CN118146972A

CN118146972A - Lactobacillus helveticus and probiotic composition for improving oral health

Info

Publication number: CN118146972A
Application number: CN202310835052.XA
Authority: CN
Inventors: 陶纯长; 金庭飞; 黄志钰
Original assignee: Guangdong Yike Weisheng Technology Co ltd
Current assignee: Guangdong Yike Weisheng Technology Co ltd
Priority date: 2023-07-10
Filing date: 2023-07-10
Publication date: 2024-06-07

Abstract

The invention discloses a lactobacillus helveticus and probiotics composition for improving oral health, which is lactobacillus helveticus K6 with a preservation number of CGMCC No. 15702. The invention also discloses a probiotic traditional Chinese medicine fermentation product and a probiotic product containing lactobacillus helveticus K6. The probiotic composition provided by the invention can effectively coagulate harmful bacteria in the oral cavity (comprising streptococcus mutans and/or porphyromonas gingivalis), improve the micro-ecological environment of the oral cavity, prevent the occurrence of dental caries, slow the swelling and pain of the gum and reduce the bad breath. The probiotics traditional Chinese medicine fermentation product provided by the invention can effectively inhibit the growth of harmful bacteria in the oral cavity, balance the microecological health of the oral cavity, and the traditional Chinese medicine composition can exert the effects of diminishing inflammation and easing pain, and the probiotics and the traditional Chinese medicine composition have synergistic effects, so that the oral cavity problem can be effectively relieved.

Description

Lactobacillus helveticus and probiotic composition for improving oral health

Technical Field

The invention relates to the technical field of probiotics, in particular to lactobacillus helveticus for improving oral health and a probiotic composition for improving oral health.

Background

The oral environment is a complex micro-ecological system consisting of a variety of microorganisms, including bacteria, fungi, protozoa, mycoplasma, viruses, etc., wherein the bacterial species in the oral cavity exceeds 700. The balance and imbalance of the oral micro-ecological environment are closely related to oral health. Caries, periodontal disease and oral mucosal disease are common oral problems. The modern people have fast life rhythm, disordered diet, bad breath, decayed tooth, gum swelling and pain, dental ulcer and other oral problems frequently occur, and the normal life is influenced.

Caries is commonly known as caries and refers to a chronic progressive disease of teeth under the influence of a variety of factors, mainly bacteria. While bad breath is mostly caused by the digestion of the retained substances in the mouth by oral microorganisms to produce volatile sulfides and other off-flavor substances (e.g., indoles, diamines).

Probiotics are living microorganisms beneficial to human health, and the probiotics effects are mainly exerted through the processes of secreting antibacterial substances, competing with pathogenic bacteria for colonization, regulating virulence related gene expression, regulating host immune response, regulating oxidative stress response, participating in nitrate- & gt nitrite- & gt nitric oxide metabolic circulation path, regulating the pH value of a biological membrane and the like. The research shows that the probiotics therapy can reduce the risk of caries, improve periodontal condition and improve the treatment effect of oral mucosa diseases, and is hopeful to be a potential way for preventing and treating oral diseases.

The probiotic bacterial strains commonly used at present mainly comprise lactobacillus, bifidobacterium, lactobacillus plantarum, lactobacillus casei, lactobacillus mucilaginosus, lactobacillus joint, bacillus, saccharomycetes and the like.

The Chinese herbal medicine has long history of use in China, and records of the treatment of oral diseases by the Chinese herbal medicine are available in ancient books. The traditional Chinese medicine has three demonstration on odontopathy, namely, one day fire, two days insect and three days kidney deficiency. Chinese herbal medicine extracts are often added to modern mouthwashes for oral care. The Chinese herbal medicines commonly used for oral health comprise flos Lonicerae, melaleuca alternifolia, radix Zanthoxyli, herba Taraxaci, flos Caryophylli, rhizoma Zingiberis recens, flos Chrysanthemi Indici, etc.

Fermentation is one of the methods for processing Chinese herbal medicines, and can improve the drug property of the Chinese herbal medicines and reduce the toxicity while obtaining fermentation metabolites. At present, the traditional Chinese medicine is fermented by probiotics and becomes an important branch of the modern fermented traditional Chinese medicine, so that the innovation and development of the traditional Chinese medicine are promoted while the types of the traditional Chinese medicine are enriched and the new functions of the traditional Chinese medicine are developed.

Patent CN103298924a discloses a probiotic composition for oral health. The composition comprising effective amounts of Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480 has the characteristics of antagonism of oral pathogens, strong colonization of the oral cavity and low acidification. However, the two strains are separated from saliva of children aged 0-5 in tropical south America development area, are all lactobacillus and are not native strains in China. The composition has single strain species and does not have the effects of detumescence and pain relief.

Patent CN113813341a discloses a traditional Chinese medicine probiotics fermented extract for improving oral problems, a preparation method and application. The obtained Chinese herbal medicine extract is fermented by saccharomycetes and lactobacillus. The Chinese medicinal composition comprises radix rehmanniae, radix rehmanniae Preparata, radix asparagi, radix Ophiopogonis, folium Eriobotryae, radix astragali, herba Dendrobii, etc. However, the capability of the Chinese herbal medicine fermentation extract obtained by the patent for inhibiting and eliminating common caries pathogenic bacteria is not studied, and the extract can only be used for oral hygiene products and oral health products.

Today's society, people's awareness of oral health care is gradually increasing. Various oral products with remarkable efficacy and small side effects are beginning to be touted by people. The development of probiotics capable of improving the micro-ecological environment of the oral cavity, preventing and treating decayed teeth and improving the breath and the composition thereof has wide development prospect and potential.

Disclosure of Invention

In order to overcome the defects of the prior art, one of the purposes of the invention is to provide lactobacillus helveticus K6 for improving oral health.

It is a second object of the present invention to provide a probiotic composition for improving oral health.

The invention aims at providing a probiotic traditional Chinese medicine fermented product for improving oral health.

It is a fourth object of the present invention to provide a probiotic product that improves oral health.

One of the purposes of the invention is realized by adopting the following technical scheme:

A Lactobacillus helveticus for improving oral health, which is Lactobacillus helveticus K6 with a preservation number of CGMCC No. 15702.

As a preferred embodiment of the present invention, the lactobacillus helveticus K6 is used for improving oral health, comprising:

inhibit the growth of Streptococcus mutans and/or Porphyromonas gingivalis,

And/or, improving the co-aggregation ability against Streptococcus mutans and/or Porphyromonas gingivalis,

And/or preventing the binding or colonization of Streptococcus mutans and/or Porphyromonas gingivalis in the oral cavity,

And/or strong adhesion to gingival epithelial cells.

The second purpose of the invention is realized by adopting the following technical scheme:

A probiotic composition for improving oral health comprising lactobacillus helveticus K6 with a preservation number of CGMCC No.15702, and at least one of the following probiotics: lactobacillus acidophilus, lactobacillus crispatus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus gasseri, lactobacillus helveticus, bifidobacterium longum subsp longum, bifidobacterium longum subsp. Infantis, bifidobacterium breve, bifidobacterium bifidum, bifidobacterium adolescentis, bifidobacterium animalis subsp.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises lactobacillus helveticus K6 with a preservation number of CGMCC No.15702, and at least one of the following probiotics: lactobacillus acidophilus K43 with the preservation number of CGMCC No.15705, lactobacillus paracasei K9 with the preservation number of CGMCC No.15025, lactobacillus casei K35 with the preservation number of CGMCC No.15704, streptococcus thermophilus W46 with the preservation number of CGMCC No.15707, pediococcus pentosaceus LN-PT16 with the preservation number of GDMCC No.62044, bifidobacterium infantis Y61 with the preservation number of CGMCC No.15710 and bifidobacterium adolescentis Y54 with the preservation number of CGMCC No. 15709.

