技术领域technical field
本发明涉及医药生物领域，具体涉及一种益生菌组合物、制剂、菌泥和用途。The invention relates to the field of medical biology, in particular to a probiotic composition, preparation, bacteria slime and application.
背景技术Background technique
医学上将围绕并覆盖在牙齿周围的软组织称为牙龈，发生于牙龈组织的急慢性炎症称为牙龈炎(gingivitis)。表现为牙龈出血，红肿，胀痛，继续发展侵犯硬组织，产生牙周炎，包括牙龈组织的炎症及全身疾病在牙龈的表现。Medically, the soft tissue that surrounds and covers the teeth is called gingiva, and the acute and chronic inflammation that occurs in the gingival tissue is called gingivitis. Manifested as gum bleeding, redness, swelling, pain, continue to develop invasion of hard tissue, resulting in periodontitis, including inflammation of gum tissue and systemic diseases in the gums.
牙周病是指发生在牙支持组织(牙周组织)的疾病，包括仅累及牙龈组织的牙龈病和波及深层牙周组织(牙周膜、牙槽骨、牙骨质)的牙周炎两大类。牙周疾病是常见的口腔疾病，是引起成年人牙齿丧失的主要原因之一，也是危害人类牙齿和全身健康的主要口腔疾病。Periodontal disease refers to diseases that occur in the tooth supporting tissue (periodontal tissue), including gingival disease involving only gingival tissue and periodontitis involving deep periodontal tissue (periodontal ligament, alveolar bone, cementum). category. Periodontal disease is a common oral disease, one of the main causes of adult tooth loss, and a major oral disease that endangers human teeth and general health.
牙周病的早期症状不易引起重视，造成牙周组织长期慢性感染，炎症反复发作，不仅损害口腔咀嚼系统的功能，还会严重影响健康。The early symptoms of periodontal disease are not easy to pay attention to, resulting in long-term chronic infection of periodontal tissue, and recurrent inflammation, which not only damages the function of the oral masticatory system, but also seriously affects health.
因此，研发一种新型的治疗牙部疾病的药物具有非常现实的意义。Therefore, researching and developing a kind of novel medicine for the treatment of dental diseases has very practical significance.
发明内容Contents of the invention
本发明的目的在于提供一种益生菌组合物，该益生菌组合物有利于破坏的牙槽骨再生，同时能抑制牙周致病菌的生长，在一定程度上可以预防和治疗牙周炎能够有效抑制牙周病，解决现代人牙齿不健康的问题。还提供了一种制剂、菌泥和用途。The object of the present invention is to provide a kind of probiotic composition, this probiotic composition is beneficial to the regeneration of damaged alveolar bone, can inhibit the growth of periodontal pathogenic bacteria at the same time, can prevent and treat periodontitis to a certain extent. Effectively inhibit periodontal disease and solve the problem of unhealthy teeth of modern people. Also provided are a preparation, bacteria sludge and application.
本发明通过下述技术方案实现：The present invention realizes through following technical scheme:
一种益生菌组合物，包括嗜酸乳杆菌、唾液链球菌M18、唾液链球菌K12。A probiotic composition comprising Lactobacillus acidophilus, Streptococcus salivarius M18 and Streptococcus salivarius K12.
以菌落数计，嗜酸乳杆菌为10-30％，唾液链球菌M18为10-50％，唾液链球菌K12为20-80％。In terms of the number of colonies, Lactobacillus acidophilus is 10-30%, Streptococcus salivarius M18 is 10-50%, and Streptococcus salivarius K12 is 20-80%.
以菌落数计，由嗜酸乳杆菌为10％，唾液链球菌M18为10％，唾液链球菌K12为80％组成。In terms of the number of colonies, it consists of 10% of Lactobacillus acidophilus, 10% of Streptococcus salivarius M18, and 80% of Streptococcus salivarius K12.
一种益生菌制剂，由前所述的益生菌组合物和药学上可接受的载体或辅料制成。A probiotic preparation is prepared from the aforementioned probiotic composition and pharmaceutically acceptable carriers or auxiliary materials.
益生菌制剂，为益生菌凝胶剂或者益生菌口颊片。The probiotic preparation is a probiotic gel or a probiotic buccal tablet.
一种益生菌菌泥，由前所述的益生菌组合物经培养离心洗涤制得。A kind of probiotic bacteria slime, which is prepared from the aforementioned probiotic bacteria composition through culture and centrifugal washing.
益生菌制剂为益生菌凝胶剂时，由前所述的益生菌组合物制备成菌泥后或经培养后离心洗涤制得的菌泥与凝胶基质混合制得。When the probiotic preparation is a probiotic gel, it is prepared by mixing the aforementioned probiotic composition into a bacteria sludge or the bacteria sludge obtained by centrifuging and washing after cultivation and gel matrix.
