Lactobacillus casei, probiotic preparation for reducing generation of VSCs and application of probiotic preparation

Abstract

The invention discloses a lactobacillus casei KC88, and the preservation number of the lactobacillus casei KC88 is CGMCC (China General Microbiological Culture Collection Center) No.32107. Meanwhile, the invention also discloses a screening process and application of the strain. According to the lactobacillus casei KC88 provided by the invention, the expression of a Cdl gene with fusobacterium nucleatum can be down-regulated, and the expression of a Mgl gene of porphyromonas gingivalis can be down-regulated. At the same time, the lactobacillus casei KC88 can inhibit the production of pathogenic bacteria biological membranes and reduce the production of volatile sulfides. And the inactivated surface protein of the casei casei KC88 can inhibit the formation of a pathogenic bacterium biological membrane. Therefore, the lactobacillus casei KC88 viable bacteria and the thermally inactivated bacteria have the effects of relieving breath and improving gastrointestinal functions, and can be applied to the fields of medicines, health care products and the like.

Description

Technical Field

[0001] The invention belongs to the field of probiotics, and in particular relates to Lactobacillus casei, a probiotic preparation comprising the Lactobacillus casei and applications thereof. Background Art

[0002] The human oral environment is a complex micro-ecological system, with more than 700 types of bacteria in the mouth. Oral microorganisms maintain the balance of the oral microecology through interactions such as symbiosis, competition and antagonism. The "Fourth Oral Disease Epidemiological Survey Report" shows that the prevalence of oral diseases in my country is as high as 96.7%, about 30% of people suffer from halitosis to varying degrees, 65% of people can perceive halitosis themselves, and 35% of people need to be informed by others. Most of the bad breath comes from the decomposition of sulfur-containing amino acids (cystine, cysteine, methionine) or serum by bacteria in the mouth to produce metabolites such as volatile sulfides (VSCs). In addition, by forming a biofilm, it provides a suitable growth environment for other anaerobic odor-causing bacteria, enhancing the strain's resistance to stress, which in turn causes bad breath. Microorganisms that cause bad breath exist in gingival crevicular fluid, saliva, dental plaque and the back of the tongue. Among them, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia are considered to be the three main odor-causing bacteria in the oral cavity. They can degrade sulfur-containing amino acids to produce unpleasant-smelling VSCs, especially hydrogen sulfide (H2S) and methyl mercaptan (CH3SH).

[0003] At present, the methods for preventing and treating halitosis in clinical practice mainly include mechanical method, chemical method and probiotic method. Mechanical method mainly includes brushing teeth, brushing tongue, scraping tongue and using dental floss. Chemical method mainly uses antibacterial agents to prevent and treat oral diseases. Commonly used antibacterial agents are chlorhexidine, metal ions, etc. However, such antibacterial agents not only have the function of inhibiting pathogenic bacteria but also inhibit beneficial bacteria in the mouth. Long-term use may increase the drug resistance of pathogenic bacteria; most antibacterial agents easily irritate the oral mucosa, seriously affecting the consumer's use experience; and most consumers use special flavored chewing gum, mints and mouthwash to improve halitosis, which actually only obtains a momentary oral fragrance and is difficult to truly inhibit pathogenic bacteria. Probiotic method has a good therapeutic effect on oral diseases. It has the characteristics of mildness, safety and not easy to be repeatedly infected. Therefore, it is urgent to screen out probiotics that can improve halitosis, especially strains that reduce volatile sulfides (VSCs) in the mouth.

[0004] The Chinese patent application with the publication number CN 110713960 A discloses a Lactobacillus plantarum LN66 and its application in products for reducing the risk of halitosis. The LN66 strain has good tolerance to lysozyme, adapts to the oral environment, and does not kill Fusobacterium nucleatum. The mechanism of reducing halitosis is mainly that the LN66 strain can downregulate the biofilm-related genes of Fusobacterium nucleatum, especially the genes encoding outer membrane adhesion proteins, including transport proteins radD, fap2, aim1 and cmpA, the heat-modified protein gene fomA that mediates adhesion and stimulation of host immune cells, and the bacterial surface adhesion protein gene fadA. In addition, LN66 can also reduce the secretion of extracellular polymers (extracellular proteins and exopolysaccharides) of Fusobacterium nucleatum and reduce the biofilm of Fusobacterium nucleatum. However, the Lactobacillus plantarum LN66 disclosed in the above application cannot reduce the production of volatile substances (hydrogen sulfide and methyl mercaptan) produced by pathogenic bacteria, and it is not disclosed that it can reduce the amount of VSCs produced by Fusobacterium nucleatum.

