CN113913348B - Klebsiella grignard SA34 and application thereof - Google Patents
Klebsiella grignard SA34 and application thereof Download PDFInfo
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- CN113913348B CN113913348B CN202111413968.3A CN202111413968A CN113913348B CN 113913348 B CN113913348 B CN 113913348B CN 202111413968 A CN202111413968 A CN 202111413968A CN 113913348 B CN113913348 B CN 113913348B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/26—Klebsiella (G)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
Abstract
The invention discloses Klebsiella grignard SA34 and application thereof, wherein the preservation number of the Klebsiella grignard SA34 is CGMCC No.21719. The Klebsiella grignard SA34 can generate hydrogen sulfide, methyl mercaptan and other gases, can be applied to screening of functional additives or buccal cigarette products for inhibiting gas production, and has good application prospects in research of novel functional additives, research and development of buccal cigarette products and formula adjustment of the buccal cigarette products.
Description
Technical Field
The invention relates to the field of oral microorganisms, in particular to Klebsiella grignard SA34 and application thereof.
Background
The oral microbiota is a collection of microorganisms that are designated to be planted in the oral cavity of a human body, and these microorganisms often form a complex community in the form of a biofilm that performs the physiological functions of the microorganisms. The structural features of the oral salivary microflora and its homeostasis are closely related to the oral and systemic health of the host.
The oral cavity connects the inside of the human body with the outside, and the community composition of saliva microorganism is complex and the variety is rich. Research reports show that various microorganisms existing in the oral cavity can generate chemical components related to halitosis such as hydrogen sulfide, methyl mercaptan, methyl sulfide and the like in the metabolic process, and the microorganisms mainly comprise clostridium, haemophilus, veillonella, clostridium perfringens and the like. The oral gas-producing bacteria are often used as clinical monitoring bacteria for preventing and treating oral diseases.
Because saliva microecology is very complex, a great number of saliva microorganisms, particularly gas-producing microorganisms, are difficult to separate and identify, so that the variety of gas-producing strains required for carrying out experiments related to halitosis, gas-producing inhibition screening and the like is not abundant enough.
Therefore, how to provide a saliva gas-producing microorganism with a strong gas production capacity for a gas production inhibition screening experiment becomes a technical problem to be solved in the art.
Disclosure of Invention
The invention aims to provide a novel technical scheme of Klebsiella grignard SA34 with strong gas production capacity for gas production inhibition screening experiments.
According to a first aspect of the present invention, there is provided klebsiella grisea (Klebsiella grimontii) SA34 having a preservation number of CGMCC No.21719.
According to a second aspect of the present invention, there is provided a microbial preparation comprising klebsiella grisea SA34 according to the present invention.
According to a third aspect of the invention, there is provided the use of klebsiella grisea SA34 or a microbial preparation according to the invention for screening functional additives or buccal tobacco products for inhibiting gas production.
According to a fourth aspect of the invention, there is provided an application of Klebsiella grignard SA34 in screening functional additives or buccal tobacco products for inhibiting gas production, wherein Klebsiella grignard (Klebsiella grimontii) SA34 with a preservation number of CGMCC No.21719 is prepared into a bacterial suspension, and then the bacterial suspension and a test sample solution are placed in a centrifuge tube containing a Dunaliella for co-culturing for 1d-7d.
Optionally, the method comprises the following steps:
step (1): streaking Klebsiella grignard SA34 on a TSA culture medium plate for activation culture;
step (2): picking upSingle colony obtained by streak culture is inoculated in TSB culture medium and shake cultured to OD 600 The value is between 0.6 and 1.0, and the strain culture solution is obtained;
step (3): diluting the strain culture solution to prepare a bacterial suspension;
step (4): preparing a test sample into a solution or a suspension;
step (5): the bacterial suspension and the test sample are prepared into solution or suspension, and the solution or suspension is transferred into a centrifuge tube containing a Du's tubule and placed into a constant temperature incubator for culturing for 1d to 7d.
Optionally, the activation culture temperature in the step (1) is 36 ℃ and the time is 16-24 h.
Optionally, the parameters of the shake culture in the step (2) are as follows: 36℃at 150rpm for 16-24 h.
Optionally, the step (4) specifically includes:
and adding the test sample and sterile physiological saline into a sterile homogenizing bag to prepare a test sample solution or suspension.
Optionally, the temperature of the culturing in step (5) is 36 ℃.
The Klebsiella grignard SA34 can generate hydrogen sulfide, methyl mercaptan and other gases, can be applied to screening of functional additives or buccal cigarette products for inhibiting gas production, and has good application prospects in research of novel functional additives, research and development of buccal cigarette products and formula adjustment of the buccal cigarette products.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail. It should be noted that: the relative arrangement of the components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless it is specifically stated otherwise.
