CN113667623A - Bacillus belgii preparation for degrading acetaldehyde and preparation method and application thereof - Google Patents
Bacillus belgii preparation for degrading acetaldehyde and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to bacillus for biologically degrading acetaldehyde, which can generate an enzyme for catalyzing and degrading acetaldehyde, wherein the bacillus is a Bacillus belgii YW-01 strain. The invention also relates to a Bacillus belgii preparation for degrading acetaldehyde, which contains one or more of bacterial cells, spores and crude enzymes of the Bacillus. Research results show that the bacillus for biologically degrading acetaldehyde and the enzyme generated by the bacillus for biologically degrading acetaldehyde are safe to human bodies, can efficiently biologically degrade acetaldehyde, and have important application prospects in the aspect of removing acetaldehyde and using the acetaldehyde for human hangover alleviation.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a bacillus beiLeisi preparation for degrading acetaldehyde, and a preparation method and application thereof.
Background
Drinking is a daily life habit of people of all countries in the world, China has a long history of wine culture, and the daily life and festivals of households cannot be drunk. According to statistics, the number of people drinking wine in China breaks through 5 hundred million, 600 million jin of white spirit is drunk in one year, and the wine is called a big drinking country. However, the health problems caused by long-term drinking or excessive drinking are more and more concerned by people, the main component alcohol (ethanol) in the wine has low toxic effect on people, but after people drink the wine, the ethanol can directly enter blood in vivo, is catalyzed and converted into acetaldehyde by alcohol dehydrogenase generated by liver, then converts the acetaldehyde into acetic acid under the catalysis of the acetaldehyde dehydrogenase, and finally is decomposed into carbon dioxide and water to be discharged out of the body. Acetaldehyde, compared to ethanol and acetic acid, is the most toxic to humans and is the major cause of intoxication and intoxication in humans. Acetaldehyde is not only produced by the intake conversion of alcohol, but also can be taken into human bodies by being used for preparing fruit essence or alcohol essence. The acetaldehyde is accumulated in blood too much to cause blood vessel expansion, red face and red ear, heart beat acceleration, headache and brain distension of people, namely the alcoholism state, so the acetaldehyde is the culprit of causing intoxication of people, the toxicity of the acetaldehyde is more than 30 times of that of ethanol, not only can destroy organs such as muscles, brains, livers, kidneys and the like to cause DNA damage and chromosome rearrangement, but also can cause hematopoietic stem cells to be damaged to cause blood diseases and cancers, and the acetaldehyde is listed in a 'primary carcinogen' list by a world health organization.
Whether the acetaldehyde accumulated in the body after drinking can be rapidly decomposed and converted is the key for human health, wherein acetaldehyde dehydrogenase plays a key conversion role. Acetaldehyde dehydrogenase produced in most people in China has low activity or even no activity, so most people are drunk soon. At present, the medicaments or foods for degrading and removing acetaldehyde in a human body are mainly extracted from Chinese herbal medicines, and the main effect is to relieve the toxicity of acetaldehyde, so the effect of relieving the alcoholism is not obvious and side effects are caused. Compared with other methods, the method for removing acetaldehyde by microbial degradation has the advantages of low cost, strong safety and the like, and is a very promising method for removing acetaldehyde by degradation, so that scholars at home and abroad carry out a great deal of research work in the field, and the method comprises reports of bacteria such as Lactobacillus rhamnosus (Lactobacillus rhamnous) and Bacillus coagulans (Bacillus coagulousns) for biodegradation of acetaldehyde, and genetic engineering strains of escherichia coli for biodegradation of acetaldehyde dehydrogenase expressed by genetic recombination, wherein the acetaldehyde biodegradation capacity is very low no matter whether the strains are naturally screened or genetically recombined, and the daily acetaldehyde degradation capacity is not more than 350 mg/L.
