CN106834414A - The quantitative detecting method of hydrogen sulfide producing strains and application in a kind of oral cavity - Google Patents
The quantitative detecting method of hydrogen sulfide producing strains and application in a kind of oral cavity Download PDFInfo
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- CN106834414A CN106834414A CN201710127320.7A CN201710127320A CN106834414A CN 106834414 A CN106834414 A CN 106834414A CN 201710127320 A CN201710127320 A CN 201710127320A CN 106834414 A CN106834414 A CN 106834414A
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- 238000000034 method Methods 0.000 title claims description 30
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 title claims description 27
- 229910000037 hydrogen sulfide Inorganic materials 0.000 title claims description 27
- 210000000214 mouth Anatomy 0.000 title claims description 18
- 241000894006 Bacteria Species 0.000 claims description 32
- 239000001963 growth medium Substances 0.000 claims description 25
- 239000007864 aqueous solution Substances 0.000 claims description 24
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 22
- 108010024636 Glutathione Proteins 0.000 claims description 18
- 230000001954 sterilising effect Effects 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 17
- 229920001817 Agar Polymers 0.000 claims description 16
- 239000008272 agar Substances 0.000 claims description 16
- 238000004659 sterilization and disinfection Methods 0.000 claims description 15
- OOMYACICIIMLQI-UHFFFAOYSA-L lead(2+);diacetate;hydrate Chemical compound O.[Pb+2].CC([O-])=O.CC([O-])=O OOMYACICIIMLQI-UHFFFAOYSA-L 0.000 claims description 14
- 229940046892 lead acetate Drugs 0.000 claims description 13
- 238000012360 testing method Methods 0.000 claims description 13
- 210000003296 saliva Anatomy 0.000 claims description 12
- 210000004556 brain Anatomy 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 238000001802 infusion Methods 0.000 claims description 9
- 235000010469 Glycine max Nutrition 0.000 claims description 6
- 230000002829 reductive effect Effects 0.000 claims description 6
- 244000068988 Glycine max Species 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 3
- 230000002906 microbiologic effect Effects 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 235000009508 confectionery Nutrition 0.000 claims 1
- 206010006326 Breath odour Diseases 0.000 description 22
- 208000032139 Halitosis Diseases 0.000 description 20
- 239000000523 sample Substances 0.000 description 20
- 239000000047 product Substances 0.000 description 13
- 229940034610 toothpaste Drugs 0.000 description 10
- 239000000606 toothpaste Substances 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 7
- 229910052739 hydrogen Inorganic materials 0.000 description 7
- 239000001257 hydrogen Substances 0.000 description 7
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 6
- 238000009472 formulation Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000004073 vulcanization Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 229960003180 glutathione Drugs 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 241000186046 Actinomyces Species 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- -1 Dichlorodiphenyl Acetate lead Chemical compound 0.000 description 2
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 description 2
- 241000605862 Porphyromonas gingivalis Species 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000002845 discoloration Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000012898 sample dilution Substances 0.000 description 2
- 210000000697 sensory organ Anatomy 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 244000235858 Acetobacter xylinum Species 0.000 description 1
- 241000606749 Aggregatibacter actinomycetemcomitans Species 0.000 description 1
- 241000239290 Araneae Species 0.000 description 1
- 241000606125 Bacteroides Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000258920 Chilopoda Species 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000186394 Eubacterium Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000192035 Peptostreptococcus anaerobius Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000605008 Spirillum Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241000589892 Treponema denticola Species 0.000 description 1
- 241001148134 Veillonella Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229940029329 intrinsic factor Drugs 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229940074571 peptostreptococcus anaerobius Drugs 0.000 description 1
- 230000003239 periodontal effect Effects 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108010009004 proteose-peptone Proteins 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 150000003464 sulfur compounds Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000016776 visual perception Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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- Chemical & Material Sciences (AREA)
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Abstract
The present invention discloses quantitative detecting method and the application of hydrogen sulfide producing strains in a kind of oral cavity, the quantitative detecting method is the culture medium based on tryptose soya agar culture medium or brain heart infusion agar culture medium, and it is down flat plate after adding lead acetate, hypo reduced glutathione, cultivated after the saliva sample or tongue fur sample spread plate in patient oral cavity, by colony counting so as to realize the quantitative determination of hydrogen sulfide producing strains in oral cavity.The present invention is capable of achieving the quantization detection to hydrogen sulfide producing strains in oral cavity, and data reliability is simple, with low cost using equipment, less demanding to operating personnel, can be applied to the preparation and evaluation and test of the oral care product for the treatment of halitosis.
