J Korean Soc Appl Biol Chem (2013) 56, 247−250 DOI 10.1007/s13765-012-3197-8
SHORT COMMUNICATION
Occurrence of Issatchenkia orientalis Exhibiting Inhibitory Effects against Soybean Lipoxygenase in Korean nuruk Yasir Anwar · Seong-Yeol Baek · Soo-Hwan Yeo · Heui-Dong Park
Received: 6 September 2012 / Accepted: 30 January 2013 / Published Online: 30 April 2013 © The Korean Society for Applied Biological Chemistry and Springer 2013
Abstract Three yeast strains (designated SHA, SHC, and SHD) exhibiting strong inhibitory effects against soybean lipoxygenase were isolated from Korean nuruk and identified as Issatchenkia orientalis. Total 80 yeast isolates from two major Korean nuruk samples were analyzed by polymerase chain reaction-restriction fragment length polymorphism and phylogenetic analysis. The most abundant yeast was identified as Pichia anomala, comprising 47 out of 80 strains, followed by 15 strains of I. orientalis. All 15 I. orientalis strains showed significant inhibitory effects against soybean lipoxygenase, higher than that of Saccahromyces boulardii used as a positive control. Keywords anti-inflammation · I. orientalis · lipoxygenase inhibition · nuruk
Inflammation is a part of the complex biological response of vascular tissues to harmful stimuli such as pathogens, damaged cells or irritants in order to remove the injurious stimuli and to initiate the healing process (Ferrero-Miliani et al., 2007). At the first step of inflammation, the conversion of arachidonic acid to 5HPETE (5-hydroperoxyeicosatetraenoic acid) takes place by 5lipoxygenase action. Therefore, inhibition of 5-lipoxygenase activity has been one of the most important targets for anti-inflammation (Yokomizo et al., 1997). Saccharomyces boulardii used as a biotherapeutic agent, after performing several clinical trials, displayed
Y. Anwar · H. D. Park () Department of Food Science and Biotechnology, Kyungpook National University, 80 Daehakro, Daegu 702-701, Republic of Korea E-mail:
[email protected] S. Y. Baek · S. H. Yeo Department of Agro-food Resource, National Academy of Agricultural Science, RDA, Suwon 441-853, Republic of Korea
beneficial effects in the prevention and the treatment of intestinal infects and the maintenance of inflammatory bowel disease (Galliano et al., 2009; Pothoulakis, 2009). Some other inhibitors from different plants and vegetables also show inhibition against soybean lipoxygenase, including ascorbic acid, 6-palmitoylascorbic acid, and trolox (Rodrigues et al., 2000; Koulkoulitsa et al., 2007). Nuruk, a cultured wheat enzyme used in two major Korean traditional rice wines such as takju and yakju, contains amylolytic enzymes produced by natural fungi grown in it (Yu et al., 2004; Jeon et al., 2007; Lee et al., 2009). Nuruk is also a natural yeast inoculator for rice wine. Korean rice wine has been reported to inhibit proliferation of the B16BL6 mouse melanoma cell line and exerts an anti-metastatic effect on the cells injected in C57/BL6 mouse (Chung et al., 1998). It has been shown to exhibit much higher anti-proliferation effects on murine Lewis lung carcinoma cells than red wine (Kim et al., 2004). In the present study, a wild yeast exhibiting strong inhibitory effects against soybean lipoxygenase was isolated, and occurrence of the strain was assayed in Korean nuruk by molecular biological methods. Yeast cells were generally grown at 30oC in YPD medium composed of 1% yeast extract, 2% peptone, and 2% glucose in distilled water. Nuruk samples of two largest companies in Songhak and Sangju, Korea were collected for the isolation of wild yeasts. For the soybean lipoxygenase test, yeast cells were cultivated in at 30oC for 48 h in YPD liquid medium. After the cultivation, the culture broth was centrifuged to remove the culture supernatant, which was used as the sample for the inhibition test. The enzyme inhibition assay was performed as previously reported by Rickert and Klinman (1999). The mixture of 850 µL of 0.1 M Tris-HCl (pH 8.5), 20 µL sample or distilled water (as a control), and 80 µL soybean lipoxygenase (Sigma, USA), except the substrate solution, was mixed throughly and incubated at room temperature for 5 min. Subsequently, the reaction was started with the addition of 50 µL of 1.8 µg/mL α-linolenic acid (Sigma) as a substrate and kept at room temperature for 5 min. The optical density was
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J Korean Soc Appl Biol Chem (2013) 56, 247−250
Fig. 1 Phylogenetic tree of yeast isolates exhibiting inhibitory effects against soybean lipoxygenase based on the ITS I-5.8S-ITS II sequences.
