L-苯丙氨酸对苹果醋发酵乙酸-2-苯乙酯合成途径的影响
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摘要
为了初步研究食品添加剂L-苯丙氨酸对苹果醋发酵过程中特征性香气成分乙酸-2-苯乙酯合成途径的影响,采用顶空固相微萃取-气相色谱质谱(HS-SPME-GC-MS)联用和酶联免疫吸附试验,检测各合成相关基质浓度和关键酶活性的变化。结果表明,当L-苯丙氨酸添加量为8 g/L时,β-苯乙醇、乙酸、乙酸-2-苯乙酯、乙酰辅酶A质量浓度分别提高了19.2%、35.9%、51.6%、10.86%;L-苯丙氨酸对乙醇脱氢酶(ADH)、醇酰基转移酶(AAT)酶活性均有一定的促进作用,却对酯酶酶活性有抑制作用。因此,本试验初步将8 g/L作为L-苯丙氨酸的最佳添加量,初步推测L-苯丙氨酸通过醇酰基转移酶途径促进乙酸-2-苯乙酯的合成及其相关基质生成量和关键酶的活性关键酶活的生成量,为高品质苹果醋的技术研究和指导生产实践提供一定的理论依据。
In order to preliminarily study the effects of food additive L-phenylalanine on the synthetic pathway of characteristic aroma components acetic acid-2-phenyl ethyl ester in apple vinegar fermentation, the related matrix concentration and key enzyme activities were detected by HS-SPMEGC-MS and enzyme linked immunosorbent assay. Results showed that when L-phenylalanine 8 g/L was added, the mass concentration of β-phenyl alcohol, acetic acid, acetic acid-2-phenyl ethyl ester and acetyl coenzyme A increased by 19.2%, 35.9%, 51.6% and 10.86%, respectively. The enzymatic activities of alcohol dehydrogenase(ADH) and alcohol acyltransferase(AAT) were promoted by L-phenylalanine, however, the esterase enzyme activity was inhibited. Therefore, the optimum L-phenylalanine addition was 8 g/L. It was preliminarily speculated that the synthesis of acetic acid-2-phenyl ethyl ester and its related matrix production and key enzyme activities were promoted by L-phenylalanine through alcohol acyltransferase pathway. The paper provided theoretical basis for the technical research and production guidance of high quality apple vinegar.
In order to preliminarily study the effects of food additive L-phenylalanine on the synthetic pathway of characteristic aroma components acetic acid-2-phenyl ethyl ester in apple vinegar fermentation, the related matrix concentration and key enzyme activities were detected by HS-SPMEGC-MS and enzyme linked immunosorbent assay. Results showed that when L-phenylalanine 8 g/L was added, the mass concentration of β-phenyl alcohol, acetic acid, acetic acid-2-phenyl ethyl ester and acetyl coenzyme A increased by 19.2%, 35.9%, 51.6% and 10.86%, respectively. The enzymatic activities of alcohol dehydrogenase(ADH) and alcohol acyltransferase(AAT) were promoted by L-phenylalanine, however, the esterase enzyme activity was inhibited. Therefore, the optimum L-phenylalanine addition was 8 g/L. It was preliminarily speculated that the synthesis of acetic acid-2-phenyl ethyl ester and its related matrix production and key enzyme activities were promoted by L-phenylalanine through alcohol acyltransferase pathway. The paper provided theoretical basis for the technical research and production guidance of high quality apple vinegar.
引文
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[26]陈燕,李小惠,张惟广.不同地区甘薯的酿酒特性及其发酵酒风味物质的分析[J].中国酿造,2016,35(10):120-125.
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[30]ETSCHMANN M M W,SELL D,SCHRADER J.Production of 2-phenylethanol and 2-phenylethylacetate from L-phenylalanine by coupling whole-cell biocatalysis with organophilic pervaporation[J].Biotechnol Bioeng,2005,92(5):624-634.
[2]GULLA M,VERZELIONI E,CANONICO M.Aerobic submerged fermentation by acetic acid bacteria for vinegar production:process and biotechnological aspects[J].Process Biochem,2014,49(10):1571-1579.
[3]薛淑琴,谢思芸,肖仔君,等.完全发酵与适度发酵苹果醋主要成分的差异性分析[J].食品科学,2017,38(12):137-143.
[4]陆军.微生物转化L-苯丙氨酸生成2-苯乙醇的研究[D].无锡:江南大学,2008.
[5]张传志.谷氨酸棒杆菌L-苯丙氨酸合成途径系统改造及发酵优化[D].无锡:江南大学,2014.
[6]杜闪,王雪花,杨政茂,等.生物转化合成β-苯乙醇代谢途径及其调控的研究[J].食品与发酵工业,2014,40(1):168-173.
