CN109251877A - 一种具有减脂等功能的纳豆芽孢杆菌Bn-SJLH001益生作用及其应用 - Google Patents
一种具有减脂等功能的纳豆芽孢杆菌Bn-SJLH001益生作用及其应用 Download PDFInfo
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Abstract
本发明涉及一株自主筛选的具有减少脂肪累积功能的纳豆芽孢杆菌（Bacillus natto SJLH001，简称为Bn‑SJLH001）的体内外功能验证、作用研究及其在益生菌食品中的应用。该菌株具有良好的体外分解纤维蛋白、抗凝血和抗氧化等体外益生活性，并有显著的减重、降低高脂膳食小鼠血清甘油三酯水平、减少三个不同部位白色脂肪（附睾脂肪、肾周脂肪和肠系膜脂肪）的重量、减小脂肪细胞直径的作用。同时，纳豆芽孢杆菌Bn‑SJLH001能够调节肠道优势菌数量，改变肠道营养物质、金属离子和代谢物转运相关基因的表达，降低脂肪生成因子基因（GSK‑3α/β）的表达，降低肾周脂肪组织尿酸的累积。此外，基于纳豆芽孢杆菌Bn‑SJLH001能显著减脂等的活性功能，本发明还利用其开发多种形式的益生食品，如益生菌压片糖果、益生菌固体饮料。
Description
技术领域
本发明涉及一种具有减脂等功能的益生菌——纳豆芽孢杆菌Bn-SJLH001的筛选、鉴定、该菌株在减少脂肪累积方面的功能验证、作用研究及其在益生菌产品（益生菌压片糖果和固体饮料）中的应用，属于食品微生物工程领域。
背景技术
肠道菌群的组成结构是决定肥胖形成的关键要素。肠道中存在着一个复杂的微生态系统，大约有100兆细菌与宿主共同生活和进化。肥胖会导致肠道菌群多样性的减少和丰富度的降低，产生内毒素的有害菌增加，具有抗炎作用的有益菌（如乳杆菌和双歧杆菌）减少。使用无菌动物模型进行菌群移植试验的结果显示，移植了胖人菌群的小鼠会表现出肥胖和代谢综合征表型，而移植了瘦人菌群的小鼠则表现出瘦体型。
益生菌被定义为当摄入一定数量时能对宿主健康起有益作用的活的微生物。补充益生菌被认为是调节肠道菌群结构的有效方式。益生菌的摄入可以增加肠道菌群的丰富度和多样性，促进肠道蠕动，使食物快速通过肠道，减少停留时间。有的益生菌还可以调节神经系统，促进饱腹感激素的分泌，增加饱腹感，从而降低食欲，改善肥胖。还有一些益生菌可以在肠道内直接黏附脂类物质，随着粪便排出体外，减少脂类的吸收。但是益生菌的作用不仅仅局限于这几种现有机制，同时益生菌具有特异性，不同的菌株作用机理不同，我们将从新的机制发掘益生菌的功能。
本发明以中国本地的传统发酵制品——豆酱中分离的纳豆芽孢菌为目标菌，对其体内减少脂肪累积的作用及机制进行了研究，并进一步开发了其在益生菌产品中的应用。
发明内容
本发明的目的之一，在于提供一种纳豆芽孢杆菌Bn-SJLH001菌株；所述纳豆芽孢乳杆菌Bn-SJLH001是从传统发酵豆酱中分离的益生菌，该纳豆芽孢杆菌Bn-SJLH001已于2018年4月20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（国家专利局指定专利微生物保藏中心，CGMCC）；保藏地址：北京市朝阳区北辰西路1号院3号；保藏号CGMCCNo.15635。
本发明的目的之二，在于验证所述纳豆芽孢杆菌Bn-SJLH001在降低宿主血清甘油三酯水平的作用。
本发明的目的之三，在于验证所述纳豆芽孢杆菌Bn-SJLH001菌株在减少宿主白色脂肪累积方面的作用。
本发明的目的之四，在于验证所述纳豆芽孢杆菌Bn-SJLH001在增加宿主肠道菌群丰度中的作用：该纳豆芽孢杆菌可以增加高脂膳食小鼠肠道细菌的丰度，并增加肠道有益菌副干酪乳杆菌的丰度。
本发明的目的之五，在于验证所述纳豆芽孢杆菌Bn-SJLH001在改善肠道基因表达中的作用：该纳豆芽孢杆菌可以调节肠道金属离子和脂质的转运来影响肠道脂肪的吸收和到脂肪组织的转运。
本发明的目的之六，在于验证所述纳豆芽孢杆菌Bn-SJLH001在改善白色脂肪组织蛋白表达和减少肾周脂肪组织尿酸累积中的作用。
本发明的目的之七，在于所述纳豆芽孢杆菌Bn-SJLH001在益生菌压片糖果制品中的应用研究。
