CN114885983B - 一种产生风味成分的乳酸菌发酵工艺 - Google Patents
一种产生风味成分的乳酸菌发酵工艺 Download PDFInfo
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Abstract
本发明公开了一种产生风味成分的乳酸菌发酵工艺，属于乳酸菌发酵技术领域，包括以下步骤：S1、往牛奶或者奶粉复原乳中接种乳酸菌在适宜的温度下开始发酵，间隔一定时间连续测定发酵过程中产生的乙醛和丙酮酸的含量，直到发酵终止；S2、根据测定的丙酮酸的含量制作丙酮酸随时间变化的曲线，记录该批次发酵发酵液中丙酮酸的含量逐渐积累的起始时间点；S3、进行另外一批次乳酸菌发酵，保持发酵条件相同，从S2中得到的丙酮酸的含量逐渐积累的起始时间点开始，在发酵液中添加丙酮酸脱羧酶，使积累的丙酮酸转变成乙醛成分。本发明提供的乳酸菌发酵工艺，能够在发酵完成后获得浓郁的青苹果风味，使得风味地道，口感好，且步骤简便，易于操作。
Description
技术领域
本发明涉及乳酸菌发酵技术领域，具体涉及一种产生风味成分的乳酸菌发酵工艺。
背景技术
糖酵解在细胞溶胶中进行，是葡萄糖经过酶催化作用降解成丙酮酸，并伴随生成NADH的过程，它是动物、植物和微生物细胞中葡萄糖分解的共同代谢途径。如果供氧不足，NADH不会进入呼吸链，而是在有些生物中把丙酮酸还原成乳酸，在有些生物中丙酮酸会转变为乙醇，并释放出二氧化碳，这是由于不同的生物细胞中含有的酶种类不一样。
乳酸菌发酵因菌种不同，代谢途径不同，生成的代谢产物不同，将乳酸发酵分为同型乳酸发酵、异型乳酸发酵和双歧杆菌发酵。其中，同型乳酸发酵是指嗜酸乳杆菌(L.acidophilus)、德氏乳杆菌(Lnc.delbriikii)等乳酸杆菌在无氧条件下利用葡萄糖经糖酵解途径生成乳酸的过程，因为乳酸杆菌大都没有丙酮酸脱羧酶，所以糖酵解途径产生的丙酮酸就不能通过脱羧作用而生成乙醛，只能在乳酸脱氢酶的催化作用下以丙酮酸作为受氢体，发生还原反应而生成乳酸，异型乳酸发酵是葡萄糖经HMP途径发酵后除主要产生乳酸外还产生乙醇、乙酸、二氧化碳等多种产物的发酵。
酸奶是一种以牛奶为原料， 经发酵后获得的一种发酵乳制品。酸奶具有独特的风味， 其风味物质包括挥发性物质和非挥发性物质。在挥发性物质中，羰基化合物是发酵乳的主要风味成分之一，其中乙醛是所有羰基化合物中对发酵乳风味产生的最关键因素，乙醛是发酵乳发酵过程中含量要远远高于其他挥发性芳香化合物，是发酵乳中重要的特征风味化合物。
但是乳酸菌代谢途径中有分支代谢途径，最后发酵得到的发酵乳中乙醛的含量比较低，导致发酵乳的青苹果风味还不是很浓郁，市场上的青苹果风味发酵乳制品品类繁多，青苹果的滋味大多都是添加天然青苹果香精或青苹果浓缩汁而来的，并不是发酵过程产生的风味成分形成的，而且风味不是很地道，口感不好。
发明内容
为了克服现有技术的不足，本发明的目的在于提供一种产生风味成分的乳酸菌发酵工艺，该发酵工艺能够在发酵完成后获得浓郁的青苹果风味，使得风味地道，口感好，且步骤简便，易于操作。
本发明的目的采用如下技术方案实现：
一种产生风味成分的乳酸菌发酵工艺，包括以下步骤：
S1、往牛奶或者奶粉复原乳中接种乳酸菌，并在适宜的温度下进行发酵，然后间隔一定时间连续测定发酵过程中产生的乙醛和丙酮酸的含量，直到发酵终止；
S2、根据测定的所述丙酮酸的含量制作丙酮酸随时间变化的曲线，记录该批次发酵发酵液中丙酮酸的含量开始积累的起始时间点；
