CN112877173A - 一种黑茶复合茯苓液态发酵菌丝保健醋及其制备方法 - Google Patents
一种黑茶复合茯苓液态发酵菌丝保健醋及其制备方法 Download PDFInfo
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- CN112877173A CN112877173A CN202110337164.3A CN202110337164A CN112877173A CN 112877173 A CN112877173 A CN 112877173A CN 202110337164 A CN202110337164 A CN 202110337164A CN 112877173 A CN112877173 A CN 112877173A
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Abstract
本发明属食醋生产技术领域,提供一种黑茶复合茯苓液态发酵菌丝保健醋及其制备方法,将黑茶茶叶或黑茶茶叶碎片、茶末、茶渣下脚料与藜麦、高粱糖化液混合,加大曲和酵母混菌酒精发酵;酒醪、茯苓菌丝枸杞发酵液、酶解红枣汁混合,加优良巴氏醋杆菌和乳酸菌进行有氧醋酸乳酸发酵;结束后再加乳酸菌,进行厌氧二次乳酸发酵制备而成。本发明对发酵原料底物、菌种配比进行了优化;对发酵过程中的多酚类活性物质的保留、积累,及茯苓多糖、三萜类功能物质的生成进行了监测,产品中:不挥发性酸≥2.0g/100mL,多酚≥300 mg/100mL,黄酮≥300 mg/100mL,多糖≥6 g/L、三萜≥100mg/L,降血脂、降血糖、增强免疫等功效。
Description
技术领域
本发明属于保健醋制备技术领域,具体涉及一种黑茶复合茯苓液态发酵菌丝保健醋及其制备方法。
背景技术
谷物醋的酿造与使用在我国有着悠久的历史。长期以来,食醋主要被作为调味品来使用,随着科技的发展,果醋、复合醋、保健醋、醋饮等得到进一步开发。茶醋是一种新型的原生态复合醋,兼具茶和醋的保健功效,具有很好的开发前景。
申请号为CN200810121568.3的发明专利“茶醋及酿造方法”,将茶叶、白砂糖和水,选用葡萄糖发酵酵母发酵制取茶原酒;由茶原酒接种醋酸菌,选用醋酸菌发酵酿造茶原醋,发酵时间为45天;在茶原醋中加入白砂糖或蜂蜜,过滤制得茶醋。
申请号为CN201610338501.X的发明专利“普洱茶醋饮料发酵加工工艺”,将醋酸菌和普洱熟茶发酵菌一起加入到含糖普洱茶汁液中,密封发酵,过滤、灭菌,即得到所述的普洱茶醋饮料。
申请号为CN CN201710505597.9的发明专利“一种茶醋及其制备方法”,将绿茶或红茶茶叶加乙醇进行提取处理,调节pH,加入醋酸杆菌进行醋酸第一次发酵,调节酒精度,再加入醋酸杆菌进行醋酸第二次发酵,陈酿,杀菌,澄清,即得成品,该茶醋兼具茶和醋的香味和疗效。
申请号为201810103284.5的发明专利“一种黑茶茶醋饮料及其制备方法”,采用黑茶汁和糯米配合,加入酒曲,密封酒精发酵得酒精发酵液,再加入黑茶茶汁稀释后,加入醋酸菌发酵,即得黑茶原醋。将黑茶原醋与黑茶汁、甜味剂、酸味剂、香精、水按照一定比例调配,即得成品黑茶茶醋饮。
综上所述,大多数相关茶醋的发明为利用茶叶水浸提液、酒精浸提液配合糯米,分两步进行发酵或直接加醋酸菌进行醋酸发酵而成,由于原料单一,使用的工艺传统,菌种没有经过优选配比,因此发酵时间较长(30~50天),发酵风味单调、缺少层次感。另外,大多数发明没有对发酵过程中多酚、黄酮类生物活性物质的保留、富集情况进行测定及工艺优化,最终产品的保健效果欠佳。
发明内容
本发明提供了一种黑茶复合茯苓液态发酵菌丝保健醋及其制备方法,将黑茶茶叶或黑茶茶叶碎片、茶末、茶渣等下脚料与藜麦、高粱糖化液混合,加入大曲和酵母进行混菌酒精发酵;酒精发酵结束后,将酒醪、茯苓菌丝枸杞发酵液、酶解红枣汁混合,添加优良巴氏醋杆菌和乳酸菌进行有氧醋酸乳酸发酵;有氧醋酸乳酸发酵结束后,再添加乳酸菌,进行厌氧二次乳酸发酵制备而成。
