Lactobacillus plantarum and its fermentation products, probiotic preparations for preventing dental caries

Abstract

The present invention belongs to the field of microbial technology, and the present invention provides a plant lactobacillus (Lactiplantibacillus plantarum) PPL19, and its deposit number is CGMCCNO.32110. The plant lactobacillus provided by the present invention can ferment orange peel, and obtain a quorum sensing quencher of pathogenic bacteria. After the plant lactobacillus ferments orange peel of the present invention, the quorum sensing quencher obtained after fermentation and extraction interferes with the mutans streptococcus ComCDE system, down-regulates the expression of ComD and ComE genes, and can down-regulate the relative expression of LuxS, a key gene of the interspecies AI-2 signal transduction system, to inhibit the formation of mutans streptococcus biofilm, thereby preventing the occurrence and development risks of dental caries. The present invention also provides a method for preparing a probiotic preparation and a probiotic oral tablet candy. The preparation method of the probiotic preparation is simple and can be mass-produced.

Description

Technical Field

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

[0002] Dental caries is a common disease that endangers oral health and may cause unnecessary health complications, seriously affecting people's physical and mental health and quality of life. Dental caries is mainly caused by a variety of factors such as specific oral microorganisms, host factors, diet, time, etc. In the process of dental caries development, dental plaque biofilm is one of the key pathogenic factors.

[0003] The main pathogenic bacteria that cause dental caries are Streptococcus mutans, which has acid resistance, acid production and the ability to form glucan, thereby forming a stable biofilm. In addition, the biofilm of Streptococcus mutans continuously creates a low pH environment on the tooth surface, causing tooth demineralization. If sucrose is ingested in the diet, it will significantly increase the adhesion and aggregation of Streptococcus mutans and accelerate the formation of cariogenic biofilms. Streptococcus mutans promotes the formation of biofilms by releasing glucosyltransferases and using sucrose to synthesize glucans. Compared with plankton, pathogenic bacterial biofilms can tolerate broad spectrum antibacterial agents and extreme external environments. Among them, chlorhexidine (broad spectrum antibacterial drug) can control cariogenic biofilms in the mouth, but it will have adverse side effects on patients, such as tooth discoloration and affecting the patient's taste, etc. It is not suitable for long-term use. Traditional single antibiotic therapy is no longer suitable for the urgent needs of the market. Therefore, it is necessary to explore emerging control strategies to inhibit the formation of Streptococcus mutans biofilms and explore their potential mechanisms.

[0004] Quorum sensing (QS) is a way for bacteria to communicate chemically. Bacteria synthesize and output autoinducers (AI) signal molecules. At a specific threshold or "group" concentration of extracellular AI molecules, bacterial biofilms receive signals, resulting in changes in related gene expression, leading to biofilm formation, and promoting the expression of a large number of virulence factors. This change in gene expression and biofilm growth characteristics caused by the accumulation of AI is called quorum sensing. In recent years, scientists have explored a new method to treat biofilm-mediated diseases in response to the quorum sensing phenomenon of dental caries biofilms. A large number of studies have shown that plant extracts and probiotics are a safe and long-lasting method that can stimulate quorum sensing quenching and effectively interrupt the bacterial communication network that causes virulence. Relevant studies have screened, so it is urgent to explore a model of combining probiotics and medicine and food to prevent the formation of Streptococcus mutans biofilms and assist in preventing the occurrence of dental caries.

[0005] The Chinese invention patent with the publication number of CN109182218A discloses a strain of Lactobacillus salivarius and its application in down-regulating the gene expression of biofilm formation of Streptococcus mutans. The Lactobacillus salivarius ZM06 of the present invention has good tolerance to lysozyme and adapts to the oral environment. It is not intended to kill Streptococcus mutans, but to reduce the expression level of genes related to biofilm formation in Streptococcus mutans. The genes directly related to biofilm formation of Streptococcus mutans include: glucosyltransferase gene gtfB, glucosyltransferase gene gtfC, fructosyltransferase gene ftf, gene gbpB encoding glucan binding protein and gene gbpC encoding glucan binding protein, specifically inhibiting the formation of biofilm of Streptococcus mutans to reduce the risk of occurrence and development of dental caries. The signal pathway involved in regulating the biofilm formation of Streptococcus mutans includes: a two-component signal transduction system TCS and an interspecies signal transduction system mediated by AI-2 signal molecules. The genes in the two-component signal transduction system TCS include: VicR / VicK genes in TCS-1, CiaR / CiaH genes in TCS-2, HK11 / RR11 genes in TCS-11, and ComD / ComE genes in TCS-13. The gene in the interspecies signal transduction system mediated by the AI-2 signal molecule is the luxS gene. However, the above application did not explore the key components of the quorum sensing system regulating pathogens. Summary of the invention

