CN114836342A - 一种乳酸菌gs-3及其在富硒中的应用 - Google Patents
一种乳酸菌gs-3及其在富硒中的应用 Download PDFInfo
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- CN114836342A CN114836342A CN202210411911.8A CN202210411911A CN114836342A CN 114836342 A CN114836342 A CN 114836342A CN 202210411911 A CN202210411911 A CN 202210411911A CN 114836342 A CN114836342 A CN 114836342A
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- selenium
- lactcaseibacillus
- chiayiensis
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- lactic acid
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Abstract
本发明涉及微生物技术领域，具体涉及一种乳酸菌GS‑3及其在富硒中的应用。本发明提供的乳酸菌(Lacticaseibacillus chiayiensis)GS‑3，其保藏编号为CGMCC No.24264。本发明的乳酸菌GS‑3，其细胞生物量大，有机硒的转化效率高，合成的有机硒能被生物体有效的转化吸收和利用，可用于生产富硒农产品、富硒蛋奶、饲料添加剂和人体富硒补充剂等。
Description
技术领域
本发明涉及微生物技术领域，具体涉及一种乳酸菌GS-3及其在富硒中的应用。
背景技术
硒被医药界和营养学界尊称为“生命的火种”，享有“长寿元素”、“抗癌之王”、“心脏守护神”“天然解毒剂”等美誉。硒在人体组织内含量为千万分之一，但它却决定了生命的存在，对人类健康的巨大作用是其他物质无法替代的。缺硒会直接导致人体免疫能力下降，临床医学证明，威胁人类健康和生命的四十多种疾病都与人体缺硒有关，如癌症、心血管病、肝病、白内障、胰脏疾病、糖尿病、生殖系统疾病等等。据专家考证，人需要终生补硒。无论是动物实验还是临床实践，都说明了应该不断从饮食中得到足够量的硒，不能及时补充，就会降低祛病能力。
硒是人和动物体内一种重要的微量元素，在人和动物的生长、免疫、繁殖、抗氧化以及激素代谢中有着重要作用。其功能的发挥主要通过参与合成硒蛋白参与动物体的代谢，目前发现的硒参与合成的蛋白有30多种，是哺乳动物体内许多酶活性中心的必需组分，如谷胱甘肽过氧化物酶、脱碘酶等，可防止因氧化而引起的衰老、组织硬化、最少也可以减慢其变化的速度；并且它还具有活化免疫系统、预防癌症的功效，是必要的微量元素。硒参与构成很多酶类，特别是谷胱甘肽过氧化酶，可以保护细胞和组织，维持其正常功能。硒还有解毒功效，因为硒和金属有很好的亲和性。硒有很强的抗氧化性，具有保护心血管和抗肿瘤的作用。对甲状腺激素的调节作用；维持正常免疫功能；抗艾滋病作用；以及维持正常生育功能。
硒有三种形态，一种是无机硒，如硒酸钠和亚硒酸钠；另一种是有机硒，比如硒代蛋氨酸和硒代半胱氨酸；第三种是硒单质。另外，不同形式的硒，其毒性不一样，无机硒、硒代氨基酸和硒单质其毒性大小关系为亚硒酸盐＞硒代半胱氨酸＞硒酸盐≈硒代蛋氨酸＞单质硒。有机硒的补硒效率高，更容易被机体转化吸收利用，且毒性比无机硒低1000倍左右，因而有机硒作为补充硒源更具有应用和推广价值。
当前常用的补硒方法是日粮中添加亚硒酸钠，但无机硒利用率低，毒性大，对环境有潜在污染。有机硒的生物利用率高，毒性小，适口性好，能显著提高动物组织、奶和蛋中的硒水平，提高动物的生产性能。目前市场上广泛使用的是富硒酵母，但富硒酵母存在破壁较难，许多有机硒不能被有效释放，另外其在转化过程中会有12％-15％左右的无机硒残留。因而，采用更容易破壁且对人体有益的益生菌作为有机硒源已成为该研究领域的重要发展方向。
乳酸菌作为一种常用的微生态制剂菌种，可以通过细菌代谢，将无机硒转化成有机硒，更有利于动物体的吸收利用，在动物饲养方面有着非常好的研究前景。富硒乳酸菌相较于富硒酵母菌的优势即乳酸菌大量生长繁殖产氨基酸、有机酸等营养代谢物质可促进宿主的消化和营养吸收，其中有机酸还能使钙、铁、磷等元素处于离子状态，提高其吸收利用率，而酵母难以破壁，成本和消化吸收不高。硒和乳酸菌两者相辅相成，协同作用，其具有安全、发酵周期短、合成效率高、工艺简单等巨大的优势和吸引力，堪称优良的富硒发酵载体。
发明内容