本发明的目的之八，在于所述纳豆芽孢杆菌Bn-SJLH001在益生菌固体饮料制品中的应用研究。
下面结合附图和实施例对本发明作进一步描述。
附图说明
本发明有如下附图：
图1：纳豆芽孢杆菌Bn-SJLH001 MRS平板菌落图
图2：纳豆芽孢杆菌Bn-SJLH001革兰氏染色图
图3：纳豆芽孢杆菌Bn-SJLH001的纤维蛋白原分解活性
图4：纳豆芽孢杆菌Bn-SJLH001的抗凝活性
图5：纳豆芽孢杆菌Bn-SJLH001的DPPH抗氧化活性
图6：纳豆芽孢杆菌Bn-SJLH001的ABTS+清除率
图7：纳豆芽孢杆菌Bn-SJLH001干预降低高脂小鼠体重（HF：高脂膳食组；BS：纳豆芽孢杆菌干预组；NC：正常对照组）
图8：纳豆芽孢杆菌Bn-SJLH001降低小鼠血清甘油三酯水平（HF：高脂膳食组；BS：纳豆芽孢杆菌干预组；NC：正常对照组）
图9：纳豆芽孢杆菌Bn-SJLH001减少小鼠附睾脂肪（eWAT）、肾周脂肪（pWAT）和肠系膜脂肪（mWAT）的重量（HF：高脂膳食组；BS：纳豆芽孢杆菌干预组；NC：正常对照组）
图10：纳豆芽孢杆菌Bn-SJLH001减少小鼠附睾脂肪（eWAT）、肾周脂肪（pWAT）和肠系膜脂肪（mWAT）细胞的直径
图11：纳豆芽孢杆菌Bn-SJLH001干预组增加小鼠肠道乳酸片球菌（Pediococcus acidilactici）和副干酪乳杆菌（Lactobacillus paracasei） 等益生菌的数量
图12：纳豆芽孢杆菌Bn-SJLH001对肠道基因表达的调节
图13：纳豆芽孢杆菌Bn-SJLH001对脂肪组织蛋白表达的调节作用
图14：纳豆芽孢杆菌Bn-SJLH001降低肾周脂肪组织的尿酸浓度
实施例1：纳豆芽孢杆菌Bn-SJLH001的分离、鉴定及体外功能评价
1、纳豆芽孢杆菌的分离和鉴定
从东北家庭制作的发酵豆酱里分离得到一株耐热的芽孢菌（NB培养基55℃培养48h），分离单菌落，于MRS培养基37℃培养48h，离心提取细菌DNA后进行细菌16S通用引物PCR，经16SrDNA比对鉴定为纳豆芽孢杆菌（SEQ ID NO: 1所示），与标准菌株Bacillus subtilisstrain CICC 10366同源性为99%。
本发明所述的纳豆芽孢杆菌菌株Bn-SJLH001，单菌落接种到MRS固体培养基上，37ºC需氧生长良好，菌落呈圆形，直径大小2.0 mm-3.0 mm，表面不光滑，呈乳白色（图1）；革兰氏染色呈阳性，菌体较平直或稍弯，两端钝圆，成单或双存在，有芽孢（图2）。
、体外纤维蛋白原分解活性鉴定
纤维蛋白平板法：准确称取1.000g 纳豆芽孢菌Bn-SJLH001冻干粉样品，1:10（w/v）加入PBS缓冲液，即纳豆芽孢菌样品液浓度为100mg/mL。进一步稀释，分别制成2.5 mg/mL和1mg/mL两种浓度的纳豆芽孢菌悬浮液。取5 mL融化的0.8% （w/v）的琼脂糖，待温度降至50°C左右时，加入5 mL 0.4% (w/v)的纤维蛋白原溶液，充分混匀后，迅速加入1 mL 200 U/mL的凝血酶溶液，再次充分混匀，然后立即倒平板，尽量避免产生气泡。待其冷却凝固后，即制成纤维蛋白平板。用打孔器打三个直径为3 mm的平板孔，其中两个孔中分别加入10 μL2.5mg/mL和1 mg/mL的纳豆芽孢杆菌样液，另一个孔中加入10 μL PBS缓冲液作为对照，于37°C恒温培养箱中放置12 h， 观察有无透明圈形成。
实验结果如图3所示。加入纳豆芽孢杆菌Bn-SJLH001样液的两个琼脂孔产生了透明圈，表明纤维蛋白发生了分解，而加入对照培养基的琼脂孔没有产生透明圈，说明纳豆芽孢杆菌Bn-SJLH001具有良好的纤维蛋白分解活性，证明其在溶解血栓方面的应用潜力。
、体外抗凝活性评价
