CN116445319A - Bacillus amyloliquefaciens with antibacterial and allergen degrading effects and application thereof - Google Patents
Bacillus amyloliquefaciens with antibacterial and allergen degrading effects and application thereof Download PDFInfo
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- CN116445319A CN116445319A CN202211389554.6A CN202211389554A CN116445319A CN 116445319 A CN116445319 A CN 116445319A CN 202211389554 A CN202211389554 A CN 202211389554A CN 116445319 A CN116445319 A CN 116445319A
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- 241000193744 Bacillus amyloliquefaciens Species 0.000 title claims abstract description 52
- 239000013566 allergen Substances 0.000 title claims abstract description 51
- 230000000593 degrading effect Effects 0.000 title claims description 6
- 230000000844 anti-bacterial effect Effects 0.000 title abstract description 5
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 16
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 14
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
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- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 description 1
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 description 1
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 description 1
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Classifications
-
- 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
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/381—Microorganisms
-
- 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
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of microorganisms, and discloses a bacillus amyloliquefaciens strainBacillus amyloliquefaciens) YHX002J and application thereof, the strain has better bacteriostasis and allergen degradation effects, and is specifically expressed in the following steps: (1) The bacillus amyloliquefaciens YHX002J has strong antibacterial activity and strong inhibition effect on common pathogenic microorganisms such as escherichia coli, staphylococcus aureus, candida albicans and aspergillus niger; (2) Bacillus amyloliquefaciens YHX002J can effectively degrade indoor common allergen and reduce allergy risk caused by indoor allergen, becauseThe bacillus amyloliquefaciens can be used for preparing a household environment microorganism cleaning agent.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus amyloliquefaciens and application thereof in inhibiting common pathogenic microorganisms and degrading allergens.
Background
With the development of economy and the improvement of living standard of people, the requirements of people on the household sanitary environment are also higher and higher, and the market of cleaning agents is also activated. The cleaning agent commonly used in the market at present mainly comprises chemical cleaning agent, and the chemical cleaning agent mainly comprises surfactant, builder, additive and the like. However, the long-term use of the chemical cleanser substance easily causes allergic reaction of human body, weakens the immunity of human body, and simultaneously causes secondary pollution when the chemical cleanser is used for a long time, so that bacteria generate drug resistance and super bacteria are easy to generate. The existing cleaning agents on the market have single functions, and still take sterilization and disinfection as main functions, and have poor cleaning effects on other pollutants in the home environment, such as allergens, etc., so that the development of the environment-friendly and multi-effect cleaning agents for the home environment is imperative.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a bacterium of the genus Bacillus, typically 0.7-0.8X2.0-3.0 μm in size, and is a gram-positive bacterium. Bacillus amyloliquefaciens is a multifunctional microorganism, and can form dominant flora through nutrition competition or space occupying effect, so that the pathogenic microorganism can be effectively prevented from propagating, and the bacillus amyloliquefaciens has a certain effect in the antibacterial field. In addition, the bacillus amyloliquefaciens has a stronger enzyme system, can generate various enzymes such as protease, lipase and the like, and can effectively remove protein pollutants (such as allergens) in the environment, so that the bacillus amyloliquefaciens can be effectively applied to the production of microbial detergents to overcome the problems of chemical detergents.
Disclosure of Invention
On the one hand, the invention protects bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YHX002J, and the strain is preserved in China Center for Type Culture Collection (CCTCC) in the 3 rd month 15 days of 2022, and the preservation number is CCTCC NO: m2022265, the preservation address is China, university of Wuhan.
In another aspect, the invention provides the use of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YHX002J for inhibiting common pathogenic microorganisms, including escherichia coli, staphylococcus aureus, candida albicans, and aspergillus niger.
The invention also protects the application of the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YHX002J in degrading allergen substances, wherein the allergens comprise dust mite allergens, house dust mite allergens, cat allergens and dog allergens.
The beneficial effects of the invention include the following aspects: (1) Bacillus amyloliquefaciens YHX002J has strong antibacterial activity against common pathogenic microorganisms, including: coli, staphylococcus aureus, candida albicans and aspergillus niger; (2) The bacillus amyloliquefaciens YHX002J can effectively degrade common indoor allergens (house dust mite allergens, cat allergens and dog allergens) and reduce the risk of allergy caused by indoor allergens.
Drawings
FIG. 1 is a colony morphology of Bacillus amyloliquefaciens YHX002J.
FIG. 2 is a microscopic morphology of Bacillus amyloliquefaciens YHX002J vegetative cells.
FIG. 3 is a photomicrograph of Bacillus amyloliquefaciens YHX002J spore.
