冷胁迫对异常威克汉姆酵母代谢物组的影响
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摘要
通过甲氧胺、双(三甲基硅烷基)三氟乙酰胺(含1%三甲基氯硅烷)对6株抗冷冻异常威克汉姆酵母(Wickerhamomyces anomalus)菌株的培养液进行衍生化,采用气相色谱-质谱法测定冷胁迫处理(4℃)对6株耐冷冻W. anomalus酵母代谢物的影响。质量浓度超过10 mg/L的代谢物为49种,其中糖、糖醇、糖酸、糖苷34种,反映了W. anomalus复杂的糖代谢能力,且包括大量具有益生元活性的糖类;此外,还含有角鲨烯、百里香酚-β-D-葡萄糖苷、β-羟基-苯丙酸、异长叶烯,表明W. anomalus具有萜类、苯丙素物质代谢途径。采用代谢物组学数据处理方法识别W. anomalus在冷胁迫时的差异代谢物,甘油、角鲨烯、赤藓糖醇、半乳糖、L-抗坏血酸-2,6-二棕榈酸酯、甘露糖、百里香酚-β-D-葡萄糖苷可能是W. anomalus冷胁迫相关的特征性代谢物。本研究可为酵母冷胁迫机制、酵母发酵食品食味品质形成机制、酵母新产品研发提供理论参考。
The effect of cold stress(4 ℃) on metabolites of six strains of the cold resistant yeast Wickerhamomyces anomalus was investigated in this paper. The fermentation broths of these yeast strains was derivatized with methoxamine and BSTFA-TMS, and then the products were determined by gas chromatography-mass spectrometry(GC-MS). A total of 49 metabolites with more than 10 mg/L concentrations were identified, including 34 saccharides, sugar alcohols and sugar acids,several of which had prebiotic activity. These results indicate that the W. anomalus has complex saccharide metabolic ability.In addition, W. anomalus produced squalene, thymol-β-D-glucoside, β-hydroxy-phenylpropionic acid, and isolongifolene,suggesting that this yeast has metabolic pathways of terpenoids and phenylpropanoids. According to the results of metabolome analysis, glycerol, squalene, erythritol, galactose, L-ascorbic acid-2,6-dipalmitate, mannose, and thymol-β-Dglucoside were the bio-markers of W. anomalus under cold stress. This study provides a theoretical basis for the studying the response mechanism of yeast to cold stress and the mechanism underlying flavor formation in yeast-fermented foods, and for the development of new products.
The effect of cold stress(4 ℃) on metabolites of six strains of the cold resistant yeast Wickerhamomyces anomalus was investigated in this paper. The fermentation broths of these yeast strains was derivatized with methoxamine and BSTFA-TMS, and then the products were determined by gas chromatography-mass spectrometry(GC-MS). A total of 49 metabolites with more than 10 mg/L concentrations were identified, including 34 saccharides, sugar alcohols and sugar acids,several of which had prebiotic activity. These results indicate that the W. anomalus has complex saccharide metabolic ability.In addition, W. anomalus produced squalene, thymol-β-D-glucoside, β-hydroxy-phenylpropionic acid, and isolongifolene,suggesting that this yeast has metabolic pathways of terpenoids and phenylpropanoids. According to the results of metabolome analysis, glycerol, squalene, erythritol, galactose, L-ascorbic acid-2,6-dipalmitate, mannose, and thymol-β-Dglucoside were the bio-markers of W. anomalus under cold stress. This study provides a theoretical basis for the studying the response mechanism of yeast to cold stress and the mechanism underlying flavor formation in yeast-fermented foods, and for the development of new products.
引文
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[24]李玉冬,吴清平,张菊梅,等.常见食源性致病菌胞外代谢轮廓分析[J].现代食品科技,2016,32(1):37-43.DOI:10.13982/j.mfst.1673-9078.2016.1.006.
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[27]PANADERO J,PALLOTTI C,RODRíGUEZ-VARGAS S,et al.A downshift in temperature activates the high osmolarity glycerol(HOG)pathway,which determines freeze tolerance in Saccharomyces cerevisiae[J].Journal of Biological Chemistry,2006,281(8):4638-4645.DOI:10.1074/jbc.M512736200.
[28]杨文洲,薛永常,窦少华,等.不同碳源对酵母代谢有机酸的影响[J].中国酿造,2007,26(10):20-22.DOI:10.3969/j.issn.0254-5071.2007.10.006.
