CN111647534A

CN111647534A - Bacillus megaterium, preparation and application thereof

Info

Publication number: CN111647534A
Application number: CN202010548105.6A
Authority: CN
Inventors: 林雅琳; 郑美大; 郑伯欣; 刘明刚
Original assignee: Corist Fujian Biotechnology Co ltd
Current assignee: Corist (Fujian) Biotechnology Co.,Ltd.; FUJIAN LUODONG BIO-TECHNOLOGY Co.,Ltd.
Priority date: 2020-06-16
Filing date: 2020-06-16
Publication date: 2020-09-11

Abstract

The invention relates to a bacillus megaterium preserved in CGMCC with the preservation number of CGMCC NO.19820, which has strong inhibition effect on escherichia coli and mites; in addition, the air purifier can also effectively remove formaldehyde, ammonia, benzene series and total volatile organic compounds in the air, and has the function of purifying the air. It can be effectively applied to the production of non-chemical antibacterial disinfectants and air purification products.

Description

Bacillus megaterium, preparation and application thereof

Technical Field

The invention relates to the field of microorganism application, and particularly relates to bacillus megaterium, and a preparation and application thereof.

Background

With the continuous development of social economy, people pay more and more attention to living environment, especially indoor environment air quality. When harmful chemical factors, physical factors and/or biological factors enter indoor air and the pollution level is increased, sub-health reactions of human bodies can be generated and even life safety can be threatened. Therefore, improvement of indoor ambient air quality should be emphasized.

The kinds of indoor air pollutants are more than 900, and are mainly classified into 3 types: gaseous pollutants, microbial pollutants, respirable particulate matter. The gaseous pollutants include Volatile Organic Compounds (VOCs), O3, CO2, etc., of which volatile organic compounds are the most predominant component. When the indoor ventilation condition is poor, the gas pollutants can be accumulated in the room, and the concentration of the gas pollutants can exceed the sanitary standard by tens of times, so that the indoor air is seriously polluted. The microbial pollutants comprise allergic reactants, viruses, bacteria which are easy to grow in indoor humid places and the like.

According to the analysis of indoor environment detection reports in recent years, indoor air pollutants mainly come from: building and interior decoration materials, outdoor pollutants, combustion products, and human activities. At present, the main cause of indoor air pollution is pollution of indoor decoration materials and furniture. The national health, construction and environmental protection departments have performed one-time spot check on the interior decoration materials, and as a result, 68% of the materials have toxic gas pollution. These decorating materials volatilize more than 300 kinds of volatile organic compounds, wherein organic compounds such as formaldehyde, ammonia, benzene, toluene and the like can cause adverse reactions such as headache, nausea, vomiting, convulsion, dyspnea and the like after being contacted with a human body, and can cause allergic reactions such as asthma, allergic rhinitis, dermatitis and the like after being repeatedly contacted with the decorating materials, and can cause cancers (lung cancer, leukemia), abortion, fetal deformity, growth and development retardation and the like after being contacted with the decorating materials for a long time.

At present, the market isThe method for treating indoor air pollutants uses sunlight, halogen tungsten lamp, mercury lamp, etc. as ultraviolet light source, and uses anatase type nano TiO₂As a method of photocatalytic degradation of a catalyst. Although this method can effectively treat organic materials, it has many drawbacks. The absorption range of ultraviolet light is narrow, the utilization rate of light energy is low, and the efficiency is also limited by the properties of a catalyst, the wavelength of the ultraviolet light and a reactor; photocatalysis requires solving the problem of light transmittance; the used catalyst is mostly nano particles, and the recovery is difficult; the method is not suitable for rooms, schools, offices, hospitals, hotels and other places.

Therefore, the development of a green and safe biological air purifying agent is an urgent need.

Disclosure of Invention

The inventor provides the bacillus megaterium which can effectively purify air and inhibit growth and reproduction of escherichia coli and mites through research, screening and verification on the bacillus megaterium, and the bacillus megaterium strain can be effectively applied to production of non-chemical air purifiers and disinfection insecticides.

In order to achieve the first object of the present invention, the present invention provides a microorganism deposited in the China general microbiological culture Collection center (CGMCC), wherein the preservation 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 megaterium, with the deposit number: CGMCC NO.19820, and the preservation date of the Bacillus megaterium KJDYBGJ-A3 is 05-month-2020 and 14-day-2020. The bacillus megaterium has strong bacteriostatic 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 megaterium which is preserved in CGMCC and has the preservation number of CGMCC NO.19820 in removing formaldehyde, ammonia, benzene series and total volatile organic matters in the air.

In order to achieve the third purpose of the invention, the invention provides the application of the bacillus megaterium which is preserved in CGMCC and has the preservation number of CGMCC NO.19820 in the aspect of inhibiting the growth and the propagation of escherichia coli.

In order to achieve the fourth object of the invention, the invention provides the application of the bacillus megaterium which is preserved in CGMCC and has the preservation number of CGMCC NO. in the aspect of inhibiting the growth and the reproduction of mites.

