CN113957006B - 一种植物乳杆菌n13及其在预防或治疗龋齿和牙周炎上的应用 - Google Patents
一种植物乳杆菌n13及其在预防或治疗龋齿和牙周炎上的应用 Download PDFInfo
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- CN113957006B CN113957006B CN202111137085.4A CN202111137085A CN113957006B CN 113957006 B CN113957006 B CN 113957006B CN 202111137085 A CN202111137085 A CN 202111137085A CN 113957006 B CN113957006 B CN 113957006B
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Abstract
本发明涉及微生物技术领域,具体涉及一种植物乳杆菌N13及其在预防或治疗龋齿和牙周炎上的应用。本发明提供的植物乳杆菌N13具有较强的抑制变异链球菌生长的能力。对溶菌酶有较高的耐受浓度,易于在口腔中定植。植物乳杆菌N13还具有较强自聚能力,以及与变异链球菌的共聚能力。本发明提供植物乳杆菌N13在制备功能食品、药物或护理用品上的应用。所述功能食品包含但不限于固体饮料、口含片、饼干、软糖、茶、咖啡及发酵乳制品等。所述药物可以为药学上可接受的剂型,包括喷雾剂、片剂、胶囊剂、口服液或冻干粉等。所述护理用品包含但不限于为口气清新喷雾、抑菌液、牙膏、漱口水、涂氟剂或洁牙粉等。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一种植物乳杆菌N13及其在预防或治疗龋齿和牙周炎上的应用。
背景技术
口腔是微生物进入人体的有效通道,口腔微生物群是人体第二大微生态系统,含有大量病原微生物,造成了多种口腔疾病,常见的有龋齿、牙周炎,口腔溃疡等。随着现代生活水平的提高,人们摄取的营养越来越丰富,营养丰富的食物残渣在口腔里成为了微生物生长的沃土,其中不乏大量病原微生物的过度生长。变异链球菌(Streptococcusmutans)就是其中的一种,变异链球菌被公认为是口腔内主要的致龋菌,该菌不仅具有较强的产酸耐酸性,而且能够利用蔗糖产生不溶性胞外多糖,进而促进大量菌群在牙齿表面聚集,形成牙菌斑生物膜,导致龋齿,由于龋齿经常疼痛,导致咀嚼功能降低,胃肠消化吸收减弱,造成机体营养不良,影响身心健康。如果不及时治疗龋齿还会引发感染性疾病如关节炎,肾炎,心肌炎,长期低热等。而牙龈卟啉单胞菌是研究广泛且证据充足引发牙周病的一种主要细菌,它们会把对牙齿有益的细菌挤走,并取代有益菌的位置。口腔内含有牙龈卟啉单胞菌对健康非常不利。牙龈卟啉单胞菌会引起牙周发炎,出现口臭,牙龈出血,牙龈流脓,伴随牙槽骨萎缩,牙周病严重者还会引发冠心病,消化道疾病,关节炎,虹膜炎等。