凝胶基质为高分子凝胶材料、生物粘附材料、保湿剂、蒸馏水组成，所述的高分子材料为卡波姆、泊洛沙姆、羟丙基纤维素、羟丙基甲基纤维素钠、壳聚糖中的一种或几种；所述的生物粘附材料为透明质酸钠、羟丙基甲基纤维素、海藻酸钠中的一种或几种；所述的保湿剂为甘油、丙二醇聚乙二醇、丙三醇、山梨醇中的一种或几种。The gel matrix is composed of polymer gel material, bioadhesive material, humectant, and distilled water, and the polymer material is carbomer, poloxamer, hydroxypropyl cellulose, hydroxypropyl methylcellulose One or more of sodium and chitosan; the bioadhesive material is one or more of sodium hyaluronate, hydroxypropylmethylcellulose, and sodium alginate; the moisturizing agent One or more of glycerin, propylene glycol polyethylene glycol, glycerol, and sorbitol.
益生菌制剂为益生菌口颊片时，将前所述的益生菌组合物制备得益生菌组合物冻干粉，再将辅料与益生菌冻干粉混合后直接压片制得。When the probiotic preparation is a probiotic buccal tablet, the aforementioned probiotic composition is prepared to obtain a probiotic composition freeze-dried powder, and then the excipients are mixed with the probiotic freeze-dried powder, and then directly compressed into tablets.
一种如前所述的益生菌组合物在制备治疗牙周炎或牙周病药物上的用途。A use of the aforementioned probiotic composition in the preparation of medicines for treating periodontitis or periodontal disease.
本发明与现有技术相比，具有如下的优点和有益效果：Compared with the prior art, the present invention has the following advantages and beneficial effects:
本发明混合益生菌相互之间的激发和抑制作用给予了牙齿健康生存的口腔环境，混合益生菌在抑制有害菌的同时，还赋予各个益生菌成员之间正向激励的作用，使得牙齿生长的口腔健康环境得以平衡，激发和促进人本体的口腔免疫功能，对牙周病和牙龈炎均有防治作用。The stimulating and inhibiting effects of the mixed probiotics of the present invention give teeth a healthy oral environment. While inhibiting harmful bacteria, the mixed probiotics also endow each probiotic member with a positive incentive effect, so that the teeth grow faster. The oral health environment can be balanced, stimulate and promote the oral immune function of the human body, and have a preventive effect on periodontal disease and gingivitis.
附图说明Description of drawings
此处所说明的附图用来提供对本发明实施例的进一步理解，构成本申请的一部分，并不构成对本发明实施例的限定。在附图中：The drawings described here are used to provide a further understanding of the embodiments of the present invention, constitute a part of the application, and do not limit the embodiments of the present invention. In the attached picture:
图1为本发明的对比实验结果小鼠牙槽骨丧失图。Fig. 1 is the mouse alveolar bone loss diagram of the comparative experimental results of the present invention.
图2为本发明的厌氧菌计数图。Fig. 2 is the anaerobic count diagram of the present invention.
具体实施方式Detailed ways
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例和附图，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples and accompanying drawings. As a limitation of the present invention.
实施例1Example 1
嗜酸乳杆菌为4×108CFU，唾液链球菌M18为4×108CFU，唾液链球菌K12为2×108CFU。Lactobacillus acidophilus is 4×108 CFU, Streptococcus salivarius M18 is 4×108 CFU, and Streptococcus salivarius K12 is 2×108 CFU.
实施例2Example 2
嗜酸乳杆菌为1×108CFU，唾液链球菌M18为1×108CFU，唾液链球菌K12为8×108CFU。Lactobacillus acidophilus is 1×108 CFU, Streptococcus salivarius M18 is 1×108 CFU, and Streptococcus salivarius K12 is 8×108 CFU.
由实施例1-2的益生菌组合物和药学上可接受的载体可制成益生菌制剂。实施例1-2中的益生菌组合物的单位不局限于108CFU，在107CFU～1010CFU。A probiotic preparation can be made from the probiotic composition of Examples 1-2 and a pharmaceutically acceptable carrier. The unit of the probiotic composition in Example 1-2 is not limited to 108 CFU, but ranges from 107 CFU to 1010 CFU.
如一种实施为益生菌凝胶剂，另一种实施为益生菌口颊片。For example, one is implemented as a probiotic gel, and the other is implemented as a probiotic buccal tablet.
为进一步说明本发明的有益效果，发明人特意做了几组对比实验。对比内容如下：In order to further illustrate the beneficial effect of the present invention, the inventor has deliberately done several groups of comparative experiments. The comparison content is as follows:
用以下对比例作为对比组。The following comparative examples were used as comparison groups.
对比例1Comparative example 1
嗜酸乳杆菌1×108CFULactobacillus acidophilus 1×108 CFU
对比例2Comparative example 2
唾液链球菌K12为8×108CFUStreptococcus salivarius K12 is 8×108 CFU
对比例3Comparative example 3
唾液链球菌M18为1×108CFUStreptococcus salivarius M18 is 1×108 CFU
对比例4Comparative example 4
嗜酸乳杆菌3.5×108CFU、唾液链球菌M18为5.5×108CFU、唾液链球菌K12为1×108CFU。实验内容如下：Lactobacillus acidophilus is 3.5×108 CFU, Streptococcus salivarius M18 is 5.5×108 CFU, and Streptococcus salivarius K12 is 1×108 CFU. The content of the experiment is as follows:
1、实验材料1. Experimental materials
1.1实验设备和器械1.1 Experimental equipment and instruments
厌养菌培养箱、台式离心机、光学显微镜、显微照相系统型、牙科专用手术器械一套。Anaerobic bacteria incubator, desktop centrifuge, optical microscope, photomicrograph system type, and a set of special dental surgical instruments.