Bacillus velezensis is a kind of spore-producing gram-positive bacteria, and has the advantages of wide antibacterial spectrum, rapid growth, easy separation and culture, strong stress resistance, high biological safety and the like. In recent years, research on bacillus belgii has been mainly focused in the fields of feed, medical treatment, textile, aquatic products, sewage treatment, plant protection, and the like. As a novel biological control microbial factor, the Bacillus belgii can prevent and control plant diseases and promote plant growth, so far, no research report that the Bacillus belgii has toxic effect exists, and no literature report exists on the aspect of using the Bacillus belgii to biodegrade acetaldehyde, so that the characteristic of using the Bacillus belgii to efficiently degrade acetaldehyde is utilized, and the preparation of the biological preparation for relieving alcoholism at the cell, spore and enzyme levels has important research significance and application value.
Disclosure of Invention
One of the purposes of the invention is to provide a bacillus for biodegradation of acetaldehyde aiming at the problems that the effect of a drug or food for degrading and removing acetaldehyde is not obvious and side effects exist in the prior art, wherein bacterial cells, spores and enzymes generated by the bacterial cells and spores are safe to a human body, can efficiently biodegrade acetaldehyde, and has important application prospect in the aspect of biodegradation and acetaldehyde removal.
The second purpose of the invention is to provide a bacillus belief preparation for degrading acetaldehyde and an application thereof, wherein the bacillus belief preparation is prepared from the bacillus for biologically degrading acetaldehyde and can efficiently biologically degrade acetaldehyde.
To this end, the present invention provides, in a first aspect, a Bacillus for biodegrading acetaldehyde, which is capable of producing an enzyme that catalyzes the degradation of acetaldehyde, said Bacillus being Bacillus belgii strain YW-01, having a accession number of CGMCC No. 22814.
In some embodiments of the invention, the bacterial cells of Bacillus belgii strain YW-01 are capable of degrading all acetaldehyde at an initial concentration of 1000mg/L within 10 hours.
In other embodiments of the invention, the crude enzyme of Bacillus belgii YW-01 strain is capable of degrading all acetaldehyde at an initial concentration of 1000mg/L within 2 hours at a protein concentration of 4.3 mg/mL.
In a second aspect, the invention provides a bacillus belgii preparation for degrading acetaldehyde, which contains one or more of bacterial cells, spores and crude enzymes of the bacillus as described in the first aspect of the invention; preferably, the bacillus belgii preparation contains spores of the bacillus as described in the first aspect of the invention.
In some of the present inventionIn embodiments, the acetaldehyde degrading bacillus beijerinckii preparation is a liquid preparation; preferably, in the acetaldehyde-degrading liquid preparation, the concentration of bacterial cells and/or spores of bacillus cells is (1-5) × 1010Per mL; and/or, in the liquid preparation for degrading acetaldehyde, the protein concentration of the crude enzyme of bacillus is 1-10 mg/mL.
In other embodiments of the invention, the acetaldehyde-degrading bacillus beijerinckii formulation is a solid powder formulation; preferably, in the acetaldehyde-degrading solid powder preparation, the content of bacterial cells and/or spores of bacillus cells is (1-5). times.1010(2-4). times.10 is more preferable10(ii)/g; and/or, in the solid powder preparation for degrading acetaldehyde, the protein content of the bacillus crude enzyme is 500mg/g, more preferably 300mg/g and 100 mg/g.
In a third aspect of the present invention, there is provided a method for producing a bacillus belgii preparation for degrading acetaldehyde according to the second aspect of the present invention, comprising:
b, inoculating the fermentation strain into a fermentation culture medium for fermentation culture to obtain a fermentation culture of bacillus;
c, carrying out centrifugal separation treatment on the fermentation culture of the bacillus to obtain bacterial cells and/or spores of the bacillus;
wherein the fermentation strain is obtained by seed culture of corresponding strains;
the corresponding strain of the fermenting species is a strain having at least 90% homology to the 16S rDNA of the strain of Bacillus according to the first aspect of the invention; preferably the corresponding strain of the species fermentum is a strain having at least 95% homology to the 16S rDNA of the strain of bacillus according to the first aspect of the invention; it is further preferred that the corresponding strain of a fermentative species is a strain of a bacillus according to the first aspect of the present invention.
According to the invention, the fermentation medium comprises, based on 1L of water, the following components in 1L of water:
5-10g of beef extract; preferably 8 to 10 g;
5-10g of peptone; preferably 8 to 10 g; and
3-8g of NaCl; preferably 4-6 g;
preferably, the pH value of the fermentation medium is 7-8;
further preferably, in step B, the temperature of the fermentation culture is 18-40 ℃, preferably 36-38 ℃.
