Construction Of Engineering Yeast Strain For Pear Wine And The Research On The Natural Alternatives Of Sulfur Dioxide

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.