本发明对发酵原料底物、菌种配比进行了优化;对发酵过程中的多酚类活性物质的保留、积累,及茯苓多糖、三萜类功能物质的生成进行了监测;最终产品中富含多酚、不挥发性酸、多糖、三萜等功能成分,具有降血脂、降血糖、增强免疫等功效。
本发明由如下技术方案实现的:一种黑茶复合茯苓液态发酵菌丝保健醋的制备方法,藜麦高粱混合糖化液中加入质量体积比为15%~20%的黑茶茶叶粉,混合均匀,加入质量体积比为30%的大曲粉和0.1%~0.3%的酵母发酵剂进行混菌酒精发酵,醪液中酒精度在9%~10%时,发酵醪降温至25℃,维持2天结束酒精发酵,过滤得酒精发酵醪液;
按酒精发酵醪液:茯苓菌丝枸杞发酵液:酶解红枣汁体积比为2:1:1混合,加入混合液质量体积比8%~10%的醋酸乳酸发酵剂进行有氧醋酸乳酸发酵,发酵至总酸为5~6g/100mL,得醋酸乳酸发酵液;
醋酸乳酸发酵结束后,按醋酸乳酸发酵液的体积,接入3%~5%的植物乳杆菌CGMCC15731种子液作为作为醋酸乳酸发酵剂,进行静置厌氧二次乳酸发酵,发酵2天后结束;
发酵结束后过滤,滤液100℃煮制2~3min,常温后熟10~20天,硅藻土过滤澄清得黑茶复合茯苓液态发酵菌丝原醋;将原醋、红枣酶解汁、糖按比例调配、超高温瞬时灭菌,即为黑茶复合茯苓液态发酵菌丝保健醋。
所述黑茶茶叶粉的制备方法为:选用安化黑砖、花砖、茯砖、湘尖,云南普洱茶,湖北青砖,广西六堡茶,四川方包、金尖中的任意一种或几种以任意比例混合作为黑茶茶叶,将黑茶茶叶或茶叶加工产生的黑茶茶叶碎片、茶末、茶渣下脚料粉碎后过50目筛,得黑茶茶叶粉。
藜麦高粱混合糖化液的制备方法为:粉碎为四至六瓣的藜麦、高粱,按照质量比为1:1混合,加入4~5倍体积的水常温润料4h,升温到65℃加入0.2%的高温淀粉酶,继续升温到90~95℃维持40~50min,降温到30℃,即为藜麦高粱混合糖化液。
所述的酵母发酵剂的制备方法为:将活化的酿酒酵母CGMCC 15729接入到麦芽汁培养基中,30℃静置培养24h,菌体浓度达108cfu/mL,8000r/min,离心10min得菌体细胞,采用无菌生理盐水洗涤菌体细胞2次,添加3倍菌体细胞质量的保护剂,浓缩发酵液与保护剂的体积比为1:3混合,真空冷冻干燥即为酵母直投式发酵剂,活菌数为1010cfu/g;所述酿酒酵母CGMCC 1572为分离自山西老陈醋酒醪中的优良菌株,产醇量达7.0%、产酯量达6.33 g/100mL,保藏于中国微生物菌种保藏中心;所述保护剂配方为w/v:海藻糖1.5 %,甘油0.5 %,山梨醇2 %,麦芽糊精1 %,脱脂奶10 %。
所述的茯苓菌丝枸杞发酵液的制备方法为:茯苓菌种(Wolfiporia cocos)CGMCC5.137转接至PDA培养基上,28℃培养5d,然后转接至100 mL液体发酵培养基中,28℃、160r/min培养5d,作为二级种子;
10L反应器中装入7.2~7.5 L液体发酵培养基,121℃灭菌20 min后,接种10~15% v/v的二级种子,28℃、160r/min振荡培养5d,8000r/min,离心10min得茯苓发酵上清液和菌丝体;发酵上清液115℃,15min灭菌;菌丝体打碎成浆液,浆液加3倍体积的水,85~95℃水浴浸提3h,过滤,滤渣重新浸提一次,合并滤液及发酵上清液得茯苓菌丝枸杞发酵液;
所述液体发酵培养基配方为w/v:枸杞子粉末1~2%,玉米浆1%,蛋白胨0.5%,麸皮6%,葡萄糖4%,MgSO4 0.05%,磷酸二氢钾0.1%,其余为水,121℃灭菌20min。
所述红枣酶解汁的制备方法为:红枣清水冲洗去表面的泥沙和杂质,加入8倍重量的水,煮制1~1.2 h,破碎打浆,加入w/v 为0.2~0.4%的果胶酶于45℃酶解2~3h,即得红枣酶解汁。
所述的醋酸乳酸发酵剂为植物乳杆菌CGMCC 15731种子液:巴氏醋杆菌CGMCC15730种子液以1:1的体积比混合制成得复合发酵剂。