According to some embodiments of the invention, the method of preparing further comprises:
step K, carrying out cell disruption treatment on the cell suspension of the bacillus under a low-temperature condition to obtain a cell-free disruption solution of the bacillus;
step L, carrying out centrifugal separation on cell-free broken liquid of the bacillus, and taking supernatant cell-free extracting solution as crude enzyme of the bacillus;
wherein the low temperature is 0-4 ℃.
In a fourth aspect, the present invention provides a use of the acetaldehyde-degrading bacillus beijerinckii preparation according to the second aspect of the present invention or the acetaldehyde-degrading bacillus beijerinckii preparation prepared by the preparation method according to the third aspect of the present invention in preparing an anti-hangover medicine, comprising:
d, cleaning the bacterial cells and/or spores of the bacillus by using normal saline to obtain pure bacterial cells and/or spores of the bacillus;
step E, in a physiological saline solution system, under the low temperature condition, breaking pure bacterial cells of the bacillus by adopting ultrasonic waves, centrifuging, and taking supernate to obtain cell-free extracting solution as a crude enzyme pure product of the bacillus;
step F, one or more of bacterial cells, spores and crude enzymes of the bacillus are frozen and dried, and the frozen and dried bacillus belius preparation is diluted to prepare the medicament for relieving alcoholism;
wherein the low temperature is 0-4 ℃.
In some embodiments of the present invention, in step F, the lyophilized bacillus belief formulation is diluted with a physiological saline to prepare an anti-hangover preparation, and the anti-hangover preparation is prepared as a liquid anti-hangover preparation.
In other embodiments of the present invention, in step F, the bacillus belief agents are diluted with freeze-dried edible starch to form a solid anti-hangover preparation.
In some preferred embodiments of the present invention, the anti-hangover agent is an oral preparation.
Researches show that the bacillus for biodegrading acetaldehyde and the produced enzyme are safe to human bodies, can biodegrade acetaldehyde, and have important application prospects in the aspect of efficiently removing acetaldehyde and relieving alcoholism.
Drawings
For the present invention to be readily understood, the following description is made with reference to the accompanying drawings.
FIG. 1 shows a 16S rDNA-based molecular evolutionary tree of Bacillus belgii YW-01.
FIG. 2 shows the growth curve of Bacillus belgii YW-01 and the kinetics of biodegradation of acetaldehyde.
FIG. 3 shows the kinetics of the crude enzyme catalyzed acetaldehyde degradation by Bacillus belgii YW-01.
Strain preservation
Bacillus velezensis (Bacillus velezensis), isolated and identified by Beijing university of science and technology, has been deposited in China general microbiological culture Collection center (CGMCC, address: China academy of sciences, institute of microbiology, No. 3, West Lu No. 1, Beijing area of the rising of the republic of China), with the preservation date: year 2021, month 07, 05, accession number: CGMCC No. 22814. The strain is named as Bacillus velezensis strain YW-01 in the invention.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Term of
The term "cell" as used herein refers to a live cell and/or a dead cell of a bacterium.
The term "spore" in the invention refers to a dormant body with very strong stress resistance formed by bacillus under certain conditions.
The term "crude enzyme" as used herein refers to a cell-free extract obtained by crushing bacterial cells of Bacillus and then centrifuging the cell-free extract to obtain a supernatant.
The term "pure crude enzyme" as used herein means a cell-free extract obtained by crushing a pure Bacillus cell and then centrifuging the supernatant, as opposed to a crude enzyme (i.e., a cell-free extract obtained by crushing a Bacillus cell and then centrifuging the supernatant).
The terms "crude enzyme of Bacillus belgii strain YW-01" and "crude enzyme of Bacillus belgii strain YW-01" as used herein are used interchangeably.
The term "microbial preparation" as used herein refers to preparations of various forms which are prepared from microorganisms having medical and research values as raw materials by conventional techniques or modern biotechnology and which are useful for the prevention (health care), treatment and diagnosis of various physiological symptoms of the human body.
The term "edible starch" as used herein refers to starch that meets the national standard for edible starch (GB 31637-2016 national food safety standard edible starch).