The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.
Techniques, methods, and apparatus known to one of ordinary skill in the relevant art may not be discussed in detail, but are intended to be part of the specification where appropriate.
In all examples shown and discussed herein, any specific values should be construed as merely illustrative, and not a limitation. Thus, other examples of exemplary embodiments may have different values.
The invention provides Klebsiella grignard (Klebsiella grimontii) SA34 which is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.21719, the strain name is Klebsiella grignard, the strain number is SA34, the classification is Klebsiella grimontii, and the preservation time is 2021, 01 and 15 days.
The invention also provides a microbial preparation comprising the klebsiella grisea SA34.
The invention also provides application of the Klebsiella grignard SA34 or a microbial preparation in screening of functional additives or buccal cigarette products for inhibiting gas production.
The invention also provides application of the Klebsiella grignard SA34 in screening functional additives or buccal cigarette products for inhibiting gas production, wherein the Klebsiella grignard SA34 with the preservation number of CGMCC No.21719 is firstly prepared into bacterial suspension, and then the bacterial suspension and a test sample solution are placed into a centrifuge tube containing a Du's tubule to be co-cultured for 1d-7d.
The above application may comprise the steps of:
step (1): klebsiella grignard SA34 was streaked onto TSA medium plates for activation culture.
The activation culture temperature in the step (1) can be 36 ℃ and the time is 16-24 hours.
Step (2): picking single colony obtained by streak culture, inoculating into TSB culture medium, shake culturing to OD 600 The value is between 0.6 and 1.0, and the strain culture solution is obtained.
The parameters of the shake culture in step (2) may be as follows: 36℃at 150rpm for 16-24 h.
Step (3): diluting the strain culture solution to prepare a bacterial suspension.
Step (4): the test sample is formulated as a solution or suspension.
Step (4) may be specifically as follows:
and adding the test sample and sterile physiological saline into a sterile homogenizing bag to prepare a test sample solution or suspension.
Step (5): the bacterial suspension and the test sample are prepared into solution or suspension, and the solution or suspension is transferred into a centrifuge tube containing a Du's tubule and placed into a constant temperature incubator for culturing for 1d to 7d.
The temperature of the cultivation in step (5) may be 36 ℃.
The experimental procedures used in the examples below are conventional, and the materials and reagents used, unless otherwise indicated, are commercially available, and the equipment used in the experiments, unless otherwise indicated, are well known to those skilled in the art.
1. Screening and isolation of klebsiella grisea SA34.
1. Culture medium
Brain heart extract agar medium (BHI), trypticase soy agar medium (TSA), trypticase Soy Broth (TSB), anaerobic agar medium, anaerobic broth, blood agar plate (beijing land bridge technologies inc.); EG medium (Haibo).
2. Apparatus and device
Incubator (memmerert company, germany); ultra clean bench (sutake air technologies limited); autoclaving (Pinus koraiensis Co., japan); a synergy microplate reader (BioTek company, usa); electronic balance (Sidoris Corp., germany); du Shixiao pipe (Kangtai laboratory equipment Co., haimei); a conventional PCR instrument (APPLIED BIOSYSTEMS); a gel imaging system (Azure Biosystems US); electrophoresis apparatus (Galileo bioscience).
3. Strain selection and isolation
(1) Saliva collection: referring to the HMP standard saliva collection regimen, 10 volunteers forbidden food and drink water 1h before saliva collection, and at least 1mL of saliva was collected into a sterile centrifuge tube, placed at 4℃and saliva sample pretreatment was completed within 1 h.
(2) Pretreatment of saliva samples: saliva from 10 volunteers was diluted with 10-fold gradient using sterile physiological saline (0.9% NaCl), respectively.
(3) Coating the culture medium: select 10, 10 2 、10 3 、10 4 The saliva sample with multiple dilution gradient is respectively absorbed into 150 mu L of diluent, and is sequentially coated with BHI culture medium, TSA culture medium, anaerobic agar culture medium, EG culture medium and blood agar plate, and each culture medium is respectively coated with 4 dishes of parallel for each dilution; then the sterile film cut into proper shape is lightly stuck on the culture medium, 2 dishes of each plate treatment are placed in parallel in a constant temperature incubator at 37 ℃ to be respectively subjected to aerobic culture (normally placed in the constant temperature incubator) and anaerobic culture (placed in an anaerobic gas producing tank and then placed in the constant temperature incubator).
(4) Gas production observation and strain separation: and (3) observing the growth condition of the bacterial colony on the flat plate at regular time, if bubbles appear around the bacterial colony, slightly lifting the film, picking up the gas-producing bacterial colony by using a sterile needle, and carrying out streaking separation on a corresponding culture medium until a pure bacterial strain is obtained.