所述糖为木糖醇、蜂蜜、低聚果糖、白砂糖一种或几种,使终糖度调整为3~4oBrix。
所述酒精发酵条件为:前两天为敞口发酵,之后为封口密闭发酵,发酵温度为30℃~32℃,发酵6~7天,醪液中酒精度在9%~10%时,发酵醪降温至25℃,维持2天结束酒精发酵;
所述醋酸乳酸发酵条件为:发酵温度为30℃,170r/min,发酵进行6~8天,发酵至总酸为5~6g/100mL结束醋酸乳酸发酵;
所述二次乳酸发酵条件为:发酵温度为40℃,发酵时间为2天;
所述超高温瞬时灭菌条件为115~130℃,5~15S。
本发明还提供了利用上述制备方法得到的黑茶复合茯苓液态发酵菌丝保健醋。
本发明所述的植物乳杆菌CGMCC 15731,为本实验室从山西老陈醋醋醅中分离出的具有耐受性强,产不挥发性,多酚能力强,发酵风味佳的优良菌株。所述的植物乳杆菌CGMCC 15731种子液的制备方法为:将活化的植物乳杆菌CGMCC 15731按3%的接种量接入到MRS液体培养基中,37℃静置培养24h,菌体浓度达108cfu/mL,8000r/min,离心10min得菌泥,用0.85%的生理盐水洗涤菌体两次后重悬菌体,使浓度达到108cfu/mL,即乳酸菌种子液。
所述的巴氏醋杆菌CGMCC 15730,为本实验室从山西老陈醋醋醅中分离出的具有耐受性强、产酸高和发酵风味佳的优良菌株。所述的巴氏醋杆菌CGMCC 15730种子液的制备方法为:将活化的巴氏醋杆菌CGMCC 15730按3%的接种量接入到醋酸菌培养基中,30℃,170r/min培养24h,菌体浓度达108cfu/mL,8000r/min,离心10min得菌泥,用0.85%的生理盐水洗涤菌体两次后重悬菌体,使浓度达到108cfu/mL,即巴氏醋杆菌CGMCC 15730种子液。所述的醋酸菌培养基配方组成:葡萄糖2g,酵母膏1g,无水碳酸钙1g,水100mL,121℃,20min灭菌,冷却后加入2mL无水乙醇。
本发明所制备产品中:总固形物≥12.0g/100mL,总酸≥5.0g/100mL,不挥发性酸≥2.0g/100mL,多酚≥300 mg/100mL, 黄酮≥300 mg/100mL,多糖≥6 g/L、三萜≥100mg/L。色泽为红棕色;具有红枣醋的果香和茶香;酸味柔和纯正、自然、浓郁醇厚、回味绵长、无其他异味;体态清亮透明。经试验证明具有降血脂、降血糖、增强免疫等功效。
与现有技术相比,本发明具有以下有益效果:
(1)本发明将黑茶、藜麦、高粱、茯苓菌丝发酵液和红枣酶解汁进行一定比例混合,进行共发酵,各种发酵原料底物中的营养因子得到了互相补充,更加有利于菌体细胞的增殖,使发酵更充分,更加有利于原料中多酚、黄酮、多糖、有机酸、萜类等活性成分的发酵累积,丰富了产品的风味,克服了以往单独采用茶叶或茶叶配合糯米发酵风味单调、发酵时间长的问题。
(2)黑茶属于后发酵茶,以较成熟的鲜叶为原料,经杀青、渥堆发酵、蒸压等工序制成,成品茶的外观呈黑色的一大类茶叶的总称,属六大类茶之一。其香气纯正,带有陈香,滋味醇和,并且富含多种活性成分包括:茶多酚,茶色素,茶多糖及咖啡碱,茶氨酸等,具有降血脂、降血压和抗氧化等功效。
本发明采用黑茶茶叶或者黑茶茶叶碎片、茶末、茶渣等下脚料为发酵原料,避免了资源浪费,体现了农产品加工副产物综合利用价值。
(3)本发明的醋酸发酵底物中复合了茯苓菌丝枸杞发酵液。茯苓是我国传统中药,是珍贵的药食两用菌。相比于传统松木栽培(10~12个月),茯苓液态发酵,具有菌丝体增殖块,生产效率高,方法简便,成本低廉等优点;并且,茯苓液态发酵液和菌丝中含有与固态培养法生产的茯苓相同的药用和保健价值,主要包括茯苓多糖和三萜类化合物茯苓酸、松苓酸等,并能产生发酵香味。将枸杞和茯苓共发酵,枸杞中的一些小分子活性物质,可以促进茯苓真菌的生长和次级代谢产物的合成与分泌,也有可能产生新的活性物质。茯苓菌丝枸杞发酵液的加入为本发明产品增加了营养保健功效,也对茯苓的现代生物产业和加产品开发提供新的思路。
