Introduction
Yeasts are heterotrophic and have relatively simple nutritional requirements. Besides fermented foods [1, 2], yeasts are distributed in natural habitats including flowers or plant debris in soils. Many species have been isolated from fermented foods and raw materials [3]. Recently, we isolated various wild yeast strains from flowers and soils [4-7] and their various phenotypes were investigated [8].
Gamma-aminobutyric acid (GABA) is a neurotransmitter in the central nervous system and is synthesized through decarboxylation by glutamate decarboxylase using the cofactor, pyridoxal-5-phosphate. GABA has hypotensive, tranquilizing, and diuretic effects, and is involved in the prevention of diabetes [9-12]. GABA is widely distributed in microorganisms, plants, and animals [13].
GABA production has been reported in yeasts such as Saccharomyces cerevisiae [14], Debaryomyces hansenii, and Rhodotorula mucilaginosa [15]. However, in these species, GABA production was relatively low and other associated physiological characteristics have not been studied.
This study was performed to obtain potent GABA-producing yeast strains for application to medicinal foods or agents, through screening of GABA production in nonpathogenic yeasts from wild flowers. Furthermore, microbiological characteristics of these strains were studied.
Materials and Methods
Strains and chemicals
Non-pathogenic yeast strains (182) were isolated from wild flowers in Korea and were used in this study [4, 7, 16-18].
Pyridoxal-5-phosphate and polyvinylidene fluoride membrane, used as a non-heating sterilization filter were purchased from Sigma-Aldrich (St. Louis, MO, USA) and thin-layer chromatography (TLC) plates were purchased from Merck KGaA (Darmstadt, Germany). Unless otherwise specified, all chemicals were of analytical grade.
Determination of GABA concentration by TLC
GABA content of cell-free extracts was determined by method described by Holdiness [19] as follows. After incubating the yeast stains at 30°C for 48 hr, each cell-free extract was obtained by centrifugation at 9,000 × g for 10 min, with subsequent sonication of the cell pellets at 20 Hz for 5 min. Each cell-free extract was dissolved in distilled water and 20 μL was spotted on the TLC plate, which was then developed using the typical developing solvent, n-butanol-acetic acid-H2O [4:1:1 (v/v/v)]. Developed TLC plates were dried at 60°C after spraying with ninhydrin solution (0.2%, w/v ethanol) for color development. The GABA spot was confirmed by comparison with a sample GABA standard spot.
Microbiological characteristics of the selected yeasts
The morphological and cultural characteristics of selected yeast strains were investigated according to Han et al. [20]. To assess ascospore formation, yeasts were cultured in yeast extract-peptone-dextrose (YPD) medium at 30°C for 24 hr and subsequently cultured for 5 days in ascospore-forming medium containing potassium acetate (1%), yeast extract (0.1%), and dextrose (0.05%). The strain was then observed using a microscope to assess ascospore formation. Yeast was successively cultured at 30°C for 7days in YPD medium, yeast extract-malt extract medium, potato-dextrose medium, and glucose-peptone-yeast extract agar containing glucose (4%), peptone (0.5%), and yeast extract (0.5%). Pseudomycelium formation was determined by observing the shape of the cell in culture.
For examination of the detailed structure of selected yeasts by scanning electron microscopy (SEM) [20], selected yeasts were cultured in YPD medium and maintained in a 20% glycerol stock. The stock was diluted using a 0.05 M cacodylate buffer (pH 8.2). The diluted solution was centrifuged at 1,300 rpm for 1 min to obtain the yeast cell pellet, which was used for fixation. The strain was also cultured in potato-dextrose-broth (PDB) medium at a shaking speed of 150 rpm in the dark at 30°C for 48 hr. The sample was fixed with 2.5% paraformaldehydeglutaraldehyde buffer with 0.05 M phosphate (pH 7.2) for 2 hr, washed with cacodylate buffer, post-fixed in 1% osmium tetroxide (in the same buffer) for 1 hr, and washed again with the same buffer. The sample was then dehydrated in graded ethanol followed by isoamyl acetate, and then dried under a fume hood. Finally, the samples were covered in gold using a sputter coater and observed with the Hitachi S4700 (Hitachi, Tokyo, Japan) field emission scanning electron microscope.
Results and Discussion
Screening of potent GABA-producing yeasts
GABA content of cell-free extracts from 182 wild yeast strains was investigated by TLC. Among the wild yeasts, Kazachstania unispora SY14-1 and Metschnikowia reukaufii SY20-7 from Seonyudo, Nakazawaea holstii 63-J-1 and Pichia guilliermondii 89-J-1 from Jeju island, and Pichia scolyti YJ14-2 and Pichia silvicola UL6-1 from Yokjido and Ulleungdo exhibited GABA production (Table 1).
Using a TLC plate, GABA production was also detected for Sporobolomyces carnicolor 73-D-3 and 374-CO-1 from Gyejoksan and Oseosan and Sporobolomyces carnicolor 402-JB-1 and Sporobolomyces ruberrimus 121-Z-3 from Baekamsan (Table 1).
Among aforementioned GABA-producing yeasts, we selected Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1 for enhanced production, assessed by stronger intensity on TLC plates.
Phylogenetic tree of Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1
The phylogenetic tree for the selected yeast strains is shown in Fig. 1. Pichia silvicola UL6-1 was closely related to Nakazawaea holstii 63-J-1 in this study, and Sporobolomyces carnicolor 402-JB-1 was closely related to Sporobolomyces ruberrimus 73-D-3 and 121-Z-3. The tree was generated by the neighbor-joining method, using MEGA v5.1.
Morphological and cultural characteristics of Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1
The morphological and cultural characteristics of Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1 are presented in Table 2.
Pichia silvicola UL6-1 was oval-shaped and employed a budding system for vegetative reproduction. The strain formed ascospores and pseudomycelia, and grew well in YPD medium, yeast extract-malt extract medium, potatodextrose medium, and 5% NaCl-containing YPD medium.
Few studies on halophilic yeasts have been performed with the exception of Zygosaccharomyces rouxii from soybeans [21] and halotolerant protease-producing Saccharomyces lipolytica [22] and Hansenula polymorpha S-9 from traditional meju [21, 23]. It is known that halophilic microorganisms produce enzymes with advantages such as preventing microbial contamination in the enzyme industry and enhancing the flavor of salted foods during aging [21]. Therefore, Pichia silvicola UL6-1, identified in this study, should be very useful in preparing halotolerant enzymes or bioactive compounds for the food and medical industries.
Sporobolomyces carnicolor 402-JB-1 was oval-shaped and used a budding system for vegetative reproduction. Furthermore, the strain did not formed ascospores and pseudomycelia, and grew well in YPD medium and yeast extract-malt extract medium.
Fig. 1. Phylogenetic tree of Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1 based on the nucleotide sequences of large subunit 26S ribosomal DNA. T, Type strain.
Structural characteristics
Fig. 2 shows features of Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1, during different media and cultural conditions, identified by optical microscopy and electron scanning microscopy (SEM). The typical shapes of vegetative cells of these strains were ellipsoidal to oval, commonly showing single cell forms and budding systems (Fig. 2).