The Lactobacillus helveticus K6 has good bile salt tolerance, can inhibit the growth of various pathogenic bacteria, has the functions of relieving hypertension, improving cerebral tension and improving memory and learning ability, and can also maintain intestinal flora balance, promote calcium absorption, improve sleep quality and the like.

Lactobacillus acidophilus K43 can secrete antibiotics (acidophilus hormone (acidolin), acidophilin (acidophilin) and lactobacillus (1) aetocidon)) to antagonize pathogenic bacteria.

Lactobacillus paracasei K9 can effectively inhibit various pathogenic bacteria, and simultaneously secreted metabolites can improve skin problems. The self-cohesiveness and copolymerization are strong, and the pathogenic bacteria can be well antagonized.

Lactobacillus casei K35 is a probiotic and is able to withstand host body defenses including enzymes in the oral cavity, low pH environment in gastric juice and bile acids of the small intestine etc. Has the functions of reducing blood pressure and cholesterol, promoting cell division, generating antibody immunity, enhancing human immunity, preventing cancer, inhibiting tumor growth and the like; also has the health-care effects of relieving lactose intolerance, allergy and the like; can inhibit and kill many spoilage bacteria and pathogenic bacteria.

Streptococcus thermophilus W46 can secrete a plurality of extracellular polysaccharides, has good gastric acid and bile salt tolerance and has obvious inhibition capability on most pathogenic bacteria. Can improve microenvironment, regulate blood pressure, and activate immune system to resist tumor.

Pediococcus pentosaceus LN-PT16 can inhibit food-borne pathogenic bacteria, is helpful for improving immunity, reducing organism cholesterol, and regulating organism metabolism.

The bifidobacterium infantis Y61 can enhance the immunity of organisms, and has the effects of resisting allergy and tumors, regulating microecology, improving organism nutrition and the like.

The bifidobacterium adolescentis Y54 can treat chronic diarrhea, antagonizes pathogenic bacteria and has strong colonisation ability. Has remarkable anti-aging effect, and can obviously increase the activity and content of superoxide dismutase (SOD) in blood, thereby reducing the oxidation reaction of free radicals and the damage to human cells, and having the effect of prolonging life.

The 8 strains have excellent acid resistance and bile salt resistance, have obvious inhibition effects on oral pathogenic bacteria such as Porphyromonas gingivalis and the like, and have no obvious antagonism on common symbiotic bacteria in the oral cavity. Has good cohesive force, can effectively interfere pathogenic bacteria to generate biological film, and reduces the formation of dental plaque.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises lactobacillus helveticus K6 and at least one of the following probiotics mixed with lactobacillus helveticus K6 in an arbitrary ratio: lactobacillus acidophilus K43, lactobacillus paracasei K9, lactobacillus casei K35, streptococcus thermophilus W46, pediococcus pentosaceus LN-PT16, bifidobacterium infantis Y61 and bifidobacterium adolescentis Y54.

As a preferred scheme of the invention, the probiotic composition for improving oral health comprises lactobacillus helveticus K6 with the preservation number of CGMCC No.15702 and streptococcus thermophilus W46 with the preservation number of CGMCC No. 15707.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises lactobacillus helveticus K6 and streptococcus thermophilus W46 mixed in any ratio. Preferably, the weight ratio of the components is 1: (0.1-8) Lactobacillus helveticus K6 and Streptococcus thermophilus W46. More preferably, the weight ratio is 1:1 Lactobacillus helveticus K6 and Streptococcus thermophilus W46.

As a preferred scheme of the invention, the probiotic composition for improving oral health comprises lactobacillus helveticus K6 with the preservation number of CGMCC No.15702, lactobacillus paracasei K9 with the preservation number of CGMCC No.15025, streptococcus thermophilus W46 with the preservation number of CGMCC No.15707 and bifidobacterium infantis Y61 with the preservation number of CGMCC No. 15710.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46 and bifidobacterium infantis Y61 mixed in any ratio. The four strains are used cooperatively, so that the oral health can be better maintained.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises 1: (0.1-8): (0.1-8): (0.1-8) Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, bifidobacterium infantis Y61.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises 1: (0.2-7): (0.3-6): (0.5-5) Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, and Bifidobacterium infantis Y61.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises 1: (0.3-6): (0.5-5): (0.5-3) Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, bifidobacterium infantis Y61.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises 1: (0.5-5): (0.5-3): (0.5-2) Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, bifidobacterium infantis Y61.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises 1: (0.3-4.5): (0.6-3): (0.5-4) Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, and Bifidobacterium infantis Y61.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises 1: (1.5-4.5): (2-3): (0.5-4) Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, and Bifidobacterium infantis Y61.

As a preferred embodiment of the present invention, the probiotic composition for improving oral health comprises 1:4.5:3:1.5 Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, bifidobacterium infantis Y61.

As a preferred embodiment of the present invention, the active ingredient of the probiotic composition for improving oral health comprises one or more combinations of probiotic live bacteria, probiotic inactivated bacteria, probiotic fermented products and probiotic lysate.

As a preferred embodiment of the present invention, the total amount of viable and/or inactivated bacteria is 1×10 ³~1×10¹¹ colony forming units (colony forming unit, CFU), preferably 1×10 ⁵~1×10¹⁰ CFU/mL (CFU/g), compared to the total mass of the probiotic composition for improving oral health.

As a preferred embodiment of the present invention, the probiotic composition is for improving oral health, comprising: preventing, alleviating and/or treating dental caries,

And/or, preventing, alleviating and/or treating periodontal disease,

And/or, preventing, alleviating and/or treating swelling and pain of gums,

And/or, improving the smell of the oral cavity,

And/or, regulating the oral cavity micro-ecological environment.

The third purpose of the invention is realized by adopting the following technical scheme:

A probiotic Chinese medicinal fermented product for improving oral health is prepared by fermenting a Chinese medicinal composition with Lactobacillus helveticus K6, which is one of the purposes, wherein the Chinese medicinal composition comprises the following components: 10-30 parts of moringa oleifera leaf, 10-30 parts of poria cocos, 5-25 parts of lophatherum gracile, 1-20 parts of dried orange peel, 10-30 parts of Chinese yam, 1-30 parts of honeysuckle and 5-30 parts of magnolia officinalis.

A probiotic traditional Chinese medicine ferment for improving oral health, which is fermented from a probiotic composition according to any one of the two purposes, to a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises: 10-30 parts of moringa oleifera leaf, 10-30 parts of poria cocos, 5-25 parts of lophatherum gracile, 1-20 parts of dried orange peel, 10-30 parts of Chinese yam, 1-30 parts of honeysuckle and 5-30 parts of magnolia officinalis.

As a preferred embodiment of the present invention, the Chinese medicinal composition comprises: 10-20 parts of moringa oleifera leaf, 10-25 parts of poria cocos, 10-20 parts of lophatherum gracile, 5-20 parts of dried orange peel, 15-30 parts of Chinese yam, 5-15 parts of honeysuckle and 5-15 parts of magnolia officinalis.

The moringa leaves have the effects of resisting oxidation, promoting diuresis, relaxing bowels and improving immunity, and are high in protein content and beneficial to fermentation. Poria has effects of removing dampness, promoting diuresis, and strengthening spleen and stomach, and can regulate intestinal health, and is beneficial for eliminating halitosis caused by dyspepsia. Lophatherum gracile has effects of clearing heat, purging pathogenic fire, promoting urination, and removing stomach fire and heart fire, thereby having effect of refreshing breath. Chen Pike has effect of refreshing breath. The Chinese yam has the effects of strengthening spleen, tonifying lung qi, strengthening kidney qi and tonifying essence, and can effectively improve the digestion function of organisms.

As a preferred scheme of the invention, the traditional Chinese medicine composition further comprises one or more of eucommia ulmoides, yucca and chrysanthemum.

