CN113197312A - 嗜热链球菌mn002在免疫调节产品中的用途及膳食补充剂 - Google Patents
嗜热链球菌mn002在免疫调节产品中的用途及膳食补充剂 Download PDFInfo
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- CN113197312A CN113197312A CN202010676506.XA CN202010676506A CN113197312A CN 113197312 A CN113197312 A CN 113197312A CN 202010676506 A CN202010676506 A CN 202010676506A CN 113197312 A CN113197312 A CN 113197312A
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- streptococcus thermophilus
- dietary supplement
- product
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Abstract
本发明公开了一种嗜热链球菌MN002在制备提高免疫的产品中的用途及膳食补充剂，该膳食补充剂包括嗜热链球菌MN002，所述嗜热链球菌MN002(Streptococcus thermophilus)于2010年5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，保藏编号为CGMCC No.3817，保藏地址为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。本发明具有更优良的耐热和pH稳定性，品质易控制，不受生产、运输等外界条件影响，不受抗生素影响，无耐药基因转移风险，且显著提高机体免疫力的效果。
Description
技术领域
本发明涉及微生物领域，具体涉及微生物菌种在免疫调节产品中的用途，并涉及该一种含有该菌种的膳食补充剂。
背景技术
免疫调节是维持机体内环境稳定的关键，如果免疫调节功能异常，对自身成分产生强烈的免疫攻击，造成细胞破坏，功能丧失，就会发生自身免疫病。如果对外界病原微生物感染不能产生适度的反应(反应过低可造成严重感染，反应过强则发生过敏反应)，也可造成对机体的有害作用。
巨噬细胞来源于血液单核细胞，可非特异性吞噬细菌、异物等，参与天然抗感染、抗肿瘤等过程，是天然免疫系统的重要组成部分。巨噬细胞在免疫过程中的具体作用为：1)免疫起始阶段，巨噬细胞可作为抗原递呈细胞摄取处理抗原并递呈给辅助性T细胞，启动特异性免疫应答；2)免疫应答阶段，活化的巨噬细胞趋化到病灶周围，更有效地吞噬细菌、杀伤靶细胞；同时，分泌多种活性物质，如溶菌酶、补体、细胞因子等，发挥相应的生物学功能。
目前临床上用于调节免疫的手段主要有以下两类：
一类是免疫调节药，通过影响机体的免疫应答反应和免疫病理反应来增强或抑制机体免疫功能的药物，包括免疫抑制药和免疫增强药，多以环多肽混合物、大环内酯类抗生素、多核苷酸肽等为基本成分，针对特异性的免疫缺陷和免疫亢进疾病有一定的疗效，但不良反应较多，导致发热、疲乏、食欲下降、恶心、呕吐、头晕、精神混乱、白细胞减少、肝功能降低、过敏反应等。
另一类是益生菌产品，均为活菌制剂产品，该类活菌制剂产品存在以下众多不足之处而影响产品的功效，包括：(1)在肠管未成熟、炎症或免疫功能不全时，活菌会突破肠壁侵入机体，进而引发炎症；(2)易受抗生素影响，也易造成耐药性基因转移；(3)长期使用活菌制剂使得噬菌体感染与爆发的风险增加；(4)不耐热、易污染、产品不稳定性等加工特性差；(5)低温、避光等产品保存条件较苛刻。
而现有技术中公开的益生菌产品中，能够达到免疫调节的菌种种类通常是乳酸杆菌和双歧杆菌等，并且还需要是活菌产品。
发明内容
