WO2021103534A1 - 一种唾液乳杆菌ls97及其应用 - Google Patents

一种唾液乳杆菌ls97及其应用 Download PDF

Info

Publication number
WO2021103534A1
WO2021103534A1 PCT/CN2020/099923 CN2020099923W WO2021103534A1 WO 2021103534 A1 WO2021103534 A1 WO 2021103534A1 CN 2020099923 W CN2020099923 W CN 2020099923W WO 2021103534 A1 WO2021103534 A1 WO 2021103534A1
Authority
WO
WIPO (PCT)
Prior art keywords
streptococcus
lactobacillus salivarius
strain
caries
group
Prior art date
Application number
PCT/CN2020/099923
Other languages
English (en)
French (fr)
Inventor
方曙光
吴明科
朱建国
王思清
占英英
Original Assignee
江苏微康生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏微康生物科技有限公司 filed Critical 江苏微康生物科技有限公司
Priority to US17/777,347 priority Critical patent/US20220409681A1/en
Publication of WO2021103534A1 publication Critical patent/WO2021103534A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • This application relates to the field of microorganisms, and in particular to a Lactobacillus salivarius LS97 and its applications.
  • Streptococcus sobrinus was once called Streptococcus sobrinus or Streptococcus pilarius, because it has a layer of hairy under the electron microscope.
  • Streptococcus cousinus has been regarded as an important cariogenic bacteria. Compared with Streptococcus mutans, Streptococcus cousinus has stronger acid production and acid resistance, and is more closely related to high caries. Therefore, it has received increasing attention from researchers.
  • the methods to prevent and treat dental caries caused by Streptococcus cousinus mainly include drug methods, diet control methods, and mechanical removal methods.
  • drug methods for example, the use of compounds containing fluoride, phenolic compounds or strong oxidants can be used to kill the cousin chain.
  • the purpose of cocci, but excessive use of drugs can easily lead to an increase in bacterial resistance, and is accompanied by corresponding side effects.
  • diet control is difficult to achieve due to the complex composition of modern diets (contains many natural sugars, refined sugars or sugar substitutes, etc.). Therefore, finding new prevention and treatment programs has become a research hotspot.
  • Probiotics is a general term for a class of active microorganisms that are beneficial to the host, and its application in intestinal health has been extensively studied. In terms of antagonizing pathogenic bacteria, probiotics can achieve the purpose of inhibiting pathogenic bacteria through the production of bacteriocins, hydrogen peroxide, organic acids and other forms.
  • bacteriocins hydrogen peroxide
  • organic acids organic acids and other forms.
  • Streptococcus mutans as an example, Chen et al. (Chinese Publication No. CN109908185A) screened out a strain of Lactobacillus plantarum CCFM8724 that can effectively inhibit the formation of Streptococcus mutans biofilm and can reduce the production of Streptococcus mutans extracellular polysaccharides by about 80%. Zhao et al.
  • This application provides a Lactobacillus salivarius LS97 that can efficiently inhibit the growth of Streptococcus cousin and its application.
  • the first aspect of this application provides a Lactobacillus salivarius, named LS97, which has been deposited in the General Microbiology Center of the China Microbial Culture Collection and Management Committee on December 10, 2018. The deposit number is CGMCC NO.16922, and the classification is named as saliva milk. Lactobacillus salivarius, deposited at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.
  • the second aspect of the present application provides an application of Lactobacillus salivarius LS97 in the preparation of a medicine for relieving oral caries.
  • the medicine includes a live strain, a dried strain or a metabolite of Lactobacillus salivarius LS97.
  • the medicine includes a pharmaceutically acceptable carrier.
  • the dosage form of the medicine is a tablet, a capsule, an oral liquid or a lyophilized powder.
  • Lactobacillus salivarius LS97 can inhibit the growth and reproduction of cousin Streptococcus. In addition, it has good copolymerization ability with planktonic cousin Streptococcus, and can eliminate cousin Streptococcus through antagonism and copolymerization.
  • LS97 can tolerate up to 1 mg/mL lysozyme and its acid production capacity is much lower than that of cousin Streptococcus.
  • Figure 1 is a comparison diagram of the self-polymerization and copolymerization performance of different lactic acid bacteria strains with cousin Streptococcus in this application;
  • Figure 2 is a comparison diagram of the acid production performance of different lactic acid bacteria strains in this application during the growth process
  • Figure 3 is a graph showing the results of the growth inhibition ability of Lactobacillus salivarius LS97 on Streptococcus mutans and Streptococcus sanguis in the present application;
  • Figure 4 is a comparison diagram of the dental caries scores of rats in this application.
  • the sterilized inoculating loop was used to pick the aforementioned different single colonies, streaked and purified them on MRS solid medium, and cultured them anaerobicly at 37°C for 48 hours to obtain a total of 11 pure lactic acid bacteria strains, named A1-A11, respectively.
  • the double-layer cup and saucer method was used to investigate the inhibitory effect of the isolated strains A1-A11 on the growth of Streptococcus sobrinus ATCC27352 (purchased from ATCC) strain.
  • the mixture (10 7 CFU/mL) was poured on the lower medium. After coagulation, take out the Oxford cup, add 100 ⁇ L of the culture supernatant of Lactobacillus to each well, and culture the above plate in an environment of 37° C. (5% CO 2) overnight.
  • lactic acid bacteria A5 has the strongest inhibitory ability, and its inhibition zone diameter is 23.1 ⁇ 1.1mm. It is higher than other strains' ability to inhibit Streptococcus cousinus.
  • ND means that no inhibition zone has been detected
  • Self-aggregation capacity (A 0 -A 24 )/A 24 ⁇ 100.
