CN114948843A

CN114948843A - Composition for increasing oral immunoglobulin A content and inhibiting pathogenic bacteria and application thereof

Info

Publication number: CN114948843A
Application number: CN202110219200.6A
Authority: CN
Inventors: 何协勋; 林文扬; 郭易纬; 黄彦宇; 林家弘; 林杞辉; 刘承叡; 陈淑慧
Original assignee: Jinqiao Biotechnology Co ltd
Current assignee: Jinqiao Biotechnology Co ltd
Priority date: 2021-02-26
Filing date: 2021-02-26
Publication date: 2022-08-30

Abstract

Composition for increasing oral immunoglobulin A content and inhibiting pathogenic bacteria and its application are provided. The invention provides application of probiotics in preparation of a composition for increasing oral immunoglobulin A content and inhibiting oral pathogenic bacteria, wherein the probiotics comprise lactobacillus plantarum (LPL 28) as an effective ingredient, can effectively increase oral IgA content and/or inhibit oral pathogenic bacteria, and has the potential of preventing dental caries and/or periodontal disease.

Description

Composition for increasing oral immunoglobulin A content and inhibiting pathogenic bacteria and application

Technical Field

The invention relates to application of probiotics in preparation of an oral composition, in particular to application of probiotics in preparation of a composition for improving oral IgA content and inhibiting oral pathogenic bacteria.

Background

In the human oral cavity, there are tens of thousands of bacteria which decompose saccharides in the oral cavity as a substrate and adhere to the oral mucosa, the tooth surface or the tissues around the teeth to form a tenacious biofilm, thereby affecting the health of the oral cavity, wherein aerobic bacteria such as Streptococcus mutans (Streptococcus mutans) preferentially adhere to the tooth surface, secrete acidic substances and corrode the enamel of the teeth, thereby causing cavities in the teeth. In addition, anaerobic bacteria such as Porphyromonas gingivalis (Porphyromonas gingivalis), Bacillus nuciferus (Fusobactrium tuberculosis), and Actinobacillus actinomycetemcomitans (Aggregatibacter actinomycetemcomitans) are preferably attached to gingival sulcus or periodontal pocket, and release toxins to stimulate periodontal tissues, resulting in swelling of periodontal inflammation, destruction of alveolar bone and tissues, and further symptoms such as tooth loosening, halitosis, and bleeding during tooth brushing.

The oral cavity is covered with mucous membrane as one of the pathways connecting the human body to the outside to protect against pathogenic bacteria, among which Immunoglobulin a (IgA) plays an important role in the mucosal immune system and the humoral immune system. IgA in saliva can maintain ecological balance of oral microorganisms, prevent pathogenic bacteria (such as respiratory tract fusion virus, rotavirus, influenza virus and novel coronavirus) from invading respiratory tract, and prevent oral pathogenic bacteria from adhering to oral cavity.

In addition to the immune system, probiotics may also assist the body in defending against oral pathogens. The probiotic bacteria not only compete with oral pathogenic bacteria for oral nutrients and living space, but also secrete antibacterial components (such as antibacterial peptide, short chain fatty acid and H) ₂ O ₂ ) Can inhibit the growth of pathogenic bacteria in oral cavity, and improve inflammation caused by pathogenic bacteria infection in oral cavity (such as: red swelling, bleeding and ulceration). In addition, some probiotics (whether active or not) and their metabolites (prebiotics) can also increase IgA content in saliva, thereby increasing oral immunity.

In view of the above, it is desirable to provide a probiotic for preparing a composition for increasing the content of immunoglobulin a in the oral cavity and inhibiting oral pathogenic bacteria, so as to increase the oral immunity and inhibit the oral pathogenic bacteria.

Disclosure of Invention

Accordingly, an aspect of the present invention provides a probiotic for preparing a composition for increasing immunoglobulin a (IgA) content in an oral cavity, wherein the probiotic comprises a specific strain as an effective ingredient.

In another aspect, the present invention provides the use of a probiotic for the manufacture of a composition for inhibiting oral pathogenic bacteria, wherein the composition comprises the probiotic bacterial strain as described above as an active ingredient.

In another aspect, the present invention provides a composition for increasing IgA content in the oral cavity and inhibiting pathogenic bacteria in the oral cavity, wherein the composition comprises the probiotic bacterial strain as an active ingredient to increase IgA content in the oral cavity.

According to the aspect of the present invention, the use of a probiotic for preparing a composition for increasing IgA content is provided, wherein the probiotic can be used as an active ingredient, for example, and the probiotic can comprise Lactobacillus plantarum LPL28, wherein the Lactobacillus plantarum LPL28 is deposited in the china general microbiological culture collection center at 2019, 6 and 18 months, and the deposition number is cgmccno. 17954, so as to increase the IgA content in the oral cavity.

In one embodiment of the invention, the probiotic bacteria may optionally comprise Lactobacillus salivarius (L. salivarius) AP-32 and/or Lactobacillus paracasei (L. paracasei) ET-66. The Lactobacillus salivarius AP-32 is preserved in China center for type culture Collection in 2011, 4 and 10 months, and the preservation number is CCTCCNO: M2011127. The lactobacillus paracasei ET-66 is preserved in the China general microbiological culture collection center in 2016, 12 and 29, and the preservation number is CGMCC number 13514.

In one embodiment of the invention, the composition may be, for example, a food composition or an oral topical composition.

In one embodiment of the present invention, the food composition can be, for example, a dairy product, a non-dairy beverage, and a dentifrice.

In one embodiment of the present invention, the oral composition for external use may be, for example, an oral care composition or a breath freshening composition.

According to another aspect of the present invention, there is provided the use of a probiotic, which may for example be an active ingredient, for the manufacture of a composition for inhibiting oral pathogenic bacteria, wherein the probiotic may comprise Lactobacillus plantarum LPL28 for inhibiting the growth of oral pathogenic bacteria. The preservation number of the lactobacillus plantarum LPL28 is CGMCC 17954.

