CN114149939B - 一种能够缓解龋齿的益生菌组合物及其应用 - Google Patents
一种能够缓解龋齿的益生菌组合物及其应用 Download PDFInfo
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- CN114149939B CN114149939B CN202111135303.0A CN202111135303A CN114149939B CN 114149939 B CN114149939 B CN 114149939B CN 202111135303 A CN202111135303 A CN 202111135303A CN 114149939 B CN114149939 B CN 114149939B
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Abstract
本发明公开了一种能够缓解龋齿的益生菌组合物及其应用，属于微生物技术领域以及医药技术领域。本发明的唾液乳杆菌LS97、植物乳杆菌N13、副干酪乳杆菌LC86和嗜酸乳杆菌LA85的组合物能够预防和/或治疗龋齿，具体体现在：（1）这一组合物可以使大鼠口腔中引起龋齿的主要病原菌变异链球菌数量减少；（2）这一组合物可以使大鼠龋齿程度减轻；（3）这一组合物可以降低大鼠血清炎症因子TNF‑ɑ、IL‑1β、IL‑6的水平。因此，含有唾液乳杆菌LS97、植物乳杆菌N13、副干酪乳杆菌LC86和嗜酸乳杆菌LA85的组合物在预防和/或治疗龋齿方面具有很大的应用前景。
Description
技术领域
本发明涉及一种能够缓解龋齿的益生菌组合物及其应用，属于微生物技术领域以及医药技术领域。
背景技术
龋齿病是由口腔致龋细菌引起的慢性感染疾病，变异链球菌(Streptococcusmutans,S.mutans)是口腔中的最常见致龋细菌。世界范围内龋齿病和牙周病的患病率约为80％～90％，国内3-6岁儿童的乳牙患龋齿率约为66.0％，中年人的蛀牙患病率为88.1％，而老年人的蛀牙患病率为98.4％。龋齿会引起咀嚼功能下降，进而加重肠胃负担，并导致营养不良。在儿童时期，乳牙的龋损会影响说话发音及身体生长发育，前牙的龋损也会影响面部美观。龋齿严重到损伤牙髓时会产生难以忍受的疼痛感，影响人们的日常生活和睡眠。此外，当口腔牙龈及牙根部发炎肿胀时，口腔局部免疫力下降会让细菌从口腔中侵入，从而引发败血症等其他身体疾病。
目前，国内外主要利用抗生素或药物杀灭或抑制致龋细菌的生长，以及机械去除牙菌斑。抗生素疗法应用于龋齿病的防治上很容易造成抗生素滥用，长时间使用会导致耐药率升高的问题。传统机械去除牙菌斑的方法主要依赖于含氟牙膏刷牙和漱口，对于成年人具有一定的可行性，但是对于儿童特别是幼儿来说存在配合度不高问题。此外含氟牙膏能够虽然能够一定程度上减少龋齿发生，但是在使用的过程中必须注意氟的过量摄入，儿童摄入过量会导致氟斑牙。因此有寻找一种简单有效的龋齿病防治手段显得尤为迫切。
研究发现，口腔内存在着一个多样化的口腔微生物群，当口腔内微生物群稳态被打破，特别是某些条件致病菌竞争性生长导致口腔菌群由低致龋菌群转化为高致龋菌群时，龋齿病的发生概率大大增加。益生菌因其良好的益生特性、安全性、拮抗变异链球菌、调节微生物菌群和口腔健康作用而被广泛研究。卫生部印发的《可用于食品的菌种名单》中包含唾液乳杆菌、植物乳杆菌和副干酪乳杆菌。
发明内容
本发明要解决的技术问题是提供一种能够缓解龋齿的益生菌组合物。
为解决上述问题，本发明提供了一种益生菌组合物，由唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85组成。
唾液乳杆菌(Lactobacillus salivarius)LS97，已于2018年12月10日保藏于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.16922。
植物乳杆菌(Lactobacillus plantarum)N13，已于2020年8月6日保藏于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.20496。
副干酪乳杆菌(Lactobacillus paracasei)LC86，已于2020年7月20日保藏于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.1.12731。
嗜酸乳杆菌(Lactobacillus acidophilus)LA85，已于2021年2月1日保藏于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.21802。
作为一种优选，所述益生菌组合物包括如下重量份的菌株：
20-30份唾液乳杆菌(Lactobacillus salivarius)LS97、5-10份植物乳杆菌(Lactobacillus plantarum)N13、5-10份副干酪乳杆菌(Lactobacillus paracasei)LC86和5-10份嗜酸乳杆菌(Lactobacillus acidophilus)LA85。
在本发明的一种实施方式中，上述益生菌的冻干菌粉的制备方法，包括如下步骤：
