摘要
本论文以青稞和从拉萨市场购买的优良青稞酒曲为主要原料,应用传统微生物发酵技术结合现代纯种制曲发酵技术、现代分子生物学技术、现代仪器分析技术,采用理化指标分析结合感官评定技术,对西藏青稞酒曲及传统青稞酒的品质,青稞酒曲及青稞酒发酵过程中的微生物区系,优势发酵菌株的筛选和原生质体诱变,纯种制曲工艺,青稞酒纯种曲酿造工艺条件,青稞酒的纯种曲发酵与传统曲发酵过程中各成分的动态变化等方面进行了系统深入的研究。在优势发酵菌株的选育,以纯种曲发酵技术生产青稞酒,改良传统西藏青稞酒的酿造工艺,提高青稞酒的品质等方面取得了较大的研究进展。主要研究结果为:
1.应用理化分析、气-质联用和气相色谱并结合感官分析技术,对西藏青稞酒曲及传统青稞酒的品质进行了分析研究,得出青稞酒曲及传统青稞酒的品质特点如下:(1)西藏青稞酒曲属于小曲类型,外观形态不一、质量良莠不齐。(2)传统青稞酒属于发酵酒范畴,各项理化指标值基本是在黄酒和清酒之间,传统优质青稞酒氨基酸含量110.912 mg/100mL,含有较丰富的矿物质和维生素B_1,维生素B_2,维生素C等;并从中检测出48种香气成分,包括醇,醛,酸,酯,酮类及少量酚等;香气值(含量/阈值)最大的是乙醛41.67,依次是乙酸40.38、异戊醇13.38、己酸4.65、乳酸乙酯3.07、正丁酸2.94、乙酸乙酯2.65、β-苯乙醇2.14、3-羟基丁酮1.36,因此这9种成分对青稞酒的风味形成影响最大。
2.应用经典的微生物分离纯化鉴定技术结合现代分子生物学技术,对青稞酒曲及青稞酒发酵过程中的微生物区系进行了系统的研究,结果表明:
(1)从青稞酒曲中,鉴定了优势霉菌130株。根霉属的有4种共81株,其中,米根霉(Rhizopusoryzae)39株,黑根霉(Rhizopus nigricans)18株,华根霉(Rhizopus chinensis)15株,少根根霉(Rhizopusarrhizus)9株。毛霉属有37株,其中18株为爪哇毛霉(Mucor javanicus),11株为总状毛霉(Mucorracermosus),8株为鲁氏毛霉(Mucor rouxians),还有8株为犁头霉属(Absidia spp.)。其余丝状真菌的量非常少,分别属于曲霉属(Aspergillus spp.),青霉属(Penicillium spp.),总状共头霉(Syncephalastrumracemosum)等。经过初筛,复筛,青稞试饭等试验,筛选出糖化功能强的菌株ZM2,糖化酶活力为520.27mg/h·g。霉菌ZM2经ITS序列分析和构建系统发育树鉴定为米根霉(Rhizopus oryzae)。
(2)从青稞酒曲中,鉴定到属的优势酵母菌有580株,分别属于酵母属(Saccharomyces)、复膜孢酵母属(Saccharomycopsis)、假丝酵母属(Candida)、毕赤氏酵母属(Pichia)、德巴利酵母属(Debaryomyces)及隐球酵母属(Cryptococcus)6个属。其中,318株鉴定至种,分别是酿酒酵母(Saccharomyces cerevisiae)、扣囊复膜孢酵母(Saccharomycopsis fibuligera)、清酒假丝酵母(Candidasake)、粉状毕赤氏酵母(Pichia forinosa)、汉逊德巴利酵母(Debaryomyces hansenii)和地生隐球酵母(Cryptococcus terreus)。经TTC法和杜氏管法初筛,发酵法复筛,玉米糖化醪筛选,得到优良酵母菌株ZY2和糖化力较强的酵母ZY28。酵母菌株ZY2在玉米糖化醪上产酒精度5.7%,口感与香气好,酸度正常。酵母ZY28在青稞试饭试验中表现出浓郁愉悦的甜香、蜜香。ZY2及ZY28经ITS序列分析和构建系统发育树分别鉴定为布拉酵母(Saccharomyces boulardii)与扣囊复膜孢酵母(Saccharomycopsis fibuligera)。