因此，本发明要解决的技术问题在于，提供一种新的能够有效活化巨噬细胞增强宿主非特异性免疫调节的菌种，即嗜热链球菌(Streptococcus thermophilus)MN002，该MN002菌种可以有效活化巨噬细胞提高白介素和TNF-α的表达水平进而显著提高机体的免疫力，本发明还公开了利用嗜热链球菌MN002的灭活菌制备膳食补充剂。
一种嗜热链球菌MN002在制备增强宿主非特异性免疫调节的产品中的用途，所述嗜热链球菌MN002的保藏编号为CGMCC No.3817。所述嗜热链球菌MN002于2010年5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，保藏编号为CGMCC No.3817，保藏地址为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。该嗜热链球菌MN002又名嗜热链球菌MN-ZLW-002。
所述产品用于活化巨噬细胞提高白介素和TNF-α的表达水平，所述嗜热链球菌MN002为灭活的嗜热链球菌MN002。
所述产品为膳食补充剂，所述灭活嗜热链球菌MN002在该膳食补充剂中的含量≥100亿个/g；优选的，所述灭活嗜热链球菌MN002在该膳食补充剂中的含量≥140亿个/g。
所述产品为微生物制剂、食品、药品或保健品。
一种膳食补充剂，包括灭活后的嗜热链球菌MN002。
所述嗜热链球菌MN002的添加量≥100亿个/g，优选的，所述嗜热链球菌MN002的添加量≥140亿个/g。
所述膳食补充剂还包括益生元、食品原料和助剂中的至少一种；
所述益生元包括低聚异麦芽糖、低聚果糖、水苏糖、乳糖醇中至少一种；所述食品原料包括脱脂乳粉、抗性糊精、果蔬粉中的至少一种；所述助剂包括柠檬酸、麦芽糊精中的至少一种。
一种膳食补充剂，包括：
一种膳食补充剂，包括：
一种膳食补充剂的制备方法，包括将原料混合均匀后分装即得。
优选的，所述混合步骤为：
(1)称取脱脂乳粉和果蔬粉，混合均匀记为A料；
(2)称取麦芽糊精、抗性糊精与灭活MN002菌粉混合均匀记为B料；
(3)称取低聚异麦芽糖、低聚果糖、水苏糖、乳糖醇和柠檬酸；
(4)把A料、B料和称取的低聚异麦芽糖、低聚果糖、水苏糖、乳糖醇和柠檬酸混合均匀。
本发明技术方案，具有如下优点：
1、本发明提供了一种嗜热链球菌MN002的新用途，其可以活化巨噬细胞提高白介素和TNF-α的表达水平，进而达到增强宿主非特异性免疫调节的作用。
2、本发明首创的采用灭活后的嗜热链球菌MN002作为功效成分；采用灭活后的菌种与活菌相比，具有更优良的耐热和pH稳定性，易加工，不受食品形式限制；同时具有含菌量稳定、品质易控制，且不污染生产线的优点；采用灭活的菌种不用冷藏，不受生产、运输等外界条件影响；也不受抗生素影响，且无耐药基因转移风险，效果十分显著；
并且，本发明通过将该嗜热链球菌MN002灭活后作为活性成分后，不仅仅能带来上述灭活菌种的效果，还能刺激巨噬细胞产生生理性炎症反应，使其大量产生白介素和TNF-α，显著提高机体免疫力，效果十分显著。
3、本发明中采用灭活的嗜热链球菌MN002开发了一种具有免疫活化功能的膳食补充剂；其中，主要功效成分为灭活嗜热链球菌MN002，同时采用低聚异麦芽糖、低聚果糖、水苏糖和乳糖醇作为益生元；通过各主要功效成分与益生元之间的协同增效，进一步刺激巨噬细胞产生生理性炎症反应，使其大量产生IL-6和TNF-α，从而提高机体的免疫力。
4、本发明中增添了麦芽糊精、脱脂乳粉、柠檬酸和果蔬粉等成分，在满足主要功效成分之间的协同增效作用的前提下，能进一步的提升口感，并延长保质期限。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明，并不局限于所述最佳实施方式，不对本发明的内容和保护范围构成限制，任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品，均落在本发明的保护范围之内。
实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
下述实施例中的灭活的嗜热链球菌MN002菌粉的制备方法，包括：
