CN114621898B - 肠膜明串珠菌及其用于制备抑制牙龈卟啉单胞菌的口腔护理用品、药品 - Google Patents
肠膜明串珠菌及其用于制备抑制牙龈卟啉单胞菌的口腔护理用品、药品 Download PDFInfo
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Abstract
具有潜在口腔益生功能的肠膜明串珠菌及其在制备抑制口腔牙龈卟啉单胞菌的口腔护理用品、药品、的应用，属于微生物技术领域。本发明所述的肠膜明串珠菌LVBH107保藏编号为CGMCC No.21362，分类命名为Leuconostoc mesenteroides，进一步由该菌可以制备肠膜明串珠菌LVBH107菌剂。本发明所述菌株和菌剂可以抑制牙龈卟啉单胞菌的生长，抑制牙龈卟啉单胞菌生物膜的形成，降低细胞中肿瘤坏死因子‑α、白细胞介素‑6、白细胞介素‑1β、前列腺素E2、一氧化氮的含量，可以作为功能性益生菌应用于口腔护理用品、药品等领域，具有非常好的产业前景。
Description
技术领域
本发明属于微生物技术领域，具体涉及一株具有潜在口腔益生功能的肠膜明串珠菌及其在制备抑制口腔牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）的口腔护理用品、药品中的应用。
背景技术
牙周炎是一种常见的口腔慢性感染性疾病，由口腔致病微生物菌斑引起的牙周支持组织的炎症和破坏，常导牙龈出血、牙周袋形成、牙槽骨丧失、牙齿松动移位甚至有可能引起发音功能和面部形象变化。越来越多的临床和实验证据表明，牙周炎可增加患多种系统性疾病（包括糖尿病、类风湿性关节炎、阿尔茨海默氏病、癌症等）的风险。牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）是目前公认的导致侵袭性牙周炎和进展为系统性疾病的重要病原体，并与牙周炎的发展密切相关。牙龈卟啉单胞菌与多种致病性或非致病性细菌通过粘附、共聚集等形成成熟的牙菌斑生物膜，黏附在牙齿表面及龈沟内，是牙周炎发病的关键因素。
目前牙周炎的治疗方法主要包括牙周基础治疗、药物治疗和外科手术治疗。牙周基础治疗即机械清除牙齿表面菌斑和牙石，尽管它可以有效降低临床炎症指标，但无法完全清除某些部位的菌斑和感染性细胞。罗红霉素、阿莫西林和甲硝唑等抗生素的药物治疗会引起细菌耐药等。手术治疗可改善龈袋深度促进牙周软组织再附着于牙面，但无法降低炎症因子并可能导致牙龈萎缩。针对牙周炎的治疗，不仅要彻底清除牙周组织上的菌斑结石，也要充分降低促炎因子的表达含量，因此寻找一种更健康的牙周治疗方案对牙周炎的治疗至关重要。
益生菌是一种有益于宿主健康的微生态调节剂，已广泛应用于食品、功能食品、药品等领域。益生菌能够改善肠道菌群结构、提高机体免疫力、降低血清胆固醇、抑制有害菌的生长等，对于高血压、高血脂、心脏病、糖尿病和癌症的防治有着重要意义。同样，益生菌在维持口腔生态系统的健康平衡中发挥重要作用，有研究显示乳酸菌在牙周病发病机制中具有保护作用。
对益生菌作为口腔护理产品抑制牙龈卟啉单胞菌的开发将有助于预防及改善由牙龈卟啉单胞菌引起的口腔疾病，研究结果可能为预防和改善牙周炎提供理论依据。因而益生菌口腔护理产品的开发应用将成为益生菌应用的一个研究热点，并将成为口腔护理产品发展的重要领域。
发明内容
本发明的目的是提供一株具有潜在口腔益生功能的肠膜明串珠菌及其在制备抑制口腔牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）药物中的应用。
本发明所述的肠膜明串珠菌LVBH107以从传统发酵食品中分离鉴定的乳酸菌为研究对象，经大量的实验筛选获得。肠膜明串珠菌LVBH107具有耐酸、耐胆盐、疏水性、粘附性等益生特性，肠膜明串珠菌LVBH107可抑制牙龈卟啉单胞菌的生长和生物膜的形成。
本发明所述的肠膜明串珠菌LVBH107，于2020年12月14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（CGMCC，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），保藏编号为CGMCC No. 21362，分类命名为肠膜明串珠菌（Leuconostoc mesenteroides）。
本发明还保护一种肠膜明串珠菌LVBH107菌剂，所述肠膜明串珠菌LVBH107菌剂中，肠膜明串珠菌LVBH107的浓度为106CFU/g以上，具体可为106~1010CFU/g。所述肠膜明串珠菌LVBH107菌剂为由含有肠膜明串珠菌LVBH107的菌液制备得到的粉剂，制备粉剂的方法可为冷冻干燥、包埋或其它工艺。
本发明所述的肠膜明串珠菌LVBH107和肠膜明串珠菌LVBH107菌剂可以作为功能性益生菌应用于口腔护理用品、药品领域，具有良好的产业前景，所述口腔护理用品可为牙膏、漱口水、凝胶、敷料等。
本发明所述的肠膜明串珠菌LVBH107和肠膜明串珠菌LVBH107菌剂可以用于制备抑制口腔牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）的药物，所述药物功能为如下（a1）至（a14）中的至少一项，具有广阔的应用前景。
（a1）抑制牙龈卟啉单胞菌的生长（图4A-C）；
（a2）抑制牙龈卟啉单胞菌/牙龈卟啉单胞菌生物膜的形成（图4A-C）；
（a3）抑制牙龈卟啉单胞菌生物膜基本结构的形成（图4A-C）；
（a4）抑制牙龈卟啉单胞菌生物膜的形成量（图4D） ；