[0005] The Chinese invention application with publication number CN 118726190 A discloses a Streptococcus salivarius MY-6 and its application. The Streptococcus salivarius provided by the present invention can effectively remove bad breath, remove odorous gases (ammonia and volatile sulfides), inhibit the growth of bad breath-producing bacteria Fusobacterium nucleatum and Porphyromonas gingivalis, and inhibit the growth of biofilms of odor-producing bacteria. The cell-free extract of Streptococcus salivarius MY-6 can down-regulate the expression of genes related to the outer membrane adhesion of Fusobacterium nucleatum, among which the expression of cmpA, aim and fomA genes is significantly down-regulated, and the relative expression level is reduced to about 0.2; in addition, it can also down-regulate the expression of genes related to the outer membrane adhesion and virulence of Porphyromonas gingivalis, among which the expression of fimA and hagA genes is significantly down-regulated. However, Streptococcus salivarius MY-6 cannot reduce the amount of VSCs produced by Fusobacterium nucleatum. Summary of the invention

[0006] The first object of the present invention is to provide a Lactobacillus casei KC88 that can reduce the production of VSCs by Fusobacterium nucleatum. The second object of the present invention is to provide a probiotic preparation comprising the Lactobacillus casei KC88.

[0007] The third object of the present invention is to provide a method for preparing the probiotic preparation.

[0008] The fourth object of the present invention is to provide an application of the probiotic preparation.

[0009] The present invention is achieved through the following technical solutions.

[0010] A Lactobacillus casei KC88, whose Latin name is Lacticaseibacillus casei. The bacterium was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 29, 2024. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101; the collection number is CGMCC No. 32107. The bacterium was alive when it was deposited.

[0011] A fermentation product is obtained by fermenting the Lactobacillus casei KC88.

[0012] A probiotic preparation comprising the Lactobacillus casei KC88 according to claim 1 and the fermentation product according to claim 2.

[0013] A method for preparing the probiotic preparation comprises the following steps:

[0014] Inoculating the Lactobacillus casei KC88 into the culture medium and fermenting to obtain probiotic bacterial slurry;

[0015] The probiotic mud is mixed with a freeze-drying protective agent and then freeze-dried to obtain the product.

[0016] The culture medium comprises the following components by weight percentage: 1.0-1.5% soy peptone, 0.5-1.5% yeast extract powder, 2.0-4.0% glucose, 0.05-0.10% Tween, 0.1-0.2% dipotassium hydrogen phosphate heptahydrate, 0.4-0.6% sodium acetate trihydrate, 0.02-0.04% magnesium sulfate heptahydrate, 0.02-0.04% manganese sulfate monohydrate, 0.05-0.15% L-cysteine, and the balance water.

[0017] The freeze-drying protective agent comprises, by weight percentage, 10-25% trehalose, 1-3% sodium thiosulfate, 1.0-1.5% maltodextrin, 1-2% sorbitol, 5-10% whey protein powder and the balance water.

[0018] The pH value of the fermentation is 5.9, and the fermentation time is 12 to 18 hours;

[0019] The pH of the lyophilization protective agent is 6.0-6.5.

[0020] An application of the probiotic preparation is characterized in that it is used to prepare a drug for down-regulating the expression of the Cdl gene of Fusobacterium nucleatum. An application of the probiotic preparation is characterized in that it is used to prepare a drug for down-regulating the expression of the Mgl gene of Porphyromonas gingivalis.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a strain of Lactobacillus casei KC88, which can significantly reduce the expression of genes (Cdl genes) related to the production of hydrogen sulfide by Fusobacterium nucleatum, and the relative expression amount is about 0.42, which can inhibit the generation of Fusobacterium nucleatum biofilm. Moreover, the Lactobacillus casei KC88 (live bacteria and heat-killed bacteria) can significantly reduce the expression of genes related to the production of methyl mercaptan by Porphyromonas gingivalis, mainly by reducing the expression of the Mgl gene, and the relative expression amount is about 0.35, which can inhibit the production of pathogenic bacteria biofilm, thereby reducing the production of volatile sulfide. Heat-killed bacteria mainly have a very significant inhibition of pathogenic bacteria biofilm due to their surface proteins, thereby inhibiting Porphyromonas gingivalis from producing volatile substances. Therefore, it can achieve the function of improving bad breath. Both live bacteria and heat-killed bacteria can significantly reduce the plaque index of mice with halitosis. The plaque index returned to the normal control level, with an index of 1. There was a thin plaque near the gingival margin, which was invisible to the naked eye and could be scraped out with the side of the probe tip. The halitosis index was also restored to normal levels, with a decrease of 46.10%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A schematic diagram showing the adhesion ability of dominant bacteria and competing bacteria to hydroxyapatite;