  • Lactic acid bacteria A5 has excellent self-polymerization and copolymerization capabilities, and its self-polymerization and copolymerization capabilities are much higher than other strains, indicating that it can also eliminate cousin Streptococcus through copolymerization.
  • the double-layer cup and saucer method was used to investigate the strain’s tolerance to lysozyme.
  • the specific operation is as follows: 1.5% (W/V) agar medium is used to prepare the lower medium, and after solidification, the sterile Oxford cup is fixed in a suitable Place, take 100 ⁇ L of probiotic bacteria liquid adjusted to OD 600 to 0.5 and add it to MRS medium containing 0.8% agar (W/V) cooled to about 50°C, mix well and pour the plate.
  • strain A1 The experimental results are shown in Table 2. Except for strain A1, the other strains can grow normally in a 1 mg/mL lysozyme solution, and strain A7 can tolerate a lysozyme concentration of 10 mg/mL. Under normal circumstances, the concentration of lysozyme contained in the oral cavity is 1-57 ⁇ g/mL. The above results indicate that the tolerance of the above-mentioned strains to lysozyme far exceeds the concentration of lysozyme in the normal oral cavity.
  • the strain A5 was screened for the ability to inhibit Streptococcus cousinus, strong self-polymerization and copolymerization performance, combined with lysozyme tolerance and acid production performance analysis of the isolated strain.
  • the identification method is as follows:
  • the obtained strain A5 was identified by 16s rDNA sequencing and physiological and biochemical identification.
  • the 16s rDNA universal primer sequence 27F is: 5'-AGAGTTTGATCCTGGCTCAG-3' (Seq ID No: 1)
  • 1492R is: 5'-GGTTACCTTGTTACGACTT-3'(Seq ID No: 2).
  • the 16S rDNA gene sequence of strain A5 was amplified and sequenced, and the PCR amplified product was sent to Shanghai Shenggong Bioengineering (Shanghai) Co., Ltd. for sequencing.
  • the nucleotide sequence of 16S rDNA of strain A5 is shown in Seq ID No: 3 .
  • strain A5 was identified as a strain of Lactobacillus salivarius and named Lactobacillus salivarius LS97. Its physiological and biochemical properties are shown in Table 3. Shown.
  • the above plate was cultured overnight at 37°C (5% CO 2 ), and a previously reported strain of Streptococcus salivarius having the ability to inhibit the above two strains was used as a control strain (Streptococcus salivarius K12, purchased from ATCC).
  • the experimental results are shown in Figure 3. Lactobacillus salivarius LS97 can effectively inhibit the growth of Streptococcus mutans and Streptococcus sanguis, and the diameter of the inhibition zone is 24.2 ⁇ 1.9 and 18.5 ⁇ 1.2mm, respectively. It has the ability to inhibit the above two cariogenic bacteria. Higher than the control strain Streptococcus salivarius K12.
  • LS97 LS97 group
  • chlorhexidine is an ionic drug with good antibacterial effect, which serves as a positive control group.
  • the cariogenic bacteria S.sobrinus and LS97 were cultured overnight for 12 hours. After adjusting the bacterial concentration to 1 ⁇ 10 8 CFU/mL, use a sterile cotton swab to dip 0.2 mL on the molars of the rat and wipe it, that is, a cotton swab that saturates and absorbs the bacterial solution.
  • the uniform action time of the sons on each quarter of the rat’s oral teeth is 15s.
  • the rats’ oral teeth were sampled for the first, second and third times respectively, and 12 ⁇ g/mL vancomycin was added. Coat the MRS solid medium to count the cousin Streptococcus. After the dental caries model group and the intervention group were infected with the cousin Streptococcus, the cousin Streptococcus in the rat oral cavity was in the range of 2 ⁇ 4 ⁇ 10 5 CFU/mL, and there was no significant difference between the groups, as shown in Table 6 for the first time The sampling results for the count of cousin Streptococcus are shown.
  • the cousin Streptococcus in the two groups decreased by 1 to 2 orders of magnitude.
  • the number of cousin Streptococcus in the Lactobacillus salivarius LS97 group decreased more significantly.
  • the chlorhexidine group was in the third time The increase in the number of cousin Streptococcus during the test may be due to the development of drug resistance in the strain.
  • the above results show that both chlorhexidine and Lactobacillus salivarius LS97 can inhibit the number of cousin Streptococcus, but the intervention effect of Lactobacillus salivarius LS97 is better than that of the chlorhexidine group.
  • the rat was killed and the molar teeth were taken out.
  • the molars were placed in 0.4% ammonium urethane staining solution and soaked overnight for 12 hours for staining, rinsed with water and dried naturally, and then observed under a stereo microscope.
  • the molar caries of rats in the group were scored by Keyes caries scoring method.
  • E grade caries which only affects enamel
  • Ds grade caries enamel caries and caries affect less than 1/4 of the range
  • the outer layer of dentin Dm grade caries, caries affect 1/4 to 3/4 of the dentin thickness
  • Dx grade caries caries affect more than 3/4 of the dentine thickness
  • both the chlorhexidine group and the LS97 group can effectively reduce the occurrence of molar enamel caries (E), superficial dentin caries (Ds) and dental caries (Dm) in rats.
  • E molar enamel caries
  • Ds superficial dentin caries
  • Dm dental caries
  • the chlorhexidine group can effectively reduce E caries and Ds caries, but the effect on Dm caries is not obvious.
  • the effect of LS97 group on reducing the occurrence of dental caries is higher than that of chlorhexidine, indicating that Lactobacillus salivarius LS97 is better than chlorhexidine in the treatment of dental caries.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Mycology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Wood Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Cosmetics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)

