【作者】 陈娟；

【导师】 阚建全；

【作者基本信息】 西南大学 ， 农产品加工及贮藏工程， 2009， 博士

【摘要】 桑椹为桑（Morus alba Linn.）的成熟果穗,是国家卫生部1988年首批公布的医食同源植物品种之一,我国桑椹产量位居世界首位。桑椹属于热性浆果,极不耐贮,易腐烂、变质。桑椹富含糖、酸、维生素、矿物元素,还含有花色苷、酚酸和黄酮等多种活性物质。本文将天然蜂蜜与桑椹结合,酿制营养全面的复合型果酒,不仅符合了酒类行业的发展趋势,而且还将打造出绿色的保健果酒品牌,迎合当今消费及市场需求。目前,我国几乎没有蜂蜜桑椹酒的酿造技术和品质特征方面的系统性研究。本论文以蜂蜜和桑椹为主要原料,应用传统的微生物分离技术,结合生物技术和仪器分析技术,采用理化指标分析结合感官评定技术,对桑椹和桑园土壤中优良酵母菌的分离、筛选和鉴定,蜂蜜桑椹酒酿造酵母菌株的构建,蜂蜜桑椹酒的发酵工艺及澄清条件,蜂蜜桑椹酒的基本化学成分和香气成分分析等方面进行了系统深入的研究。在蜂蜜桑椹酒的专用菌株、发酵工艺及品质特征等方面取得了较大的研究进展。主要研究结果为:1、从成熟桑椹和桑园土壤的富集培养液,以及蜂蜜桑椹汁的自然发酵液中,共分离得到112株酵母菌。经逐级筛选,优选出两株酵母菌,命名为HM和SM。HM分别在酒精浓度16%,SO₂浓度250 mg/L,pH2.01时,1d内产气;SM在酒精浓度14%时,1.5d内产气,在SO₂浓度200 mg/L时,1d内产气,在pH1.50时,2d内产气。HM和SM发酵蜂蜜桑椹汁,酒精度分别为9.7%,7.0%（V/V）,总酯产量分别为1890.5,2143.8（mg/L）,感官综合得分分别为84.9,83.1（分）,发酵酒具有素雅的桑果香和怡人的酒香,典型性突出。鉴定结果表明,酵母菌HM为酵母属的酿酒酵母（Saccharomyces cerevisive）,酵母菌SM为有孢圆酵母属的德尔布有孢圆酵母（Torulaspora delbrueckii）。2、对原生质体融合的各种技术条件进行了系统研究,结论如下:（1）确定以Y₁（n）(单倍体Y₁(a₁₂)和HM（n）（单倍体HM（a₄））为原生质体融合的两个亲本菌株。确定含2.0mg/mLCuSO₄的完全培养基为选择性培养基,在此培养基上,Y₁（n）能生长而HM（n）不能生长。（2）原生质体形成和再生条件:将Y₁（n）在YEPD中培养4h（28℃和180r/min）后,用含0.1%β-巯基乙醇的预处理剂处理10min（30℃和180r/min）,再用含1.0%蜗牛酶的酶解液处理1.0h（30℃和120r/min）,原生质体形成率达到94.2%,再生率达到27.1%;将HM（n）在YEPD中培养5h（28℃和180r/min）后,用含0.1%β-巯基乙醇的预处理剂处理10min（30℃和180r/min）,再用含1.5%蜗牛酶的酶解液处理1.5h（30℃和120r/min）,原生质体形成率达到95.4%,再生率达到22.2%。在原生质体形成时用0.7mol/LKCl来配制酶解溶液,而在原生质体再生时,用17%蔗糖来配制稀释液和高渗再生培养基。（3）Y₁（n）原生质体灭活条件:60℃恒温水浴处理20min,灭活率达到100%。（4）两亲本菌株按1:1比例混合（浓度均为1×10⁷个/mL）,于30℃和180r/min条件下培养15 min,加入促融剂溶液,振荡10 min,再静止10 min,融合率达到4.85×10^-6。（5）筛选及鉴定:通过TTC显色法和杜氏管发酵法初步筛选出10株发酵能力较强的融合子;通过酿造酒复筛试验,结合模糊综合评判法优选出F₆和F₁₀两株融合子,以F₆最佳。细胞的形态、大小、DNA含量和抗药性分析结果表明,融合子F₆和F₁₀为两亲本菌株真正的融合子。（6）遗传稳定性检验:融合子F₆和F₁₀经连续传代20次后,其主要性能仍然很稳定,没有发生回复突变现象,说明两融合子具有遗传稳定性。（7）通过基本生理特性和发酵性能比较,揭示了融合子和亲本之间的相互关系。3、融合子F₆的发酵工艺条件包括:桑椹最佳果胶酶酶解条件为酶解温度35℃,酶用量为每kg桑椹浆添加0.4mL诺维信酸性果胶酶BEC,酶解时间100min,自流汁得率为14.826%。初始糖度以24%（还原糖220 g/L左右）为宦。采用浊汁发酵,在后期通过下胶对原酒进行澄清处理。研究了发酵温度、接种量、初始pH值、SO₂添加量和装液量五个因素影响主发酵过程的具体情况;建立了主发酵质量预测模型为Y=88.64936-6.19732X₁+1.49109X₂+2.88565X₃-4.08817X₁²-2.49718X₃²+1.62500X₁X₂+1.37500X₂X₃（X₁-发酵温度,X₂-接种量,X₃-初始pH值）,优化的主发酵条件为温度23℃,接种量10%（V/V）和pH值3.5。后发酵温度选择10～15℃。澄清试验结果表明,以壳聚糖0.6g/L+PVPP1.0g/L复合的澄清效果最好,透光率达到60.6%,色度值为12.8,澄清处理后的酒样在酒度、糖含量和酸含量上没有变化,酚类物质和蛋白质被澄清剂絮凝去除而减少,提高了蜂蜜桑椹酒的稳定性。4、分别以大十桑棋、红果2号桑椹和红果1号桑椹和农用桑椹为原料,发酵得到四种蜂蜜桑椹酒,分析了它们含有的各种成分。四种酒样在总糖、总酸、pH值、酒度、挥发酸和单宁等成分上差异不大,但蛋白质含量差异较大。各种矿物元素的含量因品种不同而存在一定差异,但均表现出高钾低钠的突出特点,镁元素含量很高,硒元素含量5.0～8.5μg/L。氨基酸总含量和必需氨基酸含量以红果1号桑椹蜂蜜发酵酒最高,而农用桑椹蜂蜜发酵酒最低;必需氨基酸含量占氨基酸总量的比例在四种酒样间差异不明显;从氨基酸种类含量来看,四种酒样在氨基酸组分上的差异显著（p＜0.05）。蜂蜜桑椹酒的总二氧化硫、游离二氧化硫及有害微生物指标的检测结果符合国家发酵酒卫生标准。对蜂蜜桑椹酒中多酚类物质的情况进行了较详细的研究。结果表明,农用桑椹蜂蜜发酵酒的总酚、总黄酮和总花色苷含量最高,而红果1号桑椹蜂蜜发酵酒最低,大十桑椹和红果2号桑椹蜂蜜发酵酒居中。进一步对酒样中7种酚酸和5种黄酮醇进行了分析,建立了高效液相色谱分析方法,采用Agilent Analytical C18色谱柱（250mm×4.6mm,5.0μm）,酚酸测定方法为:流动相为甲醇-甲酸溶液进行梯度洗脱,流速1.0mL/min,柱温30℃,检测波长260nm,进样量20μL:黄酮醇测定方法为:流动相为甲醇-乙酸溶液进行梯度洗脱,流速1.0mL/min,柱温30℃,检测波长360nm,进样量20μL。测定结果表明,蜂蜜桑椹酒中原儿茶酸、槲皮素和桑色素的含量很高,这可能是其区别于其它果酒的重要特征;各种酚酸和黄酮醇类物质在不同酒种之间存在一定程度的差异,以酚酸中的咖啡酸和黄酮醇中的芦丁含量差异最大,这两种物质可能是区别不同酒种的重要依据。5、从大十桑椹、红果2号桑椹、红果1号桑椹和农用桑椹四种果汁中共检出64种挥发性成分,主要成分包括亚油酸、棕榈酸、肉豆蔻酸、油酸、亚油酸乙酯、亚油酸甲酯、亚麻酸乙酯、油酸甲酯、3-甲基-丁醇、苯乙醇、2,3-丁二醇、己醛、壬醛和3-羟基-2-丁酮等。以四种桑椹为原料,发酵得到四种蜂蜜桑椹酒,从四种原酒中共检出93种挥发性成分,主要成分包括3-甲基-1-丁醇、2-甲基-1-丙醇、苯乙醇、2,3-丁二醇、4-羟基苯乙醇、乙酸乙酯、丁二酸单乙酯、乳酸乙酯、油酸甲酯、乙酸、葵酸、辛酸、3-羟基-2-丁酮等;从四种陈酿酒中共检出76种挥发性成分,主要成分包括3-甲基-1-丁醇、2-甲基-1-丙醇、苯乙醇、2,3-丁二醇、1,2,3-丁三醇、4-羟基苯乙醇、3-甲基-丁酸乙酯、2-羟基-丙酸乙酯、乙酸乙酯、3-羟基-2-丁酮等。桑椹从果汁经历发酵和陈酿作用,香气成分的种类和相对百分含量变化很大,总体变化趋势为:低级脂肪酸酯上升,高级脂肪酸酯下降;低级脂肪醇上升,高级脂肪醇变化小,芳香醇上升;低级脂肪酸先升后降,高级脂肪酸大幅下降;酮类上升,醛类下降,酚类先升后降。本文首次采用原生质体融合技术,结合模糊综合评判方法,构建得到适合蜂蜜桑椹酒酿造用的新菌株,该菌株发酵性能优良,且遗传稳定性好;首次对不同品种蜂蜜桑椹酒中各种基本化学成分进行了较全面的研究,着重分析了含有的多酚类物质的情况,初步建立了蜂蜜桑椹酒的酚类物质识别因子;首次采用有机溶剂提取、GC-MS定性分析的方法,研究了不同品种的桑椹原料、蜂蜜桑椹发酵原酒和陈酿六个月的酒中香气成分的组成和比例关系,分析了从原料到陈酿酒过程中香气成分的变化规律。更多还原