[0006] The first object of the present invention is to provide a strain of Lactobacillus plantarum PPL19 that can prevent dental caries.

[0007] The second object of the present invention is to provide a fermentation product of Lactobacillus plantarum PPL19 that can prevent dental caries.

[0008] The third object of the present invention is to provide a probiotic preparation comprising the Lactobacillus plantarum PPL19.

[0009] The fourth object of the present invention is to provide a method for preparing a probiotic preparation comprising the Lactobacillus plantarum PPL19. The fifth object of the present invention is to provide an application of the Lactobacillus plantarum PPL19.

[0010] The present invention is achieved through the following technical solutions:

[0011] The present invention provides a strain of Lactobacillus plantarum PPL19, whose Latin name is Lactiplantibacillus plantarum, which was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee on September 29, 2024, with a deposit unit code of CGMCC, and an address of the deposit center: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Postal Code 100101, and a deposit number of CGMCC No. 32110. During the deposit, the bacteria are alive.

[0012] A fermentation product,

[0013] The method is obtained by fermenting orange peel with the plant lactobacillus PPL19.

[0014] Includes quorum sensing quenchers;

[0015] The structure of the quorum sensing quencher is as follows:

[0016]

[0017] A probiotic preparation comprising the Lactobacillus plantarum PPL19 according to claim 1, the fermentation product according to claim 2 and a lyophilization protectant.

[0018] The freeze-drying protective agent comprises, by weight percentage, 10-30% trehalose, 10-20% skim milk powder, 2-10% sucrose, 1-5% maltodextrin, 2-5% modified starch, 2-5% glycerol, 0.1-1% inulin, 0.5-1% arginine, 0.5-1% glutamine, and the balance is water.

[0019] The preparation method of the probiotic preparation comprises the following steps:

[0020] The probiotic bacterial slurry is evenly mixed with the freeze-drying protective agent, and freeze-dried to obtain probiotic freeze-dried powder.

[0021] The preparation method of the probiotic preparation comprises the steps of streaking the probiotics and then picking out single bacteria into a liquid MRS culture medium, culturing at 37° C., performing two rounds of activation, and centrifuging to obtain probiotic sludge.

[0022] The culture medium comprises the following components by weight percentage: 1.0-1.5% yeast peptone, 0.5-1.5% yeast extract powder, 2.0-6.0% glucose, 0.05-0.15% Tween, 0.1-0.2% dipotassium hydrogen phosphate heptahydrate, 0.4-0.6% sodium acetate trihydrate, 0.02-0.06% magnesium sulfate heptahydrate, 0.02-0.03% manganese sulfate monohydrate, 0.10-0.20% L-cysteine, 0.10-0.20% calcium chloride, 0.3-0.5% orange peel extract, and the balance distilled water.

[0023] The preparation method of the probiotic preparation further comprises the following steps:

[0024] Dissolve 1g of orange peel freeze-dried powder in 9mL PBS, take 1mL of orange peel solution and place it in 9mL of MRS liquid culture medium, inoculate 3% of Lactobacillus plantarum PPL19, and ferment at 37°C for 20-26h;