抗凝活性评价：准确称取1.0g 纳豆芽孢菌Bn-SJLH001冻干粉样品，1:10（w/v）加入PBS缓冲液（50 mM，pH 6.5），即纳豆芽孢菌样品液浓度为100mg/mL。进一步稀释至2.5、1、0.5、0.25、0.1、0.05mg/mL，制成纳豆芽孢菌浓度梯度液。依次向试管中加入1.4 mL硼砂缓冲液（50 mmol/L, pH 8.5）和0.4 mL纤维蛋白原溶液（0.72%, w/v），37°C预热5 min后，加入0.1mL凝血酶溶液（20 U/mL），37°C水浴中反应 10 min，再分别加入0.1 mL 0.05、0.1、0.25、0.5、1、2.5mg/mL系列浓度梯度的纳豆芽孢杆菌样液，于37°C水浴中反应60 min（于20min、40 min 时各振荡5 s）。最后加2 mL 0.2 M TCA溶液，于37°C静置20 min后，10000 rpm离心10 min。取上清液于275 nm下测定吸光值，以PBS溶液加TCA和酶液进行同样反应后的上清液作为对照。酶活定义：275 nm下，与对照相比，样品吸光值每分钟增加0.01相当于一个酶活单位。X(FU/mL) = (Ar-Ac) × N/(60×0.01×0.1)，式中: X为样品的酶活力，FU/mL；Ar为样品吸光值；Ac为对照吸光值；N为稀释倍数；0.1为参加反应的酶液是 0.1 mL；60为反应时间60 min，以1 min计。
实验结果如图4所示。与对照相比，纳豆芽孢杆菌Bn-SJLH001抗凝酶活随着纳豆芽孢菌浓度的上升显著上调，证明纳豆芽孢杆菌Bn-SJLH001具有良好的体外抗凝血活性。
、体外抗氧化活性评价
对纳豆芽孢杆菌Bn-SJLH001的DPPH自由基清除活性进行评价：纳豆芽孢菌浓度梯度液制作同上。取2 mL不同浓度纳豆芽孢杆菌样品液（0.05、0.1、0.25、0.5、1、2.5、5、10 mg/mL）分别加入2 mL浓度为0.2 mM的DPPH，以蒸馏水加DPPH作为空白对照，37 °C避光反应20min，5000 g离心后测定OD519，计算自由基清除率。抗氧化性（%）=[1-（A-B）/C]×100%。A：含有样品和DPPH溶液；B：含有样品溶液不含DPPH溶液；C：不含样品，含DPPH溶液。
对纳豆芽孢杆菌的ABTS+自由基清除活性进行评价：准确量取100μL ABTS 溶液（7.4mmol/L）和100μL K2S2O8氧化剂溶液（2.6mmol/L），制成200μL 的ABTS 工作母液。将配制好的ABTS 工作母液，室温避光存放12-16h，使用前用80%的乙醇进行稀释，调整到其在734nm 处的吸光度为0.7±0.02。准确量取10μL 的各浓度的纳豆芽孢杆菌样品溶液（0.05、0.1、0.25、0.5、1、2.5mg/mL）与200μL 的ABTS 工作液于96 孔板中，轻轻混匀，反应2min-6min 后于734nm 处测定其吸光度值。实验以Trolox 为阳性对照，同时设试剂空白和样品空白，实验重复3 次。按下式计算ABTS的清除率：ABTS+清除率(%)=1‐(At‐B) / A0×100%，式中：A0 为未加样的；ABTS+的吸光度值；At 为样品与ABTS+反应后的吸光度值；B 为样品空白的吸光度值。
实验结果如图5、6所示。与对照相比，纳豆芽孢杆菌Bn-SJLH001的DPPH自由基清除能力和ABTS+清除率随着纳豆芽孢菌浓度的提高而明显上升，证明纳豆芽孢杆菌Bn-SJLH001的具有较强的DPPH抗氧化活性和ABTS+清除率，表明其具有良好的抗氧化能力。
实施例2：纳豆芽孢杆菌Bn-SJLH001调节血清甘油三酯和减少三种白色脂肪累积的体内功能验证
实验分组如表1所示，每组8只小鼠（C57BL/6J雄性，初始体重20.0±2.0g，SPF级），正常组饲喂正常饲料（脂肪供能比22%、碳水化合物供能比53%、蛋白质供能比25%），高脂组和高脂益生菌干预组饲喂高脂饲料（配方中脂肪供能比60%、碳水化合物供能比21%、蛋白质供能比19%），高脂益生菌干预组每天灌胃纳豆芽孢杆菌Bn-SJLH001 1.0×109CFU/只，高脂对照组和正常对照组在同样的时间分别灌胃等量生理盐水。实验进行28周。
每周五上午10点钟进行小鼠体重称重。实验结果如图7所示，正常饲喂组（NC组）体重最低，与高脂组（HF组）相比，高脂+益生菌干预组（BS组）从第13周开始表现出体重降低，在第25、26、27、28周体重达到显著性降低（p<0.05），表明纳豆芽孢杆菌Bn-SJLH001可以有效降低高脂膳食小鼠的体重。
