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Mixed-Species Biofilm Formation by Direct Cell-Cell Contact Between Biosci. Biotechnol. Biochem., 74 (11), 2316–2319, 2010 Note Mixed-Species Biofilm Formation by Direct Cell-Cell Contact between Brewing Yeasts and Lactic Acid Bacteria y Soichi FURUKAWA,1; Kanako YOSHIDA,1 Hirokazu OGIHARA,1 y y Makari YAMASAKI,2; and Yasushi MORINAGA1; 1Department of Food Bioscience and Biotechnology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan 2Advanced Research Institute for the Sciences and Humanities, Nihon University, 12-5, 5-bancho, Chiyoda-ku, Tokyo 102-8251, Japan Received May 10, 2010; Accepted July 22, 2010; Online Publication, November 7, 2010 [doi:10.1271/bbb.100350] Mixed-species biofilm was remarkably formed in a have high ability for mixed-species biofilm formation, in static co-culture of Lactobacillus plantarum ML11-11 actual traditional fermentation processes. and Saccharomyces cerevisiae Y11-43 isolated from We focused on the combinations of yeasts and LAB brewing samples of Fukuyama pot vinegar. Mixed- isolated from brewing samples of Fukuyama pot species biofilm is probably formed by direct cell-cell vinegar, one of the most primitive types of rice vinegar contact between ML11-11 and S. cerevisiae including in Japan.15–18) Fermentation of Fukuyama pot vinegar is Y11-43 and laboratory yeast strains. Scanning electron conducted in roughly capped large pots laid in the open- microscopic observation suggested that the mixed- air without any refined culture-manipulation. Recently, species biofilm had a thick, bi-layer structure. we investigated the microbiological and biochemical transition of the fermentative process of Fukuyama pot Key words: mixed-species biofilm; Lactobacillus plan- vinegar to determine the mechanism of the establish- tarum; Saccharomyces cerevisiae ment of acetic acid fermentation through diverse microbes.19) In Fukuyama pot vinegar, three reaction Some traditional fermentation processes start from steps, saccharification, alcohol fermentation, and acetic solid biomaterials e.g., rice in rice wine (sake), vegeta- acid fermentation, proceed sequentially and partly in bles in pickled foods such as sauerkraut and kimchi, parallel, suggesting the existence of indispensable cereals (soy bean and wheat) in soy sauce, and marine interactions. These might include mixed-species biofilm products in traditional Asian condiments such as nam formation among the microorganisms catalyzing the pla.1) We hypothesized that the formation of a biofilm, a reaction steps. microbial community on the solid-liquid interface,2–4) is We obtained yeasts isolates and LAB isolates from important in the traditional fermentation process. autumn-2006 samples of Fukuyama pot vinegar fermen- Coexistence or symbiosis of yeasts and lactic acid tation collected on the 5th and 11th brewing day. We bacteria (LAB) in traditional fermented foods, such as investigated mixed-species biofilm formation between sake, wine (malolactic fermentation by LAB), lambic the yeast and LAB. To prepare seed cultures, LAB were beer, whiskey, kefir, sourdough, and pickles is well grown in DeMan, Rogosa, Sharpe broth (MRS; Becton, known.1,5) Dickinson, Franklin Lakes, NJ) at 28 C for 24 h in Several investigators have also done pioneering work static aerobic culture. Yeasts were grown in YPD broth on mixed-species biofilm formation.6–13) Especially, (YPD; Becton, Dickinson) at 28 C for 24 h in static Hogan and Kolter reported initially on pathogenic aerobic culture. Cells were cultured to the stationary bacteria and yeast co-aggregative interaction.10,12) They phase. found that Pseudomonas aeruginosa forms a dense Biofilm formation and assay protocol was almost as in biofilm on Candida albicans filaments and kills the our previous study.20) To assay biofilm formation, fungus, and, in contrast, P. aeruginosa neither binds to stationary phase