鉴于龋齿病和牙周炎的发生与口腔菌群失调密切相关,利用益生菌来调整改善口腔微生态平衡,减少病原菌的生长、聚集,是预防和(或)治疗龋齿和牙周炎的的有效途径。目前龋齿,牙周炎通常是局部口腔外科治疗并配合使用抗生素,而常用抗生素药对身体副作用很大,并且会产生抗药性;而常规的牙周刮治,翻开牙龈刮除牙结石,过程痛苦,也只能延长牙周炎复发的周期,同样无法避免其复发,因为口腔污垢很容易通过萎缩的牙龈重新进入龈下,再次形成牙结石,牙周炎仍会复发。综上,龋齿和牙周炎在外科治疗上对患者而言过程痛苦,使用药物疗法效果不明显还会产生细菌抗药性,因此在有效预防和(或)治疗龋齿和牙周炎方面需要继续研究。
发明内容
本发明的目的在于提供一种植物乳杆菌N13及其在预防或治疗龋齿和牙周炎上的应用。
本发明涉及的植物乳杆菌N13,其微生物保藏号为CGMCCNo .20496;分类命名为:Lactobacillusplantarum;保藏时间:2020年08月06日;保藏地址:北京市朝阳区北辰西路1号院3号;保藏单位:中国微生物菌种保藏管理委员会普通微生物中心。
实验证明,本发明提供的植物乳杆菌N13具有较强的抑制变异链球菌生长的能力。
本发明提供的植物乳杆菌N13在口腔应用特性测定显示对溶菌酶有较高的耐受浓度,远高出人口腔唾液中溶菌酶的浓度1~57μg/mL,该菌株具备在口腔环境良好存活的能力,易于在口腔中定植。
本发明提供的植物乳杆菌N13还具有较强自聚能力,以及与变异链球菌的共聚能力。
本发明所提供的植物乳杆菌N13能抑制病原菌变异链球菌和牙龈卟啉单胞菌的生物膜的形成,显示该菌株能有效降低病原菌在口腔的黏附作用。
本发明提供的植物乳杆菌N13能显著降低牙周炎造模引起的大鼠体重下降,减少牙周炎造模引起的口腔内牙龈卟啉单胞菌数量的上升。
本发明提供植物乳杆菌N13在预防和(或)治疗龋齿和牙周炎上的应用。
特别的,本发明提供植物乳杆菌N13在制备功能食品上的应用。所述功能食品保护但不限于固体饮料、口含片、饼干、软糖、茶、咖啡及发酵乳制品等。
特别的,本发明提供植物乳杆菌N13在制备预防或治疗龋齿和牙周炎的药物上的应用。所述的预防或治疗龋齿和牙周炎的药物可以为药学上可接受的剂型,包括喷雾剂、片剂、胶囊剂、口服液或冻干粉等。
特别的,本发明提供植物乳杆菌N13在制备预防或治疗龋齿和牙周炎的护理用品上的应用。所述的预防或治疗龋齿和牙周炎的护理用品包含但不限于为口气清新喷雾、抑菌液、牙膏、漱口水、涂氟剂或洁牙粉等。
本发明的有益效果是:
本发明提供的植物乳杆菌N13具有对口腔致病菌变异链球菌较强的抑制能力,对溶菌酶有较高的耐受能力。同时具有较强自聚能力,以及与变异链球菌的共聚能力,能抑制病原菌生物膜的形成,显示该菌株能有效降低病原菌在口腔的黏附作用;上述植物乳杆菌N13通过大鼠牙周炎造模实验,有效降低牙龈卟啉单胞菌的定植量,因此,该菌株具有改善口腔的微生态体系,预防和(或)治疗龋齿和牙周炎。
附图说明
图1植物乳杆菌N13的自聚能力。
图2植物乳杆菌N13与病原菌的共聚能力。
图3植物乳杆菌N13对大鼠体重的影响。
图4不同组的牙周炎大鼠口腔中牙龈卟啉单胞菌定植情况。
具体实施方式
下面结合实施例对本发明做进一步详细的阐述,但本发明的实施方式不限于此。
实施例一植物乳杆菌N13抑制口腔病原菌的实验
本实施例中所用的变异链球菌ATCC25175,购自中国普通微生物菌种保藏管理中心。牙龈卟啉单胞菌ATCCBAA-308购自广东省微生物菌种保藏中心。