[0024] Figure 2 A schematic diagram of the self-aggregation ability of different strains is shown;

[0025] Figure 3 Schematic diagram of the growth curves of different strains is shown. DETAILED DESCRIPTION

[0026] The Lactobacillus casei KC88 provided by the present invention is screened from the saliva of healthy women without bad breath problems, because most adults are susceptible to bad breath, and the saliva of healthy women without bad breath contains probiotics that inhibit VSCs, so it is screened from the saliva of healthy women without bad breath. Specifically, the screening method of the Lactobacillus casei KC88 is as follows:

[0027] Initial screening: Six strains of primary screening bacteria were selected through initial screening of saliva samples, infant feces, breast milk and other samples (Fusobacterium nucleatum inhibition zone ability, acid production, Fusobacterium nucleatum biofilm inhibition rate), which have stronger antibacterial and acid production properties than the positive control strains;

[0028] Rescreening: The strains obtained in the initial screening were rescreened (adhesion ability to hydroxyapatite, self-aggregation ability), so as to screen out the rescreened strains, among which Lactobacillus casei KC88 (TY1-18) had an adhesion ability to hydroxyapatite of up to 91.01%;

[0029] Final screening: Lysozyme tolerance, gastrointestinal fluid tolerance, growth curve, Porphyromonas gingivalis inhibition zone and pathogenic bacteria biofilm inhibition rate were evaluated. Combined with oral-gastric-intestinal triple tolerance screening, it was found that Lactobacillus casei KC88 could tolerate 1 mg / mL lysozyme, with a gastric fluid tolerance survival rate of up to 98.13% and an intestinal fluid tolerance survival rate of up to 85.86%. For the growth curve, Lactobacillus casei KC88 (TY1-18) can enter the logarithmic phase the earliest and the OD600 value in the stable phase is close to 2.5.

[0030] Specifically, the present invention is further described in conjunction with the following examples.

[0031] Example 1

[0032] 1. Evaluation of probiotic functional characteristics of primary screening strains

[0033] Inhibition zone: Take 200μL of the bacterial solution of Fusobacterium nucleatum ATCC 25586 (from Shanghai Collection of Microorganisms Center), spread it evenly on the blood agar plate, and leave it for 30 minutes. After the bacterial solution on the surface is fixed, use an Oxford cup to punch holes on the surface of the plate, take 200μL of lactic acid bacteria solution (OD600 is about 1.0) and inject it into the hole. Use physiological saline as a control, and culture at 37℃ for 24 hours, observe the results, and measure the diameter of the inhibition zone.

[0034] Evaluation of acid production capacity: Two dominant strains were inoculated into MRS liquid medium and cultured at 37°C for 24 h. After activation for three generations, the fermentation supernatant of each strain was collected and the pH of each fermentation supernatant was tested.

[0035] Pathogenic bacteria biofilm inhibition rate: adjust the 24h culture of pathogenic bacteria to 10 8 CFU / mL, add 100μL of pathogenic bacteria suspension to each well of a 96-well plate. At the same time, add the supernatant of all lactic acid bacteria to the 96-well plate, 100μL per well, and use MRS as a blank control. Incubate the 96-well plate at 37°C for 24h. After 24h biofilm formation, wash 3 times with PBS to carefully remove planktonic cells, and add 100μL of methanol to each well for fixation for 30 minutes. After discarding the methanol, the biofilm was stained with 0.1% crystal violet for 5 minutes. Discard the crystal violet, wash 3 times with PBS, add 200μL of 36% glacial acetic acid to each well, and measure the absorbance of each well at 590nm using an enzyme reader.

[0036] Copolymerization ability: Adjust the cultured lactic acid bacteria and Fusobacterium nucleatum to OD600 = 0.5 ± 0.05. Take equal amounts of lactic acid bacteria and Streptococcus mutans suspension, mix them evenly, and incubate them at 37°C for 24 hours. Measure the absorbance of the supernatant at 600nm. Calculate the copolymerization rate according to the following formula

[0037]

[0038] Where: Ax, initial OD value of lactic acid bacteria; Ay, initial OD value of Streptococcus mutans; Am, OD value of the supernatant after 24 h of incubation.