（a5）降低细胞中肿瘤坏死因子-α的含量（图6A）；
（a6）降低细胞中白细胞介素-6的含量（图6B）；
（a7）降低细胞中白细胞介素-1β的含量（图6C）；
（a8）降低细胞中前列腺素E2的含量（图6D）；
（a9）降低细胞中一氧化氮的含量（图6E）；
（a10）降低细胞中炎症相关基因TNF-α的表达水平（图7A）；
（a11）降低细胞中炎症相关基因IL-1β的表达水平（图7B）；
（a12）降低细胞中炎症相关基因IL-6的表达水平（图7C）；
（a13）降低细胞中炎症相关基因COX-2的表达水平（图7D）；
（a14）降低细胞中炎症相关基因iNOS的表达水平（图7E）。
附图说明
图1 肠膜明串珠菌LVBH107的生长曲线，图1A为肠膜明串珠菌LVBH107在含有0.3%胃蛋白酶（pepsin）（pH=2、pH=3、pH=5）和牛胆盐（oxgall）（0.3%、0.5%，wt/vol）的MRS培养基中培养20 h的生长曲线，图1B为肠膜明串珠菌LVBH107在含有溶菌酶（lysozyme，100 μg/mL、200 μg/mL）和过氧化氢（H2O2，0.08 mM、0.8 mM）的MRS培养基中培养20 h的生长曲线，Control组为肠膜明串珠菌LVBH107在液体MRS培养基（无任何添加）的生长情况；横坐标为生长时间，纵坐标为600nm菌的紫外吸收值。
图2 肠膜明串珠菌LVBH107的自聚特性（A）和共聚特性（B）柱形图；纵坐标为自凝聚率（图2A）和交互凝聚率（图2B），横坐标为时间。
图3 肠膜明串珠菌LVBH107的表面疏水性（A）和粘附性（B）柱形图；
图3A表示为在二甲苯（Xylene）、氯仿（Chloroform）及乙酸乙酯（Ethyl acetate）中疏水性率。图3B表示为在人结肠癌细胞（HT-29）及人牙龈上皮细胞（HGE）的粘附率。
图4 肠膜明串珠菌LVBH107对牙龈卟啉单胞菌生物膜形成的影响照片；显微镜下观察结晶紫染色的牙龈卟啉单胞菌生物膜，培养48小时，图4A为空白对照组（仅添加MRS培养基），图4B为肠膜明串珠菌LVBH107菌液组，图4C为肠膜明串珠菌LVBH107上清液组（菌液离心过滤后的上清液），标尺20 μm，图4D为肠膜明串珠菌LVBH107对牙龈卟啉单胞菌生物膜形成的抑制率柱形图；横坐标为菌液（Culture medium）和上清液（Supernatant），纵坐标为抑制率。
图5 肠膜明串珠菌LVBH107对巨噬细胞RAW264.7增殖率的影响柱形图；*表示与空白对照组比较具有显著性差异（P<0.05），#表示肠膜明串珠菌LVBH107活菌（L-LVBH107）与热灭活菌（HK-LVBH107）比较具有显著性差异（P<0.05）；横坐标为对照组（Control）及不同浓度的菌液，纵坐标为细胞增殖率。
图6 肠膜明串珠菌LVBH107对巨噬细胞RAW264.7分泌炎症因子TNF-α（A）、IL-6（B）、IL-1β（C）、PGE2（D）、NO（E）浓度影响的柱形图，纵坐标为试剂盒检测各组TNF-α、IL-6、IL-1β、PGE2、NO的浓度，横坐标为不同浓度菌液及脂多糖（P.gingivalisLPS）。*表示与P.gingivalis脂多糖模型组比较具有显著性差异（P<0.05），#表示肠膜明串珠菌LVBH107活菌与热灭活菌比较具有显著性差异（P<0.05）；
图7 肠膜明串珠菌LVBH107对巨噬细胞RAW264.7分泌炎症因子TNF-α（A）、IL-6（B）、IL-1β（C）、COX-2（D）、iNOS（E）表达影响的柱形图，以β-actin作为内对照基因归一化处理实验数据，对各组TNF-α、IL-6、IL-1β、COX-2、iNOS基因mRNA的水平进行相对定量，横坐标为不同浓度菌液及脂多糖（P.gingivalisLPS），纵坐标为相对基因表达水平，*表示与P.gingivalis脂多糖模型组比较具有显著性差异（P<0.05），#表示肠膜明串珠菌LVBH107活菌与热灭活菌比较具有显著性差异（P<0.05）。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
液体MRS培养基：溶剂为水，含蛋白胨10g/L、牛肉膏10 g/L、酵母膏5 g/L、KH2PO42g/L、乙酸钠5 g/L、柠檬酸钠5 g/L、MgSO4·7H2O 0.2 g/L、MnSO4·4H2O 0.05 g/L、吐温-801mL/L、葡萄糖20 g/L，pH6.6。
固体MRS培养基与液体MRS培养基的区别仅在于在液体MRS培养基中加入15 g/L的琼脂。
牙龈卟啉单胞菌培养基：溶剂为水，含胰蛋白胨10.0 g/L、牛心浸粉17.5 g/L、氯化钠5.0 g/L、葡萄糖2.0g/L、Na2HPO4·12 H2O 2.5 g/L、酵母粉5.0 g/L、L-盐酸半胱氨酸0.4g/L，加水至1 000 mL，115 ℃灭菌20 min；冷却后加入维生素K1 0.01g/L，氯化血红素5.0 g/L。
牙龈卟啉单胞菌固体培养基：哥伦比亚血琼脂培养基（海博生物，货号：HB0124）。
实施例1：菌株的分离、鉴定和保藏
一、菌株的分离和纯化
样本取材：传统发酵食品辣白菜。
取辣白菜2.0 g用研钵研碎，加10.0 mL生理盐水稀释，取200 uL涂布于固体MRS培养基平板后37℃厌氧（85% N2，10% H2，5% CO2，均为vol/vol）培养24~48 h。挑取产黄圈的单菌落接种于固体MRS培养基平板上继续培养，并反复划线培养纯化，得到多株纯培养菌种。