[0025] After the fermentation, 5 mL of 90% ethanol was added, and the mixture was heated to 45°C in a water bath. The ethanol-soaked clear liquid was collected and loaded onto a HP750 macroporous adsorption resin column at a rate of 4.0 BV / h for adsorption. Then, 5% ethanol solution was used to remove impurities such as sugars and some flavanones, and then 80% ethanol aqueous solution was used for elution at a flow rate of 4.0 BV / h. 5 BV was eluted and the eluate was collected. 1 mL of the extract was taken and filtered through a 0.22 μm filter membrane, and then ultra-high performance liquid chromatography was performed. ; The chromatographic conditions used for the detection are: the chromatographic column is a C18 chromatographic column, double distilled water (ddH2O) containing 2‰ formic acid is used as the mobile phase A (aqueous phase), and chromatographic grade methanol is used as the mobile phase B (organic phase) to gradiently elute and separate the flavonoids adsorbed on the chromatographic column. The elution conditions are 0min, 10% (B phase); 0.5min, 20% (B phase); 5.0min, 70%; 7.0min, 80%; 9.0min, 90%; 11min, 10%. Flow rate: 0.5mL / min; column temperature 45℃; single needle injection volume 1μL, 2 injections for each sample to reduce errors; detection wavelength: 330nm;

[0026] The fraction with a retention time of 3.65 min was collected. An application of Lactobacillus plantarum PPL19,

[0027] For use in the preparation of solid beverages; or

[0028] Used in the preparation of compressed candies; or

[0029] For use in the preparation of mouthwash; or

[0030] Used in the preparation of toothpaste; or

[0031] Used in preparing tooth cleaning powder; or

[0032] Used in the preparation of antibacterial solution; or

[0033] Used in the preparation of oral fluoride coating.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The plant lactobacillus provided by the invention has good probiotic functions, including antibacterial ability to mutans streptococci, ability to significantly inhibit the biofilm of mutans streptococci, copolymerization ability, adhesion ability to saliva-coated hydroxyapatite, and ability to tolerate lysozyme.

[0036] The fermentation product provided by the present invention contains a pathogenic bacteria population quencher, which can inhibit the biofilm formation of Streptococcus mutans by interfering with the ComCDE system of Streptococcus mutans and down-regulating the expression of ComD and ComE genes, and can also down-regulate the relative expression level of LuxS, a key gene of the interspecies signal transduction AI-2 system. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A schematic diagram of animal experiments is shown. DETAILED DESCRIPTION

[0038] Strain screening

[0039] The invention provides a Lactobacillus plantarum PPL19, which is screened from the saliva of healthy children without dental caries. Because healthy children are prone to dental caries, and the oral saliva of healthy children without dental caries contains a large amount of probiotics that inhibit pathogenic bacteria, it is screened from the saliva of children.

[0040] Example 1

[0041] 1. Evaluation of probiotic functional characteristics of primary screening strains

[0042] Inhibition zone: Take 200μL of the bacterial solution of Streptococcus mutans ATCC25175 (from Shanghai Collection of Microorganisms Center), spread it evenly on the blood agar plate, leave it for 30 minutes, and 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, and use physiological saline as a control. After culturing at 37℃ for 24h, observe the results and measure the diameter of the inhibition zone.

[0043] Evaluation of acid production capacity: Two dominant strains were inoculated into MRS liquid culture 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.

[0044] Pathogenic bacteria biofilm inhibition rate: adjust the 24h culture of Streptococcus mutans to 10 8CFU / mL, add 100L 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.

[0045] Copolymerization ability: Adjust the cultured lactic acid bacteria and Streptococcus mutans 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.

[0046]

[0047] Wherein: Ax represents the initial OD value of lactic acid bacteria; Ay represents the initial OD value of Streptococcus mutans; Am represents the OD value of the supernatant after 24 h of incubation.

[0048] After screening through the above steps, a strain with good probiotic properties was obtained, and its properties are as follows:

[0049] Table 1 Antibacterial and copolymerization abilities of dominant bacteria and competing strains

[0050]

[0051] Adhesion ability to saliva-coated hydroxyapatite

[0052] A saliva-coated hydroxyapatite model (SHA) was established. 100 μL of lactic acid bacteria suspension (mixed with saline after centrifugation) (OD600 = 0.5 ± 0.05) was mixed evenly 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 once with PBS. 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 labeled bacterial solution was the total fluorescence value. The adhesion ability of lactic acid bacteria to SHA was calculated according to the following formula.

[0053]

[0054] Lysozyme resistance:

[0055] The concentration of lysozyme in the human oral cavity (1-57 μg / mL) was determined by adding 200 μL of 0, 1, 3, 5, and 10 mg / mL lysozyme solution into the wells of the Oxford cup, spreading 100 μL of probiotic fermentation liquid on the plate, and determining the diameter of the inhibition zone using the Oxford cup inhibition zone method.