[0039] Dental plaque biofilm is a prerequisite for the occurrence of halitosis. Only when pathogenic bacteria aggregate and adhere to form biofilm and produce a large amount of acid, dental plaque begins to produce and produce volatile substances, eventually causing halitosis. Therefore, inhibiting the formation of biofilm is crucial for the prevention and treatment of dental caries. Therefore, the present invention evaluates the ability of the primary screening bacteria to inhibit the biofilm of Fusobacterium nucleatum. Specifically, the present invention determines the inhibitory effect of 19 strains of lactic acid bacteria on the biofilm of Streptococcus mutans by crystal violet staining. The specific results are shown in Table 1.

[0040] Table 1 Evaluation of the probiotic function of the predominant bacteria in the initial screening

[0041]

[0042]

[0043] After probiotics enter the oral cavity, they can copolymerize with planktonic pathogenic bacteria, and the copolymers formed are easily discharged from the oral cavity with the flow of saliva, thereby achieving the purpose of inhibiting pathogens in the oral cavity. Therefore, the copolymerization ability of probiotics and pathogens also needs to be evaluated. Based on the comprehensive evaluation of indicators such as the diameter of the inhibition zone of Fusobacterium nucleatum, the biofilm inhibition ability of Fusobacterium nucleatum, the acid production ability, and the copolymerization ability, the following six strains were selected as the subsequent screening link: TY1-18 (Lactobacillus paracasei), TY4-8 (plant), TY21-1 (plant), 24-32m4 (plant), 33m1 (plant), 31mc4 (plant), among which Streptococcus salivarius K12 was the positive control strain.

[0044] Example 2

[0045] Evaluation of adhesion and growth ability of rescreened strains

[0046] Adhesion ability to saliva-coated hydroxyapatite

[0047] The saliva-coated hydroxyapatite model (SHA) was established. 100 μL of lactic acid bacteria suspension (mixed with physiological saline after centrifugation) (OD600 = 0.5 ± 0.05) was mixed with 10 μL of acridine orange fluorescent dye (0.01% dye) and incubated at 37°C in the dark for 30 minutes. 200 μL of the labeled bacterial suspension was added to a 96-well plate pre-placed with 1 mg of SHA, and anaerobically cultured at 37°C for 30 minutes. The supernatant was removed, and the plate was resuspended after washing with PBS once. The fluorescence value was measured by a fluorescence microplate reader, which was the fluorescence value of the adhered bacteria. The negative control group was the well with only hydroxyapatite and PBS buffer, and the well with only the labeled bacterial solution was the total fluorescence value.

[0048] The adhesion ability of lactic acid bacteria to SHA was calculated according to the following formula.

[0049]

[0050] Hydroxyapatite is the main component of tooth hard tissue. The acquired film formed by salivary glycoprotein on the tooth surface is the main adhesion site for pathogenic microorganisms. Therefore, saliva-coated hydroxyapatite (SHA) is often used as an in vitro model to simulate the oral environment. Therefore, the adhesion ability of the initial screening strains to hydroxyapatite was compared. The adhesion ability was from large to small, TY21-1>33m1>TY1-18>31mc4, and their adhesion ability was better than that of the competing bacteria.

[0051] Self-aggregation ability:

[0052] Use sterile PBS (pH 7.0) to adjust the OD600 of lactic acid bacteria to 0.5 ± 0.05, take 4 mL of bacterial suspension, vortex for 10 seconds, and let it stand at room temperature. Measure the change of absorbance of the supernatant at different time intervals (2h, 4h, 8h, 18h, 24h). Calculate the self-aggregation rate (%) according to the following formula.

[0053]

[0054] Where: At, absorbance at time t; A0, absorbance at time t = 0

[0055] The self-aggregation ability of lactic acid bacteria is positively correlated with its ability to adhere to epithelial cells. In order to ensure the colonization of lactic acid bacteria in the oral environment to further alleviate bad breath, it is necessary to screen lactic acid bacteria strains with strong self-aggregation ability. In addition, because the later product is designed as a lozenge, it is necessary to find strains with strong early self-aggregation ability. The strains with better early self-aggregation rate (0-8h) are: TY21-1, TY1-18, TY4-8, 24-32M4.

[0056] Growth curve:

[0057] The growth kinetics function module of the microplate reader was used to inoculate the dominant strain into a 24-well plate, 2 mL per well, with an inoculation volume of about 2%, the temperature set at 37°C, and the time set at 24 h.

[0058] Depend on Figure 3 It can be seen that the strains that entered the logarithmic phase earliest and had higher OD values ​​in the stable phase were TY1-18, TY4-8, TY21-1, and 33m1. Gastrointestinal fluid tolerance:

[0059] Artificial gastric juice tolerance test: The treated bacterial liquid was inoculated into artificial gastric juice with pH 3.0, and cultured in a 37°C water bath for 3 hours. The number of live bacteria was determined and the survival rate was calculated.