Abstract

一种唾液乳杆菌LS97及其应用，该菌株已于2018年12月10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.16922，分类命名为唾液乳杆菌(Lactobacillus salivarius)，保藏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，其可高效抑制表兄链球菌生长。

Description

一种唾液乳杆菌LS97及其应用 技术领域
本申请涉及微生物领域，尤其涉及一种唾液乳杆菌LS97及其应用。
背景技术
表兄链球菌(Streptococcus sobrinus)曾称为远缘链球菌或茸毛链球菌，因其在电镜下有一层茸毛层而得名。长期以来，表兄链球菌一直被认为是一种重要的致龋齿菌，与变异链球菌相比较，表兄链球菌的产酸性能及耐酸性能更强，与高度龋齿性的关系更为密切，因此日益受到研究者的重视。
目前，防治表兄链球菌引起的龋齿的方法主要包括药物法、饮食控制法及机械去除法等，如可通过使用含氟化物、酚类化合物或强氧化剂等化合物，来达到杀灭表兄链球菌的目的，但是药物的过度使用易导致细菌耐药性的增加，且伴随有相应的副作用等。而饮食控制则受到现代饮食成分复杂(含有众多的天然糖、精制糖或糖类替代品等)等问题而难以实现，因此寻找新的防治方案已成为研究的热点。
益生菌是一类对宿主有益的活性微生物的总称，其在肠道健康方面的应用已被广泛研究。在拮抗致病菌方面，益生菌可通过产生细菌素、过氧化氢、有机酸等多种形式达到抑制致病菌的目的。以变异链球菌为例，陈等(中国公开号CN109908185A)筛选出一株可高效抑制变异链球菌生物膜形成的植物乳杆菌CCFM8724，可使变异链球菌胞外多糖产生量减少80％左右。赵等(中国公开号CN108048347A)筛选出一株鼠李糖乳杆菌LR863，对变异链球菌的抑菌圈直径达15.35±0.06mm。但需要指出的是，目前选育可拮抗口腔龋齿菌变异链球菌的菌株报道较多，而筛选能高效抑制表兄链球菌的菌株的报道则几乎没有，相关菌株资源几乎空白。
发明内容
本申请提供了一种可高效抑制表兄链球菌生长的唾液乳杆菌LS97及其应用。
本申请第一方面提供一种唾液乳杆菌，命名为LS97，已于2018年12月10 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.16922，分类命名为唾液乳杆菌(Lactobacillus salivarius)，保藏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。
本申请第二方面提供一种唾液乳杆菌LS97，在制备用于缓解口腔龋齿的药物中的应用。
进一步地，所述药物中包括唾液乳杆菌LS97的活菌株、干菌株或代谢物。
进一步地，所述药物包括药学上可接受的载体。
进一步地，所述药物的剂型为片剂、胶囊剂、口服液或冻干粉。
借由上述方案，本申请至少具有以下优点：
1、唾液乳杆菌LS97可抑制表兄链球菌的生长繁殖，此外，其与浮游状表兄链球菌具有良好的共聚能力，可通过拮抗与共聚作用等清除表兄链球菌。
2、对唾液乳杆菌LS97抑制其它致病菌的能力进行考察发现，LS97可高效抑制变异链球菌和血链球菌。
3、利用体外实验进行菌株产酸及溶菌酶耐受性能评价，LS97可耐受高达1mg/mL的溶菌酶且产酸能力远低于表兄链球菌等。
4、大鼠实验结果显示，唾液乳杆菌LS97可有效缓解龋齿的发生，效果优于洗必泰。
上述说明仅是本申请技术方案的概述，为了能够更清楚了解本申请的技术手段，并可依照说明书的内容予以实施，以下以本申请的较佳实施例详细说明如后。
附图说明
图1是本申请中不同乳酸菌菌株自聚及与表兄链球菌共聚性能对比图；
图2是本申请中不同乳酸菌菌株生长过程产酸性能对比图；
图3是本申请中唾液乳杆菌LS97对变异链球菌和血链球菌生长抑制能力结果图；
图4是本申请中大鼠龋齿得分对比图。
具体实施方式
下面结合实施例，对本申请的具体实施方式作进一步详细描述。以下实施 例用于说明本申请，但不用来限制本申请的范围。
实施例1唾液乳杆菌LS97的选育获得
1)菌株筛选
提取来自中国广西壮族自治区河池市巴马镇健康婴儿消化道提前液(采集前人群均未服用过抗生素类药物，无益生菌服用史，无胃肠病史)的稀释液5mL，10倍系列稀释至10 -6，每个稀释浓度取200μL涂布于选择性MRS培养基，37℃厌氧培养48-72h，分离获得平板上不同的单菌落。用灭菌后的接种环挑取前述不同单菌落在MRS固体培养基上划线纯化，37℃厌氧培养48h，得到纯乳酸菌菌株共11株，分别命名为A1-A11。
2)分离菌株拮抗表兄链球菌的试验