In one embodiment of the present invention, the probiotic bacteria may optionally comprise Lactobacillus salivarius AP-32 and/or Lactobacillus paracasei ET-66. The preservation numbers of the Lactobacillus salivarius AP-32 and the Lactobacillus paracasei ET-66 are CCTCC M2011127 and CGMCC 13514 respectively.

In one embodiment of the invention, the oral pathogenic bacteria comprise Streptococcus mutans (Streptococcus mutans) and/or periodontal pathogenic bacteria.

In one embodiment of the present invention, the periodontal pathogens may include Porphyromonas gingivalis (Porphyromonas gingivalis), Bacillus tuberculosis (Fusobacterium tuberculosis), and Actinobacillus actinomycetemcomitans (Aggregatobacterium actinomycetemcomitans).

According to another aspect of the present invention, a composition for increasing oral IgA content and inhibiting oral pathogenic bacteria is provided, which comprises probiotic bacteria as an active ingredient, wherein the probiotic bacteria comprise, for example, lactobacillus plantarum LPL28, lactobacillus salivarius AP-32, and lactobacillus paracasei ET-66. The preservation numbers of the lactobacillus plantarum LPL28, the lactobacillus salivarius AP-32 and the lactobacillus paracasei ET-66 are CGMCC17954, CCTCC M2011127 and CGMCC 13514 respectively.

The composition can obviously improve the IgA content in saliva and/or inhibit the growth of oral pathogenic bacteria, thereby maintaining the oral health.

Biological material preservation

Lactobacillus plantarum LPL28(Lactobacillus plantarum) was deposited in China General Microbiological Culture Collection Center (CGMCC) 6.18.2019 at the address of No. 3 of the national institute of Microbiological Culture Collection, North Jing, Onyang, China, zip code: 100101, and the deposition number is CGMCC No. 17954.

Lactobacillus salivarius AP-32(Lactobacillus salivarius subsp. salivarius) is deposited at 2011-4-10-day China Center for Type Culture Collection (CCTCC) (address: Wuhan university of Wuhan, China, Zip code: 430072) with the preservation number of CCTCC NO: M2011127. Lactobacillus paracasei ET-66(Lactobacillus paracasei) is deposited in China General Microbiological Culture Collection Center (CGMCC) in 2016, 12 and 29 at the address of No. 3 of the national institute of Microbiological Culture Collection, North Jing, Onyang, China, No.1 Hopkin, Japan, zip code: 100101), and the Collection number is CGMCC number 13514. In addition, Lactobacillus plantarum LPL28(Lactobacillus plantarum) was also deposited at 2011, 12/27 in taiwan, china, new bamboo food road, No. 331, consortium french, human food industry development research institute, bio-resource center, with the deposit number BCRC 910536.

Lactobacillus salivarius AP-32 was deposited in BCRC at 30.07/2009 with accession number BCRC 910437.

Lactobacillus paracasei (Lactobacillus paracasei) ET-66 was deposited in BCRC at 2016, 11, 03, with accession number BCRC 910753.

Drawings

The foregoing and other objects, features, and advantages of the invention will be apparent from the following more particular description of the embodiments of the invention, as illustrated in the accompanying drawings in which:

fig. 1 is a graph showing the percent inhibition in vitro of a single strain of different probiotics by different oral pathogens according to one embodiment of the invention.

FIGS. 2A-2E are diagrams illustrating an implementation of H according to an embodiment of the present invention ₂ O ₂ Measurement of secretion amount H of the strains AP-32 (FIG. 2A), ET-66 (FIG. 2B), LPL28 (FIG. 2C), and three strains (FIG. 2D) tested ₂ O ₂ Content and color chart (fig. 2E).

Fig. 3 is a bar graph of the bacteriostatic percentage of the three-strain post-natal lozenge against oral pathogens according to an embodiment of the present invention.

Fig. 4A-4D are bar graphs of the percentage change in oral aerobic bacteria (fig. 4A), streptococcus mutans (fig. 4B), bifidobacterium (fig. 4C), and lactobacillus (fig. 4D), respectively, according to an embodiment of the invention.

Fig. 5 is a diagram of the variation NGS of the oral bacterial phases of the three bacterial probiotics buccal tablets according to an embodiment of the invention.

FIG. 6 is a box plot of relative percentage of IgA content of saliva versus time for different buccal lozenge administrations in accordance with one embodiment of the present invention.

FIG. 7 is a plot of percentage change in salivary IgA over time for a troche for a period of time after administration of three strains according to one embodiment of the present invention.

Detailed Description

In view of the above, the present invention provides a use of probiotic bacteria for preparing a composition for increasing the content of immunoglobulin a (IgA) in the oral cavity and inhibiting pathogenic bacteria in the oral cavity, wherein the probiotic bacteria can be used as an active ingredient, for example. The probiotic bacteria may include, but are not limited to, Lactobacillus plantarum (Lactobacillus plantarum). In one embodiment, the Lactobacillus plantarum is Lactobacillus plantarum LPL28(Lactobacillus plantarum) deposited at 18.6.2019 in China General Microbiological Culture Collection Center (CGMCC) (address: institute No. 3, postal code: 100101, west way 1, sunny region, Beijing, China), with the Collection number of CGMCC No. 17954.

In addition, the probiotic bacteria may optionally comprise lactobacillus salivarius (l.salivariaus) and/or lactobacillus paracasei (l.paracasei). In one embodiment, the Lactobacillus salivarius is CN 108338361, and is deposited in China Center for Type Culture Collection (CCTCC) at No. 4/10 2011 in China Wuhan university (postal code: 430072) with the preservation number of M2011127 Lactobacillus salivarius AP-32(Lactobacillus salivarius subsp. In one embodiment, the Lactobacillus paracasei is Lactobacillus paracasei ET-66(Lactobacillus paracasei) which is deposited in China General Microbiological Culture Collection Center (CGMCC) at 2016 (12.29.10.2016 (China General Microbiological Culture Collection Center) (accession No. 3 of Xilu 1 of the sunward area of Beijing, China, zip code: 100101) and has a Collection number of CGMCC No.13514 in CN 108338361. In one embodiment, the probiotic is composed of strains of Lactobacillus plantarum LPL28, Lactobacillus salivarius AP-32, and Lactobacillus paracasei ET-66.