"Water" used in the medium or fermentation culture process of the present invention means, unless otherwise specified, sterile pure water obtained by filtration through a 0.22 μ filter.
II, embodiments
As mentioned above, the existing medicines or foods for degrading and removing acetaldehyde in human body are not satisfactory, and have the problems of the medicines or foods; for example, although preparations extracted from Chinese herbs can alleviate the toxicity of acetaldehyde, they have insignificant anti-hangover effects and side effects. The capacity of biologically degrading acetaldehyde is very low whether the strains are screened or genetically recombinant strains, and the acetaldehyde is degraded by no more than 350mg/L in day. In view of this, the present inventors have made extensive and intensive studies on biodegradation of acetaldehyde.
On the basis of long-term microbial research, a microbial pure strain capable of efficiently biodegrading acetaldehyde is successfully screened from cellar mud, and the strain and enzyme generated by the strain can efficiently biodegrade acetaldehyde, so that the method has very important research value and has important application prospect in the aspect of efficiently biodegrading and removing acetaldehyde.
Accordingly, the present invention relates to a bacillus for biodegrading acetaldehyde, which is capable of producing an enzyme that catalyzes the degradation of acetaldehyde.
The inventor successfully screens out a bacillus strain from a wine cellar. The strain was identified as Bacillus velezensis sp by extracting genomic DNA, PCR-amplifying and 16S rDNA sequencing-based molecular identification, and based on the above, the strain was identified and named Bacillus velezensis strain YW-01 (Bacillus velezensis strain YW-01). The strain is preserved in the China general microbiological culture Collection center, and the preservation number is as follows: CGMCC No. 22814.
The inventors have found that when Bacillus bleekii YW-01 strain is subjected to fermentation culture, cells and spores produced during the fermentation culture contain one or more enzymes capable of catalyzing the degradation of acetaldehyde, and a mixture of these enzymes is referred to as a crude enzyme or Bacillus bleekii YW-01 crude enzyme in the present invention.
Further research shows that cell-free broken liquid after bacterial cells are broken is centrifugally separated, and supernatant cell-free extracting solution is taken as crude enzyme of Bacillus beiLeisi YW-01; the spores can resist gastric acid and are converted into cells under the condition of pH neutrality when entering the small intestine of a human body, and further, crude enzyme of Bacillus belgii YW-01 capable of degrading acetaldehyde is generated; from this, it is easily understood that the bacterial cells, spores of Bacillus belgii strain YW-01 and crude enzyme of Bacillus belgii YW-01 are all capable of catalyzing the degradation of acetaldehyde.
The research result shows that the bacterial cells and/or spores of the Bacillus belgii YW-01 strain can completely degrade and remove the acetaldehyde with the initial concentration of 1000mg/L within 10 hours.
The crude enzyme produced by the Bacillus belgii YW-01 strain can completely degrade acetaldehyde with the initial concentration of 1000mg/L within 2 hours under the protein concentration of 4.3 mg/mL.
Based on the above, the second to fourth aspects of the present invention further provide a use or application of the bacillus for biodegrading acetaldehyde as described in the first aspect of the present invention.
Specifically, the second aspect of the present invention provides a bacillus belief preparation for biodegradation of acetaldehyde, which belongs to a microbial preparation for biodegradation of acetaldehyde, and which contains one or more of bacterial cells, spores and crude enzymes of the bacillus according to the first aspect of the present invention.
In some preferred embodiments of the invention, the bacillus belgii preparation contains spores of the bacillus as described in the first aspect of the invention.
According to some embodiments of the invention, the bacillus beijerinckii preparation that biodegrades acetaldehyde is a liquid preparation.
In some embodiments of the invention, the concentration of bacterial cells and/or spores of bacillus cells in the liquid formulation for biodegrading acetaldehyde is (1-5) × 1010/mL。
In other embodiments of the present invention, the crude enzyme of Bacillus has a protein concentration of 1-10mg/mL in the liquid formulation for biodegrading acetaldehyde.
According to other embodiments of the present invention, the bacillus belgii preparation for biodegradation of acetaldehyde is a solid powder preparation.
