J. Agric. Food Chem. 2008, 56, 99–105
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Immunoreactivity and Amino Acid Content of Fermented Soybean Products JUANA FRIAS,† YOUNG SOO SONG,‡ CRISTINA MARTÍNEZ-VILLALUENGA,† ´ ELVIRA GONZALEZ DE MEJIA,‡ AND CONCEPCION VIDAL-VALVERDE*,† Department of Food Science and Human Nutrition, University of Illinois at Urbana–Champaign, 228 ERML, MC-051, 1201 West Gregory Drive, Urbana, Illinois 61801, and Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
Food allergy has become a public health problem that continues to challenge both the public and the food industry. The objective of this research was the detection and quantification of the major human allergenic soy proteins and to study the reduction in immunoreactivity and improvement of amino acid content after fermentation of soybean flour. Fermentation was carried out in the solid state of cracked seeds inoculated with Aspergillus oryzae, Rhizopus oryzae, and Bacillus subtilis and in the liquid state of milled soybean flours fermented naturally by microorganisms present only in the seeds or by inoculation with Lactobacillus plantarum. ELISA and Western blot were used to quantify IgE antibody response, and HPLC was used to identify and quantify total amino acids. L. plantarum fermented soy flour showed the highest reduction in IgE immunoreactivity (96–99%) depending upon the sensitivity of the plasma used. Among the solid fermented products, the lowest reduction in immunoreactivity was obtained when mold strains, R. oryzae and A. oryzae, were used (66 and 68%, respectively, for human plasma 97.5 kUA/L). Among the solid fermented products, those inoculated with B. subtilis yielded a 81 and 86% reduction in immunoreactivity against both human plasma 97.5 IgE kUA/L and human pooled plasma samples, respectively. When soybean was subjected to liquid fermentation, most of the total amino acids increased significantly (p e 0.05). In solid fermentation with R. oryzae, only Ala and Thr content improved. Fermentation can decrease soy immunoreactivity, and there is potential of developing nutritious hypoallergenic soy products. KEYWORDS: Soybean; fermentation; immunoreactivity; IgE immunoreactivity; amino acids; hypoallergenic foods; antigenicity
INTRODUCTION
Food allergy is a relatively rare and violent reaction of the immune system toward food proteins. It is defined as an immunologically based adverse reaction in response to dietary antigens (1). Small regions in the allergenic proteins, called epitopes, provoke the immunoglobulin E (IgE)-mediated allergenic response (2). The allergen provokes an initial IgE antibody response followed by a secondary IgE antibody response, which signals an allergic reaction (3, 4). Food proteins bind to the allergen-specific IgE molecules residing in the mast cells and basophils, causing them to release inflammatory mediators, including histamine (1). Food allergy affects 3% of the adult population and up to 6–8% of infants. Allergenic conditions directly affect many millions of people worldwide (5–7). The estimated prevalence of soybean allergies is about 0.5% of the total U.S. population (8–10). * To whom correspondence should be addressed. Telephone: +34915622900 Ext. 241. Fax: +34-915644853. E-mail:
[email protected]. † Instituto de Fermentaciones Industriales (CSIC). ‡ University of Illinois at Urbana–Champaign.
