CN111621442A - Bacillus pumilus, preparation, composition and application - Google Patents
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Abstract
The invention relates to a Bacillus pumilus preserved in CGMCC with the preservation number of CGMCC No.19821, which has stronger bacteriostatic activity and is used for treating common pathogenic microorganisms: the salmonella, the staphylococcus aureus, the candida albicans and the pseudomonas have strong inhibiting effect; in addition, the bacillus pumilus has strong vitality and short generation time, can quickly occupy space, further enhances the bacteriostatic efficacy, and can be effectively applied to the production of non-chemical bacteriostatic disinfectants.
Description
Technical Field
The invention relates to the field of microorganism application, and particularly relates to bacillus pumilus, a preparation, a composition of the bacillus pumilus and application of the bacillus pumilus.
Background
Along with the development of national economy and the improvement of living standard of people, the sanitation literacy and the sanitation standard of people are higher and higher, the comfort and tidiness of the household living environment are pursued, and the market of the cleaning agent is activated. The chemical cleaning agent commonly used in the market at present mainly comprises a surfactant, a builder, an additive and the like. The commonly used surfactants are mainly anionic, such as Linear Alkylbenzene Sulfonate (LAS), fatty alcohol sulfate, fatty alcohol polyoxyethylene ether sulfate, alkyl sulfonate, and the like. The surfactant can damage the oily protective layer on the surface of the skin, further corrode the skin, and butterfly spots can appear on the face after long-term use. If there is a wound on the skin, the permeability is increased by more than 10 times, and the chemical toxin entering the human body can reduce the calcium ion concentration in the blood and acidify the blood. Builders include alkaline materials and acidic materials. Wherein the acidic substance can absorb water in skin tissue to coagulate protein; and alkaline substances synthesized by sodium alkyl benzene sulfonate, sodium sulfate, sodium toluene iodate, sodium tripolyphosphate and carboxymethyl cellulose can denature tissue protein and destroy cell membranes besides absorbing water, and if a human body contacts the alkaline substances for a long time, the alkaline substances can cause harm to the liver and the hematopoietic system. In addition, chemical substances in various detergents can cause anaphylactic reaction of human bodies, and fluorescent agent and whitening agent components contained in some detergents are not easily decomposed like common chemical components after entering the human bodies, but are accumulated in the human bodies, so that the immunity of the human bodies is greatly reduced.
Meanwhile, chemical components in the cleaning agent not only cause a series of health hazards to human bodies, but also pollute the environment, so that bacteria generate drug resistance and are easy to generate super bacteria. The discharge of waste water during the use of detergent is the root of environmental pollution. The discharge of a large amount of washing wastewater easily causes eutrophication of water bodies, thereby causing influence on organisms in the water and causing damage to food chains and ecological systems.
Bacillus pumilus (Bacillus pumilus) is a bacterium of the genus Bacillus with a fine rod-like shape, and is generally 0.6 to 0.7. mu. m.times.2.0 to 3.0. mu.m, gram-positive. The bacillus pumilus is a multifunctional microorganism, can form a dominant flora through nutrition competition or space site competition, effectively prevents the propagation of pathogenic microorganisms, and has a certain function in the field of bacteriostasis, but not all the bacillus pumilus have strong bacteriostasis.
Disclosure of Invention
The inventor provides the bacillus pumilus which can effectively inhibit the growth and the propagation of bacteria through research, screening and verification on the bacillus pumilus, and the bacillus pumilus strain preparation and the composition containing the components thereof can be effectively applied to the production of non-chemical antibacterial disinfectants.
In order to achieve the first object of the present invention, the present invention provides a culture collection management center (CGMCC) for common microorganisms in china, wherein the collection addresses are as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing; the classification is named as: bacillus pumilus, with the deposit number: CGMCC NO.19821, Bacillus pumilus KDXYBG-B1 with preservation date of 2020, 05 and 14. The bacillus pumilus disclosed by the invention has strong antibacterial activity and can effectively inhibit the growth and reproduction of bacteria.
In order to achieve the second purpose of the invention, the invention provides the application of the Bacillus pumilus which is preserved in CGMCC and has the preservation number of CGMCC No.19821 in the aspect of inhibiting the growth and the reproduction of bacteria.
Further, the bacteria include candida albicans, salmonella, pseudomonas and/or staphylococcus aureus.
In order to achieve the third object of the present invention, the present invention provides a candida albicans growth inhibitor, which comprises bacillus pumilus deposited in CGMCC with the collection number of CGMCC No.19821 as an active ingredient.