(4)本发明采用藜麦、高粱配合黑茶作为酒精发酵底物,相比于大多发明采用糯米为原料,营养价值更高。藜麦的蛋白质含量高达16~22%,富含多种氨基酸,比例适当且易于吸收,富含不饱和脂肪酸,类黄酮,B、E族维生素,胆碱,叶酸,β‒葡聚糖等,被国际营养学家称为“营养黄金”和“超级谷物”。
(5)本发明采用优良植物乳杆菌CGMCC 15731(菌株耐受性强、产不挥发性酸、多酚能力强、发酵风味佳)和巴氏醋杆菌CGMCC 15730(产酸能力强)联合进行醋酸乳酸发酵,并且在发酵结束后,补加植物乳杆菌CGMCC 15731进行二次乳酸发酵,增加了产品中不挥发性酸的含量,进一步提升了产品的口感和功能。
具体实施方式
下面通过具体的实施方案叙述本发明。实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言,在不背离本发明实质和范围的前提下,对实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
实施例1:茯苓菌丝枸杞发酵液的制备及多糖和三萜的测定
取保藏的茯苓菌种,转接至马铃薯葡萄糖PDA琼脂平板,在28℃下培养5 d,然后转接至装有100 mL发酵培养基的250 mL摇瓶中,于28℃、160 r/min培养5 d,作为二级种子。在10 L反应器中装入7.5 L发酵培养基,于121℃灭菌20 min后,接种15%(v/v)的二级种子,于28℃、160 r/min振荡培养5d,8000r/min,离心10min得茯苓发酵上清液和菌丝体。发酵上清液115℃,15min灭菌;菌丝体则倒入组织捣碎机中,打碎成浆液,将浆液加3倍体积的水,90℃水浴浸提3h,过滤,滤渣重新浸提一次,合并滤液及发酵上清液得茯苓菌丝枸杞发酵液。所述的发酵培养基组成为:枸杞子粉末2%,玉米浆1%,蛋白胨0.5%,麸皮6%,葡萄糖4%,MgSO4 0.05%,磷酸二氢钾0.1%,其余为水,121℃灭菌20min。
测定结果表明:发酵培养基中添加2%的枸杞子粉末,制备的茯苓菌丝发酵液(茯苓发酵上清液与菌丝提取液合并制得)中,多糖的含量为3.74g/L,三萜的含量为250mg/L。
比不添加枸杞子粉末的发酵培养基制备的茯苓菌丝发酵液中多糖含量提高了120%,三萜的含量提高了50%。枸杞中含有丰富的多糖及生物碱、黄酮等一些小分子的活性物质,可以促进茯苓次级代谢产物的合成与分泌,亦有可能产生新的活性物质。
实施例2:黑茶复合茯苓液态发酵菌丝保健醋的制备
(1)原料处理:将安化茯砖黑茶茶叶或茶叶加工产生的黑茶茶叶碎片、茶末、茶渣等下脚料粉碎后过50目筛得茶粉;将粉碎至四至六瓣的藜麦,高粱,按照质量比为1:1混合,加入5倍体积水常温润料4h后,升温到65℃时加入0.2%的高温淀粉酶,继续升温到90~95℃维持40 min,降温到30℃即为藜麦高粱混合糖化液。
(2)酒精发酵:在藜麦高粱混合糖化液中加入质量体积比为20%的黑茶茶叶粉,混合均匀,加入30%的大曲粉和0.1%的酵母发酵剂进行混菌酒精发酵,前两天为敞口发酵,之后为封口密闭发酵,发酵温度为30℃~32℃,当发酵6天,醪液中酒精度在9% 左右时,发酵醪降温至25℃,维持2天结束。
大曲粉为市售的山西老陈醋发酵专用大曲。
酵母发酵剂为直投式冻干粉发酵剂:采用的酿酒酵母CGMCC 1572分离自山西老陈醋酒醪中的优良菌株,产醇量高达7.0%、产酯量高达6.33 g/100mL,保藏于中国微生物菌种保藏中心。将活化的酿酒酵母CGMCC 15729接入到麦芽汁培养基中,30℃静置培养24h,菌体浓度达108cfu/mL,8000r/min,离心10min得菌体细胞,采用无菌生理盐水洗涤菌体细胞2次,添加3倍菌体细胞质量的保护剂[海藻糖1.5 %(w/v),甘油0.5 %(w/v),山梨醇2 %(w/v),麦芽糊精1 %(w/v),脱脂奶10 %(w/v)],浓缩发酵液与保护剂的体积比为1:3混合,真空冷冻干燥即为酵母直投式发酵剂,活菌数为1010cfu/g。