Soybean is important both from economical and nutritional standpoints. It is one of the most important grains of the legume family, and one of the main protein sources for livestock. Acceptance of soybean protein products has increased because of the low cost and high nutritional quality for human consumption and also as protein source for animal meals. In the United States, the Food Allergen Labeling and Consumer Protection Act of 2004 (P.L. 108-282) (11) includes soy in its definition of the “big 8”, which comprises those foods that cause the most allergenic reactions (12–14). Food allergens are complex mixtures of potentially immunoreactive proteins. Currently, there have been identified 33 IgE-binding allergenic proteins in soybean (15). According to Wilson et al. (16), soybean allergens comprise proteins with molecular masses from 7.0 to 71 kDa. A few of these 33 proteins are responsible for a majority (∼90%) of the allergenic responses, making it important to identify and purify these key proteins. Several studies have confirmed the degradation of soybean allergens during fermentation by microbial proteolytic enzymes in soy sauce, miso, soybean ingredients, and feed-grade soybean meals (17–19). Fermentation has the capacity to improve
10.1021/jf072177j CCC: $40.75 2008 American Chemical Society Published on Web 12/12/2007
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nutritional and functional properties compared to original products. Fermentation is an important area of research, which entails the ability to hydrolyze soy protein into smaller peptides. Soy sauce possesses microbial proteolytic enzymes able to degrade soybean proteins, including major soybean allergen (Gly m Bd 30K) into peptides and amino acids (18). Altering the structure of their epitopes can lower the immunoreactivity of soybean-containing products during processing. Moreover, the fermentation process affects the nutritional quality of legumes by improving the protein content and digestibility as a consequence of the partial degradation of complex stored proteins into more simple and soluble products (20). The objectives of this study were the detection and quantification of soybean allergens, to produce hypoallergenic soycontaining ingredients by natural and induced fermentation with Lactobacillus plantarum, Bacillus subtilis, Aspergillus oryzae, and Rhizopus oryzae and to assess the type and level of amino acids produced during this process. MATERIALS AND METHODS Chemicals. Trizma-hydrochloride, 2-mercaptoethanol (2-ME), sodium chloride, sodium carbonate, ammonium sulfate, phosphatebuffered saline-Tween buffer (PBST) at pH 7.4, bovine serum albumin (BSA), Tween-20, Tris-buffered saline (TBS) tablets, para-nitrophenyl phosphate (PNPP), ethylenediaminetetraacetic acid (EDTA), deoxycholate, and Corning Sterile syringe filter pore size of 0.2 µm were obtained from Sigma-Aldrich (St. Louis, MO). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) was performed with Phastsystem (Amersham-Pharmacia, Inc., NJ). Reagents used for SDS-PAGE included Laemmli blue dye, running buffer with TG/SDS, 12% precast polyacrylamide-gradient minigels (Amersham), buffer strips for Phastsystem minigels (Amersham), Coommassie Blue biosafe dye, and prestained SDS-PAGE molecular-weight broad-range standards, which were purchased from BioRad (Hercules, CA). The immunoblot PVDF membrane (for protein blotting, 0.2 µm) 7 × 8.4 cm was purchased from BioRad. The Bradford assay was conducted using a Biorad Bradford reagent. Other reagents, such as analyticalgrade NaOH, H2SO4, and HCl, used occasionally to adjust buffers and stop the color substrate reaction, were purchased from Sigma Chemical (St. Louis, MO). Biological Materials. Soybeans (Glycine max L. cv. merit) were provided by the Mang Fong Pacific Trading, S.A. Seeds were cleaned and stored in darkness in polyethylene containers at 4 °C until use. A. oryzae 2094T (ATCC 1011), R. oryzae CECT 2340 (ATCC 24563), B. subtilis CECT 39T (ATCC 6051), and L. plantarum CECT 748T (ATCC 14917) were purchased from the Spanish Type Culture Collection (CECT) and used as inocula, as in Fernández-Orozco et al. (17). Stock cultures were grown and maintained as follows: A. oryzae and R. oryzae were grown for 7 days on potato dextrose agar (Difco Laboratories, Detroit, MI) at 30 °C, and the spores were collected and washed twice in sterile saline solution and used as inocula. B. subtilis was grown aerobically in BHI broth (Difco Laboratories, Detroit, MI) for 18 h at 30 °C. The pelleted cells were washed twice in sterile saline solution and used as inocula. L. plantarum was grown in MRS broth (Difco Laboratories, Detroit, MI) for 18 h at 30 °C. The cells were washed twice in sterile saline solution (8.5% NaCl) and used as inocula. The characteristics of human sera used in the immunological experiments, provided by PlasmaLab International (Everett, WA), are described in Table 1. A pooled plasma sample for Western blot analysis was prepared by combining equal volumes of human plasma samples A, B, C, D, E, and F. Soybean Fermentation. Different fermentation processes of soybean were conducted as previously described (21). Solid-state fermentations were carried out using cracked seeds suspended in sterile distilled water (1:2, w/v) for 16 h and subsequently autoclaved at 121 °C for 15 min. Further, A. oryzae (105 spores/g), R. oryzae (105 spores/g), or B. subtilis (105 CFU/g) starter cultures were inoculated at 5% (v/w) in cracked seeds. The microbial suspensions
Frias et al. Table 1. Characteristics of Human Plasma Used in Immunological Experimentsa plasma number A B C D E F G
age (years)
gender
36 m 36 m 42 f 48 f 63 m 59 m WHO standard 75/502 pooled plasma IgEb
soybean-specific IgE by inmunoCAP analysis (kUA/L)a 98.7, 97.5 71.6 34.2 11.3 8.0
