CN112868768A - 一种发酵型保健奶醋及其制备方法 - Google Patents
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Abstract
本发明公开了一种发酵型保健奶醋饮品的制备方法,为两阶段发酵,一次发酵为酵母菌和乳酸菌的混菌厌氧发酵产酒精阶段;二次发酵为醋酸菌的好氧发酵产醋酸阶段。本发明所使用的奶醋发酵菌株Saccharomyces cerevisiae S1,30℃发酵22h后,酒精产量保持稳定,保证了发酵过程和发酵产品的稳定性。发酵菌株植物乳杆菌Lactobacillus plantarum RS13,37℃培养24h,能够产胞外粗多糖95.33mg/g,赋予了奶醋抗氧化保健功能;另外乳酸发酵产生了乳酸为主的有机酸,可以抑制腐败菌和致病菌的生长。第一阶段采用酵母菌和乳酸菌混菌发酵,二者不仅可以协同生长,而且代谢产物酒精和有机酸之间可以发生酯化反应,对改善奶醋的风味和香气发挥了重要作用。
Description
技术领域
本发明属于奶制品加工技术领域,具体涉及一种发酵型保健奶醋及其制备方法。
背景技术
牛奶是最古老的天然饮料之一,被誉为白色血液。牛奶中含有丰富的蛋白质、脂肪、维生素和矿物质等营养物质,而且乳蛋白中含有人体所必须的全部氨基酸;乳脂肪多为短链和中链脂肪酸;钾、磷、钙等矿物质配比合理,可以说牛奶是世界各国公认的营养保健食品。目前牛乳的主要加工产品有纯牛奶、酸奶、乳饮料、奶片、奶酪等,而市场上的奶醋产品极其少见。
奶醋是一种新型的发酵饮品,相较于配制型奶醋,发酵型奶醋不仅利用了微生物对大分子营养物质的生物转化,产生了更易人体吸收的小分子营养,而且发酵菌株本身也会产生一些功能活性物质,大大增强了产品的营养保健功能。可以说,奶醋糅合了牛奶、保健醋以及功能活性物质的营养、风味和保健功能,是一种非常有市场潜力的功能性保健饮品,可以满足人们对口味、营养、保健等多层次的需求,对于保健食品产业的发展及人民群众的身体健康具有十分重要的意义。
发明内容
本发明的目的在于提供一种以牛乳为原料,利用活性酵母、富含EPS的乳酸菌和醋酸杆菌进行两阶段发酵工艺制备的抗氧化奶醋保健饮品。
本发明发酵型奶醋的制备工艺为两阶段发酵,一次发酵为酵母菌和乳酸菌的混菌厌氧发酵产酒精阶段;二次发酵为醋酸菌的好氧发酵产醋酸阶段。本发明选择适宜奶醋发酵的菌株:酿酒酵母为S.cerevisiae S1,在1L牛奶中添加蔗糖100-150g的原料中,30℃发酵,酒精产量保持在<8g/100g;乳酸菌为植物乳杆菌L.plantarum RS13,菌株的筛选鉴定参见文献(红树莓酵素发酵过程中优势菌株的分离鉴定;魏东东,常曼曼,阴芳冉,于宏伟,郭润芳,河北农业大学学报),产胞外粗多糖95.33mg/g;醋酸杆菌产酸能力强、最适生长温度为30℃。
本发明公开的一种发酵型保健奶醋饮品的制备方法,按照以下步骤进行:
(1)菌株活化及种子液制备:划取4℃下保存的酿酒酵母,植物乳杆菌,和醋酸菌分别置于YPD,MRS和LB肉汤培养基中,酿酒酵母和醋酸菌于30℃摇床培养24h,植物乳杆菌于37℃静置培养24h;
(2)原料处理:鲜牛奶用四层纱布过滤除去杂质和异物,添加白砂糖,添加量为鲜牛奶质量的15%,搅拌溶解,并置于65℃条件下灭菌30min,冷却至室温,待发酵用;
(3)酒精发酵:酿酒酵母和植物乳杆菌混菌发酵,酿酒酵母与植物乳杆菌的接种比例为2:1,两种菌的接菌量为4%,30℃条件下发酵8h,得到的发酵汁酒精量为5-6;
(4)醋酸发酵:在酒精发酵的基础上,进行醋酸发酵,醋酸菌接菌量为5%、发酵时间为22h、装液量为40%;