纯培养菌种接种到液体MRS培养基中培养，连续活化3代后，菌液按照1:1（vol/vol）加入60%（vol/vol）甘油配制成的菌种甘油保藏液，-80℃冰箱保存。
二、菌株的鉴定
1. 生化鉴定
经革兰氏染色阳性、过氧化氢酶试验阴性、吲哚反应阴性、产硫化氢试验阴性、明胶液化试验阴性、水解淀粉试验阴性和硝酸盐还原试验阴性从菌种甘油保藏液中筛选出多株乳杆菌，将其中1株菌株命名为LVBH107。
肠膜明串珠菌LVBH107的生理生化鉴定结果：革兰氏阳性，过氧化氢酶阴性；不运动的杆菌；在15℃和45℃能够生长；耐受6.5%（wt/vol） NaCl水溶液；不水解淀粉，不液化明胶，不产生硫化氢；发酵葡萄糖产酸不产气；联苯胺试验阴性，吲哚试验阴性，乙酰甲基甲醇试验阳性。
肠膜明串珠菌LVBH107在液体MRS培养基中呈均匀浑浊生长，久置菌体呈白色沉淀。
肠膜明串珠菌LVBH107的最适生长温度37~42℃，适宜pH为5.0~7.0。
肠膜明串珠菌LVBH107的API 50CH（法国梅里埃公司）鉴定结果如表1所述。
表1：肠膜明串珠菌LVBH107的API 50CH鉴定结果
2. 分子生物学鉴定
采用PCR方法对肠膜明串珠菌LVBH107菌株进行16s rDNA鉴定。具体操作步骤如下：
（1）DNA的提取
按照TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0（货号：9763）说明书操作提取肠膜明串珠菌LVBH107菌株的DNA，将获得的菌株基因组DNA置于-20℃保存。
（2）PCR扩增引物
根据乳酸菌的16s rDNA序列的保守区域，采用16s通用引物，序列如下：
16s-Forward：5＇-AGAGTTTGATCCTGGCTCAG-3＇；
16s-Reverse：5＇-AGAAAGGAGGTGATCCAGC-3＇。
（3）PCR反应
按照TaKaRa EmeraldAmp® MAX PCR Master Mix（货号：RR320Q）说明书操作添加各组分，并进行PCR扩增反应，如表2和表3所述。
表2：PCR扩增反应体系
表3：PCR扩增程序
（4）检测16s rDNA扩增结果及纯化
预先制备2%（wt/vol）的琼脂糖凝胶，加入0.01%（vol/vol）的绿色荧光核酸染料（索莱宝，货号：G8140）。PCR扩增结束后，取5 µL PCR扩增产物和1 µL的6× DNA Loadingbuffer（索莱宝，货号：D1010）混合均匀，用移液器加入到琼脂糖凝胶板的点样孔中进行电泳，检测16srDNA扩增产物长度。目标片段长度约为1 500 bp，使用DL 2000的DNA Marker（索莱宝，货号：D1060）。电泳条件：1×TAE作为电泳液，电泳电压为100 V，电泳时间为40min。
取其余PCR扩增产物，按照TaKaRa MiniBEST DNA Fragment Purification KitVer.4.0（货号：9761）说明书进行纯化。
（4）16s rDNA基因序列分析
将纯化后的产物送到上海生工测序。获得菌株的16s rDNA序列通过BLAST程序与GenBank库中已知菌株的序列信息进行同源性比对分析，鉴定待测菌株。以16s rDNA的序列同源性大于99%为标准进行属种归类。
16s rDNA序列见序列1。
鉴定结果表明，菌株LVBH107为肠膜明串珠菌（Leuconostoc mesenteroides）。
三、菌株的保藏
肠膜明串珠菌（Leuconostoc mesenteroides）LVBH107，已于2020年12月14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），保藏编号为CGMCC No. 21362，分类命名为肠膜明串珠菌（Leuconostoc mesenteroides）。
实施例2：肠膜明串珠菌LVBH107的益生特性
一、肠膜明串珠菌LVBH107的耐受性
取培养16 h的肠膜明串珠菌LVBH107菌液，分别按3%（vol/vol）的接种量接种于含有0.3%（wt/vol）胃蛋白酶（pH=2、pH=3、pH=5）、牛胆盐（0.3%、0.5%，wt/vol）、溶菌酶（100 μg/mL、200 μg/mL）、过氧化氢（0.08 mM、0.8 mM）的液体MRS培养基中，厌氧培养20 h。分别取培养0 h、2 h、4 h、6 h、8 h、10 h、12 h、14 h、16 h、18 h、20 h菌液，紫外分光光度计测定菌液在600 nm处吸光值，其结果如图1所示。
从图1A可以看出，肠膜明串珠菌LVBH107在pH=5的0.3%胃蛋白酶、牛胆盐（0.3%、0.5%，wt/vol）中培养20 h后均能存 活。而在pH=2和pH=3的0.3%胃蛋白酶下，0~10 h肠膜明串珠菌LVBH107生长缓慢，但仍有一部分菌株存活。此外，肠膜明串珠菌LVBH107对溶菌酶（100 μg/mL、200 μg/mL）和过氧化氢（0.08 mM、0.8 mM）表现出良好的耐受能力（图1B）。Control组为肠膜明串珠菌LVBH107在液体MRS培养基（无任何添加）的生长情况。
二、肠膜明串珠菌LVBH107的自聚和共聚特性
取培养16 h的肠膜明串珠菌LVBH107菌液离心（4 000 r/min，4℃离心10 min），菌泥沉淀用PBS（0.01M，pH7.2-7.4）洗涤3次，重悬于PBS并调整600 nm波长处光密度值A0为1.0，以PBS为空白对照。37℃静置孵育2 h、4 h、6 h、8 h，每组样品做3个平行。紫外分光光度计测定上清液600 nm处吸光值，其自凝聚率结果如图2A所示。肠膜明串珠菌LVBH107菌株的自凝聚率计算公式：