Detailed Description
EXAMPLE 1 isolation of Bacillus amyloliquefaciens YHX002J
The culture medium is a nutrient agar culture medium, and the preparation method comprises the following steps: respectively weighing 10.0g of peptone, 3.0g of beef extract powder, 5.0g of sodium chloride and 15.0g of agar powder, placing into a 1000ml beaker, adding 1000ml of purified water to dissolve, boiling, cooling to 60 ℃, adjusting the pH value to 7.2-7.4 by using 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution, subpackaging into 250ml triangular bottles, sterilizing at 121 ℃ under high pressure for 20min, cooling, and pouring into a preparation plate for later use.
Bacillus amyloliquefaciens YHX002J is obtained by separating and screening the bacillus amyloliquefaciens from the soil of an organic farm by adopting a dilution flat plate coating method, and comprises the following specific steps: collecting soil with fertile soil and healthy crop growth from organic farm, placing in sterile sampling bag, taking 5g soil sample, placing into sterile 250 containing appropriate amount of glass beadsA soil suspension was prepared by adding 45mL of sterile water to a mL Erlenmeyer flask, shaking it on a constant temperature shaker at 37℃for 2h, followed by heat treatment in a water bath at 80℃for 15min. Sucking 1mL of soil suspension into a sterile test tube by a micropipette, adding 9mL of sterile water, uniformly mixing, sucking 1mL of diluent from the mixture, adding the diluent into a 2 nd sterile test tube, adding 9mL of sterile water, and the like to prepare 10 -1 、10 -2 、10 -3 、10 -4 The gradient of the soil suspension was diluted. Respectively draw 100 mu L10 -2 、10 -3 、10 -4 The soil suspensions of 3 dilution gradients are evenly spread on a nutrient agar medium and placed in a biochemical incubator at 37 ℃ for inversion culture for 48 hours. After 72 hours of cultivation, colonies with good growth vigor, similar colony morphology and the greatest number were selected, and the single bacteria were isolated and purified until the colony morphology was consistent, and the number of the strain was YHX002J.
EXAMPLE 2 identification of Bacillus amyloliquefaciens YHX002J Strain
Characteristics of the cell morphology: on nutrient agar plate medium, the colony is a beige opaque colony, and the surface has halo or no bulge and irregular edge (shown in figure 1). The bacterial cells were rod-shaped and gram-positive under a microscope, and the cell length was 0.5-0.8X1.8-3.0. Mu.m (FIG. 2). After 24 hours of culture, spores can be seen to be oval, both ends of the spores are round, the spores are not expanded, and the spores grow from the middle to the secondary end (shown in figure 3).
Molecular biological identification of strains: molecular biology identification is carried out on the strain Zongwuhan Jin Kairui biological engineering Co., ltd, the 16s rRNA and gyrA gene sequences are respectively determined, the sequencing results are shown as SEQ ID NO.1 and 2, the strain is determined to be bacillus amyloliquefaciens (Bacillus amyloliquefaciens) according to sequence comparison, and the strain is submitted to China center for preservation in type culture collection (CCTCC NO) in 3 and 15 days of 2022: m2022265.
EXAMPLE 3 inhibition of pathogenic microorganisms by Bacillus amyloliquefaciens YHX002J
The medium used was as follows:
nutrient agar medium: as in example 1, for E.coli and Staphylococcus aureus activation and plate counting.
Nutrient broth medium: respectively weighing 3.0g of beef extract, 10.0g of peptone and 5.0g of sodium chloride, placing in a 1000ml beaker, adding 1000ml of purified water to dissolve, boiling, cooling to 60 ℃, adjusting the pH value to 7.2-7.4 by using 1mol/L sodium hydroxide or 1mol/L hydrochloric acid, subpackaging into 500ml triangular bottles, filling 98ml of each bottle, and plugging a ventilation plug. And (3) sterilizing at 121 ℃ for 20min for later use, and performing independent liquid culture on escherichia coli and staphylococcus aureus, and co-culturing the escherichia coli with bacillus amyloliquefaciens and staphylococcus aureus with bacillus amyloliquefaciens.
Crystal violet neutral red bile salt agar (VRBA): respectively weighing 7.0g of peptone, 3.0g of yeast extract, 10.0g of lactose, 5.0g of sodium chloride, 1.5g of bile salt, 0.03g of neutral red, 0.002g of crystal violet and 15g of agar powder, placing into a 1000mL beaker, adding 1000mL of purified water to dissolve, adjusting the pH value to 7.3-7.5 by using 1mol/L sodium hydroxide or 1mol/L hydrochloric acid, boiling for 2min, melting the culture medium, and pouring the culture medium into a flat plate at the temperature of 45-50 ℃. Prepared temporarily, not more than 3 hours before use, for e.coli counts.
Staphylococcus aureus 3M test piece: colony counts for staphylococcus aureus.