In order to achieve the fourth object of the present invention, the present invention provides a salmonella growth inhibitor comprising bacillus pumilus deposited in CGMCC with the collection number of CGMCC No.19821 as an active ingredient.
In order to achieve the fifth object of the present invention, the present invention provides a pseudomonas growth inhibitor comprising bacillus pumilus deposited in CGMCC with the collection number of CGMCC No.19821 as an active ingredient.
In order to achieve the sixth object of the present invention, the present invention provides a staphylococcus aureus growth inhibitor, which comprises bacillus pumilus deposited in CGMCC with the collection number of CGMCC No.19821 as an active ingredient.
In order to achieve the seventh object of the invention, the invention provides an antibacterial and disinfectant composition, which contains bacillus pumilus which is preserved in CGMCC (China general microbiological culture collection center) and has the preservation number of CGMCC No.19821 as an effective component.
Further, the composition is used for inhibiting the growth of candida albicans, salmonella, pseudomonas and/or staphylococcus aureus.
The beneficial effects of the invention at least comprise the following: the bacillus pumilus preserved in CGMCC with the preservation number of CGMCC No.19821 has stronger bacteriostatic activity and is used for treating common pathogenic microorganisms: the salmonella, the staphylococcus aureus, the candida albicans and the pseudomonas have strong inhibiting effect; in addition, the biological antibacterial disinfectant has strong vitality and short generation time, can quickly occupy space, further enhances the antibacterial effect, and can be effectively applied to the production of non-chemical antibacterial disinfectants.
Drawings
FIG. 1 is a graph showing the change in the number of Staphylococcus aureus on the floor of a toilet;
FIG. 2 is a graph showing the change in the number of Staphylococcus aureus in the toilet tank;
FIG. 3 is a graph showing the change in the number of Pseudomonas bacteria in bathroom floor.
Detailed Description
To explain technical contents, structural features, achieved objects and effects of the technical solutions in detail, the following detailed description is given in conjunction with the accompanying drawings 1-2 of the specification.
EXAMPLE 1 screening and identification of Bacillus pumilus (KDXYBG-B1)
The bacillus pumilus preserved in CGMCC with the preservation number of CGMCC No.19821 is obtained by separating and screening the bacillus pumilus in soil by a dilution plate coating method by the inventor, and comprises the following specific steps:
1. collecting and separating strains: and separating the target strain from the soil sample obtained from the garbage field by adopting a dilution plate coating method. Weighing 5g of soil sample, putting the soil sample into a sterile triangular flask filled with a proper amount of small glass balls, adding 45mL of sterile water to prepare a soil suspension, and placing the soil suspension on a constant temperature oscillator at 30 ℃ for 2 h. Sucking 1mL of soil suspension into a sterile test tube by using a pipettor, adding 9mL of sterile water, uniformly mixing, sucking 1mL of diluent from the sterile test tube, adding into the 2 nd sterile test tube, adding 9mL of sterile water, and the like to prepare 10－1、10－2、10－3、10－4、10－5、10－66 dilution gradients of soil suspension. Suck 200. mu.L of 10 cells each－4、10－5、10－6And 3 soil suspensions with dilution gradient are uniformly coated on a beef extract peptone agar culture medium and are placed in a biochemical incubator at 28 ℃ for inverted culture for 72 hours. And after culturing for 72 hours, selecting colonies with good growth vigor, similar colony morphology and the largest number, separating and purifying until single bacteria with consistent colony morphology exist, and preserving strains.
2. Identification of strains
Morphological characteristics: the observation and detection show that the cell is a thin rod-shaped, gram-positive bacterium, has the body length of 0.6-0.7 mu m multiplied by 2.0-3.0 mu m and can move. On LB culture medium, the colony morphology is nearly circular, with wrinkles on the surface, opacity, and uneven edges.
3. Generation time determination
(1) Preparation of culture medium
Weighing 10.0g of peptone, 5.0g of yeast extract, 10.0g of glucose and 5.0g of NaCl; adding several milliliters of pure water, heating to completely dissolve, cooling, adjusting pH to 7.0-7.2, adding water to a constant volume of 1000mL, subpackaging in triangular flasks at 100 mL/bottle and 121 ℃, and sterilizing for 20 min.