Description
Technical field
The present invention relates to oral care techniques, more particularly to halitosis, implication research, and in particular to vulcanize in a kind of oral cavity
The quantitative detecting method of hydrogen producing strains and application.
Background technology
Implication, also known as halitosis, are common a kind of diseases in crowd, and pathogenic factor has whole body and local, it was reported that
The patient that pathogenic factor belongs to oral cavity intrinsic factor accounts for 80%, and wherein periodontitis and oulitis patient respectively accounts for 50%.Bacterium in oral cavity
It is the main cause for causing halitosis to be acted on the protein in saliva, bacterial plaque and to form the volatile sulfur compoundses such as hydrogen sulfide.Halitosis gas
The Main Ingredients and Appearance of taste is volatile sulfur compounds, wherein 90% is hydrogen sulfide (H2) and methyl mercaptan S.Bacterium in oral cavity mainly has：
Staphylococcus, streptococcus, Strep.salivarius, actinomyces, corynebacteria, model forever coccus, Bacillus acidi lactici, fusiform bacilarmature, spirillum, how
Plucked instrument bacterium and bacterium actinomycetem comitans etc., wherein quantity are most, be most importantly streptococcus, staphylococcus and Bacillus acidi lactici.
Producing the bacterium of hydrogen sulfide mainly includes porphyromonas gingivalis, tooth dirt conveyor screw and the fertile bacterium of middle Prey.Tongue fur
The upper bacterium for producing hydrogen sulfide is mainly the fertile Pseudomonas of Veillonella, actinomyces and Prey, these bacteriums in halitosis patient oral cavity
Quantity apparently higher than healthy population.The bacterium of hydrogen sulfide is produced in cysteine mainly peptostreptococcus anaerobius, small
Prey irrigates bacterium, mucus Eubacterium, periodontal centipede bacterium, the crescent monad of spider shape and Bacteroides, and vulcanization is produced in serum
The bacterium of hydrogen has middle Prey to irrigate bacterium, Rockwell Prey fertile bacterium, porphyromonas gingivalis and treponema denticola.
Halitosis can bring many puzzlements to life, and the method that halitosis is evaluated at present mainly has three kinds:(1) sense organ of people is commented
Valency, scoring is provided mainly by way of hearing according to the order of severity of testee's implication, but the method needs are experienced
People can just provide accurate scoring, and the sense organ of people varies with each individual, and with very big subjectivity, data accuracy is low；(2) it is thin
Bacterium analytic approach, takes tongue fur or back sample, uses special media culture, identifies some special strains and colony counting, but should
Method will carry out strain idenfication to the bacterium colony on different choice culture medium, and complex steps, experimental period is long；(3) sulfide is determined
Method, mainly determines the content of sulfide, but instrument using portable sulfide analyzer Halimeter or gas chromatography
Equipment funds input is larger, and operating personnel is required high.
The research report for being related to simple halitosis experimental model refer to Lead acetate paper method, and demonstrate lead acetate method with
The correlation of Halimeter methods, experiment prove the sulfide content that Lead acetate paper discoloration and Halimeter are measured have compared with
Good positive correlation, it is a kind of reliable method to illustrate that lead acetate method is said in principle, but this report merely by
Visual perception's Dichlorodiphenyl Acetate lead indicating paper discoloration of people is evaluated simply to judge halitosis, it is impossible to realize bacterium
Quantitative statisticses and analysis, so as to cannot be effectively treated to halitosis.
The content of the invention
Present invention aim in view of the shortcomings of the prior art, there is provided a kind of simple to operate, data are reliable, with low cost
, the method that quantitative determination can be carried out to the bacterium of generation hydrogen sulfide in oral cavity.
It is a further object to provide the application of above-mentioned quantitative detecting method.
Above-mentioned purpose of the invention is achieved by following scheme：
A kind of quantitative detecting method of hydrogen sulfide producing strains in oral cavity, the detection method comprises the following steps：
Step 1
Tryptose soya agar culture medium or brain heart infusion agar culture are based on 115 DEG C, 20 points of high-temp steam sterilizing
Zhong Hou, it is standby；
Prepare lead acetate water solution, the hypo aqueous solution and the reduced glutathione aqueous solution；
Step 2
Lead acetate water solution, the hypo aqueous solution and the reduced glutathione that step 1 is prepared are water-soluble
Liquid is down flat plate respectively through being added in the culture medium after step 1 sterilization treatment after filter bacteria removing after being well mixed；
Step 3
Flat board prepared by testing sample application step 2, after 37 DEG C of incubated 36~48h, carries out colony counting.