取28周空腹12h小鼠的血液离心得到血清，使用生化仪进行血清甘油三酯水平的测定。实验结果如图8所示，BS组与HF组相比，血清甘油三酯水平显著降低（p<0.05），与NC组无显著差异（p>0.05），证明纳豆芽孢杆菌Bn-SJLH001可以显著降低高脂膳食小鼠血清甘油三酯水平。
取28周小鼠三种不同部位的白色脂肪组织（附睾脂肪、肾周脂肪和肠系膜脂肪）进行称重。实验结果如图9所示。HF组的三种白色脂肪组织都比BS组有所增加（图9A/B）。BS组与HF组相比，三种白色脂肪组织重量均显著降低（p<0.05，图9C），均显著高于NC组重量（p<0.05，图9C），证明纳豆芽孢杆菌Bn-SJLH001可以显著降低高脂膳食小鼠白色脂肪重量。
取饲养28周小鼠三种不同部位的白色脂肪组织（附睾脂肪、肾周脂肪和肠系膜脂肪）进行石蜡包埋切片和HE染色。实验结果如图10所示。结果表明，与HF组相比，BS组三种白色脂肪细胞直径均显著降低（p<0.05），证明纳豆芽孢杆菌Bn-SJLH001可以显著降低高脂膳食小鼠白色脂肪直径。
表1 动物试验分组情况
| 组 别 | 实验动物数量 | 灌服制剂 |
| 高脂对照组（HF） | 8 | 生理盐水 |
| 高脂+活菌干预组（BS） | 8 | Bn-SJLH001活菌体脱脂乳液 |
| 正常对照组（NC） | 8 | 生理盐水 |
实施例3：纳豆芽孢杆菌Bn-SJLH001体内调节肠道菌群功能分析
取高脂组和高脂益生菌干预组小鼠结肠内容物，液氮速冻后于-80℃冷冻保藏。称取0.1g内容物后用PBS连续进行10倍稀释（10-1、10-2、10-3、10-4、10-5和10-6），吸取100μL10-6稀释液用玻璃棒涂布血平板。37℃厌氧培养72h后对血平板上的菌落进行质谱鉴定（MALDI-TOF MS）。实验结果如图11所示，与HF组相比，BS组的总的菌落数（图11A）以及Pediococcus acidilactici（乳酸片球菌）和Lactobacillus paracasei （副干酪乳杆菌）数量均显著升高（p<0.05，图11B），表明纳豆芽孢杆菌 Bn-SJLH001可以显著增加肠道可培养菌群的丰度以及肠道有益菌副干酪乳杆菌和乳酸片球菌的数量。
实施例4：Bn-SJLH001调节肠道基因表达的功能分析
实验小鼠分组同表1。取饲养28周高脂组和高脂益生菌干预组小鼠结肠同一部位组织，利用Trizol提取组织RNA，经二代转录组测序后比对基因的表达谱。实验结果如图12所示。与HF组相比，BS组多种肠道转运相关基因发生显著改变（p<0.05），这些基因包括：阳离子转运相关基因 [钠离子（Slc9a3、Fxyd4、Mfsd2a和Scn4a）、钾离子（Atp12a和Kcnh3）、钙离子（Trpv1 和Trpv3）、铁离子（Slc40a1）、镁离子（Trpm6）和锌离子（Slc30a10和Bhmt）]、阴离子转运相关基因[氯离子（Clic6和Clcn2）、碳酸氢根离子（Best2）、铵根离子（Trpv3）、磷酸根离子（Slc34a2）]、脂质转运[脂肪酸（Alb）、甾醇（Abcg5和Abcg8）、胆固醇（Apoa2和Apoa4）、Vitamin A（Ttr）和Vitamin D（Gc）]基因、氨基酸转运（Slc15a1）基因和核酸转运（Slc28a3）基因。这些结果表明，纳豆芽孢杆菌Bn-SJLH001可以调节肠道营养物质的转运和转运速度（肠道运动速度决定基因Clcn2）以及影响肠道物质合成催化因子的转运，推测纳豆芽孢杆菌Bn-SJLH001可以避免物质过多地转运到肠道，保持肠道所需营养物质和离子的动态平衡。此外，纳豆芽孢杆菌Bn-SJLH001还影响了脂质代谢（Lipid metabolic process）、蛋白分解（Proteolysis）、炎症形成（Inflammation）、脂质下调（Lipid-reducing）、肽酶活性（Peptidase activity）、血管生成（ Angiogenesis ）、肠道黏膜完整性和运动性（Mucusand motility）相关基因的表达（p<0.05）（图12）。这些途径均对肠道脂肪的生成和转运具有明显改善作用。
实施例5：Bn-SJLH001调节脂肪组织蛋白表达和代谢功能分析