(5) And (3) verifying and screening the gas production capacity: pure strains are selected and respectively inoculated into corresponding liquid culture mediums for placing Du's fermentation tubes, the culture is carried out for 48 hours in a constant temperature incubator at 37 ℃, the gas production time and gas production quantity of gas-producing microorganisms are recorded, and the gas production capacity is judged, and the obtained strain is named SA34.
2. Identification of Strain SA34.
1. Morphological and physiological Biochemical identification
On TSA plates, colonies were small, round, smooth in surface, clean in edge, yellowish. Strain morphology: gram staining was negative, short bar. Physiological and biochemical characteristics: glucose fermentation positive, arginine ditolylase negative, urease test positive, gelatin hydrolysis test negative, beta-galactosidase positive, glucose, arabinose, mannose and maltose assimilation positive, sodium citrate and adipic acid assimilation negative.
2. Molecular biological identification
SA34 genomic DNA was extracted using a bacterial genome extraction kit, and the 16S rRNA gene was amplified using amplification primers 20F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1500R (5'-GGTTACCTTGTTACGACT-3') of the 16S rRNA gene sequence under the following PCR amplification conditions: 94 ℃ for 4min;94℃30s,55℃30s,72℃90s,32 cycles; and at 72℃for 5min. After sequencing the PCR products, an on-line comparison analysis was performed in NCBI database (https:// www.ncbi.nlm.nih.gov /). The result shows that the PCR product has 1442bp in length and has very high homology (99%) with Klebsiella grignard (Klebsiella grimontii) in NCBI database. Based on the above results, SA34 was identified as Klebsiella grisei. The sequence table of the SrDNA of the Klebsiella grignard SA34 is SEQ ID NO. 1,GenBank NO.MW521132.
3. Analysis of carbon source metabolism
The carbon source metabolism condition of the strain SA34 is inspected by using a BIOLOG Gen III identification plate, and the result shows that when the strain SA34 is cultivated for 24 hours, the average change rate (AWCD) of the color of the carbon source reaches the highest value, then gradually drops along with the time, and the cultivation is smooth when the strain SA34 is cultivated for 72 hours to 96 hours. The strain SA34 has carbon source of 77.5% and carbon source utilization rate of 77.5%, and has highest metabolism ability to saccharides, and the second has carboxylic acid and its esters and amino acids, wherein the saccharides comprise 23 kinds of D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-melibiose, stachyose, and raffinose, and the amino acids comprise 6 kinds of D-serine, L-alanine, L-aspartic acid, L-glutamic acid, L-histamine, and L-serine, and the carboxylic acid and esters comprise 16 kinds of D-galacturonic acid, L-galacturonic acid lactone, D-gluconic acid, D-glucuronic acid, and mucic acid.
4. Gas production test
Using Oralchroma TM The gas produced by the strain SA34 was tested by a portable gas chromatograph, and the result shows that the strain is cultured for 48 hours at 37 ℃ in a TSB culture medium and can be used for a large amount of two gases, namely hydrogen sulfide and methyl mercaptan.
3. Use of klebsiella grignard SA34 in screening for inhibiting gas production.
1. Strain activation and bacterial suspension preparation: (1) Strain activation: after the strain SA34 stored in the glycerol pipe is quickly melted in water bath, streaking and inoculating on a TSA culture medium plate, and performing activation culture in a 36 ℃ incubator for 16-24 hours; picking single colony obtained by streak culture, inoculating into a culture medium containingShake culturing at 36 deg.C and 150rpm in 5mL centrifuge tube of TSB culture medium for 16-24 hr to OD 600 The value is between 0.6 and 1.0; then the strain culture solution is inoculated and cultured for the second time to OD at the concentration of 1% (v/v) 600 The value is between 0.6 and 1.0; (2) preparation of bacterial suspension: sequentially diluting the culture solution of the secondary inoculated strain with TSB culture medium at 10 times dilution gradient to obtain bacterial suspension with cell concentration of 1.0X10 5 CFU/mL。
2. Test grouping: three groups, including a blank control group, a positive control group and a test sample group, were divided into 3 groups each. Bacterial suspension without test sample was used as positive control and sterile liquid medium was used as blank control.
3. Test sample preparation: (1) Taking 5g of solid test sample, weighing 45mL of sterile physiological saline, adding the solid test sample into a sterile homogenizing bag, placing the sterile physiological saline in a flapper for flapping for 1-2 min to prepare test sample solution or suspension, filtering the test sample solution or suspension by a 0.22 mu m sterile filter in an ultra-clean workbench, collecting filtrate, and then performing double serial dilution on the filtrate by using sterile distilled water to obtain test solutions with different concentrations; (2) liquid test sample: 5mL of liquid test sample is taken and diluted into test solutions with different concentrations in double serial by sterile distilled water.