采用双层杯碟法考察所分离菌株A1-A11对表兄链球菌(Streptococcus sobrinus ATCC27352，购自ATCC)菌株生长的抑制作用。将1.5％(w/v)的BHI琼脂培养基倒入平皿中，凝固后将无菌的牛津杯固定在适宜的位置，然后将0.7％(w/v)的BHI培养基与表兄链球菌的混合物(10 7CFU/mL)倾倒在下层培养基上。待凝固后取出牛津杯，在每个孔中加入100μL乳酸杆菌的培养上清液，将上述平板在37℃(5％CO 2)的环境中培养过夜。
实验结果如表1所示，除A1、A3、A7和A11外，其余乳酸菌均可有效抑制表兄链球菌的生长，其中乳酸菌A5抑制能力最强，其抑菌圈直径达23.1±1.1mm，高于其它菌株对表兄链球菌的抑制能力。
表1不同乳酸菌对表兄链球菌的抑制能力考察
菌株 抑菌圈直径(mm)
A1 ND
A2 10.9±0.6
A3 ND
A4 13.2±1.0
A5 23.1±1.1
A6 16.8±0.9
A7 ND
A8 14.3±1.1
A9 15.9±1.3
A10 9.4±0.5
A11 ND
注：ND表示未检测出抑菌圈
3)分离菌株自聚和共聚性能分析
分别取厌氧条件下过夜培养的不同菌株与表兄链球菌，3000r/min离心10min，PBS(10mmol/L，pH 7.2)清洗2次，重悬，调节菌浓度至OD 600至0.5。共聚实验时，分别取表兄链球菌和不同菌株500μL并分别测定初始OD 600值Ax 0和Ay 0，混匀后37℃厌氧放置24h后测定OD 600值A 24，共聚能力＝[(Ax 0+Ay 0)-2×A 24)]/(Ax 0+Ay 0)×100；自聚实验时，分别取不同乳酸菌菌液1000μL，测定初始OD 600值A 0，37℃厌氧放置24h后测定OD 600值A 24。自聚能力＝(A 0-A 24)/A 24×100。
结果如图1所示，乳酸菌A5具有优异的自聚和共聚能力，其自聚和共聚能力远高于其它菌株，表明其还可通过共聚等作用清除表兄链球菌。
4)分离菌株溶菌酶耐受性能分析
采用双层杯碟法考察菌株对溶菌酶的耐受情况，具体操作如下：采用1.5％(W/V)的琼脂培养基制备下层培养基，待凝固后将无菌的牛津杯固定在适宜的位置，取100μL OD 600调节至0.5的益生菌菌液加入冷却至50℃左右含琼脂0.8％(W/V)的MRS培养基，混合均匀后倒平板。待凝固后取出牛津杯，向孔中加入50μL不同浓度的溶菌酶溶液(分别为0.2mg/mL、0.4mg/mL、0.6mg/mL、0.8mg/mL、1.0mg/mL和10.0mg/mL)，37℃厌氧培养48h测量抑菌圈直径。
实验结果如表2所示，除菌株A1外，其余菌株均可在1mg/mL的溶菌酶溶液中正常生长，其中菌株A7可耐受达10mg/mL的溶菌酶浓度。正常情况下，口腔中所含溶菌酶浓度为1-57μg/mL，以上结果表明上述菌株对溶菌酶的耐受能力远远超过正常口腔内溶菌酶浓度。
表2不同溶菌酶浓度对菌株的抑制性能考察
Figure PCTCN2020099923-appb-000001
5)分离菌株产酸性能分析
测定不同乳酸菌菌株及表兄链球菌细菌悬液的吸光度，并用磷酸盐缓冲液调节OD 600值至0.5±0.02，分别按2％(v/v)的接种量分别接种到MRS或BHI培养基中，37℃(5％CO 2)培养24h，测定终点培养液中的pH值，结果如图2所示。致龋齿菌表兄链球菌产酸性能强，终点pH值最低。相比之下，菌株A5及A6产酸性能较弱。益生菌发酵后可产生有机酸，降低环境pH值，这虽在一定程度上可抑制一些致病菌的繁殖，但在口腔环境内，有机酸特别是乳酸的产生也容易带来更大的龋齿风险，因此从产酸性能来讲产酸性相对较弱的菌株越有利于口腔防护的应用。
综合以上不同的筛选方案的结果，以对表兄链球菌抑制能力、自聚和共聚性能强，结合溶菌酶的耐受及分离菌株产酸性能分析，筛选获得菌株A5。
实施例二 菌株鉴定：
根据实施例一获得的菌株，鉴定方法如下：
对获得的菌株A5利用16s rDNA测序鉴定和生理生化鉴定，16s rDNA通用引物序列27F为：5’-AGAGTTTGATCCTGGCTCAG-3’(Seq ID No：1)，1492R为：5’-GGTTACCTTGTTACGACTT-3’(Seq ID No：2)。对菌株A5的16s rDNA基因序列进行扩增和测序，PCR扩增产物送上海生工生物工程(上海)股份有限公司测序，菌株A5的16S rDNA的核苷酸序列如Seq ID No：3所示。经过16s rDNA基因对比，与Genebank中的Lactobacillus salivarius菌株比对相似率达100％，结合生理生化鉴定结果菌株A5鉴定为一株唾液乳杆菌，命名为唾液乳杆菌LS97，其生理生化性质如表3所示。
表3菌株A5生理生化鉴定结果
生化试验项目 鉴定结果 生化试验项目 鉴定结果
水杨苷 - 甘露糖 -
麦芽糖 + 葡萄糖酸盐 -
苦杏仁苷 - pH 3.5 +
半乳糖 + 明胶液化 -
阿拉伯通 - 厌氧生长 +
甘露糖 - 15℃生长 -
果糖 + 精氨酸产氨 -
注：+：阳性结果；-：阴性结果
实施例三唾液乳杆菌LS97对变异链球菌和血链球菌生长的抑制能力分析
