Composite probiotic postbiotic composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a composite probiotic postbiotic composition as well as a preparation method and application thereof. The composition disclosed by the invention comprises a saliva combined lactobacillus metagen, a lactobacillus paracasei metagen, a lactobacillus rhamnosus metagen and a lactobacillus acidophilus metagen. According to the invention, saliva is combined with the lactobacillus after-element, the lactobacillus paracasei after-element, the lactobacillus rhamnosus after-element and the lactobacillus acidophilus after-element, so that the anti-caries effect is achieved through multiple ways. On one hand, the composition of the composite probiotic metagen provided by the invention can be combined with cariogenic bacterium surface receptors, interference bacteria are adhered to tooth surfaces, and biofilm formation is reduced; and on the other hand, the organic acid in the composition of the composite probiotic metagen provided by the invention can reduce the pH value of the oral cavity and inhibit the growth of acid-producing bacteria, and meanwhile, certain protein and peptide components can enhance the hardness of enamel and improve the acid resistance of teeth.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a composite probiotic and postbiotic composition, a preparation method and an application thereof. Background Art

[0002] Dental caries is a common oral disease primarily caused by bacteria such as Streptococcus mutans. These cariogenic bacteria adhere to the tooth surface and metabolize carbohydrates in the diet to produce acid. These acids gradually destroy the hard tissues of the teeth, leading to the development of dental caries. According to the World Health Organization, 60% to 90% of school-age children and nearly 100% of adults worldwide suffer from dental caries. Dental caries not only affects tooth structure and function, causing pain and difficulty chewing, but can also lead to complications such as pulpitis and periapical periodontitis, severely impacting quality of life.

[0003] Currently, the primary treatment for bacterial caries relies on fillings and root canal therapy. Fillings require the removal of decayed tissue and the application of a filling material, but they cannot fundamentally prevent further bacterial attack. Root canal therapy, reserved for severe cases of infection, is complex, expensive, and severely damaging to the tooth. Prevention, primarily through brushing and the use of fluoride toothpaste, is effective but cannot completely suppress the growth of oral bacteria.

[0004] Postbiotics are a general term for the bacterial components and metabolites of probiotics after fermentation, culture, and inactivation. These include bacterial lysates, cell wall components (such as peptidoglycan), short-chain fatty acids (SCFAs), enzymes, and bacteriocins. Studies have found that postbiotics have positive effects in regulating the intestinal microbiome and enhancing immunity. However, their application in oral health, particularly for bacterial caries, is relatively limited. Summary of the Invention

[0005] The purpose of the present invention is to solve the problem that the existing technology lacks the technology of using probiotics and postbiotics to prevent and treat dental caries, and to provide a composite probiotics and postbiotics composition and its preparation method and application, which can significantly improve bacterial caries and enhance the anti-caries ability of teeth.

[0006] In order to achieve the above object, the present invention provides a composite probiotic postbiotic composition, which comprises a Lactobacillus salivarius postbiotic, a Lactobacillus rhamnosus postbiotic, a Lactobacillus rhamnosus postbiotic and a Lactobacillus acidophilus postbiotic;

[0007] The saliva-associated Lactobacillus postbiotics include inactivated bacteria of saliva-associated Lactobacillus and metabolites of saliva-associated Lactobacillus; the saliva-associated Lactobacillus is saliva-associated Lactobacillus LS86;

[0008] The Lactobacillus paracasei postbiotics include inactivated bacteria of Lactobacillus paracasei and metabolites of Lactobacillus paracasei; the Lactobacillus paracasei is Lactobacillus paracasei LPC28;

[0009] The Lactobacillus rhamnosus postbiotics include inactivated Lactobacillus rhamnosus bacteria and metabolites of Lactobacillus rhamnosus; the Lactobacillus rhamnosus is Lactobacillus rhamnosus LR22;

[0010] The Lactobacillus acidophilus postbiotics include inactivated bacteria of Lactobacillus acidophilus and metabolites of Lactobacillus acidophilus; the Lactobacillus acidophilus is Lactobacillus acidophilus LA11-Onlly.

[0011] Preferably, in the composition, the mass percentage of saliva combined with Lactobacillus postbiotics is 20% to 30%; the mass percentage of Lactobacillus paracasei postbiotics is 20% to 30%; the mass percentage of Lactobacillus rhamnosus postbiotics is 20% to 30%; and the mass percentage of Lactobacillus acidophilus postbiotics is 20% to 30%.

[0012] Preferably, the inactivated bacterial concentrations of the saliva-associated Lactobacillus postbiotics, Lactobacillus paracasei postbiotics, Lactobacillus rhamnosus postbiotics and Lactobacillus acidophilus postbiotics are ≥1.0×10 11 Pieces / gram.

[0013] Preferably, the dosage form of the composition includes powder, aqueous solution, emulsion or gel; the powder includes lyophilized agent.

[0014] The present invention also provides a method for preparing the composite probiotic postbiotic composition, comprising the following steps:

[0015] The saliva combined Lactobacillus, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus acidophilus were inoculated into fermentation culture fluids respectively for fermentation to obtain four kinds of fermentation fluids;

[0016] The four fermentation broths are respectively subjected to inactivation treatment to obtain four inactivated fermentation broths;

[0017] The four inactivated fermentation broths are centrifuged separately to obtain four inactivated and centrifuged emulsions;

[0018] The four inactivated and centrifuged emulsions are freeze-dried to obtain four postbiotic raw materials;

[0019] The four postbiotic raw materials are mixed to obtain the composite probiotic postbiotic composition.

