CN110373347A - 一株富硒乳酸菌及其筛选方法和富含白藜芦醇和有机硒的风味酸奶的制备方法 - Google Patents
一株富硒乳酸菌及其筛选方法和富含白藜芦醇和有机硒的风味酸奶的制备方法 Download PDFInfo
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Abstract
本发明公开了一株富硒乳酸菌及其筛选方法和富含白藜芦醇和有机硒的风味酸奶的制备方法,特点是该乳酸菌分类命名为发酵乳杆菌PDD‑4株,于2019年03月25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,保藏编号为CGMCC No.17434;起风味酸奶制备方法包括富硒花生芽汁的制备步骤;花生芽牛奶液的制备步骤;菌株的活化以及乳酸菌发酵剂的制备步骤;最后接种发酵剂发酵、成熟制得富含白藜芦醇和有机硒的风味酸奶制品,优点是风味独特、营养丰富,而且具有抗氧化和免疫调节功能。
Description
技术领域
本发明属于食品加工领域,尤其是涉及一株富硒乳酸菌及其筛选方法和富含白藜芦醇和有机硒的风味酸奶的制备方法。
背景技术
白藜芦醇是一类是一种含有芪类结构酚类物质,天然白藜芦醇作为一种植物抗生素,在植物受到外界生物或非生物胁迫时发挥重要的作用。目前已发现70多种植物中含有白藜芦醇,其中花生、葡萄、虎杖等植物中含量较高。研究发现白藜芦醇具有顺式和反式两种构型,最大光吸收波长分别为306nm和286nm,其反式异构体的生物活性强于顺式,单体强于糖苷,且更为广运的存在。现代医学通过体内外试验发现白藜芦醇具有多种生物学活性,包括抗氧化、抗衰老、抗炎、抗癌、抗血小板凝集、免疫调节、心血管保护以及神经系统保护等。但是,在我国,白藜芦醇并不属于食品添加剂的范畴,不能直接加入食品中,因此其生物活性的利用也受到了一定程度的限制。通过使用花生芽、葡萄等制品是白藜芦醇摄入的常规途径,通过风味酸奶的研制也属于一种能够维持白藜芦醇活性,增加白藜芦醇摄入量的一种方法。
花生芽是花生生芽后产生的一种食疗兼备的食品,也叫长寿芽。它不但能够生吃,而且营养还特别丰富。花生芽的能量,蛋白质和粗脂肪含量居各种蔬菜之首,并富含维生素、钾、钙、铁、锌等矿物质及人体所需的各种氨基酸和微量元素,被誉为“万寿果芽”,研究发现,花生芽的白藜芦醇含量比花生的要高5倍,堪比葡萄酒中的白藜芦醇含量。白藜芦醇具有抑制癌细胞、降血脂、防治心血管疾病、延缓衰老等作用,保健价值极高。花生芽可以使花生中的蛋白质水解为氨基酸,易于人体吸收;油脂被转化为热量,脂肪含量大大降低,害怕肥胖的人可以长期使用,与此同时,提高了各种人体必需的微量元素利用率。
硒是人体必需的微量元素之一,具有多种生物活性功能,人体每天需要摄入一定量的硒,以保持身体的正常机能,有利于减少疾病的发生中国营养学会推荐每人每天的硒摄取量为50~200μg,但是部分人群日常生活中对硒的摄入量远远达不到营养学会推荐的摄入量。由于硒摄入不足而导致的疾病对人类健康造成了较大的威胁。如大骨节病、克山病、恶性肿瘤和机体免疫力减退等。因此,通过生物富硒手段强化食品中硒的含量,从而增加人体对硒元素的摄入,是目前功能性食品的热点研究方向之一。