1.2实验试剂1.2 Experimental reagents
厌氧血琼脂平板、PBS磷酸盐缓冲剂、4％多聚甲醛溶液、亚甲蓝染色剂。Anaerobic blood agar plate, PBS phosphate buffer, 4% paraformaldehyde solution, methylene blue staining agent.
1.3实验方法1.3 Experimental method
1.3.1动物实验及分组1.3.1 Animal experiments and grouping
将SPF级C57BL/6雄性小鼠80只，随机分成8组即:空白组(未处理组)，模型组(结扎牙线后给予溶剂组)及给药组1-6(结扎牙线后给予益生菌组；给药组1对应对比例1，给药组2对应对比例2，给药组3对应对比例3，给药组4对应实施例1，给药组5对应实施例2，给药组6对应对比例4。With 80 SPF level C57BL/6 male mice, be divided into 8 groups at random namely: blank group (untreated group), model group (give solvent group after ligating dental floss) and administration group 1-6 (give solvent group after ligating dental floss) Probiotics group; administration group 1 corresponds to comparative example 1, administration group 2 corresponds to contrast example 2, administration group 3 corresponds to contrast example 3, administration group 4 corresponds to embodiment 1, administration group 5 corresponds to embodiment 2, and administration group 5 corresponds to embodiment 2. Medicine group 6 corresponds to comparative example 4.
各组小鼠均用4％水合氯醛(0.1ml/10g，腹腔注射)麻醉，正常对照组不作干预待其苏醒，其余7组均采用丝线围绕左上第二磨牙结扎。从实验当天起空白组喂食水不做其他处理；模型组给予溶剂0.07ml/次，一天两次；The mice in each group were anesthetized with 4% chloral hydrate (0.1ml/10g, intraperitoneal injection). The normal control group was not intervened until they woke up. The remaining 7 groups were ligated around the left upper second molar with silk thread. From the day of the experiment, the blank group was fed with no other treatment; the model group was given solvent 0.07ml/time, twice a day;
1.3.2结扎定位1.3.2 Ligation positioning
小鼠用丝线结扎上颌左侧第二磨牙，同时相对一侧不做处理，作为骨高度测量基线，且保持实验全程丝线不脱落。每周检查一次丝线，失去丝线的小鼠进行标记并重新结扎，所有小鼠共观察4周，在第4周处死小鼠，观察牙釉质牙骨质界至牙槽嵴顶距离，并取丝线培养细菌。Mice were ligated with silk thread on the left maxillary second molar, while the opposite side was left untreated, which was used as the baseline for bone height measurement, and the silk thread was kept from falling off throughout the experiment. The silk thread was checked once a week, and the mice that lost the silk thread were marked and ligated again. All the mice were observed for 4 weeks, and the mice were killed at the 4th week. The distance from the enamel cementum junction to the alveolar crest was observed, and the silk thread was taken cultivate bacteria.
1.3.3骨丧失观察1.3.3 Bone loss observation
将小鼠处死，取其上颚并于显微镜下去其软组织后固定于4％多聚甲醛溶液中固定后，牙齿于1％亚甲蓝中染色。在尼康SMZ800显微镜40×光镜下观察骨丧失情况，即釉牙骨质界至牙槽嵴顶的距离，同时照片记录小鼠上颌骨骨丧失情况。The mice were sacrificed, and the palate was removed and its soft tissues were removed under a microscope and fixed in 4% paraformaldehyde solution. After fixing, the teeth were stained in 1% methylene blue. The bone loss was observed under Nikon SMZ800 microscope 40×light microscope, that is, the distance from the enamel cementum junction to the alveolar crest, and the bone loss of the maxillary of the mice was recorded by photos.
1.3.4细菌测定1.3.4 Determination of bacteria
将实验小鼠处死，取下丝线用PBS磷酸盐缓冲液冲洗掉食物残渣后，加入1ml PBS离心。再将细菌提取液稀释几倍后种于血琼脂培养基中，37℃厌氧环境下培养7d，记录下CFU数据，通过丝线对应长度计算CFU/mm。The experimental mice were sacrificed, the silk threads were removed and food residues were washed away with PBS phosphate buffered saline, and then 1 ml of PBS was added for centrifugation. Then the bacterial extract was diluted several times and planted in blood agar medium, cultured in an anaerobic environment at 37°C for 7 days, recorded the CFU data, and calculated CFU/mm according to the corresponding length of the silk thread.
1.3.5数据分析方法1.3.5 Data analysis method
应用InStat方差分析和多重比较检验法，采用t检验比较各组间骨吸收的差异。以P<0.05为差异有统计学意义。InStat analysis of variance and multiple comparison test were used, and t test was used to compare the differences in bone resorption among the groups. P<0.05 was considered statistically significant.