Potato dextrose agar medium (PDA): respectively weighing 12g of potato extract powder and 20g of glucose, placing 15g of agar in a 1000mL beaker, adding 1000mL of purified water to dissolve, boiling, cooling to 60 ℃, subpackaging, and sterilizing at 121 ℃ for 20min for activating candida albicans and aspergillus niger and counting by a flat plate, and adding 34 mug/mL kanamycin before use.
Glucose liquid medium (SDB): weighing 5g of beef extract, 5g of peptone and 20g of glucose respectively, placing in a 1000ml beaker, adding 1000ml of purified water to dissolve, boiling, cooling to 60 ℃, subpackaging into 500ml triangular flasks, filling 98ml of each flask, adding a ventilation plug, and sterilizing at 121 ℃ under high pressure for 20min for standby, and is used for separate liquid culture of candida albicans and aspergillus niger and co-culture of candida albicans, bacillus amyloliquefaciens and aspergillus niger and bacillus amyloliquefaciens.
The bacteriostasis experiment of the escherichia coli and staphylococcus aureus is as follows:
(1) Preparation of bacterial suspension: bacillus amyloliquefaciens YHX J, escherichia coli or Staphylococcus aureus were inoculated into sterilized 100ml nutrient broth medium, and cultured at 37+ -1deg.C for 24 hr. The cultured bacterial suspension is streaked on a nutrient agar plate by an inoculating loop, and is cultured for 24 hours at 37+/-1 ℃. Taking single colony of each bacterium, mixing uniformly in sterilized nutrient broth culture medium, and culturing at 37+ -1deg.C for 24 hr.
(2) Dilution count of bacterial suspension: respectively diluting and counting the cultured bacterial liquid, firstly preserving bacterial suspension in a refrigerator at 4 ℃ for standby, and respectively adjusting three bacteria to 10 after the counting result comes out 6 -10 7 CFU/mL。
(3) Co-cultivation: under aseptic operation, 1mL of escherichia coli or staphylococcus aureus suspension is sucked by an experimental group and respectively inoculated to 98mL of nutrient broth culture medium, and after uniform mixing, 1mL of bacillus amyloliquefaciens is added. The control group absorbs 1mL of escherichia coli or staphylococcus aureus suspension, respectively inoculates to 98mL of nutrient broth culture medium, and after uniform mixing, 1mL of sterile water is added. The two sets of mixed cultures were allowed to stand at 37.+ -. 1 ℃ for 24 hours and the numbers of E.coli and Staphylococcus aureus were counted.
(4) And (3) calculating results:
wherein:
x-antibacterial rate;
a-the number of plate colonies of the blank control group after co-culture;
b-number of plate colonies of test group after co-cultivation.
The bacteriostasis experiments of candida albicans and aspergillus niger were performed as follows:
(1) Preparation of bacterial suspension: bacillus amyloliquefaciens YHX J, candida albicans and aspergillus niger are respectively inoculated into sterilized 100ml of glucose liquid culture medium, and are cultured for 24 hours at the temperature of 30+/-1 ℃. The cultured bacterial suspension is streaked on a PDA culture medium plate by an inoculating loop, and is cultured for 24 hours at the temperature of 30+/-1 ℃. Taking single bacterial colonies, uniformly mixing the bacterial colonies with sterilized glucose liquid culture medium, and culturing the bacterial colonies at 30+/-1 ℃ for 24 hours.
(2) Dilution count of bacterial suspension: respectively diluting and counting the cultured bacterial liquid, firstly preserving bacterial suspension in a refrigerator at 4 ℃ for standby, and respectively adjusting three bacteria to 10 after the counting result comes out 6 -10 7 CFU/mL。
(3) Co-cultivation: under aseptic operation, 1mL of candida albicans or aspergillus niger suspension is sucked into a test group and inoculated into 98mL of glucose liquid culture medium, and then mixed uniformly, and 1mL of bacillus amyloliquefaciens is added into the test group to serve as the test group. 1ml of candida albicans or aspergillus niger suspension is taken as a control group and inoculated into 98ml of glucose liquid culture medium, and 1ml of sterile water is added after uniform mixing. The two sets of mixed cultures were allowed to stand at 30.+ -. 1 ℃ for 48 hours and the numbers of Candida albicans and Aspergillus niger were counted.
(4) And (3) calculating results:
wherein:
x-antibacterial rate;
a-the number of plate colonies of the blank control group after co-culture;
b-number of plate colonies of test group after co-cultivation.
TABLE 1 inhibition of Bacillus amyloliquefaciens YHX002J
As can be seen from Table 1, bacillus amyloliquefaciens YHX002J has strong inhibitory effects on common pathogenic bacteria such as Escherichia coli, staphylococcus aureus, candida albicans and Aspergillus niger.