The probiotic is fermented by a fermentation-fermentation culture medium, so that fermentation products can be obtained, wherein the type of the fermentation-fermentation culture medium is not limited, and the fermentation-fermentation culture medium can be, for example, a commercial bacterial culture medium, and the fermentation products contain active thalli. The fermentation step can be, for example, a single strain fermentation step using a probiotic strain to obtain a single strain fermentation. In one embodiment, the fermentation-fermentation step can be performed, for example, by simultaneously performing a multi-strain fermentation step on a plurality of probiotic strains to obtain a multi-strain fermentation product, wherein the probiotic strains can be selected from the group consisting of Lactobacillus plantarum LPL28, Lactobacillus salivarius AP-32, Lactobacillus paracasei ET-66, and any combination thereof. In one embodiment, the fermentation step can, for example, utilize a simultaneous three-strain fermentation step of Lactobacillus plantarum LPL28, Lactobacillus salivarius AP-32, and Lactobacillus paracasei ET-66 to obtain a three-strain fermentation.

Subsequently, post-treating the fermentation product (such as a single-strain fermentation product or a multi-strain fermentation product) to obtain postbiotics (such as single-strain postbiotics or three-strain postbiotics), wherein the postbiotics comprise thalli of probiotics and metabolites thereof generated after decomposing a culture medium. The method of the above-mentioned post-treatment is not limited, and may include a heat-killing step and/or a solid-liquid separation step. The heat-killing step described above can inhibit the activity of the cells to form inactive cells, and the solid-liquid separation step can remove the active cells and/or inactive cells. In one embodiment, the active ingredient may be selected from the group consisting of active bacteria, inactive bacteria, metagens and any combination thereof of probiotic bacteria, for example. In one embodiment, the heat-killing step may be performed by wet heat sterilization (e.g., high temperature and high pressure steam sterilization, microwave heating, water-proof heating), and/or dry heat sterilization. In one embodiment, the solid-liquid separation step may be performed, for example, using a centrifugation step and/or a filtration step.

The probiotic bacteria have effects of increasing IgA content in oral mucosa and saliva and inhibiting oral pathogenic bacteria, wherein IgA content is related to oral health, so that the increase of IgA content in saliva can reduce diseases caused by oral pathogenic bacteria, such as growth inhibition of oral pathogenic bacteria.

By "oral pathogenic bacteria" is meant microorganisms that can affect oral health, wherein oral pathogenic bacteria can include, but are not limited to, cariogenic and/or periodontal pathogenic bacteria. The cariogenic bacteria may include Streptococcus mutans (Streptococcus mutans), and the periodontal pathogenic bacteria may include Porphyromonas gingivalis (Porphyromonas gingivalis), Bacillus tuberculosis (Fusobacterium tuberculosis), and Actinobacillus actinomycetemcomitans (Aggregatibacter actinomycetemcomitans). In addition, Porphyromonas gingivalis, Bacillus nuclease and Actinomyces actinomycetemcomitans all produce sulfides, thereby causing halitosis. In addition to affecting oral health, oral pathogens have been associated with diseases other than oral, such as: the nucleic acid bacillus is related to colon cancer, the actinobacillus actinomycetemcomitans is closely related to colorectal cancer, and the content of the porphyromonas gingivalis and the actinobacillus are positively related to the probability of pancreatic cancer. Thus, inhibiting the growth of oral pathogens not only maintains oral health, but also reduces the incidence of the above-mentioned diseases.

When the above composition is used, the route of administration is not particularly limited, and it can be administered orally or absorbed orally, for example, depending on the actual requirement and dosage form. The effective dosage of the above composition can also be flexibly adjusted according to requirements. In an embodiment, the effective dose of the probiotic may be, for example, greater than 10 ⁶ CFU/g, however, 10 ⁷ CFU/g to 10 ¹¹ CFU/g is preferred.

In one embodiment, the composition optionally comprises a food or pharmaceutically acceptable excipient, diluent or carrier, and the like. In one embodiment, the composition may be, for example, a food composition or an oral topical composition, wherein the food composition may be, for example, a dairy product (e.g., yogurt, cheese, or fermented milk drink milk powder), a non-dairy drink (e.g., tea drink, coffee, health drink), or a dentifrice (e.g., chewing gum, lozenge, fondant, and jerky for pets), and the oral topical composition may be, for example, an oral care composition (e.g., toothpaste, dental floss, mouthwash, dentifrice, denture cleanser, or oral cream), an oral freshening composition (e.g., breath freshener), or other composition (e.g., tooth whitener).

In vitro experiments prove that the lactobacillus plantarum LPL28 can effectively improve the IgA content in the oral cavity and inhibit oral pathogenic bacteria by matching with other strains (such as lactobacillus salivarius AP-32 and lactobacillus paracasei ET-66), and the probiotics have the potential of reducing dental caries and/or periodontal disease so as to maintain oral health.

It should be added that in vitro experiments, compared with the fermentation product with a single strain, the fermentation product with three strains can secrete more H ₂ O ₂ . Secondly, the three strain metazoan can increase IgA content in the oral cavity compared to the individual single strain metazoan, showing that the three strain fermentations of lactobacillus plantarum LPL28 and lactobacillus salivarius AP-32 and lactobacillus paracasei ET-66 and/or the metazoan maintain oral health better than the single strain metazoan fermentation and/or the metazoan of lactobacillus plantarum LPL 28.

The following examples are provided to illustrate the present invention, but not to limit the invention, and those skilled in the art should understand that they can make various changes and modifications without departing from the spirit and scope of the invention.

