【作者】 张林；

【导师】 吴重德；

【作者基本信息】 四川大学 ， 发酵工程， 2021， 硕士

【摘要】 高盐稀态酱油因发酵时间长,生产环境开放及高盐弱酸的发酵条件,使微生物演替规律和相互作用关系难以剖析,产品品质受外界干扰大。选用耐盐功能菌群强化酱油发酵,是提高产品品质的有效策略之一。本研究以高盐稀态酱油为对象,基于多相检测技术分析主要代谢产物和微生物群落的动态变化,分析其潜在的相关性。然后以乙醇和挥发性组分为导向,筛选耐盐酵母菌并用于酱醪发酵,探究外源酵母菌株的差异性和接种顺序对酱醪微生物群落和代谢组分的影响。最后探讨了工艺条件对酱油品质的影响。得到主要结论如下:(1)酱醪发酵过程中代谢组分的变化与微生物群落演替密切相关Weissella、Staphylococcus、Tetragenococcus和Aspergillus、Zygosaccharomyces、Issatchenkia分别是酱醪中的优势细菌和真菌且相对丰度随发酵进行而变化显著。发酵1-3个月,理化指标的动态变化与优势属的演替相吻合。与初始酱醪相比,发酵6个月后,酱醪中L-苹果酸、焦谷氨酸和乳酸分别增加了11.79、11.39和3.19倍。挥发性组分的变化分为两个阶段,多数香味物质在发酵4-6个月大量生成。其中3-(甲硫基)-1-丙醇、辛酸乙酯、4-乙基愈创木酚(4-EG)、2(5)-乙基-4-羟基-5(2)-甲基-3(2H)-呋喃酮(HEMF)、苯乙醛、和4-乙稀基愈创木酚(4-VG)等物质的气味活度值(OAV)大于100,构成了酱醪的主体风味。酱醪中还检出了氨基甲酸乙酯(EC)和七种生物胺(BAs),均在第3个月酱醪中达到峰值,随后含量下降,其中大多数生物胺由前体氨基酸在相应脱羧酶作用下生成,这与发酵0-3个月高丰度的Weissella、Staphylococcus和Leuconostoc等产酸细菌有关。(2)定向筛选高产乙醇和挥发性组分的酱油酿造用酵母菌从高盐稀态酱醪中筛选得到2株糖代谢、产乙醇及风味组分的能力均高于常用商业酵母的同源分离株,结合显微镜观察和分子生物学鉴定均为Z.rouxii,分别命名为Z.rouxii QH-1和Z.rouxii QH-25。但分离株间具有差异,Z.rouxii QH-1产乙醇的量为3.03 g/100m L略高于Z.rouxii QH-25的2.83 g/100m L,且前者产挥发性酯和酸的量分别为1811.90μg/kg和150.93μg/kg,略强于后者的1727.17μg/kg和62.62μg/kg;而后者产挥发性醇、酚类的量分别为9181.62μg/kg和275.06μg/kg高于前者的7347.17μg/kg和217.54μg/kg。(3)耐盐酵母属/株差异性和接种顺序对酱醪酿造微生物群落和代谢组分影响显著以Z.rouxii QH-1和Z.rouxii QH-25和C.versatilis为研究对象,研究酵母菌属/株的差异和接种顺序对酱醪微生物群落及代谢组分的影响规律。结果表明,不同株的Z.rouxii及与C.versatilis的接种顺序影响了酱醪还原糖的代谢和乙醇的含量。酵母菌强化使有机酸和游离氨基酸的总含量分别增加了4.07%～25.97%和-4.58%～17.96%,其中乳酸、乙酸和草酸等有机酸和谷氨酸和精氨酸等氨基酸的含量则因接种酵母菌和顺序的不同而差异显著。Z.rouxii先接种更有利于挥发性组分的形成,其中Z.rouxii QH-25和C.versatilis顺序共培酱醪(No.4)的挥发性组分总含量增加了161.04%,主要是醇类、酯类、酚类和酮类。群落结构分析结果表明,Z.rouxii QH-1单培养的酱醪中Staphylococcus丰度降低,而接种了Z.rouxii QH-25的酱醪中均增加。接种酵母菌显著改变了酱醪真菌群落的β多样性,其中Z.rouxii QH-1强化酱醪中Aspergillus相对丰度均超过90%,而No.4酱醪中Zygosaccharomyces丰度最高。共培养酱醪中Zygosaccharomyces和Candida的菌群互作节点比单培养更多,增强了Zygosaccharomyces与Staphylococcus的正相关性。冗余分析结果表明,Staphylococcus、Zygosaccharomyces和Candida与4-EG、HEMF、3-甲基-1-丁醇和己酸乙酯等高OAV值物质均呈正相关。同时还探索了不同酱醪中特征代谢产物的代谢途径调控的差异。接种酵母均降低了酱醪中BAs的含量,共培组中No.4酱醪的效果最佳,降低了69.72%;但因增强了乙醇发酵,均使得EC的含量略增。(4)探讨了工艺条件对酱油品质的影响温度在30℃～35℃,乙醇浓度低于2.50 g/100g时更有利于减少酱油中EC的生成。酱油中BAs主要在发酵阶段形成,而EC主要在灭菌时生成,其中灭菌温度、时间、乙醇和尿素浓度均与EC含量呈正相关。以90℃,20 min为条件灭菌后,酱油有机酸含量无显著变化,但酱油色泽更加浓郁。灭菌后,强化发酵和自然发酵酱油中氨基酸含量分别降低了6.66%和2.24%,而挥发性组分的含量分别增加了9.84%和30.75%。灭菌后自然发酵生酱油加强了花果味、蜂蜜味、焦香味和一定的酸性刺激;而强化发酵酱油的麦芽香、蘑菇香、洋葱香、烟熏味和焦糖香气更浓郁。更多还原

【Abstract】 It is difficult to analyze the law of microbial succession and interaction relationship,and the product quality is greatly disturbed by the external conditions during the fermentation of high-salt dilute-state soy sauce because of the extreme environment of high salt and weak acid,long fermentation time,and open environment.Therefore,the application of salt-tolerant functional microbes to fortify soy sauce fermentation is one of the effective strategies to improve product quality.In this study,the dynamics of various metabolites and microbial communities during the process were investigated by multiphase detection approaches to analyze their potential interactions by using the high-salt dilute-state soy sauce moromi.On this basis,with ethanol and flavor components as the guide,functional strains were screened and used for the moromi fermentation,and the influence of strain-specificity and inoculation sequence on