S3、根据S1，进行另外一批次乳酸菌发酵，保持发酵条件相同，然后从所述S2中得到的所述丙酮酸的含量开始积累的起始时间点开始，在发酵液中添加丙酮酸脱羧酶，使积累的丙酮酸转变成乙醛成分，提高发酵终止后的发酵液中乙醛的含量。
进一步地，所述发酵终止后的发酵液中乙醛的含量为23～41 mg/L。
进一步地，所述S3中，进行多批次乳酸菌发酵，每批次发酵条件相同，从所述丙酮酸的含量开始积累的起始时间点开始，在多批次发酵液中，分别添加一定梯度含量的所述丙酮酸脱羧酶，在所述多批次乳酸菌发酵完成后，分别检测每批次发酵样品中乙醛的含量。
进一步地，进行10批次乳酸菌发酵，所述丙酮酸脱羧酶的酶量梯度设定为5U、10U、15U、20U、25U、30U、35U、40U、45U、50U，在10批次发酵完成后，分别检测所述乙醛的含量。
进一步地，所述S1中，发酵液的pH为4.2～4.5时，发酵终止。
进一步地，所述S1中，往牛奶或者奶粉复原乳中接种乳酸菌，并在42℃~45℃下开始发酵。
进一步地，所述S1中，然后间隔3～5h连续测定发酵过程中产生的乙醛和丙酮酸的含量，直到发酵终止。
进一步地，所述乳酸菌为保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、德氏乳杆菌中的一种或两种以上的组合物。
相比现有技术，本发明的有益效果在于：
（1）本发明提供的一种产生风味成分的乳酸菌发酵工艺，保持发酵条件不变，确定发酵发酵液中丙酮酸的含量开始积累的起始时间点，通过在该时间点在发酵液中添加丙酮酸脱羧酶，使积累的丙酮酸转变成乙醛成分，提高发酵终止后的发酵液中乙醛的含量，能够在发酵完成后获得浓郁的青苹果风味，使得风味地道，口感好，且步骤简便，易于操作，最终制备得到的发酵乳制品，发酵乳制品中乙醛含量高，青苹果风味浓郁，口感好。
（2）将发酵终止后的发酵液中乙醛的含量控制为23～41 mg/L，可以降低发酵液中乙醛的含量偏高带来的一种不愉快的味道，使风味更地道。
附图说明
图1是本发明提供的以吸光值为纵坐标、丙酮酸溶液浓度为横坐标建立的丙酮酸标准曲线。
具体实施方式
下面，结合具体实施方式，对本发明做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
实施例1
本实施例提供的一种产生风味成分的乳酸菌发酵工艺，包括以下步骤：S1、发酵开始后间隔4h连续测定发酵过程中产生的乙醛和丙酮酸的含量，直到发酵终止；
S2、根据测定的丙酮酸的含量制作丙酮酸的随时间的变化曲线，记录该批次发酵过程中丙酮酸的含量开始积累的时间点；
S3、从丙酮酸的含量开始积累的时间点开始，在发酵液中添加丙酮酸脱羧酶，使积累的丙酮酸转变成乙醛成分，使发酵终止后的发酵液中乙醛的含量高。
实施例2丙酮酸的含量测定
本实施例采用DNP法测定发酵液中丙酮酸含量，具体方法如下：
2,4-二硝基苯肼是重要的羰基试剂，可以与醛、酮和酮酸发生反应，生成黄色或红色的苯腙。丙酮酸能与2,4-二硝基苯肼结合，生成丙酮酸-2,4-二硝基苯腙，在碱性条件下丙酮酸-2,4-二硝基苯踪呈棕色，其在439 nm-530 nm都存在较强的吸收光谱，可用于测定丙酮酸的含量。
（1）DNP法标准曲线
首先准确称取2.5g丙酮酸，充分溶解于去离子水中，定容至250mL，配置成10g/L的标准丙酮酸溶液，必须当天配置当天使用，然后按照表1配制不同浓度的丙酮酸标准溶液：
表1 丙酮酸标准溶液
| 编号 | 0 | 1 | 2 | 3 | 4 | 5 |
