CN111000874A - Lactobacillus plantarum with dental caries prevention and treatment effect - Google Patents
Lactobacillus plantarum with dental caries prevention and treatment effect Download PDFInfo
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- CN111000874A CN111000874A CN201910675412.8A CN201910675412A CN111000874A CN 111000874 A CN111000874 A CN 111000874A CN 201910675412 A CN201910675412 A CN 201910675412A CN 111000874 A CN111000874 A CN 111000874A
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Abstract
The invention discloses lactobacillus plantarum with an effect of preventing and treating dental caries, and belongs to the technical field of microorganisms. The invention establishes a rat dental caries model, and determines that the lactobacillus plantarum CCFM8724 has the functions of preventing and treating dental caries by comparing the serum biochemical indexes, oral flora, tooth demineralization and dental caries conditions of mice of a blank control group, a model group, an intervention group and a prevention group, and the application effect of the lactobacillus plantarum CCFM8724 is superior to that of the traditional anti-caries medicine chlorhexidine, so that the rat dental caries model has wide market application prospect. Meanwhile, the lactobacillus plantarum CCFM8724 can be applied to probiotic functional foods and daily chemical products such as bacteria powder, yoghourt, chewable tablets, chewing gum, toothpaste, mouthwash and the like, and is expected to open a new functional market for the prevention and treatment of dental caries by probiotics.
Description
Technical Field
The invention relates to a probiotic for preventing and treating decayed teeth caused by streptococcus mutans (hereinafter referred to as mutans) and candida albicans (hereinafter referred to as candida albicans) infection, in particular to a lactobacillus plantarum with the efficacy of preventing and treating decayed teeth, belonging to the technical field of microorganisms.
Background
Caries is a bacterial infectious disease that occurs in both enamel and dentin, and the formation of plaque biofilm is a prerequisite for caries development. The mature dental plaque biomembrane is a three-dimensional ecological environment formed by embedding oral bacteria in a matrix mainly containing insoluble polysaccharide, is rich in gaps and pipeline systems, and has a certain thickness. Once this ecological imbalance is caused by changes in host, diet, microbial growth, etc., cariogenic microbes in the plaque biofilm become dominant microflora, which rapidly metabolise breakdown of carbohydrates, especially sucrose, producing organic acids, lowering the pH below 5.5, leading to demineralization of the enamel and destruction of the hard tissue structure of the tooth to form caries cavities.
Mutans streptococci are recognized as the major cariogenic bacteria. Although S.mutans is not the most abundant bacterium in the oral cavity, it rapidly coordinates the formation of cariogenic biofilms and, in combination with other cariogenic bacteria, forms a more virulent cariogenic biofilm. Unlike planktonic bacteria in the oral cavity, bacteria in biofilms are generally nutritionally restricted, in a near dormant state where they are more resistant to antibiotics and bacteriostats than planktonic metabolically active bacteria.
The association of candida albicans with streptococcus mutans enhances the pathogenicity of streptococcus mutans and enhances the formation of cariogenic biofilms. Candida albicans has a weak ability to adhere to the tooth surface, but after binding to Streptococcus mutans, the oral colonization ability is greatly enhanced. Glucosyltransferase (GTF) produced by S.mutans binds to mannan on the cell wall of Candida albicans, recruiting it to the early plaque biofilm (Ellepola K et al, Journal of dental research,2017,96(10): 1129-1135.). Candida albicans having a larger surface area may provide binding sites for more S.mutans, which in turn increases the number of Candida albicans. The two biofilms have higher biomass and cell numbers than the single biofilm, and studies have demonstrated that biofilms of candida albicans and streptococcus oralis are more resistant to antibiotics than single species (Shirtliff M E et al, FEMS microbiology letters,2009,299(1):1-8.), and these properties of the two biofilms undoubtedly increase the difficulty of caries treatment resulting therefrom.
The traditional treatment method of the decayed tooth is a surgical mode, which can not achieve the effect of radically reducing the incidence rate of the decayed tooth except for mechanically excavating and filling the decayed tooth part. The other treatment method is a bactericide, but almost all drugs kill most microorganisms through a physical and chemical method, and the residual oral microorganisms cannot maintain the balance of oral flora. Also, many therapeutic agents lose their effectiveness by binding to exopolysaccharides in the biofilm prior to their action on the bacteria. It is also one of the reasons why the antibacterial agent and antibiotic have poor effect of preventing and treating dental caries clinically. In addition, the occurrence of dental caries is an irreversible process, and once enamel is damaged, only artificial teeth are filled or planted by physical means, so that prevention of dental caries is more than treatment. Traditional tooth brushing and cleaning methods are not capable of resisting stubborn dental plaque, and children are limited in their willingness to go. When the common Chinese herbal medicine product is used for prevention, although cariogenic flora can be inhibited, the Chinese herbal medicine product has weakening effect on other dominant flora for maintaining oral cavity balance, and the use of the Chinese herbal medicine product also has the risk of dental pigment deposition.
Disclosure of Invention
[ problem ] to provide a method for producing a semiconductor device
The prior art lacks probiotics which can have both prevention and treatment effects in vivo.