[0020] Preferably, the raw materials of the fermentation culture medium include: 10 g / L glucose, 10 g / L lactose, 10 g / L peptone, 10 g / L beef extract powder, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 1 g / L cysteine ​​hydrochloride, 0.058 g / L magnesium sulfate, 0.02 g / L manganese sulfate, 801 mL / L Tween and the balance water; the fermentation culture temperature is 35-39° C., and the fermentation culture time is 16-20 h.

[0021] Preferably, the temperature of the inactivation treatment is 75-85°C, and the time of the inactivation treatment is 55-65 min.

[0022] Preferably, the solid contents of the four inactivated centrifuged emulsions are 40% to 80% respectively; the centrifugal speed is 6000 to 8000 r / min; the centrifugal flow rate is 1 to 3 T inactivated fermentation liquid / h; and the centrifugal discharge interval is ≤1200s.

[0023] The present invention also provides the use of the composition or the composition prepared by the preparation method described above in preparing a product for inhibiting pathogens; the pathogens include Streptococcus mutans and / or Porphyromonas gingivalis.

[0024] The present invention also provides the use of the above-mentioned composition or the composition prepared by the preparation method in preparing a product for preventing and / or treating bacterial caries.

[0025] The beneficial effects of the present invention are:

[0026] The invention provides a composite probiotic postbiotic composition, which comprises a Lactobacillus salivarius postbiotic, a Lactobacillus rhamnosus postbiotic, a Lactobacillus rhamnosus postbiotic and a Lactobacillus acidophilus postbiotic; the Lactobacillus salivarius postbiotic comprises an inactivated bacterial body of Lactobacillus salivarius and a metabolite of Lactobacillus salivarius; the Lactobacillus salivarius is Lactobacillus salivarius LS86, with a preservation number of CGMCC No. 6403; the Lactobacillus paracasei postbiotic comprises an inactivated bacterial body of Lactobacillus paracasei and a metabolite of Lactobacillus paracasei; the Lactobacillus paracasei is Lactobacillus paracasei LPC28, with a preservation number of CNCM No. 6403. I-4475; the rhamnosus Lactobacillus postbiotics include inactivated cells of rhamnosus Lactobacillus and metabolites of rhamnosus Lactobacillus; the rhamnosus Lactobacillus is rhamnosus LR22, with a deposit number of CNCM I-4474; the acidophilus Lactobacillus postbiotics include inactivated cells of acidophilus Lactobacillus and metabolites of acidophilus; the acidophilus Lactobacillus is acidophilus LA11-Onlly, with a deposit number of CGMCC No. 2106. The present invention combines saliva-associated Lactobacillus postbiotics, Lactobacillus paracasei postbiotics, rhamnosus Lactobacillus postbiotics, and acidophilus postbiotics to exert an anti-caries effect through multiple pathways. On the one hand, the composition of the composite probiotic postbiotic provided by the present invention can bind to the surface receptors of cariogenic bacteria, interfere with bacterial adhesion to the tooth surface, and reduce biofilm formation; on the other hand, the organic acid in the composition of the composite probiotic postbiotic provided by the present invention can reduce the pH value of the oral cavity, inhibit the growth of acid-producing bacteria, and at the same time, certain proteins and peptide components can enhance the hardness of tooth enamel and improve the acid resistance of teeth. A synergistic effect is generated between the components of the composition of the probiotic postbiotic of the present invention, which can inhibit the growth and reproduction of (Streptococcus mutans and Porphyromonas gingivalis) by regulating the oral microecology, significantly improve bacterial caries, and enhance the anti-caries ability of teeth. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0028] Figure 1 The relative abundance of Streptococcus mutans in the oral saliva of rats in Experimental Example 2 of the present invention at 0, 7, and 21 days after administration;

[0029] Figure 2The relative abundance of the opportunistic pathogen Porphyromonas gingivalis in the oral saliva of rats in Experimental Example 2 of the present invention at 21 days after administration; different letters represent significant differences among different groups, p < 0.05. DETAILED DESCRIPTION

[0030] The invention provides a composite probiotic postbiotic composition, which comprises a Lactobacillus salivarius postbiotic, a Lactobacillus rhamnosus postbiotic, a Lactobacillus rhamnosus postbiotic and a Lactobacillus acidophilus postbiotic; the Lactobacillus salivarius postbiotic comprises an inactivated bacterial body of Lactobacillus salivarius and a metabolite of Lactobacillus salivarius; the Lactobacillus salivarius is Lactobacillus salivarius LS86, with a preservation number of CGMCC No. 6403; the Lactobacillus paracasei postbiotic comprises an inactivated bacterial body of Lactobacillus paracasei and a metabolite of Lactobacillus paracasei; the Lactobacillus paracasei is Lactobacillus paracasei LPC28, with a preservation number of CNCM No. 6403. I-4475; the rhamnosus Lactobacillus postbiotics include inactivated bacteria of rhamnosus Lactobacillus and metabolites of rhamnosus Lactobacillus; the rhamnosus Lactobacillus is rhamnosus Lactobacillus LR22, with a preservation number of CNCM I-4474; the acidophilus Lactobacillus postbiotics include inactivated bacteria of Lactobacillus acidophilus and metabolites of Lactobacillus acidophilus; the acidophilus Lactobacillus acidophilus is Lactobacillus acidophilus LA11-Onlly, with a preservation number of CGMCC No.2106.

