Study On The Microorganisms And Fermentation Technology Of Highland Barley Wine

In this study,for the first time,general properties of Highland Barley Wine and its starters bought from Lasha have been studied.Mold,yeast and lactic acid bacteria about Highland barley wine fermentation have been separated and identified on a large scale.By screening these microorganisms,two yeast strainsZY2 and ZY28,one mold strain ZM2 and one lactic acid bacteria strain ZB12 were separated as superior strains.Original strains ZM2 and ZY2 were breeded by protoptast induced mutation and DES mutagenesis.Pure cultured starters processing technology was achieved using optimum design of breeding technology by RSM.This paper provided a set of processing technique with the maintenance of general quality of traditional fermented barley wine which has subtle taste,low alcohol and typical flavour.The main results are as following:1.Quality and flavor characteristics of the barley wine and its starter were identified by analyzing Physiochemical Indexes,GC-MS and sensory index,comparing with Chinese Rice Wine and Japanese sake.(1)Tibet barley wine starter was identified as Chinese yeast with different forms and qualities.The traditional barley wine was identified as fermentation wine and its physiochemical indexes of were between Rice Wine and Sake.The total content of amino acid in Quality Highland Barley Wine was 110.912 mg/100ml and the wine is also rich in vitamin B₁,B₂,C and minerals.(2)There are 48 flavor components in barley wine including 16 kinds of alcohol,5 kinds of ester,6 kinds of acid,10 kinds of aldehyde,7 kinds of ketone and orther phenols.GC-MS shows that:highest content/threshold is aldehyde 41.76,then acetic acid 40.38,Isoamyl alcohol 13.38,caproic acid 4.65, ethyl lactate 3.07,Butyric acid 2.94,ethyl acetate 2.65.β-phenylethanol 2.14,3-hydroxy butanone1.36, these 9 compounds are most responsible for the flavor.2.Microorganisms of Barley wine starter and microorganisms flora during fermentation were studied using routine isolation,purification and identification method together with molecular biological technique,including PCR-Clone Analysis.It shows that:(1)The identification of dominant mold shows:there were 130 dominant mold strains,including 81 strains of Rhizopus.SPP:Rhizopus oryzae 39 strains,Rhizopus chinensis 15 strains,Rhizopus arrhizus 9 strains,39 strains of Mucor SPP:including Mucor javanicus 18 strains,Mucor racermosus 11 strains, Mucor rouxians 8 strains,Absidia spp 8 strains.ZM2 was screened from all these mold strains with glucoamylase activity 520.27mg/h·g.ZM2 is indentified as Rhizopus oryzae through ITS sequence analysis and Phylogenetic Tree Construction.(2)The identification of dominant yeast shows:there are dominant yeast 580 strains,belonging to 6 species of Saccharomyces,Saccharomycopsis,Candida,Pichia,Debaryomyces,Cryptococcus,including 186 strains of Saccharomyces cerevisiae,Candida sake 38 strains,Pichia farinosa 16 strains, Debaryomyces hansenii 12 strains,Cryptococcus terreus 5 strains.ZY2 was screened from all these yeast strains for its high alcohol production ability,while ZY28 strain was screened by its ability to produce glucoamylase and good flavour.ZY2 and ZY28 are identified as Saccharomyces boulardii and Saccharomycopsis fibuligera,respectively through ITS sequence analysis and Phylogenetic Tree Construction.(3)The identification of dominant lactic acid bacteria shows:38 strains in 3 species were dominant lactic acid bacteria,including Streptococcus faecalis,Pediococcus pentosaceus,Pediococcus acidialactici,Lactobacillus.johnsonii and Lactobacillus sake.ZB12 strain was screened for further research. It was identified as Pediococcus pentosaceus through ITS sequence analysis and Phylogenetic Tree Construction.3.ZY2 and ZM2 were breeded by protoplast induced mutation with UV and DES combination.It shows that:(1)UV-Y2 was obtained with higher alcohol production by UV irradiation of ZY2 protoplast for 50s. The protoplast of UV-Y2 was treated with 1.5%DES for 30 minutes,then the final mutant strain UV-DES-Y2 was obtained which can produce more than 33.33%alcohol after a series of screening by TTC plating screen,Durham fermentation and erlenmeyer flask fermentation.etc.(2)UV-M4 was obtained with higher alcohol production by UV irradiation of ZM2 protoplast for 30s. The protoplast of UV-Y2 was treated with 0.9%DES 50 minutes,then the final mutant strain UV-DES-M4 was obtained which can produce more than 50.9%Glucoamylase.The optimum of enzymatic production for starter 1 is achieved by Using RSM:highland barley powder with 40%water,8.06%soya flour,1.0%inoculation, cultivating at 30.05℃for 42h.and the Glucoamylase is 895.32 mg/h·g dry starter.(3)the optimal processes of making yeast solid starter:highland barley powder50%glucose-bean sprout liquid medium(GBM)whose initial pH is 3.5(for ZY2)or 5(for ZY28),15%inoculating ratio which were previously breeded in GBM for 24h,cultivating at 32℃(for ZY2)or 25℃(for ZY28)for 30h,so the starter 2(ZY2)and starter 3(ZY28)were obtained.4.The optimization experiments of brewing technique with pure strain fermentation were carried with the following results.Using Tibet barley 320 as raw materials,soaked or no soaked with the ratio of 1:2 water,steamed 2h,with the inoculum's concentration of 2%pure starter 1,2%pure starter 2 and1%pure starter 3,fermented on the condition of 28℃and 72h.5.The change of compositions of the barley wine with pure strain and the traditional barley wine during its fermentation process are studied.The results were showed as follows:The changing tendency of compositions during two kinds of fermentation process was similar:pH declined,temperature increase slowly and declined slowly with the highest temperature 36℃.The activities of the saccharifying enzymes and the reducing sugar content increased significantly(P＜0.05)during the first period of fermentation to 24h,then decreased gradually.The total sugar content decreased significantly (P＜0.05)during all the fermentation while the alcoholicity increased significantly(P＜0.05).The alcoholicity during the fermentation with pure strain process reached to 10.3%higher than traditional fermentation.Both the ammonia-N and the acid proteinase increased significantly(P＜0.05)up to 24h of fermentation;and then decreased slowly.The total content of barley wine during the fermentation with pure strain reached to 80.923 mg/100ml without Methionine.