[0060] Test of tolerance to artificial intestinal fluid: After treating bacteria with high survival rate in artificial gastric fluid for 3 hours, they were inoculated into artificial intestinal fluid (containing 0.3% bile salt) and cultured in a 37°C water bath for 3 hours. After that, the number of live bacteria was determined and the survival rate was calculated.

[0061] Lysozyme tolerance: The concentration of lysozyme in the human oral cavity (1-57 μg / mL). Add 200 μL of 0, 1, 3, 5, and 10 mg / ml lysozyme solution into the wells of an Oxford cup. Spread 100 μL of probiotic fermentation liquid on the plate and determine the diameter of the inhibition zone using the Oxford cup inhibition zone method.

[0062] Table 2 Effect of different strains on lysozyme tolerance

[0063]

[0064] Lysozyme in saliva has a strong dissolving effect on the acetylamino polysaccharide components on the wall of Gram-positive cocci, thus causing bacterial death. As shown in Table 2, all six lactic acid bacteria can tolerate lysozyme at a concentration of 1 mg / mL, which is much higher than the concentration of lysozyme in the human mouth (1-57 μg / mL); for 10 mg / mL lysozyme, the smaller the inhibition zone, the stronger the strain tolerance, and it can be seen that the lysozyme tolerance is: TY21-1>31mc4>33m1>TY1-18.

[0065] Table 3 Tolerance of dominant bacteria in gastrointestinal fluid

[0066]

[0067] The gastrointestinal fluid tolerance of the above four strains was tested, and the two strains with the strongest gastrointestinal fluid tolerance were TY1-18 and TY21-1. The inhibition zone of Porphyromonas gingivalis and its biofilm inhibition rate:

[0068] Subsequently, the antibacterial ability of another pathogenic bacterium causing bad breath was evaluated. The results showed that TY1-18 had a strong antibacterial ability against Porphyromonas gingivalis, with an inhibition zone of 14.32 mm and a biofilm inhibition rate of up to 92.39%.

[0069] Table 4 Antibacterial ability of dominant bacteria against Porphyromonas gingivalis

[0070]

[0071] Example 3

[0072] 3.16S rDNA identification

[0073] Using the bacterial genomic DNA extraction kit, the extracted genomic DNA of the strain was used as a template for PCR amplification, and the lactic acid bacteria primers were universal primers:

[0074] 27F (SEQ ID No. 1): AGAGTTTGATCCTGGCTCAG;

[0075] 1492R (SEQ ID No. 2): GGTTACCTTGTTACGACTT;

[0076] After electrophoresis detection of the amplified product, it was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing peak graph was integrated to cut off the 800bp region of the sequencing result; the result was obtained by comparing it with the GenBank gene library through the BLAST program. The strain source and identification results are shown in Table 4.

[0077] Table 4 Strain sources and identification results

[0078]

[0079] The 16S DNA sequence of Lactobacillus casei KC88 is shown below:

[0080] ACTATGGGTAACATACTAGTACTGTTAGTCGAACGAGTTCTCGTTGATGATCGGTGCTT

[0081] GCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCT

[0082] GCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAA

[0083] CCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGG

[0084] CGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTG

[0085] AGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCA

[0086] GCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTG

[0087] AAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAAC

[0088] TGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGC

[0089] CGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGC

[0090] AGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGA

[0091] AACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAAT

[0092] GCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGAC

[0093] GCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCC

[0094] GTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACG

[0095] CATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACG

[0096] GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTT

[0097] ACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAA

[0098] ATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC

[0099] GCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGA

[0100] CTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTAT

[0101] GACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGG

[0102] TCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACAC

[0103] GAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGG

[0104] CCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCG

[0105] TAACCCTATAGGAACGAACTGTCTCATAGAGTACTACTTG

[0106] Example 4 Probiotic Preparation Method

[0107] A method for preparing the probiotic preparation comprises the following steps:

[0108] The culture medium comprises the following components by weight percentage: 1.2% soy peptone, 1.05% yeast extract powder, 2.0% glucose, 0.05% Tween, 0.1% dipotassium hydrogen phosphate heptahydrate, 0.4% sodium acetate trihydrate, 0.02% magnesium sulfate heptahydrate, 0.024% manganese sulfate monohydrate, 0.0515% L-cysteine, and the balance distilled water. During the fermentation process, the constant pH is 5.9, the fermentation time is 16 hours, and the probiotic mud is obtained after centrifugation.