(2) Growth log phase assay
Inoculating the purified single colony into a 100 ml/bottle culture medium, placing the culture medium in a shaking culture (220r/min) at 37 ℃, sampling every 1h to determine OD value and colony count (1, 2, 3, 4, 5, 6 and 7h), drawing a curve by taking sampling time as abscissa and OD value and bacteria count as ordinate, and determining log phase.
(3) Generation time determination
When the strain grows into a logarithmic phase, selecting 3 time points t0, t1 and t2 in the logarithmic phase to count the viable bacteria on a flat plate, carrying out operation according to an enterprise standard, sampling 1ml, wherein the measured bacteria number is Mo, M1 and M2 respectively, and the unit CFU/ml, and calculating the generation time of two time periods (t0 → t1, t1 → t2) according to the following formula:
first time period generation time:
G1＝(t1-t0)×Lg2/(LgM1-LgM0)
in the formula, G1 represents the generation time of stage t0 → t1, M0 represents the number of cells at t0 (CFU/G), and M1 represents the number of cells at t1 (CFU/G).
Generation time of second period:
G2＝(t2-t1)×Lg2/(LgM2-LgM1)
in the formula, G2 represents the generation time of stage t1 → t2, M1 represents the number of cells at t1 (CFU/G), and M2 represents the number of cells at t2 (CFU/G).
(4) time-OD measurement value:
(5) OD viable count:
(6) generation time (min)
The logarithmic growth phase of Bacillus pumilus is 5h → 6h from the value measured by time-OD; from the results of the generation time measurement, the logarithmic growth phase of Bacillus pumilus was 4h → 5h, and the generation time was 12 min.
Therefore, the Bacillus pumilus (KDXYBG-B1) which is preserved in CGMCC and has the preservation number of CGMCC No.19821 has strong vitality and short generation time, can quickly occupy space, and forms high-efficiency competitive inhibition on bacteria.
Example 2A preparation containing Bacillus pumilus (Bacillus pumilus with a spore number of 1.48 × 10)6CFU/mL)
1. And (3) activation: performing streak activation on the stored bacillus pumilus by using an NA culture medium at the temperature of 37 ℃ for 24 hours;
the nutrient agar medium (NA) formula is as follows: 10.0g of peptone, 3.0g of beef extract powder, 5.0g of sodium chloride, 17.0g of agar and 1L of pure water are added, the pH value is 7.3, the temperature is 121 ℃, and the high-temperature sterilization treatment is carried out for 25 min.
2. Preparing a seed solution: inoculating the activated single colony in a nutrient broth culture medium, placing the culture medium at 37 ℃ for shaking culture for 24-48h, sampling, and performing identification such as dyeing to prevent pollution;
3. fermentation: fermenting for 48h at normal temperature by using a stainless steel fermentation tank and a fermentation culture medium thereof with the inoculation amount of 5%, observing the spore rate under a microscope, and stopping stirring when the spore rate reaches more than 90%;
4. adsorption and drying: adding an adsorption carrier into the bacillus pumilus fermentation liquor, and drying to obtain bacillus pumilus powder, wherein the addition amount of the carrier is based on that each gram of the dried bacillus pumilus contains 1000 hundred million CFU, and the carrier comprises 30% of bran, 30% of defatted rice bran and 40% of montmorillonite;
5. mixing, according to the counting result obtained by detection, preparing a preparation containing the bacillus pumilus according to the formula of 2.5 percent of 1-methoxy-2-propanol, 1.0 percent of subtilisin, 1.0 percent of amylase, 5.0 percent of bacillus pumilus powder and 90.5 percent of pure water, wherein the content of bacillus pumilus spores in the preparation is 1.48 × 106CFU/mL。
Example 3: example 2 Effect of Bacillus pumilus-containing preparations on Candida albicans, Salmonella, and Pseudomonas aeruginosa
The substance to be tested: PVC (linoleum), ceramic tile, glass (area is 30X 30cm)
The test strains were: candida albicans, salmonella and pseudomonas aeruginosa
Pathogen suspension density: 109-108CFU/ml
Detergent test concentration: 1 percent of
The experimental method comprises the following steps: the surface of the test object was first cleaned, rinsed, disinfected and finally rinsed thoroughly with a neutral solution of GLOSS XPRESS supplied by Medical Institute of Ostrava, and a pathogen suspension (50uL) was inoculated onto the surface of the test object. Wherein Candida albicans is inoculated on the PVC; inoculating salmonella on the ceramic tile; the glass is inoculated with pseudomonas aeruginosa. The surfaces of the test subjects inoculated with the pathogens were cleaned daily for five consecutive days with probiotic cleansers and the survival of the pathogens was monitored by plate counting.