酸奶是采用优质纯鲜牛奶加入白糖均质,经超高温灭菌后接入乳酸菌发酵后制成的一种发酵型乳制品。在发酵过程中,鲜牛奶中的酪蛋白遇酸凝固,成为有弹性的凝块,颜色乳白、气味清香、酸甜可口。与牛乳相比,酸奶的营养成分更趋完善和更易消化吸收,内含的乳糖被部分分解成半乳糖和葡萄糖,后者进而转化为乳酸等有机酸,并且酸奶由于乳酸菌的发酵作用,在营养成分得到改善的同时,也产生了一些生理活性物质如有机酸、芳香物质、抗生物质、SOD、细胞壁外多糖、活性酶、乳酸菌增殖因子及乳酸菌等,对机体功能有显著的调节作用,这些物质在维持肠道正常菌群平衡,调节肠道有益菌群达到正常水平、胆固醇含量降低、防衰老和延年益寿等方面都有重要的功能,因此酸奶是一种对人体十分有益的营养保健食品,对于酸奶的研究十分有意义。目前,市售酸奶大多是以牛奶或奶粉为原料经乳酸菌发酵制成,种类和功能比较单一,未见利用生物转化法生产同时富含有机硒和白藜芦醇等功能因子的特色风味酸奶研究报道。
发明内容
本发明所要解决的技术问题是提供一株富硒乳酸菌及其筛选方法,同时提供一种不仅风味独特、营养丰富,而且具有抗氧化和免疫调节功能的富含白藜芦醇和有机硒的风味酸奶的制备方法。
本发明解决上述技术问题所采用的技术方案为:
1、一株富硒乳酸菌,该乳酸菌分类命名为发酵乳杆菌(Lactobacillus fermentum)PDD-4株,于2019 年03月25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(地址为北京市朝阳区北辰西路1号院3号),保藏编号为CGMCC No.17434。花生发芽过程中转化无机硒为有机硒,同时产生大量的白藜芦醇和黄酮类物质。
2、上述富硒乳酸菌的筛选方法,包括以下步骤:
(1)乳酸菌的初步筛选及分离纯化
挑选传统发酵食品泡菜为样品进行稀释后,涂布于含有溴甲酚紫的MRS固体培养基平板,倒置培养于37~43℃培养箱36~48h,挑选出橙黄色的菌落,将筛选得到的具有典型乳酸菌菌落特征的单一菌落初步鉴定为乳酸菌,将初步确定的乳酸菌菌落在MRS固体平板上连续划线几次,挑取出形态单一的菌落活化后置于甘油保藏管中存放在-80℃冰箱中;
(2)目标菌株的筛选
将步骤(1)初步筛选的乳酸菌菌株经分离纯化后涂布于含有20-100mg/L Na2SeO3的MRS固体培养基平板上,倒置培养于37-43℃培养箱24~36h,挑选出在同等Na2SeO3浓度下菌落颜色呈现出乳白色或微红色的菌落;再涂布于含有20~50mg/L Na2SeO3 的MRS固体培养基平板上,按上述条件培养,挑选出在同等Na2SeO3浓度下菌落颜色呈现出乳白色或微红色的菌落,继续分离纯化至单一菌落,进行革兰氏染色和生理生化特性鉴定,选取革兰氏阳性、H2O2酶为阴性的菌,即为富硒乳酸菌,该菌株分类命名为发酵乳杆菌(Lactobacillus fermentum)PDD-4株,于2019年03月25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,保藏编号为CGMCC No.17434。
上述含有溴甲酚紫的MRS固体培养基的配制方法如下:将MRS肉汤52.24g,溴甲酚紫0.04g,琼脂粉0.1g~0.2g,溶于1L蒸馏水中,于121℃灭菌20min即可;所述的含有Na2SeO3的MRS固体培养基的配制方法如下:将MRS肉汤52.24g,琼脂粉0.1g~0.2g,溶于1L蒸馏水中,于121℃灭菌20min,待培养基冷却至55℃时,加入一定量Na2SeO3灭菌培养液,即可配制成所需浓度的固体培养基。
3、一种富含白藜芦醇和有机硒的风味酸奶的制备方法,包括以下步骤:
(1)富硒花生芽汁的制备
选取颗粒、大小一致、成熟饱满的花生种子,用75%乙醇浸泡消毒 15min后,用灭菌后的蒸馏水反复冲洗3遍,将消毒冲洗后的种子至于亚硒酸钠溶液中恒温浸泡后,置于双层滤纸平皿上于恒温培养箱中避光发芽5d,每隔24h用消毒后的蒸馏水润洗数次并更换滤纸,取富硒发芽结束后的鲜花生芽,按花生芽和牛奶质量体积比1g:2ml的比例榨汁,混匀过单层灭菌纱布,取匀浆,得到富硒花生芽汁;