In some embodiments of the invention, the content of bacterial cells and/or spores of bacillus cells in the solid powder preparation for biodegrading acetaldehyde is (1-5) × 1010Per g, preferably (2-4). times.1010/g。
In other embodiments of the invention, the protein content of the crude enzyme of Bacillus in the solid powder preparation for biodegradation of acetaldehyde is 100-500mg/g, preferably 200-300 mg/g.
In a third aspect of the present invention, there is provided a method for producing a bacillus belgii preparation for biodegradation of acetaldehyde as described in the second aspect of the present invention, comprising:
b, inoculating the fermentation strain into a fermentation culture medium, and performing fermentation culture for 3-5 days at 18-40 ℃, preferably 36-38 ℃ and at the rotating speed of a shaking table of 100-;
c, carrying out centrifugal separation treatment on the fermentation culture of the bacillus to obtain bacterial cells and/or spores of the bacillus;
wherein the fermentation strain is obtained by seed culture of corresponding strains.
As known to those skilled in the art, the 16S rRNA is currently used internationally for molecular identification of bacteria, and thus, 16S rRNA can be used for alignment to obtain homology in similarity comparison. Therefore, the fermentation strain used in the present invention is not limited to the field isolate used in the present invention, and 16S rDNA is a DNA sequence corresponding to the coding rRNA on the chromosome of the bacterium and exists in the chromosomal genome of all bacteria. FIG. 1 shows a molecular evolutionary tree based on 16S rDNA, the Bacillus of the present invention being Bacillus belgii strain YW-01.
Thus, in the present invention, the corresponding strain of the fermentative species is a strain having at least 90% homology to the 16S rDNA of the strain of bacillus according to the first aspect of the present invention; preferably the corresponding strain of the species fermentum is a strain having at least 95% homology to the 16S rDNA of the strain of bacillus according to the first aspect of the invention; it is further preferred that the corresponding strain of a fermentative species is a bacillus strain according to the first aspect of the present invention. That is, a person skilled in the art can obtain a strain highly homologous to the 16S rDNA of Bacillus belgii YW-01 of the present invention and a strain having the same or similar acetaldehyde degrading function, by simply screening or mutagenizing Bacillus belgii YW-01 of the present invention, without changing the 16S rDNA of Bacillus belgii YW-01.
In the step C, the centrifugal separation treatment includes subjecting the liquid fermentation culture to centrifugal separation to obtain a precipitate (i.e., bacillus cells and/or spores), resuspending and washing the precipitate with physiological saline, and then subjecting the precipitate to centrifugal separation to obtain bacillus cells and/or spores.
The conditions for the centrifugation in the step C are not particularly limited in the present invention, and in some embodiments of the present invention, the substance to be separated may be centrifuged for 10min at 8000-.
According to the method, the fermentation culture is shaking table or fermentation tank fermentation culture of strains, and the fermentation strains are inoculated into a fermentation culture medium in the form of seed liquid. The inoculation amount of the seed liquid is 0.1-1% (v/v); preferably, the inoculation amount of the seed liquid is 0.2-0.5% (v/v); further preferably, the amount of the seed liquid to be inoculated is 0.5% (v/v).
Specifically, the fermentation medium comprises the following components in 1L of water in terms of 1L of water:
5-10g of beef extract;
5-10g of peptone; and
NaCl 3-8g。
preferably, the fermentation medium comprises the following components in 1L of water, based on 1L of water:
8-10g of beef extract;
8-10g of peptone; and
NaCl 4-6g。
in some embodiments of the invention, the initial pH of the fermentation medium is adjusted using 40% (wt/v) sodium hydroxide solution and 36% (v/v) hydrochloric acid solution, the pH of the fermentation medium being 7-8.
According to some embodiments of the present invention, the method for preparing a bacillus according to the present invention further comprises step a: the Bacillus belgii YW-01 strain monoclonal colony provided by the invention is selected and inoculated into 100mL fermentation liquid culture medium, and after shaking culture is carried out for 3 days at the temperature of 38 ℃ and the rotating speed of 200r/min, a fermentation strain (seed solution) is prepared.
The inventors studied the effect of different temperatures on the growth of Bacillus beijerinckii YW-01 and found that Bacillus beijerinckii YW-01 grew fast at a temperature of 38 ℃.