determined at 234 nm using a UV-visible spectrophotometer (Shimadzu, Japan). The inhibition activity was expressed as the percentage of the decrease of absorbance in the presence of the sample compared with the absorbance in the absence of the sample. Yeast chromosomal DNAs were isolated from the cells grown in YPD broth at 30oC for 48 h by general methods (Kaiser et al., 1994). PCR primers used for the amplification of yeast ITS I5.8S-ITS II region were ITS1 (5'-CATTTAGAGGAACTAAAAG TCG-3') and ITS4 (5'-CCTCCGCTTATTGATATGC-3'). Amplification was performed in 50 µL mixture containing 5 µL of 10 × PCR buffer, 1 µL of 10 mM dNTPs, 10 pmol each of forward and reverse primers, 2 µL of template DNA, and 0.25 µL Taq DNA polymerase. The temperature program was composed of an initial denaturation at 95oC for 6 min, 30 cycles at 94oC for 1 min, 55oC for 1 min, 72oC for 1 min, and a final extension at 72oC for 5 min. After the polymerase chain reaction, the amplified products were checked in a 1.5% agarose gel. Appropriate amounts of amplicons were digested at 37oC for 1 h with 0.5 µL each of Hinf I and Hae III endonucleases (Takara, Japan). The digested DNA fragments were resolved on 1.5% agarose gel with 0.5 × TBE buffer according to the general methods (Sambrook and Russel, 2001). One hundred bp ladders were used as markers for checking the DNA size. Nucleotide sequences of the ITS I-5.8S-ITS II region were compared with those available in the GenBank database by using the BLAST method to determine their approximate phylogenetic affiliation and their sequence similarities at the National Center for Biotechnology Information, USA (Altschul et al., 1997; http:// ncbi.nlm.nih.gov/BLAST). The sequences of the related taxa were
acquired from the same web site. Nucleotide sequences were initially aligned using the CLUSTAL X program (Thompson et al., 1997) and then manually adjusted. Distance matrices were calculated, and phylogenetic tree for the data set was created according to the Kimura two-parameter model and neighborjoining method by using the Mega4 (ver. 4.02, Molecular Evolutionary Genetics Analysis, USA) software package obtained from the web site (www.megasoftware.net). Over 100 wild yeasts were isolated from Korean nuruk samples based on the colony shape and microscopic observation. After isolation, all the isolates were tested for inhibitory activities against soybean lipoxygenase using their culture supernatants(?). Among the isolates, three strains (designated SHA, SHB, and SHD) exhibiting strong inhibitory effects against lipoxygenase were selected. Their ITS I-5.8S-ITS II sequences were identical to those of Issatchenkia orieatalis ATCC 2410 strain. Phylogenetic analysis based on the sequences revealed that all three strains were closely related to I. orientalis (Fig. 1). Several I. orientalis yeasts have been isolated as malic acid-degrading yeast from a Korean Campbell Early grape wine pomace (Seo et al., 2007) and as a urease producing yeast from Korean nuruk (Lee and Park, 2012), indicating that the yeast has been well adapted to Korean environment. Therefore, distribution of the yeast species was assayed in two different nuruk samples obtained from the biggest nuruk manufacturing companies, which are located over 200 km apart from each other in Korea. Each 40 random yeast isolates from the 2 nuruks were first grouped by PCR-(spell out)restriction fragment length polymorphism of ITS I-5.8S-ITS II regions (Fig. 2), and representatives of each group were identified by phylogenetic
J Korean Soc Appl Biol Chem (2013) 56, 247−250
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Fig. 2 PCR-RFLP of yeasts isolated from two Korean major nuruk designated SJ (a) and SH (b). PCR fragments amplified from the chromosomal DNA of the isolates using IT1 and ITS4 primers were digested with restriction endonucleases Hae III and Hinf I. The Digested DNA fragments were resolved on 1.5% agarose gel. Lane M represents 100 bp DNA ladder used as a DNA size marker.
analysis based on their sequences (Table 1, Supplement 1). Average yeast populations of SJ and SH were very similar, which were about 6.10×105 and 6.12×105 cfu/g, respectively. Among 40 isolates from SJ nuruk, 23 isolates were identified as Pichia anomala and 12 were I. orientalis. The others were 1 Torulopsis delbrueckii, 1 S. cerevisiae, 1 Hanseniaspora uvarum, 1 Clavispora lustiniae, and 1 Zygoascus hellenicus var. hellenicus. However, 24 P. anomala, 9 T. delbrueckii, 3 I. orientalis, 3 P. fermentans, and 1 S. cerevisiae were identified from SH nuruk. Total 15 strains were identified as I. orientalis from 80 isolates of 2 nuruk samples. Interestingly, the region for SJ nuruk growth is very famous for vineyard in Korea, in which several I. orientalis strains have been isolated recently (Lee and Park, 2012). In particlar, 12 strains out of 40 were I. orientalis in SJ nuruk, whereas only 3 strains in SH nuruk. All the 15 strains showed significant inhibitory effects against soybean lipoxygenase (Table 1). Their activities were higher than S. boulardii, a positive control strain which has been reported to contain anti-inflammation effects (Lee et al., 2005; Sougioultzis et al., 2006; Samir and Daniel, 2007; Galliano et al., 2009; Pothoulakis, 2009; Grijó et al., 2010; Zanello et al., 2011). However, no inhibitory effects were detected in the other strains. Effects of I. orientalis isolated from Korean nuruk in the present study on the inflammation have yet to be elucidated. Acknowledgment This study was carried out with the support of Cooperative Research Program for Agricultural Science & Technology Development (200901AFT154192334), RDA, Republic of Korea.
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