In above-mentioned steps 1, the tryptose soya agar culture medium uses commercially available tryptose soya agar culture
Base is to be capable of achieving the present invention, it is also possible to which the conventional formulation according to tryptose soya agar culture medium is prepared：Every liter of tryptone
Soy agar culture medium contains tryptone 15g, soy peptone 5g, sodium chloride 5g and agar 15g, pH7.3 ± 0.2,25 DEG C.
In above-mentioned steps 1, the brain heart infusion agar culture medium can be real using commercially available brain heart infusion agar culture medium
The existing present invention, it is also possible to which the conventional formulation according to brain heart infusion agar culture medium is prepared：Every liter of brain heart infusion agar culture medium contains
There is bovine brain to soak powder 4g, beef heart infusion 4g, peptone 5g, casein peptone 16g, sodium chloride 5g, glucose 2g, sulfuric acid disodium hydrogen 2.5g
With agar 13.5g, 7.4 ± 0.2,25 DEG C of pH value.
In above-mentioned steps 1, lead acetate, hypo and reductive glutathione are commercially available prod.
In above-mentioned steps 1, lead acetate water solution is the lead acetate water solution that lead acetate mass percent concentration is 10%, five
Hydration sodium thiosulfate solution is the hypo that hypo mass percent concentration is 10%
The aqueous solution, the reduced glutathione aqueous solution is the reduced form gluathione that reduced glutathione mass percent concentration is 25%
The peptide aqueous solution；In above-mentioned steps 2, by lead acetate water solution, the hypo aqueous solution and reduced glutathione water
Solution respectively through being added in the culture medium after step 1 sterilization treatment after filter bacteria removing, the lead acetate water solution and training
The volume ratio for supporting base is 1:50, the volume ratio of the hypo aqueous solution and culture medium is 1:100, the reduction
The volume ratio of the type glutathione aqueous solution and culture medium is 1:100.
In above-mentioned steps 2, the filtration sterilization is to carry out filtration sterilization using 0.22 μm of filter membrane.
In above-mentioned steps 3, testing sample is patient's saliva of buccal cavity or tongue fur sample.
In above-mentioned steps 3, testing sample draws 100 μ L and is coated putting down after needing first to dilute 10~100 times before the spread plate
Plate, dilution ratio during concrete operations is that the microbe quantity magnitude contained according to saliva or tongue fur sample determines that control is final
Plate microbiological bacterium colony is between 30~300.The testing sample dilution is to treat test sample using 0.85% physiological saline
Product are diluted.
The present inventor's research discovery, flat board prepared by step 2,37 DEG C of incubated 36~48h are applied to by testing sample
Afterwards, many colony growths are had, but the bacterial clump for only producing hydrogen sulfide is black, because hydrogen sulfide reacts life with lead acetate
Into vulcanized lead black precipitate, and remaining non-bacterial clump for producing hydrogen sulfide is for white or faint yellow, colouring discrimination clearly, because
Black colonies need to only be counted by this, you can realize the accurate metering of the bacterial clump to producing hydrogen sulfide.
Quantitative detecting method of the invention can be used for different formulations or different brands oral care product to problem of bad breath
Regulation effect detection, such as inventor's research finds, hydrogen sulfide bacterial number produced in the saliva of general halitosis patient and is averagely existed
2300cfu/mL or so, used the toothpaste of removing foul breath after two weeks, hydrogen sulfide bacterial number majority is produced in saliva and drops to 50cfu/
Below mL, therefore, the oral care product of different formulations or different brands is tried out, if after on probation two weeks, patient's saliva
Hydrogen sulfide bacterial number is produced in liquid can drop to below 50cfu/mL, then illustrate that the formula or the brand have certain treatment mouthful
Smelly effect.
Quantitative detecting method of the invention can also be used to instruct the research and development of oral care product, such as by different formulations
Quantitative determination after oral care product is on probation, accurately to judge which formula can effectively treat halitosis.