In order to achieve the fifth object of the present invention, the present invention provides an air purifying agent comprising as an active ingredient bacillus megaterium deposited in CGMCC with the collection number of CGMCC No. 19820.

In order to achieve the sixth object of the present invention, the present invention provides an escherichia coli growth inhibitor comprising bacillus megaterium deposited in CGMCC with the collection number of CGMCC No.19820 as an active ingredient.

In order to achieve the seventh object of the present invention, the present invention provides a mite growth inhibitor, which comprises bacillus megaterium as an active ingredient, wherein the bacillus megaterium is deposited in CGMCC No. 19820.

The beneficial effects of the invention at least comprise the following: the bacillus megaterium is preserved in CGMCC with the preservation number of CGMCC NO.19820 and has extremely strong inhibition effect on escherichia coli and mites; in addition, the air purifier can also effectively remove formaldehyde, ammonia, benzene series and total volatile organic compounds in the air, and has the function of purifying the air. It can be effectively applied to the production of non-chemical antibacterial disinfectants and air purification products.

Detailed Description

In order to explain technical contents, structural features, and objects and effects of the technical means in detail, the following detailed description is given with reference to specific embodiments.

The media mentioned in the present invention are as follows:

nutrient agar medium (NA): 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.

Nutrient broth culture medium: 5.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride, and 1L of pure water with pH 7.0, 121 ℃ and 25min for high-temperature sterilization.

Example 1 Collection, isolation and characterization of Bacillus megaterium (KJDYBGJ-A3)

The bacillus megaterium preserved in CGMCC with the preservation number of CGMCC NO.19820 is obtained by separating and screening the inventor in orchid, and the specific steps are as follows:

1. screening and separating strains: and (4) washing dirt on the surface of the orchid, and then transferring the orchid into a sterile bottle. The orchid is firstly soaked in mercury bichloride with the concentration of 0.1% for five minutes by oscillation, then washed for three times by purified water, then soaked in 75% alcohol for five minutes by oscillation, and finally washed for five times by purified water. Shearing the treated orchid, transferring the orchid to a sterile mortar, adding physiological saline and quartz sand for grinding, centrifuging for ten minutes in a centrifugal machine with the rotating speed of 2000r/min, taking the supernatant, coating the supernatant on an NA culture medium plate, and inversely placing the NA culture medium plate in a constant-temperature biochemical incubator at 30 ℃ for culturing for 48 hours. And after 48h, selecting colonies with good growth vigor, similar colony morphology and the largest number, separating and purifying the single bacteria until the colony morphology is consistent, and preserving the strains.

2. The strain morphological characteristics are as follows: the cell is a long rod-shaped gram-positive bacterium, has the body length of 1.2-4.0 mu m, can move, and is cultured at 37 ℃ for 2-3 days to form spores, the size of the spores is 0.6-1.5 mu m, and the spores are oval. The colony surface is smooth and opaque, and is dirty white.

Example 2A preparation containing Bacillus megaterium (Bacillus megaterium spore number 3.0 × 10)⁷CFU/ml)

1. And (3) activation: marking and activating the preserved bacillus megaterium by using an NA culture medium at 37 ℃ for 24 hours;

2. preparing a seed solution: inoculating the activated single colony into 100mL nutrient broth culture medium, performing shake culture at 37 deg.C for 24-48h, sampling, and performing identification such as staining to prevent contamination;

3. fermentation: fermenting for 48h at normal temperature by using a stainless steel fermentation tank and a fermentation medium thereof, wherein the inoculation amount is 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 megaterium fermentation liquor, and drying to obtain bacterial powder, wherein the addition amount of the carrier is based on that each gram of the dried bacillus megaterium contains 1000 hundred million CFU, and the carrier comprises 30% of bran, 30% of defatted rice bran and 40% of montmorillonite;

5. and (3) detection: counting the obtained bacillus megaterium according to an SN/T4624.3-2016 method;

6. mixing, namely proportioning the bacterial powder obtained by the fermentation of the bacillus megaterium and ultrapure water according to the counting result obtained by detection, wherein the proportion of the bacterial powder to the ultrapure water is 10 percent and 90 percent of the ultrapure water, and finally obtaining the preparation containing the bacillus megaterium, wherein the number of the bacillus megaterium in the preparation is 8.0 × 10⁷CFU/ml。

Example 3: application of preparation containing Bacillus megaterium 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) crystal violet neutral red bile salt agar (VRBA): the plate colony used for coliform bacteria is counted, and the manufacturer is Guangdong Huanji microbial technology limited company.