[0031] As an embodiment, in the composition of the present invention, the mass percentage of saliva-combined Lactobacillus postbiotics is 20% to 30%; as an optional embodiment, the mass percentage of the saliva-combined Lactobacillus postbiotics can be any one of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% and 30%. As an embodiment, the mass percentage of Lactobacillus paracasei postbiotics in the composition is 20% to 30%; as an optional embodiment, the mass percentage of Lactobacillus paracasei postbiotics can be any one of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% and 30%. As an embodiment, the mass percentage of the Lactobacillus rhamnosus postbiotic in the composition is 20% to 30%; as an optional embodiment, the mass percentage of the Lactobacillus rhamnosus postbiotic can be any one of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% and 30%. As an embodiment, the mass percentage of the Lactobacillus acidophilus postbiotic in the composition is 20% to 30%; as an optional embodiment, the mass percentage of the Lactobacillus acidophilus postbiotic can be any one of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% and 30%.

[0032] As an embodiment, the inactivated bacteria concentrations of the saliva combined Lactobacillus postbiotics, Lactobacillus paracasei postbiotics, Lactobacillus rhamnosus postbiotics and Lactobacillus acidophilus postbiotics of the present invention are ≥1.0×10 11 As a preferred embodiment, the inactivated bacteria concentration of the saliva-combined Lactobacillus postbiotic, Lactobacillus paracasei postbiotic, Lactobacillus rhamnosus postbiotic and Lactobacillus acidophilus postbiotic of the present invention is 1.0×10 11 Pieces / gram.

[0033] As an embodiment, the dosage form of the composition of the present invention includes powder, aqueous solution, emulsion or gel. As a preferred embodiment, the dosage form of the composition of the present invention is powder, and the powder includes freeze-dried powder.

[0034] The present invention also provides a method for preparing the composite probiotic postbiotic composition as described above, comprising the following steps:

[0035] The saliva combined Lactobacillus, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus acidophilus were inoculated into fermentation culture fluids respectively for fermentation to obtain four kinds of fermentation fluids;

[0036] The four fermentation broths are respectively subjected to inactivation treatment to obtain four inactivated fermentation broths;

[0037] The four inactivated fermentation broths are centrifuged separately to obtain four inactivated and centrifuged emulsions;

[0038] The four inactivated and centrifuged emulsions are dried separately to obtain four postbiotic raw materials;

[0039] The four postbiotic raw materials are mixed to obtain the composite probiotic postbiotic composition.

[0040] The present invention inoculates Lactobacillus saliva, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus acidophilus into fermentation broth for fermentation and culture, respectively, to obtain four fermentation broths. As an embodiment, the raw materials of the fermentation broth include: 10g / L glucose, 10g / L lactose, 10g / L peptone, 10g / L beef extract powder, 5g / L yeast extract, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate, 1g / L cysteine ​​hydrochloride, 0.058g / L magnesium sulfate, 0.02g / L manganese sulfate, 1mL / L Tween 80 and the remainder water. As an embodiment, the temperature of the fermentation culture is 35-39°C. As an optional embodiment, the temperature of the fermentation culture can be 35°C, 36°C, 37°C, 38°C or 39°C. As an embodiment, the fermentation culture time is 16-20h. As an optional embodiment, the fermentation culture time can be 16 hours, 17 hours, 18 hours, 19 hours or 20 hours.

[0041] After obtaining the four fermentation broths, the present invention performs an inactivation treatment on the four fermentation broths respectively to obtain four inactivated fermentation broths. As an embodiment, the temperature of the inactivation treatment of the present invention is 75 to 85°C. As an optional embodiment, the temperature of the inactivation treatment of the present invention can be 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C or 85°C. As an embodiment, the time of the inactivation treatment is 55 to 65 minutes. As an optional embodiment, the time of the inactivation treatment can be 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes or 65 minutes.

[0042] After obtaining the four inactivated fermentation broths, the present invention centrifuges each of the four inactivated fermentation broths to obtain four inactivated centrifuged emulsions. In one embodiment, the present invention uses a disc centrifuge for centrifugation. In one embodiment, the solids content of the four inactivated centrifuged emulsions is 40% to 80%; in another embodiment, the solids content of the four inactivated centrifuged emulsions can be 40%, 50%, 60%, 70%, or 80%. In one embodiment, the centrifuge rotates at a speed of 6,000 to 8,000 r / min; in another embodiment, the centrifuge rotates at a speed of 6,000, 7,000, or 8,000 r / min. In one embodiment, the centrifuge has a flow rate of 1 to 3 tons of inactivated fermentation broth per hour; in another embodiment, the centrifuge has a flow rate of 1, 2, or 3 tons of inactivated fermentation broth per hour. In another embodiment, the centrifuge has a discharge interval of ≤1,200 seconds. It should be noted that the volume of the inactivated fermentation broth of the present invention is very large, and the centrifugation operation cannot completely separate the supernatant and the precipitate. Some supernatant still exists in the "precipitate", so the precipitate after centrifugation appears in the form of an emulsion. Therefore, the precipitate product after centrifugation is named the emulsion after inactivation centrifugation.

[0043] After obtaining the four inactivated, centrifuged emulsions, the present invention then separately dries the four inactivated, centrifuged emulsions to obtain four postbiotic raw materials. In one embodiment, the drying comprises freeze-drying, spray-drying, or vacuum drying. In a preferred embodiment, the drying is freeze-drying, and the freeze-drying time is 48 to 72 hours. In an alternative embodiment, the freeze-drying time can be 48 hours, 60 hours, or 72 hours.

[0044] After obtaining the four postbiotic raw materials, the present invention mixes the four postbiotic raw materials according to weight percentage to obtain the composite probiotic postbiotic composition. In the composition, the weight percentage of saliva combined with Lactobacillus postbiotics is 20% to 30%, the weight percentage of Lactobacillus paracasei postbiotics is 20% to 30%, the weight percentage of Lactobacillus rhamnosus postbiotics is 20% to 30%, and the weight percentage of Lactobacillus acidophilus postbiotics is 20% to 30%. The remaining features are the same as described above and will not be repeated here.