(1)唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillusplantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85分别划线于MRS固体培养基上，37℃条件下培养48h，得到单菌落。
(2)挑取单菌落分别接种于MRS液体培养基中，37℃条件下厌氧培养16h进行活化，得到一级种子液。
(3)将一级种子液按接种量为2％(v/v)分别转接至种子培养基中得到二级种子液，培养条件为37℃，厌氧培养16h。
(4)将二级种子液分别接入发酵培养基中进行高密度发酵，接种量为2％(v/v)，培养条件为35～37℃，厌氧培养8～18h。
(5)待发酵液中菌体密度达到109CFU/mL，分别收集发酵液；将所述发酵液分别离心收集菌体。
(6)收集菌体后分别用冻干保护剂重悬，得到重悬液。
(7)将重悬液采用真空冷冻法进行冻干，得到唾液乳杆菌(Lactobacillussalivarius)LS97、植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85冻干粉。
在本发明的一种实施方式中，所述培养基为MRS培养基(g/L)：蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L。
在本发明的一种实施方式中，所述冻干保护剂和菌体的质量比为2:1。
在本发明的一种实施方式中，所述保护剂包含100g/L的海藻糖。
本发明还提供了一种上述益生菌组合物在制备预防和/或治疗龋齿的保健食品、药品或口腔护理用品上的应用。所述益生菌组合物，包括唾液乳杆菌(Lactobacillussalivarius)LS97、植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85的组合物。
在本发明的一种实施方式中，所述保健食品、药品或口腔护理用品中，唾液乳杆菌(Lactobacillus salivarius)LS97活菌数不低于2×108CFU/mL或2×108CFU/g，植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85的活菌数为不低于1x108CFU/mL或1x108CFU/g。
本发明的有益效果在于：
1、本发明提供了含有唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85的组合物，此组合物能够预防和/或治疗龋齿，具体体现在：
(1)降低了大鼠口腔中引起龋齿的主要病原菌变异链球菌数量；
(2)减轻了大鼠龋齿程度；
(3)降低大鼠血清炎症因子TNF-ɑ、IL-1β、IL-6的水平。
因此，本发明的唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85的组合物在预防和/或治疗龋齿方面具有很大的应用前景。
2、唾液乳杆菌(Lactobacillus salivarius)、植物乳杆菌(Lactobacillusplantarum)、副干酪乳杆菌(Lactobacillus paracasei)和嗜酸乳杆菌(Lactobacillusacidophilus)目前均已被纳入卫生部下发的《可用于食品的菌种名单》，因此，本发明的唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillusacidophilus)LA85对人体而言，健康且无毒副作用。
附图说明
图1为不同处理组大鼠龋齿得分对比图。
图2为不同处理组大鼠血清肿瘤坏死因子α(Tumornecrosisfactorα，TNF-α)水平相比于空白对照的变化。
图3为不同处理组大鼠血清白细胞介素-1β(Interleukin-1β,IL1β)水平相比于空白对照的变化。
图4为不同处理组大鼠血清白细胞介素-6(Interleukin-6,IL6)水平相比于空白对照的变化。
具体实施方式
下面结合附图对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例中涉及的培养基如下：
MRS培养基(g/L)：蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L。
实施例1：唾液乳杆菌(Lactobacillus salivarius)LS97冻干粉的制备
(1)唾液乳杆菌(Lactobacillus salivarius)LS97划线于MRS固体培养基上，37℃条件下培养48h，得到单菌落。
(2)挑取单菌落接种于MRS液体培养基中，37℃条件下厌氧培养16h进行活化，得到一级种子液。
(3)将一级种子液按接种量为2％(v/v)转接至种子培养基中得到二级种子液，培养条件为37℃，厌氧培养16h。
(4)将二级种子液分别接入发酵培养基中进行高密度发酵，接种量为2％(v/v)，培养条件为35～37℃，厌氧培养8～18h。
(5)待发酵液中菌体密度达到109CFU/mL，收集发酵液；将所述发酵液分别离心收集菌体。