[ technical solution ] A
The invention provides lactobacillus plantarum with the functions of preventing and treating dental caries, wherein the lactobacillus plantarum is lactobacillus plantarum CCFM8724 with the preservation number of CGMCC No.5492, which is disclosed in the patent application CN102533618A lactobacillus plantarum CCFM8724 and application thereof.
The lactobacillus plantarum CCFM8724 can be used for preventing and treating dental caries caused by streptococcus mutans and candida albicans infection.
The invention also provides application of the lactobacillus plantarum CCFM8724 in preparing functional food and daily chemical products for preventing dental caries.
The invention also provides the yoghourt with the functions of preventing and treating the decayed teeth, and the preparation steps of the yoghourt comprise: mixing milk powder, inulin, stevioside and water, homogenizing, making into fermentation raw material, sterilizing, cooling, inoculating mixed powder of Lactobacillus bulgaricus and Streptococcus thermophilus, fermenting, adding freeze-dried powder of Lactobacillus plantarum CCFM8724, stirring, canning, and aging to obtain probiotic yogurt with dental caries preventing and treating effects.
Specifically, the preparation method of the yoghourt comprises the following steps: mixing milk powder, inulin, stevioside and water in a weight ratio of: 20: 5: 5: 75, mixing and homogenizing to prepare a fermentation raw material; sterilizing at 121 ℃ for 300s at ultrahigh temperature, cooling to 42 ℃, and inoculating mixed bacterial powder of zymocyte lactobacillus bulgaricus and streptococcus thermophilus, wherein the inoculation amount of the zymocyte is 0.3 percent of the total weight of fermentation raw materials; fermenting at 42 deg.C for 12 hr, and blending; cooling the fermentation product to 37 deg.C, adding freeze-dried powder of Lactobacillus plantarum CCFM8724 with CFU 10 as raw material9And/ml yoghourt, stirring, canning, preserving at 4 ℃ for 2 days, naturally completing after-ripening, and preparing the probiotic yoghourt.
The invention also provides a chewing gum with the functions of preventing and treating dental caries, which is prepared by the following steps: softening chewing gum base, adding Lactobacillus plantarum CCFM8724, and processing by 1010Benefit toAdding raw bacteria/chewing gum at a certain proportion, stirring, cooling, extruding, and cutting to obtain probiotic chewing gum.
In one embodiment of the invention, the chewing gum may further comprise a substance having an inhibitory effect on streptococcus mutans, such as cranberry extract.
Specifically, the preparation steps of the chewing gum comprise: softening chewing gum base in 60 deg.C oven for 4 hr, uniformly cooling to 37 deg.C, adding Lactobacillus plantarum CCFM8724, and pressing at 10%10Adding probiotics/granulated chewing gum according to the proportion, uniformly stirring and cooling, putting into a noodle cutting machine for extruding for 3 times, keeping the temperature at 20 ℃ and the relative humidity at 56%, and aging for 12-22 h to prepare the probiotic chewing gum. The lactobacillus plantarum CCFM8724 freeze-dried powder is added into the chewing gum, and the number of probiotics in the freeze-dried powder is 1012Per gram.
The invention also provides a toothpaste with the functions of preventing and treating dental caries, which is prepared by the following steps: stirring the toothpaste base material, natural mineral salt and probiotics to obtain a mixture, vacuumizing the mixture, degassing to obtain paste, and grinding to obtain the probiotics toothpaste.
In one embodiment of the invention, substances which have an inhibitory effect on streptococcus mutans, such as cranberry extract, may also be added to the toothpaste.
Specifically, the preparation steps of the toothpaste comprise: weighing the following raw materials in parts by weight: adding 100 parts of toothpaste base material, 3 parts of natural mineral salt, 7 parts of probiotics and 40 parts of cranberry extract (concentrated to 1000 mu g/mL) into a stirrer, stirring to obtain a mixture, vacuumizing the mixture, degassing to obtain paste, and grinding to obtain the probiotic toothpaste. The lactobacillus plantarum CCFM8724 freeze-dried powder is added into the chewing gum, and the number of probiotics in the freeze-dried powder is 1012Per gram.
The invention also provides a rat dental caries model, and the construction method of the model comprises the following steps: selecting SPF Wistar rats, male rats, 3 weeks old rats and 40-50 g of body weight, and infecting the rats with variable chains and white idea once a day for 5days continuously. The specific operation method of infection is that 200 mu L of each bacterial suspension of streptococcus mutans and candida albicans is saturated and absorbed by a sterile cotton stick, the bacterial suspension is applied to the occlusal surfaces of molar teeth of upper and lower jaws of rats and acts for 15s, and after the teeth are applied, the oral cavity is fasted and the water is forbidden for half an hour.
The method for screening substances with the functions of preventing and treating decayed teeth by applying the rat decayed tooth model comprises the steps of dividing rats of the rat decayed tooth model into a model group and a pre-treatment group, wherein the model group is fed with a decayed tooth-causing feed and added with cane sugar in diet, and the pre-treatment group is fed with the decayed tooth-causing feed and added with cane sugar in diet and is treated by using the substances to be screened; in addition, a blank control group and an intervention group are set, wherein the blank control group is not infected by streptococcus mutans and candida albicans, the blank control group is fed with a cariogenic feed and is added with cane sugar in the diet, the intervention group is firstly treated with a substance to be screened and is then infected with the streptococcus mutans and candida albicans, and the intervention group is fed with the cariogenic feed and is added with cane sugar in the diet; during the feeding process, the weight, the serum biochemical indexes and the oral flora of the rat are monitored, after the feeding is finished, the tooth demineralization condition of the rat is detected by utilizing Micro-CT, and the tooth caries condition of the rat is observed by utilizing a body type microscope.