(3)从青稞酒曲中,鉴定了优势乳酸菌38株,分别属于乳杆菌属,链球菌属和片球菌属3个属。其中有5株鉴定到种,分别是粪链球菌(Streptococcus faecalis)、戊糖片球菌(Pediococcuspentosaceus)、乳酸片球菌(Pediococcus acidialactici)、约氏乳杆菌(Lactobacillus.johnsonii)和米酒乳杆菌(Lactobacillus sake)。筛选到一株戊糖片球菌ZB12作为后续研究菌株。ZB12经过16S rDNA序列分析和构建系统发育树,鉴定为戊糖片球菌,与常规生理生化反应的鉴定结果一致。
3.应用原生质体诱变技术对ZY2,ZM2进行UV+DES复合诱变,并经单因素实验和响应面法优化制曲工艺,研究结果如下:
(1)经UV+DES复合诱变ZY2的原生质体,得到突变株UV-DES-Y2,其产酒精能力比出发菌株ZY2提高了33.3%,发酵15Bx的麦芽汁,酒度可达8.0%(v/v);在耐热性,发酵能力,酒精耐受性,发酵温度范围,发酵速率和降糖速率方面均优于ZY2菌株。以pH值3.5的葡萄糖豆芽汁与青稞粉按1.0mL:2.0g混合,接入15%的24h液体培养酵母UV-DES-Y2,32℃培养24～28h,30℃风干,得到酵母纯种曲2,菌数达9.2×10~8cfu/g。
(2)经UV+DES复合诱变ZM2的原生质体,得到高产糖化酶菌株UV-DES-M4,其产糖化酶活力达到846.27±79.24mg/h.g,比出发菌株ZM2提高了50.9%。应用SAS8.2软件,通过响应面(Box-Behnken设计法)对霉菌制曲条件进行了优化,确定UV-DES-M4产糖化酶的最佳条件为:制曲温度为30.05℃,豆饼粉加入量为8.06%,接种量为1%,培养30h,得到纯种曲1,糖化酶活力高达895.32 mg/h·g。
(3)自然pH值的葡萄糖豆芽汁与青稞粉按1.0mL:2.0g混合,接入15%的24h液体培养酵母ZY28,25℃培养30h,制得纯种曲3。
4.对青稞酒纯种曲酿造工艺条件进行了优化实验研究,结果表明,青稞酒的最佳纯种曲发酵工艺条件为:选择“藏青320”青稞原料,淘洗干净后,浸泡或者不浸泡但加水1:2,进行常压蒸煮2 h,摊凉至35℃,接入青稞原料重量2%的纯种曲1,2%纯种曲2及1%纯种曲3,在28℃下发酵72h。按此工艺得到的青稞酒,其理化指标和感官指标与传统曲发酵的青稞酒基本一致。
5.对青稞酒的纯种曲发酵与传统曲发酵过程中各成分的动态变化进行了研究,结果表明,青稞酒的纯种曲发酵过程中,表现出类似传统曲发酵过程中各成分的动态变化趋势,如pH值明显下降和酸度上升;发酵品温先缓升,再缓降,最高达36℃左右;还原糖含量和糖化酶活力值都在24h内显著增加(P＜0.05),达到最大值后逐渐下降;总糖含量呈显著性下降趋势,而酒精度显著增高(P＜0.05),纯种发酵的酒精度可达到10.3%,比传统发酵的提高57.7%;酒醅中酸性蛋白酶活力及氨态氮含量同时呈显著性增高(P＜0.05),24h后,蛋白酶活力及氨态氮含量逐渐降低;纯种发酵的青稞酒氨基酸含量只有80.923 mg/100mL,缺乏蛋氨酸。
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 B_1、B_2、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.
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