[0045] The present invention also provides the use of the composition or the composition prepared by the preparation method described above in preparing a product for inhibiting pathogens; the pathogens include Streptococcus mutans and / or Porphyromonas gingivalis.

[0046] In one embodiment, the diseases that can be caused by the mutans Streptococcus include dental caries. In one embodiment, the diseases that can be caused by the Porphyromonas gingivalis include dental caries, periodontitis and halitosis.

[0047] The present invention also provides the use of the above-mentioned composition or the composition prepared by the preparation method in preparing a product for preventing and / or treating bacterial caries.

[0048] To further illustrate the present invention, the following is a detailed description of a composite probiotic postbiotic composition provided by the present invention, its preparation method, and application, in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents or equipment used in the present invention are conventional reagents in the art and can be purchased; unless otherwise specified, the methods used in the present invention are conventional operations in the art.

[0049] Example 1 A composite probiotic postbiotic composition

[0050] The composition is composed of 25% of saliva-associated Lactobacillus LS86 postbiotics, 25% of Lactobacillus paracasei LPC28 postbiotics, 25% of Lactobacillus rhamnosus LR22 postbiotics, and 25% of Lactobacillus acidophilus LA11-Onlly postbiotics, calculated by mass percentage. The specific preparation method is as follows:

[0051] (1) Lactobacillus saliva LS86, Lactobacillus paracasei LPC28, Lactobacillus rhamnosus LR22, and Lactobacillus acidophilus LA11-Onlly were inoculated into fermentation culture medium for fermentation at a temperature of 37° C. and for 18 hours to obtain four fermentation broths;

[0052] The fermentation broth is composed of: 10 g / L glucose, 10 g / L lactose, 10 g / L peptone, 10 g / L beef extract powder, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 1 g / L cysteine ​​hydrochloride, 0.058 g / L magnesium sulfate, 0.02 g / L manganese sulfate, 1 mL / L Tween 80, and the balance water;

[0053] The postbiotics are composed of inactivated probiotic bacteria and metabolites;

[0054] The Lactobacillus salivarius LS86 has a deposit number of CGMCC No. 6403, which is disclosed in patent CN108157973A; the Lactobacillus paracasei LPC28 has a deposit number of CNCM I-4475, which is disclosed in patent CN106234891A; the Lactobacillus rhamnosus LR22 has a deposit number of CNCM I-4474, which is disclosed in patent CN108157973A; and the Lactobacillus acidophilus LA11-Onlly has a deposit number of CGMCC No. 2106, which is disclosed in patent CN106234891A.

[0055] (2) performing heat inactivation treatment on the four fermentation broths respectively, wherein the heat inactivation temperature is 80° C. and the heat inactivation time is 60 min, to obtain four inactivated fermentation broths;

[0056] (3) The four inactivated fermentation broths were centrifuged separately in a disc centrifuge at a centrifugal speed of 7000 r / min; a centrifugal flow rate of 2 T inactivated fermentation broth / h; and a centrifugal discharge interval of 1200 s to obtain four milky inactivated centrifuged emulsions, each of which had a solid content of 60%;

[0057] (4) The four inactivated and centrifuged emulsions were pre-frozen at -40°C for 4 h and then vacuum-dried for 48 h to obtain four postbiotic freeze-dried powders. The bacterial concentration in the freeze-dried powders was 1.0 × 10 11 Pieces / gram;

[0058] (5) The four postbiotic freeze-dried powders are mixed according to weight percentage to obtain the composite probiotic postbiotic composition.

[0059] Example 2 A composite probiotic postbiotic composition

[0060] Calculated by mass percentage, the composition consists of 20% of saliva-associated Lactobacillus LS86 postbiotics, 30% of Lactobacillus paracasei LPC28 postbiotics, 20% of Lactobacillus rhamnosus LR22 postbiotics and 30% of Lactobacillus acidophilus LA11-Onlly postbiotics.

[0061] The preparation method of the composition is the same as that of Example 1, except that the four inactivated and centrifuged emulsions are mixed according to weight percentage to prepare an aqueous solution, and the bacterial concentration in the aqueous solution is 2.0×10 11 Pieces / gram.

[0062] Example 3 A composite probiotic postbiotic composition

[0063] Calculated by mass percentage, the composition consists of 30% of saliva-associated Lactobacillus LS86 postbiotics, 20% of Lactobacillus paracasei LPC28 postbiotics, 30% of Lactobacillus rhamnosus LR22 postbiotics and 20% of Lactobacillus acidophilus LA11-Onlly postbiotics.

[0064] The preparation method of the composition is the same as that of Example 1, except that the emulsions obtained by centrifugation after inactivation of the four fermentation broths are mixed according to weight percentage to prepare an emulsion, and the bacterial cell concentration in the emulsion is 3.0×10 11 Pieces / gram.

[0065] Example 4 A composite probiotic postbiotic composition

[0066] Calculated by mass percentage, the composition consists of 20% of saliva-associated Lactobacillus LS86 postbiotics, 20% of Lactobacillus paracasei LPC28 postbiotics, 30% of Lactobacillus rhamnosus LR22 postbiotics and 30% of Lactobacillus acidophilus LA11-Onlly postbiotics.

[0067] The preparation method of the composition is the same as that of Example 1, except that the four inactivated and centrifuged emulsions are mixed according to weight percentage to prepare a gel, and the bacterial concentration in the gel is 1.0×10 11 Pieces / gram.

[0068] Comparative Example 1 A composite probiotic postbiotic composition

[0069] Calculated by mass percentage, the composition consists of 33.3% of saliva-associated Lactobacillus LS86 postbiotics, 33.3% of Lactobacillus paracasei LPC28 postbiotics and 33.3% of Lactobacillus acidophilus LA11-Onlly postbiotics.