(6)收集菌体后用海藻糖浓度为100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1)，得到重悬液。
(7)将重悬液采用真空冷冻法进行冻干，得到唾液乳杆菌(Lactobacillussalivarius)LS97冻干粉。
实施例2：植物乳杆菌(Lactobacillus plantarum)N13冻干粉的制备
(1)植物乳杆菌(Lactobacillus plantarum)N13划线于MRS固体培养基上，37℃条件下培养48h，得到单菌落。
(2)挑取单菌落接种于MRS液体培养基中，37℃条件下厌氧培养16h进行活化，得到一级种子液。
(3)将一级种子液分别转接至种子培养基中得到二级种子液，培养条件为37℃，厌氧培养16h。
(4)将二级种子液分别接入发酵培养基中进行高密度发酵，接种量为2％(v/v)，培养条件为35～37℃，厌氧培养8～18h。
(5)待发酵液中菌体密度达到109CFU/mL，收集发酵液。
(6)将所述发酵液分别离心，收集菌体后用海藻糖浓度为100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1)，得到重悬液。
(7)将重悬液采用真空冷冻法进行冻干，得到植物乳杆菌(Lactobacillusplantarum)N13冻干粉。
实施例3：副干酪乳杆菌(Lactobacillus paracasei)LC86冻干粉的制备
(1)副干酪乳杆菌(Lactobacillus paracasei)LC86划线于MRS固体培养基上，37℃条件下培养48h，得到单菌落。
(2)挑取单菌落接种于MRS液体培养基中，37℃条件下厌氧培养16h进行活化，得到一级种子液。
(3)将一级种子液分别转接至种子培养基中得到二级种子液，培养条件为37℃，厌氧培养16h。
(4)将二级种子液分别接入发酵培养基中进行高密度发酵，接种量为2％(v/v)，培养条件为35～37℃，厌氧培养8～18h。
(5)待发酵液中菌体密度达到109CFU/mL，收集发酵液。
(6)将所述发酵液分别离心，收集菌体后用海藻糖浓度为100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1)，得到重悬液。
(7)将重悬液采用真空冷冻法进行冻干，得到副干酪乳杆菌(Lactobacillusparacasei)LC86冻干粉。
实施例4：嗜酸乳杆菌(Lactobacillus acidophilus)LA85冻干粉的制备
(1)嗜酸乳杆菌(Lactobacillus acidophilus)LA85划线于MRS固体培养基上，37℃条件下培养48h，得到单菌落。
(2)挑取单菌落接种于MRS液体培养基中，37℃条件下厌氧培养16h进行活化，得到一级种子液。
(3)将一级种子液分别转接至种子培养基中得到二级种子液，培养条件为37℃，厌氧培养16h。
(4)将二级种子液分别接入发酵培养基中进行高密度发酵，接种量为2％(v/v)，培养条件为35～37℃，厌氧培养8～18h。
(5)待发酵液中菌体密度达到109CFU/mL，收集发酵液。
(6)将所述发酵液分别离心，收集菌体后用海藻糖浓度为100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1)，得到重悬液。
(7)将重悬液采用真空冷冻法进行冻干，得到嗜酸乳杆菌(Lactobacillusacidophilus)LA85冻干粉。
实施例5：含有唾液乳杆菌LS97、植物乳杆菌N13、副干酪乳杆菌LC86和嗜酸乳杆菌LA85益生菌组合物的制备
称取麦芽糊精50重量份、唾液乳杆菌LS97菌粉20重量份、植物乳杆菌N13菌粉10重量份、副干酪乳杆菌LC86菌粉10重量份和嗜酸乳杆菌LA85菌粉10重量份，混合均匀，即得到含有唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillusplantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85益生菌组合物。
实施例6：含有唾液乳杆菌LS97、植物乳杆菌N13、副干酪乳杆菌LC86和嗜酸乳杆菌LA85益生菌组合物的制备
称取麦芽糊精50重量份、唾液乳杆菌LS97菌粉30重量份、植物乳杆菌N13菌粉5重量份、副干酪乳杆菌LC86菌粉10重量份和嗜酸乳杆菌LA85菌粉5重量份，混合均匀，即得到含有唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillusplantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85益生菌组合物。
实施例7：乳杆菌组合物对变异链球菌感染的大鼠口腔变异链球菌清除作用
取3周龄左右的SPF级雌性Wistar大鼠饲养于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，之后按体重随机分组，将大鼠按体重随机分为4组，每组12只，其中一组设置为空白对照组(空白组)，一组设置为龋齿模型组(模型组)，一组设置为实验组，一组设置LS97组。实验开始前7天，对龋齿模型组、实验组和LS97组连续7天擦拭变异链球菌。确定变异链球菌在龋齿模型组和实验组大鼠口腔成功定植，即造模成功之后，实验组每天擦拭实施例5制备的含有唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85乳酸菌组合物1次直至第10周实验结束，LS97组每天擦拭实施例1制备的唾液乳杆菌(Lactobacillus salivarius)LS97。