[0056] Table 2 Adhesion and lysozyme tolerance of dominant bacteria and competing bacteria

[0057] Dental caries-dominant bacteria Adhesion rate with hydroxyapatite (%) 3mg / mL lysozyme tolerance (mm) Lactobacillus plantarum PPL19 91.82 8.21 Streptococcus salivarius K12 70.26 14.43

[0058] Lactobacillus plantarum PPL19 had a strong adhesion ability to saliva-coated hydroxyapatite, with an adhesion rate of up to 91.82%, and a smaller inhibition zone to 3 mg / mL lysozyme, indicating that it had stronger tolerance.

[0059] 2.16S rDNA identification

[0060] The bacterial genomic DNA extraction kit was used to extract the DNA of the strain, and the extracted genomic DNA of the strain was used as a template for PCR amplification. The lactic acid bacteria primers were universal primers:

[0061] 27F (SEQ ID No. 2): AGAGTTTGATCCTGGCTCAG;

[0062] 1492R (SEQ ID No. 3): GGTTACCTTGTTACGACTT;

[0063] 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 3.

[0064] Table 3 Strain sources and identification results

[0065] Strain number Accession number Sample source 16S rDNA identification results PPL19 CGMCC No.32110 Healthy children's saliva Lactobacillus plantarum

[0066] The 16S DNA sequence of Lactobacillus plantarum PPL19 is as follows:

[0067] AGGGTACCCTCCAGAGTGATCGAGCCTCGAGCTGTGGTCCAGGGTGACCCTTCAATC

[0068] AGATTTTGATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAG

[0069] AAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCA

[0070] TGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATT

[0071] AGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGG

[0072] GTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTA

[0073] GGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAA

[0074] GGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTC

[0075] AGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGG

[0076] TAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCG

[0077] GTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTG

[0078] GGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTA

[0079] GATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGA

[0080] GGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAA

[0081] CGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAA

[0082] GCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGC

[0083] CCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAG

[0084] GTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATAC

[0085] AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC

[0086] GAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCC

[0087] GGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCT

[0088] GGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAG

[0089] CTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAG

[0090] TCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTT

[0091] GTACACACCGCCCGTCACACCATGAGAGTTTGTAACATAGAAAGTCGCTGGGGTCAGTAAGTGGTACGGAAGGTACTCATAGAGTATCCCC。

[0092] Example 2

[0093] The fermentation experiment of Lactobacillus plantarum PPL19 was carried out, and the specific steps were as follows:

[0094] (1) Fermentation

[0095] Because orange peel contains a high content of tangeretin, there are literature reports that tangeretin has the ability to inhibit Streptococcus mutans, so prepare orange peel solution (1g orange peel freeze-dried powder dissolved in 9mL PBS), aspirate 1mL orange peel solution into 9mL MRS liquid culture medium, the inoculation amount of Lactobacillus plantarum PPL19 is 3%, the fermentation temperature is 37°C, the rotation speed is 100r / min, and the fermentation time is 20-26h.

[0096] (2) Extraction

[0097] After the fermentation, add 5 mL of 90% ethanol, heat to 45°C in a water bath, shake repeatedly for more than 20 minutes, collect the ethanol-soaked clear liquid and load it onto an HP750 macroporous adsorption resin column for adsorption at a rate of 4.0 BV / h, then use 5% ethanol solution to remove impurities such as sugars and some flavanones, then elute with 80% ethanol aqueous solution at a flow rate of 4.0 BV / h, elute 5 BV, collect the eluate, and the above is the tangerine peel fermentation extract, which is freeze-dried and administered to animals.

[0098] Example 3

[0099] Effects of extracts on gene expression in Streptococcus mutans biofilms by transcriptome analysis

[0100] (1) Pathogenic bacteria culture: The pathogenic bacteria were inoculated into sterilized BHI medium at a ratio of 2%, 3% goat serum and 100 mg / L bovine serum albumin were added, and the medium was incubated at 37° C. in an anaerobic incubator for 24 h. The OD of the pathogenic bacteria culture was adjusted to about 0.8.