实验小鼠分组同表1。取28周正常组、高脂组和高脂益生菌干预组小鼠（每组随机取三只）的三种不同部位的白色脂肪组织（附睾脂肪、肾周脂肪和肠系膜脂肪）通过以下步骤进行蛋白免疫印记实验（Western-blot）。
1）提取组织总蛋白：称取50mg组织于带磨珠的匀浆管中，加入组织蛋白TPR匀浆液和蛋白酶抑制剂，使用组织研磨器Mini-Beadbeater-24将组织充分研磨。在冰上静置30min后，匀浆液经12000g，4℃离心30min后上清液转移到EP管。最后分装-80冻存；
2）蛋白定量：BCA蛋白定量试剂与蛋白溶液反应后，使用酶标仪对其蛋白浓度进行测定；
3）SDS-PAGE凝胶电泳：蛋白样品与5×蛋白上样缓冲液（碧云天生物技术有限公司）混合后首先95℃加热5min使其变性。蛋白上样量为100μg，浓缩胶部分电泳恒压80V，30min，分离胶恒压120V，70min；
4）湿法转膜：先用转膜缓冲液润湿滤纸和PVDF膜（Millipore，提前用甲醇润湿30s）。按滤纸层—分离胶—PVDF膜—滤纸层“三明治”的结构放置，设置220mA恒流转移1-2h。预染Marker用于判断蛋白分子量位置；
5）封闭：将PVDF膜用含5%的脱脂牛奶TBST溶液覆盖后，常温摇床孵育2h；
6）孵育一抗与洗涤：将一抗按比例稀释（稀释比1:1000），放在摇床上4℃过夜孵育。一抗孵育后的膜用TBST洗3次×10分钟；
7）孵育二抗与洗涤：按比例稀释酶标二抗（1:10000），放在摇床上室温孵育1小时。二抗孵育后的膜用TBST洗3次×10分钟；
8）化学发光成像：取等量Immobilon ECL的A液、B液（Millipore公司），混合后均匀覆盖膜1min，至于化学曝光仪器中进行曝光，最后得到曝光条带。
实验结果如图13所示。与HF组相比，BS组肠系膜脂肪组织脂肪生成因子GSK-3α/β的表达下调，同时肾周脂肪组织激素敏感性甘油三酯脂肪酶（HSL）和自噬相关Beclin-1蛋白水平也下调，而脂肪酸合酶（fatty acid synthase，FAS）表达无显著变化。证明纳豆芽孢菌 Bn-SJLH001可以显著降低肠系膜脂肪组织GSK-3α/β、肾周脂肪组织HSL和自噬相关Beclin-1蛋白的表达，对脂肪组织中脂肪的累积产生抑制作用。
另外取三种白色脂肪组织进行匀浆，高速离心后取上清液，采用比色法对尿酸浓度进行测定。实验结果如图14所示。与HF组相比，BS组肾周脂肪组织尿酸浓度显著降低（p<0.05），而附睾脂肪组织和肠系膜脂肪组织尿酸浓度无显著差异（p>0.05）。证明益生菌Bn-SJLH001可以显著降低肾周脂肪组织尿酸的累积，与肠道转运尿酸的机制相符合。
实施例6：高活性Bn-SJLH001菌粉的制作
1. 菌种活化和种子液制备：冻存的菌种使用MRS固体培养基划线培养后，挑取单菌落在试管中活化三代达到活性最强（MRS液体培养基，37 ºC培养24 h）。接着接入500mL大瓶MRS液体培养基进行扩培（120r/min摇床37 ºC培养24 h）。
2. 发酵：以1×106 CFU/mL的接种量接入发酵罐进行培养（37 ºC培养24 h）。
3. 离心和喷雾干燥：发酵液经过离心（5000r/min, 15min）得到菌泥。发酵菌泥与脱脂乳（20%，w/v）按照1：2混匀进入喷雾干燥机进行喷雾干燥（70℃，蠕动泵30r/min），获得菌粉。
实验结果：经PBS倍比稀释和MRS平板计数，纳豆芽孢杆菌菌粉的活菌量大于1×1011 CFU/g。
实施例7：Bn-SJLH001压片糖果的制作
1.原料配方（以基本100g计）
一种高活菌含量的益生菌压片糖果，其制作步骤如下：
A. 原料配制：脱脂奶粉25g-35g、益生菌菌粉3g-4g、水果粉25g-35g、乳糖3g-5g、甘露醇10g-12g、低聚半乳糖粉10g-15g、木糖醇3.5g-4.5g、微晶纤维素5g-8g、硬脂酸镁0.5g-1g，以上各成分按照重量进行配比。
2. 制作方法：
A、原料按比例配制。
B、混料：将经步骤A的原料倒入混合机（900r/h、30min），混匀。
C、过筛和压片：经步骤B混匀后的原料通入筛选机，过80目筛后，进入压片机，得益生菌压片产品。
3. 检验：益生菌片破碎后倍比稀释经平板计数，纳豆菌粉的活菌量大于1×109CFU/g。益生菌压片糖果在片重差异、硬度、脆碎度方面符合国标。