其中致龋细菌变异链球菌在TSB培养基中37℃过夜培养12h，将菌液经8000g离心5min收集菌体，生理盐水重悬后离心洗涤，重复洗涤3次，生理盐水再次重悬调节菌浓度到1×108CFU/mL。乳酸菌组合物生理盐水悬浮调节菌浓度到5×108CFU/mL，唾液乳杆菌(Lactobacillus salivarius)LS97生理盐水悬浮调节菌浓度到5×108CFU/mL。用无菌棉签蘸取0.2mL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，除空白对照组喂食正常饲料和饮用水外，其他组均饲以致龋饲料Diet2000#和添加有5％蔗糖的饮用水实验周期维持10周。其中，致龋饲料Diet2000#主要成分如表1所示。
表1致龋饲料Diet2000#主要成分
| 成分 | 重量百分比(％) |
| 全麦粉 | 6 |
| 蔗糖 | 56 |
| 精炼奶粉 | 28 |
| 苜蓿叶粉 | 3 |
| 脱水全干粉 | 1 |
| 酵母 | 4 |
| 氯化钠 | 2 |
在实验的第3、7、10周(分别对应实验的早、中和后期)给大鼠口腔牙齿分别进行第1次、第2次和第3次取样，在MMS固体培养基(添加有200μg/mL硫酸链霉素的MitisSalivarius Agar)上涂布进行变异链球菌计数。实验结果如表2所示。
表2大鼠口腔中变异链球菌(S.mutants)计数结果
| 取样次数 | 空白组 | 模型组 | 实验组 | LS97组 |
| 1 | <30a | 5.68±0.11 | 4.46±0.24 | 4.85±0.16 |
| 2 | <30a | 5.54±0.14 | 3.21±0.19 | 4.08±0.23 |
| 3 | <30a | 5.71±0.10 | 2.81±0.23 | 3.71±0.11 |
由表2可知，实验组中的变异链球菌下降约2个数量级，模型组中变异链球菌则无明显下降，较组合物处理的实验组大鼠，LS97组变异链球菌下降较少。由此可见，本发明中含有唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillusplantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85益生菌组合物有对口腔变异链球菌有较好的清除作用，组合物对变异链球菌的清除作用优于唾液乳杆菌(Lactobacillus salivarius)LS97单菌。
实施例8：乳杆菌组合物对变异链球菌感染大鼠龋齿的缓解作用
SD大鼠分组、造模及处理方法同实施例7。
大鼠实验结束后，杀死大鼠并取出大鼠臼齿，之后置于高压蒸汽锅中在115℃条件下处理20分钟。然后用手术刀剥离软组织，自然干燥后将放置于臼齿置于0.4％的紫脲酸铵染液中过夜浸泡12h染色，清水漂洗并自然干燥后在体视显微镜下观察各组大鼠臼齿龋损情况，利用Keyes龋齿计分法给大鼠牙齿进行龋齿打分。根据Keyes经典评估龋齿的方法，将牙齿龋损破坏程度分为四个等级：E级龋，龋坏仅累及牙釉质；Ds级龋，牙釉质龋坏及龋损累及范围不超过1/4的牙本质外层；Dm级龋，龋损累及范围处于1/4～3/4的牙本质厚度；Dx级龋，龋损累及范围超过3/4的牙本质厚度，结果如图1所示。
从图1可知，与龋齿模型组相比，用含有唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillusparacasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85益生菌组合物处理的实验组有效减轻了大鼠磨牙釉质龋(E)、牙本质浅龋(Ds)和牙本质中龋(Dm)的发生，且组合物对龋齿的缓解作用明显优于唾液乳杆菌(Lactobacillus salivarius)LS97单菌。
这些结果表明，含有唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85益生菌组合物在预防和/或治疗龋齿中有较好的作用。
实施例9：乳杆菌组合物对大鼠血清炎症因子指标的影响
SD大鼠分组、造模及处理方法同实施例6。
血清中相关细胞因子的检测：TNF-α、IL-1β和IL-6含量的测定采用ELISA试剂盒，方法参照说明书。将空白对照组血液中炎症因子TNF-α、IL-1β和IL-6的水平设为1，龋齿模型组、实验组和LS97组血液中的炎症因子TNF-α、IL-1β和IL-6水平均与空白对照组比较，所得比值反应相较于正常生理状态的炎症因子升高情况。
由图2可知，与空白对照组比较，龋齿模型组大鼠血清TNF-α水平明显升高。与龋齿模型组比较，组合物干预处理的实验组大鼠血清TNF-α水平升高程度减轻，且效果好于唾液乳杆菌(Lactobacillus salivarius)LS97单菌干预处理的LS97组。