4. Sample addition and culture: respectively taking 9mL of bacterial suspension and 1mL of test sample with different dilution concentrations, adding the test sample into a 50mL sterile centrifuge tube, fully and uniformly mixing, placing the test sample into the centrifuge tube containing the Du's tubule, placing the test sample into a constant temperature incubator at 36 ℃ for culturing for 1-7 d, observing the generation condition of bubbles in a Du Shixiao tube at regular time, and recording the occurrence time of the bubbles in a Du Shixiao tube. After 7d incubation, the size of the air bubbles in the dolphin tubule was measured using vernier calipers and recorded. The positive control group is 9mL of bacterial suspension and 1mL of sterile distilled water; the blank was 9mL of sterile liquid medium and 1mL of sterile distilled water.
5. And (3) result judgment: the longer the time for starting to generate bubbles in the Du's tubule and the shorter the length of the bubbles, the stronger the ability to suppress gas generation.
While certain specific embodiments of the invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the invention. It will be appreciated by those skilled in the art that modifications may be made to the above embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.
Claims (9)
1. Klebsiella grignardKlebsiella grimontii) SA34, which is characterized in that the preservation number is CGMCC No. 2171.
2. A microbial preparation comprising klebsiella grisedge SA34 according to claim 1.
3. Use of klebsiella grisedge SA34 of claim 1 or the microbial preparation of claim 2 for screening for functional additives inhibiting gas production, said gas comprising hydrogen sulfide and methyl mercaptan.
4. The application of Klebsiella grignard SA34 in screening functional additives for inhibiting gas production is characterized in that Klebsiella grignard with preservation number of CGMCC No.21719 is firstly treatedKlebsiella grimontii) SA34 is prepared into bacterial suspension, and then the bacterial suspension and a test sample solution are placed in a centrifuge tube containing a Dunaliella tubule for co-culturing for 1d-7d, wherein the gas comprises hydrogen sulfide and methyl mercaptan.
5. The use according to claim 4, characterized in that it comprises the following steps:
step (1): streaking Klebsiella grignard SA34 on a TSA culture medium plate for activation culture;
step (2): picking single colony obtained by streak culture, inoculating into TSB culture medium, shake culturing to OD 600 The value is between 0.6 and 1.0, and the strain culture solution is obtained;
step (3): diluting the strain culture solution to prepare a bacterial suspension;
step (4): preparing a test sample into a solution or a suspension;
step (5): the bacterial suspension and the test sample are prepared into a solution or suspension, the solution or suspension is transferred into a centrifuge tube containing a Du's tubule and placed in a constant temperature incubator for culturing 1d-7d.
6. The use according to claim 5, wherein the activation culture temperature in step (1) is 36℃and the time is 16h-24h.
7. The use according to claim 5, wherein the parameters of the shake culture in step (2) are as follows: 36 ℃,150rpm,16h-24h.
8. The use according to claim 5, wherein said step (4) is specifically as follows:
and adding the test sample and sterile physiological saline into a sterile homogenizing bag to prepare a test sample solution or suspension.
9. The use according to claim 5, wherein the temperature of the cultivation in step (5) is 36 ℃.
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| JP2019135951A (en) * | 2018-02-08 | 2019-08-22 | 株式会社マンダム | Screening method of halitosis inhibiting component |
| WO2021122741A2 (en) * | 2019-12-17 | 2021-06-24 | Fundación Para El Fomento De La Investigación Sanitaria Y Biomédica De La Comunitat Valenciana | Prebiotic and probiotic treatment to reduce oral dysbiosis and promote eubiosis |
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| WO2006022471A1 (en) * | 2004-08-25 | 2006-03-02 | Jong Suk Oh | Lactic acid bacteria inhibiting halitosis |
| NZ538235A (en) * | 2005-02-15 | 2008-07-31 | Syft Technologies Ltd | Evaluation of bacterial growth and antibiotic sensitivity in blood cultures using selected ion flow tube mass spectrometry |
| EP2133414A1 (en) * | 2008-06-11 | 2009-12-16 | Basf Se | Uses and methods for preventing and /or treating oral malodour |
| US10329591B2 (en) * | 2014-09-01 | 2019-06-25 | Evonik Degussa Gmbh | Method and microorganism for methionine production by fermentation with improved methionine efflux |
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| JP2019135951A (en) * | 2018-02-08 | 2019-08-22 | 株式会社マンダム | Screening method of halitosis inhibiting component |
| WO2021122741A2 (en) * | 2019-12-17 | 2021-06-24 | Fundación Para El Fomento De La Investigación Sanitaria Y Biomédica De La Comunitat Valenciana | Prebiotic and probiotic treatment to reduce oral dysbiosis and promote eubiosis |
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