EXAMPLE I isolation, preservation and microbiological Properties of Lactobacillus plantarum LPL28

Lactobacillus plantarum LPL28 (hereinafter referred to as LPL28) is a strain isolated from miso. After isolation, strain LPL28 was stored at-80 ℃ in MRS medium (brand: DIFCO, Dorkstol, Inc.) containing 20% glycerol. Before use, strain LPL28 was inoculated into MRS broth containing 0.05% cysteine at 37 ℃ and cultured for 24 hours and subcultured once more in the same way to ensure better activity of strain LPL 28.

After activation of the strain LPL28, it was spread on MRS agar medium and cultured at 37 ℃ until colonies grew (about 48 hours). Morphological characteristics of the activated strain LPL28 were observed, and the results are shown in Table 1.

TABLE 1

Next, the microbial identification kit API 50CHL (bioMerieux, France) was used to analyze and identify the species of the strain LPL28, and the results are shown in Table 2, in which "+" indicates positive, "-" indicates negative, and "? "indicates that the reaction is weak. According to the results of table 2, the biochemical properties of the strain LPL28 were similar to those of lactobacillus plantarum, and thus it was judged that the strain LPL28 was lactobacillus plantarum.

TABLE 2

Next, RNA purification and reverse transcription-polymerase chain reaction (RT-PCR) were performed to obtain 16S rDNA of strain LPL28, and the sequences were identified as SEQ ID NOs: 1 and 2, performing PCR on the upstream primer and the downstream primer to obtain a primer with sequences shown as SEQ ID NO: 3, and a fragment of 145 bp. The Alignment was performed using Basic Local Alignment Search Tool (BLAST), and the strain LPL28 was indeed Lactobacillus plantarum. The separated strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC 17954.

EXAMPLE II evaluation of the efficacy of Lactobacillus plantarum LPL28 in vitro in inhibiting oral pathogens

The efficacy of probiotics to inhibit oral pathogens was evaluated using the double agar method, wherein the probiotic strains used are listed in table 3, and exemplary oral pathogenic strains are listed in table 4. Notably, the strain LGG is a commercial probiotic that inhibits streptococcus mutans, referred to herein as a positive control strain. It should be noted that the Fenghua technology is provided by Fenghua Biotechnology GmbH, Tainan, Taiwan, China. In Table 3, Lactobacillus rhamnosus LGG is deposited at the American Type Culture Collection (ATCC) Of Manassas, Vaginia, USA, Bifidobacterium lactis BB-12 and Lactobacillus acidophilus LA-5 are deposited at the German Collection Of Microorganisms and Cell Cultures GmbH, DSMZ, and the oral pathogenic strains Of Table 4 are deposited at the Culture Collection Of University Of Goodburg (Culture Collection Of Goynburg, CCUG) or the American Type Culture Collection Of Goodpasture, Goodburg, Sweden. The oral pathogenic strains of table 4 were also deposited at the biological resources conservation and Research Center (BCRC) of the institute of food industry development, institute of new bamboo treasury, taiwan.

TABLE 3

TABLE 4

First, the probiotic strains shown in table 3 were cultured using MRS broth, streptococcus mutans, porphyromonas gingivalis, and mycobacterium pleomorphus were cultured using TSB broth, and actinobacillus was cultured using BHI (brain heart infusion broth), to obtain probiotic and disease solution, respectively. Adjusting the bacterial contents of probiotic liquid and pathogenic bacteria liquid to 1 × 10 respectively ⁹ cfu/g and 1x10 ⁷ cfu/g to 1x10 ⁹ cfu/g。

The probiotic solution was coated in a line-drawn manner on the first agar medium, so that after culturing in an anaerobic environment at 37 ℃ for about 48 hours, a probiotic band of about 2 cm was formed. The first agar culture medium is prepared from MRS culture solution.

Next, second agar layers of 45 ℃ were added to the first agar medium, respectively. After the second layer of culture agar is cooled and solidified, respectively coating germ liquid of oral pathogenic strains on the second layer of culture agar, and culturing for 48 hours at 37 ℃, wherein the second layer of culture agar of streptococcus mutans, porphyromonas gingivalis and mycobacterium mononucleosis is prepared from TSB culture solution, and the second layer of culture agar of actinobacillus parasuis is prepared from BHI culture solution. If the probiotic has bacteriostatic ability, then on the second layer of agar, a zone of bacteriostatic activity will form on both sides of the zone of probiotic. The width of the zone is used to quantify the bacteriostatic ability, wherein when the zone is 0 cm to 1 cm, 1 cm to 2 cm, 2 cm to 3 cm and greater than 3 cm, the bacteriostatic scores are respectively defined as 0 score, 1 score, 2 score and 3 score.

The antibacterial scores of the probiotic strains against different oral pathogenic strains and the average of each antibacterial score are listed in table 5. As shown in Table 5, the bacterial strain LPL28 has a higher bacteriostatic score than the positive control bacterial strain LGG, and shows that the active thallus of the bacterial strain LPL28 has good bacteriostatic ability.

TABLE 5

EXAMPLE III evaluation of the in vitro Effect of metazoan of Lactobacillus plantarum LPL28 on inhibition of oral pathogens

The method comprises the steps of performing single-strain fermentation on probiotic strains LPL28, Lactobacillus salivarius AP-32 and Lactobacillus paracasei ET-66 to obtain single-strain fermentation, wherein the single-strain fermentation is performed by using MRS culture solution at 37 ℃ for 48 hours. Lactobacillus salivarius AP-32 (accession number: CCTCC M2011127) (hereinafter referred to as AP-32) and Lactobacillus paracasei ET-66 (accession number: CGMCC 13514) (hereinafter referred to as ET-66) are described in the strain of patent number TW I639389B, and are not described herein again.