[0101] (2) Lactobacillus plantarum PPL19 and its citrus peel extract were co-cultured with Streptococcus mutans in non-contact manner, and the key genes of the quorum sensing gene system involved in regulating biofilm formation in Streptococcus mutans were investigated by fluorescence quantitative PCR. The control group was without probiotics and extracts, and the differences in gene expression between the control group and the experimental group were compared.

[0102] (3) RNA of Streptococcus mutans was extracted and reverse transcribed into cDNA, and the synthesized cDNA template was stored at -80°C for future use. Fluorescence quantitative PCR was performed according to the primers obtained in the table, and the 16SrRNA gene was used as the internal reference gene. The primer sequences are shown in Table 4.

[0103] The ComCDE system is important for the initiation of biofilm formation. The quorum sensing feedback loop ComCDE mainly includes ComC (precursor of competence stimulating peptide CSP), ComD (transmembrane histidine kinase signal transduction protein) and ComE (response regulatory protein). ComC is first truncated into a 21-amino acid protein by ABC transporters, and then further processed into CSP by the extracellular membrane-bound protease SepM, which is an 18-amino acid AI]. The binding of CSP to ComD leads to the phosphorylation of ComE, which causes the transcription of another σ factor ComX from the comX gene, thereby inducing the expression of genetically competent mutans Streptococcus.

[0104] AI-2 is a signaling molecule used for interspecies communication. The synthesis of AI-2 depends on an enzyme called LuxS, which catalyzes the conversion of S-ribosylhomocysteine ​​to homocysteine, the final step of an important pathway responsible for bacterial protein and nucleic acid methylation. As AI-2 accumulates outside the cell, a periplasmic receptor binds to AI-2 to initiate signal transduction, which in turn enables gene transcription and virulence. Therefore, the expression of key genes in the ComABCDE two-component signaling system and the AI-2 interspecies signaling system was evaluated.

[0105] Table 4 Primer information

[0106]

[0107] Table 5 Signal transduction systems regulating biofilm formation in Streptococcus mutans

[0108]

[0109] The QQ extract group and Lactobacillus plantarum can significantly reduce the expression of ComD, ComE and luxS genes, among which the down-regulation amount of QQ extract is significantly lower than that of the probiotic group, especially for the expression of LuxS gene, the relative expression amount of LuxS gene in the QQ extract group can be down-regulated to about 0.32, indicating that QQ extract can play a key role in interfering with the formation of mutans streptococcus biofilm, especially down-regulating the relative expression amount of LuxS, a key gene of the interspecies AI-2 signal transduction system, thereby interfering with the formation of mutans streptococcus biofilm and achieving the effect of quorum sensing quenching. Therefore, this orange peel extract is defined as a quorum sensing quencher QQ.

[0110] Example 4

[0111] Detection of orange peel extract (quorum sensing quencher, QQ)

[0112] 1 mL of the extract was drawn with a 1 mL sterile syringe, filtered through a 0.22 μm filter membrane into a sample vial, and then tested by ultra-performance liquid chromatography (UPLC). The chromatographic conditions used for the test were: the chromatographic column was a C18 column, double distilled water (ddH2O) containing 2‰ formic acid was used as the mobile phase A (aqueous phase), and chromatographic grade methanol was used as the mobile phase B (organic phase) to gradiently elute and separate the flavonoids adsorbed on the chromatographic column. The elution conditions were 0 min, 10% (B phase); 0.5 min, 20% (B phase); 5.0 min, 70%; 7.0 min, 80%; 9.0 min, 90%; 11 min, 10%. Flow rate: 0.5 mL / min; column temperature 45 °C; single injection volume 1 μL, 2 injections per sample to reduce errors; detection wavelength: 330 nm.

[0113] Among the substances extracted by the above method, the substance with a retention time of 3.65min showed the effect of quorum sensing quenching. Its spectral data are as follows:

[0114] 1H NMR (300MHz, CD3CN, δ, ppm):

[0115] 11.65(1H insec amine),10.1(1H in indole),9.48(2H in alcohol),7.14(1Hin indole),6.88(1H in 1-benzene),6.56(1H in indol),6.53(1H in indol),6.40(1Hin 1-ethylene),6.31(1H in 1-benzene),3.95(3H in methyl),2.48(3H in methyl).

