【作者】 张曼；

【导师】 杜木英；

【作者基本信息】 西南大学 ， 食品科学， 2022， 硕士

【摘要】 李子是蔷薇科植物李的果实,在世界范围内被广泛种植和食用,特别是中国、美国和欧洲。李子是一种呼吸跃变型果实,成熟期集中,采摘后不耐贮藏,易造成资源浪费。李子酒是近年来李子精深加工的主要方向之一,目前市面上的商品李子酒多采用商业酿酒酵母（Saccharomyces cerevisiae,Sc）酿造,而过度使用商业酿酒酵母会给果酒带来风味同质化、果香不突出、缺乏典型性等负面影响。越来越多的研究表明采用非酿酒酵母（non-Saccharomyces cerevisiae）与酿酒酵母混合发酵能够丰富果酒香气,提高果酒品质。然而,目前关于混合发酵的研究多集中在对葡萄酒香气的提升效应,关于李子酒的报道和应用鲜少。此外,不同非酿酒酵母具有的生理特性各不相同,在混合发酵过程中发挥的作用也不尽相同,关于李子酒在酿造期间的风味形成及变化规律的研究不够系统和全面。因此,本研究首先评估了4株非酿酒酵母的生理特性及其与酿酒酵母（D254）混合发酵特性,然后通过优化接种顺序和接种比例以放大混合发酵对李子酒的品质提升效应,最后在采用最佳混合发酵参数下对李子酒的整个酿造过程（包括主发酵和陈酿）中主要成分的变化规律进行了系统探究,以期为混合发酵李子酒的工业化应用提供科学依据。主要研究结果如下:（1）比较了热带假丝酵母（Candida tropicalis,Ct）、东方伊萨酵母（Issatchenkio orientalis,Io）、美极梅奇酵母（Metschnikowia pulcherrima,Mp）以及葡萄汁有孢汉逊酵母（Hanseniaspora uvarum,Hu）在生理特性、与酿酒酵母混合发酵特性两方面的差异。结果显示:4株非酿酒酵母均能在24 h内达到最大生物量后进入稳定期。4株非酿酒酵母都能够耐受350 g/L葡萄糖、3.5%柠檬酸、6%乙醇以及400 mg/L SO₂,均符合酿造初期要求。在混合发酵实验中,与酿酒酵母单独发酵（10 d）相比,混合发酵延缓了发酵进程（12～13 d）。不同菌株组合发酵得到的李子酒的理化指标均符合国标GB15037—2006要求。除Mp-Sc外,混合发酵对其余李子酒的色度和色调均有显著影响（P<0.05）。香气成分结合主成分分析结果表明,Mp-Sc香气组成结构优于其它酒样,特别是该组中辛酸乙酯、癸酸乙酯以及芳樟醇的显著提高（P<0.05）且香气活度值（Odor Activity Value,OAV）>1,表明该组合能够明显增强酒体中的果香、花香。Mp-Sc更适用混合发酵李子酒,有利于增加果酒香气复杂性,提升产品品质。（2）在上一章的试验基础上,本章研究了不同接种顺序包括同时接种（sim）和顺序接种（seq）以及不同初始接种比例包括M.pulcherrima:S.cerevisiae=10:1、1:1、1:10对Mp-Sc混合发酵李子酒品质和风味的影响,分别记作10:1sim、1:1sim、1:10sim、10:1seq、1:1seq以及1:10seq。结果表明:在发酵过程中,乙醇含量和酿酒酵母均能够抑制M.pulcherrima的生长。与同时接种（10:1sim、1:1sim、1:10sim）相比,接种相同酵母比例的顺序接种（10:1seq、1:1seq、1:10seq）消耗的糖更少,因而产生的乙醇含量更低。不同样品中有机酸的含量和种类因采取的接种策略的不同而不同。与顺序发酵相比,同时发酵样品中的乙酸和琥珀酸的含量较高。在所有李子酒样品中共鉴定出48种挥发性成分,其中1:1seq样品中酯类和萜类含量丰富,醇类、挥发酸类和酚类含量相对较低。主成分分析表明,10:1seq和1:1seq与酯类、萜类物质相关性较高。此外,除1:10sim外不同接种策略对李子酒的香气均有不同程度的影响。定量描述感官分析（Quantitative Descriptive Analysis,QDA）结果显示1:1seq具有最高得分的“整体香气”、“果香”和“醇香”。1:1seq是Mp-Sc混合发酵的最佳接种策略。（3）在前两章实验基础之上,本章进一步研究了李子酒在酿造期间（主发酵和陈酿）的基本理化指标、有机酸、活性成分、抗氧化活性以及香气成分的形成及动态变化规律。结果表明,发酵后苹果酸含量下降,乳酸、乙酸、柠檬酸和琥珀酸含量增加,但在陈酿阶段有机酸的种类和含量变化不显著（P>0.05）。总酚和总黄酮整体都呈现先上升后下降至稳定的趋势,均在发酵第1 d达到最大值,含量分别为3.86 mg/m L和1.71mg/m L。从所有样品中共检测出6种主要单体酚,以芦丁和槲皮素为主。在发酵阶段,DPPH、ABTS阳离子以及羟基自由基抗氧化活性明显提高（P<0.05）,但在陈酿阶段整体变化不明显（P>0.05）。香气成分结合电子鼻结果显示,发酵前后李子酒风味组成显著改变（P<0.05）。李子汁中醛、酸、酮类成分含量丰富,发酵后酯类大量形成,尤其是在陈酿第40 d时,酯类和萜烯类物质在陈酿期间达到最大值,苯乙醇、辛酸乙酯、芳樟醇、大马士酮等是其主要特征风味成分。综上,建议采用M.pulcherrima与S.cerevisiae混合发酵李子酒按照1:1的初始接种比例进行顺序接种,主发酵11 d,陈酿40 d,此时能够获得品质更佳、风味更好、活性成分含量更高的李子酒,终产品的酒精度为11.99%,可溶性固形物为6.30°Brix,总酸含量为5.94 g/L,总酚含量为2.1 mg/m L,总黄酮含量为0.45 mg/m L,口感酸甜适宜,风味浓郁,香气突出。更多还原

【Abstract】 Plum（Prunus salicina L.）,a member of the Rosaceae family,is an edible fruit consumed mainly in America,China,and Europe.However,the plum is respiration climacteric fruit,and storage-tolerance is poor after harvesting.What’s more,plum is highly seasonal and the mature period is relatively concentrated.Wine-making is one of major directions for plum.At present,the market plum wine is brewed by commercial yeast.However,excessive use of commercial yeast will bring flavor homogenization,lack