【作者】 张大为；

【导师】 董文宾；

【作者基本信息】 陕西科技大学 ， 应用化学， 2012， 博士

【摘要】 梨是我国仅次于苹果和柑橘的第三大水果，产量已经跃居世界第一，但是目前以鲜食为主，出现了供过于求的矛盾。对梨进行深加工是解决矛盾的有效手段，而开发梨酒是一种行之有效的解决方法。由于我国对于梨酒的加工技术不够完善，故加大研究力度已经成为研究者和生产者的当务之急。另外，SO2是传统果酒酿造常用的抗氧化剂及抗菌剂，至今仍然被使用。但是大量研究表明SO2会对人体造成危害，各国都要求降低SO2在果酒中的含量，故找到能够替代SO2的安全、高效的方法已经引起了国内外的关注。本文从梨皮及梨园土壤中分离得到了梨酒专用酵母，并通过原生质体融合技术构建了适合于梨酒酿造的优良酵母工程菌。另外，从中草药中筛选出了能够替代SO2的天然替代物，并探讨了天然替代物的抗氧化机理。同时对加入天然替代物后的梨酒香气成分进行了分析。以下为本论文主要内容：1.本研究以梨园土壤及果皮为分离源，分离得到了279株酵母，通过TTC染色法筛选得到产酒精能力及产香能力较强的酵母46株。然后再利用杜氏管发酵法二级筛选及三角瓶三级筛选得到了产酒率最高的菌株YDJ05和产香能力最强的菌株YS03，其产酒率和总酯含量分别为：8.8%（v/v）和0.38g/L。通过形态观察及26SrDNA D1/D2区序列分析，两株酵母分别鉴定为酿酒酵母和东方伊莎酵母，命名为Saccharomyces cerevisiae YDJ05和Issatchenkia orientalis YS03。2．为了得到性能更加优良、更适合于梨酒酿造的菌株，本课题通过原生质体融合技术构建梨酒酵母工程菌，分别以菌株Saccharomyces cerevisiae YDJ05及Issatchenkia orientalis YS03为融合亲本X和Y。通过EMS诱变亲本菌株Y，得到了一株精氨酸营养缺陷型菌株，以此作为遗传标记。亲本菌株X和Y采用蜗牛酶液在35℃下处理100min，得到最佳的原生质体形成率及再生率，分别为：93.6%和94.2%，相对应的原生质体再生率分别为27.8%和31.6%。以加热的方式灭活亲本菌株X，确定灭活时间为14min。在以PEG为促融剂的条件下对两株菌进行原生质体融合，得到DJ01^DJ06六株融合子，其中DJ02的产酒精率、产香率等指标最优，分别达到了9.87%和0.37g/L。通过26sDNA D1/D2区序列分析，融合子DJ02鉴定为酿酒酵母，命名为Saccharomyces cerevisiae DJ02。3．本部分以构建的酵母工程菌Saccharomyces cerevisiae DJ02作为酿造菌株，并对其发酵条件进行详细的优化，结果如下：利用单因素实验初步确定了基本发酵条件为：装液量80mL、接种量11%、初始pH值5.5、培养温度20℃、发酵时间7d。通过添加中草药刺激因子，发现砂仁、枸杞、杜仲、苦参、厚朴和陈皮六种中草药对该菌体出酒率有显著的影响，其中砂仁的刺激作用最为明显，出酒率达到了10.57%。通过Plackett-Burman实验发现，在培养温度、装液量、初始pH值、接种量、发酵时间、苦参浓度、砂仁浓度、杜仲浓度、陈皮浓度、厚朴浓度、枸杞浓度等因素中五个影响显著的因素分别为：培养温度、装液量、初始pH值、发酵时间以及砂仁浓度。通过Box-Behnken中心组合设计对影响出酒率显著的5个因素进一步优化，采用响应面方法对试验结果进行分析，得到培养温度、装液量、初始pH值、发酵时间以及砂仁浓度的最佳值分别为：23.2℃、76.5mL、5.77、7.6d和1.0%，此时出酒率为11.94%，比未优化之前提高了21%。4．从抗菌及抗氧化两个角度出发，寻找能够替代SO₂的中草药，并对其抗氧化机理进行探讨。以53种具有潜在抗菌性能的中草药作为研究对象，通过单因素和组合实验筛选出地菜子、栝楼、酸枣仁和虎杖四种中草药具有较强的抗菌性能，与SO₂相接近。研究表明其最适添加量为3mL/100mL（3%）。挑选出40种具有潜在抗氧化能力的中草药，通过总还原力、DPPH·的清除实验、O₂^-·的清除和·OH的清除实验，发现花生壳、槐花等9种中草药具有较强的抗氧化性。将上述中草药加入到梨酒中，利用福林酚法测定梨酒的多酚含量，发现花生壳、丁香和丹参在梨酒内具有较强的抗氧化能力。将具有较强的抗菌及抗氧化能力的中草药组合，分析其抗氧化性和抗菌性能，再通过酿造菌种的适应性实验，得出“酸枣仁+丁香”同时具有较强的抗氧化和抗菌能力，其添加量为3%（各1.5%），作为梨酒中SO₂的天然替代物。通过定量实验分析，能替代70%的SO2。分析SO₂天然替代物的抗氧化机理发现，梨酒中加入该替代物后，其氧化还原电位明显降低，低的氧化还原电位可以减慢多酚类物质的氧化。另外，梨酒在加入“酸枣仁+丁香”后，多酚的相对聚合度明显降低，从而减少羟基自由基的产生，进而抑制梨酒中多酚的氧化。5．梨酒中加入SO₂天然替代物后，采用GC-MS分析得到该梨酒的主要香气成分，结果如下：含有正丁醇、苯乙醇等醇类12种，占总香气成分的17.92%；含有甲基丙醇二酸、己酸等酸类物质20种，占总香气成分的9.14%；含有辛酸乙酯、琥珀酸氢乙酯、磷酸三丁酯等酯类物质为19种，占总香气量的14.23%；含有2,3-二羟基苯并呋喃、3-羟基-1-（4-羟基-3-甲氧基苯基）-1-丙酮等8种酮类物质，含量为1.22%；含有十五烷、2,6,10-三甲基十四烷等烷烃类物质有14种，含量为1.66%；含有3种苯类物质，分别为1-乙基丁基苯、二丁羟基甲苯、2-丙烯基-1,4二甲氧基-3-甲基苯，其含量为0.36%；含有丁香酚、对特辛基苯酚等多酚类物质6种，其含量高达22.45%；含有4种酰胺类物质，分别为油酸酰胺、芥酸酰胺、N, N-二甲基十二酰胺和十二烷基酰胺，含量为16.57%；还含有苯乙烯、2,6-二异丙基-茴香醚、1,6-二甲基-4-异丙基-萘和1-十九碳烯四种物质，含量为总香气成分的4.07%。与传统酿造中全部加入SO₂的对照梨酒相比较，香气成分的种类及含量差别不大，滋味、口感等感官指标相近。故可以得出结论：“酸枣仁+丁香”作为SO₂替代物是可行的，可以大大减少了SO₂的用量，具有重要的理论和现实意义。更多还原

【Abstract】 In China, pear is the third largest citrus fruit after apple and orange,and production has been the first in world. However at present theeating fresh fruit is the main consumption ways, appeared thecontradiction of supply exceeds demand. Processing of pear is aneffective means of resolving contradictions. The developing pear wineis an effective solution. Because the processing technology of pear wineare not perfect. It is urgent for the researchers and producers to increasethe strength of researching of the moment. In addition, the sulfurdioxide is used as antioxidants and antibacterial agents in traditionalwine brewing. Nowadays it is still be used. But a lot of research showsthat the sulfur dioxide will cause harm to human body, which leadedthat the sulfur dioxide is required to reduce in wine in many countries.Finding some safety, high efficiency, non-toxic method can substitutethe sulfur dioxide has caused the attention of both at home and abroad.Two kinds of pear special