(6) Preparation of a suspension of bacterial propagules of Escherichia coli (CICC 10389): 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 a3 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 the escherichia coli bacterial suspension is sucked and inoculated into 100ml of nutrient broth culture medium, 1ml of the preparation containing the bacillus megaterium prepared in the example 2 is added, and the mixture is uniformly mixed; adding 1ml of the same test bacterial suspension into 100ml of nutrient broth culture medium as a blank control group, mixing uniformly, measuring the number of Escherichia coli 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 with 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) The bacteriostatic effect is as follows:

example 4: the CMA monitoring report for the bacillus megaterium-containing formulation prepared in example 2 reports the monitoring categories: indoor air location: college of food and bioengineering colleges of college university

Monitoring items: formaldehyde, ammonia, benzene series (benzene, toluene, xylene), Total Volatile Organics (TVOC) were monitored according to:

1. formaldehyde: GB/T18204.2-2014 public place sanitation inspection method part 2: chemical pollutants (phenol reagent spectrophotometry)

2. Ammonia: GB/T18204.2-2014 public place sanitation inspection method part 2: chemical pollutants (Nashi reagent spectrophotometry)

3. Benzene series (benzene, toluene, xylene): determination of HJ 584-2010 ambient air benzene series by activated carbon adsorption/carbon disulfide desorption-gas chromatography

4. Total Volatile Organics (TVOC): GB/T18883-2002 national standard test method for indoor air:

the untreated base paper was suspended in a blank test chamber A and the decontaminated material was sprayed (sample spray amount 50 ml/m)³) Suspended in the sample group test chamber B.

Containers with fitted contaminant release sources a1 and B1 were placed in blank test chamber a and sample test chamber B, respectively, and the chamber doors were immediately closed.

And starting fans in the blank test chamber A and the sample test chamber, stirring for 1min to uniformly mix the air in the chamber with the pollutants in the release source, simultaneously closing the fans, sampling the air in the blank chamber, and measuring the initial concentration of the pollutant concentration value in the blank chamber.

And sampling air pollutants in the two chambers after 24 hours, and performing analysis and test, wherein the concentration value is the concentration value of the A, B chambers in a certain time period.

The experiment is repeated twice, and a blank control in the first experiment is marked as a control sample 1, and a test sample is marked as a test sample 1; the blank in the second run was designated control 2 and the test run was designated test run 2.

And (3) monitoring results:

example 5: the bacillus megaterium-containing formulation prepared in example 2 was monitored for formaldehyde, ammonia, TVOC removal according to:

3. Total Volatile Organics (TVOC): GB/T18883-2002 national standard experiment method for indoor air:

the sample is placed at 1.5m³The test is carried out in the experimental cabin, and the detected spraying amount is 50ml/m³Detecting concentration values of formaldehyde, ammonia and TVOC after acting for 24h, calculating a relevant removal rate, and detecting a result:

the results show that twenty-four hours after using the bacillus megaterium-containing formulation, the concentrations of formaldehyde, ammonia and TVOC in the test chamber are significantly reduced compared to the blank control. Wherein, the removal rate of the preparation containing the bacillus megaterium to formaldehyde is 90.5 percent; the removal rate of ammonia was 91.7%; the removal rate of TVOC was 81.7%.

Example 6 use of bacillus megaterium-containing formulations prepared in example 2 to inhibit the growth of mites

In a sealed aerated plastic container at 28 ℃ and 70% humidity, dust mites were inoculated and hatched, the bottom of the container was padded with woven cloth without additional feed. On the surface of the woven fabric in the container, a preparation containing bacillus megaterium is used for half, and purified water with the same quantity is used for half. The preparation containing Bacillus megaterium was used for seven consecutive days, and the change in the number of dust mites on both sides was observed and recorded.

The observation results are:

from the above experimental results, it was found that the number of dust mites which were added daily with the preparation containing Bacillus megaterium was greatly decreased, while the number of dust mites which were added with purified water was slightly increased, and the preparation containing Bacillus megaterium had a certain inhibitory effect on the propagation and growth of dust mites, as can be seen from the comparison between the two groups. Therefore, the preparation containing the bacillus megaterium can effectively inhibit the growth and the reproduction of mites in daily life.

The test results show that the bacillus megaterium preserved in CGMCC with the preservation number of CGMCC NO.19820 has extremely strong inhibition effect on escherichia coli and mites; in addition, the air purifier can also effectively remove formaldehyde, ammonia, benzene series and total volatile organic compounds in the air, and has the function of purifying the air.

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.

Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all the modifications of the equivalent structure or equivalent flow path using the present specification, or the direct or indirect application to other related fields are included in the scope of the present invention.

Claims

1. Is preserved in CGMCC with the preservation number of CGMCC NO. 19820.

2. The application of the bacillus megaterium preserved in CGMCC with the preservation number of CGMCC NO.19820 in removing formaldehyde, ammonia, benzene series and total volatile organic compounds in the air.

3. The application of the bacillus megaterium preserved in CGMCC with the preservation number of CGMCC NO.19820 in inhibiting the growth and reproduction of escherichia coli.

4. The application of the bacillus megaterium preserved in CGMCC with the preservation number of CGMCC NO.19820 in inhibiting the growth and reproduction of mites.

5. An air purifying agent characterized in that the purifying agent contains the Bacillus megaterium of claim 1.

6. An Escherichia coli growth inhibitor comprising the Bacillus megaterium of claim 1.

7. A mite growth inhibitor characterized by comprising the Bacillus megaterium of claim 1.