EXAMPLE 4 degradation of indoor common allergens by Bacillus amyloliquefaciens YHX002J
House dust mite allergen Der f1, house dust mite allergen Der p1, cat allergen Fel d1, dog allergen Can f1 detection kit is purchased from Indoor Biotechnologies, house dust mite allergen detection kit Der f1 product number is EL-DP1, house dust mite allergen detection kit Der p1 product number is EL-DF1, cat allergen detection kit Fel d1 product number is EL-FD1, dog allergen Can f1 detection kit product number is EL-CF1. The specific operation method is as follows: bacillus amyloliquefaciens YHX002J stored in glycerol tubes were streaked onto nutrient agar plates, incubated at 37+ -1deg.C for 24 hours, and then single colonies were picked and inoculated into nutrient broth for overnight 12-14 hours. Then, 90. Mu.L of the cultured overnight bacterial liquid (the viable count was 2X 10) 8 CFU/mL) into a sterile test tube, 10. Mu.L of the prepared allergen solution was added thereto, the initial allergen concentration was 25ng/mL, and the mixture was allowed to stand at 37.+ -. 1 ℃ for 72 hours. The control group was prepared by adding 10. Mu.L of the prepared allergen solution to 90. Mu.L of sterile water for the same period of time. After 72h of action, the allergen concentration in the solution was tested by the double antibody sandwich ELISA method, as described with reference to the detection kit. The results were calculated as follows:
P＝(T C -T t )/T C ×100％
p: allergen-removing rate (%); t (T) C : concentration of allergen in control group (ng/mL) 72h after the action; t (T) t : concentration of allergen solution (ng/mL) in the test group after 72h of action.
TABLE 2 allergen degradation Effect
As can be seen from Table 2, bacillus amyloliquefaciens YHX002J has a good degrading effect on common dust mite allergens (house dust mite allergens, dust mite allergens) and pet allergens (cat allergens, dog allergens).
Preparation method of bacillus amyloliquefaciens YHX002J bacterial liquid preparation
(1) Activating strains: the preserved bacillus amyloliquefaciens YHX002J is streaked and activated by using a nutrient agar culture medium, wherein the temperature is 37 ℃ and the culture time is 24 hours, and the formula of the nutrient agar culture medium is the same as that of example 1;
(2) Preparing seed liquid: inoculating the activated single colony into nutrient broth culture medium, shaking culturing at 37deg.C for 24-48 hr, sampling, and performing identification such as dyeing to prevent pollution;
(3) Fermentation: fermenting and culturing with 60L stainless steel fermenter with inoculum size of 3%, fermenting at 37deg.C for 24 hr, observing spore rate under microscope, and stopping fermentation when spore rate reaches above 90%; the fermentation medium consisted of: 10g/L of glucose, 10g/L of peptone, 5g/L of yeast extract powder, 1.36g/L of potassium dihydrogen phosphate, 1.79g/L of dodecahydrate and disodium hydrogen phosphate, 0.27g/L of magnesium chloride hexahydrate, 0.17g/L of manganese chloride tetrahydrate and 0.4g/L of calcium chloride.
(4) Membrane filtration and concentration: transferring the fermentation liquor to a material barrel of membrane filtration equipment (the pore diameter of a filter membrane is 200 nm) after fermentation is finished, performing membrane filtration and concentration on thalli to obtain concentrated liquor, and performing spore counting;
(5) Diluting and filling: according to the spore count result of the membrane filtration concentrate and the final filling concentration (4×10) 6 CFU/mL), after determining the appropriate dilution factor, a proper amount of concentrate is measured, added with sterilized purified water for dilution and split-filled into small bottles (100 mL/bottle or 300 mL/bottle) by a small filling machine.
In conclusion, the bacillus amyloliquefaciens YHX002J has a strong inhibition effect on common pathogenic microorganisms such as escherichia coli, staphylococcus aureus, candida albicans and aspergillus niger, and has a good degradation effect on indoor common allergens (house dust mite allergens, cat allergens and dog allergens), so that the strain has a great application potential in household cleaning products.
Claims (4)
1. Bacillus amyloliquefaciens @Bacillus amyloliquefaciens) YHX002J, characterized in that the preservation number is CCTCC NO: m2022265.
2. Bacillus amyloliquefaciens @Bacillus amyloliquefaciens) YHX002J inhibits Escherichia coli, staphylococcus aureus,
Candida albicans or aspergillus niger.
3. Bacillus amyloliquefaciens @Bacillus amyloliquefaciens) The application of YHX002J in degrading allergen, which is characterized in that the allergen comprises dust mite allergen, house dust mite allergen, cat allergen and dog allergen.
4. The bacillus amyloliquefaciens liquid preparation is characterized in that the preservation number of the bacillus amyloliquefaciens is CCTCC NO: m2022265.
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