| 丙酮酸溶液浓度（g/L） | 0.0 | 2 | 4 | 6 | 8 | 10 |
| 10g/L的标准丙酮酸体积（mL） | 0.0 | 2 | 4 | 6 | 8 | 10 |
| 定容至（mL） | 10.0 | 10.0 | 10.0 | 10.0 | 10.0 | 10.0 |
试管中依次加入1mL DNP试剂和100μL标准丙酮酸溶液，37℃水浴恒温反应20min，再加入10mL的0.4 moL的NaOH溶液，摇匀，以0号为参照，测520nm处的吸光值，以吸光值OD520nm为纵坐标，丙酮酸溶液浓度为横坐标，建立丙酮酸标准曲线，如附图1所示。标准曲线方程为：C=0.1206×OD520nm-0.0013，线性相关系数为0.9999。
（2）发酵液中丙酮酸溶液浓度的DNP法测定
实施例1提供的每次间隔4h的发酵液样品经10000 rpm高速离心10min后，取上清液，适当稀释，使其浓度处于标准曲线范围内。分别取1mL DNP试剂和100μL的稀释样品加入到试管中，37℃水浴恒温反应20min，再加入10mL的0.4 moL的NaOH溶液，摇匀，以空白为对照，测520nm处吸光值，每次发酵液样品测试重复三次。根据标准曲线方程可计算得样品中的丙酮酸浓度，计算结果如表2所示。
表2 发酵液样品中的丙酮酸浓度测试结果
| 时间 | 4h | 8h | 12h | 16h | 20h | 24h | 28h | 32h | 36h | 40h |
| 丙酮酸浓度（g/L） | 0 | 0 | 2.5±0.2 | 3.6±0.3 | 4.5±0.5 | 5.8±0.2 | 6.5±0.4 | 7.8±0.5 | 8.6±0.5 | 9.5±0.8 |
从表2可以看出从发酵开始后的12h后，发酵液中丙酮酸的含量在积累，可以在该时间点添加一定量的丙酮酸脱羧酶，使积累的丙酮酸转变成乙醛成分，使发酵终止后的发酵液中乙醛的含量高。
实施例3
本实施例提供发酵液中乙醛含量的测定方法，具体方法如下：
精确量取2.0mL 1%的NaHSO3溶液分别加入3个250mL具塞三角瓶中，分别加入实施例1提供的每次间隔4h的发酵液样品10mL，摇匀，在室温下放置1h后，加入1mL 1%淀粉溶液，用0.10mol/L的碘标准溶液滴至接近终点，再用0.01mol/L的碘标准溶液滴定至终点（淡蓝紫色，30s内不褪色），不计数。再加20mL 1mol/L的NaHCO3，振荡混匀至溶液蓝色消失，用0.01mol/L的碘标准溶液滴定至终点，记录消耗的标准碘液的体积，同时做空白试验。
计算公式：乙醛含量（mg/L）=（V1-V2）×C×0.022×106/10
V1-样品滴定消耗I2标准溶液的体积mL；
V2-空白滴定消耗I2标准溶液的体积mL；
C -1/2 I2标准溶液的浓度mol/L；
0.022-乙醛化学反应基本单位 g；
10-发酵液样品的体积mL。
进行10批次乳酸菌发酵，实施例3的发酵条件和实施例2相同，从实施例2发酵开始后的12h时分别在这10批次的发酵液中依次添加5U、10U、15U、20U、25U、30U、35U、40U、45U、50U量的丙酮酸脱羧酶，直到在10批次发酵完成后，分别检测每批次发酵液中的乙醛的含量，每批次发酵液样品的乙醛的含量测定结果如表3所示：
表3 10批次发酵完成后发酵液中的乙醛含量测定结果（n=3，x±SD， mg/L）
| 酶量（U） | 5 | 10 | 15 | 20 | 25 | 30 | 35 | 40 | 45 | 50 |
