CN113943665B - 两株降解生物胺的野生酵母及其在果酒酿造中的应用 - Google Patents
两株降解生物胺的野生酵母及其在果酒酿造中的应用 Download PDFInfo
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Abstract
本发明公开了一株酿酒酵母和一株葡萄汁有孢汉逊酵母,以及它们在降低果酒中生物胺含量的应用。所述两株酵母保藏于中国微生物菌种保藏管理委员会普通微生物中心,其中酿酒酵母的保藏编号为CGMCC No.2350523505,葡萄汁有孢汉逊酵母的保藏编号为CGMCC No.23504。两株酵母经过检测都具有较强的生物胺降解能力,能够降低葡萄酒、蓝莓酒、桑椹酒、蓝靛酒、樱桃酒、越橘酒、红树莓酒、山楂酒、草莓酒等果酒发酵过程中色胺、β‑苯乙胺、腐胺、尸胺、组胺、酪胺、亚精胺和精胺的含量。
Description
技术领域
本发明涉及微生物技术领域。本发明具体涉及一株酿酒酵母和一株葡萄汁有孢汉逊酵母,以及它们在降低葡萄酒、桑椹酒、蓝莓酒等果酒发酵过程中生物胺含量的应用。
背景技术
生物胺是一类低分子量的碱性含氮有机化合物,主要由酶作用于游离氨基酸发生脱羧反应,或通过醛、酮的胺化和转胺作用产生,其在发酵食品中广泛存在。目前果酒中已经检测出20多种生物胺,主要为组胺、酪胺、腐胺及尸胺,也含有少量的色胺、β-苯乙胺、精胺和亚精胺。适量的生物胺能够作为激素或神经递质在人体中发挥重要的生理功能,正常情况下可以被人体肠道中的单胺氧化酶和二胺氧化酶代谢,但是如果这些酶在抑制剂如酒精或某些抗抑郁药物的存在下出现功能失调,或者摄入的生物胺含量超出了脱毒系统的能力,生物胺就会进入体液循环并对机体的心肺、神经、消化和免疫系统产生毒性作用,导致严重的人类健康问题。
果酒中生物胺水平是评价果酒安全性和品质的重要指标,高浓度的生物胺会影响果酒的感官特性,产生金属,肉和腐败的风味。此外,果酒中的乙醇和乙醛能够抑制胺氧化酶的活性,促进生物胺穿过肠壁,从而增强生物胺的潜在毒性,使得控制果酒中生物胺水平这一问题受到极大关注。生物胺在果酒酒精发酵过程中的产生是不可避免的,一些酿酒酵母能够产生少量的多胺,此外,一些非酿酒酵母能够合成组胺和尸胺,这些都表明在果酒酿造中酵母选择的重要性。目前,在果酒发酵过程中降低生物胺含量的方法主要有三种:1)减少可利用的游离氨基酸水平;2)控制产氨基酸脱羧酶微生物的生长及氨基酸脱羧酶的活性;3)接种具有生物胺降解能力的微生物作为发酵剂。但是,由于氨基酸是果酒中香气物质形成的重要前体,并且发酵中理化条件的改变可能会影响果酒的感官特性,因此选择高生物胺降解能力的微生物作为发酵剂是有效控制果酒中生物胺积累的重要策略。研究发现,葡萄园生态系统中的一些真菌能够降解培养基中的组胺、酪胺和腐胺,然而,这些微生物的生物胺降解能力受到葡萄酒基质的影响,不同菌株之间有很大差异。在这种情况下,筛选和利用生物胺降解能力强的酵母作为发酵剂酿造果酒可能是一种具有应用潜力的降低果酒中生物胺含量的方法。
随着中国果酒产业的迅速发展,果酒中生物胺含量较高的问题逐渐突出,但是针对具有生物胺降解作用的本土酵母的研究工作尚未开展。此外,目前各酒厂对进口商业酵母过度依赖,且品牌选择集中,导致我国果酒缺乏地域特色,风格单一化问题严重,而本土优质酵母在酿造产区和品种特色果酒中的巨大潜力未受到重视。因此,筛选和利用具有高生物胺降解活力的本土优质酵母资源,不仅能够降低果酒中生物胺含量,提高果酒的安全品质,也有助于更好地呈现中国果酒的本土特色,对推动果酒产业发展具有重要意义。
发明内容
因此,本发明的目的是提供两株本身不产生物胺,且能够高效降解已存在的生物胺的酵母,一株酿酒酵母,一株葡萄汁有孢汉逊酵母,两株酵母单菌株/顺序接种具有应用于葡萄酒、桑椹酒、蓝莓酒等果酒发酵中,降低果酒发酵期间8种生物胺含量的能力。
本发明的另一目的是提供一种高通量筛选具有生物胺降解能力酵母的方案。
针对上述目的,本发明提供的技术方案如下:
本发明提供的酿酒酵母(Saccharomyces cerevisiae),名称为HL17,属于酵母属(Saccharomycessp.),分离自河北怀来百花谷酒庄成熟的红色酿酒葡萄品种——“赤霞珠”(Cabernet Sauvignon)葡萄果实。该菌株已于2021年9月28日保藏于“中国微生物菌种保藏管理委员会普通微生物中心”,保藏地址:北京市朝阳区大屯路3号中国科学院微生物研究所,保藏号为:CGMCCNo.23505,建议分类命名为:酿酒酵母(Saccharomyces cerevisiae)。