(3)有氧醋酸乳酸发酵:酒精发酵结束后,按酒精发酵醪液:茯苓菌丝枸杞发酵液:酶解红枣汁体积比为2:1:1混合,加入10%的醋酸乳酸发酵剂进行有氧醋酸乳酸发酵,发酵温度为30℃,170r/min,发酵进行6天,发酵至总酸为5~6g/100mL,得醋酸乳酸发酵液。
茯苓菌丝枸杞发酵液的制备方法,同实施例1。
红枣酶解汁的制备方法为:红枣清水冲洗去表面的泥沙和杂质,加入8倍重量的水,煮制1 h,破碎打浆,加入质量体积比为0.3%的果胶酶于45℃酶解2.5h,即得红枣酶解汁。
醋酸乳酸发酵剂制备方法:植物乳杆菌CGMCC 15731,为本实验室从山西老陈醋醋醅中分离出的具有耐受性强,产不挥发性,多酚能力强,发酵风味佳的优良菌株。巴氏醋杆菌CGMCC 15730,为本实验室从山西老陈醋醋醅中分离出的具有耐受性强、产酸高和发酵风味佳的优良菌株。
将活化的植物乳杆菌CGMCC 15731按3%的接种量接入到MRS液体培养基中,37℃静置培养24h,菌体浓度达108cfu/mL,8000r/min,离心10min得菌泥,用0.85%的生理盐水洗涤菌体两次后重悬菌体,使浓度达到108cfu/mL,即乳酸菌种子液;将活化的巴氏醋杆菌CGMCC15730按3%的接种量接入到醋酸菌培养基中,30℃,170 r/min培养24h,菌体浓度达108cfu/mL,8000r/min,离心10min得菌泥,用0.85%的生理盐水洗涤菌体两次后重悬菌体,使浓度达到108cfu/mL,即巴氏醋杆菌CGMCC 15730种子液;植物乳杆菌CGMCC 15731种子液:巴氏醋杆菌CGMCC 15730种子液以1:1的体积比混合制得复合醋酸乳酸发酵剂。
(4)厌氧乳酸二次发酵:有氧醋酸乳酸发酵结束后,按醋酸乳酸发酵液的体积,接入5%的植物乳杆菌发酵剂(植物乳杆菌CGMCC 15731种子液),进行静置厌氧二次乳酸发酵,发酵温度为40℃,发酵时间为2天。
(5)过滤、后熟、调配、杀菌、灌装:发酵结束后,纱布过滤,得到的滤液100℃煮制3min,常温后熟20天后,经硅藻土过滤澄清得红枣薏米原醋。在原醋中添加2%(w/v)木糖醇、3%(w/v)的低聚果糖、2%(w/v)的蜂蜜,115~130℃,5S,无菌灌装,即得成品黑茶复合茯苓液态发酵菌丝保健醋。
对发酵过程中的总酸、不挥发性酸的生成,及发酵原料中多酚、黄酮、多糖、三萜类功能物质的保留、生成累积进行了动态监测,结果如表1所示:经厌氧乳酸二次发酵后,总酸达到6.20 g/100mL,不挥发性酸达到2.51 g/100mL。在酒精发酵阶段,多酚和黄酮的含量显著增加,分别从酒精发酵初期的200.12 mg/100mL和103.34mg/100mL上升到酒精发酵末期的290.14mg/100mL和156.76mg/100 mL,分析原因是因为随着酒精发酵的进行,原料中的多酚和黄酮逐渐溶出,并且酒精度越高越易溶出;到醋酸乳酸发酵阶段初期,由于富含多酚、黄酮的原料–红枣酶解汁的加入,使得多酚、黄酮的含量进一步上升,但在此之后整个醋酸乳酸发酵阶段二者含量无显著变化。并且,经醋酸乳酸发酵和厌氧乳酸二次发酵后,多糖和三萜的含量逐渐累积上升,最终,多糖的含量达到7.25 g/L,三萜的含量达到122.15 mg/L。
表1 黑茶复合茯苓液态发酵菌丝原醋制备过程中成分指标的测定
对比例1:黑茶复合茯苓液态发酵菌丝保健醋的制备(不添加藜麦):原料中不添加藜麦,即将藜麦高粱混合糖化液替换为高粱糖化液。其余制备方法、操作步骤同实施例2。
对比例2:原料中不添加茯苓菌丝发酵液的保健醋的制备:酒精发酵结束后,不添加茯苓菌丝发酵液,即按酒精发酵醪液:酶解红枣汁体积比为1:1混合,加入10%的醋酸乳酸发酵剂进行有氧醋酸乳酸发酵。其余制备方法、操作步骤同实施例2。