[7]宋山,潘丽军,吴学凤.乙醇脱氢酶过表达对醋酸菌发酵产酸影响研究[J].食品科技,2016,41(4):13-18.
[8]李树萍,邱诗棋,吴宛芹,等.气相色谱-嗅闻-质谱联用分析红枣白兰地风味成分[J].食品科学,2017,38(4):187-191.
[9]焦娇,李凯,李树萍,等.HS-SPME-GC-O-MS分析红枣发酵酒中的挥发性成分[J].食品科学,2017,38(4):197-203.
[10]范丽.苹果酒酵母中合成酯类化合物关键酶的研究[D].无锡:江南大学,2005.
[11]JI H L,CHAO Z,JU Y K,et al.Evaluation on anticancer effect against HL-60 cells and toxicity in vitro and in vivo of the phenethyl acetate isolated from a marine bacterium Streptomyces griseus[J].Fish Aquat Sci,2015,18(1):35-44.
[12]YOSHIOKA K,HASHIMOTO N.Ester formation by alcohol acetyltransferase from brewers yeast[J].Agr Biol Chem,1981,45(10):2183-2190.
[13]于立梅,刘俊梅,冯卫华,等.催陈方式对山竹果酒理化特性和香气成分的影响[J].现代食品科技,2013,29(10):2442-2446.
[14]TOMASSETTI M,FAN魳M,BIANCHINI G,et al.Retention of stereochemistry in the microwave assisted synthesis of 1H-tetrazole bioisosteric moiety from chiral phenyl-acetic acid derivatives[J].Tetrahedr Lett,2013,54:6247-6250.
[15]MARTIN V,BOIDO E,GIORELLO F,et al.Effect of yeast assimilable nitrogen on the synthesis of phenolic aroma compounds by Hanseniaspora vineae strains[J].Yeast,2016,33(7):323-328.
[16]李维,崔丹瑶,王建辉,等.HS-SPME-GC-MS分析高产酯低产高级醇酿酒酵母发酵酒的风味物质[J].现代食品科技,2018,34(3):248-254.
[17]张峻松,张文叶,刘程毅,等.生物技术制备的天然山楂香料的化学成分分析[J].精细化工,2006,23(10):977-979.
[18]陈玉香,叶汶坤,何冬萍,等.酵母菌对红曲黄酒风味物质形成的影响[J].中国食品学报,2015,15(7):218-224.
[19]刘志彬,张雯,陈玉香,等.异常维克汉姆酵母的乙酸苯乙酯合成途径[J].中国食品学报,2015,15(11):48-53.
[20]范丽,徐岩,王栋.法尔皮有孢汉生酵母(Hanseniaspora valbyensis)醇乙酰基转移酶的分离纯化及特性[J].食品科学,2006,27(2):49-53.
[21]STRIBNY J,QUEROL A,P魪REZTORRADO R.Differences in enzymatic properties of the Saccharomyces kudriavzevii and Saccharomyces uvarum alcohol acetyltransferases and their impact on aroma-active compounds production[J].Front Microbiol,2016,7:897-910.
[22]宋娟,张霁红,康三江,等.乙酸-2-苯乙酯的合成途径与调节研究[J].西南大学学报(自然科学版),2018,40(8):25-31.
[23]宋娟,张霁红,康三江,等.苹果醋发酵过程中L-苯丙氨酸促进乙酸-2-苯乙酯的合成[J].现代食品科技,2019,35(1):1-8.
[24]牛明福,李亚恒,陈金帅,等.马克斯克鲁维酵母生物转化2-苯乙醇工艺优化及耐高温特性分析[J].食品与发酵工业,2018,44(2):15-20.
[25]李江涌,王如福,焦玉双.山西老陈醋熏醅前后香气成分的变化分析[J].中国酿造,2016,35(9):47-50.
[26]陈燕,李小惠,张惟广.不同地区甘薯的酿酒特性及其发酵酒风味物质的分析[J].中国酿造,2016,35(10):120-125.
[27]吴霏霏.枇杷果实香气检测及AAT1基因克隆表达与酶活性分析研究[D].扬州:扬州大学,2015.
[28]中华人民共和国国家卫生和计划生育委员会.GB 5009.225-2016酒中乙醇浓度的测定[S].北京:中国标准出版社,2016.
[29]何炯灵.新型果醋固定化发酵工艺探索[D].杭州:浙江工商大学,2018.
[30]ETSCHMANN M M W,SELL D,SCHRADER J.Production of 2-phenylethanol and 2-phenylethylacetate from L-phenylalanine by coupling whole-cell biocatalysis with organophilic pervaporation[J].Biotechnol Bioeng,2005,92(5):624-634.
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