodium chloride and sodium nitrite. The original strain of L. gasseri JCM1131 T did not grow on MRS plates containing 3.3% sodium chloride and 200 ppm sodium nitrite, but several mutants (e.g., 1131-M8) generated by UV irradiation grew on these plates (Fig. 1). Their resistance was well maintained after at least 20 transfers in MRS broth with or without sodium chloride and sodium nitrite. The pH changes of the model sausages fermented with L. gasseri strains were compared (Table 1). The pH drop of meats was utilized for estimating the growth of Lactobacillus strains. A mutant strain (1131-M8) demonstrated satisfactory pH decline caused by lactic acid production in meat containing 3.3% sodium chloride and 200 ppm sodium nitrite. Proteins extracted from the cell surface of 1131M8 strain were slightly different from those of the original strain (Fig. 2). However, other biochemical characteristics (e.g., carbohydrate fermentation ability, acid and bile-tolerance) of 1131-M8 were indistinguishable from those of the original strain (data not shown). Many functions of bacterial cell surface proteins have been reported. In lactobacilli these proteins are believed to control the transfer of nutrients and provide a protective barrier for the cell (Lotal, 1993; Moschl et al., 1993). Thus, one possibility is that mutants became resistant to sodium chloride and sodium nitrite due to changes in the surface structure (proteins) of cells. Intestinal lactobacilli, such as L. gasseri, have been reported to show beneficial health properties,
K. Arihara, M. Itoh / International Journal of Food Microbiology 56 (2000) 227 – 230
229
Fig. 1. Colonies of Lactobacillus gasseri JCM1131 T (A, B) and UV-induced strain 1131-M8 (C, D) formed on MRS agar plates with (B, D) and without (A, C) 3.3% sodium chloride and 200 ppm sodium nitrite. Table 1 Resistance of Lactobacillus gasseri strains (original strain of JCM1131 T and UV-induced strain 1131-M8) to sodium chloride and sodium nitrite in model sausages Strains of L. gasseri
JCM1131 T 1131-M8 a
pH of sausages after 24-h fermentation a Without NaCl and NaNO 2
With 3.3% NaCl and 200 ppm NaNO 2
4.3 4.4
5.8 4.6
The pH of sausages prior to fermentation was 6.0.
and have been utilized as probiotics and dairy starter cultures (Arihara and Luchansky, 1994). In recent years, several meat microbiologists have highlighted the possibility of developing healthy meat products
by using probiotic and / or intestinal lactic acid bacteria (Jessen, 1995; Hugas and Monfort, 1997; ¨ Hammes and Hertel, 1998; Incze, 1998; Lucke, 1999). However, to date, there are few published studies on this area (Arihara et al., 1996, 1998; Sameshima et al., 1998). Since meat products with such bacteria may develop new markets, research and development in this area is expected to increase. In our results, 1131-M8 strain generated from L. gasseri JCM1131 T exhibited satisfactory tolerance against sodium chloride and sodium nitrite for meat fermentation. Since L. gasseri has been reported to be one of the predominant lactic acid bacteria in human intestinal tracts (Benno et al., 1989), this mutant strain could be an appropriate probiotic starter culture for meat fermentation.
230
K. Arihara, M. Itoh / International Journal of Food Microbiology 56 (2000) 227 – 230
Fig. 2. SDS–PAGE of cell surface proteins from Lactobacillus gasseri strains (original strain of JCM1131 T and UV-induced strain 1131-M8). Arrows indicate major difference in SDS–PAGE patterns.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (No. 11660272) from the Ministry of Education, Science and Culture of Japan, and the School of Veterinary Medicine and Animal Sciences, Kitasato University.
References Arihara, K., Luchansky, J.B., 1994. Dairy lactobacilli. In: Hui, Y.H., Khachatourians, G.G. (Eds.), Food Biotechnology: Microorganisms, VCH Publishers, New York, pp. 609–643. Arihara, K., Ota, H., Itoh, M., Kondo, Y., Sameshima, T., Yamanaka, H. et al., 1996. Utilization of probiotic lactic acid bacteria for meat products. Proc. Int. Cong. Meat Sci. Technol. 42, 501–502.
Arihara, K., Ota, H., Itoh, M., Kondo, Y., Sameshima, T., Yamanaka, H., 1998. Lactobacillus acidophilus group lactic acid bacteria applied to meat fermentation. J. Food Sci. 63, 544–547. Benno, Y., Endo, K., Mizutani, T., Namba, Y., Komori, T., Mitsuoka, T., 1989. Comparison of fecal microflora of elderly persons in rural and urban areas of Japan. Appl. Environ. Microbiol. 55, 1100–1105. Hammes, W.P., Hertel, C., 1998. New developments in meat starter cultures. Meat Sci. 49 (Suppl. 1), S125–S138. Havenaar, R., Huis in’t Veld, J.H.J., 1992. Probiotics: a general view. In: Wood, B.J.B. (Ed.), The Lactic Acid Bacteria in Health and Disease, The Lactic Acid Bacteria, Vol. 1, Elsevier Applied Science, London, pp. 151–170. Holzapfel, W.H., Haberer, P., Snel, J., Schillinger, U., Huis in’t Veld, J.H.J., 1998. Overview of gut flora and probiotics. Int. J. Food Microbiol. 41, 85–101. Hugas, M., Monfort, J.M., 1997. Bacterial starter cultures for meat fermentation. Food Chem. 59, 547–554. Incze, K., 1998. Dry fermented sausages. Meat Sci. 49 (Suppl. 1), S169–S177. Jessen, B., 1995. Starter cultures for meat fermentations. In: Campbell-Platt, G., Cook, P.E. (Eds.), Fermented Meats, Blackie Academic and Professional, Glasgow, pp. 130–159. Klaenhammer, T.R., Kullen, M.J., 1999. Selection and design of probiotics. Int. J. Food Microbiol. 50, 45–57. Lotal, S., 1993. Crystalline surface-layers of the genus Lactobacillus. In: Beveridge, T.J., Koval, S.F. (Eds.), Advances in Bacterial Paracrystalline Surface Layers, Plenum Press, New York, pp. 57–65. ¨ Lucke, F.-K., 1999. Utilization of microbes to process and to preserve meat. Proc. Int. Cong. Meat Sci. Technol. 45, 538– 547. Moschl, A., Schaffer, U., Sleytr, U.B., Messner, P., 1993. Characterization of the S-layer glycoproteins of two lactobacilli. In: Beveridge, T.J., Koval, S.F. (Eds.), Advances in Bacterial Paracrystalline Surface Layers, Plenum Press, New York, pp. 281–284. Mukai, T., Arihara, K., 1994. Presence of intestinal lectin-binding glycoproteins on the cell surface of Lactobacillus acidophilus. Biosci. Biotech. Biochem. 58, 1851–1854. Sameshima, T., Magome, C., Takeshita, K., Arihara, K., Itoh, M., Kondo, Y., 1998. Effect of intestinal Lactobacillus starter cultures on the behaviour of Staphylococcus aureus in fermented sausage. Int. J. Food Microbiol. 41, 1–7. Schillinger, U., 1999. Isolation and identification of lactobacilli from novel-type probiotic and mild yoghurts and their stability during refrigerated storage. Int. J. Food Microbiol. 47, 79–87.