对比例3:醋酸乳酸发酵剂菌种只有巴氏醋杆菌CGMCC 15730,不添加乳酸菌的保健醋的制备:实施例2中所述的醋酸乳酸菌发酵剂为:植物乳杆菌CGMCC 15731种子液:巴氏醋杆菌CGMCC 15730种子液以1:1的体积比混合制得复合醋酸乳酸发酵剂;对比例2中的醋酸乳酸菌发酵剂为:巴氏醋杆菌CGMCC 15730种子液(菌体浓度为108 cfu/mL);即只添加巴氏醋杆菌CGMCC 15730进行单纯醋酸发酵。
种子液制备方法同实施例2。其余制备方法、操作步骤同实施例2。
实施例2与对比例1、对比例2和对比例3所得醋的成分检测结果见表2。
表2 黑茶复合茯苓液态发酵菌丝保健成品醋成分指标的测定
通过实施例2与对比例相比较,表明按照实施例2中方案制备的黑茶复合茯苓液态发酵菌丝保健成品醋的中总酸、不挥发性酸、多酚、黄酮、多糖和三萜的含量显著高于1、2和3。
与对比例1中只采用高粱糖化液配合黑茶作为酒精发酵底物相比,本发明的优选例–实施例2中采用藜麦、高粱混合糖化液配合黑茶作为酒精发酵底物,由于藜麦中富含多种氨基酸和B、E族维生素,胆碱,叶酸等,可提供给微生物更丰富的生长因子,从而促进微生物生长,使产酸量升高;另外藜麦中还富含类黄酮、β‒葡聚糖等,因此提升了最终产品中的多酚、黄酮和多糖的含量。
与对比例2中只添加酶解红枣汁进行醋酸乳酸发酵相比,本发明的优选例–实施例2中除添加酶解红枣汁之外,还添加了茯苓菌丝发酵液进行醋酸乳酸发酵,茯苓菌丝发酵液中主要的活性成分为茯苓多糖和茯苓素,属于小分子的四环三萜类化合物,这些活性成分显著提升了产品中多糖和三萜的含量;并且,茯苓菌丝在生长过程中会产生种类丰富的糖基水解酶、糖脂水解酶及多糖裂解酶等纤维素水解酶,溶解植物组织、细胞壁及淀粉等结构,有利于有效成分的溶出,从而会显著增加发酵液中多酚、黄酮的含量;另外,茯苓菌丝发酵液中富含丰富的氨基酸,如异亮氨酸、缬氨酸、丙氨酸等11种氨基酸,其作为生长因子,显著促进了乳酸菌、醋酸菌的生长,从而提升了产品中不挥发性酸和总酸的含量。
与对比例3中只添加巴氏醋杆菌CGMCC 15730进行单纯醋酸发酵相比,本发明的优选例–实施例2中则采用体积比为植物乳杆菌CGMCC15731种子液:巴氏醋杆菌CGMCC 15730种子液为1: 1的复合发酵剂进行协同醋酸乳酸发酵,显著提升了产品中总酸和不挥发性酸的含量,缓冲了醋酸形成的刺激性风味,使产品酸味更加柔和;并且产品中多糖的含量也显著提高,乳酸菌普遍具有产多糖的能力,在乳品工业中,常使用产胞外多糖的乳酸菌以提高酸奶的粘稠度,改善产品质地;本发明中植物乳杆菌利用酶解红枣汁和茯苓菌丝发酵液等发酵底物中丰富的糖类物质进一步生成多糖。
实施例3:黑茶复合茯苓液态发酵菌丝保健醋的降血糖、降血脂功效实验
动物实验:健康SD大鼠(150 ± 20)g 30只,随机分为正常对照组10只,模型组10只,给醋组10只,饲养温度22±1℃,相对湿度55±5%。前6周,正常对照组饲喂基础饲料,模型组和给醋组均饲喂高糖高脂饲料,给醋组每天灌胃黑茶复合茯苓液态发酵菌丝保健醋(制备方法见实施例2),灌胃剂量为5mL/kg体重,模型组与正常对照组则灌胃等量生理盐水。在第7周时,模型组和给醋组采用链脲佐菌素(STZ)(40 mg/kg) 腹腔注射诱导,正常对照组注射生理盐水,继续饲养到13周试验结束后,处死大鼠,留取标本,采用试剂盒测定大鼠血糖、血脂及激素水平。
高糖高脂饲料:玉米粉(45%),维生素(1%),猪油(20%),酪蛋白(20%),骨粉(4%),蔗糖(10%)。普通饲料:玉米粉(50%),麸皮(25%),豆料(20%),骨粉(2%),鱼粉(2%),维生素(1%)。
黑茶复合茯苓液态发酵菌丝保健醋对糖尿病大鼠空腹血糖及糖耐受性影响结果见表3,如表3所示,在第6周末,饲喂高糖高脂饲料并且经STZ处理组大鼠的葡萄糖耐受能力明显被降低,与正常对照组相比,血药浓度-时间曲线下面积(area under the curve,AUC)值显著升高。给醋组大鼠在第13周的空腹血糖值相比第6周显著降低,而正常对照组大鼠和模型组大鼠第6周和第13周的空腹血糖值差异不大。试验结果说明黑茶复合茯苓液态发酵菌丝保健醋可以显著改善高糖高脂饲料-STZ诱导的Ⅱ型糖尿大鼠的葡萄糖耐受性。