[ PROBLEMS ] the present invention
The invention provides the application of lactobacillus plantarum CCFM8724 in preventing and treating dental caries, and compared with other treatment means, the probiotics has the advantage far exceeding other means. The probiotics can play a preventive role after being planted in the oral cavity, and the probiotics do not need to be used for a long time like other medicines.
The prevention capability of the lactobacillus plantarum CCFM8724 is also suitable for the situation that dental plaque or a decayed cavity exists or the decayed cavity is filled, and the healthy oral flora can be maintained, so that the decayed tooth is not aggravated or secondary decayed tooth occurs.
The lactobacillus plantarum CCFM8724 can maintain the development of oral flora from the root and stop the generation of caries from the source, thereby achieving the purpose of preventing and treating the caries. This is not achieved by the existing means for treating dental caries because the existing means for treating dental caries can only passively treat the dental caries after the onset of disease, cannot achieve the preventive effect, and can cause recurrence after one treatment.
The lactobacillus plantarum CCFM8724 has good prevention and treatment effects on serious dental caries caused by the action of streptococcus mutans and candida albicans.
Drawings
FIG. 1: experimental flow chart.
FIG. 2: graph of body weight trend in rats.
FIG. 3: counting the oral flora of the rat; a. counting results of the variable chains; b. counting results of the white minds; c. counting results of breast stems.
FIG. 4: Micro-CT scans the molars of rats.
FIG. 5: volume of molar enamel in rats treated with Micro-CT.
FIG. 6: a. a schematic diagram of division of a rat lower jaw molar buccal lingual scoring unit; b. schematic diagram of division of units of rat mandibular molar fossa groove score.
FIG. 7: rat molar staining pattern under a body type microscope; A. b, C, D, E, F, G, H are photographs of the smooth surface of rat molar in a blank control group, a dental caries model group, a chlorhexidine intervention group, a chlorhexidine prevention group, a CCFM8724 intervention group, a CCFM8724 prevention group, a CCFM634 intervention group and a CCFM634 prevention group respectively; a. b, c, d, e, f, g, h are photographs of the corresponding pit and furrow surfaces (longitudinal sections) stained.
Indicates significant differences between treatment groups and model groups (P < 0.05).
Detailed Description
MRS culture medium: 5.0g/L yeast powder, 10.0g/L beef extract, 10.0g/L peptone, 20.0g/L glucose, 2.0g/L anhydrous sodium acetate, 2.0g/L hydrogencitrate diamine, 2.6g/L dipotassium hydrogen phosphate, 0.25g/L manganese sulfate monohydrate, 0.5g/L magnesium sulfate heptahydrate and Tween-801 mL, and the pH value is 6.2-6.4.
TSBY medium: tryptone soy broth medium supplemented with 0.6% yeast powder. The yeast extract contains 6.0g/L of yeast powder, 3.0g/L of soybean protein, 17.0g/L of tryptone, 2.5g/L of glucose, 5g/L of sodium chloride and 2.5g/L of dipotassium hydrogen phosphate, and the pH value is 7.0-7.4.
YPD medium: 1.0g/L of yeast powder, 2.0g/L of glucose and 2.0g/L of peptone.
BIGGY culture medium: 1.0g/L of yeast extract, 10.0g/L of glycine, 10.0g/L of glucose, 5.0g/L of bismuth ammonium citrate, 3.0g/L of sodium sulfite, 16.0g/L of agar and pH of 6.6-7.0.
MS (mitis salivarius agar) medium: 15.0g/L of casein peptone, 5.0g/L of animal tissue pepsin hydrolysate, 1.0g/L of D-glucose, 50.0g/L of sucrose, 4.0g/L of dipotassium phosphate, 0.075g/L of trypan blue, 0.0008g/L of crystal violet, 15.0g/L of agar and pH 6.8-7.2.
Lactobacillus plantarum CCFM8724 bacterial liquid: inoculating Lactobacillus plantarum CCFM8724 in MRS liquid culture medium at an inoculum size of 2%, culturing in an incubator at 37 deg.C for 24h, and regulating the bacterial suspension concentration to 1 × 109cfu/ml。
Streptococcus mutans suspension: culturing Streptococcus mutans strain ATCC 25175 in TSBY medium at 37 deg.C for 12h, and adjusting the concentration of bacterial suspension to 1 × 109cfu/ml。
Candida albicans suspension: culturing Candida albicans in YPD medium at 37 deg.C for 12 hr, and regulating the bacterial suspension concentration to 1 × 106cfu/ml。
Example 1 animal experiments on the use of Lactobacillus plantarum CCFM8724 for the prevention and treatment of dental caries
Experimental animals and strains:
SPF-grade Wistar rats, male, 3 weeks old, with a weight of 40-50 g, were purchased from Beijing Wintonlihua laboratory animal technology Co., Ltd. (production license number SCXK (Jing) 2012-0001).