the disturbance of the microbial community and metabolic components of fermented moromi was explored.Finally,The effect of process conditions on the quality of soy sauce was discussed.These results are as follows:(1)The changes of metabolic components were closely related to the microbial community during the soy sauce fermentationWeissella,Staphylococcus,Tetragenococcus and Aspergillus,Issatchenkia,Zygosaccharomyces were the dominant bacteria and fungi,respectively,and their relative abundance significantly was changed with fermentation time.The dynamics of physicochemical properties were consistent with the succession of dominant microbes after 1-3 months of fermentation.Compared with the initial moromi,the contents of L-malic acid,pyroglutamic acid,and lactic acid were increased by 11.79,11.39,and 3.19 times(p < 0.05),respectively,after 6 months of fermentation.The change of volatile components was divided into two stages,most of which were produced in large quantities during 4-6 months of fermentation.The main flavor profile of the sauce mash was composed of 3-(methyl thiol)-1-propanol,ethyl octanoate,4-ethyl guaiacol(4-EG),2(5)-ethyl-4-hydroxy-5(2)-methyl-3(2H)-furanone(HEMF),phenylacetaldehyde,4-ethyl-guaiacol(4-VG),etc due to their OAV were greater than 100 for at least one fermentation period.The ethyl carbamate(EC)and seven kinds of biogenic amines(BAs)were detected in moromis,and their contents reached the maximum in the third month,then their contents decreased.Most BAs were produced by precursor amino acids under the action of corresponding decarboxylase,which was associated with a higher abundance of acid-producing bacteria such as Weissella,Staphylococcus,and Leuconostoc during the 0-3 months of fermentation(2)The yeasts for soy sauce brewing were selected based on high-yield ethanol and volatile componentsTwo salt-tolerant yeasts were screened from high-salt dilute-state soy sauce moromi,and their ability of sugar metabolism,ethanol production,and flavor components production were higher than those of common commercial yeasts.They were identified as Z.rouxii by microscopic observation and molecular biological identification and were named Z.rouxii QH-1 and Z.rouxii QH-25,respectively.However,there were differences between the two isolated yeasts.The ethanol productivity of Z.rouxii QH-1 was 3.03 g/100 m L,which was slightly higher than that of Z.rouxii QH-25(2.83 g/100 m L).The contents of volatile esters and acids produced by Z.rouxii QH-1 were 1811.90 μg/kg and 150.93 μg/kg,respectively,which were slightly higher than those of Z.rouxii QH-25(1727.17 μg/kg and 62.62μg/kg).However,the contents of volatile alcohols and phenols produced by the latter were 9181.62 μg/kg and 275.06 μg/kg,respectively,which were higher than those of the former(7347.17 μg/kg and 217.54 μg/kg).