[0116] 13C NMR (300MHz, CD3CN, δ, ppm)

[0117] Protocol of the C-13 NMR Prediction:(Lib=S)

[0118] 182.1,153.9,145.0,142.8,145.5,136.5,129.6,124.9,124.5,124.3,119.9,117.8,115.5,105.8,102.9,102.4,97.2,55.8,19.9.

[0119] From the above data, we can know that the structure of the substance is as follows:

[0120]

[0121] Example 5

[0122] The preparation method of probiotics is as follows:

[0123] Probiotic fermentation culture: after streaking the probiotics, single bacteria are picked out and put into liquid MRS, cultured at 37°C, activated twice, and centrifuged to obtain probiotic sludge; the culture medium comprises the following components in weight percentage: 1.0-1.5% yeast peptone, 0.5-1.5% yeast extract powder, 2.0-6.0% glucose, 0.05-0.15% Tween, 0.1-0.2% dipotassium hydrogen phosphate heptahydrate, 0.4-0.6% sodium acetate trihydrate, 0.02-0.06% magnesium sulfate heptahydrate, 0.02-0.03% manganese sulfate monohydrate, 0.10-0.20% L-cysteine, 0.10-0.20% calcium chloride, 0.3-0.5% orange peel extract, and the balance distilled water;

[0124] Preparation of probiotic freeze-dried powder: the probiotic mud is mixed with a freeze-dried protective agent evenly, and freeze-dried to obtain the probiotic freeze-dried powder; the freeze-dried protective agent comprises the following components in weight percentage: trehalose 10-30%, skim milk powder 10-20%, sucrose 2-10%, maltodextrin 1-5%, modified starch 2-5%, glycerol 2-5%, inulin 0.1-1%, arginine 0.5-1%, glutamine 0.5-1%, and water as the balance.

[0125] The probiotic is Lactobacillus plantarum PPL19.

[0126] Preparation method of probiotic compressed candy: formula of 1000 oral compressed candy: sorbitol 40g, oligofructose 10.0g, xylitol 10.0g, magnesium stearate 2.0g, probiotic freeze-dried powder 20g, QQ extract 5000mg.

[0127] The tablet candy is prepared by crushing the above ingredients into fine powder and then tableting. The weight of a single tablet candy is 1.0 g.

[0128] Example 6

[0129] Animal experiments

[0130] (1) Preparation of lactic acid bacteria

[0131] Before being used in animal experiments, Lactobacillus plantarum PPL19 (from healthy children) was centrifuged at 6000r for 5min, the supernatant was removed, and the cells were washed twice with sterile saline and resuspended. The OD value was adjusted to about 0.8 by dilution.

[0132] (2) Animal experiment grouping and dental caries model establishment

[0133] Female SPF Wistar rats (21 days old) were selected as the research subjects. Before the experiment, the rats were treated with ampicillin (0.5 μg / mL) and streptomycin (200 μg / mL) to inhibit the oral flora of the rats for 3 days. After 3 days of adaptive feeding, all the rats were randomly divided into 6 groups, including blank control group (CK group, 8 rats), model group (M group, 8 rats), probiotic treatment group (OD=0.8, T1 group, 8 rats), QQ extract treatment group (T2, 100 μg / mL, 8 rats), dominant bacteria prevention group (OD=0.8, P1 group, 8 rats), and QQ extract prevention group (P2, 100 μg / mL, 8 rats).

[0134] Blank control group (CK group): rats were given normal diet and distilled water throughout the experiment; all other groups were fed with cariogenic diet Diet2000 (6% whole wheat flour, 56% sucrose, 28% skim sugar-free milk powder, 4% yeast, 3% alfalfa powder, 1% liver powder, 2% salt), and free to drink distilled water solution supplemented with 5% (w / v) sucrose;

[0135] Dental caries model group (M group): Starting from the first day of the experiment, the patients were infected with Streptococcus mutans for 5 consecutive days. The specific operation was as follows: Streptococcus mutans was cultured for 16 hours, centrifuged and diluted to 10 with physiological saline. 8 CFU / mL, use a sterile cotton swab to apply the bacterial suspension on the rat's mouth, apply it on the teeth for 15 seconds, and prohibit eating and drinking within half an hour after tooth coating to ensure microbial colonization. On the 6th to 7th day, the colonization of Streptococcus mutans in the mouth was tested, and the colony unit (CFU) and rat weight were recorded.