The streptococcus mutans adopts streptococcus mutans ATCC 25175, and is purchased from China general microbiological culture collection center CGMCC. Candida albicans was isolated from the oral cavity of volunteers.
Lactobacillus plantarum CCFM634(CGMCC No.9740) has a good effect of inhibiting double-bacterium biofilm in vitro (24h mediated biofilm inhibition amount is more than 60%) and is also used for comparison in animal experiments. For the experiment of inhibiting the double-bacterial biofilm, the mediation group is that 50 mu L of each streptococcus mutans and Candida albicans suspension is added into a 96-well plate, 100 mu L of lactobacillus plantarum supernatant is added into a 96-well plate, the culture is carried out for 24h at 37 ℃, the biofilm is carefully cleaned by PBS for 2 times when the double-bacterial biofilm grows to 24h, 100 mu L of lactobacillus plantarum supernatant is added, and the culture is carried out for 24h at 37 ℃; in the double-bacteria biomembrane control, blank MRS liquid culture medium is used for replacing lactobacillus plantarum supernatant, and 6 parallels are arranged in each group; after the culture, the biological membrane is carefully cleaned by PBS for 2 times, the biological membrane is kept stand at room temperature and dried in the air, then 100 mu L of 0.1% crystal violet solution is added into each hole, the biological membrane is dyed for 30min, the biological membrane is cleaned by PBS for 2 times after the dyeing is finished, each hole is dissolved by 95% ethanol, and the absorbance value is read under an enzyme-labeling instrument OD600 nm. The reduction after biofilm mediation was calculated as reduction (%) ═ (biofilm double control-biofilm amount after mediation)/biofilm double control. The lactobacillus plantarum CCFM8724 mediated double bacterial biofilm reduction was 85.32%, and the lactobacillus plantarum CCFM634 mediated double bacterial biofilm reduction was 65.57%.
The experimental procedure is described in FIG. 1.
Experimental protocol and grouping:
TABLE 1 Experimental grouping of rats
Groups division of experimental wistar rats
(Note: cariogenic feed Diet Diet 2000 comprises the following main components of 6% of whole wheat flour, 56% of sucrose, 28% of refined milk powder, 3% of alfalfa leaf powder, 1% of dehydrated whole dry powder, 4% of yeast and 2% of salt)
The rats were randomly divided into 8 groups according to body weight, and referring to table 1, the non-carious control group (i.e., blank control) was normally Diet during the entire experiment, and all rats in the other groups were fed cariogenic Diet 2000 (purchased from south ton troffel fodder science co., ltd) supplemented with distilled water supplemented with 5% sucrose.
The caries model group and intervention group are infected with Candida albicans and variable chain for 5 consecutive days (1-5 days) (the specific operation method of infection is that sterile cotton stick is used to absorb 200 μ L of bacterial suspension of Streptococcus mutans and Candida albicans each, and the bacterial suspension is applied to molar teeth of upper and lower jaws of ratOcclusal surface and applying for 15s, fasting and water-deprivation for half an hour after applying teeth), wiping a sample from the teeth of the rat with a cotton swab, placing the cotton swab in 1mL of physiological saline so that bacteria on the cotton swab are transferred to the physiological saline, diluting and coating the 1mL of physiological saline and counting the number of variable chains and white monies, and counting the result>102After CFU/mL is successfully planted and chain-changing and Candida albicans planting is ensured (planting is successfully planted on about 6-7 days), the intervention group is continuously mediated for 5days (8-12 days) by lactobacillus plantarum CCFM8724, CCFM634 or 0.02% Chlorhexidine (CHX), and then is mediated for 6 weeks three times per week. The specific operation of all infection and mediation is that 200 mu L of bacteria suspension or 200 mu L of Chlorhexidine (CHX) with 0.02% is saturated and absorbed by a sterile cotton stick, applied to the occlusal surfaces of molar teeth of upper and lower jaws of rats and acted for 15s, and fasted and forbidden for half an hour after tooth application.
In particular, the experiment also provided a prophylactic group, which preferentially colonized the oral cavity and characterized its prophylactic effect. A prevention group adopting lactobacillus plantarum CCFM8724 or lactobacillus plantarum CCFM8724 is characterized in that a sterile cotton stick is used for saturated absorption of 200 mu L of lactobacillus suspension on 5 consecutive days of 1-5 days of an experiment, the lactobacillus suspension is applied to occlusal surfaces of molar teeth of upper and lower jaws of a rat and acts for 15s, after tooth application, fasting and water prohibition are performed for half an hour to fix lactobacillus, and the number of lactobacillus is counted by sampling and applying the teeth of the rat>102And (4) the CFU is successfully planted. Then, infection chain changing and whiting are carried out for 5 consecutive days on 8-12 days, wherein the infection (infection) mode is that 200 mu L of bacteria suspension is saturated and absorbed by a sterile cotton stick, the bacteria suspension is applied to the occlusal surfaces of molar teeth of upper and lower jaws of rats and acts for 15s, and the rats are fasted and are forbidden to water for half an hour after tooth application. The prevention group using chlorhexidine is that 0.02% chlorhexidine is used for 5 consecutive days, then variable chain and white mince are infected for 5 consecutive days, and the variable chain and white mince are counted by sampling and coating on days 13 and 14. Then, sucrose was fed for 6 weeks.