【Abstract】 Mulberry was one of varieties which were proclaimed as Herb-Food-Homologous plant by National Ministry of Health in 1988.China is the biggest producer of mulberry in the world. Mulberry which is easy to putresce and deteriorate belongs to hot berries.Mulberry is rich in sugar, acid,vitamin and mineral,furthermore,there are some bioactive constituents such as anthocyanin, phenolic acid,flavonoid and so on in mulberry.In this study,mulberry and honey were combined to brew out a new type of fruit wine with plenty of nutrients.This not only accorded with developing tendency of wine industry,but aslo could creat a green and healthy brand of fruit wine,in order to satisfy consumption requirment.At present,there were scarcely any systematical studies about fermentation technology and quality characteristics of honey-mulberry wine.In this study,mulberry and honey were used as raw materials,and routine microorganism isolation method together with biological technique and instrument analysis technique were applied. For the first time,excellent yeast strains from mature mulberry and soil in mulberry orchard were isolated,screened and identified.Then,the new strain suitably brewing honey-mulberry wine was constructed through protoplast fusion technique.The optimum fermentation condition and clarification condition of honey-mulberry wine were discussed.Lastly,fundamental chemical components and flavor components of honey-mulberry wine were analyzed.1、From both the enrichment culture medium and natural fermentaion medium,112 yeast strains were separated.These strains were screened step by step,and two yeast strains were selected out and named HM and SM.It was proved that,the HM could start to ferment within one day respectively at the alcohol concentration of 16%,the SO₂ concentration of 250 mg/L and at the pH of 2.01;on the other hand,the SM could start to ferment within one and half days at the alcohol concentration of 14%,within one day at the SO₂ concentration of 200mg/L,and within two days at the pH of 1.50. Through fermentation of HM and SM,the alcohol concentration respectively reached 9.7%and 7.0%,the total ester concentration respectively came to 1890.5 mg/L and 2143.8 mg/L,and the sensory evaluation score respectively were 84.9 and 83.1.Wine fermented by these two stains had excellent fragrance and distinctive characteristics.Based on experiment results and relative references,the HM was identified as Saccharomyces cerevisive,and the SM was identified as Torulaspora delbrueckii.2、Systematical researches on conditions of the protoplast fusion were carried out,the results were showed as follows:（1） At first,Y_1（a12） haploid having intense fermentability and HM_（a4） haploid having good flavor were used as parental strains,and were named robe Y₁（n） and HM（n） respectively.After hereditary marker screening of parental strains,the complete culture medium having 2.0mg/mLCuSO₄ was regarded as the selective medium.Y₁（n） could grow but HM（n） couldn’t grow on this selective medium.