As a preferred scheme of the invention, the probiotic traditional Chinese medicine fermented product for improving oral health is a fermented product containing traditional Chinese medicine and living bacteria, which is obtained by extracting the traditional Chinese medicine composition, adding the lactobacillus helveticus K6 (or adding the probiotic composition of any one of the two purposes) for fermentation. The traditional Chinese medicine probiotics fermentation composition contains living bacteria, not only can effectively inhibit the growth of harmful bacteria in the oral cavity and maintain the oral environment, but also can exert the effects of clearing heat and detoxicating, diminishing inflammation and easing pain, and can relieve the oral problems such as gum swelling and pain and improve halitosis.

The invention also provides a preparation method of the probiotic traditional Chinese medicine fermented product for improving oral health, which comprises the following steps:

(1) Extracting traditional Chinese medicine: cleaning the medicinal materials of the traditional Chinese medicine composition, drying, crushing, adding water for extraction, filtering, and concentrating the filtrate to obtain a traditional Chinese medicine extract;

(2) Activating strains: inoculating probiotics in the probiotic composition into an MRS liquid culture medium respectively, and culturing into strain seed liquid under anaerobic conditions;

(3) Preparing a liquid culture medium: adding a carbon source, a nitrogen source and inorganic salt into the traditional Chinese medicine extract obtained in the step (1), dissolving the traditional Chinese medicine extract in sterile water, uniformly mixing, adjusting the pH value of a culture medium, and sterilizing for later use;

(4) Fermentation: inoculating the probiotic composition seed liquid activated in the step (2), and fermenting and culturing under anaerobic conditions to obtain a fermentation composition (liquid state);

(5) And (3) drying: and (3) adding a filler and/or a protective agent into the product obtained in the step (4), and performing vacuum freeze drying or spray drying to obtain a fermented solid product.

(6) Sieving: carrying out superfine grinding and sieving on the product subjected to vacuum freeze drying in the step (5) to obtain a probiotic traditional Chinese medicine fermentation composition (powder); or directly sieving the spray-dried product in the step (5) to obtain the probiotic fermented composition (powder).

As a preferable scheme of the invention, the extraction method in the step (1) is boiling extraction, the water addition amount is 5-15 times of the mass of the traditional Chinese medicinal materials, the extraction time is 1-2 h times each time, and the extraction times are 2-3 times each time; or extracting with microwave with water 5-15 times of the Chinese medicinal materials, the extraction power of 800-1800 w, the extraction times of 2-3 times, and the extraction time of 0.5-2 h each time; or ultrasonic extraction with water content 5-15 times of the quality of the Chinese medicinal materials, extraction temperature 25-50deg.C, ultrasonic power 1000-1500 w, working frequency 2-7-s stop 1-5 s, and extraction time 0.5-2 h each time.

As a preferable scheme of the invention, the inoculation amount of probiotics in the probiotic composition in the step (2) is 2-5%, the fermentation temperature is 30-38 ℃, the fermentation time is 12-48 h, the culture condition is stationary culture, and the viable count of the strain seed liquid is 10 ⁷-10⁹ cfu/mL.

As a preferable scheme of the invention, the weight ratio of the traditional Chinese medicine extract, the carbon source, the nitrogen source and the inorganic salt in the step (3) is (50-95): (1-20): (1-20): (1-10); the carbon source can be one or more of molasses, glucose, maltose and sucrose; the nitrogen source can be one or more of beef extract, peptone and yeast extract powder, and the inorganic salt can be one or more of sodium chloride, calcium chloride, magnesium sulfate and zinc sulfate; the pH of the culture medium is adjusted to be 5.0-6.5.

As a preferred embodiment of the present invention, the total inoculum size of the probiotic composition in step (4) is 2-10%; the fermentation temperature is 28-39 ℃, the fermentation time is 12-150 h, the anaerobic condition is static culture, and when the pH value of the fermentation liquor reaches 4.0-4.5, the fermentation end point can be judged.

As a preferred embodiment of the present invention, the weight ratio of the filler and/or protecting agent in step (5) to the fermented composition obtained in step (4) is (20-50): (50-80); the filler can be one or more of maltodextrin, corn starch, resistant dextrin, soluble rice flour, microcrystalline cellulose, silicon dioxide and zeolite powder; the protective agent can be one or more of skimmed milk powder, trehalose, sodium alginate, modified tapioca starch, beta-cyclodextrin and gelatin.

As a preferable scheme of the invention, in the step (5), the vacuum freeze-drying process is carried out at the temperature of-45 to-35 ℃ for 1.5-3 h, at the temperature of-5 ℃ for 15-25 h, at the temperature of 25-35 ℃ for 10-20 h, the total freeze-drying time is 35-48 h, and the shelf temperature and the material temperature at the end point are not more than 5 ℃. The air inlet temperature of the spray drying process is 60-78 ℃, the air outlet temperature is 50-69 ℃, the atomizer pressure is 180-220 kpa, and the feeding rate is 13-20 mL/min.

As a preferred embodiment of the present invention, the number of the sieve in the step (6) is 60 to 80 mesh. Sieving to obtain probiotic fermented product (powder) of Chinese medicinal materials.

As a preferred embodiment of the present invention, the probiotic Chinese medicine ferment is used for improving oral health, comprising: preventing, alleviating and/or treating dental caries,

And/or, preventing, alleviating and/or treating periodontal disease,

And/or, preventing, alleviating and/or treating swelling and pain of gums,

And/or, improving the smell of the oral cavity,

And/or, regulating the oral cavity micro-ecological environment.

The probiotics traditional Chinese medicine fermented product for improving oral health provided by the invention can clear heat and detoxify, diminish inflammation and relieve pain, and relieve swelling and pain of gum, and has better effect than a pure traditional Chinese medicine composition or a pure compound probiotics.

The fourth purpose of the invention is realized by adopting the following technical scheme:

a probiotic product for improving oral health comprising lactobacillus helveticus K6 as described in one of the objects.

A probiotic product for improving oral health comprising a probiotic composition of any one of the two purposes.

A probiotic product for improving oral health comprising the probiotic traditional Chinese medicine ferment of any one of the three purposes.

As a preferred embodiment of the present invention, the probiotic product is for improving oral health, comprising: preventing, alleviating and/or treating dental caries,

And/or, preventing, alleviating and/or treating periodontal disease,

And/or, preventing, alleviating and/or treating swelling and pain of gums,

And/or, improving the smell of the oral cavity,

And/or, regulating the oral cavity micro-ecological environment.

As a preferred aspect of the present invention, the probiotic product for improving oral health provided by the present invention may be used for preventing and/or treating oral problems caused by oral pathogenic bacteria, including micro-ecological disorders or abnormalities caused by bacteria, fungi, etc. in the oral cavity, including humans and animals. Such conditions may be severe pathological conditions as well as minor discomfort, such as caries, periodontitis, gingivitis, herpes, ulcers, and halitosis, etc.; problems such as swelling and aching of gums can also be included.

As a preferred embodiment of the present invention, the present invention provides a probiotic freeze-dried powder for improving oral health, comprising the lactobacillus helveticus K6 of the object, and/or comprising the probiotic composition of any one of the objects, and/or comprising the probiotic Chinese herbal ferment of any one of the three objects. Preferably, the probiotic freeze-dried powder may be administered orally.

As a preferred embodiment of the present invention, the present invention provides a probiotic lysate for improving oral health, which is formed by lysing lactobacillus helveticus K6 according to the object, and/or by lysing a probiotic composition according to any one of the objects, and/or by lysing a probiotic Chinese medicinal fermentation according to any one of the three objects. The lysate can be taken orally or applied externally.

As a preferred embodiment of the invention, the probiotic lysate for improving oral health does not mean that the final product comprises only the composition after lysis, and other ingredients may be added according to the actual application. Such as mixing the lysate with a probiotic broth or the like.