of typicalness and characteristic,and other negative effects to plum wine.In recent years,more and more studies have shown that the mixed culture fermentation of non-Saccharomyces cerevisiae with S.cerevisiae can improve the quality of the fruit wine.However,most of current study concentrated on grape wine,plum wine has not yet been thoroughly explored as a subject of research.More importantly,different non-S.cerevisiae has different characteristic.And the research on the formation and change regular of plum wine during brewing is not systematic and comprehensive.Therefore,at first,this experiment assessed biochemical characteristics and mixed culture fermentation characteristics of four non-S.cerevisiae to select a match of mixed culture fermentation with huge potential.Then optimized inoculation strategy to enlarge the improvement effect of mixed culture fermentation.Finally,the dynamic changes of major components of plum wine during wine-making process were conducted in order to provide a scientific basis for the industrial application of mixed culture fermentation for plum wine.Results were showed as follow:（1）Biochemical characteristics and mixed culture fermentation characteristics Candida tropicalis,Issatchenkio orientalis,Metschnikowia pulcherrima,Hanseniaspora uvarum were compared.The results showed that:four non-S.cerevisiae attached maximum biomass and entered to stable phase within 24 h.All of four non-S.cerevisiae were able to withstand 350g/L glucose,3.5%citric acid,6%ethanol and 400 mg/L SO₂,which met basic brewing requirements.Compared with S.cerevisiae pure fermentation（10 d）,mixed fermentation delayed the fermentation process（12～13 d）.The basic physical and chemical properties of different wines all met the national standards.Among all samples,Io-Sc had the highest volatile acid content（0.78 g/L）.In addition to Mp-Sc,mixed culture fermentation had a significant effect on the chrominance and hue of plum wine（P<0.05）.The analysis of aroma components combined with principal components showed that the aroma components of Mp-Sc were better than other wine samples.Among them,ethyl caprylate,ethyl caprate,ethyl laurate,and linalool were significantly improved and the odor?activity?value>1,which was beneficial to enhance the fruit flavor and floral.In summary,Mp-Sc was more suitable for brewing plum wine,which was beneficial to increase the complexity of aroma and improve quality.（2）This study describes the effect of variable factors on aroma and quality of plum wine include inoculum ratio（M.pulcherrima:S.cerevisiae=10:1,1:1,and 1:10）,as well as simultaneous versus sequential inoculation,with tested trials spanning 10:1sim,1:1sim,1:10sim,10:1seq,1:1seq,and 1:10seq,respectively.Results showed that during fermentation,M.pulcherrima growth was inhibited by S.cerevisiae.Compared to each simultaneous analog,sequential