Quantitative detecting method of the invention can also be used to realize the accurate treatment of halitosis patient, such as remove halitosis on the market
Oral care product it is a lot, but be not that each product can be effective, so by not for patient individual
After with brand try out, using quantitative detecting method of the invention, can accurately judge that the product of which brand can be effective
The quantity of product vulcanization hydrogen bacteria in patient oral cavity is reduced, so that for patient's amount suitable MC for removing halitosis of body selection
Product.
Compared with prior art, the present invention has the advantages that：
1. the present invention is by the selection to culture medium, lead acetate, hypo and reductive glutathione
Selection and consumption, and condition of culture selection, it is ensured that the bacterium for producing hydrogen sulfide can form black colonies, rather than produce hydrogen sulfide
Bacterium will not produce black colonies, so as to clearly exactly will produce hydrogen sulfide bacterium and other bacteriums be distinguished, protect
The present invention has been demonstrate,proved to producing the accuracy of hydrogen sulfide bacteria quantified detection.
2nd, also hypo and reductive glutathione are added in selection in basal medium of the invention, so that
Can effectively protect lead acetate not oxidized, realize the stabilization performance of lead acetate effect, ensure the accuracy of quantitative determination.
3. of the invention by bacterium colony culture and colony counting, realize the quantitative statisticses of product vulcanization hydrogen bacteria in oral cavity and divide
Analysis, it is achieved thereby that being effectively treated to halitosis.
4. present invention testing equipment used is simple, with low cost, less demanding to operating personnel, and data reliability, favorably
In large-scale promotion.
Specific embodiment
The present invention is further described through with reference to specific embodiment, but specific embodiment is not appointed to the present invention
What is limited.
Embodiment 1
Experimental subjects：The people of halitosis patient 6.
The present embodiment is separately sampled to 6 halitosis patients, and every patient will take four testing samples, institute specific as follows
Show：
Sample 1：Saliva first to patient before the patient uses removing foul breath toothpaste samples, and the saliva sample is the patient's
Sample 1；
Sample 2：Tongue fur first to patient before the patient uses removing foul breath toothpaste is sampled, and the tongue fur sample is the patient's
Sample 2；
Sample 3：The patient is allowed to use the toothpaste of a removing foul breath after two weeks, the saliva to the patient samples, the saliva sample
Product are the sample 3 of the patient；
Sample 4：The patient is allowed to use the toothpaste of a removing foul breath after two weeks, the tongue fur to the patient is sampled, the tongue fur sample
Product are the sample 4 of the patient.
6 patients are used with a removing foul breath toothpaste, and the application method of toothpaste is all respectively to brush teeth the daily morning, noon and afternoon
Once, two weeks by a definite date.
Then all testing samples are carried out into quantitative determination according to the method for the present invention, is concretely comprised the following steps：
Step 1
Brain heart infusion agar culture is based on 115 DEG C, high-temp steam sterilizing is standby after 20 minutes；
Prepare the lead acetate water solution that lead acetate mass percent concentration is 10%；
Prepare the hypo aqueous solution that hypo mass percent concentration is 10%；
Prepare the reduced glutathione aqueous solution that reduced glutathione mass percent concentration is 25%；
Step 2
The lead acetate water solution that step 1 is prepared is carried out after filtration sterilization according to 1 using 0.22 μm of filter membrane:50 volume
Than being added in the culture medium after step 1 sterilization treatment, the hypo aqueous solution that step 1 is prepared uses 0.22
μm filter membrane carry out after filtration sterilization according to 1:100 volume ratio is added in the culture medium after step 1 sterilization treatment, is just walked
The rapid 1 reduced glutathione aqueous solution prepared is carried out after filtration sterilization according to 1 using 0.22 μm of filter membrane:100 volume ratio
It is added in the culture medium after step 1 sterilization treatment, plate is down flat after then culture medium is well mixed；
Step 3
By testing sample dilution 10 times draw flat board prepared by 100 μ L application steps 2 afterwards after, after 37 DEG C of incubated 48h,
Carry out the counting of black colonies.
In above-mentioned steps 1, brain heart infusion agar culture medium, lead acetate, hypo and reproducibility gluathione
Peptide is commercially available prod.
Result is as shown in table 1.
Table 1 produces H2S counts of bacteria
From the results shown in Table 1, this removing foul breath toothpaste is best to No. 4 and No. 6 removal halitosis effects of patient, and
For No. 3 patients, this obvious therapeutic effect of removing foul breath toothpaste is less desirable, so No. 3 patients need to consider to change other
Brand removing foul breath toothpaste.
