Suppressive Effect of Wild Saccharomyces Cerevisiae and Saccharomyces Paradoxus Strains on Ige Production by Mouse Spleen Cells
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Food Sci. Technol. Res., 19 (6), 1019–1027, 2013 Suppressive Effect of Wild Saccharomyces cerevisiae and Saccharomyces paradoxus Strains on Ige Production by Mouse Spleen Cells 1* 1 2 1 2 2 Takeshi Kawahara , Daichi NaKayama , Keisuke toda , Shyuichiro iNagaKi , Katsumi taNaKa and Hisako yaSui 1 Laboratory of Food Bioscience, Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Kamiina, Nagano 399-4598, Japan 2 Kiso Town Resources Institute, 2326-6 Fukushima Kisomachi, Kiso-gun, Nagano 397-8588, Japan Received February 18, 2013; Accepted July 17, 2013 The genus Saccharomyces includes industrial yeasts that are used for bread and alcoholic beverage production. Saccharomyces strains isolated from natural resources, referred to as “wild” yeasts, are used for making products with strain-specific flavors that are different from those of the “domesticated” in- dustrial yeasts. The physiological effects of wild yeast are poorly understood. In this study, we isolated 2 Saccharomyces cerevisiae strains (S02 − 03) and 5 Saccharomyces paradoxus strains (P01 − 02, S01, S04 − 05) from natural resources in the Kiso area and investigated the effect of these fungal strains on IgE production by mouse spleen cells. Culturing spleen cells with heat-killed yeasts resulted in elevated IFN-γ and IL-12 levels followed by significant reduction in IgE levels. The S03 and P01 strains induced IL-12 p40 and IL-10 expression in RAW264 cells. Thus, wild strains of S. cerevisiae and S. paradoxus regulate macrophage cytokine production to improve the Th1/Th2 immune balance and suppress IgE production. Keywords: Saccharomyces cerevisiae, Saccharomyces paradoxus, wild yeast, IFN-γ, IL-12, IgE, allergy Introduction tained from common industrial yeast. To date, some of the The yeasts belonging to the genus Saccharomyces sensu favorably fermented foods obtained by using wild Saccharo- stricto have long been used for fermented food and alcoholic myces strains have been introduced into the market and have beverage production because of their metabolic properties become popular (Oda et al., 2010). that enable conversion of sugar into ethanol and CO2 (Rainieri Despite their contribution in sensory function, physi- et al., 2003; Sicard and Legras, 2011). In particular, Sac- ological functions provided by Saccharomyces yeasts have charomyces cerevisiae, S. bayanus, and S. pastorianus (S. been reported only for a single special strain. Saccharomyces carlsbergensis) have been empirically considered non-patho- cerevisiae var. boulardii (S. boulardii) has been extensively genic to humans, and are commonly used as starter cultures studied for its role in attenuating the toxicity of several en- (brewer’s yeast, baker’s yeast, and wine yeast, respectively) teric bacterial pathogens (Gedak, 1999; Wu et al., 2008), for food and beverage production on an industrial scale. maintaining epithelial barrier integrity (Dahan et al., 2003; Moreover, Saccharomyces yeasts are consumed as nutritional Klingberg et al., 2008), treating and preventing gastrointes- supplements. tinal diseases (Zanello et al., 2009), and reducing inflamma- On the other hand, non-industrial yeast strains have been tory responses triggered by bacterial infection (Lee et al., isolated from many natural resources such as fruits, tree sap, 2005). The probiotic properties of S. boulardii have been flowers, and soil. These yeasts are frequently referred to as determined by double-blind, randomized, and placebo-con- “wild” yeasts in contradistinction to “domesticated” indus- trolled studies (Bleichner et al., 1997; Sazawal et al., 2006). trial yeasts and have been extensively used for alcoholic Notably, S. boulardii is the only probiotic yeast strain that is fermentation. Some wild Saccharomyces yeasts produce utilized as a functional food ingredient today. molecules with unique sensory flavors, which are not ob- S. boulardii stimulates intestinal immunity by enhanc- ing immunoglobulin (Ig)A production. Buts et al. (1990) *To whom correspondence should be addressed. reported that orally administered S. boulardii induced mu- E-mail: [email protected] cosal secretory IgA in the small intestine of rat. In addition, 1020 t. Kawahara et al. Lessard et al. (2009) reported that the intake of S. boulardii myces yeasts as follows: Sample resources were submerged in modulated the development of intestinal IgA secretion and 10 − 50 mL of isolation medium and incubated at 29℃ for 1 reduced intestinal bacterial translocation in pigs. Thus, these week, following which, 1 mL of the forming culture superna- reports imply that orally administered Saccharomyces yeasts tant was mixed with 13 mL of yeast-peptone-sucrose (YPS) have the potential to regulate Ig production. However, little is broth (1% yeast extract, 2% peptone, and 2% sucrose; pH 5.8) known about the effect of Saccharomyces strains, including supplemented with 3% ethanol, 2% lactic acid, 0.2% sodium various