fermentation which was inoculated with the same yeast ratio consumed less sugar and produced lower ethanol content.Organic acids in each sample varied with the use of different inoculation strategies.A total of 48 aroma compounds were quantified,and 1:1seq sample was observed to be abundant in esters and terpenes but relatively low in higher alcohols,fatty acids,and phenols.Sensory evaluation of the variously-prepared samples revealed that 1:1seq sample featured a highly intense‘global aroma’,‘fruity’,and‘alcohol’notes.PCA illustrated that different inoculation protocols of mixed culture fermentation influenced plum wine aroma except 1:10sim.Ultimately,1:1seq was identified as a promising candidate for production of high-quality plum wine.（3）On the basis of the first two chapters,this chapter further studied the basic physio-chemical,organic acid,active ingredient,antioxidant activity,and aroma components,the formation and dynamic variation of aroma components during plum wine brewing process,including alcohol fermentation and aging.The results showed that after alcohol fermentation,the malic acid content decreased,lactic acid,acetic acid,citric acid and succinic acid content increased,but the type and content of organic acids in the aging stage were not significant（P<0.05）.During the brewing of plum wine,total phenol and total flavonoids showed the first decline in the rise,all of which reached the maximum at the end of the fermentation of the first day,respectively,from 3.86 mg/m L and 1.71 mg/m L.6 major phenolic substances were detected,which were mainly rutin and quercetin.After fermentation,DPPH,ABTS cation,and hydroxy free radical anti-oxidation activity of plum wine increased significantly,but the overall change in the ageing stage was not obvious（P>0.05）.The results of GC-MS and the PCA diagram of the electronic nose showed that,the plum wine flavor composition was significantly changed before and after fermentation（P<0.05）.The plum juice was rich in aldehyde,acid,and ketone substances,and after fermented ester substances were formed.Especially in plum wine sample when the aging was 40 d,ester and terpene substances reached maximum,phenylethyl alcohol,ethyl octoate,aromatic alcohol,and damascenone etc.were characteristic flavor.In summary,it was recommended to use the mixed fermentation of M.pulcherrima and S.cerevisiae with the initial vaccination ratio at 1:1 and sequence inoculation,the alcohol fermentation was 11 d,and the aging time was 40 d.Based on these condition,plum wine with better quality and higher activity component was acquired.The alcohol of the final product is 11.99%,the soluble solid is 6.30°Brix,the total acid content is 5.94 g/L,the total polyphenol content is 2.1 mg/m L,the total flavonoid content is 0.45 mg/m L,product taste was suitable and rich in flowers,and fruit flavor.更多还原