And then, carrying out post-treatment on the single-strain fermentation medium to obtain single-strain metazoan, wherein the post-treatment comprises a thermal killing step and a centrifugation step. The heat killing step is heating in a water-proof way at 100 ℃ to inactivate thalli in the single-strain fermentation medium, and the centrifugation step is centrifugation at 4000rpm for 10 minutes, wherein separated supernatant comprises single-strain metazoan.

Adding 100 μ l of single strain metazoan into 4.8ml TSB culture solution or BHI culture solution, and respectively adding 100 μ l of germ solution 10 ⁶ CFU/mL (Streptococcus mutans, Porphyromonas gingivalis, and Bacillus nucleatus pleomorphus were added to the TSB broth, and Agileobacter actinomycetemcomitans were added to the BHI brothIn (b), culturing at 37 ℃ (Streptococcus mutans for 20 hours, Porphyromonas gingivalis, Bacillus nuclease-derived subspecies polymorpha, and Actinobacillus actinomycetemcomitans for 4 days) to obtain a postbiotic culture. Control cultures were prepared in the same manner as the postbiotic cultures described above, except that no postbiotic was added.

The post-natal and control culture sequences were diluted and plated on TSB agar medium (streptococcus mutans, porphyromonas gingivalis, and subspecies polymorpha with nucleic acid bacillus) or BHI agar medium (aggregatibacter actinomycetemcomitans), respectively, and cultured at 37 ℃ for 48 hours, and the number of colonies was counted to calculate the percent inhibition, where the percent inhibition was 100% minus the percent survival, and the percent survival was the percentage of the number of bacteria of the post-natal culture divided by the number of bacteria of the control culture. Thus, the percent inhibition of the control culture was 0%.

Fig. 1 is a graph showing the percent inhibition of individual strains of metazoan of different probiotics against different oral pathogens in vitro, according to an embodiment of the invention, wherein the horizontal axis represents the metazoan of the probiotic strain and the vertical axis represents the percent inhibition, and the graph numbers "," "and" "respectively indicate that the metazoan cultures have statistically significant differences (p <0.05, p <0.001) in the percent inhibition compared to the control culture, after statistics in t-test.

As shown in FIG. 1, the percentage of inhibition of oral pathogens by single strain metagens of LPL28, AP-32 and ET-66 was higher compared to control cultures without added metagens, indicating that single strain metagens of LPL28, AP-32 and ET-66 have better inhibitory activity.

Example four measurement of H of different probiotic strains ₂ O ₂ Amount of secretion

H ₂ O ₂ Can inhibit the growth of bacteria, and whiten teeth, thereby promoting the growth of H ₂ O ₂ And (3) measuring the secretion amount, so that the bacteriostatic ability of the probiotic strain can be evaluated. Firstly, respectively carrying out the single-strain fermentation on the strains AP-32, ET-66 and LPL28 by using MRS culture solution so as to obtain single-strain fermentation products. On the other hand, MRS culture solution is used for simultaneously treating the strain AMixing P-32, ET-66 and LPL28 to perform a three-strain fermentation step to obtain a three-strain fermentation product, wherein the conditions of the three-strain fermentation step are the same as those of the single-strain fermentation step, except that the number of inoculated strains is different. The total bacteria content of the single-strain fermentation material and the three-strain fermentation material is the same (about 10) ⁹ CFU/mL), wherein in the fermentation product of the three strains, the bacterial quantity ratios of the strains AP-32, ET-66 and LPL28 are 1: 1: 1. respectively centrifuging 0.1mL of single-strain fermentation and three-strain fermentation at 4500rpm for 5 min to obtain thallus, and then using 4.9mL of piperazine-1, 4-diethylsulfonic acid (2-ethanesulfonic acid), PIPES]The cells were redissolved in a buffer (100mM) and reacted at 37 ℃ at 220rpm for 5 hours to obtain a PIPES culture. Then, centrifugation was carried out at 4500rpm, and 10. mu.L of the supernatant was taken to H ₂ O ₂ The reaction was carried out on a test paper (manufacturer: Merck), and the color change of the test paper was observed after 10 seconds. H can be obtained by contrasting the colors of the test paper and the color comparison card ₂ O ₂ And (4) concentration. The results are recorded in fig. 2A to 2E.

FIGS. 2A-2E illustrate an implementation of H according to an embodiment of the present invention ₂ O ₂ Measurement of secretion amount H of the strains AP-32 (FIG. 2A), ET-66 (FIG. 2B), LPL28 (FIG. 2C), and three strains (FIG. 2D) tested ₂ O ₂ Contents and color chart (fig. 2E). As shown in FIGS. 2A-2D, the PIPES cultures of the fermentation fermentates of strains AP-32, ET-66 and LPL28 contained 5mg/L, 0mg/L and 2mg/L H, respectively ₂ O ₂ H of PIPES culture of fermentation of the three strains ₂ O ₂ The content of H in PIPES culture of which is 10mg/L and is greater than that of fermentation medium of single strain ₂ O ₂ The content sum shows that compared with the single-strain fermentation medium obtained after the single-strain fermentation step is respectively carried out on the strains AP-32, ET-66 and LPL28, the H of the three-strain fermentation medium obtained after the three-strain fermentation step is simultaneously carried out on the strains AP-32, ET-66 and LPL28 ₂ O ₂ The content is higher.

Example five evaluation of the efficacy of the three strains of metazoan buccal tablets in inhibiting pathogenic bacteria in the oral cavity in vitro

Preparing placebo buccal tablets and three-strain postnatal buccal tablets. The placebo is suckedThe tablet comprises food additives such as sweetener (such as D-sorbitol, erythritol and sucralose), fructo-oligosaccharide, lactose, spice, magnesium stearate and silicon dioxide, wherein the content of the food additives can be adjusted according to the requirements. The above food additives are all known ingredients, and can be adjusted at will according to actual requirements, and the adjustment does not affect the evaluation of the bacteriostatic effect, so it is not repeated herein. In addition, the components of the three-strain post-growth buccal tablet comprise food additives contained in the placebo buccal tablet and the three-strain post-growth. The three-strain post-growth medium is obtained by performing the three-strain fermentation step and the post-treatment on strains AP-32, ET-66 and LPL28, wherein the bacteria content of the three-strain fermentation medium is 1x10 ⁹ CFU/mL, and the content of the three strains of metazoan in the buccal tablet is 50 mg/g.