[0070] The preparation method of the composition is the same as that of Example 1.

[0071] Comparative Example 2: A probiotic postbiotic

[0072] The preparation was carried out according to the preparation method described in Example 1, except that the raw materials used to prepare the probiotic postbiotics only included Lactobacillus paracasei LPC28.

[0073] Comparative Example 3: A probiotic postbiotic

[0074] The preparation was carried out according to the preparation method described in Example 1, except that the raw materials used to prepare the probiotic postbiotics only included Lactobacillus rhamnosus LR22.

[0075] Comparative Example 4: A probiotic postbiotic

[0076] The preparation was carried out according to the preparation method described in Example 1, except that the raw materials used to prepare the probiotic postbiotics only included Lactobacillus saliva LS86.

[0077] Comparative Example 5: A probiotic postbiotic

[0078] The preparation was carried out according to the preparation method described in Example 1, except that the raw materials used to prepare the probiotic postbiotics only included Lactobacillus acidophilus LA11-Onlly.

[0079] Experimental Example 1 Antibacterial experiment of the composition of probiotic single bacteria postbiotics and compound probiotic postbiotics

[0080] In this experimental example, Streptococcus mutans and Porphyromonas gingivalis were used as indicator bacteria to conduct antibacterial experiments to verify the antibacterial effects of different probiotic single-bacterial postbiotics or composite probiotic postbiotics.

[0081] The deposit number of the mutans Streptococcus is ATCC 35668, and the deposit number of the Porphyromonas gingivalis is ATCC BAA-308 (W83).

[0082] In this experimental example, the postbiotic composition described in Example 1 was prepared into a lyophilized powder, aqueous solution, emulsion, or gel. The application effects were not significantly different. Therefore, the lyophilized powder of the composition described in Example 1 was used as the experimental group, and the probiotic postbiotics described in Comparative Examples 1 to 5 were prepared into lyophilized powders, which were used as controls 1 to 5. The culture medium included brain heart infusion broth (BHI) medium (used for culturing Streptococcus mutans) and modified GlfuAnaerobic Medium (GAM) broth medium (used for culturing Porphyromonas gingivalis).

[0083] The components of BHI medium are as follows: 10.0 g of tryptone, 5.0 g of sodium chloride, 17.5 g of beef heart extract powder, 2.0 g of glucose, 2.5 g of disodium hydrogen phosphate (12H2O), and 1 L of distilled water.

[0084] The components of modified GAM medium are as follows: Peptone 15.0g, glucose 3.0g, tryptone 10.0g, sodium dihydrogen phosphate 2.5g, soytone 3.0g, sodium chloride 3g, yeast extract powder 5.0g, soluble starch 0.3g, beef powder 2.0g, L-cysteine ​​0.3g, digested serum powder 13.5g, sodium thioglycolate 0.15g, bovine liver extract powder 1.2g, distilled water 1L.

[0085] The experimental instruments and materials used in this experiment are: Oxford cup (inner diameter 6.0 mm, outer diameter 7.8 mm, height 10.0 mm), sterile culture dish (inner diameter 90 mm, height 16 mm), McFadden tube, anaerobic culture jar, anaerobic culture bag, microaerobic gas production bag, nitrogen, standard mixed gas (10% CO2 + 10% H2 + 80% N2), vernier caliper, alcohol lamp, inoculation loop and centrifuge tube.

[0086] The test method is as follows:

[0087] Cultivate the indicator bacteria solution and dilute the two pre-cultured indicator bacteria solutions to 1×10 8 CFU / mL, and obtain the indicator bacteria suspension for later use.

[0088] Preparation of double-layer test plates: Pour 10 mL of heated and melted 2% sterile agar into a sterile culture dish. After it is fully cooled and solidified, take 500 μL of pre-diluted Streptococcus mutans indicator bacteria suspension or Porphyromonas gingivalis indicator bacteria suspension and add it to 5 mL of pre-melted and cooled to 50°C semi-solid culture medium (containing 0.7% agar). Mix well with a shaker and quickly pour it onto the solidified lower plate. Spread the upper semi-solid culture medium evenly to make the upper layer bacterial concentration of 1×10 7 CFU / mL double-layer detection plate.

[0089] Sample preparation: 1.024 g of the sample shown in the experimental group and control groups 1 to 5 was weighed and dissolved in 2 mL of deionized water. Then, six samples with decreasing concentrations were prepared in sequence using a two-fold gradient dilution method, namely 0.512 g / mL, 0.256 g / mL, 0.128 g / mL, 0.064 g / mL, 0.032 g / mL, and 0.016 g / mL. The sample numbers are shown in Table 1.

[0090] Table 1 Concentration gradient dilution table of each sample

[0091]

[0092] Experimental Procedure: The experimental procedure was based on the national standard GB / T 38483 and optimized based on the differences in growth conditions for different indicator bacteria: After loading the plates with Streptococcus mutans, the plates were directly placed in a constant temperature incubator and incubated at 37°C until a zone of inhibition appeared; after loading the plates with Porphyromonas gingivalis, the plates were placed in an anaerobic jar, the air in the jar was evacuated using a vacuum pump, and then the jar was flushed twice with nitrogen and then injected with the mixed gas. Finally, the anaerobic jar was placed in a constant temperature incubator at 37°C and incubated until a zone of inhibition appeared; a double-layer test plate with an equal amount of deionized water was used as the control group.

