Study On Fermentation Technology And Quality Characteristic Of Honey-mulberry Wine

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.