[29]SPANOVA M,CZABANY T,ZELLNIG G,et al.Effect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast Saccharomyces cerevisiae[J].Journal of Biological Chemistry,2010,285(9):6127-6133.DOI:10.1074/jbc.M109.074229.
[30]BLAGOVI?B,RUP?I?J,MESARI?M,et al.Lipid analysis of the plasma membrane and mitochondria of brewer’s yeast[J].Folia Microbiologica,2005,50(1):24-30.DOI:10.1007/BF02931290.
[2]WU M Y,ZHANG C Y,SUN X,et al.Effects of NTH1 gene deletion and overexpressing TPS1 gene on freeze tolerance in baker’s yeast[J].Lecture Notes in Electrical Engineering,2014,249:447-454.DOI:10.1007/978-3-642-37916-1_46.
[3]HESPEELS B,XIANG L,FLOT J F,et al.Against all odds:trehalose-6-phosphate synthase and trehalase genes in the bdelloid rotifer adineta vaga were acquired by horizontal gene transfer and are upregulated during desiccation[J].PLoS ONE,2015,10(7):e0131313.DOI:10.1371/journal.pone.0131313.
[4]MAHMUD S A,HIRASAWA T,SHIMIZU H.Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses[J].Journal of Bioscience&Bioengineering,2010,109(3):262-266.DOI:10.1016/j.jbiosc.2009.08.500.
[5]BANDARA A,FRASER S,CHAMBERS P J,et al.Trehalose promotes the survival of Saccharomyces cerevisiae,during lethal ethanol stress,but does not influence growth under sublethal ethanol stress[J].FEMS Yeast Research,2009,9(8):1208-1216.DOI:10.1111/j.1567-1364.2009.00569.x.
[6]YU S,YUTAKA H,KEISUKE H,et al.Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker’s yeast[J].Microbial Cell Factories,2012,11(1):1-8.DOI:10.1186/1475-2859-11-40.
[7]SASANO Y,HAITANI Y,HASHIDA K,et al.Simultaneous accumulation of proline and trehalose in industrial baker’s yeast enhances fermentation ability in frozen dough[J].Journal of Bioscience&Bioengineering,2012,113(5):592-595.DOI:10.1016/j.jbiosc.2011.12.018.
[8]TSOLMONBAATAR A,HASHIDA K,SUGIMOTO Y,et al.Isolation of baker’s yeast mutants with proline accumulation that showed enhanced tolerance to baking-associated stresses[J].International Journal of Food Microbiology,2016,238:233-240.DOI:10.1016/j.ijfoodmicro.2016.09.015.
[9]SHI X J,MIAO Y L,CHEN J Y,et al.The relationship of freeze tolerance with intracellular compounds in baker’s yeasts[J].Applied Biochemistry&Biotechnology,2014,172(6):3042-3053.DOI:10.1007/s12010-014-0744-2.
[10]KOBAYASHI Y,IWATA H,YOSHIDA J,et al.Metabolic correlation between polyol and energy-storing carbohydrate under osmotic and oxidative stress condition in Moniliella megachiliensis[J].Journal of Bioscience and Bioengineering,120,4:405-410.DOI:10.1016/j.jbiosc.2015.02.014.
[11]ASLANKOOHI E,REZAEI M N,VERVOORT Y,et al.Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation[J].PLoS ONE,2015,10(3):e0119364.DOI:10.1371/journal.pone.0119364.
[12]PATEL A K,BHARTIYA S,VENKATESH K V.Analysis of osmoadaptation system in budding yeast suggests that regulated degradation of glycerol synthesis enzyme is key to near-perfect adaptation[J].Systems&Synthetic Biology,2014,8(2):141-154.DOI:10.1007/s11693-013-9126-2.
[13]宋振玉,李楠,张姝,等.低糖适应性耐冻酵母菌与普通酵母菌细胞构成成分的比较[J].中国酿造,2009,28(6):57-61.DOI:10.3969/j.issn.0254-5071.2009.06.019.
[14]MARIA L M,AMPARO Q,MANUEL G J.Metabolomic comparison of Saccharomyces cerevisiae and the cryotolerant species S.bayanus var.uvarum and S.kudriavzevii during wine fermentation at low temperature[J].PLoS ONE,2013,8(3):e60135.DOI:10.1371/journal.pone.0060135.