[0093] Results were determined by measuring the diameter of the inhibition zone for each indicator bacteria group using a vernier caliper. The plates were placed upside down on a black background without reflective light, and reflected light was used for illumination to provide high contrast for the inhibition zone. The diameter of the zone of complete inhibition was defined as the zone of inhibition, with no visible bacterial growth visible at the edge. Three sets of data were measured for each inhibition zone, and the diameters of the inhibition zones for each sample were recorded. The results for the different groups are summarized in Tables 2 to 7.

[0094] Table 2 Inhibitory effect of control group 2 on various indicator bacteria (mm)

[0095] serial number Streptococcus mutans Porphyromonas gingivalis comparison A0 22.95±0.77 6.00±00 6.00±00 A1 21.43±0.26 6.00±00 6.00±00 A2 20.14±0.50 6.00±00 6.00±00 A3 16.83±1.08 6.00±00 6.00±00 A4 11.58±0.48 6.00±00 6.00±00 A5 6.00±00 6.00±00 6.00±00

[0096] As shown in Table 2, Lactobacillus paracasei LPC28 had a good antibacterial effect against Streptococcus mutans, with an average diameter of 22.95 mm at the maximum concentration A0, and a minimum inhibitory concentration of 0.032 g / mL in the experimental group. It had no significant inhibitory effect on Porphyromonas gingivalis.

[0097] Table 3 Inhibitory effects of the three control groups on the indicator bacteria (mm)

[0098] serial number Streptococcus mutans Porphyromonas gingivalis comparison B0 26.78±1.04 22.17±0.54 6.00±00 B1 24.78±0.28 16.53±0.67 6.00±00 B2 18.75±0.43 12.68±0.49 6.00±00 B3 10.36±0.11 9.54±0.18 6.00±00 B4 6.00±0.00 6.00±00 6.00±00 B5 6.00±0.00 6.00±00 6.00±00

[0099] As shown in Table 3, Lactobacillus rhamnosus LR22 exhibited a strong antibacterial effect against Streptococcus mutans. The average diameter of the inhibition zone for the maximum concentration sample B0 was 26.78 mm, and the minimum inhibitory concentration was 0.064 g / mL. Lactobacillus rhamnosus LR22 also exhibited a strong antibacterial effect against Porphyromonas odontoides. The average diameter of the inhibition zone for the maximum concentration sample B0 was 22.17 mm, and the minimum inhibitory concentration in the experimental group was 0.064 g / mL, resulting in an average inhibition zone diameter of 9.54 mm.

[0100] Table 4 Inhibitory effect of the four control groups on the indicator bacteria (mm)

[0101] serial number Streptococcus mutans Porphyromonas gingivalis comparison C0 17.85±0.9 6.00±00 6.00±00 C1 15.20±0.36 6.00±00 6.00±00 C2 10.48±0.18 6.00±00 6.00±00 C3 6.00±00 6.00±00 6.00±00 C4 6.00±00 6.00±00 6.00±00 C5 6.00±00 6.00±00 6.00±00

[0102] As shown in Table 4, Lactobacillus salivarius LS86 was highly effective against Streptococcus mutans. The average diameter of the inhibition zone for sample C0, the highest concentration, was 17.85 mm, and the minimum inhibitory concentration in the experimental group was 0.128 g / mL. Lactobacillus salivarius LS86 had no inhibitory effect on Porphyromonas gingivalis.

[0103] Table 5 Inhibitory effects of the control group and the five groups on the indicator bacteria (mm)

[0104] serial number Streptococcus mutans Porphyromonas gingivalis comparison D0 22.16±0.61 6.00±0.00 6.00±0.00 D1 20.65±0.46 6.00±0.00 6.00±0.00 D2 18.77±1.04 6.00±0.00 6.00±0.00 D3 15.23±0.23 6.00±0.00 6.00±0.00 D4 10.36±0.16 6.00±0.00 6.00±0.00 D5 6.00±0.00 6.00±0.00 6.00±0.00

[0105] As shown in Table 5, Lactobacillus acidophilus LA11-Only exhibited a strong inhibitory effect against Streptococcus mutans. The average diameter of the inhibition zone for sample D0, the highest concentration, was 22.16 mm, and the minimum inhibitory concentration in the experimental group was 0.032 g / mL. Lactobacillus acidophilus LA11-Only had no inhibitory effect against Porphyromonas gingivalis.

[0106] Table 6 Inhibitory effect of experimental group on each indicator bacteria (mm)

[0107] serial number Streptococcus mutans Porphyromonas gingivalis comparison Ⅱ0 27.78±0.54 23.67±0.43 6.00±0.00 Ⅱ1 25.45±0.67 16.76±0.54 6.00±0.00 Ⅱ2 19.45±0.55 14.34±0.34 6.00±0.00 Ⅱ3 17.34±0.56 11.54±0.43 6.00±0.00 Ⅱ4 12.20±0.40 10.32±0.51 6.00±0.00 Ⅱ5 10.36±0.31 6.00±0.00 6.00±0.00

[0108] As shown in Table 6, the composite probiotic and postbiotic composition prepared by the present invention has a good antibacterial effect on Streptococcus mutans. The average diameter of the inhibition zone of the maximum concentration sample II0 is 27.78 mm, and the minimum sample concentration II5 still has an inhibitory effect on Streptococcus mutans, with an average inhibition zone diameter of 10.36 mm. It also has a good antibacterial effect on Porphyromonas odontoides. The average diameter of the inhibition zone of the maximum concentration sample II0 is 23.67 mm. The minimum inhibitory concentration in the experimental group is 0.032 g / mL, and the average inhibition zone diameter is 10.32 mm.