As a preferred embodiment of the present invention, the preparation process of the probiotic lysate comprises: the lactobacillus helveticus K6 of the first aspect, and/or the probiotic composition of any one of the second aspect and/or the probiotic traditional Chinese medicine fermentation of any one of the third aspect are fermented and then subjected to lysis treatment.

As a preferred embodiment of the present invention, the preparation process of the probiotic lysate comprises: the lactobacillus helveticus K6 of the first purpose and/or the probiotic composition of any one of the second purpose and/or the probiotic traditional Chinese medicine fermentation product of any one of the third purpose are prepared into powder, and the powder is prepared by adding purified water for suspension and then is subjected to lysis treatment.

As a preferred embodiment of the present invention, the lysis treatment method includes, but is not limited to, high-pressure homogenization lysis, ultrasonic treatment lysis and enzymolysis lysis.

As a preferred embodiment of the present invention, the probiotic product for improving oral health provided by the present invention may comprise lactobacillus helveticus K6 as described in one of the objects, and/or the probiotic composition as described in any one of the objects, and/or the probiotic Chinese herbal fermented product as described in any one of the three objects. In addition to this, one or more other active agents and/or daily acceptable excipients (in the case of daily use compositions), pharmaceutically acceptable excipients (in the case of pharmaceutical compositions) or suitable edible ingredients (in the case of edible compositions) may be included.

As a preferred scheme of the invention, the probiotic product for improving oral health can be food, cosmetic products, health products and pharmaceutical products.

As a preferred embodiment of the present invention, the probiotic product for improving oral health provided by the present invention can be used for various foods, such as probiotic freeze-dried powder, dairy products, yogurt, curd, cheese, milk powder, milk-based fermented products, ice cream, chocolate, fillings, candy, baked goods, fruit juice, chewing gum, etc.

As a preferred embodiment of the invention, the probiotic product for improving oral health provided by the invention can also be used for animal foods, such as animal feeds and animal health products. The animal is preferably, but not limited to, livestock, poultry or pets.

As a preferred embodiment of the present invention, the probiotic product for improving oral health provided by the present invention may be used in daily chemical products such as toothpastes, dentifrices, tooth powders, topical oral gels, mouth rinses, mouth sprays, dental strips, chewing gums, and the like.

As a preferred embodiment of the present invention, the probiotic product for improving oral health provided by the present invention may be in solid, liquid or gaseous form, in particular in the form of a powder, tablet, film preparation, solution, aerosol, granule, lozenge, pill, suspension, emulsion, capsule, syrup, liquid, elixir, extract, tincture or fluid extract, or in a form particularly suitable for oral administration.

The invention provides a probiotic product for improving oral health, which comprises probiotics capable of effectively condensing harmful bacteria in the oral cavity and improving the microecological environment of the oral cavity, can prevent decayed teeth and reduce bad breath.

Compared with the prior art, the invention has the beneficial effects that:

(1) The probiotic composition provided by the invention can effectively coagulate harmful bacteria in the oral cavity (comprising streptococcus mutans and/or porphyromonas gingivalis), improve the microecological environment in the oral cavity, prevent the occurrence of dental caries, slow the swelling and pain of the gingiva, and reduce the bad breath in the oral cavity.

(2) The probiotics traditional Chinese medicine fermentation product provided by the invention can effectively inhibit the growth of harmful bacteria in the oral cavity, balance the microecological health of the oral cavity, and the traditional Chinese medicine composition can exert the effects of diminishing inflammation and easing pain, and the probiotics and the traditional Chinese medicine composition have synergistic effects, so that the oral cavity problem can be effectively relieved.

(3) The probiotics for improving oral health, the probiotic composition and the probiotic traditional Chinese medicine fermented product provided by the invention can be applied to various products of human beings and animals, and have a wide application range. Meanwhile, the composition can be prepared into various forms of substances including but not limited to probiotic freeze-dried powder, lysate and the like, and has wide application prospect.

Biological material preservation information

Lactobacillus helveticus K6 with a preservation number of CGMCC NO.15702 is classified and named as: lactobacillus helveticus Lactobacillus helveticus was deposited in China general microbiological culture Collection center (address: institute of microbiology, national academy of sciences of China, the university of China, the Korean area North Star, the West way 1, the address: CGMCC, for 28 th, and the abbreviation of the deposit unit is CGMCC).

Lactobacillus casei K35 with a preservation number of CGMCC NO.15704, and is classified and named as follows: lactobacillus casei Lactobacillus casei was deposited in China general microbiological culture Collection center (address: no. 3 of the institute of microorganisms, national academy of sciences of China, the Korean area North Star, the Beijing) at 28 th month in 2018, and the abbreviation of the deposit unit is CGMCC.

Streptococcus thermophilus W46 has a preservation number of CGMCC NO.15707 and is classified and named as follows: streptococcus thermophilus Streptococcus thermophilus was deposited at China general microbiological culture Collection center (address: north Star, west Lu 1, 3, national academy of sciences of China, postal code: 100101) at 28 th month 04 in 2018, and the preservation unit is abbreviated as CGMCC.

Bifidobacterium infantis Y61 has a preservation number of CGMCC NO.15710 and is classified and named as follows: bifidobacterium infantis Bifidobacterium infantis was deposited in China general microbiological culture Collection center (address: no. 3 of the institute of microorganisms, national academy of sciences of China, having the name of CGMCC: north Star, kogyo, beijing) at 28 th month of 2018, and the abbreviation of the deposit unit is CGMCC.

Drawings

FIG. 1 is a diagram showing the results of a test for verifying the co-aggregation capability of a probiotic to oral harmful bacteria in an embodiment of the present invention;

FIG. 2 is a graphical representation of the results of an adhesion test of a second probiotic of the verification system to oral cells according to an embodiment of the present invention;

FIG. 3 is a diagram showing the results of an experiment for verifying the effect of three traditional Chinese medicine extracts on the growth of probiotics in the system according to the embodiment of the invention;

FIG. 4 is a graphical representation of plaque assay results from a three animal assay of the verification system of the present invention;

FIG. 5 is a diagram showing the results of the oral volatile gas concentration detection test in a three-animal test of the verification system according to the embodiment of the invention;

FIG. 6 is a diagram showing the results of gum redness and swelling tests in three animal experiments of the verification system of the embodiment of the invention.

Description of the embodiments

The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined to form new embodiments. The raw materials, equipment, etc. used in the following examples are available in a purchase manner except for special restrictions.

The probiotics used in the test process of the embodiment of the invention comprise lactobacillus acidophilus K43, lactobacillus helveticus K6, lactobacillus helveticus HH-L PH17, bifidobacterium infantis Y61, bifidobacterium adolescentis Y54, lactobacillus casei K35, lactobacillus paracasei K9, streptococcus thermophilus W46 and pediococcus pentosaceus LN-PT16.

The information of lactobacillus acidophilus K43 is shown in patent CN114848685A, an antiallergic probiotic composition, application of the composition and probiotic Chinese medicinal fermented product.

The information of Lactobacillus helveticus HH-LPH 17 is shown in patent CN114262671A, which is a Lactobacillus helveticus strain for preventing and treating cholestatic liver diseases and application thereof.

The information of Bifidobacterium adolescentis Y54 is shown in patent CN115678804A, which is a probiotic composition for inhibiting melanin formation and application thereof.

The information of lactobacillus paracasei K9 is shown in patent CN114568663A, namely a Yu Ning heart-relieving probiotic fermented product, and a preparation method and application thereof.

The information of the Pediococcus pentosaceus LN-PT16 is shown in the patent CN114504623A in detail, and the preparation method of the traditional Chinese medicine probiotics composite product for improving qi deficiency constipation.