(3)The effects of microbial communities and metabolites in moromi were significantly influenced by different homologous isolates of Z.rouxiiThe effect of starters,consisted of different homologous isolates of Z.rouxii and C.versatilis,on the microbial community and metabolic components was investigated.The results showed that the concentration of reducing sugars and ethanol were affected by different homologous isolates of Z.rouxii and inoculation sequence with C.versatilis.The total contents of organic acids and free amino acids increased by4.07% ～ 25.97% and-4.58% ～ 17.96%,respectively in all samples of yeasts inoculation,among which the contents of organic acids such as lactic acid,acetic acid,oxalic acid and amino acids such as Glu and Arg varied significantly with the difference of Z.rouxii and inoculation sequence.Inoculation of Z.rouxii prior to C.versatilis was more conducive to the formation of volatile components,of them,the total content of volatile components was increased by 161.04%,mainly alcohols,esters,phenols and ketones in the No.4 by sequential inoculation of Z.rouxii QH-25 and C.versatilis.The results obtained by the community diversity analysis showed that Z.rouxii QH-1 single inoculation decreased the relative abundance of Staphylococcus,while the abundance of Staphylococcus in the moromi of Z.rouxii QH-25 inoculation were increased.The yeast inoculation significantly changed the β-diversity of the fungal community.The relative abundance of Aspergillus in the moromi of Z.rouxii QH-1 inoculation was more than 90%,while the No.4 moromi had the highest abundance of Zygosaccharomyces.In co-cultured moromi,the nodes of the interacting relationship between Zygosaccharomyces and Candida was increased,as well as enhanced the positive correlation of Zygosaccharomyces with Staphylococcus,which compared with fortified three strains alonely.Redundancy analysis showed that Staphylococcus,Zygosaccharomyces and Candida were positively correlated with high OAV substances such as 4-EG,HEMF,3-methyl-1-butanol and ethyl caproate.Meanwhile,metabolic pathways of characteristic metabolites and the abundance of relevant enzymes were differentiated.The content of BAs was reduced in the moromi by yeast inoculation,and the effect of No.4 was the best in the co-culture group,which was reduced by 69.72%.However,the content of EC was slightly increased due to the enhancement of ethanol fermentation.(4)The effect of process conditions on the quality of soy sauceThe temperature was 30℃ ～ 35℃ and the concentration of ethanol was lower than 2.50 g/100 g,which was more conducive to reduce the formation of EC in soy sauce.BAs were mainly formed during the soy sauce fermentation stage,while EC was mainly formed during sterilization.Of them,sterilization temperature,sterilization time,ethanol and urea concentrations were all positively correlated with EC content.At 90℃ for 20 min,the main physicochemical property and organic acids were not affected,while the color of soy sauce was more intense.FAA contents were decreased by 6.66% and 2.24%,while volatile components were increased by 9.84%and 30.75% after sterilization respectively,in the fortified and natural fermentation soy sauce.The effect of sterilization on the characteristic flavor was closely related to the fermentation type.After sterilization,the natural fermentation soy sauce had enhanced the fruit,honey,burnt flavor and some acid stimulation,while the fortified fermentation soy sauce had more malt,mushroom,onion,smoky and caramel aromas.更多还原