[0109] The freeze-drying protective agent comprises 15% trehalose, 1.2% sodium thiosulfate, 1.2% maltodextrin, 1.0% sorbitol and 5% whey protein powder, and the pH is adjusted to 6.0 before freeze-drying.

[0110] The probiotic bacterial slurry is evenly mixed with the freeze-drying protective agent, and freeze-dried to obtain probiotic freeze-dried powder.

[0111] Example 5 Study on the mechanism of Lactobacillus casei KC88 inhibiting the production of volatile substances by Porphyromonas gingivalis 1. Preparation of live bacteria, heat-killed bacteria and cell-free supernatant of Lactobacillus casei KC88 Lactobacillus casei KC88 Lactobacillus casei KC88 was cultured overnight, centrifuged at 10,000×g for 5 minutes, washed three times with PBS, and then the bacterial solution was resuspended to an OD600 of about 0.8, which was live bacteria; according to the above procedure, the bacterial suspension with an OD600 of about 0.8 was inactivated in a 70°C water bath for 1 h to obtain heat-killed bacteria; according to the above method, the bacteria were centrifuged and the resuspended liquid was stirred at 100 r / min for more than 2 h, then centrifuged at 10,000×g for 5 minutes, and filtered with a 0.22 μm sterile filter membrane to obtain a cell-free supernatant, and the live bacteria, heat-killed cells and cell-free supernatant were stored at 4°C.

[0112] 2. Pathogenic bacteria biofilm inhibition rate: The above samples were used to evaluate the inhibition rate of Porphyromonas gingivalis biofilm.

[0113] 3. Lactobacillus casei KC88 can inhibit the expression of gas-producing genes of Porphyromonas gingivalis

[0114] Methods: The control group (PBS), live bacteria, heat-killed bacteria, and cell-free supernatant were co-cultured with Porphyromonas gingivalis, and the expression of key gas-producing genes was investigated by fluorescence quantitative PCR. The RNA of the odor-causing bacteria was extracted and reversely transcribed into cDNA, and the synthesized cDNA template was stored in -80. Fluorescence quantitative PCR was performed according to the primers obtained in Table 5, and the 16S rRNA gene was used as the internal reference gene. SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) was used for amplification, and 2 -ΔΔCT The target gene was relative quantified and the cycle threshold (Ct) was determined.

[0115] Table 5 Fluorescence quantitative PCR primer information

[0116]

[0117]

[0118] The odor-causing bacteria in the oral cavity can degrade sulfur-containing amino acids to produce unpleasant VSCs, especially hydrogen sulfide (H2S) and methyl mercaptan (CH3SH). Among them, L-cysteine ​​desulfurization hydrolase acts on L-cysteine ​​to produce pyruvate, ammonia and hydrogen sulfide, while L-methionine α-deamino-γ-mercaptomethane lyase (METase) produces α-ketobutyrate, ammonia and methyl mercaptan from L-methionine. Porphyromonas gingivalis produces a large amount of methyl mercaptan through METase, and this process is encoded by the Mgl gene. Studies have found that a mutant strain of Porphyromonas gingivalis with a defect in the Mgl gene produces significantly less methyl mercaptan than the wild type. mgl is not only responsible for the production of methyl mercaptan, but also related to the pathogenicity of pathogenic bacteria. In addition, Fusobacterium nucleatum produces a large amount of hydrogen sulfide through L-cysteine ​​desulfurization hydrolase, and this process is encoded by the Cdl gene.

[0119] Therefore, it is necessary to evaluate the influence of Lactobacillus casei KC88 on the expression of Mgl and Cdl genes. As shown in Table 6, the inhibition rate of pathogenic bacteria biofilm by live bacteria of Lactobacillus casei KC88 is higher than that of the heat-inactivated group, but there is no significant difference. The heat-inactivated cell group can significantly reduce the expression of the Mgl gene, and the relative expression amount is about 0.35. It can be seen from this that the heat-inactivated thalline of Lactobacillus casei KC88 has a stronger inhibitory effect, can significantly reduce the expression of Mgl and Cdl genes and inhibit the generation of pathogenic bacteria biofilm, thereby inhibiting the production of volatile substances by Porphyromonas gingivalis.

[0120] Table 6 Effects of different groups on pathogenic bacteria biofilm inhibition rate and MGL gene expression

[0121]

[0122] Example 6

[0123] Inactivated bacteria contain a variety of components, including teichoic acid, surface protein, peptidoglycan, capsular polysaccharide, etc. In order to find out which component inhibits the adhesion of pathogenic bacteria and their gas production, the following experimental verification is carried out.