2实验结果2 Experimental results
2.1牙槽骨骨吸收结果2.1 Results of alveolar bone resorption
图1显示，空白组大鼠牙周组织正常，釉牙骨质界至牙槽嵴顶的距离非常低，而模型组与空白组相比，第二磨牙牙槽骨骨吸收明显高于基线水平。给药组1、2、3、6其釉牙骨质界至牙槽嵴顶的距离与模型组相比有一定降低，给药组4、5与模型组相比釉牙骨质界至牙槽嵴顶的距离大大减少。给药组1、2、3与给药4、5相比釉牙骨质界至牙槽嵴顶的距离明显增加，给药组6与给药组4、5相比釉牙骨质界至牙槽嵴顶的距离增加很多，说明第一：实施例1和2给出的益生菌组合物相对于单个组份具有显著的有益效果；第二，非本发明配比范围的组合物并不一定能够达到单组份的效果，因为给药组6与给药组2相比釉牙骨质界至牙槽嵴顶的距离有所增加。Figure 1 shows that the periodontal tissue of the rats in the blank group was normal, and the distance from the enamel cementum junction to the alveolar crest was very low. Compared with the blank group, the alveolar bone resorption of the second molar in the model group was significantly higher than the baseline level . Compared with the model group, the distance from the enamel cementum junction to the alveolar crest of the drug treatment groups 1, 2, 3, and 6 decreased to a certain extent. The distance between the crests of the grooves is greatly reduced. Compared with administration 4 and 5, the distance from enamel cementum junction to alveolar crest in administration group 1, 2, 3 was significantly increased; in administration group 6, compared with administration group 4, 5, the distance from enamel cementum junction to The distance of alveolar crest increases a lot, illustrates the first: the probiotic composition that embodiment 1 and 2 provides has significant beneficial effect with respect to single component; The single-component effect must be achieved, because the distance from the enamel cementum junction to the alveolar crest in the administration group 6 was increased compared with the administration group 2.
2.2细菌计数2.2 Bacteria count
由图2，给药组1-6厌氧菌数量较低，其中给药组1、3、6有显著差异(*P＜0.05)，给药组2、4、5有极显著差异(**P＜0.01)，且给药组4、5抑菌效果最佳，给药组均显示出较强的牙周厌氧致病菌抑制作用。From Figure 2, the number of anaerobic bacteria in administration groups 1-6 is low, wherein administration groups 1, 3, and 6 have significant differences (*P<0.05), and administration groups 2, 4, and 5 have extremely significant differences (* *P<0.01), and the antibacterial effect of administration group 4 and 5 was the best, and the administration groups all showed strong inhibitory effect on periodontal anaerobic pathogenic bacteria.
由以上的对比实验总结可知，牙周炎是细菌菌斑生物膜导致的一种以骨吸收、软组织炎症为主要表现的慢性、破坏性疾病，治疗不及时，最终导致牙齿拔除。本实验进行了4周的动物实验，通过丝线结扎小鼠左上颌第二磨牙牙颈部的方法创建模型，观察牙槽骨骨吸收程度。实验包括8组：空白组、模型组和给药组1-6，在第4周并比较各组釉牙骨质界至牙槽嵴顶的距离作为牙槽骨丧失的指标。给药组釉牙骨质界至牙槽嵴顶的距离与模型组相比大大降低，尤其是给药组(实施例1组)、给药组5(实施例2组)有显著差异，可见混合益生菌组合对丝线结扎诱导的小鼠牙槽骨丧失有显著的缓解作用，使牙槽骨能够再生。From the summary of the above comparative experiments, it can be seen that periodontitis is a chronic and destructive disease caused by bacterial plaque biofilm, mainly manifested by bone resorption and soft tissue inflammation. If the treatment is not timely, it will eventually lead to tooth extraction. In this experiment, animal experiments were carried out for 4 weeks. The model was established by ligating the neck of the left maxillary second molar of the mouse with silk thread, and the degree of bone resorption of the alveolar bone was observed. The experiment included 8 groups: blank group, model group and administration group 1-6. At the 4th week, the distance from the enamel cementum junction to the alveolar crest was compared in each group as an index of alveolar bone loss. The distance between the enamel-cementum junction and the alveolar crest of the administration group is greatly reduced compared with the model group, especially the administration group (Example 1 group) and Administration group 5 (Example 2 group) have significant differences, as can be seen The mixed probiotic combination significantly alleviated the alveolar bone loss induced by silk ligation in mice, enabling alveolar bone regeneration.