100 mu L of germ solution is respectively added into 4.8mL of TSB culture solution (Streptococcus mutans, Porphyromonas gingivalis and Bacillus nucleatum subspecies pleomorph) and BHI culture solution (Actinomyces actinomycetemcomitans), and then one placebo buccal tablet or three bacterial strain postnatal buccal tablets are respectively added, and culture is carried out at 37 ℃ (Streptococcus mutans is cultured for 20 hours, Porphyromonas gingivalis, Bacillus nucleatus subspecies pleomorph and Actinomyces actinomycetemcomitans are cultured for 4 days) so as to obtain the buccal tablet culture of the placebo buccal tablets and the three bacterial strain postnatal buccal tablets. In addition, a control culture was prepared in the same manner as the buccal lozenge culture described above, but without the addition of buccal lozenges. The percent inhibition was then calculated as in example three and the results are recorded in figure 3.

Fig. 3 is a bar graph of the percent inhibition of oral pathogenic bacteria by three-strain post-natal lozenges according to an embodiment of the present invention, wherein the horizontal axis represents groups, the vertical axis represents percent inhibition, and the numbers "" and "" respectively represent significant statistical differences (p <0.05, p <0.01) compared to placebo buccal lozenges after statistical analysis by student's t test. As shown in fig. 3, the three-strain metazoan buccal tablet has higher antibacterial percentage against oral pathogenic strains compared to placebo buccal tablet, showing that the three-strain metazoan can effectively inhibit oral pathogenic bacteria.

EXAMPLE VI, evaluation of the efficacy of three probiotic strains in inhibiting oral pathogenic bacteria and improving oral and/or gastrointestinal health

And respectively centrifuging the single-strain fermentation filtration material and the three-strain fermentation filtration material to prepare AP-32, ET-66, LPL28 and three-strain probiotic buccal tablets, wherein the probiotic buccal tablets comprise corresponding bacteria and additives of placebo buccal tablets, and the total bacteria content of the probiotic buccal tablets is 50 mg/g.

The placebo buccal tablets and the three probiotics buccal tablets are respectively administered to the subjects for 4 weeks, wherein the administration mode is that one buccal tablet is respectively contained (not bitten) in the morning, the middle and the evening. The subject has no systemic disease and no smoking, and saliva contains about 10% of the total weight of the composition ⁵ Of streptococcus mutans in healthy adults between the ages of 20 and 40. It should be noted that each buccal tablet is about 10g, is not bitten, and can stay in oral cavity for about 10 minutes, but has individual difference according to the oral water content and body temperature of the subject.

After 2 weeks and 4 weeks of non-administration of the buccal lozenge, respectively, the inside and outside of the subject's teeth were scraped with a cotton swab to collect oral tissue samples. Next, the oral tissue sample was inoculated uniformly into 5mL of TSB medium containing 50% glycerol to obtain an oral bacteria sample. Then, according to the detected target, oral bacteria samples were cultured for 2 days at 37 ℃ using the corresponding culture conditions, respectively, and then the number of colonies was counted. Specifically, the corresponding culture conditions refer to a specific medium and an oxygen environment. The culture conditions corresponding to the oral aerobic bacteria of the oral bacteria sample are a plate counting agar culture medium and an aerobic environment. The culture conditions for Streptococcus mutans are the Streptococcus mitis agar (MSBA) medium and the facultative anaerobic environment. The culture conditions for the bifidobacteria and the lactobacilli are cysteine MRS agar culture medium, anaerobic environment or facultative anaerobic environment. The bacterial contents of the above strains are shown in FIGS. 4A to 4D.

Fig. 4A to 4D are bar graphs of the percentage change in the bacterial content of oral aerobic bacteria (fig. 4A), streptococcus mutans (fig. 4B), bifidobacterium (fig. 4C), and lactobacillus (fig. 4D), respectively, according to an embodiment of the present invention, in which the horizontal axis represents the type of buccal tablet and the administration condition, the vertical axis represents the percentage change in the bacterial content of oral cavity of 100% without administration of buccal tablet, the graphs are labeled "," "and" "represent statistically significant differences from those of oral buccal tablet administered with placebo's t-test (p <0.05, p <0.01, p <0.001) (n ═ 25), and the graphs", "#" and "#" represent statistically significant differences from those of probiotic's t-test (p < 05.05.05) compared to those of probiotic strains administered without administration of probiotic (p.05.05) p <0.001) (n 25).

As shown in fig. 4A, compared to placebo buccal tablets and buccal tablets without administration, after administration of probiotic buccal tablets, the percentage change of the oral aerobic bacteria content is significantly reduced, and after 2 weeks of administration of ET-66, LPL28 and three-strain probiotic buccal tablets, the three-strain probiotic buccal tablets have a significant effect, wherein the effect of the three-strain probiotic tablets is better than that of the AP-32, ET-66 and LPL28 buccal tablets.

As shown in fig. 4B, compared to the placebo buccal lozenge for 4 weeks and the buccal lozenge without administration, the percentage of change in the bacterial content of streptococcus mutans after 4 weeks administration of the probiotic buccal lozenge is significantly reduced, wherein the three probiotic strains have significant effect after 2 weeks administration, and the effect of the three probiotic strains is better than that of the AP-32, ET-66 and LPL28 buccal lozenge.

As shown in fig. 4C, the percentage change of the bifidobacterium content after 4 weeks of administration of the probiotic buccal tablets was significantly increased compared to 4 weeks of administration of placebo buccal tablets and no administration of the buccal tablets, wherein the effect of the three probiotic strains on buccal tablets was significant after 2 weeks, and the effect of the three probiotic strains on buccal tablets was better than that of the AP-32, ET-66 and LPL28 probiotic buccal tablets.