表3 黑茶复合茯苓液态发酵菌丝保健醋对糖尿病大鼠空腹血糖及糖耐受性影响
注:均值 (SD,标准偏差) (n = 10);**P<0.01表示给醋组与模型组相比有极显著性差异。
黑茶复合茯苓液态发酵菌丝保健醋对糖尿病大鼠激素水平的影响结果见表4,Ⅱ型糖尿病是非胰岛素依赖型糖尿病,以高血糖、胰岛素抵抗和胰岛素相对缺乏为主要特征。表4结果显示糖尿病模型组血清胰岛素、胰高血糖素和瘦素水平显著高于正常对照组(P<0.01)。灌胃黑茶复合茯苓液态发酵菌丝保健醋后,3种激素含量有所降低。糖尿病模型组胰高血糖素样肽-1(Glucagon-like peptide 1,GLP-1)和酪酪肽(peptide tyrosinetyrosine,PYY)水平较正常对照组显著降低,灌胃黑茶复合茯苓液态发酵菌丝保健醋,这两种激素水平显著提高(P<0.01)。
表4 黑茶复合茯苓液态发酵菌丝保健醋对糖尿病大鼠激素水平的影响
注:均值 (SD,标准偏差) (n = 10);**P<0.01表示给醋组与模型组相比有极显著性差异。
黑茶复合茯苓液态发酵菌丝保健醋对糖尿病大鼠血脂水平的影响结果见表5,血脂异常是糖尿病的常见特征,表5中的测定结果显示,模型组和给醋组大鼠血清中总胆固醇、甘油三酯和低密度脂蛋白-胆固醇较正常对照组明显提高,HDL-C水平三组间差异不显著。实验结果表明,灌胃黑茶复合茯苓液态发酵菌丝保健醋可以显著抑制高脂饲料诱导的糖尿病大鼠血清中总胆固醇和甘油三酯的水平。
表5 黑茶复合茯苓液态发酵菌丝保健醋对糖尿病大鼠血脂水平的影响
注:均值 (SD,标准偏差) (n = 10);**P<0.01表示给醋组与模型组相比有极显著性差异。
实施例4:黑茶复合茯苓液态发酵菌丝保健醋对机体免疫能力的影响
动物实验:健康昆明小鼠,雄性,体重20±2g,饲养温度22±1℃,相对湿度55±5%。设置正常对照组、给醋组,每组10只,自由摄食饮水。给醋组每天灌胃5mL/kg黑茶复合茯苓液态发酵菌丝保健醋(制备方法见实施例2),持续灌胃30天,正常对照组灌胃等量的生理盐水。进行小鼠延迟变态反应、NK细胞活性及小鼠脾脏指数测定。
黑茶复合茯苓液态发酵菌丝保健醋对小鼠机体免疫能力的影响结果见表6,由表6可知,给醋组与正常对照组小鼠的体重和脾脏指数二组之间并无显著差异,表明该黑茶复合茯苓液态发酵菌丝保健醋对小鼠脾脏重量无显著影响,可显著促进小鼠迟发性变态反应、NK细胞活性和抗炎因子IFN-α、IL-10的水平。证明黑茶复合茯苓液态发酵菌丝保健醋具有免疫增强作用。
表6 黑茶复合茯苓液态发酵菌丝保健醋对小鼠机体免疫能力的影响
Claims (10)
1.一种黑茶复合茯苓液态发酵菌丝保健醋的制备方法,其特征在于:藜麦高粱混合糖化液中加入质量体积比为15%~20%的黑茶茶叶粉,混合均匀,加入质量体积比为30%的大曲粉和0.1%~0.3%的酵母发酵剂进行混菌酒精发酵,醪液中酒精度在9%~10%时,发酵醪降温至25℃,维持2天结束酒精发酵,过滤得酒精发酵醪液;
按酒精发酵醪液:茯苓菌丝枸杞发酵液:酶解红枣汁体积比为2:1:1混合,加入混合液质量体积比8%~10%的醋酸乳酸发酵剂进行有氧醋酸乳酸发酵,发酵至总酸为5~6g/100mL,得醋酸乳酸发酵液;
醋酸乳酸发酵结束后,按醋酸乳酸发酵液的体积,接入3%~5%的植物乳杆菌CGMCC15731种子液作为醋酸乳酸发酵剂,进行静置厌氧二次乳酸发酵,发酵2天后结束;
发酵结束后过滤,滤液100℃煮制2~3min,常温后熟10~20天,硅藻土过滤澄清得黑茶复合茯苓液态发酵菌丝原醋;将原醋、红枣酶解汁、糖按比例调配、超高温瞬时灭菌,即为黑茶复合茯苓液态发酵菌丝保健醋。
2.根据权利要求1所述的一种黑茶复合茯苓液态发酵菌丝保健醋的制备方法,其特征在于:所述黑茶茶叶粉的制备方法为:选用安化黑砖、花砖、茯砖、湘尖,云南普洱茶,湖北青砖,广西六堡茶,四川方包、金尖中的任意一种或几种以任意比例混合作为黑茶茶叶,将黑茶茶叶或茶叶加工产生的黑茶茶叶碎片、茶末、茶渣下脚料粉碎后过50目筛,得黑茶茶叶粉。