此外，已证实的牙周致病菌包括牙龈卟啉单胞菌、伴放线聚集杆菌、福赛坦氏菌、粘放线菌、齿垢密螺旋体、具核梭杆菌、中间普氏菌等厌氧菌。给药组的厌氧菌数量较模型组厌氧菌数量大大减少，可见混合益生菌组合对小鼠口腔中的厌氧菌有较强的抑制作用。出现这样的结果可能是，组合中不同益生菌对口腔有害菌抑制作用不同，不同比例的混合益生菌之间的协同作用也各有差异，在一定比例范围内，混合益生菌之间相互作用，相互促进，起到群体的协同作用，使得其抑制有害菌的生长繁殖作用比单独使用益生菌的抑菌作用大大增强，给予了牙齿健康生存的口腔环境。In addition, the confirmed periodontal pathogens include Porphyromonas gingivalis, Aggregate actinomycetes, Forsythium, Actinomyces viscosus, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia, etc. anaerobic bacteria. The number of anaerobic bacteria in the administration group was greatly reduced compared with that in the model group. It can be seen that the mixed probiotic combination has a strong inhibitory effect on the anaerobic bacteria in the oral cavity of mice. Such a result may be that different probiotics in the combination have different inhibitory effects on oral harmful bacteria, and the synergistic effect between mixed probiotics in different proportions is also different. Within a certain proportion range, the interaction between mixed probiotics, Promote each other and play a synergistic effect of the group, which makes the antibacterial effect of inhibiting the growth and reproduction of harmful bacteria much stronger than that of probiotics alone, and provides a healthy oral environment for teeth.
综上所述，混合益生菌组合有利于破坏的牙槽骨再生，同时能抑制牙周致病菌的生长，在一定程度上可以预防和治疗牙周炎。In summary, the combination of mixed probiotics is beneficial to the regeneration of damaged alveolar bone, and can inhibit the growth of periodontal pathogenic bacteria, which can prevent and treat periodontitis to a certain extent.
实施例5益生菌凝胶剂Embodiment 5 probiotic gel
一种益生菌凝胶剂的制备方法，其特征在于包括以下步骤：A kind of preparation method of probiotic gel is characterized in that comprising the following steps:
(1)益生菌菌泥的制备：取益生菌菌种于BHI液体培养基中，5％CO2、37℃条件下，厌氧环境中培养12h，益生菌培养液以3000r/min的转速离心10min，反复用生理盐水洗涤，得到益生菌菌泥。(1) Preparation of probiotic slime: take probiotic strains in BHI liquid medium, culture in an anaerobic environment for 12 hours under 5% CO2 and 37°C, and centrifuge the probiotic culture solution at a speed of 3000r/min 10min, washed repeatedly with physiological saline to obtain probiotic bacteria slime.
(2)凝胶基质：凝胶基质由高分子凝胶材料、生物粘附材料、保湿剂、蒸馏水组成，所述的高分子材料为卡波姆、泊洛沙姆、羟丙基纤维素、羟丙基甲基纤维素钠、壳聚糖中的一种或几种；所述的生物粘附材料为透明质酸钠、羟丙基甲基纤维素、海藻酸钠中的一种或几种；所述的保湿剂为甘油、丙二醇聚乙二醇、丙三醇、山梨醇中的一种或几种。(2) Gel matrix: the gel matrix is made up of polymer gel material, bioadhesive material, humectant, distilled water, and described polymer material is carbomer, poloxamer, hydroxypropyl cellulose, One or more of hydroxypropyl methylcellulose sodium and chitosan; the bioadhesive material is one or more of sodium hyaluronate, hydroxypropyl methylcellulose and sodium alginate species; the humectant is one or more of glycerin, propylene glycol polyethylene glycol, glycerol, and sorbitol.
表1Table 1
(1)益生菌菌泥的制备：取益生菌菌种于BHI液体培养基中，5％CO2、37℃条件下，厌氧环境中培养12h，益生菌培养液以3000r/min的转速离心10min，反复用生理盐水洗涤，得到益生菌菌泥。(1) Preparation of probiotic slime: take probiotic strains in BHI liquid medium, culture in an anaerobic environment for 12 hours under 5% CO2 and 37°C, and centrifuge the probiotic culture solution at a speed of 3000r/min 10min, washed repeatedly with physiological saline to obtain probiotic bacteria slime.
(2)凝胶基质：卡波姆分散于蒸馏水中溶胀，浓度为1-10％，加入三乙醇胺调pH至4-12，加入甘油，加入透明质酸，搅拌均匀，得到凝胶基质。(2) Gel matrix: Carbomer is dispersed in distilled water and swelled at a concentration of 1-10%, adding triethanolamine to adjust the pH to 4-12, adding glycerin, adding hyaluronic acid, stirring evenly to obtain a gel matrix.
(3)将益生菌菌泥用蒸馏水分散，于凝胶基质混合，搅拌均匀，得到益生菌凝胶。(3) Disperse the probiotic bacteria slime with distilled water, mix it with the gel matrix, and stir evenly to obtain the probiotic bacteria gel.
实施例6Example 6
表2Table 2
(1)益生菌菌泥的制备：取益生菌菌种于BHI液体培养基中，5％CO2、37℃条件下，厌氧环境中培养12h，益生菌培养液以3000r/min的转速离心10min，反复用生理盐水洗涤，得到益生菌菌泥。(1) Preparation of probiotic slime: take probiotic strains in BHI liquid medium, culture in an anaerobic environment for 12 hours under 5% CO2 and 37°C, and centrifuge the probiotic culture solution at a speed of 3000r/min 10min, washed repeatedly with physiological saline to obtain probiotic bacteria slime.