(5)发酵后调配:发酵总酸不低于8g/kg为发酵终点,发酵结束后添加甜菊糖、果胶,20MPa均质;
(6)巴杀灭菌:均质后的奶醋于65℃保温30min,进行巴氏杀菌,冷却至室温,即得奶醋成品。
所述步骤(5)中菊糖的添加量为0.15%、果胶的添加量为0.2%。
本发明中,酒精度测定采用蒸馏得到酒***溶液后用酒精计测定;酸度测定采用酸碱滴定法测定;糖度测定使用手持糖度计测定;感官评价、理化指标测定参考GB/T15038-2006。
通过上述技术方案制备的奶醋产品质量特征:
(1)感官指标:奶醋色泽乳白,有奶香味和醋酸味,口感酸甜爽口;
(2)理化指标:总酸TA:8.18g/kg,总糖含量:6.9%,总固形物TSS:20.88Brix。
(3)微生物指标:菌落总数(cfu/mL)≤100;大肠菌群(MPN/100mL)≤3;致病菌:未检出。
有益效果:(1)本发明提供的奶醋发酵菌株S.cerevisiae S1,在1L牛奶中添加蔗糖100-150g,30℃发酵22h后,酒精产量保持稳定,始终维持在<8g/100g,保证了发酵过程和发酵产品的稳定性。
(2)本发明提供的奶醋发酵菌株植物乳杆菌L.plantarum RS13,37℃培养24h,能够产胞外粗多糖95.33mg/g,赋予了奶醋抗氧化保健功能;另外乳酸发酵产生了乳酸为主的有机酸,可以抑制腐败菌和致病菌的生长,保证产品的品质。
(3)本发明提供的发酵工艺中,第一阶段采用酵母菌和乳酸菌混菌发酵,二者不仅可以协同生长,而且代谢产物酒精和有机酸之间可以发生酯化反应,对改善奶醋的风味和香气发挥了重要作用。
附图说明
图1为不同蔗糖添加量的牛乳中总糖含量及产物酒精含量的变化图;
图2为乳酸菌产胞外多糖的菌株筛选图;
图3为甜菊糖添加量对发酵型奶醋感官得分及糖酸比的影响图;
图4为牛乳发酵前后抗氧化性比较图。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
实施例1酵母菌及醋酸菌的选择
1.奶醋发酵工艺流程
2.适合奶醋发酵的酵母菌筛选及酒精生成动力学
分别挑取于斜面保存的酵母菌,接种于YPD肉汤培养基中,30℃培养12h进行活化,然后按照2%的接菌量接种于牛奶基质中,30℃条件下培养48h,根据表1中色泽、口感、组织状态、风味、酒精含量等指标,可以看出,酵母菌S.cerevisiae S1菌株为最适合菌株,然后进行下一步的动力学试验。
表1感官分析分值与产乙醇量
将活化24h的S.cerevisiae S1和菌液按照2%接菌量接种于不同初始蔗糖添加量的(0,50,100,150g/L)的牛乳,30℃条件进行无氧发酵。从0h开始,每隔8h取样,测定发酵牛乳中酵母菌的底物蔗糖的含量及产物酒精的体积分数。结果如图1所示,发酵过程中的底物总糖的含量随着发酵时间的延长均逐渐降低,而酒精含量逐渐升高,说明底物糖被消耗,转化生成酒精。添加100-150g/L的蔗糖,酒精产量变化不大,发酵40h后酒精含量均维持在8%以下,确定蔗糖添加量为150g/L。
3.乳酸菌胞外多糖的含量
挑取斜面生长的供试乳酸菌,接种于液体MRS培养基,37℃培养24h进行活化,并以2%的比例转接到100mL的液体MRS中,37℃培养24h。发酵液室温条件下2000r/min离心5min。倒掉上清液,往下层菌体里面加入45%苯酚水溶液(45%苯酚加热状态下易分离,每次加入样品中时需摇匀),使固液比为1:30。微波炉加热5min,60℃浸提60min,于75℃下减压浓缩。浓缩液加5倍体积的95%的乙醇醇沉。4℃下,静置过夜。倒掉上清液,收集沉淀,金属浴干燥60min,称干重,即得胞外多糖。结果如图2所示,植物乳杆菌L.plantarum RS13菌株产生的胞外多糖含量最高,因此确定该菌株为适宜的发酵菌株。