[0109] Table 7 Inhibitory effect of control group 1 on each indicator bacteria (mm)

[0110] serial number Streptococcus mutans Porphyromonas gingivalis comparison I0 23.18±0.32 6.00±0.00 6.00±0.00 I1 21.41±0.53 6.00±0.00 6.00±0.00 I2 19.45±0.55 6.00±0.00 6.00±0.00 I3 15.31±0.47 6.00±0.00 6.00±0.00 I4 11.20±0.40 6.00±0.00 6.00±0.00 I5 6.00±0.00 6.00±0.00 6.00±0.00

[0111] As shown in Table 7, the composite probiotic postbiotic prepared in control group 1 had a good antibacterial effect on Streptococcus mutans. The average diameter of the inhibition zone of the maximum concentration sample I0 was 23.18 mm, and the minimum sample concentration I5 had no inhibitory effect on Streptococcus mutans. Control group 1 also had no inhibitory effect on Porphyromonas gingivalis.

[0112] Experimental Example 2 Evaluation of the efficacy of a composite postbiotic composition on oral bacterial caries in rats

[0113] This experiment used the composite probiotic and postbiotic composition prepared by the preparation method described in Example 1.

[0114] Animals: 40 female SD rats weighing 50-60 g, 3 weeks old.

[0115] Take the composite probiotics and postbiotics composition prepared in Example 1 and mix it with physiological saline to a bacterial concentration of 1×10 8 CFU / mL; 1×10 9 CFU / mL; 1×10 10 CFU / mL, as samples of low, medium and high doses respectively.

[0116] Grouping: 40 rats were divided into 5 groups, 8 rats in each group, and treated according to the grouping conditions shown in Table 8.

[0117] Table 8 Animal experiment grouping

[0118]

[0119]

[0120] Modeling: The experiment lasted for 28 days, and the following treatments were performed according to the number of experimental days:

[0121] The first to third days were the antibiotic interference period, during which rats in each group were fed drinking water containing antibiotics (ampicillin and streptomycin) to interfere with the original oral flora.

[0122] On days 4 to 7, the oral cavity of rats in each group was inoculated with Streptococcus mutans and Porphyromonas gingivalis (double-bacteria modeling method). A sterile cotton swab was dipped in 200 μL of the mixed bacterial solution of Streptococcus mutans and Porphyromonas gingivalis and wiped on the incisors and molars of the rats once a day for 4 consecutive days.

[0123] The treatment period was from day 8 to day 28. Rats were dipped in 200 μL of the corresponding drug with a sterile cotton swab and evenly applied to the incisors and molars once a day for 21 consecutive days. After application, the rats were deprived of water for 30 minutes.

[0124] During the treatment period, rats in all groups except the blank group drank drinking water containing 50 g / L sucrose for 21 consecutive days. On the 0th, 7th, and 21st days of treatment, oral saliva was collected from each group of rats. The relative abundance of Streptococcus mutans in the rats' oral saliva was calculated and the results are shown in Tables 9 and Figure 1 The relative abundance of Porphyromonas gingivalis in rat oral saliva was statistically analyzed, and the results are shown in Figure 2 shown.

[0125] Bacterial species identification and counting: Collect oral rinse fluid and count Streptococcus mutans and Porphyromonas gingivalis.

[0126] Table 9 Relative abundance of Streptococcus mutans at different times

[0127] Time point control group Model Group Low-dose group Medium dose group High-dose group Treatment day 0 <![CDATA[0.53±0.18 a ]]> <![CDATA[1.08±0.24 b ]]> <![CDATA[1.07±0.10 b ]]> <![CDATA[0.95±0.10 b ]]> <![CDATA[0.92±0.18 b ]]> Treatment day 7 <![CDATA[0.57±0.28 a ]]> <![CDATA[1.11±0.28 b ]]> <![CDATA[0.83±0.06 c ]]> <![CDATA[0.77±0.12 c ]]> <![CDATA[0.61±0.19 ac ]]> Treatment day 21 <![CDATA[0.39±0.28 a ]]> <![CDATA[1.11±0.28 b ]]> <![CDATA[0.58±0.18 c ]]> <![CDATA[0.44±0.16 ac ]]> <![CDATA[0.39±0.16 ac ]]>

[0128] Note: Data are expressed as Mean±SD (n=8), different letters represent significant differences among different groups, p<0.05.

[0129] Table 10 Relative abundance of Porphyromonas gingivalis at different times

[0130] Time point control group Model Group Low-dose group Medium dose group High-dose group Treatment day 0 <![CDATA[0.48±0.12 a ]]> <![CDATA[1.11±0.11 b ]]> <![CDATA[1.10±0.15 b ]]> <![CDATA[1.12±0.11 b ]]> <![CDATA[1.08±0.22 b <!-- 10 -->]]> Treatment day 7 <![CDATA[0.45±0.09 a ]]> <![CDATA[1.12±0.13 b ]]> <![CDATA[0.69±0.13 c ]]> <![CDATA[0.58±0.22 c ]]> <![CDATA[0.57±0.14 c ]]> Treatment day 21 <![CDATA[0.49±0.14 a ]]> <![CDATA[1.14±0.18 b ]]> <![CDATA[0.54±0.11 c ]]> <![CDATA[0.41±0.11 ac ]]> <![CDATA[0.42±0.15 ac ]]>

[0131] Note: Data are expressed as Mean±SD (n=8), different letters represent significant differences among different groups, p<0.05.

[0132] As shown in Tables 9 and 10, after wiping the rat incisors and molars with the double bacterial mixed solution, the relative abundance of Streptococcus mutans and Porphyromonas gingivalis increased significantly, with significant differences compared with the control group (p < 0.05), indicating that Streptococcus mutans and Porphyromonas gingivalis colonized the surface of rat teeth. Figure 1 It can be seen that the relative abundance of Streptococcus mutans in the oral cavity of rats smeared with probiotics continued to decrease. After 21 days of treatment, it returned to a relative abundance level close to that of the control group, as shown in Table 10 and Figure 2 It can be seen that after 21 days of treatment, the relative abundance of Porphyromonas gingivalis showed a significant downward trend, which indicates that the composite probiotic postbiotic composition prepared by the present invention can significantly reduce the relative abundance of Porphyromonas gingivalis in the oral cavity of rats.