Also can clearly find out the generating unit of the main halitosis of each patient from the result of table 1, such as No. 1 patient, mainly
It is more containing vulcanization hydrogen bacteria is produced in saliva, therefore, for No. 1 patient, it is possible to correspondingly controlled for this position
Treat.
In sum, the quantitative statisticses to producing vulcanization hydrogen bacteria, and this can actually be accomplished using the method for the present invention
Plant quantitative statisticses and very positive effect is played to Bad Breath Treatment really.
The announcement and teaching of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula is changed and changed.Therefore, the invention is not limited in specific embodiment disclosed and described above, to of the invention
Some modifications and changes should also be as falling into scope of the claims of the invention.Although additionally, being used in this specification
Some specific terms, but these terms are merely for convenience of description, do not constitute any limitation to the present invention.
Claims (6)
1. in a kind of oral cavity hydrogen sulfide producing strains quantitative detecting method, it is characterised in that the quantitative detecting method includes following step
Suddenly：
Step 1
Tryptose soya agar culture medium or brain heart infusion agar culture are based on 115 DEG C, high-temp steam sterilizing is after 20 minutes,
It is standby；
Prepare lead acetate water solution, the hypo aqueous solution and the reduced glutathione aqueous solution；
Step 2
Lead acetate water solution, the hypo aqueous solution and the reduced glutathione aqueous solution that step 1 is prepared point
It is added in the culture medium after step 1 sterilization treatment after not processed through filtration sterilization, plate is down flat after being well mixed；
Step 3
Black colonies after 37 DEG C of incubated 36~48h, are counted by flat board prepared by testing sample application step 2.
2. according to claim 1 in oral cavity hydrogen sulfide producing strains quantitative detecting method, it is characterised in that the step 1
In, lead acetate water solution is the lead acetate water solution that lead acetate mass percent concentration is 10%, hypo water
Solution is the hypo aqueous solution that hypo mass percent concentration is 10%, reduced form paddy Guang
The sweet peptide aqueous solution is the reduced glutathione aqueous solution that reduced glutathione mass percent concentration is 25%；The step
In 2, by lead acetate water solution, the hypo aqueous solution and the reduced glutathione aqueous solution respectively through filtering bacterium
It is added to after treatment in the culture medium after step 1 sterilization treatment, the volume ratio of the lead acetate water solution and culture medium is 1:50,
The volume ratio of the hypo aqueous solution and culture medium is 1:100, the reduced glutathione aqueous solution and
The volume ratio of culture medium is 1:100.
3. according to claim 1 in oral cavity hydrogen sulfide producing strains quantitative detecting method, it is characterised in that the step 2
In, filtration sterilization is to carry out filtration sterilization using 0.22 μm of filter membrane.
4. according to claim 1 in oral cavity hydrogen sulfide producing strains quantitative detecting method, it is characterised in that the step 3
In, testing sample is patient's saliva of buccal cavity or tongue fur sample.
5. according to claim 1 in oral cavity hydrogen sulfide producing strains quantitative detecting method, it is characterised in that the step 3
In, testing sample draws 100 μ L and is coated flat board after needing first to dilute 10~100 times before the spread plate, control final flat board micro-
Biological bacterium colony is between 30~300.
6. the quantitative detecting method of hydrogen sulfide producing strains is preparing treatment mouth in a kind of oral cavity of any one described in claim 1-5
Application in smelly oral care product.
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| CN201710127320.7A CN106834414A (en) | 2017-03-03 | 2017-03-03 | The quantitative detecting method of hydrogen sulfide producing strains and application in a kind of oral cavity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104023732A (en) * | 2011-08-05 | 2014-09-03 | 株式会社养乐多本社 | Prophylactic or therapeutic agent for oral diseases |
| CN104480029A (en) * | 2014-11-20 | 2015-04-01 | 西北农林科技大学 | Wine yeast capable of low-yielding hydrogen sulfide and ethyl carbamate as well as screening method and application of wine yeast |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104023732A (en) * | 2011-08-05 | 2014-09-03 | 株式会社养乐多本社 | Prophylactic or therapeutic agent for oral diseases |
| CN104480029A (en) * | 2014-11-20 | 2015-04-01 | 西北农林科技大学 | Wine yeast capable of low-yielding hydrogen sulfide and ethyl carbamate as well as screening method and application of wine yeast |
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