同样采用双层杯碟法考察唾液乳杆菌LS97对变异链球菌(Streptococcus mutans UA159，购自ATCC)和血链球菌(Streptococcus sanguinis SK36，购自ATCC)生长的抑制，将1.5％(w/v)的琼脂培养基倒入平皿中，凝固后将无菌的牛津杯固定在适宜的位置，然后将0.7％(w/v)的BHI培养基与含变异链球菌或血链球菌的混合物(10 7CFU/mL)倾倒在下层培养基上。待凝固后取出牛津杯，在每个孔中加入100μL乳酸杆菌的培养上清液。将上述平板在37℃(5％CO 2)的环境中培养过夜，以先前报道的一株具有抑制上述两株菌能力的唾液链球菌作为对照菌株(Streptococcus salivarius K12，购自ATCC)。实验结果如图3所示，唾液乳杆菌LS97可有效抑制变异链球菌和血链球菌的生长，抑菌圈直径 分别达24.2±1.9和18.5±1.2mm，对上述两株致龋齿菌的抑制能力高于对照菌株唾液链球菌K12。
实施例四唾液乳杆菌LS97对大鼠龋齿的缓解作用分析
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10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(龋齿组)，两组设置为干预组，干预组连续7天擦拭S.sobrinus，确定其在大鼠口腔成功定植之后，对应实验组分别连续7天擦拭洗必泰(洗必泰组)和LS97(LS97组)，之后每周3次。其中洗必泰为抑菌效果良好的离子型药物，作为阳性对照组。其中致龋细菌S.sobrinus和LS97均过夜培养12h，调节菌浓度到1×10 8CFU/mL后，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Diet 2000#和添加有5％蔗糖的饮用水，致龋饲料主要成分见表4。实验周期维持10周。
表4致龋饲料Diet 2000#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
盐 2
1)唾液乳杆菌LS97对大鼠体重的影响
大鼠实验前后体重测定结果如表5所示。大鼠实验期间，实验组与对照组之间大鼠体重增量无显著性差异(P>0.05)，说明在动物实验期间所有的实验动 物呈现出良好的身体状况。
表5大鼠实验前后体重测定结果
组别 实验前大鼠体重(g) 实验后大鼠体重(g) 大鼠体重增量(g)
空白组 64.03±3.15 205.94±21.35 140.42±23.74
龋齿组 62.98±3.56 209.32±20.12 145.12±21.75
洗必泰组 60.32±5.28 212.49±23.86 147.83±19.73
LS97组 60.13±6.03 211.51±19.75 147.09±19.94
注：每列各个组之间的体重均无显著差异(P>0.05)
2)大鼠口腔中表兄链球菌(S.sobrinus)的变化情况
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，在添加有12μg/mL万古霉素的MRS固体培养基上涂布进行表兄链球菌计数。龋齿模型组和干预组在感染表兄链球菌之后大鼠口腔中的表兄链球菌在2～4×10 5CFU/mL的范围，各组之间无显著差异，如表6中第一次取样对表兄链球菌的计数结果所示。对实验组进行干预后，两组中的表兄链球菌下降1～2个数量级，其中唾液乳杆菌LS97组中表兄链球菌数量下降更为明显，此外，洗必泰组中在第三次检验时出现表兄链球菌数量上升的情况，可能是由于菌株产生耐药性所致。以上结果显示洗必泰和唾液乳杆菌LS97均可抑制表兄链球菌数量，但唾液乳杆菌LS97干预效果优于洗必泰组。
表6不同时期各组实验大鼠口腔取样表兄链球菌计数情况(Log值)
取样次数 空白组 龋齿组 洗必泰组 LS97组
1 <30 5.41±0.12 5.53±0.23 5.51±0.21
2 <30 5.28±0.09 4.00±0.19 3.63±0.13
3 <30 5.17±0.11 4.31±0.13 3.48±0.12
3)大鼠龋齿得分
大鼠实验结束后，杀死大鼠并取出大鼠臼齿，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4所示。
从图4可知，与龋齿模型组相比，洗必泰组和LS97组均能有效减轻大鼠磨牙釉质龋(E)、牙本质浅龋(Ds)和牙本质中龋(Dm)的发生。另外，对比洗必泰组和LS97组作用结果可知，洗必泰组能有效减少E龋和Ds龋，但对Dm龋效果不明显。相比之下，LS97组对减少龋齿发生的效果要高于洗必泰，说明在治疗龋齿方面唾液乳杆菌LS97的效果胜于洗必泰。
以上所述仅是本申请的优选实施方式，并不用于限制本申请，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本申请的保护范围。