The sampling and detection refer to GB15982-2012, which discloses an object surface microorganism pollution inspection method.
The sampling method comprises the following steps: placing a 5cm × 5cm sterilization specification plate on the surface of an object to be detected, using 1 cotton swab soaked with sterile physiological saline sampling solution to smear 5 times in the specification plate in a transverse and vertical reciprocating manner respectively, rotating the cotton swabs along with the cotton swab, continuously sampling 3 specification plates, shearing off a hand contact part, and placing the cotton swabs into a test tube filled with 10mL sampling solution.
The detection method comprises the following steps: and (3) after fully shaking the sampling tube, inoculating 1.0mL of eluent with different dilution times to a plate, adding about 15mL of culture medium cooled to 45 ℃, and placing the plate in a constant-temperature incubator at 37 ℃ for culturing for 48 hours.
Calculating the formula:
the experimental results are as follows: (the unit of the number of bacteria is CFU/cm2)
Example 4: application of preparation containing Bacillus pumilus prepared in example 2 to inhibition of growth and reproduction of Escherichia coli
(1) Normative citation document: an evaluation method of antibacterial and bacteriostatic effects of a standard QB/T2738-;
(2) main apparatus and reagents: electronic balance, constant temperature incubator, high pressure steam sterilization pot, constant temperature water bath, pH meter, colony counter, super clean bench, ultrasonic cleaner, microscope and other essential instrument for microbe experiment;
(3) preparing a nutrient agar culture medium: weighing 3.0g of beef extract, 10.0g of peptone, 15.0g of agar powder and 5.0g of sodium chloride respectively, putting the beef extract, the peptone, the agar powder and the sodium chloride into a 1000ml beaker, adding 1000ml of purified water to dissolve the beef extract, 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 the beef extract into a 250ml triangular flask, carrying out autoclaving at 121 ℃ for 20min, and refrigerating for later use;
(4) preparation of nutrient broth culture medium: weighing 3.0g of beef extract, 10.0g of peptone and 5.0g of sodium chloride respectively, placing the beef extract, the peptone and the sodium chloride in a 1000ml beaker, adding 1000ml of purified water to dissolve the beef extract, 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 the beef extract in a 250ml triangular flask, sterilizing at 121 ℃ for 20min under high pressure, and refrigerating for later use;
(5) staphylococcus aureus test strip (6491): the colony count for staphylococcus aureus was obtained from minnesota mining and manufacturing.
(6) Preparation of a suspension of bacterial propagules of staphylococcus aureus (CICC 10384): taking a freeze-dried strain tube, opening the tube under aseptic operation, sucking about 0.5ml of liquid culture medium by using a sterile sucker, and completely dissolving freeze-dried strain powder in the tube. Taking a nutrient broth culture medium 5.0ml-10ml test tube, dripping a little strain suspension, placing in an incubator at 37 ℃ for culturing for 24h, taking the strain suspension of the first generation culture by using an inoculating loop, streaking and inoculating on a slant, and culturing in the incubator at 37 ℃ for 24h to obtain a 3 rd generation culture; taking a fresh culture of the strain, sucking 5ml of diluent, adding into a slant test tube, repeatedly blowing and sucking, washing off thallus Porphyrae, transferring the washing solution into another sterile test tube, and mixing with an electric mixer. The bacterial propagule suspension is stored in a refrigerator at 4 ℃ for later use, and should be used on the same day and should not be used overnight.
(7) The method comprises the following operation steps: under the aseptic operation, 1ml of staphylococcus aureus suspension is sucked and inoculated into 100ml of nutrient broth culture medium, 1ml of the preparation containing bacillus pumilus prepared in the example 2 is added, and the mixture is uniformly mixed; adding 1ml of the same test bacteria suspension into 100ml of nutrient broth culture medium as a blank control group, mixing uniformly, measuring the number of staphylococcus aureus in the broth by using a plate counting method, sucking 1ml of sample liquid (or taking 2-3 dilutions of the sample liquid after appropriate dilution), placing the sample liquid into a sterilization plate, and inoculating two sterilization plates into each sample liquid. Pouring a culture medium cooled to 40-45 ℃, uniformly mixing around the 8-shaped culture medium, after the culture medium is solidified, inversely buckling the flat plate, culturing in an incubator at 37 ℃ for 48 hours, and counting bacterial colonies serving as viable bacteria; the mixed culture was subjected to shaking culture (200r/min) at a constant temperature of 37 ℃ and 200r/min for 48 hours, and after the co-culture, the number of test bacteria was measured and counted as described above.