由图3可知，与空白对照组比较，龋齿模型组大鼠血清IL-1β水平明显升高。与龋齿模型组比较，组合物干预处理的实验组大鼠血清IL-1β水平升高程度明显减轻，且效果好于唾液乳杆菌(Lactobacillus salivarius)LS97单菌干预处理的LS97组。
由图4可知，与空白对照组比较，龋齿模型组大鼠血清IL-6水平明显升高。与龋齿模型组比较，组合物干预处理的实验组大鼠血清IL-6水平升高程度明显减轻，且效果好于唾液乳杆菌(Lactobacillus salivarius)LS97单菌干预处理的LS97组。
这些结果表明，含有唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillus plantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85益生菌组合物在有减轻变异链球菌感染导致的龋齿个体炎症反应的作用。
实施例10：乳杆菌组合物在益生菌型果蔬酵素固体饮料中的应用
唾液乳杆菌(Lactobacillus salivarius)LS97、植物乳杆菌(Lactobacillusplantarum)N13、副干酪乳杆菌(Lactobacillus paracasei)LC86和嗜酸乳杆菌(Lactobacillus acidophilus)LA85益生菌组合物可用于制备益生菌型果蔬酵素固体饮料，益生菌型果蔬酵素固体饮料的具体制备过程如下：
称取益生元33重量份，水溶性膳食纤维15重量份，糖醇15重量份，水果粉10重量份，果蔬酵素粉2重量份，唾液乳杆菌(Lactobacillus salivarius)LS9710重量份，植物乳杆菌(Lactobacillus plantarum)N135重量份，副干酪乳杆菌(Lactobacillus paracasei)LC865重量份，嗜酸乳杆菌(Lactobacillus acidophilus)LA855重量份将这些物料充分混匀，得到粉剂；将粉剂装入到小包装袋中封口，每袋2g,得到固体饮料制品。
其中，益生元包括37.5重量份低聚异麦芽糖、8重量份水苏糖和5重量份低聚木糖，水溶性膳食纤维包括20重量份聚葡萄糖和5重量份抗性糊精，糖醇包括5重量份木糖醇、8重量份赤藓糖醇和2重量份D-甘露糖醇，水果粉包括5重量份甜橙果粉、3重量份芒果果粉、1重量份针叶樱桃粉和0.5重量份桑葚粉。
以上结合附图对本发明的实施方式作了详细说明，但本发明不限于所描述的实施方式。对于本领域的技术人员而言，在不脱离本发明原理和精神的情况下，对这些实施方式进行多种变化、修改、替换和变型，仍落入本发明的保护范围内。
Claims (4)
1.一种益生菌组合物，由唾液乳杆菌（Lactobacillus salivarius）LS97、植物乳杆菌（Lactobacillus plantarum）N13、副干酪乳杆菌（Lactobacillus paracasei）LC86和嗜酸乳杆菌（Lactobacillus acidophilus）LA85组成；
所述唾液乳杆菌LS97，于2018年12月10日保藏于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CGMCCNO.16922；
所述植物乳杆菌N13，于2020年8月6日保藏于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CGMCCNo.20496；
所述副干酪乳杆菌LC86，于2020年7月20日保藏于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.1.12731；
所述嗜酸乳杆菌LA85，于2021年2月1日保藏于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CGMCCNo.21802。
2.根据权利要求1所述的益生菌组合物，其特征在于，所述益生菌组合物包括如下重量份的菌株：20-30份唾液乳杆菌LS97、5-1 0份植物乳杆菌N13、5-10份副干酪乳杆菌LC86和5-10份嗜酸乳杆菌LA85。
3.权利要求1或2所述益生菌组合物在制备预防和/或治疗龋齿的药品或口腔护理用品上的应用。
4.根据如权利要求3所述的应用，其特征在于，所述药品或口腔护理用品中，唾液乳杆菌LS97活菌数不低于2×108 CFU/mL或2×108 CFU/g，植物乳杆菌N13、副干酪乳杆菌LC86和嗜酸乳杆菌LA85的活菌数不低于1×108 CFU/mL或1×108 CFU/g。
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| Microbiome of Deep Dentinal Caries from Reversible Pulpitis to Irreversible Pulpitis;Jinxin Zheng;J Endod;第45卷(第3期);全文 * |
| 应用傅里叶红外光谱对干燥综合症伴龋齿患者口腔特征微生物的初步鉴别;周爽;实用口腔医学杂志;第37卷(第3期);全文 * |
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