wine yeast were isolated from the pear skinand soil. A kind of yeast engineering strain was structured by theprotoplast fusion technology which was suitable for the wine brewing.The natural substitutes have been found from Chinese herbs. And theantioxidant mechanism of the natural substitutes was discussed in thepaper. At the same time the pear wine aroma compositions wasanalyzing after adding natural substitute by GC-MS. The following isthe main content of this paper.1. In this paper279strains of yeast were isolated from the orchardsoil and peel.46strains yeast with strong ability in alcohol and aromaproducing through TTC stain method. Then YDJ05and YS03wereselected through the secondary fermentation by the DuShi tube and the third stage fermentation by the triangle bottles. The alcohol productionand aroma rate of YDJ05and YS03was8.8%(v/v) and0.38g/L.Through morphologic observation and analysis of26SrDNA D1/D2Regional sequence, the two strains of yeast were identified assaccharomyces cerevisiae and Issatchenkia Oriental by yeast, named asSaccharomyces cerevisiae YDJ05and Issatchenkia orientalis YS03.2. In order to getting superior performance and more suitable forpear wine brewing strains, The yeast engineering strain was structuredby the protoplast fusion technology. Saccharomyces cerevisiae YDJ05and Issatchenkia orientalis YS03were used as the fusion parents X andY. A arginine nutrition unfairness strain was selected by EMSmutagenesis from parents strains Y. The strains X and Y were dealt withthe snail enzyme liquid in35℃100min. The best protoplast formationrate and regeneration rate were93.6%,94.2%and27.8%,31.6%.Inactivate strains of the parents of X with heating them in15min. In thecircumstance with PEG as promotion melting agent DJ01~DJ06sixstrains fusant were gotten, one of them DJ02with the best alcoholproducting rate and aroma producting rate were reached to9.87%and0.37g/L. Through analysis of26S rDNA D1/D2Regional sequence,fusant DJ02is Saccharomyces cerevisiae by identification, namedSaccharomyces cerevisiae DJ02.3. Saccharomyces cerevisiae DJ02was used as brewing strains,and optimize the fermentation conditions in detail. The results are asfollows.By using single factor experiments the basic conditions offermentation were determined preliminarily. The fermentationconditions were pack fluid amount80mL, inoculumconcentration11%,initial pH5.5, culture temperature20℃, fermentation time7d.The six kinds of traditional Chinese herbs including fructus amomi,wolfberry, eucommia, sophora flavescens, mangnolia officinalisand andtangerine peel have significant effect on the rates of liquor. Fructusamomi is the most obvious stimulating effect one, it is to10.57%.Through the Plackett-Burman experiment, five factors including culture temperature, fluid amount, initial pH value, fermentation time andfructus amomi concentration were found to have significant effect onthe experiment results among nine ones including the cultivationtemperature, fluid amount, initial pH value, inoculumconcentration,fermentation time, Sophora flavescens concentration, fructus amomiconcentration, eucommia concentration, tangerine peel concentration,mangnolia officinalisand concentration, wolfberry concentration.Among them five factors of significant effects are: the culturetemperature, fluid amount, initial pH value, fermentation time andfructus amomi concentration.The five factors were optimized through the Box-Behnken centercombination design. The test results were analyzed by response surfacemethod. The best fermentation conditions are: culture temperature23.2℃, fluid amount76.5mL, initial pH value5.77, fermentation