[0136] Probiotic treatment group: After S. mutans colonization, probiotic solution (OD=0.8) was given for 5 consecutive days, and then probiotics were applied to teeth 3 times a week.

[0137] QQ extract treatment group: QQ extract was obtained according to the method of Example 2 and prepared into a concentration of 100 μg / mL. After the colonization of Streptococcus mutans, the teeth were treated with QQ extract smear for 5 consecutive days, and then the QQ extract was smeared 3 times a week.

[0138] Predominant bacteria prevention group: To test the preventive effect of lactic acid bacteria on dental caries, probiotic solution (OD=0.8) was first applied to the teeth on days 1-5. After lactic acid bacteria colonization, Streptococcus mutans was used for 5 consecutive days. No treatment was given to the prevention group within 6 weeks after the end of the experiment. The entire experimental period was 8 weeks. The weight gain of the mice was recorded every 2 days.

[0139] QQ extract prevention group: QQ extract was obtained according to the method of Example 2 and prepared at a concentration of 100 μg / mL. In order to detect the preventive effect of lactic acid bacteria on dental caries, QQ extract (100 μg / mL) was first applied to the teeth on days 1-5. After the colonization of lactic acid bacteria, Streptococcus mutans was used for 5 consecutive days. No treatment was performed in the prevention group within 6 weeks after the end of the experiment. The entire experimental period was 8 weeks. The weight gain of the mice was recorded every 2 days.

[0140] 3) Oral microbial count

[0141] The entire experimental period lasted for 56 days, during which four samplings were conducted, namely on the 6th, 13th, 27th and 57th days. The specific sampling method was to use a sterile cotton swab to take samples from rats in all groups, and scrape the surface of the rats' teeth back and forth 3-4 times. TYCSB medium plus 200U / L of bacitracin was used to count mutans streptococci; 12μg / ml of vancomycin was added to count oral lactic acid bacteria.

[0142] (4) Rats dental caries scoring

[0143] Caries scoring: Prepare 0.4% ammonium murexide dye solution, soak the upper and lower jaws in the dye solution for 12 hours, take them out and rinse them with clean water, dry them, and use diamond abrasive sheets to make sagittal hemisections along the mesiodistal direction of the upper and lower molars of the rats. Observe and evaluate the caries of the rat molars under a stereo microscope. The caries of the rat molars after staining appeared red, while the normal non-carious parts were almost not stained or the color was very light; according to the Keyes scoring standard, caries were scored and the degree of tooth caries was divided into 4 levels: E-level caries, caries only in the enamel; Ds-level caries, enamel damage and caries range not exceeding 1 / 4 of the outer layer of dentin; Dm-level caries, caries range is between 1 / 4 and 3 / 4 of the dentin; Dx-level caries, caries range exceeds 3 / 4 of the dentin.

[0144] Table 1 Colonization of Streptococcus mutans

[0145]

[0146] In the first sampling, the colonization rate of Streptococcus mutans was 5.2-5.5log (CFU / mL). After 5 days of continuous drug intervention, the Streptococcus mutans in the T1 group dropped to 4.1log (CFU / mL) and remained at 4.0-4.1log (CFU / mL) until the end of the experiment. The colonization rate of Streptococcus mutans in the T2 group dropped to 3.88log (CFU / mL) in the fourth sampling. It can be seen that the effect of QQ extract is better than that of the probiotic group. In the second sampling (5 days after administration), the colonization rate of Streptococcus mutans in the prevention group (P1 and P2) was significantly lower than that in the caries model group (p < 0.05); before the fourth sampling, the Streptococcus mutans had been infected, and the number of Streptococcus mutans continued to rise after drug treatment was stopped. The T1 group rose from 4.12log (CFU / mL) (second sampling) to 4.32log (CFU / mL), still maintaining a low level and significantly lower than the caries group. After the fourth sampling, the mutans streptococci in the prevention group dropped to about 4.1-4.2, which was not significantly different from the treatment group. For probiotics, there was no significant difference between the treatment effect and the prevention effect; while the prevention effect of QQ extraction was greater than that of treatment.