(3)花生芽牛奶液的制备:在牛奶中按体积比添加富硒花生芽汁和蔗糖,混合均匀,制成富硒花生芽牛奶液,其中富硒花生芽牛奶液中富硒花生芽汁体积百分数为30%~50%,蔗糖体积百分数为 4~8%;
(4)菌株的活化:在无菌操作条件下,将德氏乳杆菌保加利亚亚种(BNCC336436或BNCC186626或BNCC138500或BNCC187941)和嗜热链球菌(BNCC187932 或BNCC186629或BNCC175966或BNCC175937)、植物乳杆菌植物亚种PDD-1和权利要求1所述的发酵乳杆菌分别接种于MRS液体培养基中,分别在35~42℃的培养箱中培养24~36h;
(5)乳酸菌发酵剂的制备:将活化后的德氏乳杆菌保加利亚亚种和嗜热链球菌按1:1的比例进行混合的混合菌种、植物乳杆菌植物亚种PDD-1和发酵乳杆菌在无菌操作条件下,分别按一定接种量接种于在经巴氏杀菌后冷却至37~40℃的纯牛奶中,在34~40℃保温箱中培养3~6h,相应制成第一发酵剂、第二发酵剂和第三发酵剂;
(6)接种发酵剂发酵:将富硒花生芽牛奶液进行杀菌处理后,按质量或体积比添加第一发酵剂5~8%、第二发酵剂1~4%和第三发酵剂1~5%,充分搅拌使其混合均匀后,置于发酵箱内,于温度为32~39℃,发酵处理4~8h得到发酵液;
(7)酸奶的成熟:将步骤(5)得到的发酵液移入4~8℃冰箱或冷藏室内,经6~12h发酵成熟后,制得富含白藜芦醇和有机硒的风味酸奶制品。
植物乳杆菌植物亚种(Lactobacillus plantarum subsp. plantarum)PDD-1株,于2018 年06月19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,保藏编号为CGMCC No.15953。
发酵乳杆菌(Lactobacillus fermentum)PDD-4株,于2019 年03月25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,保藏编号为CGMCC No.17434。发酵乳杆菌。
与现有技术相比,本发明的优点在于:本发明首次公开了一株富硒能力较强的乳酸菌及其筛选方法和同时富含白藜芦醇和有机硒酸奶的制备方法,将富硒花生芽和蔗糖按一定比例加入纯牛奶中,杀菌,接种保加利亚乳杆菌和嗜热链球菌发酵剂及能转化无机硒的富硒乳酸菌发酵剂。在整体发酵过程中,嗜热链球菌能够分泌出乳糖酶,将乳糖分解成葡萄糖和半乳糖,除供给能源外,半乳糖可参与脑苷脂和神经物质的合成,有助于婴幼儿脑和神经系统的发育;富硒花生芽能够将无机硒转化成有机硒,提高硒的生物活性;富硒乳酸菌能够将花生发芽过程中没有转化的无机硒转化成有机硒,提高酸奶中有机硒的含量;本产品除了普通酸奶具有的维持肠道正常菌丛平衡、调节肠道有益菌群、降低胆固醇含量的作用外,由于富含白藜芦醇、有机硒和黄酮类物质等功能因子,本产品还具有抗癌、抗血小板凝集、抗氧化自由基、抗病毒和抗衰老等保健功能,为特色风味的功能性酸奶开发提供一定的思路和启发,也为白藜芦醇等非国标添加物在食品中的运用探索了一条可行的道路。最终得到的酸奶产品中白藜芦醇、有机硒和黄酮苷元的含量分别为5.10-13.10µg/mL、165.50-175.12µg/mL、75.70-105.60µg/mL。