MRS培养基成分:酵母粉5.0g/L、牛肉膏10.0g/L、蛋白胨10.0g/L、葡萄糖20.0g/L、无水乙酸钠2.0g/L、柠檬酸氢二铵2.0g/L、三水合磷酸氢二钾2.6g/L、一水合硫酸锰0.25g/L、七水合硫酸镁0.5g/L和吐温-801mL/L,pH 6.2~6.4。
BHI培养基成分:牛脑浸粉4.0g/L、牛心浸粉4.0g/L、蛋白胨5.0g/L、酪蛋白胨16.0g/L、氯化钠5.0g/L、葡萄糖2.0g/L、十二水合磷酸氢二钠2.5g/L,pH值7.2~7.4。
菌株的活化:将植物乳杆菌N13甘油管菌株以MRS液体培养基体积计按1-3%接种于MRS液体培养基中,37℃培养24h后,得到发酵液。
病原菌菌悬液制备:变异链球菌按照5%(V/V)接种至BHI液体培养基中,37℃培养16~20h后,调节菌液浓度为109CFU/ml。
植物乳杆菌N13抑制口腔病原菌的实验:将BHI固体培养基冷却至55℃左右,与病原菌菌悬液按一定比例混匀,使体系病原菌活菌数在106CFU/mL数量级,然后迅速倾注于预先放置牛津杯的平板中,待培养基冷却凝固后,取出牛津杯,于每孔注入200μL的植物乳杆菌N13发酵上清液,将平皿轻盖后正置于37℃恒温培养箱,培养适宜时间后观察,并用游标卡尺测量抑菌圈直径。
表1植物乳杆菌N13对口腔病原菌的抑菌圈直径
结果表明由表1显示,植物乳杆菌N13对变异链球菌有显著的抑制作用,说明该菌株能抑制口腔病原菌的生长,改善口腔菌群环境,可预防龋齿口腔疾病。
实施例二植物乳杆菌N13对溶菌酶的耐受性检测
将植物乳杆菌N13甘油管菌株以MRS液体培养基体积计按1~3%接种于MRS液体培养基中,37℃培养24h后,调整菌悬液浓度为108CFU/mL。取200ul上述植物乳杆菌N13菌液于MRS平板上进行涂布,待吸收后将灭过菌的牛津杯轻轻放置在涂有菌液的MRS平板上。然后在牛津杯中加入100uL不同浓度的溶菌酶溶液(0.1mg/ml,0.3mg/ml,0.5mg/ml,0 .7mg/ml,1mg/ml,2mg/ml) ,以不含溶菌酶的无菌水为对照,每组设置3个平行。将上述平板平稳放置在37℃厌氧培养箱培养16~20h,检测植物乳杆菌对溶菌酶的抑菌圈大小。抑菌圈大小表示植物乳杆菌N13可以耐受该浓度的溶菌酶。
表2植物乳杆菌N13对溶菌酶的耐受性
实验结果由表2显示,植物乳杆菌N13可以耐受质量浓度为1mg/ml的溶菌酶,而人体口腔中溶菌酶的质量浓度(1~57μg/mL) ,表明植物乳杆菌N13具备在口腔环境中存活的能力。
实施例三植物乳杆菌N13的自聚和与病原菌共聚能力
1、自聚能力:将培养好的植物乳杆菌N13菌液,离心,收集菌体,用PBS缓冲液冲洗2次后,再用PBS重悬,调至OD600=0.6。取40mL细胞悬浊液于50mL离心管中,涡旋充分混合后置于37℃条件下孵育,菌悬液孵育2h、5h、21h、24h时后,分别小心吸取上层菌液2.5mL,测定菌悬液在600nm处的吸光值OD,计算菌株自聚力。自聚力%=1-(At/Ao) ,其中Ao=起始吸光值,At=取样时的吸光值。