The experimental results are as follows:
(1) body weight
Rats were weighed each week and the growth of each group of rats was compared.
As can be seen from the results of fig. 2, the blank control group on the normal diet showed a more significant weight gain and a significantly higher weight gain than the other groups compared to the other caries diet groups, and there was no significant difference in weight gain between the caries diet groups, whether intervention, prevention or model. Suggesting that caries diet and caries model of double-bacteria infection will have an effect on body weight.
(2) Biochemistry of serum
Fasting for 14h before killing mice, killing the rats after anesthesia and neck breaking, taking blood from heart, standing the blood collecting tube for half an hour, centrifuging at 4 ℃ for 15min at 3000Xg to obtain serum, and storing at-20 ℃. Serum indices were measured by a full-automatic biochemical analyzer, and the test items included blood glucose Glu (glucose content in blood), Total Cholesterol (TC), Triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) indices, as shown in table 2.
TABLE 2 Biochemical indices of serum of rats in each group
Tab.2 Serum biochemical indexes of rats in each group
Note: a, b, c and d represent the significance difference P <0.05 between each index group;
as can be seen from table 2, the blank group and the model group have significant differences in each index, wherein only the blood glucose index is decreased after different prevention and treatment means, and the intervention group and the prevention group of CCFM8724 have better effect of decreasing the blood glucose index than the intervention group and the prevention group of chlorhexidine, indicating that CCFM8724 may reduce the side effect of sucrose diet to some extent, and may be one of the ways to prevent and treat dental caries.
(3) Oral flora count in rats
The sampling mode is that a sterile cotton stick is used for scraping a rat oral tooth surface sample in a back-and-forth rotating mode, and dilution coating counting is carried out. The whole experiment has a period of 8 weeks, four times of sampling are carried out in the period, the first sampling is used as the planting test of cariogenic bacteria or lactobacillus plantarum CCFM8724 on the 6 th day, and the second sampling planting test is carried out after the bacteria suspension is treated for another 5 consecutive days, namely the 13 th day of the experiment. The third sampling was day 28 of the experiment and the fourth sampling was the last sampling on day 56 of the experiment, i.e. before the rats were sacrificed. Colony counting is carried out on 3 solid plates in total, and MS (mitis salivaria agar) and 200U/L bacitracin are added to count the streptococcus mutans respectively; adding 20 mu g/ml vancomycin MRS solid culture medium to count lactobacillus in the oral cavity of the rat; candida albicans was counted in BIGGY solid medium (BBL, No.211027, BD Diagnostic Systems).
The counting results are shown in FIG. 3.
As can be seen from fig. 3a and 3b, the counting results of the model groups in the four samples have no significant difference. The first sampling is that after the continuous 5-day colonization of the white ants and the variable chains, the counting results of the model group and the intervention group have no significant difference, and the variable chains and the white ants are significantly reduced after the intervention group conducts the chlorhexidine and the CCFM8724 for the continuous 5-day mediation (P is<0.05). The CCFM8724 intervention group maintained low levels of variable strand in the next three samples, while chlorhexidine failed to inhibit the increase in white memory. From the condition of the prevention group, the colonization of lactobacillus inhibits the colonization of variable chains in the oral cavity, and the colonization level is obviously lower than that of the model group (P)<0.05), and at the third and fourth sampling, the chain-changing quantity of the chlorhexidine prevention group almost rises to the level of the model group, while the lactobacillus plantarum CCFM8724 prevention group always controls the chain-changing order of magnitude of 104The medicine has a better prevention effect than chlorhexidine due to the CFU/mL. The change in the number of candida albicans was not obvious in the two prevention groups, chlorhexidine was apparently not effective against candida albicans, and the initial colonization of candida albicans was not significantly affected after colonization by lactobacillus plantarum CCFM8724, but the level of candida albicans was significantly reduced in the subsequent process. From FIG. 2c the colonization of Lactobacillus plantarum CCFM8724, initially Lactobacillus plantarum CCFM8724 was colonized at 106Around CFU/mL, the existence of pathogenic bacteria does not affect the colonization of the lactobacillus plantarum CCFM8724, and the lactobacillus plantarum CCFM8724 of the intervention group is stabilized at 10 along with the change of time5CFU/mL, and the prevention group was not supplemented with Lactobacillus either after the initial colonization with Lactobacillus plantarum CCFM8724, the amount was stabilized at 104CFU/mL shows that the lactobacillus plantarum CCFM8724 has certain colonization ability in the oral cavity of a rat, and the colonization effect can be enhanced if the lactobacillus plantarum CCFM8724 is supplemented regularly.
(4) Micro-CT detection of rat tooth demineralization condition
The mandible of all rats was imaged with a Micro computerized tomography (Micro CT) system (Quantum Gx 2; Perkinelmer, Hopkinton, MA, USA). Parameters are as follows: 90kv, 88 muA; visual field range: 18 μm; collecting time: 4 min; a camera mode: high resolution. Each sample was rotated 360 ° and all images were imported into Analyze 12.0 software (Analyze direct, Overland Park, KS, USA) to reconstruct a three-dimensional image of the mandible for analysis. Enamel was separated from the entire mandible at a fixed threshold and the volume of enamel was calculated. The three-dimensional reconstruction and section views and the enamel separation are shown in fig. 4, and the volume change of enamel in each group is shown in fig. 5.