Verification system one: in vitro antibacterial screening test of probiotics on oral harmful bacteria

Streptococcus mutans (Streptococcus mutans, S.mutans) is a resident flora in the oral cavity and is also recognized as an important cariogenic bacteria. Porphyromonas gingivalis (Porphyromonas gingivalis, P.gingivalis) is also one of the resident species in the oral cavity, and is closely related to the occurrence of periodontitis, and is considered to be one of the important pathogenic bacteria causing alveolar bone resorption. Caries and periodontitis are common oral problems, so that two oral pathogenic bacteria are selected as research objects, and the in vitro antibacterial capacity of different probiotic strains on the oral pathogenic bacteria is examined.

Preliminary screening test of probiotic strains

In vitro bacteriostasis tests of the two oral pathogenic bacteria are carried out on a series of probiotic bacterial strains, wherein the in vitro bacteriostasis tests comprise lactobacillus acidophilus K43, lactobacillus helveticus K6, lactobacillus helveticus HH-L PH17, bifidobacterium infantis Y61, bifidobacterium adolescentis Y54, lactobacillus casei K35, lactobacillus paracasei K9, streptococcus thermophilus W46 and pediococcus pentosaceus LN-PT16.

The probiotic strains are respectively inoculated in MRS liquid culture medium, and cultured overnight under the condition of culture temperature suitable for the growth of each strain until the concentration of bacterial liquid is more than 10 ⁷ cfu/mL. Respectively inoculating the two oral pathogenic bacteria into tryptone soybean broth culture medium (TSB), culturing under respective proper conditions until the bacterial liquid concentration is above 10 ⁷ cfu/mL, regulating the bacterial liquid concentration to 10 ⁶ cfu/mL, taking 100 mu L of the bacterial liquid to be coated on the surface of a soybean casein blood plate culture medium, placing oxford cups at equal intervals after the surface is dried, adding 100 mu L of probiotics bacterial liquid into the oxford cups, using MRS broth culture medium as negative control, and using amoxicillin as positive control. Culturing at 36+ -1deg.C 36 h, measuring diameter of the inhibition zone with vernier caliper, and making 2 strains of probiotics. The results of the inhibition zone are shown in table 1.

TABLE 1 results recording of diameter (mm) of inhibition zone of different probiotics against 2 oral harmful bacteria

Sequence number	Strain name	P. gingivalis	S.mutans
				1	Lactobacillus acidophilus K43	26.30±0.87	28.43±0.69
2	Lactobacillus helveticus K6	29.94±0.95	29.03±0.23
				3	Lactobacillus helveticus HH-LPH 17	25.62±0.65	24.59±0.29
4	Lactobacillus casei K35	28.80±0.35	25.34±0.66
				5	Lactobacillus paracasei K9	29.56±0.11	28.26±0.39
6	Pediococcus pentosaceus LN-PT16	20.96±1.18	23.23±0.19
				7	Streptococcus thermophilus W46	25.61±0.81	25.26±0.82
8	Bifidobacterium infantis Y61	28.73±0.58	26.34±0.36
				9	Bifidobacterium adolescentis Y54	25.23±0.19	24.18±0.21
10	Negative control	×	×
				11	Positive control	41.96±0.48	37.26±0.66

Note that: and the 'X' represents that the diameter of the inhibition zone is less than or equal to 8 mm.

As can be seen from table 1, all probiotic single strains tested produced a zone of inhibition for 2 oral harmful bacteria. Wherein Lactobacillus helveticus K6, lactobacillus paracasei K9, bifidobacterium infant Bifidobacterium Y61 and Streptococcus thermophilus W46 of other bacteria have relatively large antibacterial circle on harmful bacteria in oral cavity in the respective bacteria strain, and have good antibacterial ability. And the inhibition effect of the Lactobacillus helveticus K6 on the harmful bacteria in the oral cavity is superior to that of the Lactobacillus helveticus HH-LPH 17, which shows that the strain has good performance in the aspect of oral bacteriostasis.

2. Screening test of probiotic combinations

And carrying out equal-ratio combination and compounding on the probiotic strains subjected to the test, so that the concentration of the combined compound bacterial liquid is more than 10 ⁷ cfu/mL, and verifying whether the combination of different strains can obtain better antibacterial effect. The results are shown in Table 2.

TABLE 2 results recording of diameter (mm) of inhibition zone of different probiotic combinations on 2 oral harmful bacteria

Sequence number	Strain combinations	P. gingivalis	S.mutans
				1	Lactobacillus helveticus K6+ Lactobacillus acidophilus K43	25.30±0.67	28.15±0.62
2	Lactobacillus helveticus K6+ Lactobacillus casei K35	27.86±0.63	26.35±0.36
				3	Lactobacillus helveticus K6+ Lactobacillus paracasei K9	29.50±1.02	28.98±0.82
4	Lactobacillus helveticus K6+ streptococcus thermophilus W46	32.19±0.51	29.88±1.03
				5	Lactobacillus helveticus K6+ bifidobacterium infantis Y61	29.35±1.30	27.65±0.57
6	Lactobacillus helveticus HH-LPH 17+ streptococcus thermophilus W46	25.73±1.06	25.34±0.85
				7	Lactobacillus paracasei K9+ Streptococcus thermophilus W46	33.20±0.60	29.06±0.75
8	Lactobacillus paracasei K9+ bifidobacterium infantis Y61	33.88±0.92	29.23±0.61
				9	Streptococcus thermophilus W46+ bifidobacterium infantis Y61	27.95±0.83	25.54±1.28
10	Lactobacillus helveticus K6+Lactobacillus paracasei K9+Bifidobacterium infantis Y61+Streptococcus thermophilus W46	34.73±1.26	30.60±0.49
				11	Negative control	×	×
12	Positive control	41.96±0.48	37.26±0.66

As can be seen from Table 2, the combination of Lactobacillus helveticus K6 with Lactobacillus paracasei K9 and the combination of Lactobacillus helveticus K6 with Bifidobacterium infantis Y61 showed no significant difference in the diameter of the zone of inhibition of the composition compared with the corresponding single strain, indicating that the two combinations did not show a synergistic effect. The Lactobacillus helveticus K6 is combined with the Lactobacillus acidophilus K43, the Lactobacillus helveticus K6 is combined with the Lactobacillus casei K35, and the diameter of the bacteriostasis circle of the composition is even smaller than that of the corresponding single strain, which indicates that the combined strains have no synergistic effect. Lactobacillus helveticus HH-LPH 17 in combination with Streptococcus thermophilus W46 showed no synergy in the diameter of the zone of inhibition of the composition against oral harmful bacteria compared to the corresponding single strain.

The combination of the Lactobacillus helveticus K6 and the streptococcus thermophilus W46, the combination of the Lactobacillus paracasei K9 and the bifidobacterium infantis Y61 and the antibacterial circle diameter of the 4-strain (Lactobacillus helveticus K6+ Lactobacillus paracasei K9+ bifidobacterium infantis Y61+ streptococcus thermophilus W46) compound combination are obviously larger than the corresponding antibacterial circle diameter of a single strain, which indicates that the strain combinations can play a synergistic effect and can effectively inhibit the growth of oral harmful bacteria (streptococcus mutans and porphyromonas gingivalis). In particular, the diameter of a bacteriostasis zone generated by the compound combination of 4 strains of bacteria is maximum, which indicates that the use effect of the 4 strains of bacteria is optimal.

3. Probiotic combination proportioning screening test

On the premise of ensuring the same viable count, the 4 probiotics are compounded according to different weight ratios, and the inhibition condition of the bacterial strain combinations under different ratios on 2 oral harmful bacteria (streptococcus mutans and porphyromonas gingivalis) is verified. The compounding ratio of 4 probiotics is shown in table 3.