Claims (5)

  1. 一种唾液乳杆菌LS97，其被命名为LS97，已于2018年12月10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.16922。
  2. 根据权利要求1所述的唾液乳杆菌LS97在制备用于缓解口腔龋齿的药物中的应用。
  3. 根据权利要求2所述的应用，其中，所述药物中包括唾液乳杆菌LS97的活菌株、干菌株或代谢物。
  4. 根据权利要求2所述的应用，其中，所述药物包括药学上可接受的载体。
  5. 根据权利要求2所述的应用，其中，所述药物的剂型为片剂、胶囊剂、口服液或冻干粉。
PCT/CN2020/099923 2019-11-25 2020-07-02 一种唾液乳杆菌ls97及其应用 WO2021103534A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/777,347 US20220409681A1 (en) 2019-11-25 2020-07-02 Lactobacillus salivarius ls97 and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911163718.1A CN110819569B (zh) 2019-11-25 2019-11-25 一种唾液乳杆菌ls97及其应用
CN201911163718.1 2019-11-25

Publications (1)

Publication Number Publication Date
WO2021103534A1 true WO2021103534A1 (zh) 2021-06-03

Family

ID=69558858

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/099923 WO2021103534A1 (zh) 2019-11-25 2020-07-02 一种唾液乳杆菌ls97及其应用

Country Status (3)

Country Link
US (1) US20220409681A1 (zh)
CN (1) CN110819569B (zh)
WO (1) WO2021103534A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115960775A (zh) * 2023-02-28 2023-04-14 吉林大学 一株增加雏鹅的生产性能并抗痛风的唾液乳杆菌

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110819569B (zh) * 2019-11-25 2022-12-06 微康益生菌(苏州)股份有限公司 一种唾液乳杆菌ls97及其应用
CN111471610B (zh) * 2020-03-02 2022-09-06 上海理工大学 一种用于缓解咽炎的唾液乳杆菌
CN112126605A (zh) * 2020-10-12 2020-12-25 镇江市天益生物科技有限公司 一种唾液乳杆菌及其应用
CN114149939B (zh) * 2021-09-27 2023-06-16 微康益生菌(苏州)股份有限公司 一种能够缓解龋齿的益生菌组合物及其应用
CN114621895B (zh) * 2022-03-14 2023-09-12 江南大学 一种能够抑制具核梭菌并改善口气的唾液联合乳杆菌
CN116426406B (zh) * 2022-12-16 2023-10-27 南方医科大学南方医院 一种唾液联合乳杆菌dy802及其应用
CN118147021B (zh) * 2024-05-11 2024-07-09 微康益生菌(苏州)股份有限公司 一种缓解痛风性关节炎的复合益生菌及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016076842A1 (en) * 2014-11-11 2016-05-19 Colgate-Palmolive Company In vitro oral biofilm models of interdental spaces and uses
CN106399197A (zh) * 2016-10-28 2017-02-15 中国人民解放军疾病预防控制所 一株唾液乳杆菌及其用途
CN109182218A (zh) * 2018-10-19 2019-01-11 嘉兴益诺康生物科技有限公司 一株唾液乳杆菌及在下调变形链球菌生物膜形成的基因表达中的应用
CN110819569A (zh) * 2019-11-25 2020-02-21 江苏微康生物科技有限公司 一种唾液乳杆菌ls97及其应用
CN111084754A (zh) * 2020-02-20 2020-05-01 江苏微康生物科技有限公司 一种抗口腔幽门螺杆菌的益生菌牙膏及其制备方法
CN111195266A (zh) * 2019-12-20 2020-05-26 江苏微康生物科技有限公司 具有缓解高尿酸作用的益生菌组合物及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT3412766T (pt) * 2017-06-06 2021-04-19 Casen Recordati S L Estirpe lactobacillus salivarius, composição que compreende a mesma e suas utilizações