(1)将保存的菌种MN 002用MRS液体培养基进行活化培养，37℃静置培养16-20h，培养至对数期，活菌数达到109cfu/mL以上；
(2)离心收集湿菌体，加入保护剂，在70-80℃真空干燥，得到具有灭活嗜热链球菌MN002的菌粉。
实施例1
一种膳食补充剂，由脱脂乳粉、麦芽糊精、抗性糊精、低聚异麦芽糖、低聚果糖、水苏糖、乳糖醇、柠檬酸、紫薯粉、灭活的嗜热链球菌MN002组成。
本实施例中灭活的嗜热链球菌MN002菌粉的制备方法，包括：
(1)将保存的菌种MN 002用MRS液体培养基进行活化培养，37℃静置培养18h，培养至对数期，活菌数达到109cfu/mL以上；
MRS液体培养基的配方为：
定容至1L并调节pH至6.2即可。
(2)离心收集湿菌体，加入保护剂，在75℃条件下真空干燥，得到具有灭活嗜热链球菌MN002的菌粉；本实施例中所采用的菌粉中灭活的嗜热链球菌MN002的数量约2500亿个/g。
本发明中该膳食补充剂的具体配比如表1所示。
表1
按照上述表1中的配比，采用以下方法制备获得的：
(1)按照组方量称取脱脂乳粉和紫薯粉，混合均匀记为A料；
(2)称取麦芽糊精、抗性糊精与灭活MN002菌粉混合均匀记为B料；
(3)称取低聚异麦芽糖、低聚果糖、水苏糖、乳糖醇和柠檬酸；
(4)把A料、B料和称取的低聚异麦芽糖、低聚果糖、水苏糖、乳糖醇和柠檬酸混合均匀，分装即得。
本实施例中，上述膳食补充剂的分装规格为每条2g，每条中灭活的嗜热链球菌MN002的数量均大于280亿。推荐每日摄入3条。
实施例2
本实施例中区别仅仅在于，本实施例中采用实例7中的组成和配比，区别仅仅在于，本实施例中的MN002为活菌制剂，所述MN002活菌制剂的制备方法如下：
将活化好的嗜热链球菌MN002在40℃条件下经两级培养8个小时；培养物接入增菌培养基，接菌量为2.5％，在温度为40℃，pH值在7.1条件下，搅拌发酵18小时获得嗜热链球菌发酵液；在4℃条件下将前述嗜热链球菌MN002发酵液离心18分钟，取下层沉淀备用；将沉淀与冻干保护剂充分混合后，在-50℃下真空冷冻干燥12h即可获得嗜热链球菌MN002菌粉，其中MN002菌粉的活菌数为2.5×1011cfu/g。
试验例-免疫活化试验
1、试验设计
采用上述实施例中制备得到的各类膳食补充剂，设置肽聚糖(peptidoglycan,PGN)阳性对照和空白对照，与小鼠单核巨噬细胞系RAW264.7共培养后测定细胞因子(IL-6、IL-10、IL-12、TNF-α)的分泌情况及mRNA表达水平，分析热灭活MN002膳食补充剂的免疫调节功能。
2、实验方法
2.1RAW264.7细胞培养
从液氮罐中取出冻存的RAW264.7细胞，37℃水浴解冻1-2min，待冻存液融化后吸取冻存液1ml，加入5ml完全培养基，置于15ml离心管中吹打混匀并转移至25mm2培养瓶，置于37℃、5％CO2孵箱中培养。
每天观察细胞形态和生长状况，每1-2d更换培养基。当细胞单层80％融合时进行传代，或转入75mm2培养瓶：弃去旧培养液，用PBS洗涤3次后加入4℃PBS 3ml刺激细胞3-4min，待细胞变圆、透亮时吸净PBS，用完全培养基小心吹落瓶底细胞，以1:3-5的比例传代于新培养瓶中，补充至5ml或12ml培养基，以相同条件继续培养。
2.2RAW264.7细胞与受试样共培养
采用实施例1中实例1-实例7和对比1、实施例2中的膳食补充剂，分别用灭菌生理盐水配制成质量浓度为10％的膳食补充剂悬液，再采用RPMI-1640培养基稀释10倍得到工作液。
24孔板中每孔加入0.9mL浓度为5×105个/mL的RAW264.7细胞悬液，37℃、5％CO2孵箱中培养2h使细胞贴壁。每孔再分别加入0.1mL实例1-实例7的工作液、对比1的工作液、实施例2的工作液、PGN浓度为20μg/mL的RPMI-1640培养基，每组设置3个平行孔，37℃、5％CO2孵箱中共培养24h后收集上清液。
2.3细胞因子检测