一株产β-葡萄糖苷酶乳酸菌,该乳酸菌分类命名为植物乳杆菌植物亚种(Lactobacillus plantarum subsp. plantarum)PDD-1株,于2018 年06月19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(地址为北京市朝阳区北辰西路1号院3号),保藏编号为CGMCC No.15953。PDD-1是产一株产β-葡萄糖苷酶乳酸菌,它可以将黄酮类物质转化成游离的黄酮苷元,黄酮苷元的膜渗透性强,容易透过肠黏膜细胞,相对于黄酮糖苷更易被人体吸收;发酵前黄酮苷元含量为0.030 mg/mL,经Lactobacillus plantarum subsp. Plantarum PDD-1菌株发酵后,酸奶中黄酮苷元含量为0.094 mg/mL。
一株富硒乳酸菌,该乳酸菌分类命名为发酵乳杆菌(Lactobacillus fermentum)PDD-4株,于2019年03月25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,保藏编号为CGMCC No.17434。富硒乳酸菌能够将花生发芽过程中没有转化的无机硒转化成有机硒,转化率22.0%。
具体实施方式
以下结合实施例对本发明作进一步详细描述。
一、实验测定方法
1、目的菌株的富硒能力测定
(1)硒单质析出反应:将MRS肉汤52.24g,1.5%琼脂粉(1g~2g/100mL),溶于1L蒸馏水中,于121℃灭菌20min,待培养基冷却至约55℃时,加入高浓度的Na2SeO3灭菌培养液(V/V),配置成不同浓度梯度的Na2SeO3 MRS固体培养基,制备平板,冷却,分别将100μL液样品涂布不同浓度的的Na2SeO3 MRS固体培养基平板上,倒置于37℃培养箱中培养36h.观察颜色反应,根据菌落颜色变红深浅判定乳酸菌的耐硒能力;
(2)硒标准曲线的绘制
取1μg/ml的Na2SeO3标准溶液0、2.0、4.0、6.0、8.0、10.0ml,分别移入125ml分液漏斗,加水至35ml,加入5%的EDTA~2Na溶液1ml,摇匀,调节pH值2~3,各加0.5%3.3’~二氨基联苯胺溶液4ml,摇匀,置于暗处30min。调节pH值至中性,加入10ml甲苯震荡两分钟,静置分层,弃水层,甲苯层通过棉花栓过滤于比色皿中,测定420nm波长出的吸光度值,绘制标准曲线。得方程y = 0.0098x + 0.4611 R² = 0.9981
(3)残留无机硒含量的测定
发酵液5500r/min离心20min,取2ml上清液于石墨炉消解仪中进行程序性升温消解,直至消化液清亮,如还有颜色,则冷却后加入2ml的H2O2进行除色加热,待液体冷却后移入10ml容量瓶中,蒸馏水稀释至刻度线,移入分液漏斗中,加水至35ml。以下同标准曲线。根据样品的吸光度,从标准曲线中查得相应的硒含量,乘以相应的倍数,此为残留无机硒含量的测定。
2、样品中白藜芦醇含量的测定
白藜芦醇含量的测定方法采用高效液相色谱法,将白藜芦醇标准品分别配制成0、2、4、6、8、10mg/L的梯度标准溶液经过0.45μm的微孔滤膜后选择适用的液相色谱条件进行测定,以标准品浓度x(单位mg/L)为横坐标,高效液相色谱法出峰面积y为纵坐标,绘制标准曲线,得方程:y = 80.22x + 0.92,R² = 0.9995
(1)酸奶中白藜芦醇的测定
按上述步骤发酵的酸奶,搅拌均匀取20g发酵好的酸奶样品于100mL离心管中,加入10mL无水乙醇后在高速匀浆机中匀浆提取2min后,于4000r/min离心10min,将上清液转入50mL容量瓶中,样品残渣再分别用10mL无水乙醇提取2次,合并3次提取液,用无水乙醇定容至50mL;