Table 34 strain compound proportion table

Strain name combinations	Combination 1	Combination 2	Combination 3	Combination 4	Combination 5
						Lactobacillus helveticus K6	2	1	1.5	3	1
Lactobacillus paracasei K9	3	2	3.5	1	4.5
						Streptococcus thermophilus W46	4	3	3	2	3
Bifidobacterium infantis Y61	1	4	2	4	1.5

Under the premise of ensuring that the viable count of each strain is the same, the compositions are combined according to the compound proportion of the table 3, and the obtained compositions are subjected to oxford cup bacteriostasis test, and the results are shown in the table 4.

Table 44 results of different ratio combinations of strains on diameter (mm) of inhibition zone of 2 strains of oral harmful bacteria

Sequence number	Strain combinations	P. gingivalis	S.mutans
				1	Combination 1	33.51±1.37	30.16±0.64
2	Combination 2	32.89±1.03	29.26±0.72
				3	Combination 3	33.16±0.96	29.87±0.66
4	Combination 4	30.22±1.25	28.96±1.28
				5	Combination 5	34.96±0.71	31.06±0.61
8	Empty negative control	×	×
				9	Positive control	41.96±0.48	37.26±0.66

From the results in table 4, it can be seen that the probiotic strains have the following inhibition effects on oral harmful bacteria according to different weight ratios: combination 5 > combination 1 > combination 3 > combination 2 > combination 4.

The strains of different bacteria are compounded for use, so that the synergistic effect among the strains can be better exerted. The weight ratio of the Lactobacillus helveticus K6, the Lactobacillus paracasei K9, the streptococcus thermophilus W46 and the bifidobacterium infantis Y61 in the combination 1-5 is 1 (0.1-8). In particular, when the weight ratio of lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46 and bifidobacterium infantis Y61 is 1: (1.5-4.5): (2-3): (0.5-4), the effect is better (combinations 1-3, 5), especially when the weight ratio of lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, bifidobacterium infantis Y61 is 1:4.5:3:1.5, the in vitro inhibition effect on 2 oral harmful bacteria is optimal.

And a verification system II: co-aggregation capability test of probiotics on oral harmful bacteria

The method comprises the steps of combining 1-3 and 5 of cultured lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, bifidobacterium infantis Y61 bacteria liquid and 4 strains of bacteria, regulating the concentration of bacterial suspension to 10 ⁸ cfu/mL, vortex oscillating about 10 s, standing at room temperature for 4 h, marking the OD _600nm of supernatant as A ₁, respectively taking 1mL of streptococcus mutans and porphyromonas gingivalis in a centrifuge tube, centrifuging at 6000 rpm and 4 ℃ for 10min, discarding the supernatant, re-suspending and washing the collected bacteria twice by using PBS buffer, adding 1mL PBS buffer, mixing uniformly, standing at room temperature for 4 h, and measuring the absorbance value of the bacterial suspension OD _600nm to be A ₂. The probiotics and the combined bacterial suspension thereof are uniformly mixed with bacterial suspensions of two pathogenic bacteria in equal volume respectively, and the mixture is kept stand for 4 h, and the supernatant OD _600nm is measured and is marked as A ₃. The copolymerization collection rate of probiotics and pathogenic bacteria is calculated as follows: co-aggregation rate/% = (1-2A ₃/（A₁＋A₂)). Times.100%.

As a result, as shown in FIG. 1, it can be seen that Lactobacillus paracasei K9 and Lactobacillus helveticus K6 exhibit better co-aggregation ability against Streptococcus mutans and Porphyromonas gingivalis than the other two probiotics (Streptococcus thermophilus W46, bifidobacterium infantis Y61). The probiotic compound composition 3 and 5 has better copolymerization and aggregation capability on oral pathogenic bacteria than the other two groups (combination 1 and combination 2), wherein the combination 5 has better copolymerization and aggregation capability, can effectively prevent the combination or the colonization of streptococcus mutans and porphyromonas gingivalis, and forms a copolymer with the streptococcus mutans and porphyromonas gingivalis to achieve the effect of eliminating the pathogenic bacteria, can reduce the harm of the pathogenic bacteria in the oral cavity, and maintains the health of the oral cavity.

And (3) a verification system III: adhesion test of probiotics to oral cells

HGE cells (human gingival epithelial cells) were grown to 70-80% confluence, inoculated into 12-well cell culture plates for culture so that the cell concentration per well was 1.0X10- ⁵, after the cells reached 70-80% confluence, the upper-layer culture solution was discarded, washed 3-5 times with an equal volume of PBS, the residual culture solution was removed, an equal volume of high-sugar DMEM culture solution free of diabody was added, the culture was continued for 24 h, the supernatant culture solution was discarded, washed 3-5 times with PBS, 500. Mu.L of a bacterial suspension (PBS suspension) of 1.0X10- ⁸ cfu/mL Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, and Bifidobacterium infantis Y61 was added to each well, after 2h of co-incubation of the two cells, 3-5 times with an equal volume of PBS was removed, and 2.0 mL of 1% (vol/vol) Triton X-100 was added to each well for incubation of 10 min. And collecting a mixed solution of two cells in each hole, and carrying out viable probiotic counting after the mixed solution is diluted in a PBS gradient. The calculation formula of the adhesion rate of probiotics to cells is as follows: adhesion rate/% = N ₁/N₂ x 100%, where N ₁ is the number of viable probiotic bacteria added (1.0 x 10 ⁸ cfu/mL）,N₂ viable bacteria adhered to the cells.

As shown in FIG. 2, the HGE cells are gingival epithelial cells, the adhesion of different probiotic strains to the HGE cells is different, the adhesion capability of the Lactobacillus paracasei K9 and the Streptococcus thermophilus W46 to the gingival epithelial cells is better, the adhesion sites can be competing with pathogenic bacteria in the oral cavity, and the HGE cells can stay in the oral cavity for a long time, so that the competitive advantage of the probiotic strains is improved, and the micro-ecological environment of the oral cavity is better maintained.

And a verification system IV: screening test of Chinese medicinal composition

According to research, the traditional Chinese medicine is applied to the oral cavity in ancient times, and the proper traditional Chinese medicine can play roles in inhibiting bacteria, diminishing inflammation and easing pain. The traditional Chinese medicine composition provided by the invention comprises the following components in mass: 10-30 parts of moringa oleifera leaf, 10-30 parts of poria cocos, 5-25 parts of lophatherum gracile, 1-20 parts of dried orange peel, 10-30 parts of Chinese yam, 1-30 parts of honeysuckle and 5-30 parts of magnolia officinalis. Extracting the traditional Chinese medicine with water to obtain a traditional Chinese medicine extract, and then inoculating probiotics for fermentation. The traditional Chinese medicine extract generally has a certain antibacterial effect, and the effect of fermentation may be affected by inoculating probiotics for fermentation. Therefore, the invention screens the composition of the Chinese medicinal extract to obtain the Chinese medicinal composition which does not influence the fermentation effect, and can relieve the swelling and pain of gum and improve the breath.

1. Test of the influence of traditional Chinese medicine extracts on the growth of probiotics

(1) Activating strains: respectively inoculating 2% of Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46 and bifidobacterium infantis Y61 to MRS liquid culture medium, culturing at 37 ℃ for 24h, and regulating the viable count of the bacterial liquid to 10 ⁷ cfu/mL for later use;

(2) Extracting traditional Chinese medicine: cleaning the Chinese medicinal materials, drying, mixing according to the proportion of Table 5, pulverizing, boiling and extracting with water 5-15 times of the mass of the Chinese medicinal materials for 2-3 times, and extracting for 1-2 h times each time. Filtering after extraction, concentrating the filtrate to obtain a traditional Chinese medicine extract, and sterilizing for later use;

(3) Co-cultivation: adding 50% of the extract in (2) into MRS liquid culture medium, uniformly mixing, and respectively inoculating the probiotic bacteria liquid in (1) according to 2% of inoculum size. Taking liquid MRS culture medium without extract as blank control, and standing at 37deg.C for culturing 24 h;

(4) Viable bacteria count: the cultured samples in (3) were subjected to live lactobacillus counting, and the growth rate of each test group was calculated.