3.根据权利要求1所述的一种黑茶复合茯苓液态发酵菌丝保健醋的制备方法,其特征在于:藜麦高粱混合糖化液的制备方法为:粉碎为四至六瓣的藜麦、高粱,按照质量比为1:1混合,加入4~5倍体积的水常温润料4h,升温到65℃加入0.2%的高温淀粉酶,继续升温到90~95℃维持40~50min,降温到30℃,即为藜麦高粱混合糖化液。
4.根据权利要求1所述的一种黑茶复合茯苓液态发酵菌丝保健醋的制备方法,其特征在于:所述的酵母发酵剂的制备方法为:将活化的酿酒酵母CGMCC 15729接入到麦芽汁培养基中,30℃静置培养24h,菌体浓度达108cfu/mL,8000r/min,离心10min得菌体细胞,采用无菌生理盐水洗涤菌体细胞2次,添加3倍菌体细胞质量的保护剂,浓缩发酵液与保护剂的体积比为1:3混合,真空冷冻干燥即为酵母直投式发酵剂,活菌数为1010cfu/g;所述酿酒酵母CGMCC 1572为分离自山西老陈醋酒醪中的优良菌株,产醇量达7.0%、产酯量达6.33 g/100mL,保藏于中国微生物菌种保藏中心;所述保护剂配方为w/v:海藻糖1.5 %,甘油0.5 %,山梨醇2 %,麦芽糊精1 %,脱脂奶10 %。
5.根据权利要求1所述的一种黑茶复合茯苓液态发酵菌丝保健醋的制备方法,其特征在于:所述的茯苓菌丝枸杞发酵液的制备方法为:茯苓菌种(Wolfiporia cocos)CGMCC5.137转接至PDA培养基上,28℃培养5d,然后转接至100 mL液体发酵培养基中,28℃、160r/min培养5d,作为二级种子;
10L反应器中装入7.2~7.5 L液体发酵培养基,121℃灭菌20 min后,接种10~15% v/v的二级种子,28℃、160r/min振荡培养5d,8000r/min,离心10min得茯苓发酵上清液和菌丝体;发酵上清液115℃,15min灭菌;菌丝体打碎成浆液,浆液加3倍体积的水,85~95℃水浴浸提3h,过滤,滤渣重新浸提一次,合并滤液及发酵上清液得茯苓菌丝枸杞发酵液;
所述液体发酵培养基配方为w/v:枸杞子粉末1~2%,玉米浆1%,蛋白胨0.5%,麸皮6%,葡萄糖4%,MgSO4 0.05%,磷酸二氢钾0.1%,其余为水,121℃灭菌20min。
6.根据权利要求1所述的一种黑茶复合茯苓液态发酵菌丝保健醋的制备方法,其特征在于:所述红枣酶解汁的制备方法为:红枣清水冲洗去表面的泥沙和杂质,加入8倍重量的水,煮制1~1.2 h,破碎打浆,加入w/v 为0.2~0.4%的果胶酶于45℃酶解2~3h,即得红枣酶解汁。
7.根据权利要求1所述的一种黑茶复合茯苓液态发酵菌丝保健醋的制备方法,其特征在于:所述的醋酸乳酸发酵剂为植物乳杆菌CGMCC 15731种子液:巴氏醋杆菌CGMCC 15730种子液以1:1的体积比混合制成得复合发酵剂。
8.根据权利要求1所述的一种黑茶复合茯苓液态发酵菌丝保健醋的制备方法,其特征在于:所述糖为木糖醇、蜂蜜、低聚果糖、白砂糖一种或几种,使终糖度调整为3~4oBrix。
9.根据权利要求1所述的一种黑茶复合茯苓液态发酵菌丝保健醋的制备方法,其特征在于:所述酒精发酵条件为:前两天为敞口发酵,之后为封口密闭发酵,发酵温度为30℃~32℃,发酵6~7天,醪液中酒精度在9%~10%时,发酵醪降温至25℃,维持2天结束酒精发酵;
所述醋酸乳酸发酵条件为:发酵温度为30℃,170r/min,发酵进行6~8天,发酵至总酸为5~6g/100mL结束醋酸乳酸发酵;
所述二次乳酸发酵条件为:发酵温度为40℃,发酵时间为2天;
所述超高温瞬时灭菌条件为115~130℃,5~15S。
10.一种利用权利要求1至9任意一种制备方法得到的黑茶复合茯苓液态发酵菌丝保健醋。
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