(2)将(1)中所得待测样液摇匀过0.45μm滤膜后入高效液相色谱仪。以不加富硒花生芽汁的酸奶做空白对照,以对照组和实验组中分别测得白藜芦醇含量之差作为优化的指标。
二、具体实施例
实施例1
一株富硒乳酸菌,该乳酸菌分类命名为发酵乳杆菌(Lactobacillus fermentum)PDD-4株,于2019年03月25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,保藏编号为CGMCC No.17434。
实施例2
上述实施例1中富硒乳酸菌的筛选方法,包括以下步骤:
(1)乳酸菌的初步筛选及分离纯化
挑选传统发酵食品泡菜为样品进行稀释后,涂布于含有溴甲酚紫的MRS固体培养基平板,倒置培养于37~43℃培养箱36~48h,挑选出橙黄色的菌落,将筛选得到的具有典型乳酸菌菌落特征的单一菌落初步鉴定为乳酸菌。初步确定的菌落在MRS固体平板上连续划线几次,挑取出形态单一的菌落活化后置于甘油保藏管中存放在-80℃冰箱中,以备后期试验所用。所述的含有溴甲酚紫的MRS固体培养基的配制方法如下:将MRS肉汤52.24g,溴甲酚紫0.04g,1.5%琼脂粉(1g~2g/100mL),溶于1L蒸馏水中,于121℃灭菌20min即可;所述不同Na2SeO3浓度的MRS固体培养基的配置方法如下:将MRS肉汤52.24g,1.5%琼脂粉(1g~2g/100mL),溶于1L蒸馏水中,于121℃灭菌20min,待培养基冷却至约55℃时,加入高浓度的Na2SeO3灭菌培养液(V/V),即可配置成所需浓度的固体培养基;
(2)目标菌株的筛选
将步骤(1)初步筛选的乳酸菌菌株经分离纯化后涂布于含有不同Na2SeO3浓度(20、50、100mg/L)MRS固体培养基平板上,倒置培养于37~43℃培养箱24~36h,挑选出在同等Na2SeO3浓度下菌落颜色呈现出乳白色或微红色的菌落;再涂布于20~50mg/L的Na2SeO3 MRS固体培养基平板上,按上述条件培养,挑选出在同等Na2SeO3浓度下菌落颜色呈现出乳白色或微红色的菌落,继续分离纯化至单一菌落,进行革兰氏染色和生理生化特性鉴定,选取革兰氏阳性、H2O2酶为阴性的菌,即为富硒乳酸菌,该菌株分类命名为发酵乳杆菌(Lactobacillus fermentum)PDD-4株,于2019年03月25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,保藏编号为CGMCC No.17434。
实施例3
(1)富硒花生芽的制备
选取颗粒、大小一致、成熟饱满的花生种子,用75%乙醇浸泡消毒15min后,用灭菌后的蒸馏水反复冲洗3遍,将消毒冲洗后的种子至于Na2SeO3溶液中恒温浸泡后,置于双层滤纸平皿上于恒温培养箱中避光发芽5d,每隔24h用消毒后的蒸馏水润洗数次并更换滤纸,发芽结束后取冻干粉碎后样品,分别测定总硒、有机硒、白藜芦醇含量,检测结果为总硒量 9.462μg/g,有机硒含量6.33μg/g,白藜芦醇含量37.02μg/g;
(2)富硒花生芽汁的制备
取富硒发芽结束后的鲜花生芽,去红衣,按花生芽/牛奶(M/V)1:2的比例榨汁,混匀过单层灭菌纱布,取匀浆。
实施例4
一种利用实施例1或实施例2筛选的富硒乳酸菌和实施例3获得的花生芽汁制备富含白藜芦醇和有机硒酸奶的方法,主要包含以下步骤:
(1)菌株的活化:在无菌操作条件下,将德氏乳杆菌保加利亚亚种(BNCC336436或BNCC186626或BNCC138500或BNCC187941)和嗜热链球菌(BNCC187932 或BNCC186629或BNCC175966或BNCC175937)、植物乳杆菌植物亚种(PDD-1)、权利要求1所述的发酵乳杆菌分别接种于MRS液体培养基中,分别在35~42℃的培养箱中培养24~36h;
(2)乳酸菌发酵剂的制备:将活化后的德氏乳杆菌保加利亚亚种和嗜热链球菌按1:1的比例进行混合的混合菌种、植物乳杆菌植物亚种和发酵乳杆菌在无菌操作条件下,分别按一定接种量接种于在经巴氏杀菌后冷却至37~40℃的纯牛奶中,在34~40℃保温箱中培养3~6h,制成第一发酵剂、第二发酵剂和第三发酵剂;
(3)富硒花生芽牛奶液的制备:按体积比添加富硒花生芽汁30%~50%(富硒花生芽汁/牛奶=3:7~1:1)和蔗糖 4~8%,混合均匀,制成富硒花生芽牛奶液;
(4)接种发酵剂发酵:将富硒花生芽牛奶液进行杀菌处理后,按质量或体积比添加第一发酵剂5~8%、第二发酵剂1%~4%和第三发酵剂1%~5%,充分搅拌使其混合均匀后,置于发酵箱内,于温度为32~39℃,发酵处理4~8h得到发酵液;
(5)酸奶的成熟:将步骤(4)得到的发酵液移入4~8℃冰箱或冷藏室内,经6~12h发酵成熟后,制得富含白藜芦醇和有机硒的风味酸奶制品。
对照组制备方法区别在于以不耐硒的植物乳杆菌植物亚种(PDD-1)代替保藏编号为CGMCC No.17434的发酵乳杆菌,制备得到的酸奶为对照组。酸奶中黄酮苷元含量为0.080-0.093 mg/mL。
实施例5
同上述实施例4,其区别在于:
步骤(3)采用纯牛奶作为发酵液。
对照组制备方法区别在于以不加富硒花生芽汁的酸奶做空白对照,以对照组和实验组中分别测得白藜芦醇含量,检测结果为白藜芦醇含量为7.02-10.56μg/mL。
上述说明并非对本发明的限制,本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内,做出的变化、改型、添加或替换,也应属于本发明的保护范围。
Claims (6)
1.一株富硒乳酸菌,其特征在于:该乳酸菌分类命名为发酵乳杆菌(Lactobacillus fermentum)PDD-4株,于2019 年03月25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,保藏编号为CGMCC No.17434。
2.一种权利要求1所述的富硒乳酸菌的筛选方法,其特征在于包括以下步骤:
(1)乳酸菌的初步筛选及分离纯化
挑选传统发酵食品泡菜为样品进行稀释后,涂布于含有溴甲酚紫的MRS固体培养基平板,倒置培养于37~43℃培养箱36~48h,挑选出橙黄色的菌落,将筛选得到的具有典型乳酸菌菌落特征的单一菌落初步鉴定为乳酸菌,将初步确定的乳酸菌菌落在MRS固体平板上连续划线几次,挑取出形态单一的菌落活化后置于甘油保藏管中存放在-80℃冰箱中;
(2)目标菌株的筛选
将步骤(1)初步筛选的乳酸菌菌株经分离纯化后涂布于含有20-100mg/L Na2SeO3的MRS固体培养基平板上,倒置培养于37-43℃培养箱24~36h,挑选出在同等Na2SeO3浓度下菌落颜色呈现出乳白色或微红色的菌落;再涂布于含有20~50mg/L Na2SeO3 的MRS固体培养基平板上,按上述条件培养,挑选出在同等Na2SeO3浓度下菌落颜色呈现出乳白色或微红色的菌落,继续分离纯化至单一菌落,进行革兰氏染色和生理生化特性鉴定,选取革兰氏阳性、H2O2酶为阴性的菌,即为富硒乳酸菌,该菌株分类命名为发酵乳杆菌(Lactobacillus fermentum)PDD-4株,于2019年03月25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,保藏编号为CGMCC No.17434。