实施例2奶醋发酵条件的优化
1.奶醋发酵第一阶段的酒精发酵条件优化
在确定了牛奶基质中添加150g/L蔗糖,发酵温度为30℃的基础上,根据Box-Benhnken Design(BBD)设计法对影响酒精产量和感官的菌株复合比例、接菌量以及发酵时间这三个因素,利用软件Design-Exper 8.0.6进行三因素三水平设计和实验,响应面试验设计及响应值结果如表2-4所示。
表2发酵基质Box-Benhnken中心组合设计表
表3发酵基质响应面试验设计及结果
表4回归模型方差分析及显著性分析
根据数据进行多元拟合回归分析,分别建立酒精量(Y1)及感官得分(Y2)对菌液配比(A)、接菌量(B)和发酵时间(C)的回归模型,回归方程:Y1=5.06+0.22A+0.36B+0.84C-0.050AB-0.60AC-0.075BC-0.52A2-1.49B2-0.093C2
Y2=87.00+1.00A-4.88B-11.13C+6.00AB+4.5AC-1.75BC-9.63A2-4.87B2-1.38C2
回归方程决定系数R12=0.9272,R22=0.9305。
根据方差分析和方程分析,无论是酒精量还是感官响应值,模型均表现为P<0.01,说明模型极显著,而且失拟项P>0.05,说明失拟项不显著,因此,酒精量和感官预测模型结构合理。另外,A2和B2对Y1影响显著,表明发酵条件对响应值非线性关系,并且分析各参数F值可得知FC>FB>FA,因此各参数对第一阶段的发酵影响大小依次为:发酵时间>接菌量>菌株配比。
响应面结果预测的第一阶段的最优发酵条件为:酵母菌:植物乳杆菌=2:1、混合接菌量4.08%、发酵时间为8.12h,酒精量可达到5.50%,感官得分可为84.14分。考虑到实际操作,将发酵条件参数调整为酵母菌:植物乳杆菌=2:1、混合接菌量4%、发酵时间为8h,得到发酵汁酒精量为5.68,感官评分86,酒精量与预测的结果相差3.3%,感官评分与预测值相差2.2%,结果表明实际操作结果与预测值接近。
2.奶醋发酵的第二阶段醋酸发酵条件优化
在第一阶段发酵的基础上,根据Box-Benhnken Design(BBD)设计法对影响醋酸产量和感官的装液量、接菌量以及发酵时间这三个因素,利用软件Design-Exper 8.0.6进行三因素三水平设计和实验,响应面试验设计及响应值结果如表5-7所示。
表5发酵条件Box-Benhnken中心组合设计表
表6发酵基质响应面试验设计及结果
利用软件对数据进行多元拟合回归分析,分别建立总酸含量(Y1)及感官得分(Y2)对接菌量(A)、发酵时间(B)和装液量(C)的回归模型,回归方程:
Y1=+8.24+0.31A+0.94B-0.22C-0.086AB-0.33AC-0.094BC-1.32A2-0.28B2-0.32C2
Y2=+89.00+1.75A+5.00B-4.25C-5.00AB+0.50AC+0.50BC-9.50A2-5.00B2-1.50C2
回归方程决定系数R12=0.9163,R22=0.9084
表7回归模型方差分析及显著性分析
根据方差分析和方程分析,无论是酒精量还是感官响应值,模型均表现为P<0.01,说明模型极显著,而且失拟项P>0.05,说明失拟项不显著,因此,酒精量和感官预测模型结构合理。另外,A2和B2对Y1影响显著,表明发酵条件对响应值非线性关系,并且分析各参数F值可得知FB>FC>FA,因此各参数对发酵型奶醋的影响大小依次为:发酵时间>装液量>接菌量。