[0147] Table 2 Smooth surface caries scores in different groups

[0148]

[0149] It can be seen from the caries scores that the probiotics group and the QQ extract group can significantly reduce the caries scores.

Claims

1. A Lactobacillus plantarum PPL19, characterized in that: Its accession number is CGMCCNO.32110.

2. A fermentation product, characterized in that: The method is obtained by fermenting orange peel with the plant lactobacillus PPL19 as claimed in claim 1.

3. The fermentation product according to claim 2, characterized in that: Includes quorum sensing quenchers; The structure of the quorum sensing quencher is as follows:

4. A probiotic preparation, characterized in that: The invention comprises the plant lactobacillus PPL19 as claimed in claim 1, the fermentation product as claimed in claim 2 and a freeze-drying protective agent.

5. The probiotic preparation according to claim 4, characterized in that: The freeze-drying protective agent comprises, by weight percentage, 10-30% trehalose, 10-20% skim milk powder, 2-10% sucrose, 1-5% maltodextrin, 2-5% modified starch, 2-5% glycerol, 0.1-1% inulin, 0.5-1% arginine, 0.5-1% glutamine, and the balance is water.

6. The method for preparing the probiotic preparation according to claim 5, characterized in that: The steps include: The probiotic bacterial slurry is evenly mixed with the freeze-drying protective agent, and freeze-dried to obtain probiotic freeze-dried powder.

7. The method for preparing the probiotic preparation according to claim 5, characterized in that: The method comprises the steps of streaking the probiotics, picking out single bacteria and putting them into a liquid MRS culture medium, culturing at 37°C, performing two rounds of activation, and centrifuging to obtain probiotic bacterial slurry.

8. The method for preparing the probiotic preparation according to claim 5, characterized in that: The culture medium comprises the following components by weight percentage: 1.0-1.5% yeast peptone, 0.5-1.5% yeast extract powder, 2.0-6.0% glucose, 0.05-0.15% Tween, 0.1-0.2% dipotassium hydrogen phosphate heptahydrate, 0.4-0.6% sodium acetate trihydrate, 0.02-0.06% magnesium sulfate heptahydrate, 0.02-0.03% manganese sulfate monohydrate, 0.10-0.20% L-cysteine, 0.10-0.20% calcium chloride, 0.3-0.5% orange peel extract, and the balance distilled water.

9. The method for preparing the probiotic preparation according to claim 5, characterized in that: The following steps are also included: Dissolve 1g of orange peel freeze-dried powder in 9mL PBS, take 1mL of orange peel solution and place it in 9mL of MRS liquid culture medium, inoculate 3% of Lactobacillus plantarum PPL19, and ferment at 37°C for 20-26h; After the fermentation, 5 mL of 90% ethanol was added and heated to 45°C in a water bath. The ethanol-soaked clear liquid was collected and loaded onto a HP750 macroporous adsorption resin column at a rate of 4.0 BV / h for adsorption. Then, 5% ethanol solution was used to remove sugars, some flavanones and other impurities, and then 80% ethanol aqueous solution was used for elution at a flow rate of 4.0 BV / h. 5 BV of elution was eluted and the eluate was collected. Take 1 mL of the extract, filter it through a 0.22 μm filter membrane, and then perform ultra-high performance liquid chromatography detection; The chromatographic conditions used for the detection were as follows: the chromatographic column was a C18 column, double distilled water containing 2‰ formic acid was used as mobile phase A, and chromatographic grade methanol was used as mobile phase B to gradiently elute and separate the flavonoids adsorbed on the chromatographic column, and the elution conditions were 0 min, 10%; 0.5min, 20%; 5.0min, 70%; 7.0min, 80%; 9.0min, 90%; 11min, 10%; flow rate: 0.5mL / min; column temperature 45℃; single injection volume 1μL, 2 injections per sample to reduce error; detection wavelength: 330nm; The fraction with a retention time of 3.65 min was collected.

10. An application of Lactobacillus plantarum PPL19, characterized in that: For use in the preparation of solid beverages; or Used in the preparation of compressed candies; or For use in the preparation of mouthwash; or Used in the preparation of toothpaste; or Used in preparing tooth cleaning powder; or Used in the preparation of antibacterial solution; or Used in the preparation of oral fluoride coating.

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