2、共聚能力:植物乳杆菌N13按3%接种量取菌液接种于MRS液体培养基,37℃培养24h,菌液备用。口腔病原菌按1%接种量接种至BHI培养基中,37℃培养24h,菌液待用。分别将培养好的植物乳杆菌N13和病原菌,离心,收集植物乳杆菌菌体及病原菌菌体,用PBS缓冲液冲洗2次后,再用PBS重悬,调至植物乳杆菌N13的OD600值为0.60,病原菌的OD600值为0.4,充分震荡混匀,分别测定初始OD600的值Ax、Ay,植物乳杆菌N13与病原菌各取20mL等体积至50mL离心管中混匀,涡旋震荡20S后37℃静置孵育,菌悬液孵育2h、5h、21h、24h时,分别小心吸取上层液体2.5mL,测定菌悬液在600nm处的吸光值(A) ,计算菌株与病原菌的共聚力。共聚力%=[(Ax+Ay)/2-A(x+y)]/[Ax+Ay/2]×100,其中x和y分别代表两种菌株,(x+y)代表混合物。
结果如图1、图2所示,植物乳杆菌N13具有较强的自聚能力,随着时间的延长,自聚能力增强。植物乳杆菌N13还具有较强的与口腔病原菌的共聚能力,随着时间的延长,共聚能力增强,在24h时共聚能力为67%。说明植物乳杆菌N13能够通过共聚方式去除病原菌,更有利发挥益生作用,从而达到改善口腔微生态的作用。
实施例四抗生素敏感性实验
将植物乳杆菌N13在MRS固体平板上划线活化后,挑取菌苔至生理盐水中制备菌悬液,调整菌悬液浓度为108CFU/mL,取100μL菌悬液,用无菌棉签均匀涂布于MRS固体平板上,将抗生素药敏试纸片有序置于平板表面,置于厌氧条件下,37℃培养24~36h后,用游标卡尺测量抑菌圈直径。根据美国临床和实验标准协会CLSI的评价标准,判断抗生素对植物乳杆菌的耐药性。
表3植物乳杆菌N13对抗生素的敏感性
实验结果见表3,植物乳杆菌N13对13种常用抗生素敏感,对四环素中度敏感,表明植物乳杆菌N13是安全的益生菌菌种。
实施例五植物乳杆菌N13生物膜形成能力和对病原菌生物膜抑制能力
按照上述实施例活化植物乳杆菌和病原菌,植物乳杆菌N13和病原菌的菌液浓度调节至OD600=0.5;同时将菌液分为3组,一组益生菌菌液,一组病原菌菌液,最后一组益生菌和病原菌液按1∶1的比例混合制成悬液。分别将三组菌液取200uL添加到96孔板中,于37℃培养箱厌氧培养24h,小心吸去各孔的培养基,去掉游离细菌,每孔加入200μL的PBS缓冲液清洗3次,并在室温静置晾干后每孔中加入100μL的浓度为1%结晶紫溶液,室温下染色15min,使黏附的细菌着色;倾去染色液后,用PBS缓冲液洗涤4次,干燥后每孔中加入100μL浓度为95%的乙醇溶解染色后的生物膜,25℃摇床低速水平摇晃20min进行脱色,脱色结束后,用酶标仪在OD550nm处读取吸光度值,即为所得数值。
表5植物乳杆菌N13生物膜形成能力和对病原菌生物膜抑制能力
实验结果如表5所示:植物乳杆菌N13的OD550处吸光值低于变异链球菌的吸光值;植物乳杆菌与病原菌混合液OD550处的吸光值低于变异链球菌的吸光值;而经乙醇洗脱后的生物膜上的结晶紫含量反映了生物膜的厚度,吸光值越低表明生物膜形成能力越低,从检测的数据可以看出植物乳杆菌N13形成生物膜的能力较低,而植物乳杆菌N13与口腔病原菌混合液的检测结果表明植物乳杆菌N13可以降低变异链球菌和牙龈卟啉单胞菌的生物膜形成能力,从而减少病原菌在牙齿上的附着,降低对牙齿的破坏。