本发明的目的是提供一种高活性的富硒乳酸菌株，并将其应用于功能性制品的研发。
为了实现本发明的目的，本发明的技术方案如下：
本发明提供的乳酸菌(Lacticaseibacillus chiayiensis)GS-3，该菌株于2022年1月7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(简称CGMCC，地址：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，邮编100101)，分类命名为Lacticaseibacillus chiayiensis，保藏编号为CGMCC No.24264。
进一步地，本发明还提供乳酸菌(Lacticaseibacillus chiayiensis)GS-3的发酵产物。含有乳酸菌(Lacticaseibacillus chiayiensis)GS-3或发酵产物的菌剂。
本发明所述的菌剂可以为液体菌剂或固体菌剂，可采用常规技术手段、加入微生物制剂领域允许的辅料制备得到。
本发明还提供发酵培养上述乳酸菌(Lacticaseibacillus chiayiensis)GS-3的方法，其采用含有亚硒酸钠的MRS培养基对上述乳酸菌(Lacticaseibacilluschiayiensis)GS-3进行发酵。
本发明中，MRS培养基，每升培养基包括如下成分：蛋白胨10g，牛肉浸粉5g，酵母浸粉4g，葡萄糖20g，磷酸氢二钾2g，柠檬酸三铵2g，醋酸钠5g，硫酸镁0.2g，硫酸锰0.05g，琼脂15g，吐温801g；pH值6.2±0.2。
本发明培养方法中，接种量为4.8-5.2％。
发酵温度为：36-38℃，发酵时间为58-62小时。
优选，接种量为5％；发酵温度为37℃，亚硒酸钠的添加量为4.8-5.2mM(更优选为5mM),发酵时间为60小时。
本发明通过实验证明，乳酸菌(Lacticaseibacillus chiayiensis)GS-3可以高效生产有机硒，基于此，本发明另提供上述乳酸菌(Lacticaseibacillus chiayiensis)GS-3或发酵产物或菌剂或方法在以下任一方面中的应用：
(1)发酵生产有机硒；
(2)生产富硒农产品；
(3)生产富硒补充剂；
(4)生产饲料添加剂；
(5)生产提升人体的免疫力，抵抗肠道氧化应激，保护肠道氧化还原稳态，抑制肠道氧化损伤的产品。
本发明再提供一种发酵生产有机硒的方法，其包括以上述乳酸菌(Lacticaseibacillus chiayiensis)GS-3进行发酵的步骤。
优选，所述发酵的条件如上所述。
本发明的有益效果在于：
本发明提供了一株富硒能力较好的乳酸菌菌株GS-3，该菌株不仅可以合成生物有机硒，且有机硒转化率较高。该菌株产生的生物有机硒，结合多糖、蛋白质和多肽成分可高效提高动物和人体的免疫力，抵抗肠道氧化应激，保护肠道氧化还原稳态，抑制肠道氧化损伤，效果显著优于目前在种养殖行业广泛使用的无机亚硒酸钠，可以用于生产富硒农产品、富硒蛋奶和人体富硒补充剂等。
另外，通过发酵测试发现GS-3细胞生物量大，有机硒的转化效率高，无机硒残留量低，显著低于酵母富硒的无机硒残留量，且细胞很容易破壁，合成的有机硒能被生物体有效的转化吸收和利用。
本发明的富硒乳酸菌GS-3发酵制剂还可用于微生态饲料添加剂，是良好的有机硒补充载体。
附图说明
图1为本发明乳酸菌菌株GS-3不同接种条件下的生长曲线。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
本实施例提供了一株富硒能力较好的乳酸菌菌株GS-3，并对其培养条件进行了优化。
1.选用的菌种
本发明所选用的菌种GS-3是从发酵饮品中自行分离到的，经序列扩增、比对鉴定为Lacticaseibacillus chiayiensis。保藏编号为：CGMCC 24264。
2.菌株接种量的确定
将GS-3分别以3％和5％的接种量接种到2升MRS液体培养基中，于37℃静置培养，每隔3h取样计数(结果见表1)，以培养时间为横坐标，相对应的菌数对数值为纵坐标，绘制生长曲线，见图1。通过生长曲线测定可知，按5％接种培养后细胞生物量较大，且菌株GS-3在0至10h快速增长，处于对数生长期；10至20h生长速度开始减缓但仍在增长，24h时生长达到最大值，之后呈下降趋势。
表1不同接种条件下的OD值
3.硒添加量的确定
将灭菌的亚硒酸钠Na2SeO3添加到2升MRS培养液中，以5％接种37℃培养24小时后，测定不同浓度下的菌液OD值，观察GS-3的生长情况和耐受性，结果见表2。Na2SeO3浓度在1至5毫摩尔范围内时，菌液浓度变化不大，这说明，在此范围内，细菌表现为硒耐受，硒的添加并未影响菌种的生长，同时，菌液逐渐变红，细菌在代谢过程中将无机硒转化成有机硒的能力并未受到抑制。随着培养液硒浓度的增加，菌液OD值减小，活菌数下降，逐渐表现为硒不耐受。硒浓度在5毫摩尔以上时随着硒添加量的增加，菌体生长受到抑制，同时转化硒的能力下降，故选取5毫摩尔作为亚硒酸钠添加浓度。