As shown in fig. 4D, the percentage change of the lactobacillus content was significantly increased after administration of the probiotic buccal tablets compared to administration of placebo buccal tablets and non-administration of the probiotic buccal tablets, wherein the effect was significant after 2 weeks of administration of LPL28 and three probiotic strains, and the effect of the three probiotic strains was better than that of AP-32, ET-66 and LPL 28. Therefore, when the probiotic buccal tablet is administered, oral pathogenic bacteria (streptococcus mutans) can be effectively reduced, oral beneficial bacteria (such as bifidobacteria and lactobacilli) can be improved, and compared with a single-strain probiotic buccal tablet (namely, the probiotic buccal tablet prepared from AP-32, ET-66 and LPL28), the effect of the three-strain buccal tablet is better.

In addition, after 2 weeks and 4 weeks without buccal lozenge administration, saliva samples of the subjects were collected, DNA samples in the saliva samples were extracted, and the DNA samples were then sent to tussi biotechnology company for Next Generation Sequencing (NGS). The operation method of the NGS is as follows: first, commercially available primers (shown as SEQ ID NOs:4 and 5) and a commercially available PCR kit (see

High-Fidelity PCR Master Mix; manufacturer: new England Biolabs, usa) nucleic acid fragments of V3 and V4 segments of 16S rRNA were amplified and electrophoresed, followed by Extraction with an Extraction kit [ Qiagen Gel Extraction kit; manufacturer: german Qiagen company (Qiagen)](about 400bp to 450bp) isolating the nucleic acid fragment.

Then, a reagent kit is prepared by using the whole genome sequencing sample (

DNA PCR-free sample preparation kit; manufacturer: U.S. Illumina) prepared sequencing samples and created a DNA gene library (genomic DNA library). Next, a fluorescent light detection instrument (Qubit @2.0 Fluorometer; manufacturer: American Siemens fly technologies) and sequencing analysis System [ Agilent Bioanalyzer 2100 system; manufacturer: agilent Technologies, Inc. of America]DNA gene libraries were analyzed. Finally, the DNA was sequence analyzed on the Illumina HiSeq 2500 platform. The results of NGS are recorded in fig. 5.

Fig. 5 is a graph of the variation of the phase of the oral bacteria NGS (heat map) of the buccal lozenge of three strains of probiotics according to an embodiment of the present invention, wherein the horizontal axis represents the type of buccal lozenge and the experimental time, the vertical axis represents the classification of the bacteria (which can be genus, species or subspecies), and the numbers "x" and "x" respectively represent the statistically significant difference (p <0.05, p <0.001) (n ═ 25) between the bacteria content of the buccal lozenge administered to placebo by student's t-test. As can be seen from FIG. 5, after the three probiotic strains are administered to the buccal tablets for 4 weeks, the contents of oral probiotics (such as Lactobacillus salivarius, Lactobacillus plantarum, and Bifidobacterium dentis) can be effectively increased.

In addition, changes in the physiological state of the oral cavity and intestinal tract of the subjects were investigated by questionnaire, and the results are listed in tables 6 and 7, respectively, wherein

numerals

0, 1, 2 and 3 indicate no symptom, mild symptom, moderate symptom and severe symptom (numerals 2 in over 3 days, numerals 1 in 1 to 3 days, and numerals 0 in over 1 day in 3 days in table 7), and letters "a" and "b" indicate that the subjects administered with the placebo buccal tablet had a significant statistical difference compared to the subjects administered with the placebo buccal tablet, respectively, after statistical analysis with the student's t test. As shown in table 6, the symptoms of aphtha (also called as "orosceta" and "mouth bar rupture"), pustule, watery mouth, and the like of the subjects administered with the three-strain probiotic buccal tablets were significantly reduced, and as shown in table 7, the symptoms of constipation, gastroesophageal reflux, cold, drowsiness, and the like were all significantly improved, showing that the three-strain probiotic buccal tablets not only improved the uncomfortable symptoms of the oral cavity, but also positively affected the physiological states other than the oral cavity.

TABLE 6

TABLE 7

EXAMPLE seventhly, evaluation of the ability of probiotic lozenges of Single and three strains to boost oral IgA concentration

And respectively preparing AP-32 buccal pastilles, ET-6 buccal pastilles 6 and LPL28 buccal pastilles by using the single strain fermentation of the strains AP-32, ET-66 and LPL28 in the fourth embodiment, wherein the bacterial content of the buccal pastilles is 50mg/g respectively. The placebo troche, the AP-32 troche, the ET-66 troche, the LPL28 troche and the three probiotic strains troche were administered to the subjects, respectively, saliva of the subjects was collected for 2 weeks and 4 weeks without administration, and the IgA content was measured by enzyme-linked immunosorbent assay (ELISA), and the results were recorded in fig. 6. The ELISA method is well known to those of ordinary skill in the art and will not be described herein.

Fig. 6 is a box-shaped graph of relative IgA content percentage of saliva at different times of administration of different buccal tablets according to an embodiment of the present invention, wherein the horizontal axis represents the kind and time of buccal tablets, the vertical axis represents the relative IgA content percentage of 100% of the IgA content of saliva without administration of buccal tablets, the numbers "x", and "x" represent statistically significant differences (p <0.5, p <0.01, p <0.001) (n ═ 25) from that of placebo's t test after statistical analysis, and the numbers "# #" and "n" represent statistically significant differences (p <0.01, p <0.001) (n ═ 25) from that of non-administration of buccal tablets after statistical analysis by student's t test #.