根据响应面结果得到发酵奶醋的预测最优条件为:醋酸菌接菌量为5.01%、发酵时间为22.06h、装液量为40%,预测总酸可达到8.43g/kg,感官得分可为90.03,并且具有可行性。为了便于实际操作将发酵条件参数设置为醋酸菌接菌量为5%、发酵时间为22h、装液量为40%,得到发酵奶醋中总酸为8.182g/kg,感官评分88。设计三次重复试验以验证预测条件的可行性及有效性。结果总酸量与预测的结果相差仅2.9%,感官得分与预测值相差2.3%,结果表明实际操作结果与预测值接近。
实施例3调配
发酵结束后,对发酵产品进行调配,主要是进行糖酸比以及稳定性调节。结果如图3所示,甜菊糖添加量为0.15-0.20%时,感官得分88分以上,说明此时风味好,口感容易被接受,综合考虑菊糖添加量设为0.15%。
四种稳定剂CMC-Na、瓜尔豆胶、黄原胶和果胶添加量对发酵型奶醋稳定性的影响,结果如表8所示。综合持水性和感官考虑,在奶醋中添加0.2%的果胶有利于产品品质的提高。
表8稳定剂对发酵型奶醋品质的影响
实施例4产品性质检测
1.产品理化指标
(1)感官指标:奶醋色泽乳白,有奶香味和醋酸味,口感酸甜爽口;
(2)理化指标:总酸TA:8.18g/kg,总糖含量:6.9%,总固形物TSS:20.88Brix。
(3)微生物指标:菌落总数(cfu/mL)≤100;大肠菌群(MPN/100mL)≤3;致病菌:未检出。
2.奶醋的抗氧化性能
如图4所示,牛乳经两次发酵过程,总抗氧化性能力都显著增强(P<0.01),其中发酵型奶醋总还原力是未发酵乳的提高了1.71倍,ABTS自由基清除能力提高了0.49倍,DPPH自由基清除能力提高了0.62倍。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (3)
1.一种发酵型保健奶醋饮品的制备方法,其特征在于,按照以下步骤进行:
(1)菌株活化及种子液制备:划取4℃下保存的酿酒酵母S.cerevisiae S1,植物乳杆菌L.plantarum RS13,和沪酿1.01号醋酸菌分别置于YPD,MRS和LB肉汤培养基中,酿酒酵母和醋酸菌于30℃摇床培养24h,植物乳杆菌于37℃静置培养24h;
(2)原料处理:鲜牛奶用四层纱布过滤除去杂质和异物,添加白砂糖,添加量为鲜牛奶质量的15%,搅拌溶解,并置于65℃条件下灭菌30min,冷却至室温,待发酵用;
(3)酒精发酵:酿酒酵母和植物乳杆菌混菌发酵,酿酒酵母与植物乳杆菌的接种比例为2:1,两种菌的接菌量为4%,30℃条件下发酵8h,得到的发酵汁酒精量为5-6;
(4)醋酸发酵:在酒精发酵的基础上,进行醋酸发酵,醋酸菌接菌量为5%、发酵时间为22h、装液量为40%;
(5)发酵后调配:发酵总酸不低于8g/kg为发酵终点,发酵结束后添加甜菊糖、果胶,20MPa均质;
(6)巴杀灭菌:均质后的奶醋于65℃保温30min,进行巴氏杀菌,冷却至室温,即得奶醋成品。
2.根据权利要求1所述一种发酵型保健奶醋饮品的制备方法,其特征在于,所述步骤(3)中酿酒酵母为S.cerevisiae S1,所述植物乳杆菌为L.plantarum RS13。
3.根据权利要求1所述一种发酵型保健奶醋饮品的制备方法,其特征在于,所述步骤(5)中菊糖的添加量为0.15%、果胶的添加量为0.2%。
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