实施例六植物乳杆菌N13对大鼠牙周炎的缓解作用
取雄性、5周龄左右的SPF级Wistar大鼠30只,在温度和湿度均恒定的屏障环境中,按12h光照和12h黑夜进行饲养。所有大鼠前一星期适应环境和饮食,之后按每组平均体重一致的原则随机分成3组,每组10只,分别是空白对照组(空白组)、牙周炎模型组(模型组)和益生菌干预组(益生菌组) ,实验周期维持5周。空白对照组在实验期间喂食正常饲料和饮用水,除空白组外,模型组和益生菌组均进行牙周炎造模,持续一周,造模期间,每天擦拭牙龈卟啉单胞菌菌悬液(菌悬液浓度109CFU/mL)200μl,擦拭后并停止喂食及饮水1h,确定其在大鼠口腔成功定植,即造模成功。造模成功后,益生菌干预组每周3次擦拭植物乳杆菌N13 菌悬液,直至第5周实验结束。在大鼠臼齿上擦拭,即将饱和吸收菌液的棉拭子在大鼠口腔牙齿的每四分之一部位统一作用时间为15s。实验期间,牙周炎模型组和益生菌干预组均饲喂饲料Keyes2000和添加有10%蔗糖的饮用水。其中Keyes2000饲料成分(w/w) :小麦粉6%,奶粉28%,蔗糖56%,酵母4%,苜蓿粉3%,肝粉1%,盐2%。
(1)植物乳杆菌N13对大鼠体重的影响
大鼠实验前后体重测定结果如图3所示。实验结束后,大鼠体重的顺序是空白组>益生菌组>模型组,空白组平均体重显著高于模型组,牙周炎能够明显影响模型组大鼠的进食造成体重下降;相较于模型组,益生菌组可以缓解牙周炎造成的大鼠体重下降,体重较模型组增加了81g。
(2)植物乳杆菌N13对牙周炎致病菌的定植情况
实验期间,在造模结束后的每一周分别给大鼠口腔牙齿进行取样,共取样四次。将取得样品稀释涂布于相应固体平板进行活菌计数,作为致病菌定植检验。菌落计数用到的固体平板为:添加有12μg/mL氨苄青霉素和5%(v/v)脱纤维羊血的BHI固体培养基,并结合菌落形态特征对大鼠口腔中的牙龈卟啉单胞菌进行计数。
考察不同时期取样各组实验大鼠口腔牙龈卟啉单胞菌的计数结果,由图4可知,模型组在第2次到第4次取样时,牙龈卟啉单胞菌的定植量始终保持在106CFU/mL。益生菌组在第1次到第2次取样时,牙龈卟啉单胞菌的定植量相较于模型组下降了2个数量级;在第3次到第4次取样时,可以控制牙龈卟啉单胞菌在大鼠口腔中定植的生物量在103CFU/mL。说明益生菌干预后显著降低了牙龈卟啉单胞菌在口腔中定植,有效改善口腔环境,起到预防和(或)治疗牙周炎的作用。
实施例七植物乳杆菌N13在固体饮料的应用
植物乳杆菌N13固体饮料配方:以100g固体饮料计:低聚果糖38g,植物乳杆菌N13菌粉5g,果粉35g,聚葡萄糖粉15g,麦芽糖醇粉6g,柠檬酸1g。
植物乳杆菌N13固体饮料制作方法:
(1)将上述原料按比例进行配制,混合均匀后过100目筛倒入混匀机,充分混匀。然后将混匀的原料转入罐装设备,罐装封口后即为益生菌固体饮料产品。
(2)取固体饮料产品进行活菌稀释计数的检测,植物乳杆菌N13的活菌数大于1×1010CFU/g。
实施例八植物乳杆菌N13在牙膏方面的应用
本实施例涉及预防和(或)治疗龋齿和牙周炎植物乳杆菌N13在牙膏方面的应用。