| 乙醛 | 5.6±0.8 | 8.6±0.5 | 10.5±0.2 | 15.4±0.1 | 18.5±0.2 | 23.0±0.5 | 32.1±0.5 | 41.0±0.6 | 45.2±0.8 | 46.8±0.5 |
实施例4
本实施例中招募20个酸乳爱好者对实施例3提供的10批次发酵生成的酸乳进行品尝，品尝的过程中进行盲评，每品尝一个样品后使用纯净水漱口，然后再进行下一次品尝，并根据个人的喜好度依次评分为1～10分，最后结果取平均分，结果如表4所示。
表4 对10批次乳酸菌发酵完成后的样品进行品尝的评分结果
| 酶量（U） | 5 | 10 | 15 | 20 | 25 | 30 | 35 | 40 | 45 | 50 |
| 评分 | 2.5±0.5 | 4.0±0.2 | 6.8±0.6 | 8.5±0.1 | 9.5±0.2 | 9.8±0.6 | 9.9±0.5 | 9.8±0.6 | 8.2±0.8 | 5.6±0.2 |
从表中可以看出，当酸乳中的乙醛浓度在23.0～41.0 mg/L时，酸乳中青苹果风味越来越浓，给人一种清爽的芬芳味，喜好度增大；但当浓度超过45.2 mg/L时，喜好度反而下降，有一种不愉快的味道。
上述实施方式仅为本发明的优选实施方式，不能以此来限定本发明保护的范围，本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (5)
1.一种产生风味成分的乳酸菌发酵工艺，其特征在于，包括以下步骤：
S1、往牛奶或者奶粉复原乳中接种乳酸菌，并在42℃~45℃下进行发酵，然后间隔3～5h连续测定发酵过程中产生的乙醛和丙酮酸的含量，直到发酵终止，发酵液的pH为4.2～4.5时，发酵终止；
S2、根据测定的所述丙酮酸的含量制作丙酮酸分别随时间变化的曲线，记录该批次发酵液中丙酮酸的含量开始积累的起始时间点；
S3、根据S1，进行另外一批次乳酸菌发酵，保持发酵条件相同，然后从所述S2中得到的所述丙酮酸的含量开始积累的起始时间点开始，在发酵开始12h后，在发酵液中添加丙酮酸脱羧酶，使积累的丙酮酸转变成乙醛成分，提高发酵终止后的发酵液中乙醛的含量，发酵终止后的发酵液中乙醛的含量为23～41mg/L。
2.如权利要求1所述的一种产生风味成分的乳酸菌发酵工艺，其特征在于，所述S3中，进行多批次乳酸菌发酵，每批次发酵条件相同，从所述丙酮酸的含量开始积累的起始时间点开始，在多批次发酵液中，分别添加酶量为5U梯度含量的所述丙酮酸脱羧酶，在所述多批次乳酸菌发酵完成后，分别检测每批次发酵样品中乙醛的含量。
3.如权利要求2所述的一种产生风味成分的乳酸菌发酵工艺，其特征在于，进行10批次乳酸菌发酵，所述丙酮酸脱羧酶的酶量梯度设定为5U、10U、15U、20U、25U、30U、35U、40U、45U、50U，在10批次发酵完成后，分别检测所述乙醛的含量。
4.如权利要求1所述的一种产生风味成分的乳酸菌发酵工艺，其特征在于，所述S1中，所述乳酸菌在牛奶或者奶粉复原乳中的接种量是0.5%～5％。
5.如权利要求1～4任一项所述的一种产生风味成分的乳酸菌发酵工艺，其特征在于，所述乳酸菌为保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、德氏乳杆菌中的一种或两种以上的组合物。
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