（2） The optimal formation and regeneration conditions of parental strains were:After Y₁（n） cells were cultured 4 hours in YEPD medium（28℃,180r/min）,cells were collected by centrifuing, then were pretreated with the 0.1%β-Mercaptoethanol solution for 10 minutes（30℃,180r/min）, and treated with 1.0%snailenzyme for 1 hour at 30℃lastly（120r/min）,the formation rate and regeneration rate of Y₁（n） respectively were 94.2%and 27.1%;For the other parental strain,after HM（n） cells were cultured 5 hours in YEPD medium（28℃,180r/min）,cells were collected by centrifuing,then were pretreated with the 0.1%β-Mercaptoethanol solution for 10 minutes（30℃, 180r/min）,and treated with 1.5%snailenzyme for 1.5 hours at 30℃lastly（120r/min）,the formation rate and regeneration of HM（n） respectively were 95.4%and 22.2%.The stable factor of osmotic pressure in the enzyme hydrolysis solution was 0.7 mol/L KCl,and in the regeneration cultrue mudium it was 17%sucrose..（3） The protoplast of Y₁（n） could be completely inactivated by 60℃of water bath treatment for 20 minutes.（4） The fusion method was:the mix proportion of parental strains was 1:1;culture conditions were firstly the rotary culture 15 minutes（30℃,180r/min）,and another 10 minutes after adding the fusion-accelerating agent,lastly the standing culture 10 minutes;the fusion rate could reach 4.85×10^-6.（5） Ten fusants were initially selected out through the TTC method and Du-tube method.After screening again,the phsic-chemical properties、sensory quality and major aroma components of fresh wine were analyzed.Through fuzzy comprehensive evaluation of different honey-mulberry wines fermented by fusants and parental strains,F₆ and F₁₀ with good fermentability and aroma productivirty were choosed as better ones,especially the fusant F₆ was the best one.Through results of cell volume,DNA content and drug resistance property for fusants,the F₆ and F₁₀ were identified as real hybrids of parental strains.（6） After twenty times of continuous inoculations,the principal properties of fusants were still stable,and did not mutate reversely.This results suggested that these two fusants had stable hereditary properties.（7） Through studies on fundamental physiological characteristics and fermentation properties, the relationship between parental strains and fusants were revealed.3、The fermentation technology of the strain F₆ was completely studied,and the results showed:The optimal condition of pectinase action was:the adding content was 0.4mL Novo pectinase BEC per kilograme mulberry pulp,the action temperature was 35℃,natural pH,the action time was 100 minutes.Under this condition,the automatic drops yield got to 14.826%.The fermentation processes of the F₆ varied under different initial sugar concentrations,the optimum initial sugar concentration was 24%,that is,the concentration of reducing sugar was 220 g/L.Fermentation with cloudy juice and clarification treatment to new wine was selected tobe the fermentation mode.In primary fermentation,some factors such as initial