(8) Formula for calculation
In the formula: x-bacteriostasis rate,%
A-average number of colonies in control group
B-average number of colonies in the test group
(9) Bacteriostatic effect
Example 5: application of preparation containing bacillus pumilus prepared in example 2 to household cleaning
The living room toilet and the bathroom in the bedroom of two household rooms (T and S) are respectively selected as test areas, the test areas are cleaned once every three days for two months by using the preparation prepared in example 2, the toilet and the bathroom are used as usual during the test period, the cleaning conditions of areas such as the floor of the toilet, the water tank of the toilet, the floor of the bathroom and the like are monitored once every 3-5 days, the staphylococcus aureus bacteria number and pseudomonas are detected, and the detection results are shown in figures 1-3.
As seen from FIGS. 1 to 3, the numbers of Staphylococcus aureus and Pseudomonas bacteria in 2 sets of house rapidly decreased within 2 weeks after use, and the use continued for 2 monthsThe number is less than 500CFU/m2And the number of pseudomonas colonies is less than 200CFU/m2。
Example 6: example 2 application of the preparation containing Bacillus pumilus to gymnasium
The areas of a front desk, a sitting chest expander, a sitting transverse lifter, a rotary bicycle, a front door handle of a children room, a small water tank of a male washroom, a shower of men, a corridor floor of the men and the like are selected as test areas in the gymnasium.
The bacteria count of each test area was measured by ATP technique before using probiotic cleanser, the test areas were routinely washed with the preparation containing bacillus pumilus prepared in example 2, the bacteria count of each test area was measured by ATP technique after one worship, and the bacterial contamination reduction rate was calculated from the statistical results.
The ATP fluorescence detector is based on the firefly luminescence principle and utilizes a luciferase-luciferin system to rapidly detect Adenosine Triphosphate (ATP). Since all living cells of organisms contain constant ATP, the ATP content can clearly indicate the residual amount of microorganisms and other organisms in a sample and is used for judging the sanitary condition.
The results of the experiments are shown in the following table.
As can be seen from the above table, when the preparation containing bacillus pumilus prepared in example 2 was used, ATP readings were greatly reduced because the bacillus pumilus was substantially present in the form of spores, and was attached to the surface of the equipment in a large amount after cleaning, occupying the living space of harmful bacteria, but the spores did not germinate in a short time. Spores are dormant bodies of bacteria and, unlike vegetative bodies, do not produce ATP. When the survival conditions are proper, the spores germinate to form nutrient bodies, and the ATP content is increased. Therefore, when the ATP detector is used after cleaning, the reading will be reduced, and the ATP can be used for detecting the bacterial quantity to judge the sanitary condition.
The test results show that the bacillus pumilus preserved in CGMCC with the preservation number of CGMCC No.19821 has the advantages of capability of treating common pathogenic microorganisms: the salmonella, the staphylococcus aureus, the candida albicans and the pseudomonas have strong inhibiting effect; in addition, the biological antibacterial agent has strong vitality and short generation time, can quickly occupy space, and further enhances the antibacterial effect.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.
It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.
Claims (9)
1. The Bacillus pumilus is preserved in CGMCC with the preservation number of CGMCC NO. 19821.
2. The use of the Bacillus pumilus of claim 1 for inhibiting the growth and reproduction of bacteria.
3. Use according to claim 2, wherein the bacteria comprise candida albicans, salmonella, pseudomonas and/or staphylococcus aureus.
4. A candida albicans growth inhibitor, wherein said formulation comprises the bacillus pumilus of claim 1.
5. A Salmonella growth inhibitor characterized by comprising the Bacillus pumilus of claim 1.
6. A pseudomonas growth inhibitor comprising the bacillus pumilus of claim 1.
7. A Staphylococcus aureus growth inhibitor comprising the Bacillus pumilus of claim 1.
8. A bacteriostatic disinfectant composition comprising the Bacillus pumilus of claim 1 as an active ingredient.
9. Bacteriostatic disinfecting composition according to claim 8, characterized in that said composition is used for inhibiting the growth of Candida albicans, Salmonella, Pseudomonas and/or Staphylococcus aureus.
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| CN112493259A (en) * | 2020-12-04 | 2021-03-16 | 中诚国联（河南）生物科技有限公司 | Organic-inorganic composite microbial agent for inhibiting pathogenic bacteria and preparation method thereof |
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