According to some embodiments of the invention, the method of preparing further comprises:
step K, performing cell disruption treatment on the cell suspension of the bacillus in an ice water bath (namely an ice water mixture at 0-4 ℃) to obtain a cell-free disruption solution of the bacillus;
and step L, carrying out centrifugal separation on the cell-free broken liquid of the bacillus, and taking a supernatant cell-free extracting solution as a crude enzyme of the Bacillus belgii YW-01 strain.
The conditions for the centrifugation in the step L are not particularly limited in the present invention, and in some embodiments of the present invention, the substance to be separated may be centrifuged for 10-20min at 15000-.
In a fourth aspect, the present invention provides a use of the bacillus belief formulation for biodegradation of acetaldehyde according to the second aspect of the present invention or the bacillus belief formulation for biodegradation of acetaldehyde prepared by the preparation method according to the third aspect of the present invention for preparing an anti-hangover agent, comprising:
d, cleaning the bacterial cells and/or spores of the bacillus by using normal saline to obtain pure bacterial cells and/or spores of the bacillus;
step E, in a physiological saline solution system, under the low temperature condition of 0-4 ℃, breaking pure bacteria cells of the bacillus by ultrasonic waves, centrifuging, and taking supernatant fluid to obtain cell-free extracting solution as a crude enzyme pure product of the bacillus;
and F, freeze-drying one or more of bacterial cells, spores and crude enzymes of the bacillus, and diluting the freeze-dried bacillus belief preparation to prepare the anti-alcoholism medicament.
In some embodiments of the present invention, in step F, the lyophilized bacillus belief formulation is diluted with a physiological saline to prepare an anti-hangover preparation, and the anti-hangover preparation is prepared as a liquid anti-hangover preparation.
In other embodiments of the present invention, in step F, the bacillus belief agents are diluted with freeze-dried edible starch to form a solid anti-hangover preparation.
In some preferred embodiments of the present invention, the anti-hangover agent is an oral preparation.
III, correlation detection method in the invention
(1) The cell and/or spore concentration of the present invention is determined by the following method:
the method for measuring the cell and/or spore concentration of the Bacillus belgii YW-01 comprises the steps of taking a culture of the Bacillus belgii YW-01, diluting the culture by a certain multiple of physiological saline, and directly measuring the cell and/or spore concentration in the culture by using a flow cytometer (SYSMEX, Germany).
(2) The acetaldehyde concentration in the invention is measured by the following method:
and (3) measuring the concentration of acetaldehyde, namely centrifuging a culture of Bacillus belgii YW-01, diluting a supernatant, mixing the diluted supernatant with 2, 4-dinitrophenylhydrazine according to a certain proportion, reacting in a water bath at 25 ℃ for 30 minutes, adding a NaOH solution according to a certain proportion, standing for 5 minutes, measuring the absorbance at 380nm by using a 722S visible spectrophotometer (Shanghai prismatic light), and calculating the concentration of the acetaldehyde according to a standard curve.
(3) The concentration of the crude enzyme protein is determined by the following method:
taking a cell-free extracting solution of Bacillus belgii YW-01, diluting by a certain multiple through a phosphate buffer solution, adding a Coomassie brilliant blue G-250 dye reagent in proportion, reacting for 10 minutes, measuring absorbance at 595nm by using a 722S visible spectrophotometer (Shanghai prismatic light), and calculating the protein concentration by adopting a standard curve method.
III example
The present invention will be specifically described below with reference to specific examples. The experimental methods described below are, unless otherwise specified, all routine laboratory procedures. The experimental materials described below, unless otherwise specified, are commercially available.
Example 1:
(1) preparing a growth medium of Bacillus belgii YW-01, which comprises the following components (per liter): 10.0g of beef extract, 10.0g of peptone and 5.0g of NaCl. 100ml of the prepared liquid medium was added to a 500 ml Erlenmeyer flask, and sterilized at high temperature and high pressure (121 ℃) for 20 minutes, and then further sterilized under ultraviolet irradiation in a clean bench for 20 minutes.