Growth rate/% = (number of viable bacteria of each test group sample/number of viable bacteria of the blank group) ×100%

The traditional Chinese medicine composition is shown in a table 5 according to the weight ratio.

Table 5 Chinese medicinal composition proportioning table

Chinese medicine name combination	Combination 1	Combination 2	Combination 3	Combination 4	Combination 5
						Moringa oleifera leaf	10	15	20	25	20
Poria cocos	25	20	10	15	20
						Lophatherum gracile (Lophatherum gracile)	15	20	10	12	15
Dried orange peel	5	5	20	3	10
						Chinese yam	25	30	15	15	20
Honeysuckle flower	15	5	10	15	10
						Cortex Magnolia officinalis	5	5	15	15	5

As shown in FIG. 3, it can be seen that the different Chinese medicinal composition extracts have a certain effect on the fermentation of probiotics. The fermentation growth rate of probiotics in the traditional Chinese medicine compositions 1-2 and 5 is 84-97%, and the traditional Chinese medicine compositions can be normally grown and fermented. The fermentation growth rate of probiotics in the traditional Chinese medicine combination 3 and 4 is 73-82%, which shows that part of traditional Chinese medicine extracts have a certain inhibition effect on the growth of probiotics, and each traditional Chinese medicine component should occupy proper dosage in the traditional Chinese medicine combination.

2. Preparation of probiotic Chinese medicine fermented product for improving oral health

Examples

The embodiment provides a probiotic traditional Chinese medicine fermented product for improving oral health, and the preparation method comprises the following steps:

(1) Extracting traditional Chinese medicine: weighing 10 parts of moringa leaves, 25 parts of poria cocos, 15 parts of lophatherum gracile, 5 parts of dried orange peel, 25 parts of Chinese yam, 15 parts of honeysuckle and 5 parts of magnolia officinalis. Cleaning the medicinal materials, drying, pulverizing, extracting with water, filtering, and concentrating the filtrate to obtain the traditional Chinese medicine extract.

(2) Activating strains: respectively inoculating 2% of Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46 and Bifidobacterium infantis Y61 to MRS liquid culture medium, culturing at 37deg.C for 24h, and regulating the viable count of the bacterial liquid to 10 ⁷ cfu/mL;

(3) Preparing a liquid culture medium: the traditional Chinese medicine extract obtained in the step (1) is prepared by the following steps: carbon source: nitrogen source: the weight ratio of the inorganic salt is 75:15:10:5, uniformly mixing, adjusting the pH value of the culture medium to 5.0-6.5, and sterilizing for later use;

(4) Fermentation: the weight ratio of the lactobacillus helveticus K6, the lactobacillus paracasei K9, the streptococcus thermophilus W46 and the bifidobacterium infantis Y61 is 1:4.5:3:1.5 inoculating the seed solution of the activated probiotic composition in the step (2), wherein the total inoculation amount is 8%, the fermentation time is 100 h at 35 ℃, and the anaerobic condition is used for culturing until the pH value of the fermentation solution is 4.0-4.5, so as to obtain a fermentation composition (liquid state);

(5) And (3) drying: maltodextrin is added into the product of the step (4), and the weight ratio of the maltodextrin to the traditional Chinese medicine probiotics fermentation composition (liquid state) is 35:65, uniformly mixing, and then performing vacuum freeze drying, wherein the temperature of the shelf at the end point is not more than 5 ℃ with the temperature of the material at minus 45 to minus 35 ℃ being kept at 2h, the temperature of the material at minus 5 to 5 ℃ being kept at 20h, the temperature of the material at 25 to 35 ℃ being kept at 15 h.

(6) Sieving: (5) The vacuum freeze-dried product is subjected to superfine grinding and is sieved by a 80-mesh sieve, so that the finished product is obtained.

Example 2

The present embodiment provides a probiotic Chinese medicinal fermented product for improving oral health, which is different from embodiment 1 in that the Chinese medicinal composition is specifically: 15 parts of moringa oleifera leaf, 20 parts of poria cocos, 20 parts of lophatherum gracile, 5 parts of dried orange peel, 30 parts of Chinese yam, 5 parts of honeysuckle and 5 parts of magnolia officinalis.

Example 3

The present embodiment provides a probiotic Chinese medicinal fermented product for improving oral health, which is different from embodiment 1 in that the Chinese medicinal composition is specifically: 20 parts of moringa oleifera leaf, 20 parts of poria cocos, 15 parts of lophatherum gracile, 10 parts of dried orange peel, 20 parts of Chinese yam, 10 parts of honeysuckle and 5 parts of magnolia officinalis.

3. Animal experiment

Selecting adult male rats with weight of 170-250 g, feeding into constant temperature and constant humidity room with temperature of 22-24deg.C and light and dark period of 12h respectively, and daily feeding conventional solid food with water. These rats were randomly divided into 5 groups of 8 rats each. Group A is a blank group, group B is a dental plaque model group, and groups C-E are dental plaque model+traditional Chinese medicine fermentation composition groups (probiotic traditional Chinese medicine fermentation examples 1, 2,3 groups, tested in preparing the fermentation composition (liquid state) of step 4). Bacteria used in the dental plaque model group are streptococcus mutans, the streptococcus mutans is cultured until the concentration of bacterial liquid is about 10 ⁸ cfu/mL, 1 mL bacterial liquid is dipped by a sterile cotton swab, and the bacterial liquid is uniformly smeared on teeth, oral mucosa and tongue heads of rats, and after the modeling is successful, the test is carried out. Wherein the group A, B wipes the teeth, oral mucosa and tongue of the rats with a sterile cotton swab dipped with 1 mL PBS buffer every day for the first week; groups C-E each day were rubbed against the teeth, oral mucosa and tongue of rats with swabs dipped in 1 mL different traditional Chinese medicinal fermentation compositions. Treatment was performed every two days for the second week of the test, during which all groups were fed normal feed and drinking water. All treatments were performed under sedative or anaesthetic conditions in rats.

(1) Plaque scoring:

after continuous use of the fermented traditional Chinese medicine composition 14 d, plaque status of rat teeth was recorded once on test day 0, 7 days and 14 days, and test teeth were stained with plaque disclosing agent and rinsed with pure water.

Plaque scoring criteria were as follows:

a. Coverage area: 0 minutes = dental plaque; 1 min = scattered punctate plaque at the gingival margin of the neck; 2 minutes = neck plaque width no more than 1 mm;3 minutes = neck plaque coverage width exceeding 1 mm, but below 1/3 of the tooth face; 4 minutes = plaque coverage area accounts for 1/3-2/3 of the tooth surface; 5 minutes = plaque coverage area is more than 2/3 of the tooth surface.

B. thickness: 0min = no color; 1 min = mild, punctiform or reddish; 2 minutes = medium, red; 3 minutes = severe, dark red.

C. Plaque score calculation: coverage area x thickness, arithmetic mean was calculated from the test dental plaque score.

As can be seen from fig. 4, group C-E day 14 plaque scores were significantly lower than the placebo group a and plaque model group B. The plaque score of group E on day 14 was significantly lower than that of group C, indicating that the probiotic fermented composition of traditional Chinese medicine used in group E (probiotic traditional Chinese medicine fermented product of example 3, probiotic composition 5 and traditional Chinese medicine combination 5) was effective in reducing plaque production.