LAB or yeast cultures were inoculated nor kills yeast-form C. albicans. into fresh YPD at the dilution rate of 1:100 in These pioneering works on the mixed-species biofilm monoculture, while for co-culture, stationary phase prompted us to investigate the possibility that yeasts and cultures of the LAB and yeast were inoculated into LAB can form a mixed-species biofilm in co-culture. fresh YPD at a dilution rate of 1:200. A 96-well We have found positive combinations of yeasts and polystyrene micro-titer plate (#92696, TPP AG, Adolf LAB, in which LAB produced extracellular factors Ku¨hner, Switzerland) was used for biofilm formation. supporting the formation of yeast biofilm in a single After inoculation, both mono- and co-culture samples culture.14) These yeasts and LAB were arbitrarily were incubated at 30 C for 24 h. Quantification of selected laboratory and industrial strains. In this study, biofilm formation was done by the conventional titer we tried to identify LAB and yeast combinations that plate method.14,20,21) y To whom correspondence should be addressed. Soichi FURUKAWA, Tel/Fax: +81-466-84-3973; E-mail: [email protected]; Makari YAMASAKI, E-mail: [email protected]; Yasushi MORINAGA, Tel/Fax: +81-466-84-3971; E-mail: [email protected] Mixed-Species Biofilm Formation by Yeasts and Lactic Acid Bacteria 2317 Table 1. Mixed-Species Biofilm Formation between Yeast and LAB Isolates from Fukuyama Pot Vinegar Fermentation Collected on the 5th and 11th Brewing Day Yeast Y5-1 Y5-11 Y5-26 Y5-31 Y5-43 Yeast Y11-10 Y11-15 Y11-34 Y11-43 Y11-50 LAB single 1 1 1 1 1 LAB single 1 1 1 1 1 GML5-1 1 1 1 1 1 1 ML11-1 1 1 1 1 1 1 GML5-2 1 1 1 1 1 1 ML11-2 1 1 1 1 1 1 GML5-3 1 1 1 1 1 1 ML11-3 1 1 1 1 1 1 GML5-4 1 1 1 1 1 1 ML11-4 1 1 1 1 1 1 GML5-5 1 1 1 1 1 1 ML11-5 1 1 1 1 1 1 GML5-6 1 1 1 1 1 1 ML11-6 1 4 2 2 2 3 GML5-7 1 1 1 1 1 1 ML11-7 1 1 1 1 1 1 GML5-8 1 1 1 1 1 1 ML11-8 1 1 1 1 1 1 GML5-9 1 2 2 2 2 1 ML11-9 1 1 1 1 1 1 GML5-10 1 1 1 1 1 2 ML11-10 1 1 1 1 1 1 GML5-11 1 1 1 1 1 1 ML11-11 2 4 2 2 5 3 GML5-12 1 1 1 1 1 1 ML11-12 1 1 1 1 1 1 GML5-13 1 1 1 1 1 1 ML11-13 1 1 1 1 1 1 GML5-14 1 1 1 1 1 1 ML11-14 1 1 1 1 1 2 GML5-15 1 1 1 1 1 1 ML11-15 1 1 1 1 1 1 GML5-16 1 1 1 1 1 1 ML11-16 1 1 1 1 1 1 GML5-17 1 1 1 1 1 1 ML11-17 1 1 1 1 1 1 GML5-18 1 1 1 2 1 2 ML11-18 1 1 1 1 1 1 GML5-19 2 1 1 2 2 2 ML11-19 1 1 1 1 1 1 GML5-20 2 1 1 2 1 1 ML11-20 1 1 1 1 1 1 GML5-21 2 2 2 2 2 1 ML11-21 1 1 1 1 1 1 GML5-22 1 1 1 1 1 1 ML11-22 1 1 1 1 1 1 GML5-23 1 1 3 2 2 2 ML11-23 1 1 1 1 1 2 GML5-24 1 1 1 1 1 1 ML11-24 1 1 1 1 1 1 GML5-25 1 2 1 2 2 2 ML11-25 1 1 1 1 1 1 GML5-26 1 1 1 1 1 1 ML11-26 1 1 1 1 1 1 GML5-27 1 1 1 1 1 1 ML11-27 1 1 1 1 1 1 GML5-28 1 1 1 1 1 1 ML11-28 1 3 1 2 2 1 GML5-29 1 1 3 2 2 1 ML11-29 1 1 1 1 1 1 GML5-30 1 1 1 1 1 1 ML11-30 1 1 1 1 1 1 GML5-31 1 2 1 2 2 2 ML11-31 2 2 2 1 1 1 GML5-32 1 1 1 1 1 1 GML5-33 1 1 1 1 1 1 GML5-34 1 1 1 1 1 1 GML5-35 1 1 1 1 1 1 GML5-36 1 1 1 1 1 1 GML5-37 1 1 2 1 1 1 GML5-38 1 1 1 1 1 1 GML5-39 1 1 1 1 1 1 GML5-40 2 2 1 2 2 2 GML5-41 1 1 1 1 1 1 GML5-42 1 1 1 1 1 1 GML5-43 1 1 1 1 1 1 GML5-44 2 2 2 2 2 2 GML5-45 1 1 1 1 1 1 Light gray area show that the biofilm formation slightly increased in the mixed-culture. Concentrated gray area show that the biofilm formation highly increased in the mixed-culture. 1 to 5 show the level of the biofilm formation. The criteria of the biofilm formation level were light transmissibility (thickness) and coverage of the micro-titer plate well bottom of the biofilm. Level 5: micro-titer plate well bottom was not light transmissible and fully covered by biofilm. Level 4: micro-titer plate well bottom was not light transmissible but partly not covered by biofilm. Level 3: micro-titer plate well bottom was light transmissible but fully covered by biofilm. Level 2: micro-titer plate well bottom was light transmissible and partly covered by biofilm.
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