wild strains, on the production of other classes of Igs. propionate, and 0.01% chloramphenicol and incubated at 29℃ In this study, we isolated 2 S. cerevisiae and 5 S. para- under anaerobic conditions. After 10 days, 1 mL of the form- doxus strains from fruit and soil samples and investigated the ing culture supernatant was seeded in a YPD agar plate (pH 6.8) effect of those strains on IgE production, an Ig class closely containing 1.5% (w/v) agar and incubated at 29℃ for 2 days. associated with the pathophysiology of type-I allergy (Gould The yeast-like 4970 colonies were then transferred to fresh et al., 2003). Among the isolated Saccharomyces strains and YPD broth. The yeast strains grown in the YPD broth at 29℃ type strains of the same species, S. cerevisiae S03 and S. were stored at −80℃ until use. paradoxus P01 showed the strongest IgE suppressive effect. Classification of yeast strains Isolated yeast-like strains The IgE suppressive effect of these strains was thermostable were provisionally classified using the API ID32 C identifi- at 200℃ for 40 min. These strains significantly increased cation kit (Sysmex-bioMérieux; Marcy l’Etoile, France) ac- interferon (IFN)-γ and interleukin (IL)-12 production in cording to the manufacturer’s protocols. spleen cells and increased IL-12 p40 and IL-10 mRNA ex- Among the 16 strains classified as S. cerevisiae in the pression in macrophage-like RAW264 cells. Thus, some wild API ID32 C test, seven strains that suppressed IgE produc- strains of S. cerevisiae and S. paradoxus can be used as IgE- tion were further evaluated by comparing the partial D1/D2 suppressive agents in functional foods. 26S rDNA sequences with the DNA sequences provided by Techno Suruga (Shizuoka, Japan) using the National Center Materials and Methods for Biotechnology Information (NCBI) database and the Materials The isolation medium for Saccharomyces standard nucleotide-nucleotide homology search tool BLAST yeasts was prepared as follows: Approximately, 150 g of (http://www.ncbi.nlm.nih.gov/BLAST). commercially available dried rice malt (Kojiya Mitsuemon, Preparation of heat-killed yeasts Isolated Saccharomy- Aichi, Japan) was immersed in 450 mL of distilled water ces strains were cultured in YPD medium at 30℃ for 24 h and incubated at 56℃ for 6 h for saccharification. The sac- and then harvested by centrifugation at 1000 × g for 10 min. charified liquid was then centrifuged at 3000 × g for 10 min. The yeast pellet was washed 3 times with distilled water and The supernatant was sterilized using a 0.2-μm pore cellulose then lyophilized. acetate membrane filter (Advantec, Tokyo, Japan). The fil- The lyophilized yeasts were heated at 70℃ for 30 min trate was supplemented with 2% lactic acid, 0.2% sodium in a thermal cycler (PTC-200; MJ Research, Waltham, MA) propionate, and 0.01% chloramphenicol for use as isolation and used as heat-killed yeasts. For evaluating the effect of medium (pH 3.1). baking temperatures on the bioactivity of yeasts, the lyophi- Type strains of S. cerevisiae (NBRC 10217) and S. para- lized yeasts were incubated at 200℃ for 0, 20, 30, or 40 min doxus (NBRC 10609) were purchased from the National prior to the heat inactivation. Institute of Technology and Evaluation Biological Resource Mice Specific pathogen-free male BALB/c mice aged Center (NBRC; Chiba, Japan). These strains were cultured in 6 weeks were purchased from Japan SLC (Shizuoka, Japan) yeast-peptone-dextrose (YPD) broth (1% yeast extract, 2% and housed at 23℃ ± 3℃ under a 12-h light/dark cycle. All peptone, and 2% d-glucose; pH 5.8) and stored as frozen cul- the animal protocols used in this study were approved by the tures in the medium at −80℃. Committee for Animal Experiments of Shinshu University. Mouse leukemic monocyte cell line RAW264 was ob- Mouse spleen cell culture Spleen cells were prepared tained from Riken Cell Bank (Ibaraki, Japan) and maintained from 6 to 8-wk-old mice according to a previously de- in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, scribed method (Kawahara et al., 2006). Briefly, mice were Japan) containing 10% heat-inactivated fetal bovine serum euthanized using the vertebral-dislocation method, and the (FBS; HyClone, Logan, UT, USA). spleens were aseptically harvested. A single-cell suspension Isolation and cultivation of yeast strains For yeast isola- was prepared by gentle manipulation of the spleen in RPMI tion, 208 sample resources, including fruits, flowers, leaves 1640 medium. To remove the red blood cells, the spleen cells and soil, were collected from the Kiso area (Nagano, Japan). were treated with hemolytic buffer (17 mM Tris-HCl buffer The enrichment culture method was used to isolate Saccharo- containing 0.144 M ammonium chloride; pH 7.2) for 5 min IgE-Suppressing Effect of Wild Saccharomyces Strains 1021 at room temperature, then diluted with fresh RPMI 1640 tories, Gaithersburg, MD, USA), 2 μg/mL rat anti-mouse IL- medium (same volume as the hemolytic buffer), and centri- 12 p40/70 biotin-conjugated (BD Pharmingen) and rat anti- fuged at 450 × g for 10 min to remove the hemolytic buffer.