CN108486022B (zh) * 2018-07-02 2020-08-28 四川大学 一株抗龋病植物乳杆菌及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016076842A1 (en) * 2014-11-11 2016-05-19 Colgate-Palmolive Company In vitro oral biofilm models of interdental spaces and uses
CN106399197A (zh) * 2016-10-28 2017-02-15 中国人民解放军疾病预防控制所 一株唾液乳杆菌及其用途
CN109182218A (zh) * 2018-10-19 2019-01-11 嘉兴益诺康生物科技有限公司 一株唾液乳杆菌及在下调变形链球菌生物膜形成的基因表达中的应用
CN110819569A (zh) * 2019-11-25 2020-02-21 江苏微康生物科技有限公司 一种唾液乳杆菌ls97及其应用
CN111195266A (zh) * 2019-12-20 2020-05-26 江苏微康生物科技有限公司 具有缓解高尿酸作用的益生菌组合物及其应用
CN111084754A (zh) * 2020-02-20 2020-05-01 江苏微康生物科技有限公司 一种抗口腔幽门螺杆菌的益生菌牙膏及其制备方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AZMI FAYYADHAH BT. MOHD, ZAIHAN ARIFFIN, MOHAMMAD KHURSHEED ALAM: "In Vitro Study of Antibacterial Properties of Dental Restorative Materials towards Streptococcus sobrinus and Lactobacillus salivarius", INTERNATIONAL MEDICAL JOURNAL, vol. 20, no. 4, 1 August 2013 (2013-08-01), pages 473 - 475, XP055814935 *
HAN HUI: "Effect of LPE on the Growth of Five Cariogenic Bacteria and a Variety of Cariogenic Enzymes Activity of Streptococcus.Sobrinus", MEDICINE & PUBLIC HEALTH, CHINA MASTER’S THESES FULL-TEXT DATABASE, 1 May 2012 (2012-05-01), XP055814938 *
LUMIKARI, M. ; SOUKKA, T. ; NURMIO, S. ; TENOVUO, J.: "Inhibition of the growth of Streptococcus mutans, Streptococcus sobrinus and Lactobacillus casei by oral peroxidase systems in human saliva", ARCHIVES OF ORAL BIOLOGY, PERGAMON PRESS, OXFORD., GB, vol. 36, no. 2, 1 January 1991 (1991-01-01), GB, pages 155 - 160, XP026167986, ISSN: 0003-9969, DOI: 10.1016/0003-9969(91)90078-9 *
SUN JINGHUA, NIU YU-MEI, FAN MING-WEN: "Advances on the Cariogenic Character of Streptococcus Sobrinus and Its Relationship with Anti-Caries Immunization", INTERNATIONAL JOURNAL OF STOMATOLOGY, vol. 35, no. 2, 1 March 2008 (2008-03-01), pages 167 - 175, XP055814945 *
YANG JUAN: "Isolation and Characterization of Lactobacillus as Potential Oral Probiotics", MEDICINE & PUBLIC HEALTH, CHINA MASTER’S THESES FULL-TEXT DATABASE, 1 March 2013 (2013-03-01), XP055814936 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115960775A (zh) * 2023-02-28 2023-04-14 吉林大学 一株增加雏鹅的生产性能并抗痛风的唾液乳杆菌
CN115960775B (zh) * 2023-02-28 2024-03-12 吉林大学 一株增加雏鹅的生产性能并抗痛风的唾液乳杆菌