式中：A0为600 nm波长处测得光密度值；At为不同静置时间点测得光密度值。
取培养16 h的肠膜明串珠菌LVBH107和牙龈卟啉单胞菌菌液离心（4 000 r/min，4℃离心10 min），弃上清并收集菌泥沉淀，用PBS（0.01M，pH7.2-7.4）洗涤3次，重悬于PBS并调整600 nm光密度值分别为0.5±0.05和0.6±0.05，两者各取500 μL混合后振荡（200 r/min, 5 min），37℃静置作用2h、4h、6h、8h，每组样品做3个平行。紫外分光光度计测定上清液600 nm处吸光值。其交互凝集率结果如图2B所示。肠膜明串珠菌LVBH107的交互凝集率计算公式：
式中：Ax为肠膜明串珠菌LVBH107的吸光值，Ay为牙龈卟啉单胞菌的吸光值，Amix为两种菌混合培养后的吸光值。
自聚初期（2 h）肠膜明串珠菌LVBH107的自聚集能力较弱（图2A），自凝集率为5.80%。2~8 h自凝集率逐渐增加，8 h时肠膜明串珠菌LVBH107自凝集率达到17.19%。由图2B的结果可以看出，2 h时肠膜明串珠菌LVBH107的共聚能力较低（24.73%），2~8 h的共聚能力明显增大，说明肠膜明串珠菌LVBH107开始与牙龈卟啉单胞菌发生相互凝集作用。8 h时肠膜明串珠菌LVBH107交互凝集率达到32.05%。
三、肠膜明串珠菌LVBH107的表面疏水性和粘附特性
采用微生物黏着碳氢化合物法测肠膜明串珠菌LVBH107表面疏水性，选择甲苯、二甲苯作为疏水性有机溶剂，测定肠膜明串珠菌LVBH107的表面疏水性；选择氯仿作为路易斯酸，乙酸乙酯作为路易斯碱，测定肠膜明串珠菌LVBH107表面所带电荷。将待测肠膜明串珠菌LVBH107离心（4 000 r/min，4℃离心10 min）收集菌泥，菌泥用PBS洗涤3次，重悬于KNO3缓冲液（0.1 mol/L，pH 6.2），调菌悬液的吸光度OD600为0.6，记作A0；向3 mL菌液中分别加入有机溶剂1 mL，先于室温下预孵育10 min，振荡2 min后继续在室温下孵育30 min。测定水相在600 nm处吸光度值，记为A1，疏水率的计算公式为：
肠膜明串珠菌LVBH107表面酸碱电荷的测定同上，将有机溶剂替换为乙酸乙酯和氯仿即可，其结果如图3A所示。
HT-29、HGE细胞生长状况良好时（70%～80%汇合度），以1×105个/孔的浓度接种至12孔板中培养。待细胞贴壁完全并达到70%～80%汇合度时，更换不含双抗的高糖DMEM培养液（索莱宝，货号：12110）培养24 h。PBS洗涤孔板中单层细胞3次，每个孔中加入500 μL浓度为1×108CFU/mL的肠膜明串珠菌LVBH107，孵育2 h后用PBS洗涤3次，除去游离未附着的肠膜明串珠菌LVBH107。每孔中加入2 mL 1%（vol/vol）Triton X-100（美国Sigma-Aldrich公司，货号：T8787）孵育10 min。收集每孔内溶液并分别进行10倍梯度稀释，采用平板菌落计数法检测培养后菌株的活菌数，其粘附率结果如图3B所示。肠膜明串珠菌LVBH107对细胞的黏附率计算公式：
式中：V0为添加的肠膜明串珠菌LVBH107活菌数（1×109CFU/mL），V1为粘附在细胞上的活菌数。
图3A显示的实验结果表明肠膜明串珠菌LVBH107对二甲苯的疏水率均为18.06%，具有较高的疏水性。肠膜明串珠菌LVBH107具有一定的静电作用力，对氯仿和乙酸乙酯表现出一定的黏附性，分别为30.95%和23.64%，表明菌株表面携带大量可产生静电作用力的电荷。如图3B所示，肠膜明串珠菌LVBH107对HT-29细胞的黏附率26.45%。为了进一步评价肠膜明串珠菌LVBH107在口腔内的粘附能力，考察了菌株对HGE细胞的粘附作用。肠膜明串珠菌LVBH107对口腔上皮细胞HGE的黏附率较高，黏附率为39.27%。
四、肠膜明串珠菌LVBH107的的抗生素敏感性
选择药敏纸片琼脂扩散法（K-B法）进行抗生素敏感性试验。无菌操作台内制备新的MRS琼脂培养基，室温凝固后均匀涂布1mL 1×108CFU/mL肠膜明串珠菌LVBH107菌液。将药敏纸片（比克曼生物）贴于琼脂表面，每培养皿贴4张，静置5 min之后，倒置培养皿并置于厌氧环境下培养48 h后测量抑菌圈的直径并记录，结果如表4、表5所述。
表4：药敏纸片种类、含药量及抑菌圈直径判断标准
注：表中S-susceptible，敏感；I-intermediate，中度敏感；R-resistance，耐药。
表5：肠膜明串珠菌LVBH107的药敏试验结果
注：表中S-susceptible，敏感；I-intermediate，中度敏感；R-resistance，耐药。
研究中采用药敏纸片琼脂扩散法考察乳酸菌对18种抗生素的敏感性。肠膜明串珠菌LVBH107的药敏试验结果见表5，肠膜明串珠菌LVBH107对红霉素、强力霉素、米诺环素、氨苄西林、氯霉素、头孢呋辛表现敏感，具有良好的安全性。肠膜明串珠菌LVBH107对青霉素、庆大霉素、链霉素、多粘菌素B、万古霉素、利福平、头孢唑啉、头孢他啶均表现耐药性，对四环素、哌拉西林、头孢哌酮、头孢曲松表现中度敏感。
实施例3：肠膜明串珠菌LVBH107对牙龈卟啉单胞菌生物膜的抑制作用