pH,fermentation temperature,inoculation volume, SO₂ adding concentration and loading volume would mainly affect the fermentation process, especially,the former three factors were most important.Through three-factor quadric orthogonal regression composite experimental design,a regression equation with high reliability was obtained, which was Y=88.64936-6.19732X₁+1.49109X₂+2.88565X₃-4.08817X₁²-2.49718X₃²+1.62500X₁X₂+1.3 7500X₂X₃ X₁ was fermentation temperature,X₂ was inoculum volume,X₃ was pH value).The optimum technology parameters were 23℃of fermentation temperature,10%of inoculum volume and 3.5 of initial pH value.In secondary fermentation,new wine was kept under 10～15℃,after approximately 20 days,the sensory quality of wine would became better.In addtition to,the results of clarification experiment indicated that:The optimal clarification condition was chitosan 0.6 g/L +1.0g/LPVPP.This treatment could improved the light transmittance upto 60.6%,chromaticity was 12.8,at the same time,changes of alcohol content,residual sugar content and acid content were not observed,but the amount of both phenol and protein decreased beacause they were removed by clarifying agents.After this treatment,the stability of honey-mulberry wine was improved finally.4、Various fundamental chemical components of four kinds of honey-mulberry wine were respectively analyzed,which were fermented from Dashi mulberry、Hongguol mulberry、Hongguo2 mulberry and agricultural mulberry.Among these four wine samples,the amounts of total sugar, total acid,pH,alcohol,volatile acid and tannin varied little,but the content of protein vaned greatly. The amount of each mineral varied with different wine sample,while they all had a outstanding characteristic of high K and low Na;Mg was high;The amount of Se was between 5.0μg/L and 8.5μg/L.As far as the amino acid was concerned,the amount of total amino acid and essential amino acid in wine fermented from Hongguol mulberry was the highest and the amount in wine fermented from agricultural mulberry was the lowest.Differences about the EAA/TAA（ratio of essential amino acid to total amino acid） for the four wine samples were small.The composition of amino acid varied significantly according to different wine samples.The detection results of total sulfur dioxide,free sulfur dioxide and nocuous microorganism accorded with National Hygienic Standard of Fermented Wines.Besides,the content of phenols in honey-mulberry wine were analyzed,the results showed that: As far as the total amount of phenol,flavone and anthocyanin were concerned,the highest concentration was wine fermented from agricultural mulberry,the lowest concentration was wine fermented from Hongguol mulberry,the middle were wines respectively fermented from Dashi mulberry and Honguo 2 mulberry.After a further study,a RP-HPLC method was developed for simultaneous determination of 7 phenolic acids（GAL,PRO,HYD,CHL,CAF,COU,FER） in honey-mulberry wine.A Agilent Analytical C18 column was used with formic acid and methanol as mobile phase at a flow rate of 1.0mL/min,the column temperature was 30℃and the UV detection wavelength was 260nm,injection volumn was 20μL.Another RP-HPLC method was developed for simultaneous