[0133] As shown above, after 21 days of continuous treatment with the composite probiotic and postbiotic combination in rats with bacterial dental caries (S. mutans / P. gingivalis dual-bacterial model), the relative abundance of S. mutans in the oral cavity of rats treated with the probiotics continued to decline, returning to a level close to that of the control group after 21 days of treatment. The relative abundance of P. gingivalis also showed a clear downward trend. Therefore, after 21 days of treatment, the composite postbiotic sample significantly reduced the relative abundance of S. mutans and P. gingivalis in the oral cavity of rats (p < 0.05), preventing dental caries and acidic damage to tooth enamel, and maintaining gingival and periodontal health.

[0134] In summary, the composite probiotic and postbiotic composition of the present invention has good in vitro antibacterial properties. It was verified by the rat oral bacterial caries animal model that after 21 days of treatment, the relative abundance of Porphyromonas gingivalis and Streptococcus mutans in the experimental group continued to decrease, returning to a relative abundance level close to that of the control group, significantly reducing the relative abundance of Streptococcus mutans and Porphyromonas gingivalis in the rat oral cavity (p<0.05), playing a preventive and auxiliary therapeutic role in preventing caries and acidic destruction of tooth enamel, and maintaining gum and periodontal health.

[0135] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments like this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A composite probiotic and postbiotic composition, characterized in that: The composition includes Lactobacillus salivarius postbiotics, Lactobacillus rhamnosus postbiotics, Lactobacillus rhamnosus postbiotics, and Lactobacillus acidophilus postbiotics; The saliva-associated Lactobacillus postbiotics include inactivated bacteria of saliva-associated Lactobacillus and metabolites of saliva-associated Lactobacillus; the salivarius Lactobacillus is Lactobacillus salivarius LS86; The Lactobacillus paracasei postbiotics include inactivated bacteria of Lactobacillus paracasei and metabolites of Lactobacillus paracasei; the Lactobacillus paracasei is Lactobacillus paracasei LPC28; The Lactobacillus rhamnosus postbiotics include inactivated Lactobacillus rhamnosus bacteria and metabolites of Lactobacillus rhamnosus; the Lactobacillus rhamnosus is Lactobacillus rhamnosus LR22; The Lactobacillus acidophilus postbiotics include inactivated bacteria of Lactobacillus acidophilus and metabolites of Lactobacillus acidophilus; the Lactobacillus acidophilus is Lactobacillus acidophilus LA11-Onlly.

2. The composition according to claim 1, wherein In the composition, the mass percentage of saliva combined with lactobacillus postbiotics is 20% to 30%; the mass percentage of Lactobacillus paracasei postbiotics is 20% to 30%; the mass percentage of Lactobacillus rhamnosus postbiotics is 20% to 30%; and the mass percentage of Lactobacillus acidophilus postbiotics is 20% to 30%.

3. The composition according to claim 2, wherein The inactivated bacterial concentrations of the saliva combined Lactobacillus postbiotics, Lactobacillus paracasei postbiotics, Lactobacillus rhamnosus postbiotics and Lactobacillus acidophilus postbiotics are ≥1.0×10 11 Pieces / gram.

4. The composition according to claim 1, wherein The dosage form of the composition includes powder, aqueous solution, emulsion or gel; the powder includes lyophilized agent.

5. The method for preparing the composite probiotic postbiotic composition according to any one of claims 1 to 4, characterized in that: The steps include: The saliva combined Lactobacillus, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus acidophilus were inoculated into fermentation culture fluids respectively for fermentation to obtain four kinds of fermentation fluids; The four fermentation broths are respectively subjected to inactivation treatment to obtain four inactivated fermentation broths; The four inactivated fermentation broths are centrifuged separately to obtain four inactivated and centrifuged emulsions; The four inactivated and centrifuged emulsions are freeze-dried to obtain four postbiotic raw materials; The four postbiotic raw materials are mixed to obtain the composite probiotic postbiotic composition.

6. The preparation method according to claim 5, wherein The raw materials of the fermentation culture liquid include: 10g / L glucose, 10g / L lactose, 10g / L peptone, 10g / L beef extract powder, 5g / L yeast extract, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate, 1g / L cysteine ​​hydrochloride, 0.058g / L magnesium sulfate, 0.02g / L manganese sulfate, 801mL / L Tween and the balance water; the fermentation culture temperature is 35-39°C, and the fermentation culture time is 16-20h.

7. The preparation method according to claim 5, wherein The temperature of the inactivation treatment is 75 to 85° C., and the time of the inactivation treatment is 55 to 65 minutes.

8. The preparation method according to claim 5, wherein The solid contents of the four inactivated centrifuged emulsions are respectively 40% to 80%; the centrifugal speed is 6000 to 8000 r / min; the centrifugal flow rate is 1 to 3 T of inactivated fermentation liquid / h; and the centrifugal discharge interval is ≤1200s.

9. Use of the composition according to any one of claims 1 to 4 or the composition prepared by the preparation method according to any one of claims 5 to 8 in preparing a product for inhibiting pathogens; the pathogens include Streptococcus mutans and / or Porphyromonas gingivalis.

10. Use of the composition according to any one of claims 1 to 4 or the composition prepared by the preparation method according to any one of claims 5 to 8 in preparing a product for preventing and / or treating bacterial caries.

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