[15]MURAKAMI Y,YOKOIGAWA K,KAWAI F,et al.Lipid composition of commercial bakers’yeasts having different freezetolerance in frozen dough[J].Bioscience Biotechnology Biochemistry,1996,60(11):1874-1876.DOI:10.1271/bbb.60.1874.
[16]BLAGOVI?B,MESARI?M.Changes in lipid composition during low temperature storage of baker’s yeast[C]//9th Yeast Lipid Conference,Berlin.Njema?ka,2009:21-23.
[17]PETRUJKI?B T,?EFER D S,JOVANOVI?I B,et al.Effects of commercial selenium products on glutathione peroxidase activity and semen quality in stud boars[J].Animal Feed Science&Technology,2014,197:194-205.DOI:10.1016/j.anifeedsci.2014.09.001.
[18]LóPEZ-MALO M,GARCíA-RíOS E,CHIVA R,et al.Functional analysis of lipid metabolism genes in wine yeasts during alcoholic fermentation at low temperature[J].Microbial Cell,2014,1(11):365-375.DOI:10.15698/mic2014.11.174.
[19]LóPEZ-MALO M,QUEROL A,GUILLAMON J M.Metabolomic comparison of Saccharomyces cerevisiae and the cryotolerant species S.bayanus var.uvarum and S.kudriavzevii during wine fermentation at low temperature[J].PLoS ONE,2013,8(3):60135.DOI:10.1371/journal.pone.0060135.
[20]CA?AS P M I,GARCíA-ROMERO E,MANSO J M H,et al.Influence of sequential inoculation of Wickerhamomyces anomalus,and Saccharomyces cerevisiae,in the quality of red wines[J].European Food Research&Technology,2014,239(2):279-286.DOI:10.1007/s00217-014-2220-1.
[21]YE M,YUE T,YUAN Y.Effects of sequential mixed cultures of Wickerhamomyces anomalus and Saccharomyces cerevisiae on apple cider fermentation[J].FEMS Yeast Research,2015,14(6):873-882.DOI:10.1111/1567-1364.12175.
[22]原苗苗,赵新节,孙玉霞.低温对葡萄酒香气成分和酵母代谢的影响[J].食品与发酵工业,2017,43(12):268-276.DOI:10.13995/j.cnki.11-1802/ts.014924.
[23]陈玉香.红曲黄酒中酵母产特征酯类物质合成机理研究[D].福州:福州大学,2014.
[24]李玉冬,吴清平,张菊梅,等.常见食源性致病菌胞外代谢轮廓分析[J].现代食品科技,2016,32(1):37-43.DOI:10.13982/j.mfst.1673-9078.2016.1.006.
[25]刘晓燕,张瑾,王媛媛,等.解脂耶氏酵母利用甘蔗渣发酵产赤藓醇[J].淮阴师范学院学报(自然科学版),2017,16(4):323-328.DOI:10.16119/j.cnki.issn1671-6876.2017.04.009.
[26]YAMADA R,YAMAUCHI A,ANDO Y,et al.Modulation of gene expression by cocktailδ-integration to improve carotenoid production in Saccharomyces cerevisiae[J].Bioresource Technology,2018,268:616-621.DOI:10.1016/j.biortech.2018.08.044.
[27]PANADERO J,PALLOTTI C,RODRíGUEZ-VARGAS S,et al.A downshift in temperature activates the high osmolarity glycerol(HOG)pathway,which determines freeze tolerance in Saccharomyces cerevisiae[J].Journal of Biological Chemistry,2006,281(8):4638-4645.DOI:10.1074/jbc.M512736200.
[28]杨文洲,薛永常,窦少华,等.不同碳源对酵母代谢有机酸的影响[J].中国酿造,2007,26(10):20-22.DOI:10.3969/j.issn.0254-5071.2007.10.006.
[29]SPANOVA M,CZABANY T,ZELLNIG G,et al.Effect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast Saccharomyces cerevisiae[J].Journal of Biological Chemistry,2010,285(9):6127-6133.DOI:10.1074/jbc.M109.074229.
[30]BLAGOVI?B,RUP?I?J,MESARI?M,et al.Lipid analysis of the plasma membrane and mitochondria of brewer’s yeast[J].Folia Microbiologica,2005,50(1):24-30.DOI:10.1007/BF02931290.
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