As shown in fig. 4, the carious lesion sites of the model group and the absence of stripped enamel are evident from the intact 3D reconstructed hard dental tissue, in contrast to the blank control group with no carious lesions and intact enamel, which was expected for both groups of controls. Comparing the chlorhexidine drying preparation group and the chlorhexidine prevention group, the chlorhexidine prevention group can see obvious carious lesion parts on the graph, while the chlorhexidine drying preparation group, the CCFM8724 intervention group and the CCFM8724 prevention group can not see particularly obvious carious lesion parts relatively, and the depth of the carious lesion is shallow when seen from the section view. The enamel volume was calculated from the stripped enamel to visually present the carious lesion of the enamel, as shown in fig. 5, the four treatment groups all had significant differences (P <0.05) from the enamel loss of the model group, ordered from low to high as carious lesion: CCFM8724 prevention group < CCFM8724 dry run group < chlorhexidine prevention group.
(5) Rat caries score under stereomicroscope
The rat teeth treatment procedure after the experiment was completed was as follows: after anesthesia, the neck is cut off to kill the rat, the skull of the rat is taken out, soft tissues on the jaw bone of the tooth are stripped by using a scalpel, the jaw bone is ultrasonically cleaned for 20min, residual dregs in the slits between the teeth are cleaned, the upper jaw bone and the lower jaw bone are soaked in 10 percent paraformaldehyde for 24h, and the rat is cleaned and dried after the completion. Preparing 0.4% of purple urea ammonium dye solution, soaking the upper and lower jaws in the dye solution for 12h, taking out, rinsing with clear water, air-drying, half-cutting the rat along the upper and lower parts of the rat and the mesial and sagittal parts of molar tooth surfaces, observing and evaluating the condition of the rat molar tooth caries under a body microscope, and scoring according to the Keyes (Journal of dental Research, 1958) scoring standard.
The scoring criteria were as follows: the carious lesion of the rat molars after staining appeared red, while the normal non-carious lesion was either barely stained or very light in color. The cariosity degree of teeth is divided into four grades: grade E caries, caries only in the enamel; ds-grade caries, enamel damage and outer dentin layers with carious extent not exceeding 1/4; dm level caries, the range of caries is 1/4-3/4 dentin; dx grade caries, dentin with a carious extent exceeding 3/4. Rats were divided into several tooth surface units according to buccal surface, lingual surface, proximal surface and alveolar conditions of molars, as shown in table 3. The division of the scores of the bucco-lingual surface and the fossa groove of the lower molar teeth of the rat is schematically shown in FIG. 6. The score of carious lesion which affects dentin is still added with the score of carious lesion which is deeply developed to all levels of dentinal caries on the basis of the score of caries of E level, namely the total score of teeth with deeper carious lesion is the superposition of all levels, the score of caries of E level is not influenced, and the score of caries of D level can never exceed caries of E level. The specific method is briefly described as follows: smooth facial caries include buccal caries, lingual caries, and adjacent facial caries. Wherein, the grading of buccal and lingual surfaces is to roughly estimate the proportion of enamel surface affected by caries (caries depth is detected if necessary, but the depth detection is not made because the experiment mainly studies enamel caries), i.e. the tooth surface is averagely divided into a plurality of equal parts (as shown in figure 6 a), the range of caries lesion exposure is 1 point, and small-range caries lesions with scattered positions can be roughly estimated by an overlay method; each adjacent surface is divided by 1 in one unit, but generally not counted in total because the first molar mesial surface is almost free from caries (but if caries is found, it can be regarded as one unit and divided by 1), and the third molar distal surface is regarded as a continuation of the buccal surface and included in one of the four divided units. The alveolar caries is formed by virtually spreading curves of walls of each alveolar ridge of molars on a longitudinal section, and then dividing the curves into a plurality of linear units (as shown in figure 6 b), wherein each unit is 1 minute, the numbers of the first, second and third alveolar ridges of the lower jaw are 3, 2 and 1 in sequence, and the numbers of the upper jaw are 2, 2 and 1 in sequence. The same jaw specimen is half-cut to obtain both sides of the cheek and tongue, and the groove of the section is scored separately, and the one with the larger score is finally selected as the score of the specimen. The total number of units (i.e., total score) of each carious lesion in the molars of rats is shown in table 3. Note: all scoring results are positive integers without decimal numbers.
TABLE 3 units of molar tooth surface distribution of rats
Note ① 1st2nd3rdRespectively showing the first, second and third molars of the rat
② the second molar of rat comprises a mesial and a distal proximal surface
The teeth of each group of rats were observed and photographed under a stereomicroscope, and caries were scored according to the range of caries destruction, and the scoring results are shown in table 4.