本发明提供的葡萄汁有孢汉逊酵母(Hanseniaspora uvarum),名称为FS35,属于汉逊酵母属(Hanseniaspora sp.),分离自北京市房山区龙熙堡酒庄成熟的红色酿酒葡萄品种——“赤霞珠”(Cabernet Sauvignon)葡萄果实。该菌株已于2021年9月28日保藏于“中国微生物菌种保藏管理委员会普通微生物中心”,保藏地址:北京市朝阳区大屯路3号中国科学院微生物研究所,保藏号为:CGMCC No.23504,建议分类命名为:葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)。
除非特别指明,酿酒酵母(Saccharomyces cerevisiae)HL17CGMCCNo.23505,在本文中简称HL17,葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)FS35 CGMCC No.23504,在本文中简称FS35。
本发明提供的FS35和HL17可通过单菌株/顺序接种的方式参与到果酒的酿造过程中。以葡萄酒为例,利用FS35和HL17单菌株/顺序接种酿造葡萄酒的优点在于:在筛选过程中,FS35和HL17均表现出良好的生物胺降解能力,能够降解8种生物胺(组胺、酪胺、尸胺、腐胺、色胺、β-苯乙胺、亚精胺、精胺),FS35总胺降解率可达68.98%,其中组胺、酪胺、腐胺、尸胺降解率分别为81.97%、70.40%、94.84%、97.69%;HL17总胺降解率可达60.91%,其中组胺、酪胺、腐胺、尸胺降解率分别为87.76%、94.90%、92.69%、94.39%。在随后的葡萄酒酿造当中,使用FS35和HL17顺序接种的组别能够使葡萄酒发酵完全,且在生物胺水平上显著低于进口商业酿酒酵母组,总胺降解率可达79.17%,其中组胺、酪胺、腐胺、尸胺降解率分别为93.60%、100%、99.95%、100%。花香和果香突出,口感更佳。因此,在发酵过程中,通过接种FS35和HL17,可以利用本土特色酵母资源,同时降解多种生物胺,更好地解决葡萄酒等果酒发酵期间由于氨基酸脱羧作用所引起的生物胺含量较高的难题,从而获得安全品质更高的果酒,降低果酒中过量生物胺带给消费者的健康风险,对控制食品中的生物胺水平具有重要意义。同时,通过使用本土特色酵母菌株替代商业酿酒酵母,有助于改善使用进口商业酿酒酵母带来的果酒品质趋同化等缺陷,更好地呈现国产果酒的特色,从而促进中国果酒产业的自主创新型发展。
本发明还提供了一种可以用于快速精确检测野生酵母生物胺降解能力的方法,能够在实际工业应用中简化筛菌程序,缩短筛菌时间,从而大大提高筛选具有生物胺降解活力酵母菌株的工作效率。
生物保藏说明
生物材料HL17,分类命名:酿酒酵母Saccharomyces cerevisiae,于2021年9月28日保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCC No.23505。
生物材料FS35,分类命名:葡萄汁有孢汉逊酵母Hanseniaspora uvarum,于2021年9月28日保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCC No.23504。
附图说明
图1示菌株FS35和HL17在WL培养基上的菌落形态;
图2示测试菌株小型酿酒试验后葡萄酒样品中生物胺含量;
图3示8种生物胺标准溶液及内标衍生物液相色谱图。
具体实施方式
为了更好地理解与实施,下面结合实施例和附图详细说明本发明;所举实施例仅用于解释本发明,并非用于限制本发明的范围。
除非特别指明,以下实施例中所用的方法均为常规方法。
除非特别指明,以下实施例中所用的试剂均为分析纯级别的试剂,且可从正规渠道商购获得。
除非特别指明,以下实施例中的定量试验,均设置三次重复实验,结果取统计平均值。
实施例中涉及的培养基配方如下:
YPD固体培养基(每1L):酵母粉10g,蛋白胨20g,葡萄糖20g,琼脂20g。
葡萄酒酵母WL鉴别培养基(每1L):酵母粉4g,胰蛋白胨5g,葡萄糖50g,KH2PO40.55g,KCl 0.425g,CaCl2 0.125g,MgSO4 0.125g,FeCl3 2.5mg,MnSO4 2.5mg,琼脂20g,溴甲酚绿22mg。
生物胺降解菌株筛选培养基(每1L):YCB培养基11.7g,色胺、β-苯乙胺、腐胺、尸胺、组胺、酪胺、亚精胺、精胺各50mg(色谱级)