[0124] 1. Effect of sodium periodate treatment on the inhibition of pathogenic bacterial biofilm

[0125] The overnight cultured lactic acid bacteria were washed three times with PBS, resuspended in 50 mM sodium periodate solution, incubated at 37 °C for 30 min, centrifuged at 5000 x g for 10 min, collected, washed five times with PBS, and the concentration of the bacterial solution was adjusted to 1.0 × 109 CFU / mL. After heat inactivation, the biofilm inhibition experiment was performed with the cultured pathogenic bacteria (Porphyromonas gingivalis), and the untreated strain was used as a control.

[0126] 2. Study on the inhibitory effect of lipoteichoic acid on pathogenic bacterial biofilm by bovine serum albumin treatment

[0127] The lactic acid bacteria cultured overnight were washed three times with PBS, resuspended in 2% bovine serum albumin, incubated at 37°C for 30 min, centrifuged at 5000xg for 10 min, collected, washed five times with PBS, and the concentration of the bacterial solution was adjusted to 1.0×109 CFU / mL. After heat inactivation, the biofilm inhibition experiment was performed with the cultured pathogenic bacteria (Porphyromonas gingivalis), and the untreated strain was used as a control.

[0128] 3. Lithium chloride treatment to study the inhibitory effect of surface proteins on pathogenic bacterial biofilm

[0129] The overnight cultured lactic acid bacteria were washed three times with PBS, resuspended in 5M LiCl, incubated at 37°C for 30 min, centrifuged at 5000 x g for 10 min, collected, and washed five times with PBS to adjust the bacterial solution concentration to 1.0 × 10 9 CFU / mL, after heat inactivation, the biofilm inhibition experiment was performed with cultured pathogenic bacteria (Porphyromonas gingivalis), and the untreated strain was used as a control.

[0130] Table 7 Inhibitory effects of heat-killed bacteria on pathogenic bacteria by different treatment methods

[0131]

[0132] Sodium periodate can cleave the CC bonds between sugar hydroxyl groups, so the polysaccharides on the surface of bacteria can be effectively removed by sodium periodate. After using sodium periodate to remove the extracellular polysaccharides on the surface of KC88, the inhibitory ability of pathogenic bacteria biofilm decreased, but did not reach a very significant level (P>0.01), indicating that extracellular polysaccharides are not the main factor.

[0133] Because bovine serum albumin and lipoteichoic acid have similar cell membrane receptors, bovine serum albumin can significantly inhibit the biomembrane inhibition of lipoteichoic acid. The results showed that there was no significant difference between the KC88 group and the blank group (P>0.01), indicating that lipoteichoic acid is not an important factor.

[0134] Lithium chloride was used to remove the surface protein of KC88 bacteria to verify whether the strain's inhibitory effect on pathogenic bacteria biofilm was due to the influence of its surface protein. Lithium chloride treatment could significantly reduce the inhibition rate of pathogenic bacteria biofilm, indicating that the surface protein of the strain was involved in the inhibition process.

[0135] In summary, KC88 heat-killed bacteria can significantly inhibit the biofilm of pathogenic bacteria due to its surface proteins, thereby inhibiting the production of volatile substances by Porphyromonas gingivalis.

[0136] Example 7 Animal Experiment Verification

[0137] Preparation of pathogenic bacteria solution

[0138] Fusobacterium nucleatum was inoculated into nutrient broth medium and cultured at 37°C under anaerobic conditions for 2-5 days. A single colony on the plate was picked and 5 mL of bacterial suspension was prepared with sterile PBS (50 mmol / L, pH 7.4) and adjusted to a McFarland turbidimetric concentration of 0.5 (i.e., 1 x 10 7 CFU / mL), then carboxymethyl cellulose was added and filtered through a filter membrane with a pore size of 0.22 μm to prepare a fresh pathogenic bacteria solution containing 2% carboxymethyl cellulose.

[0139] Preparation of lactic acid bacteria liquid:

[0140] The bacterial agent prepared according to Example 4 was inactivated at 110°C for 15 min, and then the OD600 of the bacterial solution was adjusted to about 0.8 using sterile PBS to obtain a lactic acid bacteria solution.