TABLE 4 rat caries score
Note: significant difference from model group (P <0.05)
The dental caries of rat mandible under stereomicroscope is shown in figure 7. From fig. 7, the smooth surface and pit and fissure plots for each group are seen, the red-stained sites are carious lesions, the severity of caries is judged according to the unit score of the red spots, and the blank control is not stained to indicate no carious lesions. From the stereomicroscope results alone, lactobacillus plantarum CCFM8724 exhibited a significant effect, both from a prophylactic and interventional perspective, compared to the severity of the model group, whereas lactobacillus plantarum CCFM634 did not have a significant effect in vivo or even comparable to the model group. From the results of caries scores of all rats (table 4), lactobacillus plantarum CCFM634 unexpectedly had no effect of preventing or interfering with caries, or even had a tendency to aggravate caries, in animal experiments compared to caries models; the other four treatment groups significantly reduced the occurrence of smooth-surfaced and alveolar enamel caries (E), and dentin superficial caries (Ds) except for the chlorhexidine prevention group, which only reduced smooth-surfaced enamel caries (E), indicating that the drug prevention effect was not significant. The lactobacillus plantarum CCMFM8724 shows a remarkable caries-relieving effect in the intervention and prevention groups, even if the caries in dentin (Dm) does not appear in the lactobacillus prevention groups, which indicates that the early blocking of the colonization of cariogenic bacteria is beneficial to the development process of later dental caries. The chlorhexidine intervention group did not significantly reduce the alveolar ridge Dm, and the treatment effect was rather superficial. Generally, the sequence according to the caries prevention and treatment effect is as follows: lactobacillus plantarum prophylaxis CCMFM8724> lactobacillus plantarum CCMFM8724 intervention > chlorhexidine prophylaxis. The lactobacillus plantarum CCFM634 has no effect in vitro experiments whether intervening or preventing, which indicates that the bacteria with effect in vitro experiments can not necessarily exert the same effect in vivo because of the complexity of oral environment and the need for in vivo experimental verification.
Example 2 preparation of a powder containing viable Lactobacillus plantarum CCFM8724 bacteria
Adding lactobacillus plantarum CCFM8724 with an inoculum size of 10% into an MRS liquid fermentation tank for culturing for 12h, controlling the pH value in the fermentation process to be 5.4-5.8, stopping fermentation, cooling to below 20 ℃, collecting thalli by using centrifugal equipment, adding a freeze-drying protective agent (4% of glycerol, 25% of skim milk powder, 4% of ascorbic acid and 1.5% of sodium carbonate) according to the mass ratio of bacterial sludge of 1:1, directly adding the protective agent into the bacterial sludge, balancing and emulsifying for 30 min. And (3) placing the emulsion added with the bacterial sludge into a freeze dryer for freeze drying for 48h at-40 ℃ under 4Pa, and carrying out low-strength crushing and sieving on the freeze-dried product to obtain the high-activity probiotic freeze-dried powder.
Example 3 preparation of yogurt containing viable Lactobacillus plantarum CCFM8724 bacteria
Milk powder, inulin, stevioside and water in a weight ratio: 20: 5: 5: 75, mixing and homogenizing to prepare a fermentation raw material; sterilizing at 121 deg.C for 300sCooling to 42 ℃, inoculating mixed bacterial powder of zymophyte lactobacillus bulgaricus and streptococcus thermophilus, wherein the inoculation amount of the zymophyte is 0.3 percent of the total weight of the fermentation raw materials; fermenting at 42 deg.C for 12 hr, and blending; cooling the fermentation product to 37 deg.C, adding lyophilized powder of Lactobacillus plantarum CCFM8724 with a feeding amount of 109And (3) stirring and canning the CFU/ml yoghourt, preserving at 4 ℃ for 2 days, naturally completing after-ripening, and preparing the probiotic yoghourt.
Example 4 preparation of chewing gum containing viable Lactobacillus plantarum CCFM8724 bacteria
Adding lactobacillus plantarum CCFM8724 into MRS liquid culture medium at an inoculation amount of 4% for culturing for 18 h; centrifugally collecting the cultured probiotics, and freeze-drying the thalli in a freeze dryer at-40 ℃ and 4Pa for 48 hours; freeze-dried powder with probiotic amount of 10 in-80 deg.C refrigerator for use12CFU/g. Softening chewing gum base in 60 deg.C oven for 4 hr, uniformly cooling to 37 deg.C, adding CCFM8724, and sealing by 1010And adding the CFU probiotics/granulated chewing gum according to the proportion, uniformly stirring and cooling, putting into a noodle cutting machine, extruding for 3 times, keeping the temperature at 20 ℃ and the relative humidity at 56%, and aging for 12-22 h to prepare the probiotics chewing gum.
Example 5 preparation of toothpaste containing live Lactobacillus plantarum CCFM8724 bacteria
Adding lactobacillus plantarum CCFM8724 into MRS liquid culture medium at an inoculation amount of 4% for culturing for 18 h; centrifugally collecting the cultured probiotics, and freeze-drying the thalli in a freeze dryer at-40 ℃ and 4Pa for 48 hours; freeze-dried powder with probiotic amount of 10 in-80 deg.C refrigerator for use12CFU/g. Weighing the following raw materials in parts by weight: 100 parts of toothpaste base material, 3 parts of natural mineral salt, 7 parts of probiotics and 40 parts of cranberry extract (concentrated to 1000 mu g/mL) are added into a stirrer to be stirred to obtain a mixture, the mixture is vacuumized and degassed to obtain paste, and the paste is ground to obtain the probiotic toothpaste.