采用结晶紫染色法对牙龈卟啉单胞菌生物膜的形成能力进行半定量检测。取培养16 h的肠膜明串珠菌LVBH107培养液（culture medium），其中一部分4℃保存备用。另一部分离心（4 000 r/min，4℃离心10 min），收集上清液，并用0.22 μm滤膜过滤得到肠膜明串珠菌LVBH107上清液（supernatant）。将肠膜明串珠菌LVBH107培养液/上清液与牙龈卟啉单胞菌菌液（1×106CFU/mL）按照1:1的比例添加于24孔Transwell培养皿（美国Corning公司，货号：3413，滤膜孔径：0.4 μm）的上室和下室，添加MRS培养基（不添加肠膜明串珠菌LVBH107）作为空白对照，37℃厌氧培养48 h。培养结束后弃去培养液，PBS轻柔清洗3次，去除没有形成生物膜的浮游牙龈卟啉单胞菌，Transwell培养皿下室底部表面黏附的即为牙龈卟啉单胞菌生物膜。牙龈卟啉单胞菌生物膜用2.5%（vol/vol）戊二醛4℃过夜进行固定，PBS清洗3次后室温干燥，每孔加入100 μL 0.1%（wt/vol）结晶紫染色15 min，弃去孔中的结晶紫，并用75%（vol/vol）乙醇缓慢冲洗除去未结合的结晶紫，室温干燥。置于显微镜下观察牙龈卟啉单胞菌生物膜结构并拍照记录。采用酶标仪在540nm波长处测定其光密度值，并计算肠膜明串珠菌LVBH107对牙龈卟啉单胞菌生物膜形成的抑制率。牙龈卟啉单胞菌生物膜的抑制率计算公式：
式中：肠膜明串珠菌LVBH107菌液/上清液处理后的牙龈卟啉单胞菌生物膜的吸光度值（AS），无处理的牙龈卟啉单胞菌生物膜作空白对照（Ac）。
通过显微镜观察结晶紫染色的牙龈卟啉单胞菌生物膜的生长特征。空白对照组是牙龈卟啉单胞菌培养48 h形成的生物膜（图4A），大量菌株紧密聚集形成高度有组织的、不均匀的结构，构成了明显的生物膜立体结构，并有类似蘑菇状的微菌落。肠膜明串珠菌LVBH107培养液明显抑制了牙龈卟啉单胞菌的聚集，生物膜由单层牙龈卟啉单胞菌菌株构成，结构松散，无膜性结构形成（图4B）。肠膜明串珠菌LVBH107上清液抑制牙龈卟啉单胞菌生物膜的形成，牙龈卟啉单胞菌菌株排列松散，有较小的蘑菇状的微菌落形成，形成的生物膜不均匀且间隙大（图4C）。肠膜明串珠菌LVBH107培养液能抑制81.31%的牙龈卟啉单胞菌生物膜形成（图4D），上清液能抑制35.28%生物膜的形成。肠膜明串珠菌LVBH107培养液的作用效果明显优于上清液的作用效果。
实施例4：肠膜明串珠菌LVBH107对P.gingivalis脂多糖诱导的巨噬细胞RAW264.7炎症模型的抗炎作用
一、肠膜明串珠菌LVBH107对巨噬细胞RAW264.7增殖作用的影响
1. 肠膜明串珠菌LVBH107菌悬液制备
（1）活菌菌悬液的制备：取传代后的肠膜明串珠菌LVBH107于4℃ 4000 r/min离心5 min，用PBS洗涤三次，用无抗无血清的DMEM培养基重悬。调整菌悬液浓度为1×109CFU/mL、1×108CFU/mL、1×107CFU/mL。
（2）热灭活菌悬液的制备：取传代后的肠膜明串珠菌LVBH107离心条件为4℃ 4000r/min离心5 min，用PBS洗涤三次，用PBS重悬，菌悬液放入100 ℃水浴中加热15 min，进行热灭活处理。4℃ 4000 r/min离心10 min，弃上清PBS，用无抗无血清的DMEM培养基重悬。调整菌悬液浓度为1×109CFU/mL、1×108CFU/mL、1×107CFU/mL。
2. 肠膜明串珠菌LVBH107对巨噬细胞RAW264.7增殖率的检测
待巨噬细胞RAW264.7生长状况良好时（70%~80%汇合度），PBS洗涤3次后，添加1 mL0.25%（wt/vol）胰蛋白酶-EDTA进行消化5 min后弃去消化液，加入1 mL完全高糖DMEM培养基终止消化。转移至15 mL离心管中离心（1000 r/min，5 min），弃掉上清液，加入2 mL完全高糖DMEM培养基，充分吹打混合均匀后分成2瓶，每个T-25培养瓶中加入4 mL完全高糖DMEM培养基，置于细胞培养箱（37℃，5% CO2，90% RH）进行传代培养。
采用CCK-8试剂盒（美国美国APExBIO，货号 K1018）检测肠膜明串珠菌LVBH107对巨噬细胞RAW264.7增殖作用的影响。将巨噬细胞RAW264.7接种于96孔板，每孔5×103个细胞，每组6个副孔。待细胞贴壁完全并达到70%～80%汇合度时，弃去培养液，添加100 μL无抗无血清的DMEM培养基，分别添加100 μL肠膜明串珠菌LVBH107活菌（L-LVBH107）及热灭活菌（H-LVBH107）悬液。空白对照组添加等体积的无抗无血清的DMEM培养基做相同处理。置于培养箱（37℃，5% CO2，90% RH）中共培养6 h后，弃去培养液，用PBS洗涤3次，并在每孔中加入300 μL的CCK-8工作液，置于细胞培养箱孵育2~4 h后，每孔等体积转移至96孔板，并使用酶标仪在450 nm波长处测量吸光度。调零组为无抗无血清的DMEM培养基（无细胞）和CCK-8工作液添加的孔。
细胞增殖率=（实验组OD450–调零OD450）/（对照组OD450–调零OD450）×100%
