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

Abstract

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.

Characterization of Bacillus Subtilis Isolated from Asian Fermented Foods

“…Media The media for screening proteolytic bacteria and enzyme production were prepared according to the method described by Phromraksa et al (2008).…”

“…Immunoblotting was performed according to Phromraksa et al (2008) with slight modifications. Proteins on the gel were electrotransferred onto a PVDF membrane (ATTO, Japan) and then blocked overnight at 4℃ with 1% skim milk (Difco, USA) in TBS-T (Tris-buffered saline, pH 7.4, containing 0.1% Tween-20).…”

“…We recently found numerous aerobic bacteria in soaked rice and rice blocks from production processing of fermented rice-noodles (Kobayashi et al, 2007). B. subtilis, isolated from the soaked rice, showed interesting properties that have the potential to reduce allergenicity of wheat allergen (gliadin) (Phromraksa et al, 2008). In Myanmar, Ikeda et al (2005) reported that B. subtilis, isolated from soaked rice from fermented rice-noodle (Mohinger) processing, showed high proteolytic activities towards the protein substrates, azocasein and azocollagen, and could digest allergenic rice protein.…”

“…Liquefaction of the medium is an indicator of bacterial enzyme activity. Uninoculated identical medium was used as a control.Phylogenetic analysis Eleven B. subtilis strains were isolated and identified by 16S rDNA sequencing in our previous study (Nagano and To, 2000;Tran and Nagano, 2002;Liu and Nagano, 2008;Phromraksa et al, 2008). The 16S rDNA sequences of bacteria were deposited in the DDBJ database under the accession numbers shown in Table 1.…”

“…These samples included 1 sample of fermented soybean seeds collected The samples were suspended in sterilized distilled water, spread on nutrient agar slants (containing 0.5% glucose (Nacalai Tesque, Japan)) and incubated at room temperature. All bacteria were maintained before screening on nutrient agar slants (containing 0.5% glucose) at 4℃.Media The media for screening proteolytic bacteria and enzyme production were prepared according to the method described by Phromraksa et al (2008).Gelatin stab medium contained 1.8% nutrient medium (Eiken Chemical, Japan) and 17% gelatin (Nacalai Tesque). The medium was sterilized at 121℃ for 15 min and kept at 4℃ until use.…”