表2不同硒浓度下的OD值
| Na<sub>2</sub>SeO<sub>3</sub>浓度(mM) | OD<sub>600</sub>(Abs) |
| 0 | 24.26 |
| 2 | 24.35 |
| 4 | 24.19 |
| 5 | 24.32 |
| 6 | 19.58 |
| 8 | 16.04 |
| 10 | 12.84 |
4.硒含量的测定
将灭菌的亚硒酸钠Na2SeO3溶液按5毫摩尔每升的浓度添加到MRS培养液中，以5％接种、37℃恒温发酵100升，在不同时间点取样测定各生长时期的硒含量。依据GB5009.268-2016方法进行硒的测定，检测结果可知，发酵上清液中的硒含量逐渐降低，菌体细胞中的硒含量逐渐增加。培养液中的亚硒酸钠逐渐被菌吸收转化利用，富集到菌体内转化成有机硒，随细胞数增长有机硒含量增加。
具体结果见表3-表4。
表3上清液中硒含量的测定结果
| 培养时长(h) | Se(mg/L) |
| 0 | 259.9 |
| 12 | 105.4 |
| 24 | 85.8 |
| 48 | 70.6 |
| 55 | 61.4 |
| 60 | 43.8 |
| 66 | 33.3 |
| 72 | 27.0 |
| 84 | 23.5 |
| 96 | 34.1 |
表4菌体中硒含量的测定结果
| 培养时长(h) | Se(mg/kg) |
| 12 | 10017.6 |
| 24 | 16715.4 |
| 48 | 19179.9 |
| 55 | 21814.5 |
| 60 | 22495.1 |
| 66 | 20888.5 |
| 72 | 18940.3 |
| 84 | 17890.9 |
| 96 | 17405.0 |
5.发酵培养时长的确定
根据表3-表4发酵检测结果可知，随着培养时间的延长，发酵上清液中的硒逐渐被菌体吸收利用，细胞富硒量逐渐提升，培养到60h时，菌体富硒量达到最大。结合生长情况，往后随着培养时间延长，菌体细胞出现裂解，一部分硒释放到培养基中，细胞富硒量减少。综合考虑，选取发酵60h作为富硒乳酸菌发酵培养时长。
最终，由上述结果本发明确定的乳酸菌菌株GS-3的富硒发酵生产最优条件为：
培养基：MRS；
接种量：5％；
培养温度：37℃；
亚硒酸钠添加浓度：5mM；
发酵时长：60h。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (10)
1.乳酸菌(Lacticaseibacillus chiayiensis)GS-3，其特征在于，保藏编号为CGMCCNo.24264。
2.权利要求1所述的乳酸菌(Lacticaseibacillus chiayiensis)GS-3的发酵产物。
3.一种菌剂，其特征在于，含有权利要求1所述的乳酸菌(Lacticaseibacilluschiayiensis)GS-3或权利要求2所述的发酵产物。
4.一种发酵培养权利要求1的乳酸菌(Lacticaseibacillus chiayiensis)GS-3的方法，其特征在于，采用含有亚硒酸钠的MRS培养基对权利要求1的乳酸菌(Lacticaseibacillus chiayiensis)GS-3进行发酵。
5.根据权利要求4所述的方法，其特征在于，接种量为4.8-5.2％。
6.根据权利要求4或5所述的方法，其特征在于，发酵温度为：36-38℃，发酵时间为58-62小时。
7.根据权利要求4-6任一项所述的方法，其特征在于，接种量为5％；发酵温度为37℃，亚硒酸钠的添加量为4.8-5.2mM,发酵时间为60小时。
8.权利要求1所述的乳酸菌(Lacticaseibacillus chiayiensis)GS-3，或权利要求2所述的发酵产物，或权利要求3所述的菌剂，或权利要求4-7任一项所述的方法在以下任一方面中的应用：
(1)发酵生产有机硒；
(2)生产富硒农产品；
(3)生产富硒补充剂；
(4)生产饲料添加剂；
(5)生产提升人体的免疫力，抵抗肠道氧化应激，保护肠道氧化还原稳态，抑制肠道氧化损伤的产品。
9.一种发酵生产有机硒的方法，其特征在于，包括以权利要求1所述的乳酸菌(Lacticaseibacillus chiayiensis)GS-3进行发酵的步骤。
10.根据权利要求9所述的方法，其特征在于，所述发酵的条件如权利要求4-7任一项所述。
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