Food Sci. Biotechnol. 22(4): 1171-1174 (2013) DOI 10.1007/s10068-013-0199-1
RESEARCH NOTE
Combined Effect of Lactic Acid Bacteria and Citric Acid on Escherichia coli O157:H7 and Salmonella Typhimurium Sheungwoo Seo, Day Jung, Xiaoyu Wang, Dong Joo Seo, Min Hwa Lee, Bog-Hieu Lee, and Changsun Choi
Received: 7 December 2012 / Revised: 1 March 2013 / Accepted: 5 March 2013 / Published Online: 31 August 2013 © KoSFoST and Springer 2013
Abstract Lactic acid-producing bacteria (LAB) and organic acids can inhibit the growth of pathogenic bacteria and spoilage organisms. Here, synergistic effect of LAB and citric acid was examined. Escherichia coli O157:H7 or Salmonella Typhimurium were treated with a 1% citric acid, 2% citric acid, LAB mixture, LAB mixture+1% citric acid, and LAB mixture+2% citric acid for 10, 20, 30, and 60 min, respectively. While LAB only, the addition of 1 or 2% citric acid caused 0.28-0.57 log reductions of E. coli O157:H7 or S. Typhimurium within 60 min, the treatment of LAB mixture+2% citric acid showed 1.96 and 6.24 log reductions of E. coli O157:H7 and S. Typhimurium, respectively. In conclusion, LAB and citric acid act synergistically and the combination showed its potential of an effective hurdle for the inactivation of foodborne pathogens. Keywords: Escherichia coli O157:H7, Salmonella Typhimurium, citric acid, lactic acid bacteria, hurdle technology
Introduction Lactobacillus, Lactococcus, Enterococcus, Oenococcus, Pediococcus, Streptococcus, and Leuconostoc belong to the group of lactic acid-producing bacteria (LAB). The genus Sheungwoo Seo, Day Jung, Xiaoyu Wang, Dong Joo Seo, Min Hwa Lee, Bog-Hieu Lee, Changsun Choi () Department of Food and Nutrition, Graduate School of Natural Science, Chung-Ang University, Anseong, Gyeonggi 456-756, Korea Tel: +82-31-670-4589; Fax: +82-31-676-8741 E-mail:
[email protected] Bog-Hieu Lee, Changsun Choi School of Food Science and Technology, College of Natural Science, Chung-Ang University, Anseong, Gyeonggi 456-756, Korea
Lactobacillus is an especially large group of LAB with over 100 species and subspecies (1). Based on the characteristics of their fermentation patterns, they are classified into homofermentive and heterofermentive species. While homofermentive LAB convert sugars mostly into lactic acid, heterofermentive LAB covert sugars into lactic acid, acetic acid, ethanol, and carbon dioxide. Because they produce lactic acid, lactobacilli can survive in weakly acidic environments at pH 5.5-6.5 (1). Lactobacillus plantarum and Lactobacillus acidophilus are considered food-grade microorganisms that are found in fermented vegetables, meats, and dairy products. They are commonly present in the human gastrointestinal tract because they can survive in highly acidic gastric conditions and colonize in the intestinal epithelium (1,2). Because LAB produce lactic acid and antimicrobial compounds including bacteriocins during fermentation or storage, the application of LAB effectively reduces Escherichia coli O157:H7 and Salmonella Typhimurium on packaged ground beef and chicken (1,3,4). Some lactobacilli strains can inhibit the growth of pathogenic fungi and several viruses, and LAB have been successfully used to control pathogenic microorganisms in the food and animal food industries (5). Organic acid such as acetic acid, lactic acid, and citric acid are also used to control foodborne pathogens in the food and feed industries. Since organic acids have long been used as food additives and preservatives to extent shelf life, they are generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (2,6,7). Organic acids are used to effectively reduce E. coli O157:H7, Listeria monocytogenes, and S. Typhimurium in broth culture models and chicken meat systems (3,7). Organic acids have been combined with sugar or plant extracts to produce additive or synergistic control over foodborne pathogens in experimental models (3,8). In order to enhance the control of pathogenic bacteria,
1172
Seo et al.
combined treatments with chemical and/or biological sanitation methods are key (9). Therefore, the objective of this study was to examine the survival of E. coli O157:H7 and S. Typhimurium after combined treatment with citric acid and lactobacilli mixture and to optimize the formula of this combination.
Materials and Methods Bacterial inoculum Escherichia coli O157:H7 ATCC 43889, E. coli O157:H7 ATCC 43890, Salmonella Typhimurium ATCC 19585, and S. Typhimurium ATCC 43174 were obtained from the American Type Culture Collection (Manassas, VA, USA). A single colony of each E. coli O157:H7 or S. Typhimurium strain was inoculated in EC broth (CM0853; Oxoid, Basingstoke, UK) and Rappaport-Vassiliadis soya peptone broth (CM0866; Oxoid), respectively. Two strains of E. coli O157:H7 or S. Typhimurium were incubated at 37oC for 24 h and then mixed for use as inoculum. Each inoculum of E. coli O157:H7 and S. Typhimurium respectively contained 7.13 and 6.65 log colony forming units (CFU)/mL. Experimental design The mixture of Lactobacillus acidophilus and L. plantarum was cultured and provided by the Laboratory of Environmental Microbiology, Gwangju Agricultural Technology Center (Korea). L. acidophilus and L. plantarum were isolated from kimchi, a traditional Korean fermented food. The identification of Lactobacillus strains was performed using an API kit (50CHL; bioMérieux, Marcy I’Etoile, France). Each strain was cultured in broth media containing 2.5% yeast extract, 1.5% glucose, and 2% sucrose at 35oC for 48 h. Strains were mixed at a 1:1 ratio, and approximately 8.33 log CFU/mL of LAB mixture was used in this study. LAB mixtures supplemented with 1 or 2%(w/v) citric acid (Sigma-Aldrich, St. Louis, MO, USA) were also prepared. Broth media was used as negative control and 1 or 2% citric acid was supplemented in broth media. A standard benchtop pH meter (PHi510; Beckman Coulter, Fullerton, CA, USA) was used to measure the pH of the 1% citric acid, 2% citric acid, LAB mixture, LAB mixture+1% citric acid, and LAB mixture+2% citric acid. E. coli O157:H7 or S. Typhimurium cocktail was treated with the same amount of LAB mixture at room temperature for 0, 10, 20, 30, and
60 min. Each of the samples was diluted 10-fold in 0.2% peptone water (LP0037; Oxoid). They were then plated onto MacConkey sorbitol agar (279100; BD, Detroit, MI, USA) or MacConkey agar (CM0115; Oxoid), respectively, and incubated at 37oC for 24 h. Statistical analysis All experiments were performed in triplicate. To investigate the inhibitions of E. coli O157:H7 and S. Typhimurium due to the mixtures of LAB and citric acid, analysis of variance (ANOVA) and Duncan’s multiple range tests were performed using SAS software (version 9.1; SAS Inc., Cary, NC, USA). The data were considered statistically significant when a p-value less than 0.01 (p