3.根据权利要求2所述的一株富硒乳酸菌的筛选方法,其特征在于所述的含有溴甲酚紫的MRS固体培养基的配制方法如下:将MRS肉汤52.24g,溴甲酚紫0.04g,琼脂粉0.1g~0.2g,溶于1L蒸馏水中,于121℃灭菌20min即可;所述的含有Na2SeO3的MRS固体培养基的配制方法如下:将MRS肉汤52.24g,琼脂粉0.1g~0.2g,溶于1L蒸馏水中,于121℃灭菌20min,待培养基冷却至55℃时,加入一定量Na2SeO3灭菌培养液,即可配制成所需浓度的固体培养基。
4.一种富含白藜芦醇和有机硒的风味酸奶的制备方法,其特征在于包括以下步骤:
(1)富硒花生芽汁的制备
选取颗粒、大小一致、成熟饱满的花生种子,用75%乙醇浸泡消毒 15min后,用灭菌后的蒸馏水反复冲洗3遍,将消毒冲洗后的种子至于亚硒酸钠溶液中恒温浸泡后,置于双层滤纸平皿上于恒温培养箱中避光发芽5d,每隔24h用消毒后的蒸馏水润洗数次并更换滤纸,取富硒发芽结束后的鲜花生芽,按花生芽和牛奶质量体积比1g:2ml的比例榨汁,混匀过单层灭菌纱布,取匀浆,得到富硒花生芽汁;
(3)花生芽牛奶液的制备:在牛奶中按体积比添加富硒花生芽汁和蔗糖,混合均匀,制成富硒花生芽牛奶液,其中富硒花生芽牛奶液中富硒花生芽汁体积百分数为30%~50%,蔗糖体积百分数为 4~8%;
(4)菌株的活化:在无菌操作条件下,将德氏乳杆菌保加利亚亚种和嗜热链球菌、植物乳杆菌植物亚种PDD-1和权利要求1所述的发酵乳杆菌分别接种于MRS液体培养基中,分别在35~42℃的培养箱中培养24~36h;
(5)乳酸菌发酵剂的制备:将活化后的德氏乳杆菌保加利亚亚种和嗜热链球菌按1:1的比例进行混合的混合菌种、植物乳杆菌植物亚种PDD-1和发酵乳杆菌在无菌操作条件下,分别按一定接种量接种于在经巴氏杀菌后冷却至37~40℃的纯牛奶中,在34~40℃保温箱中培养3~6h,相应制成第一发酵剂、第二发酵剂和第三发酵剂;
(6)接种发酵剂发酵:将富硒花生芽牛奶液进行杀菌处理后,按质量或体积比添加第一发酵剂5~8%、第二发酵剂1~4%和第三发酵剂1~5%,充分搅拌使其混合均匀后,置于发酵箱内,于温度为32~39℃,发酵处理4~8h得到发酵液;
(7)酸奶的成熟:将步骤(5)得到的发酵液移入4~8℃冰箱或冷藏室内,经6~12h发酵成熟后,制得富含白藜芦醇和有机硒的风味酸奶制品。
5.权利要求4所述的一种富含白藜芦醇和有机硒的风味酸奶的制备方法,其特征在于:所述的植物乳杆菌植物亚种(Lactobacillus plantarum subsp. plantarum)PDD-1株,于2018 年06月19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,保藏编号为CGMCC No.15953。
6.权利要求4所述的一种富含白藜芦醇和有机硒的风味酸奶的制备方法,其特征在于:所述的发酵乳杆菌(Lactobacillus fermentum)PDD-4株,于2019 年03月25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,保藏编号为CGMCC No.17434。
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