(2) Oral volatile gas concentration detection

The concentrations of volatile gases H ₂ S and CH ₃ SH in the oral cavity of rats were measured on day 14 of the test using a halitosis detector. The specific operation method comprises the following steps: after the rat is sedated or anesthetized, the mouth of the rat is firstly opened for 3-5S, then the lips of the rat are fully closed and kept for about 30S, under the condition that the lips are not fully opened after 30S, the injector is stretched into the oral cavity from gaps of premolars of the rat, the lips are closed as much as possible, then the piston is slowly pulled to extract about 7 mL gas, the gas is left for 3-5S to fully exchange, after sampling is completed, the injector is immediately pulled out and connected with a halitosis detector, and the concentration of the extracted oral gas volatile compounds H ₂ S and CH ₃ SH is measured. And repeatedly detecting each rat for 2-5 times, and preferentially selecting 2 detection values for statistical analysis. The results are calculated as the% reduction in volatile gases. The calculation formula is as follows: volatile gas reduction amplitude/% = (group a volatile gas concentration-test group oral volatile gas concentration)/group a volatile gas concentration x 100%

As shown in FIG. 5, the group A is used as a blank control group, and the reduction amplitude of the oral volatile gas of the rats in the rest test groups is calculated and changed based on the group A, so that the group C-E can effectively reduce the generation of the oral volatile gas of the rats, reduce the bad breath and freshen the breath.

(3) Gum redness and swelling test

SPF-grade healthy female SD rats with the mass of 200-220 g, complete dentition, no caries, periodontal disease and the like are selected as test animals, wherein the test animals are about 6 weeks old. The experimental groups (periodontitis modeling+drug), the negative control group (periodontitis modeling+sterile water) and the blank control group (periodontitis modeling) are randomly divided into 10 groups. And (3) carrying out intraperitoneal injection anesthesia on each group of rats by using 1% sodium pentobarbital, wherein after about 3-5 min, the orthostatic reflection of the rats disappears, and the anesthesia takes effect.

The non-absorbable thread containing the Porphyromonas gingivalis solution was tied off to the neck of the second molar of the left maxilla of the rat, and the Porphyromonas gingivalis solution was applied to the gingival margin. The postoperative high sugar diet is fed with sucrose water of 100 g/L. After the experimental periodontitis model of the rat is established successfully, ligature wires of all model rats are removed and the model rats are dosed. The test groups were as follows:

Rats in test groups 1-4 were tested with the probiotic combination 5 and probiotic traditional Chinese medicine fermentate examples 1-3 (tested with the fermented composition (liquid) of preparation step 4), the viable count content of each group was similar, and the coupon samples were applied to the left maxillary second gingival margin; the rats of the negative control group are coated with sterile water at a dose of 20 mu L each time, 3 times per day; the model set did not do anything. All rats were sacrificed and observed after 20 d of continuous dosing.

Periodontal status of the left maxillary second molars of several groups of rats was observed and gingival sulcus bleeding index was recorded. The gingival sulcus bleeding index is as follows: 0 represents that the appearance of the gingiva is normal, and the gingival sulcus is not bleeding in detection; 1 represents that the gum has normal appearance, but the gingival sulcus is diagnosed with bleeding; 2 represents bleeding from a visit, the gum color is changed but there is no swelling; 3 represents bleeding from a visit, and the change of gum color is accompanied by slight swelling; 4 represents the bleeding with obvious swelling of gum with or without color change; 5 represents the bleeding from probing, spontaneous bleeding, marked swelling of the gum with or without ulceration.

The results are shown in FIG. 6. After the molding is successful, the oral cavity of the rat is checked, so that the problems of periodontal tissue inflammation such as bleeding by probing, gingival retraction, tooth root exposure and the like can be seen after the molding of the rat. After 20 d treatment, the gum inflammation problem was relatively light in the rats in the test group, the gum color became pink and the texture was dense, compared with the negative control and model groups, wherein the gum condition of the rats in group 4 (probiotic Chinese herbal fermentate of example 3) was relatively best, and the normal appearance state was substantially recovered. The effects of groups 2-4 are superior to group 1, indicating that the effect of the probiotic Chinese medicinal fermentate is superior to the effect of a pure probiotic combination. The negative control group rats had slightly less gum redness than the model group, but bleeding was observed. The gum red swelling of the modeling group is obvious, and the bleeding is serious in the probing.

The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.

Claims

1. The lactobacillus helveticus for improving oral health is characterized in that the lactobacillus helveticus is lactobacillus helveticus K6 with a preservation number of CGMCC No. 15702.

2. Lactobacillus helveticus for improving oral health according to claim 1, wherein said lactobacillus helveticus K6 is used for improving oral health, comprising:

inhibit the growth of Streptococcus mutans and/or Porphyromonas gingivalis,

And/or strong adhesion to gingival epithelial cells.

3. A probiotic composition for improving oral health, comprising lactobacillus helveticus K6 with a preservation number of CGMCC No.15702, and at least one of the following probiotics: lactobacillus acidophilus, lactobacillus crispatus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus gasseri, lactobacillus helveticus, bifidobacterium longum subsp longum, bifidobacterium longum subsp. Infantis, bifidobacterium breve, bifidobacterium bifidum, bifidobacterium adolescentis, bifidobacterium animalis subsp.

4. A probiotic composition for improving oral health according to claim 3, comprising lactobacillus helveticus K6 with a preservation number of CGMCC No.15702, and at least one of the following probiotics: lactobacillus acidophilus K43 with the preservation number of CGMCC No.15705, lactobacillus paracasei K9 with the preservation number of CGMCC No.15025, lactobacillus casei K35 with the preservation number of CGMCC No.15704, streptococcus thermophilus W46 with the preservation number of CGMCC No.15707, pediococcus pentosaceus LN-PT16 with the preservation number of GDMCC No.62044, bifidobacterium infantis Y61 with the preservation number of CGMCC No.15710 and bifidobacterium adolescentis Y54 with the preservation number of CGMCC No. 15709.

5. A probiotic composition for improving oral health according to claim 3, comprising lactobacillus helveticus K6 with a preservation number of CGMCC No.15702 and streptococcus thermophilus W46 with a preservation number of CGMCC No. 15707;

Or comprises Lactobacillus helveticus K6 with a preservation number of CGMCC No.15702, lactobacillus paracasei K9 with a preservation number of CGMCC No.15025, streptococcus thermophilus W46 with a preservation number of CGMCC No.15707, and bifidobacterium infantis Y61 with a preservation number of CGMCC No. 15710.

6. A probiotic composition for improving oral health according to claim 5, comprising in weight ratio 1: (0.1-8) Lactobacillus helveticus K6 and Streptococcus thermophilus W46;

Or comprises the following components in percentage by weight: (0.1-8): (0.1-8): (0.1-8) Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, bifidobacterium infantis Y61.

7. A probiotic composition for improving oral health according to claim 5, comprising in weight ratio 1:1 lactobacillus helveticus K6 and streptococcus thermophilus W46;

Or comprises the following components in percentage by weight: (0.3-4.5): (0.6-3): (0.5-4) Lactobacillus helveticus K6, lactobacillus paracasei K9, streptococcus thermophilus W46, and Bifidobacterium infantis Y61.

8. A probiotic traditional Chinese medicine ferment for improving oral health, characterized in that it is fermented from the lactobacillus helveticus K6 according to any one of claims 1-2, or from the probiotic composition according to any one of claims 3-7, said traditional Chinese medicine composition comprising: 10-30 parts of moringa oleifera leaf, 10-30 parts of poria cocos, 5-25 parts of lophatherum gracile, 1-20 parts of dried orange peel, 10-30 parts of Chinese yam, 1-30 parts of honeysuckle and 5-30 parts of magnolia officinalis.

9. A probiotic product for improving oral health, comprising lactobacillus helveticus K6 according to any of claims 1-2, or comprising a probiotic composition according to any of claims 3-7, or comprising a probiotic chinese herbal ferment according to claim 8.

10. The oral health improving probiotic product of claim 9, wherein said oral health improving comprises: preventing, alleviating and/or treating dental caries,

And/or, preventing, alleviating and/or treating periodontal disease,

And/or, preventing, alleviating and/or treating swelling and pain of gums,

And/or, improving the smell of the oral cavity,

And/or, regulating the oral cavity micro-ecological environment.