肠膜明串珠菌LVBH107对巨噬细胞RAW264.7增殖的影响如图5所示，7 Log CFU/mL的肠膜明串珠菌LVBH107活菌和热灭活菌可明显提高细胞增值率，达到空白对照组细胞增值率的126.45%、116.63%（P<0.05）。8 Log CFU/mL的肠膜明串珠菌LVBH107活菌可明显提高细胞增值率，达到空白对照组细胞增值率的115.94%（P<0.05），肠膜明串珠菌LVBH107热灭活菌对细胞增值率提升不明显，达到空白对照组细胞增值率的103.76%。9 Log CFU/mL的肠膜明串珠菌LVBH107活菌和热灭活菌可明显降低细胞增值率，达到空白对照组细胞活力值增值率的87.23%、75.47%（P<0.05），抑制了巨噬细胞RAW264.7的增殖。因此选择7 Log CFU/mL和8 Log CFU/mL肠膜明串珠菌LVRAW264.7的增殖。结果表明，7 Log CFU/mL和8 LogCFU/mL肠膜明串珠菌LVBH107活菌和热灭活菌对巨噬细胞RAW264.7没有细胞毒性作用，9Log CFU/mL肠膜明串珠菌LVBH107活菌和热灭活菌可明显抑制巨噬细胞RAW264.7的增殖，因此选择7 LogCFU/mL和8 Log CFU/mL肠膜明串珠菌LVBH107活菌和热灭活菌进行后续试验。
二、肠膜明串珠菌LVBH107对P.gingivalis脂多糖诱导的巨噬细胞RAW264.7炎症模型的影响
1. 肠膜明串珠菌LVBH107对P.gingivalis脂多糖诱导的巨噬细胞RAW264.7分泌炎症因子的影响
将巨噬细胞RAW264.7接种于6孔板，每孔1×105个细胞，每组6个副孔，培养24 h贴壁后，弃去培养液。添加1.5 mL无抗DMEM培养基，分别加入肠膜明串珠菌LVBH107活菌菌悬液预处理3 h、热灭活菌菌悬液预处理6 h，再加入1 μg/mLP.gingivalis脂多糖刺激24 h（37℃，5% CO2，90% RH）。收集细胞及培养液于离心管，4℃ 3000 r/min离心10 min后取上清液保存。按照酶联免疫吸附测定（enzyme linkedimmunosorbent assay，ELISA）试剂盒说明书，根据双抗夹心法检测细胞培养液中肿瘤坏死因子α（Tumor Necrosis factor-α，TNF-α）、白细胞介素-6（Interleukin-6，IL-6）、白细胞介素-1β（Interleukin-1β，IL-1β）、前列腺素E2（Prostaglandin E2，PGE2）的浓度（江莱生物，货号：JL29365、JL18212、L18442、JL12640）。参照试剂盒说明书检测细胞培养液中一氧化氮（nitric oxide，NO）浓度（南京建成，货号：A013-2-1）。
P.gingivalis脂多糖刺激巨噬细胞RAW264.7后，明显促进细胞炎症因子TNF-α、IL-6、IL-1β的分泌（图6A、B、C）。7 Log CFU/mL和8 Log CFU/mL肠膜明串珠菌LVBH107预处理巨噬细胞RAW264.7后，不同程度的抑制炎症因子TNF-α、IL-6、IL-1β的分泌（图6A、B、C），缓解了RAW264.7细胞的炎症状态。与P.gingivalis脂多糖模型组相比，低剂量（7 Log CFU/mL）的肠膜明串珠菌LVBH107对炎症因子的抑制作用更显著。肠膜明串珠菌LVBH107活菌和热灭活菌相比，活菌的抑制作用效果优于热灭活菌（P<0.05）。
P.gingivalis脂多糖可明显增加巨噬细胞RAW264.7分泌PGE2、NO水平。7 LogCFU/mL和8 Log CFU/mL肠膜明串珠菌LVBH107预处理巨噬细胞RAW264.7后，不同程度的抑制PGE2的分泌（图6D），肠膜明串珠菌LVBH107活菌和热灭活菌相比，活菌的抑制作用效果优于热灭活菌（P<0.05）。与P.gingivalis脂多糖模型组相比，7 Log CFU/mL的肠膜明串珠菌LVBH107活菌和热灭活菌预处理巨噬细胞RAW264.7，明显抑制巨噬细胞RAW264.7分泌NO的能力（P<0.05），其中活菌对巨噬细胞分泌NO的抑制作用显著（P<0.05）（图6E）。
2. 肠膜明串珠菌LVBH107对P.gingivalis脂多糖诱导的巨噬细胞RAW264.7相关炎症因子基因表达的影响
将巨噬细胞RAW264.7接种于6孔板，每孔1×105个细胞，每组6个副孔，培养24 h贴壁后，弃去培养液。添加1.5 mL无抗DMEM培养基，分别加入肠膜明串珠菌LVBH107活菌菌悬液预处理3 h、热灭活菌菌悬液预处理6 h，再加入1 μg/mLP.gingivalis脂多糖刺激24 h（37℃，5% CO2，90% RH）。收集细胞及培养液于离心管中，4℃ 3000 r/min离心10 min后弃上清液，细胞沉淀用PBS清洗两次后，提取巨噬细胞RAW264.7的RNA，用实时荧光定量PCR方法检测巨噬细胞RAW264.7炎症相关基因TNF-α、IL-1β、IL-10、COX-2（环氧合酶-2，cyclooxygenase-2）、iNOS（一氧化氮合酶，Inducible nitric oxide synthase）的相对表达量。具体操作步骤如下：
（1）细胞总RNA提取
按照《TaKaRa Mini BEST Universal RNA Extraction Kit》（货号：9767）说明书提取巨噬细胞RAW264.7的RNA。
① 向培养细胞中加入600 μL的裂解Buffer RL（已加入50×DTT Solution），水平放置片刻，使裂解液均匀分布于细胞表面并裂解细胞，然后使用移液枪吹打细胞使其脱落。将内含细胞的裂解液转移至离心管中，用移液枪反复吹吸直至裂解液中无明显沉淀。裂解液静置2 min。
② 将gDNA Eraser Spin Column安放到2 mL的Collection Tube（试剂盒提供）上。将裂解液转移入到gDNAEraser Spin Column中。12 000 rpm，离心1 min。弃gDNAEraser Spin Column。保留2 mL Tube中的滤液。
③ 加入与液体等体积的70%乙醇（此时可能会出现沉淀），使用移液枪将溶液混合均匀。