分别采用小鼠IL-6、IL-10、IL-12、TNF-αELISA试剂盒(美国R&D公司)测定上清液中细胞因子IL-6、IL-10、IL-12和TNF-α的浓度。采用细胞总RNA提取试剂盒(成都福际生物技术有限公司)及cDNA合成试剂盒(美国BIO-RAD公司)TRIzol法提取板底细胞RNA，逆转录为cDNA后，利用qPCR试剂盒(美国BIO-RAD公司)测定IL-6、IL-10、IL-12和TNF-α的基因相对表达量(以β-actin为内参基因)。
3、实验结果
qPCR和ELISA结果分别如表2、表3所示，表2为各细胞因子的mRNA表达量(内参的倍数)，表3为各细胞因子的蛋白分泌量(pg/ml)。
表2
注：上述表1中，a与空白对照组比较，P<0.05；b与PGN组比较，P<0.05。
表3
注：上述表2中，a与空白对照组比较，P<0.05；b与PGN组比较，P<0.05；N.D表示未检出。
通过上述表1和表2可知：
灭活MN002膳食补充剂IL-10的基因表达和蛋白分泌量均低于PGN组(P<0.05)，但蛋白分泌量高于空白对照组(P<0.05)。
灭活嗜热链球菌MN002膳食补充剂诱导分泌IL-6的能力强于PGN阳性对照，其对IL-6的基因表达和蛋白分泌量均显著高于PGN阳性对照和空白对照(P<0.05)；灭活MN002膳食补充剂对TNF-α的mRNA表达量与PGN阳性对照无统计学差异，但强于空白对照(P<0.05)，灭活MN002膳食补充剂对TNF-α的的蛋白分泌量显著高于PGN阳性对照和空白对照(P<0.05)。
通过实验结果显示，灭活嗜热链球菌MN002膳食补充剂可以活化巨噬细胞RAW264.7，使其大量产生IL-6和TNF-α，具有活化巨噬细胞、增强宿主非特异性免疫调节的功能。
同时，本发明还对仅仅只有菌剂时的效果进行了验证，通过验证得知：在具有与实例7和实施例2相同菌含量的情况下，食品原料和助剂的添加对本申请效果的影响并不显著。即，直接采用相同剂量的灭活菌剂的效果与实例7中添加有食品原料和助剂的效果基本一致，直接采用相同剂量的活菌菌剂的效果与实施例2中添加有食品原料和助剂的效果基本一致。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种嗜热链球菌MN002在制备增强宿主非特异性免疫调节的产品中的用途，所述嗜热链球菌MN002的保藏编号为CGMCC No.3817。
2.根据权利要求1所述的用途，其特征在于，所述产品用于活化巨噬细胞提高白介素和TNF-α的表达水平；所述嗜热链球菌MN002为灭活的嗜热链球菌MN002。
3.根据权利要求2所述的用途，其特征在于，所述产品为膳食补充剂，所述灭活嗜热链球菌MN002在该膳食补充剂中的含量≥100亿个/g；优选的，所述灭活嗜热链球菌MN002在该膳食补充剂中的含量≥140亿个/g。
4.根据权利要求1或2所述的用途，其特征在于，所述产品为微生物制剂、食品、药品或保健品。
5.一种膳食补充剂，其特征在于，包括灭活后的嗜热链球菌MN002。
6.根据权利要求5所述的一种膳食补充剂，其特征在于，所述嗜热链球菌MN002的添加量≥100亿个/g，优选的，所述嗜热链球菌MN002的添加量≥140亿个/g。
7.根据权利要求5或6所述的一种膳食补充剂，其特征在于，还包括益生元、食品原料和助剂中的至少一种；
所述益生元包括低聚异麦芽糖、低聚果糖、水苏糖、乳糖醇中至少一种；所述食品原料包括脱脂乳粉、抗性糊精、果蔬粉中的至少一种；所述助剂包括柠檬酸、麦芽糊精中的至少一种。
8.根据权利要求5-7任一所述的一种膳食补充剂，其特征在于，包括：
9.根据权利要求8所述的一种膳食补充剂，其特征在于，包括：
10.一种膳食补充剂的制备方法，其特征在于，包括将原料混合均匀后分装即得；优选的，混合步骤为：
(1)称取脱脂乳粉和果蔬粉，混合均匀记为A料；
(2)称取麦芽糊精、抗性糊精与灭活MN002菌粉混合均匀记为B料；
(3)称取低聚异麦芽糖、低聚果糖、水苏糖、乳糖醇和柠檬酸；
(4)把A料、B料和称取的低聚异麦芽糖、低聚果糖、水苏糖、乳糖醇和柠檬酸混合均匀。
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