Hiroko Nagano

Purification and Properties of a Protease Produced by Bacillus Subtilis CN2 Isolated from a Vietnamese Fish Sauce

Bacillus subtilis CN2 isolated from a Vietnamese fish sauce produced a large quantity of an alkaline protease, when grown on a soy peptone medium. The protease was purified to an electrophoretically homogeneous state and crystallized in its pure condensed solution. The molecular weight was determined to be 27,636 Da, and the N-terminal amino acid sequence was AQSVPYGISQIKAPAL. The optimum pH and temperature were pH 10.0 and 50 °C, respectively. The protease was active over a wide pH range of pH 7.0–11.0, and also active over a broad temperature range of 30–60 °C. The enzyme was potently inhibited by 1 mM phenylmethanesulphonyl fluoride, but resistant to 1 mM sodium dodecyl sulphate (SDS).

Identification of Proteolytic Bacteria from Thai Traditional Fermented Foods and Their Allergenic Reducing Potentials

This study aimed to identify proteolytic bacteria from Thai traditional fermented foods and investigate their allergenic reducing potentials to wheat and milk allergens. Nine bacteria were isolated from fermented foods as follows: fermented soybean seeds (Thua Nao), fermented soybean paste (Thua Nao), wheat flour dough of steamed stuffed bun (Sa La Pao), and soaked rice from Thai fermented rice-noodle (Kha Nhom Jeen) processing. Both phenotypic and genotypic identifications were used in this study. It was found that all isolates were Gram-positive rods. Seven isolates were matched and identified as Bacillus subtilis by both techniques, and the remaining 2 isolates were phenotypically and genotypically identified as B. licheniformis and B. subtilis, respectively. The concentrated crude enzyme of B. subtilis DB and SR could reduce allergenicity of gliadin by hydrolyzing the allergenic gliadin fragments detected by immunoblotting. Furthermore, the enzyme of B. subtilis DB could also reduce allergenicity of beta-lactoglobulin (beta-LG) detected by hydrolyzing the major allergenic epitope of beta-LG at Gln(35)-Ser(36) position. B. subtilis DB and SR can be applied for the production of hypoallergenic wheat flour or milk food products.

Natural populations of lactic acid bacteria in douchi from Yunnan Province, China

This research was aimed at isolating and identifying the predominant lactic acid bacteria (LAB) in the traditional Chinese salt-fermented soybean food, douchi, from Yunnan, China. The predominant LAB present were isolated and identified by conventional culture-dependent methods combined with molecular biological methods. Two hundred and sixty isolates were obtained from thirty kinds of traditional fermented douchi from six cities and counties in Yunnan, and those strains were divided into twelve groups by their morphological and biochemical characteristics. Based on 16S ribosomal DNA (rDNA) sequencing and analysis, 56 representative strains were identified as belonging to 6 genera and 14 species: Lactobacillus (4 spp.), Weissella (3 spp.), Pediococcus (2 spp.), Staphylococcus (2 spp.), Enterococcus (1 sp.), and Bacillus (2 spp.). The results show that douchi contains a large natural population of LAB of diverse composition from which some strains could be selected as starters for functional fermented foods. This is the first study on the original douchi from Yunnan, and the results suggest that it may be a useful source for the isolation of LAB. This study has also laid a foundation for further research on developing functional douchi products.

Molecular Cloning and Sequence Analysis of an Extracellular Protease from Four Bacillus subtilis Strains

Four Bacillus subtilis strains were isolated from traditional fermented foods, and the sequences of their extracellular alkaline proteases (AprE) were analyzed and cloned. The recombinant enzymes synthesized by means of Escherichia coli exhibited high proteolytic activity. AprE CN2 showed hydrolytic activity 4-fold higher than that of AprE 168. This activity was also seen in the presence of relatively high NaCl concentrations.

Microsatellite Analysis of Saccharomyces cerevisiae in Cooked Bread

Bread has been a staple of the human diet since antiquity. Many Saccharomyces cerevisiae strains have been used in raising the dough during bread-making. However, the genetic relationships and characteristics of these strains are not well understood. In this study, we analyzed several S. cerevisiae strains using microsatellite polymorphism. First, we extracted DNA from cooked bread (baked, steamed, and fried) and analyzed S. cerevisiae polymorphisms using six microsatellite loci, which is half of the subset reported previously. To the best of our knowledge, this is the first report that demonstrates the possibility of analyzing S. cerevisiae microsatellite polymorphisms using cooked bread as a source of yeast DNA.