④ 立即将混合液（含沉淀）全部转入到RNA Spin Column（含2 mL CollectionTube）中。12 000 rpm，离心1 min，弃滤液。
⑤ 将500 μL的Buffer RWA加入至RNA Spin Column中，12 000 rpm离心30 s，弃滤液。
⑥ 将600 μL的Buffer RWB加入至RNA Spin Column中，12 000 rpm离心30 s，弃滤液。
⑦ 将RNA Spin Column重新安置于2 mL Collection Tube上，12 000 rpm离心2min。
⑧ 将RNA Spin Column安置于1.5 mL的RNaseFree Collection Tube上，在RNASpin Column膜中央处加入50～200 μL的RNase Free dH2O或0.1% DEPC处理水，室温静置5min。
⑨ 12 000 rpm离心2 min洗脱RNA。
（2）参照《PrimeScript™ RT reagent Kit with gDNA Eraser（Perfect RealTime）》（货号：RR047A）说明书的步骤将RNA逆转录合成cDNA。
① 去除基因组DNA反应如表6所述
表6：去除基因组DNA反应体系
| 组分 | 体积 |
| 5×gDNA Eraser Buffer | 2.0 μL |
| gDNA Eraser | 1.0 μL |
| Total RNA | 1.0 μg |
| RNase Free dH2O | 补至10 μL |
混合后充分混匀，42 ℃孵育2 min。
② 反转录反应如表7所述
表7：反转录反应体系
| 组分 | 体积 |
| 步骤1的反应液 | 10.0 μL |
| PrimeScript RT Enzyme Mix I | 1.0 μL |
| RT Primer Mix | 1.0 μL |
| 5×PrimeScript Buffer 2 | 4.0 μL |
| RNase Free dH2O | 4.0 μL |
| Total | 20.0 μL |
上述各试剂充分混匀，37 ℃反应15 min，85 ℃反应5 s后取出。取5 μL逆转录产物用DEPC水稀释至100 μL，即为DNA模板。
（3）按照《TB Green® Premix Ex Taq™ II （TliRNaseH Plus）》说明书进行实时荧光定量PCR（Quantitative Real-time PCR）反应，检测巨噬细胞RAW264.7炎症相关基因TNF-α、IL-1β、IL-10、COX-2、iNOS的相对表达量。通过2-ΔΔCt方法（∆Ct=Ct目的基因-Ct内参基因，∆∆Ct=∆Ct实验-∆Ct对照）对基因的差异表达水平进行定量分析。
① 引物序列如表8所示
表8：相关引物序列
荧光定量PCR反应如表9、表10所示
表9：荧光定量PCR反应体系
| 组分 | 体积 |
| DNA模板 | 2.0 μL |
| 上游引物（10 μM） | 0.5 μL |
| 下游引物（10 μM） | 0.5 μL |
| 2×SYBR Green qPCR Mixture | 10.0 μL |
| DEPC-ddH2O | 7.0 μL |
| Total | 20.0 μL |
表10：荧光定量PCR反应程序
如图7所示，P.gingivalis脂多糖刺激巨噬细胞RAW264.7后，促进细胞炎症因子TNF-α（图7A）、IL-6（图7B）、IL-1β（图7C）、COX-2（图7D）、iNOS（图7E）的表达。7 Log CFU/mL的肠膜明串珠菌LVBH107活菌和热灭活菌预处理巨噬细胞RAW264.7，明显抑制巨噬细胞RAW264.7炎症因子TNF-α、IL-6、IL-1β、COX-2、iNOS基因的表达，活菌对巨噬细胞炎症因子基因的抑制作用最明显（P<0.05）。8 Log CFU/mL肠膜明串珠菌LVBH107活菌预处理巨噬细胞RAW264.7后，不同程度的抑制炎症因子TNF-α、IL-6、IL-1β、COX-2、iNOS基因的表达（P<0.05）。8 Log CFU/mL肠膜明串珠菌LVBH107热灭活菌对巨噬细胞炎症因子基因没有明显的抑制作用。
序列表
<110> 吉林大学
<120> 肠膜明串珠菌及其用于制备抑制牙龈卟啉单胞菌的口腔护理用品、药品
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1497
<212> DNA
<213> Leuconostoc mesenteroides LVBH107
<400> 1
agctataatg cagtcgaacg ctttgtcttt aactgatctg acgagcttgc tctgatttga 60
ttttatctga caaagagtgg cgaacgggtg agtaacacgt gggtaaccta cctcttagca 120
ggggataaca tttggaaaca agtgctaata ccgtataata ccaacaaccg catggttgtt 180
ggttgaaaga tggttctgct atcactaaga gatggacccg cggtgcatta gctagttggt 240
aaggtaacgg cttaccaagg caatgatgca tagccgagtt gagagactga tcggccacaa 300
tgggactgag acacggccca tactcctacg ggaggcagca gtagggaatc ttccacaatg 360