As shown in FIG. 6, the percentage of salivary relative IgA content of subjects administered with the AP-32 buccal tablet, the ET-66 buccal tablet, the LPL28 buccal tablet and the three strains of probiotic buccal tablet all increased significantly after 2 and 4 weeks of administration compared to the placebo buccal tablet, showing that the strains AP-32, ET-66 and LPL28 had the efficacy of increasing salivary IgA content. However, the saliva relative IgA content percentage of the subjects administered with the three strains of probiotic buccal tablets was higher compared to the AP-32 buccal tablets, the ET-66 buccal tablets, and the LPL28 buccal tablets, showing that the effect of the three strains of fermentation medium of the strains AP-32, ET-66, and LPL28 in increasing the salivary IgA content was better compared to that of the single strain.

EXAMPLE eight evaluation of the ability of three Strain metazoan buccal tablets to boost oral IgA concentration

The three strain post-natal lozenges of example five were administered to the subjects for 4 weeks in the same manner and under the same conditions as example six. Saliva of the subjects was collected after 2 weeks and 4 weeks of non-administration, and the content of IgA in saliva was measured by the ELISA described above.

Fig. 7 is a plot of percentage change of IgA to saliva at different times for the three-strain-administered buccal tablets, wherein the horizontal axis represents time and groups, the vertical axis represents percentage of IgA content at 100% of the IgA content before administration, the plots "", and "") represent statistically significant differences (p <0.01, p <0.001) (n ═ 25) from the placebo buccal tablets, and the plot "#" represents statistically significant differences (p <0.001) (n ═ 25) from the non-three-strain-administered buccal tablets, as determined by student's t-test. As shown in fig. 7, salivary IgA content significantly increased in subjects administered three strain metazoans after 2 and 4 weeks of administration compared to placebo buccal tablets, showing that three strain metazoans of strain AP-32, strain ET-66 and strain LPL28 were effective in increasing salivary IgA content.

The results show that the lactobacillus plantarum LPL28 of the present invention can surely increase oral IgA content and suppress oral pathogenic bacteria, which means that the application of the lactobacillus plantarum LPL28 of the present invention can reduce the incidence of dental caries and/or periodontal disease and has potential for preventing dental caries and/or periodontal disease.

In summary, although the application of the lactobacillus plantarum LPL28 of the present invention to increase oral IgA content and inhibit oral pathogenic bacteria is described by way of example of a specific strain, a specific formulation, a specific subject, a specific administration method, or a specific evaluation method, it will be understood by those skilled in the art that the present invention is not limited thereto, and that the present invention may be carried out using other strains, other formulations, other subjects, other administration methods, or other evaluation methods without departing from the spirit and scope of the present invention.

While the present invention has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

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Claims

1. Use of a probiotic for preparing a composition for increasing oral immunoglobulin a (IgA) content, wherein the probiotic is used as an active ingredient, the probiotic comprises Lactobacillus plantarum (LPL 28), the Lactobacillus plantarum LPL28 is stored in the china general microbiological culture collection management center at 6/18/2019 with the storage number of CGMCC17954, so as to increase the IgA content in the oral cavity.

2. Use of probiotic bacteria for the preparation of a composition for increasing IgA content in the oral cavity according to claim 1, wherein the probiotic bacteria further comprise lactobacillus salivarius AP-32 and/or lactobacillus paracasei (l.paracasei) ET-66, the lactobacillus salivarius AP-32 being deposited at the chinese type culture collection center at 2011-4-10 days with the collection number CCTCC M2011127, and the lactobacillus paracasei ET-66 being deposited at the chinese common micro organism species collection management center at 2016-12-29 days with the collection number CGMCC 13514.

3. Use of probiotic bacteria according to claim 1 for the preparation of a composition for increasing the IgA content of the oral cavity, wherein the composition is a food composition or an oral topical composition.

4. Use of a probiotic as defined in claim 1 for the manufacture of a composition for increasing IgA content in the oral cavity, wherein the food composition is a dairy product, a non-dairy drink product or a dentifrice food product.

5. Use of probiotic bacteria according to claim 1 for the preparation of a composition for increasing the IgA content of the oral cavity, wherein the composition for external use is an oral care composition or a breath freshening composition.

6. Use of a probiotic for the preparation of a composition for inhibiting oral pathogenic bacteria, wherein the probiotic is used as an active ingredient, and the probiotic comprises Lactobacillus plantarum (LPL 28) for inhibiting the growth of the oral pathogenic bacteria, and the Lactobacillus plantarum LPL28 has a accession number of CGMCC 17954.

7. Use of probiotic bacteria according to claim 6 for the preparation of a composition for inhibiting oral pathogens, wherein the probiotic bacteria further comprise Lactobacillus salivarius AP-32 and/or Lactobacillus paracasei ET-66, the Lactobacillus salivarius AP-32 having the accession number CCTCC M2011127 and the Lactobacillus paracasei ET-66 having the accession number CGMCC 13514.

8. Use of probiotic bacteria according to claim 6 for the preparation of a composition for inhibiting oral pathogens, wherein the oral pathogens comprise Streptococcus mutans (Streptococcus mutans) and/or periodontal pathogens.

9. Use of probiotic bacteria for the preparation of a composition for inhibiting oral pathogenic bacteria according to claim 8, wherein the periodontal pathogenic bacteria comprise Porphyromonas gingivalis (Porphyromonas gingivalis), Bacillus subtilis (Fusobacterium tuberculosis) and Agrobacterium tumefaciens (Aggregatobacterium actinomycetes) bacteria.

10. Use of probiotic bacteria according to claim 6 for the preparation of a composition for inhibiting oral pathogens, wherein the composition is a food composition or an oral topical composition.

11. A composition for increasing oral IgA content and inhibiting oral pathogenic bacteria comprises probiotics as an effective component, and is characterized in that the probiotics comprise lactobacillus plantarum LPL28, lactobacillus salivarius AP-32 and lactobacillus paracasei ET-66, wherein the preservation numbers of the lactobacillus plantarum LPL28, the lactobacillus salivarius AP-32 and the lactobacillus paracasei ET-66 are CGMCC17954, CCTCC M2011127 and CGMCC 13514 respectively.