(2)凝胶基质：泊洛沙姆分散于蒸馏水中溶胀，浓度为1-10％，加入甘油，加入透明质酸，搅拌均匀，得到凝胶基质。(2) Gel matrix: the poloxamer is dispersed in distilled water and swelled at a concentration of 1-10%, adding glycerin and hyaluronic acid, and stirring evenly to obtain a gel matrix.
(3)将益生菌菌泥用蒸馏水分散，于凝胶基质混合，搅拌均匀，得到益生菌凝胶。(3) Disperse the probiotic bacteria slime with distilled water, mix it with the gel matrix, and stir evenly to obtain the probiotic bacteria gel.
实施例7Example 7
表3table 3
(1)益生菌菌泥的制备：取益生菌菌种于BHI液体培养基中，5％CO2、37℃条件下，厌氧环境中培养12h，益生菌培养液以3000r/min的转速离心10min，反复用生理盐水洗涤，得到益生菌菌泥。(1) Preparation of probiotic slime: take probiotic strains in BHI liquid medium, culture in an anaerobic environment for 12 hours under 5% CO2 and 37°C, and centrifuge the probiotic culture solution at a speed of 3000r/min 10min, washed repeatedly with physiological saline to obtain probiotic bacteria slime.
(2)凝胶基质：泊洛沙姆分散于蒸馏水中溶胀，浓度为1-10％，加入甘油，加入羟丙基甲基纤维素，搅拌均匀，得到凝胶基质。(2) Gel matrix: poloxamer is dispersed in distilled water and swelled at a concentration of 1-10%, adding glycerin and hydroxypropyl methylcellulose, and stirring evenly to obtain a gel matrix.
(3)将益生菌菌泥用蒸馏水分散，于凝胶基质混合，搅拌均匀，得到益生菌凝胶。(3) Disperse the probiotic bacteria slime with distilled water, mix it with the gel matrix, and stir evenly to obtain the probiotic bacteria gel.
实施例8Example 8
表4Table 4
(1)益生菌菌泥的制备：取益生菌菌种于BHI液体培养基中，5％CO2、37℃条件下，厌氧环境中培养12h，益生菌培养液以3000r/min的转速离心10min，反复用生理盐水洗涤，得到益生菌菌泥。(1) Preparation of probiotic slime: take probiotic strains in BHI liquid medium, culture in an anaerobic environment for 12 hours under 5% CO2 and 37°C, and centrifuge the probiotic culture solution at a speed of 3000r/min 10min, washed repeatedly with physiological saline to obtain probiotic bacteria slime.
(2)凝胶基质：卡波姆分散于蒸馏水中溶胀，浓度为1-10％，加入三乙醇胺调pH至4-12，加入甘油，加入羟丙基甲基纤维素，搅拌均匀，得到凝胶基质。(2) Gel matrix: Carbomer is dispersed in distilled water and swelled at a concentration of 1-10%, adding triethanolamine to adjust the pH to 4-12, adding glycerin, adding hydroxypropyl methylcellulose, stirring evenly to obtain a gel Gel base.
(3)将益生菌菌泥用蒸馏水分散，于凝胶基质混合，搅拌均匀，得到益生菌凝胶。(3) Disperse the probiotic bacteria slime with distilled water, mix it with the gel matrix, and stir evenly to obtain the probiotic bacteria gel.
测定实施例5-8中所获得益生菌凝胶剂的活菌含量。The live bacteria content of the probiotic gel obtained in Examples 5-8 was measured.
分别取实施例5-8中所得益生菌凝胶剂，加入50ml生理盐水，待片剂溶解后，静止5min，用生理盐水梯度稀释至合适浓度，在MRS琼脂培养基上培养，5％CO2、37℃条件下厌氧培养24小时，进行菌落计数。Take the probiotic gels obtained in Examples 5-8 respectively, add 50ml of normal saline, wait for the tablet to dissolve, stand still for 5min, dilute to a suitable concentration with a gradient of normal saline, and cultivate on MRS agar medium, 5% CO2 , Anaerobic culture at 37°C for 24 hours, and colony counting.
表5活菌含量测定结果Table 5 Viable bacteria content assay results
测定实施例5-8中所获得益生菌凝胶剂的透光率考察。Measure the light transmittance of the probiotic gel obtained in Examples 5-8.
分别取实施例5-8中所得益生菌凝胶剂，常温保存1个月、2个月、3个月、4个月、5个月、6个月后，测定透光率。Take the probiotic gels obtained in Examples 5-8 respectively, store them at room temperature for 1 month, 2 months, 3 months, 4 months, 5 months, and 6 months, then measure the light transmittance.
表6Table 6
测定实施例5-8中所获得益生菌凝胶剂的活菌存活率考察Determination of the Viable Bacteria Survival Rate Investigation of the Probiotics Gel Obtained in Examples 5-8
分别取实施例5-8中所得益生菌凝胶剂，常温保存1个月、2个月、3个月、4个月、5个月、6个月后，测定活菌含量。Take the probiotic gels obtained in Examples 5-8 respectively, store them at room temperature for 1 month, 2 months, 3 months, 4 months, 5 months, and 6 months, then measure the content of viable bacteria.