实施例1使用高通量方法筛选得到酿酒酵母HL17和葡萄汁有孢汉逊酵母FS35
本发明一株生物胺降解能力强的酿酒酵母,是从河北怀来百花谷葡萄园赤霞珠葡萄果实中得到并保存,另一株生物胺降解能力强的葡萄汁有孢汉逊酵母,是从北京市房山区龙熙堡酒庄赤霞珠葡萄果实中得到并保存,之后通过实验筛选所得,筛选过程如下:
取出在甘油管中冻存的菌株,解冻后按照10%的比例接种于液体YPD培养基中,28℃,160rpm培养12h,使用接种环蘸取少量菌液在WL鉴别培养基上进行平板划线,划线完成的平板在28℃静置培养48h;
若WL平板上没有出现杂菌,则用接种环挑取WL平板上的目标单菌落接种到液体YPD培养基中培养12h,然后用0.9%生理盐水调整其菌液浓度OD600=1.0进行后续试验,否则应该重复上述平板划线的操作,直到使菌株纯化为止;
向15mL离心管中添加适量生物胺降解菌株筛选培养基,将已调整浓度的菌液(OD600=1.0)接种到离心管中,接种量为10%,每种菌液接种3个离心管作为平行试验,记录好每个离心管所对应的单菌落编号,将离心管置于28℃恒温培养箱中培养72h;
将培养完成后的菌液在4℃、12000rpm的高速离心机中离心10min,取上清液并经过0.22μm滤膜过滤,用UPLC检测经过滤后菌液中8种生物胺的浓度,以公式(1)计算各菌株的生物胺降解率,从而对不同菌株的降胺能力进行评价。
式中:C0′:培养72h后不添加酵母的筛选培养基中各生物胺浓度;
C1′:培养72h后添加酵母的筛选培养基中各生物胺浓度;
C0:筛选培养基中各生物胺初始浓度,即50mg/L。
以不添加酵母的筛选培养基为对照,各菌株生物胺降解率的测定结果见表1,通过离心管发酵试验,共获得5株生物胺降解活力较高的菌株。其中,FS35对腐胺、尸胺、组胺和精胺的降解效果较好,且总胺降解率最高,为68.98%±0.09%;HL17对除精胺和亚精胺以外的6种生物胺降解效果较好,总胺降解率为60.91%±0.21%;虽然HL10的总胺降解率高于HL17,但HL10不能降解精胺和亚精胺,因此,选择FS35和HL17用于下一步试验。
表1试验菌株对8种生物胺的降解率(%)
实施例2野生酿酒酵母HL17、野生葡萄汁有孢汉逊酵母FS35和商业酿酒酵母红佳酿小型酿酒比较试验
将成熟赤霞珠品种酿酒葡萄除梗破碎,以500mL玻璃广口瓶作为葡萄酒发酵容器,结合前期菌株筛选结果,共设置4个发酵试验组,每组分别设置3个平行,具体分组情况见表2。酵母经活化后,接种于YPD液体培养基中,于28℃,160rpm条件下恒温振荡培养12h。向每个广口瓶中加0.4L葡萄醪,0.4mL亚硫酸。酵母按10%接种量接入葡萄醪,顺序接种发酵是在接种FS35发酵72h后再接种酿酒酵母。用蘸有亚硫酸的纱布清理广口瓶口、瓶盖,瓶盖倒放,置于25℃下发酵。葡萄醪开始发酵后,每天进行温度、糖度和比重的检测并记录。根据发酵情况适时搅拌。当葡萄酒比重不再下降时,取上层清液灌装,密封置于4℃冰箱中保存。
表2发酵试验组的设置
使用UPLC对发酵结束后葡萄酒样品中生物胺的种类及含量进行测定,结果见图2。
定量结果显示,以FS35和HL17顺序接种酿造的葡萄酒总胺降解率可达79.17%,未检出尸胺和酪胺,腐胺降解率最高,为99.95%,组胺降解率为93.60%,色胺和β-苯乙胺降解率均高于商业酿酒酵母组,分别为90.18%和97.28%,亚精胺和精胺降解率分别为28.99%和23.35%。表明FS35和HL17顺序接种具有较强的降低葡萄酒发酵期间生物胺含量的潜力,其酿造的葡萄酒安全品质较高。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一株葡萄汁有孢汉逊酵母,其特征在于,其保藏编号为CGMCC No.23504。
2.接种权利要求1所述的葡萄汁有孢汉逊酵母发酵72h后再接种酿酒酵母HL17或红佳酿的顺序接种方式在降低果酒发酵过程中色胺、β-苯乙胺、腐胺、尸胺、组胺、酪胺、亚精胺和精胺含量的应用,其中酿酒酵母HL17的保藏编号为CGMCC No.23505。
3.一种生产果酒的方法,其特征在于,接种如权利要求1所述的葡萄汁有孢汉逊酵母发酵72h后再接种酿酒酵母HL17或红佳酿发酵果实原料,得到低生物胺含量的果酒,其中酿酒酵母HL17的保藏编号为CGMCC No.23505。
4.根据权利要求3所述的方法,其特征在于,所述果实为葡萄、桑葚、蓝莓、蓝靛、樱桃、越橘、红树莓、山楂或草莓中至少一种。
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