The yogurt prepared according to the method of example 3 above was applied to carious children, the experimental method being: 30 children of 8-12 years old with dental caries confirmed are selected and randomly divided into 2 groups, the control group is given with the commercially available common yoghourt, and the experimental group is given with the yoghourt added with the CCFM8724 live bacteria. All children collected saliva samples on the dorsal side of the tongue before the experiment, and after dilution in a gradient, counted streptococcus mutans on ms (mitis salivaria agar) plates; the experimental group and the control group were fed 100g of yogurt per day. The toothpaste for two groups of children can be used for brushing teeth once every morning and evening, and has no antibacterial component. After 2 months, saliva samples were aspirated from the backs of the tongues of all children, and after dilution of the saliva samples in a gradient, S.mutans was counted on MS (mitis salivaria agar) plates. The results of the experiment are shown in table 5:
TABLE 5 chain-changing horizontal Change in oral cavity of child
| Group of | Before experiment (10)5CFU) | Experiment day 60 (10)5CFU) |
| Control group | 5.5±2.1 | 5.2±2.5 |
| Experimental group | 5.8±1.9 | 0.3±1.2* |
Note: indicates that the difference between the experimental group and the control group is significant (P <0.05)
The experimental results show that after the yoghourt containing the CCFM8724 live bacteria is eaten, the number of streptococcus mutans in the oral cavity of the decayed children is obviously reduced, and the application product of the bacteria can effectively resist the growth condition of the mutans in the oral cavity and relieve the decayed tooth condition of the children.
Similarly, experiments prove that the examples 2, 4 and 5 can reduce the level of the number of the variable chains in the oral cavity of a human body, and prove that the strain of lactobacillus plantarum CCFM8724 has the effect of remarkably preventing and treating dental caries no matter in animal experiments or human experiments.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. Application of lactobacillus plantarum CCFM8724 in preparation of articles for preventing and treating dental caries.
2. The use according to claim 1, wherein the dental caries is a dental caries caused by a streptococcus mutans and candida albicans dual infection.
3. Use according to claim 1, wherein the articles comprise functional food, daily chemical products.
4. The yoghourt with the effects of preventing and treating the decayed teeth is characterized by comprising the following preparation steps: mixing milk powder, inulin, stevioside and water, homogenizing, making into fermentation raw material, sterilizing, cooling, inoculating mixed powder of Lactobacillus bulgaricus and Streptococcus thermophilus, fermenting, adding freeze-dried powder of Lactobacillus plantarum CCFM8724, stirring, canning, and aging to obtain probiotic yogurt with dental caries preventing and treating effects.
5. A chewing gum with the functions of preventing and treating dental caries, which is characterized in that the preparation steps of the chewing gum comprise: softening chewing gum base, adding Lactobacillus plantarum CCFM8724, and processing by 1010And adding the CFU probiotics/granulated chewing gum according to the proportion, uniformly stirring, cooling, extruding and cutting to prepare the probiotic chewing gum.
6. A chewing gum for preventing and treating dental caries according to claim 5, wherein substances having inhibitory effect on Streptococcus mutans are further added to the chewing gum, and the substances include: cranberry extract.
7. A toothpaste with the effects of preventing and treating dental caries is characterized in that the preparation steps of the toothpaste comprise: stirring toothpaste base material, natural mineral salt and Lactobacillus plantarum CCFM8724 to obtain a mixture, vacuumizing the mixture, degassing to obtain paste, and grinding to obtain the probiotic toothpaste.
8. The toothpaste with dental caries preventing and treating effect as claimed in claim 7, wherein substances with streptococcus mutans inhibiting effect are further added to the toothpaste, and comprise: cranberry extract.
9. A rat dental caries model is characterized in that the model is constructed by the following method: selecting an SPF-grade Wistar rat, a male Wistar rat, a 3-week-old Wistar rat and a body weight of 40-50 g, and infecting the rat with streptococcus mutans and candida albicans once a day for 5 consecutive days; the specific operation method of infection is to apply bacterial suspension of streptococcus mutans and candida albicans to the occlusal surfaces of molar teeth of upper and lower jaws of rats, apply the bacterial suspension for 15s, and fast and forbid water for half an hour after applying the bacterial suspension to the teeth.
10. The method for screening substances having the effects of preventing and treating dental caries by using the rat dental caries model according to claim 9, wherein rats of the rat dental caries model are divided into a model group and a preparation group, wherein the model group is fed with a cariogenic feed and sucrose is added to diet, and the preparation group is fed with the cariogenic feed, sucrose is added to diet and the treatment is carried out by using the substances to be screened; in addition, a blank control group and an intervention group are set, wherein the blank control group is not infected by streptococcus mutans and candida albicans, the blank control group is fed with a cariogenic feed and is added with cane sugar in the diet, the intervention group is firstly treated with a substance to be screened and is then infected with the streptococcus mutans and candida albicans, and the intervention group is fed with the cariogenic feed and is added with cane sugar in the diet; during the feeding process, the weight, the serum biochemical indexes and the oral flora of the rat are monitored, after the feeding is finished, the tooth demineralization condition of the rat is detected by utilizing Micro-CT, and the tooth caries condition of the rat is observed by utilizing a body type microscope.
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