ggcgcaagcc tgatggagca acgccgcgtg tgtgatgaag ggtttcggct cgtaaaacac 420
tgttataaga gaagaacggc actgagagta actgttcagt gtgtgacggt atcttaccag 480
aaaggaacgg ctaaatacgt gccagcagcc gcggtaatac gtatgttcca agcgttatcc 540
ggatttattg ggcgtaaagc gagcgcagac ggttatttaa gtctgaagtg aaagccctca 600
gctcaactga ggaatggctt tggaaactgg atgacttgag tgcagtagag gaaagtggaa 660
ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct 720
ttctggactg taactgacgt tgaggctcga aagtgtgggt agcaaacagg attagatacc 780
ctggtagtcc acaccgtaaa cgatgagtgc tagatgttcg agggtttccg cccttgagtg 840
tcgcagctaa cgcattaagc actccgcctg gggagtacga ccgcaaggtt gaaactcaaa 900
ggaattgacg gggacccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa 960
gaaccttacc aggtcttgac atcccttgct aatcctagaa ataggacgtt cccttcgggg 1020
acaaggtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080
cccgcaacga gcgcaaccct tattattagt tgccagcatt tagttgggca ctctagtgag 1140
actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200
cctgggctac acacgtgcta caatggcata tacaacgagt cgccaacccg cgagggtgcg 1260
ctaatctctt aaagtatgtc tcagttcgga ttgtaggctg caactcgcct acatgaagtc 1320
ggaatcgcta gtaatcgcgg atcagcacgc cgcggtgaat acgttcccgg gtcttgtaca 1380
caccgcccgt cacaccatga gagtttgtaa cacccaaagc cggtggggta accttttagg 1440
agccagccgt ctaaggtggg acagatgatt agggtgaagt cgtagcaaga gcccggc 1497
Claims (5)
1.一种肠膜明串珠菌LVBH107，于2020年12月14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. 21362，分类命名为Leuconostoc mesenteroides。
2.权利要求1所述的肠膜明串珠菌LVBH107在制备抑制牙龈卟啉单胞菌生物膜形成的药物中的应用。
3.一种肠膜明串珠菌LVBH107菌剂，其特征在于：是由含有权利要求1所述的肠膜明串珠菌LVBH107的菌液制备得到，肠膜明串珠菌LVBH107的浓度为10 6CFU/g以上。
4.如权利要求3所述的一种肠膜明串珠菌LVBH107菌剂，其特征在于：肠膜明串珠菌LVBH107的浓度为106~1010CFU/g。
5.权利要求3或4所述的肠膜明串珠菌LVBH107菌剂在制备抑制牙龈卟啉单胞菌生物膜形成的药物中的应用。
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|---|---|---|---|---|
| JP2010053062A (ja) * | 2008-08-28 | 2010-03-11 | Sunstar Inc | 口腔用組成物 |
| CN107158040A (zh) * | 2017-04-12 | 2017-09-15 | 华南农业大学 | 一种口腔微生态制剂及其制备方法 |
| KR20190143580A (ko) * | 2018-06-21 | 2019-12-31 | 동의대학교 산학협력단 | 치주질환 예방 및 개선용 조성물 |
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| JP2010053062A (ja) * | 2008-08-28 | 2010-03-11 | Sunstar Inc | 口腔用組成物 |
| CN107158040A (zh) * | 2017-04-12 | 2017-09-15 | 华南农业大学 | 一种口腔微生态制剂及其制备方法 |
| KR20190143580A (ko) * | 2018-06-21 | 2019-12-31 | 동의대학교 산학협력단 | 치주질환 예방 및 개선용 조성물 |
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