表7Table 7
实施例9益生菌口颊片Embodiment 9 probiotic buccal tablet
总体方法如下：The overall approach is as follows:
(1)益生菌冻干粉的制备：益生菌培养液以3000r/min的转速离心10min，得到益生菌菌泥；在益生菌菌泥中添加脱脂奶粉为冻干保护剂，浓度为1-10％；冻干厚度为1.5-2cm，冷却至-80℃，预冻4h，冷冻干燥处理；出箱，得益生菌冻干粉；(1) Preparation of probiotic freeze-dried powder: the probiotic culture solution was centrifuged at a speed of 3000r/min for 10 minutes to obtain the probiotic slime; adding skimmed milk powder to the probiotic slime was used as a freeze-drying protective agent at a concentration of 1-10 %; freeze-dried with a thickness of 1.5-2cm, cooled to -80°C, pre-frozen for 4 hours, and freeze-dried; out of the box, probiotic freeze-dried powder was obtained;
(2)药用辅料：以下组分按重量份进行配制，其中辅料分别为异麦芽酮糖、甘露醇、微晶纤维素、硬脂酸镁、二氧化硅、甜菊素、薄荷提取物；(2) Pharmaceutical excipients: the following components are prepared in parts by weight, wherein the excipients are isomaltulose, mannitol, microcrystalline cellulose, magnesium stearate, silicon dioxide, stevia, and peppermint extract;
(3)粉末直接压片：将所述辅料粉碎过80目筛，益生菌冻干粉过80目筛，过筛后将辅料与益生菌粉按重量比5:100-1混合，混合后得到可直接压片的原料；(3) Powder direct tableting: the auxiliary material is crushed through an 80-mesh sieve, and the probiotic freeze-dried powder is passed through an 80-mesh sieve. After sieving, the auxiliary material and the probiotic powder are mixed in a weight ratio of 5:100-1 to obtain Raw materials that can be directly compressed into tablets;
(4)压片：将混合的原料投入压片机压片，调节压力为20-60kN，得到益生菌片剂。(4) Tablet compression: put the mixed raw materials into a tablet machine for tablet compression, and adjust the pressure to 20-60kN to obtain probiotic tablets.
具体实施为：The specific implementation is:
(1)药用辅料的配制：按重量份配制，其中异麦芽酮糖醇50份、甘露醇30份、微晶纤维素10份、薄荷提取物4份、甜菊素4份、硬脂酸镁1份、二氧化硅1份；(1) Preparation of pharmaceutical excipients: prepared in parts by weight, including 50 parts of isomalt, 30 parts of mannitol, 10 parts of microcrystalline cellulose, 4 parts of mint extract, 4 parts of stevia, and magnesium stearate 1 part, 1 part of silicon dioxide;
(2)原料混合：将所述辅料粉碎过80目筛，益生菌冻干粉过80目筛，过筛后将辅料与益生菌粉按重量比50-1混合，混合后得到可直接压片的原料；(2) Mixing of raw materials: crush the auxiliary materials through an 80-mesh sieve, and pass the probiotic freeze-dried powder through an 80-mesh sieve. After sieving, mix the auxiliary materials and the probiotic powder at a weight ratio of 50-1, and obtain tablets that can be directly compressed after mixing. raw materials;
(3)压片：将混合的原料投入压片机压片，调节压力为20kN，得到益生菌片剂。(3) Tablet compression: put the mixed raw materials into a tablet machine for tablet compression, and adjust the pressure to 20kN to obtain probiotic tablets.
实施例10:Embodiment 10:
一种含有口腔益生菌的口颊片，其制备方法包括以下步骤：A buccal tablet containing oral probiotics, the preparation method of which comprises the following steps:
(1)药用辅料的配制：按重量份配制，其中异麦芽酮糖醇20份、甘露醇50份、微晶纤维素20份、薄荷提取物4份、甜菊素4份、硬脂酸镁0.5份、二氧化硅1.5份；(1) Preparation of pharmaceutical excipients: prepared in parts by weight, including 20 parts of isomalt, 50 parts of mannitol, 20 parts of microcrystalline cellulose, 4 parts of mint extract, 4 parts of stevia, and magnesium stearate 0.5 parts, 1.5 parts of silicon dioxide;
(2)原料混合：将所述辅料粉碎过80目筛，益生菌冻干粉过80目筛，过筛后将辅料与益生菌粉按重量比50-1混合，混合后得到可直接压片的原料；(2) Mixing of raw materials: crush the auxiliary materials through an 80-mesh sieve, and pass the probiotic freeze-dried powder through an 80-mesh sieve. After sieving, mix the auxiliary materials and the probiotic powder at a weight ratio of 50-1, and obtain tablets that can be directly compressed after mixing. raw materials;
(3)压片：将混合的原料投入压片机压片，调节压力为20kN，得到益生菌片剂。(3) Tablet compression: put the mixed raw materials into a tablet machine for tablet compression, and adjust the pressure to 20kN to obtain probiotic tablets.