Also Published As

Publication number Publication date
CN110819569B (zh) 2022-12-06
CN110819569A (zh) 2020-02-21
US20220409681A1 (en) 2022-12-29

Similar Documents

Publication Publication Date Title
WO2021103534A1 (zh) 一种唾液乳杆菌ls97及其应用
EP1539927B1 (en) Probiotic bacterium: lactobacillus fermentum
KR101860553B1 (ko) 질 내 병원성 미생물에 대한 증식억제활성을 갖는 락토바실러스 속 균주
CN113832077B (zh) 鼠李糖乳杆菌及其应用
DK2555785T3 (en) Use of Streptococcus salivarius in the treatment of chronic respiratory tract infections
JP2018520677A (ja) たとえば、細菌性膣炎の治療のためのＬａｃｔｏｂａｃｉｌｌｕｓ ｒｈａｍｎｏｓｕｓ（ラクトバチルス・ラムノサス）細菌
WO2008052468A1 (fr) Nouvelle souche de lactobacillus rhamnosus, composition pharmaceutique la contenant et ses utilisations, et procede de preparation associe
CN114774315B (zh) 鼠李糖乳杆菌菌株LRa05在制备增强免疫力制品和/或缓解湿疹制品方面的用途
US11786568B2 (en) Strain of caucasus yoghurt lactobacillus MSR101 and use thereof
JP7369976B2 (ja) 乳酸菌、該乳酸菌由来の自然免疫活性化剤、感染症予防・治療薬及び飲食品
CN116396884A (zh) 鼠李糖乳杆菌和一种抑制幽门螺杆菌的组合物
CN114836349A (zh) 拮抗幽门螺旋杆菌的嗜酸乳杆菌la16及其应用
US11197898B2 (en) Fructophilic lactic acid producing bacteria
CN112546074A (zh) 一株能够抑制IL-23、Th17轴相关炎症因子释放的短双歧杆菌及其应用
KR101164512B1 (ko) 비피도박테리움 슈도카테눌라튬 ｓｐｍ１２０４ 유산균 또는 이의 배양물을 유효성분으로 하는 가축용 생균제 조성물
KR20200128887A (ko) 위장관 운동 장애 질환 개선, 치료 또는 예방용 김치 유산균 조성물
CN111139204B (zh) 一种益生菌菌剂及其制备方法和应用
LU503459B1 (en) Application of Bifidobacteriumlactis in improving intestinal flora disorder
US20220118032A1 (en) Fructophilic lactic acid producing bacteria
AU2019445706B2 (en) Fructophilic lactic acid producing bacteria
CN117089494A (zh) 一种防治幽门螺杆菌感染的副干酪乳杆菌及其组合物与应用
CN117866842A (zh) 一株粪肠球菌mb3及其在制备抗炎和抗衰食品药品中的应用
CN118109345A (zh) 一株粪肠球菌xy3及其在制备抗炎和助睡眠食品药品中的应用
CN116333913A (zh) 一种唾液乳杆菌、含有其的益生菌组合物及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20893261

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20893261

Country of ref document: EP

Kind code of ref document: A1