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Isolation and characterization of lactic acid bacteria from soils in vineyards.

important role in food technology. LAB include rod, short-rod and coccus in cell types and physiological and biochemical characteristics. Isolates of LAB have been frequently reported from a variety of environments, including milk products, fermented foods and plants. However, studies on the isolation of LAB from soil remain scarce, even though it is well known that spore-forming LAB exist in soil (Nakayama and Yanoshi, 1967a, b; Yanagida et al., 1997). Rich nutrition, such as from carbohydrates, minerals, nitrogen compounds and other substances, is necessary for the growth of LAB. However, it has not been determined whether the nutritional condition of soil is suitable for the growth of LAB. Since grapes contain a large quantity of sugar, we considered that the necessary nutrition for the growth of LAB in the soil of vineyards may be richer than that of other soil, due to fruit fall. This study aimed to isolate and identify LAB from soil, primarily from vineyards but also from other sites. Eighty-seven soil samples were collected from Yamanashi and Nagano prefectures in Japan, mainly in the rhizospheres of vines (Table 1). Two methods, viz. direct spreading and accumulation with incubation, were used for the isolation of acid-producing bacteria. Each collected sample was incubated in 5 ml of two media containing GYP (2% glucose, 1% yeast extract, 1% peptone, 1% sodium acetate and 0.5% vol/vol salts solution, adjusted to pH 7.0) and BM (2% polypeptone, 0.5% tryptose, 2% Bacto-liver extract, 0.5% yeast extract, 0.001% Tween 80, 1% glucose, 0.5% fructose, 0.1% DL-malic acid, 0.008% MnCl2· 4H2O, 25% filtered tomato juice and 75% distilled water, adjusted to pH 5.4) under anaerobic conditions (BBL GasPak, H2 CO2) at 30°C for 2–3 days. After incubation, acid-producing bacteria were isolated. These bacteria were tested with a Gram stain, and only Gram-positive bacteria were kept. The isolates were examined for their phenotypic features, including physiological and biochemical characteristics. Examinations were conducted to determine the cell shape, lactic acid isomer, fermentation type, growth temperature, ability to ferment carbohydrates, type of cell wall and GC content (Kozaki et al., 1992). The API50CHL fermentation test was performed in some isolates. Isolates were classified and identified using 16S rDNA RFLP (Restriction Fragment Length Polymorphisms) (Gurtler et al., 1991; Jang et al., 2003; Johansson et al., 1995; Ramos and Harlander, 1990; Sato et al., 2000) and 16S rDNA sequences (Sato et al., 2001). The DNA of each isolate was extracted, and PCR reactions were performed using a Gene Amp Isolation and characterization of lactic acid bacteria from soils in vineyards