Yeast produce various bioactive agents [1-4] including an antihypertensive angiotensin I-converting enzyme inhi- bitor [5]. Most of the yeasts that produce these com- pounds have been isolated from fermented foods or their raw materials [3,4,6]. Recently, we isolated and identi- fied various yeasts, including unrecorded yeasts, from wild flowers growing in cities [7,8], mountains [9], inland and coastal areas [10,11], and, islands, such as Jeju Island, Korea [12,13].
In a previous paper [13], we reported the diversity of yeasts present on wild flowers in Ulleungdo and Yokjido, Korea; we also reported other characteristics of the unre- corded yeasts isolated from Yokjido [14]. Here, we describe the mycological characteristics of the unrecorded yeasts isolated from Ulleungdo, Korea.
We investigated the morphological characteristics of the unrecorded yeasts using previously described methods [13]. The physiological functions of the yeasts were determined as follows. The selected unrecorded yeasts were cultured in yeast extract-peptone-dextrose (YPD) medium at 30°C for 2 d. After centrifugation at 10,000 ∞ g for 15 min, su- pernatants and cells were obtained. The cells were dis- rupted by vortexing with sonication and then centrifuged at 12,000 ∞ g for 20 min. Cell-free extract was obtained and the supernatant was used to determine the physiolo- gical functionalities of the yeasts.
Antihypertensive angiotensin I-converting enzyme (ACE) inhibitory activity was assayed using the method publi- shed by Cushman and Cheung [15]. Antioxidant activity was assayed using the method of Lee et al. [4] using anti- oxidant activity (DPPH) as a substrate. Tyrosinase inhibi- tory activity was assayed by the method of Kim et al. [16]. Xanthine oxidase inhibitory activity and superoxide dismutase (SOD)-like activity were determined using a modification of a previously described method [9]. α-Glucosidase inhibitory activity was assayed using the me- thod described Kang et al. [17] using ρ-nitrophenyl-β-D-glucophylanoside (PNPG) as a substrate.
The unrecorded yeasts were screened from 58 yeasts isolated in Ulleungdo by searching Keris, PubMed and other fungal taxonomy databases. The previously unre- corded yeasts were identified as Meyerozyma caribbica UL 5-1 and Pichia silvicola UL6-1; they were isolated from Duchesnea chrysantha and Rubia akane in Ulleungdo.
Mycological characteristics of the unrecorded yeasts
The morphological and cultural characteristics of the two unrecorded yeasts are summarized in Table 1 and Fig. 1. The two unrecorded yeasts were both oval in shape did not form ascospores and pseudomycelia. Both grew well in YPD, yeast extract-malt extract (YM) and potato-dextrose (PD) broth and also grew in vitamin-free me- dium. M. caribbica UL5-1 was found to be halophilic and thermotolerant, and grew well in 20% NaCl-YPD broth at 37ºC. Both yeasts were negative for urease activity.
Table 1. Microbiological and cultural characteristics of the newly reporting yeasts from wild flowers of Ulleungdo, Korea
1)O, Oval 2)B, Budding 3)W, White
Fig. 1. Morphological characterization of Meyerozyma caribbica UL5-1 (A,C) and Pichia silvicola UL6-1 (B,D). A and B, Optical microscope (scale bar-1 μm); C and D, Scanning electron microscope (scale bars=5 μm).
We previously reported about a thermotolerant yeast isolated from wild flowers [14], traditional meju [6], and halotolerant yeasts from Korean fermented soy sauce, Doenjang and Gochujang [18].
We investigated the assimilation and fermentation of the two unrecorded yeasts on various carbon sources, using previously described methods [18] (Table 2 and 3). M. caribbica UL5-1 utilized several types of hexose, pentose and sugar alcohol such as D-glucose, D-galactose, D-mal- tose, D-saccharose, D-trehalose, D-melezitose, D-raffinose, D-cellobiose, D-xylose, L-arabinose, glycerol, 2-keto-D-glu- conate, D-sorbitol, methyl-α-D-glucopyranoside and N-acetyl-glucosamine. However, it was not able to utilize D-lactose, xylitol, or inositol. Pichia silvicola UL6-1 utilized only D-glucose, D-galactose, D-trehalose, D-cellobiose, glycerol and D-sorbitol. Furthermore, M. caribbica UL5-1 fermented D-glucose, fructose, mannose, D-galactose, su- crose and raffinose, whereas P. silvicola UL6-1 fermented only D-glucose, fructose, mannose and cellobiose.
Physiological functionalities of the unrecorded yeasts
The physiological functionalities of the supernatants and cell-free extracts from the unrecorded yeasts were inve- stigated (Table 4). The antihypertensive ACE inhibitory activities of supernatants from M. caribbica UL5-1 and Pichia silvicola UL6-1 were 84.2% and 82.6%, respective- ly, approximately 50% higher than those of their cell-free extracts. These results were higher than those of Saccharomyces cerevisiae KCTC 7904 (42.1%)[13], Pichia anomala (31.0%), Pichia anomala KCCM 11473 (72.0%)[19], or Pleurotus cornucopiae (78.0%)[20].
Table 4. Physiological functionalities of unrecorded yeasts from Ulleungdo, Korea
*ACE, Angiotensin I-converting enzyme; XOD, Xanthine oxidase; SOD, Superoxide dismutase.
**n.d; not detected.
The anti-obesity α -glucosidase inhibitory activity of cell-free extract from Pichia silvicola UL6-1 was also very high 85.8%. These results were higher than those of Aspergillus oryzae N157-1 (48.3%)[17] or Pichia burtonii (90.9%)[3] isolated from Korean traditional fermented foods. Tyrosi- nase inhibitory activity and the other functionalities were either not detected or were very weak (15%).