[0141] Establishment of periodontitis and halitosis model in rats

[0142] Select SPF Wistar male rats, weighing 180-220g, healthy, active, shiny hair, good appetite, no caries, and no periodontal disease. After intraperitoneal injection of 3% sodium pentobarbital 1mg / kg, select the mandibular first molar and tie it with medical silk thread at the neck of the tooth. Put the ligature silk thread into the gingival sulcus as much as possible. If the ligature thread falls off, it needs to be re-ligated. After ligation, feed with 10% sugar water instead of drinking water, select pathogenic bacteria liquid (1×10 7 CFU / mL), and 0.1 ml of the prepared pathogenic bacteria solution was applied to the mandibular first molar periodontally every other day. After 28 days, the rat plaque index (PI) and halitosis concentration were detected. Two rats were taken, the periodontal tissues were fixed, and pathological sections were made to observe the pathological changes of periodontal tissues. The results showed that the PI value of the animals was greater than 3, and there was a large amount of inflammatory cell infiltration in the periodontal fibers, disordered arrangement of collagen fiber bundles, and alveolar bone absorption, which was judged to be a successful model.

[0143] Animal grouping and drug administration

[0144] Male rats were randomly divided into normal control group (normal rats), model group (model rats), experimental group A (live bacteria with OD of about 0.8), and experimental group B (inactivated bacteria with OD of about 0.8), with 10 rats in each group. After successful modeling, the normal control group and the model group were given 0.5% sodium carboxymethyl cellulose by gavage, and the experimental group A was smeared with lactic acid bacteria liquid in the periodontal tissue; the experimental group B was smeared with inactivated lactic acid bacteria liquid in the periodontal tissue, smeared once a day at 1ml / 100g body weight for 14 consecutive days. The spirit, activity, hair, diet, periodontal changes, etc. of the rats were observed every day.

[0145] Evaluation indicators:

[0146] A. Plaque index (PI): Check the mesial buccal, buccal, distal buccal and lingual surfaces of each tooth and score them separately. The sum of the four scores divided by 4 is the score of the tooth. 0 means no plaque near the gingival margin; 1 means thin plaque near the gingival margin, which is invisible to the naked eye and can be scraped out with the side of the probe tip; 2 means moderate plaque at the gingival margin or adjacent surface; 3 means a large amount of soft scale in the gingival sulcus or at the gingival margin and adjacent surface.

[0147] B. Halitosis index (HI): The breath analyzer Halimeter PLUS was used to measure the breath index (ppm). Table 7 Comparison of halitosis index of rats in different groups after the plaque index was completed

[0148]

[0149] Both live Lactobacillus casei KC88 and heat-killed bacteria can significantly reduce the plaque index of mice with bad breath, and the plaque index returned to the normal control level, with an index of 1. There was a thin plaque near the gingival margin, which was invisible to the naked eye and could be scraped out with the side of the probe tip. The bad breath index could also be restored to normal levels, returning to the normal control level, with a decrease of 46.10%.

Claims

1. A Lactobacillus casei KC88, characterized in that: That The deposit number is CGMCC No.32107.

2. A fermentation product, characterized in that: The method is obtained by fermenting the Lactobacillus casei KC88 according to claim 1.

3. A probiotic preparation, characterized in that: The invention comprises the Lactobacillus casei KC88 as claimed in claim 1 and the fermentation product as claimed in claim 2.

4. A method for preparing the probiotic preparation as claimed in claim 3, characterized in that: The steps include: Inoculating the Lactobacillus casei KC88 into the culture medium and fermenting to obtain probiotic bacterial slurry; The probiotic mud is mixed with a freeze-drying protective agent and then freeze-dried to obtain the product.

5. The method for preparing the probiotic preparation according to claim 4, characterized in that: The culture medium comprises the following components by weight percentage: 1.0-1.5% soy peptone, 0.5-1.5% yeast extract powder, 2.0-4.0% glucose, 0.05-0.10% Tween, 0.1-0.2% dipotassium hydrogen phosphate heptahydrate, 0.4-0.6% sodium acetate trihydrate, 0.02-0.04% magnesium sulfate heptahydrate, 0.02-0.04% manganese sulfate monohydrate, 0.05-0.15% L-cysteine, and the balance water.

6. The method for preparing the probiotic preparation according to claim 4, characterized in that: The freeze-drying protective agent comprises, by weight percentage, 10-25% trehalose, 1-3% sodium thiosulfate, 1.0-1.5% maltodextrin, 1-2% sorbitol, 5-10% whey protein powder and the balance water.

7. The method for preparing the probiotic preparation according to claim 4, characterized in that: The pH value of the fermentation is 5.9, and the fermentation time is 12 to 18 hours; The pH of the lyophilization protective agent is 6.0-6.

5.

8. An application of the probiotic preparation as claimed in claim 3, characterized in that: The method is used for preparing drugs for down-regulating the expression of Cdl gene of Fusobacterium nucleatum.

9. An application of the probiotic preparation as claimed in claim 3, characterized in that: It is used to prepare drugs for down-regulating the expression of the Mgl gene of Porphyromonas gingivalis.

Images