实施例8：Bn-SJLH001益生菌固体饮料的制作
1. 原料配方（以基本100g计）
一种高活菌含量的益生菌固体饮料的的制作步骤如下：
A:原料配制：益生菌菌粉2g-3g、水果粉25g-40g、甘露醇25g-40g、低聚半乳糖粉12g-15g、木糖醇3.5g-5g、柠檬酸0.5g，以上各成分按照重量进行配比。
2. 制作方法：
A、原料按比例配制。
B、混合：将经步骤A的原料过80目筛后倒入混合机（900r/h、30min），充分混匀。
C、灌装：经步骤B混匀后的原料倒入灌装设备，灌装塑封后得益生菌固体饮料产品。
3. 检验：益生菌固体饮料倍比稀释后经平板计数，纳豆菌粉的活菌量大于1×109 CFU/g。
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。
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<110> 北京首佳利华科技有限公司
<120> 一种具有减脂等功能的纳豆芽孢杆菌Bn-SJLH001益生作用及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1444
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 2
cgcgtgctat acatgcaagt cgagcggaca gatgggagct tgctccctga tgttagcggc 60
ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc cgggaaaccg 120
gggctaatac cggatggttg tttgaaccgc atggttcaaa cataaaaggt ggcttcggct 180
accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg ctcaccaagg 240
caacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag acacggccca 300
gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc tgacggagca 360
acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg gaagaacaag 420
taccgttcga atagggcggt accttgacgg tacctaacca gaaagccacg gctaactacg 480
tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag 540
ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg gggagggtca 600
ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg tagcggtgaa 660
atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct gtaactgacg 720
ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa 780
acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta acgcattaag 840
cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac gggggcccgc 900
acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga 960
catcctctga caatcctaga gataggacgt ccccttcggg ggcagagtga caggtggtgc 1020
atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080
ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt gacaaaccgg 1140
aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct 1200
acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca caaatctgtt 1260
ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg 1320
gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 1380
agagtttgta acacccgaag tcggtgaggt aaccttttag gagccagccg ccgaagtgac 1440
aaag 1444
Claims (8)
1.一种具有减脂等功能的纳豆芽孢杆菌菌株（Bacillus natto SJLH001，简称为Bn-SJLH001），其特征在于，所述纳豆芽孢杆菌是从传统发酵豆酱中分离获得的益生菌，该菌株已于2018年4月20日保存在中国微生物菌种保藏管理委员会普通微生物菌种保藏中心（国家专利局指定专利微生物保藏中心），地址为中国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，保藏号CGMCC No.15635。
2.如权利要求1所述的纳豆芽孢杆菌菌株Bn-SJLH001，其特征在于：所述的纳豆芽孢杆菌菌株单菌落接种到MRS固体培养基上，37 ºC需氧生长良好，菌落呈圆形，直径大小2 mm-3.0 mm，表面不光滑，呈乳白色；革兰氏染色呈阳性，菌体较平直或稍弯，两端钝圆，成单或双存在，有芽孢，经16SrDNA比对鉴定为纳豆芽孢杆菌。
3.如权利要求1、2所述的纳豆芽孢杆菌菌株Bn-SJLH001，其特征在于：所述纳豆芽孢杆菌具有显著的体外纤维蛋白原分解活性，并有良好的抗凝活性和抗氧化性等优良的生物活性，同时，其具有显著的降脂功能：减轻高脂膳食小鼠的体重、降低血清甘油三酯浓度、降低白色脂肪组织（附睾脂肪、肾周脂肪和肠系膜脂肪）的重量和减小脂肪细胞直径的功能。
4.如权利要求3所述的降脂功能，其肠道作用包含：调节肠道优势菌数量，改变肠道营养物质、金属离子和代谢物转运相关基因的表达；其脂肪组织作用包含：降低脂肪生成因子基因（GSK-3α/β）的表达，降低肾周脂肪组织尿酸的累积。
5.一种具有减脂等功能的纳豆芽孢杆菌Bn-SJLH001菌粉，其特征在于：所述纳豆芽孢杆菌菌粉是由权利要求1-4所述纳豆芽孢杆菌的发酵产物经喷雾干燥（进料蠕动泵速度30r/min，喷粉温度70℃）制备而成。
6.如权利要求5所述的纳豆芽孢杆菌Bn-SJLH001菌粉在益生菌压片糖果中的应用，其特征在于：所述纳豆压片糖果是由纳豆芽孢杆菌Bn-SJLH001菌粉经一定比例配方混合后进行压片获得，每100g压片糖果包括脱脂奶粉25g-35g、益生菌菌粉3g-4g、水果粉25g-35g、乳糖3g-5g、甘露醇10g-12g、低聚半乳糖粉10g-15g、木糖醇3.5g-4.5g、微晶纤维素5g-8g、硬脂酸镁0.5g-1g。
7.如权利要求5所述的纳豆芽孢杆菌Bn-SJLH001菌粉在益生菌固体饮料中的应用，其特征在于：所述益生菌固体饮料是由纳豆芽孢杆菌Bn-SJLH001菌粉经一定比例配方混合后进行灌装获得，每100g固体饮料包含菌粉2g-3g、水果粉25g-40g、甘露醇25g-40g、低聚半乳糖粉12g-15g、木糖醇3.5g-5g、柠檬酸0.5g。
8.如权利要求6、7所述的益生菌压片糖果和益生菌固体饮料，其特征在于：所述益生菌压片糖果和益生菌固体饮料活菌含量高，益生菌活菌数达109CFU/g，水分含量小于7%；在保质期内其活菌含量未有明显变化。
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