实施例11:Embodiment 11:
一种含有口腔益生菌的口颊片，其制备方法包括以下步骤：A buccal tablet containing oral probiotics, the preparation method of which comprises the following steps:
(1)药用辅料的配制：按重量份配制，其中异麦芽酮糖醇40份、甘露醇20份、微晶纤维素30份、薄荷提取物4份、甜菊素4份、硬脂酸镁1.5份、二氧化硅0.5份；(1) Preparation of pharmaceutical excipients: prepared in parts by weight, including 40 parts of isomalt, 20 parts of mannitol, 30 parts of microcrystalline cellulose, 4 parts of mint extract, 4 parts of stevia, and magnesium stearate 1.5 parts, 0.5 parts of silicon dioxide;
(2)原料混合：将所述辅料粉碎过80目筛，益生菌冻干粉过80目筛，过筛后将辅料与益生菌粉按重量比100-1混合，混合后得到可直接压片的原料；(2) Mixing of raw materials: crush the auxiliary materials through an 80-mesh sieve, and pass the probiotic freeze-dried powder through an 80-mesh sieve. After sieving, mix the auxiliary materials and the probiotic powder at a weight ratio of 100-1, and obtain tablets that can be directly compressed after mixing. raw materials;
(3)压片：将混合的原料投入压片机压片，调节压力为40kN，得到益生菌片剂。(3) Tablet compression: put the mixed raw materials into a tablet press for tablet compression, and adjust the pressure to 40kN to obtain probiotic tablets.
实施例12:Embodiment 12:
一种含有口腔益生菌的口颊片，其制备方法包括以下步骤：A buccal tablet containing oral probiotics, the preparation method of which comprises the following steps:
(1)药用辅料的配制：按重量份配制，其中异麦芽酮糖醇50份、甘露醇15份、微晶纤维素25份、薄荷提取物4份、甜菊素4份、硬脂酸镁1份、二氧化硅1份；(1) Preparation of pharmaceutical excipients: prepared in parts by weight, including 50 parts of isomalt, 15 parts of mannitol, 25 parts of microcrystalline cellulose, 4 parts of mint extract, 4 parts of stevia, and magnesium stearate 1 part, 1 part of silicon dioxide;
(2)原料混合：将所述辅料粉碎过80目筛，益生菌冻干粉过80目筛，过筛后将辅料与益生菌粉按重量比100-1混合，混合后得到可直接压片的原料；(2) Mixing of raw materials: crush the auxiliary materials through an 80-mesh sieve, and pass the probiotic freeze-dried powder through an 80-mesh sieve. After sieving, mix the auxiliary materials and the probiotic powder at a weight ratio of 100-1, and obtain tablets that can be directly compressed after mixing. raw materials;
(3)压片：将混合的原料投入压片机压片，调节压力为40kN，得到益生菌片剂。(3) Tablet compression: put the mixed raw materials into a tablet press for tablet compression, and adjust the pressure to 40kN to obtain probiotic tablets.
测定实施例9-12中所获得口颊片的活菌含量。The live bacteria content of the buccal tablets obtained in Examples 9-12 was measured.
分别取实施例9-12中所得口颊片，加入50ml生理盐水，待片剂溶解后，静止5min，用生理盐水梯度稀释至合适浓度，在MRS琼脂培养基上培养，5％CO2、37℃条件下厌氧培养24小时，进行菌落计数。Take the buccal tablets obtained in Examples 9-12 respectively, add 50ml of normal saline, wait for the tablet to dissolve, stand still for 5min, dilute with normal saline to a suitable concentration, and cultivate on MRS agar medium, 5% CO2 , 37 Under the condition of ℃, anaerobic culture was carried out for 24 hours, and colony counting was carried out.
表8活菌含量测定结果Table 8 Viable bacteria content assay results
实验实施例2：Experimental Example 2:
测定实施例9-12中所获得口颊片的稳定性考察Measure the stability investigation of obtained buccal tablet in embodiment 9-12
分别取实施例9-12中所得口颊片，常温保存1个月、2个月、3个月、4个月、5个月、6个月后，按照实验实施例2中方法，测定片剂中的活菌含量。Get the buccal tablets obtained in Examples 9-12 respectively, store them at room temperature for 1 month, 2 months, 3 months, 4 months, 5 months, and 6 months, and measure the tablet according to the method in Experimental Example 2. The content of live bacteria in the agent.
表9稳定性考察Table 9 Stability investigation
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the scope of the present invention. Protection scope, within the spirit and principles of the present invention, any modification, equivalent replacement, improvement, etc., shall be included in the protection scope of the present invention.
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| IT202100001169A1 (en) | 2021-01-27 | 2022-07-27 | Alfasigma Spa | BUCCAL MUCOADHESIVE GEL CONTAINING PROBIOTICS FOR USE IN THE PREVENTION AND TREATMENT OF PERIODONTAL DISEASES. |
| WO2022161893A1 (en) | 2021-01-27 | 2022-08-04 | Alfasigma S.P.A. | Buccal mucoadhesive gel containing probiotics for use in the prevention and treatment of periodontal diseases. |
| IT202100010802A1 (en) | 2021-05-03 | 2022-11-03 | Alfasigma Spa | SOLID MUCOADHESIVE FORMULATION INCLUDING PROBIOTICS FOR USE IN THE PREVENTION AND TREATMENT OF ORAL DYSBIOSIS. |
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| CN107865891B (en) | 2020-04-28 |
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