determination of 5 flavonols（RUT,QUE,QUEE,MOR,KAE） in honey-mulberry wine.A Agilent Analytical C18 column was used with acetic acid and methanol as mobile phase at a flow rate of 1.0mL/min,the column temperature was 30℃and the UV detection wavelength was 360nm,injection volumn was 20μL.The results showed that,higher substances in honey-mulberry wine were PRO,QUEE and MOR,this may be an important characteritic of honey-mulberry wine differentiating from other fruit wines.There were some differences in phenolic acids and flavonols among four kinds of wine sample,especially the content of caffeic acid and rutin,sothat,they would be important substances differentiating all kinds of honey-mulberry wine.5、Among Dashi mulberry、Honguo2 mulberry、Honguol mulberry and agricultural mulberry, 64 volatile components were detected from these four kinds of mulberry fruit.The principal aroma components included Linoleic acid、Palmitic acid、Myristic acid、Oleic acid、Linoleic acid ethyl ester、Linoleic acid methyl ester、Linolenic acid ethyl ester、Oleic acid methyl ester、1-Butanol,3-methyl-、Benzeneethanol、2,3-Butanediol、Hexaldehyde、Nonanal、3-hydroxy-2-butanonel and so on.Four kinds of honey-mulberry wine were correspondingly fermented from these four kinds of mulberry.93 volatile components were detected from the four kinds of new honey-mulberry wine.The principal volatile components included 1-Butanol,3-methyl-、1-Propanol,2-methyl-、Benzeneethanol、4-hydroxy-Benzeneethanol、Ethyl acetate、Monoethyl succinate、Ethyl lactate、Oleic acid methyl ester、Acetic acid、Decanoic acid、Octanoic acid、3-hydroxy-2-Butanonel and so on.76 volatile components were detected from the four kinds of aging honey-mulberry wine.The principal volatile component included 3-methyl-1-Butanol、2-methyl-1-Propanol、Benzeneethanol、2,3-Butanediol、1,2,3-Butanetriol、4-hydroxy-Benzeneethanol、3-methyl-Butyric acid ethyl ester、2- hydroxy- Propanoic acid ethyl ester、Ethyl acetate、3-hydroxy-2-Butanonel and so on.The kind and relative percentage varied greatly through the fermentation and storage period,the change tendency was:short chain fatty acid-ester ascended,long chainfatty acid-ester descended;short chain fatty alcohol ascended,long chain fatty alcohol changed little,aromatic alcohol ascended;low chain fatty acid ascended early and descended later,long chainfatty acid descended a lot;ketone ascended;aldehyde descended;phenol ascended early and descended later.Innovations of this paper were as follows:protoplast fusion technique were used with fuzzy comprehensive evaluation to construct a new strain suitably brewing honey-mulberry wine,which had excellent fermentation properties and stable hereditary properties.Various fundamental chemical components in honey-mulberry wine with different mulberry varieties were analyzed,especially phenols were emphasized;Phenol identifying factor of honey-mulberry wine was preliminarily constructed.The aroma components of mulberry fruit,new honey-mulberry wine and aging honey-mulberry wine with four varieties were extracted by solvent and analyzed by GC-MS.Composit ion and proportion of aroma components were analyzed and change regulation of aroma components from material to aging wine were revealed.更多还原