牙膏各组分的重量分数:山梨糖醇8份,去离子水57份,聚乙二醇8份,甘油5份,二氧化硅12份,月桂醇硫酸酯钠2份,黄原胶6份,糖精钠5份,羟基磷灰石10份,焦磷酸四钠5份,羟丙基瓜尔胶3份,硅酸镁锂3份,羟苯甲酯1份,香精1份,植物乳杆菌N136份,二氧化钛0.5 份。
制备方法:先将香料、活性添加剂、稳定剂、糖精在预溶锅中用纯净水溶解至均匀,加入制膏锅中,然后将粉料二氧化硅加入制膏锅中,再添加香精,进行刮板搅拌、均质搅拌、研磨;在捏和、研磨过程中进行抽真空,直至真空度达-0 .096Mpa为止,时间约为50分钟。捏合、研磨结束后,打出少量的膏体进行检验,合格后,将膏体打入储存锅进行陈化,使物料自然冷却至常温,同时使物料充分膨胀形成均相的粘合体,提高物料的弹性,陈化时间约为120分钟。在制膏过程中,因捏和、研磨过程会磨擦放热,故在夏季需用夹套冷却水控制温度≤45℃。将陈化后的膏体转入自动罐装线上进行罐装,罐装完成后包装入库。
实施例九植物乳杆菌N13在口腔喷雾剂方面的应用
本实施例涉及预防和(或)治疗龋齿和牙周炎植物乳杆菌N13在口腔喷雾剂方面的应用。
口腔喷雾剂的重量成分组成:植物乳杆菌N13发酵液9份,碳酸氢钠2份,苯甲酸钠2份,维生素C3份,薄荷油3份,柠檬油1份,透明质酸3份,黄原胶2份,甘油1份,羟丙基甲基纤维素1份。
口腔喷雾剂的制备方法:按照成分组成配比称取植物乳杆菌N13,碳酸氢钠,苯甲酸钠,维生素C,透明质酸,甘油于烧杯中,加适量纯水搅拌均匀至所有成分溶解,再加入薄荷油,柠檬油,黄原胶,羟丙基甲基纤维素,将以上混合物于45℃水浴锅中加热搅拌,加热过程中加水补足水至所需体积,直至所有成分完全溶解,所得溶液即为本实施例中的口腔喷雾剂。
Claims (7)
1.一种植物乳杆菌(Lactobacillus plantarum)N13,其微生物保藏号为CGMCC No.20496;分类命名为:Lactobacillus plantarum。
2.权利要求1所述的植物乳杆菌N13在制备功能食品上的应用,所述功能食品包含固体饮料、口含片、饼干、软糖、茶、咖啡或发酵乳制品。
3.根据权利要求2所述的应用,其特征在于,固体饮料中植物乳杆菌N13的活菌数大于1×1010CFU/g。
4.权利要求1所述的植物乳杆菌N13在制备预防或治疗龋齿和牙周炎的药物上的应用,所述药物选自:口腔喷雾剂、片剂、胶囊剂、口服液或冻干粉。
5.根据权利要求4所述的应用,其特征在于,所述口腔喷雾剂的重量成分组成:植物乳杆菌N13发酵液9份,碳酸氢钠2份,苯甲酸钠2份,维生素C3份,薄荷油3份,柠檬油1份,透明质酸3份,黄原胶2份,甘油1份,羟丙基甲基纤维素1份。
6.权利要求1所述的植物乳杆菌N13在制备预防或治疗龋齿和牙周炎的护理用品上的应用,所述护理用品选自:口气清新喷雾、抑菌液、牙膏、漱口水、涂氟剂或洁牙粉。
7.根据权利要求6所述的应用,其特征在于,所述牙膏各组分的重量分数:山梨糖醇8份,去离子水57份,聚乙二醇8份,甘油5份,二氧化硅12份,月桂醇硫酸酯钠2份,黄原胶6份,糖精钠5份,羟基磷灰石10份,焦磷酸四钠5份,羟丙基瓜尔胶3份,硅酸镁锂3份,羟苯甲酯1份,香精1份,植物乳杆菌N13 6份,二氧化钛0.5份。
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