time7.6dand fructus amomi concentration1.0%. And the rate of liquor was11.94%and increased21%than before optimization.4. The substitutes of the sulfur dioxide were selected on Chinese herbsfrom two aspects including antibiosis and antioxidant. Caizi, Gualou zi,Spina date seed and Tiger rod were found to have strong antibacterialproperty among53kinds of potential Chinese herbs researched bysingle factor and group experiments. Their antibacterial properties wereclose to the sulfur dioxide. Research showed that the optimal addingamount was3mL/100mL (3%).40kinds of Chinese herbs with potential antioxidant propertieswere research through the general reduction force, the clear experimentof DPPH, the clear experiment of O2and·OH.9types of ones werefound to have strong antioxidant role including peanut shells, flossophorae and so on. The peanut shells, cloves and salvia miltiorrhizaehad strong antioxidant ability in pear wine by using the Phenol flintmethod for the determination of polyphenols.The antibacterial and antioxidant properties of the above Chineseherbs were analyzed after putting into simultaneously the pear wine.Then the brewing strain adaptability experiment on Chinese herbs was done in the pear wine. The spina date seed adding clove have strongantioxidant and antibacterial ability, the add amount is3%(1.5%foreach), as a natural alternative of sulfur dioxide in pear wine. Throughthe quantitative experimental analysis, it can replace70%of sulfurdioxide.The antoxidative mechanism was analyzed in this part. the REDOXpotential of the pear wine reduced significantly with The low REDOXpotentials slowed down the polyphenols oxidation. In addition, Therelative degree of polymerization of polyphenol reduced significantlyafter adding the spina date seed and clove. Thus the generation ofhydroxyl radicals were reduced and restrained polyphenols oxidation inthe pear wine.5. After adding the natural substitute of sulfur dioxide in pear wine,the main aroma compositions were analyzed by GC-MS. The resultswere as follows. There were12kinds of alcohol including n-butylalcohol, phenethyl alcohol accounting for17.92%of the total aromacompositions. There were20species acids including methyl propanoltwo acid, caproic acid, accounting for9.14%of the total aromacompositions.There were19species ester accounting for14.23%of the total ofaroma including octylic acid ethyl ester, amber acid ethyl esterhydrogen, TBP lipid and so on. There were8kinds of ketone such asBenzofuran,2,3-dihydro-,1-Propanone,3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-. The content was1.22%.There were14species including pentadecane, Tetradecane,2,6,10-trimethyl-alkanes and so on accounting for1.66%. There were3kinds of benzene including Benzene,(1-ethylbutyl)-, ButylatedHydroxytoluene,2-Allyl-1,4-dimethoxy-3-methyl-benzene accountingfor0.36%. There were6kinds of polyphenol including Eugenol, Phenol,4-(1,1,3,3-tetramethylbutyl) and so on accounting for22.45%. Therewere9-Octadecenamide,(Z)-,13-Docosenamide,(Z)-,N-Dimethyldodecanamide and Dodecanamide in the pear wine accounting for16.57%andStyrene,2,6-Diisopropylanisole,Naphthalene,6-dimethyl-4-(1-methyleth yl)-and1-Nonadecene four material, The content was4.07%of thetotal aroma compositions.The pear wine of adding the sulfur dioxide substitution was similarto the one putting into sulfur dioxide pear wine in some ways such asthe sensory flavor and taste. So the conclusion can be drawn that thespina date seed and clove as sulfur dioxide substitution was feasible. Itcan greatly reduce the amount of sulfur dioxide, and has an importanttheoretical and practical significance.更多还原