(2) Inoculating 0.5 ml of Bacillus belgii YW-01 bacterial liquid into a triangular flask liquid culture medium under the aseptic condition in a clean workbench, carrying out batch culture for 3 days under the conditions of the temperature of 38 ℃ and the rotating speed of a shaking table of 200r/min, and then harvesting the Bacillus belgii YW-01 cells and/or spores by a method of pouring out supernatant after centrifugation (8000 r/min, 10 minutes).
Adding 20mL of the Bacillus beiLeisi YW-01 cell suspension into a 50mL glass tube, inserting the glass tube into ice water, and crushing the Bacillus beiLeisi YW-01 cells by using an ultrasonic cell crusher, wherein the conditions are as follows: ultrasonic power 400W, interval 2 seconds, ultrasonic oscillation 10 seconds, crushing time 15 minutes (each time 5 minutes). After completion of cell disruption, the cell disruption solution was centrifuged at 15000 rpm for 20 minutes, and the supernatant was slowly decanted as a cell-free extract (crude enzyme) of Bacillus beiensis YW-01.
(3) According to different concentrations of acetaldehyde, cells and/or spores of the cultured and prepared Bacillus belgii YW-01 and crude enzyme are used as a fast, safe and efficient biocatalyst and are added according to a certain proportion, so that the aim of rapidly and efficiently degrading and removing acetaldehyde is fulfilled.
FIG. 1 shows that the strain we screened has a closest relationship to B.belgii and is therefore designated B.belgii YW-01 strain.
FIG. 2 shows that acetaldehyde at an initial concentration of 1000mg/L can be completely degraded with the growth of Bacillus belgii YW-01 from 50 to 250 hundred million/mL in 10 hours, indicating that Bacillus belgii YW-01 has a strong biodegradability for acetaldehyde.
FIG. 3 shows that the cell-free extract (crude enzyme) of Bacillus belgii YW-01 can catalyze and degrade acetaldehyde at a faster rate, and can completely degrade acetaldehyde with an initial concentration of 1000mg/L for 2 hours at a protein concentration of 4.3mg/mL, so that the acetaldehyde degradation rate is higher.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Claims (10)
1. A bacillus for biologically degrading acetaldehyde, which can generate an enzyme for catalyzing and degrading acetaldehyde, wherein the bacillus is a Bacillus belgii YW-01 strain with the preservation number of CGMCC No. 22814.
2. The bacillus of claim 1, wherein the bacterial cells and/or spores of the strain YW-01 of bacillus belgii are capable of completely degrading and removing acetaldehyde at an initial concentration of 1000mg/L within 10 hours.
3. The Bacillus of claim 1, wherein the crude enzyme from Bacillus belgii strain YW-01 is capable of degrading all acetaldehyde at an initial concentration of 1000mg/L within 2 hours at a protein concentration of 4.3 mg/mL.
4. A bacillus belgii preparation for degrading acetaldehyde, comprising one or more of bacterial cells, spores and crude enzymes of the bacillus of any one of claims 1 to 3; preferably, the Bacillus belgii preparation contains spores of the Bacillus as claimed in any one of claims 1 to 3.
5. The Bacillus belgii preparation according to claim 4,
the bacillus beleisi preparation for degrading acetaldehyde is a liquid preparation; preferably, in the acetaldehyde-degrading liquid preparation, the concentration of bacterial cells and/or spores of bacillus cells is (1-5) × 1010Per mL; and/or, in the liquid preparation for degrading acetaldehyde, the protein concentration of the crude enzyme of bacillus is 1-10 mg/mL;
or the acetaldehyde-degrading Bacillus belgii preparation is a solid powder preparation; preferably, in the acetaldehyde-degrading solid powder preparation, the content of bacterial cells and/or spores of bacillus is (1-5). times.1010(2-4). times.10 is more preferable10(ii)/g; and/or, in the solid powder preparation for degrading acetaldehyde, the protein content of the bacillus crude enzyme is 500mg/g, more preferably 300mg/g and 100 mg/g.
6. A method of preparing a Bacillus belgii preparation for degrading acetaldehyde as claimed in any one of claims 4 or 5, comprising:
b, inoculating the fermentation strain into a fermentation culture medium for fermentation culture to obtain a fermentation culture of bacillus;
c, carrying out centrifugal separation treatment on the fermentation culture of the bacillus to obtain bacterial cells and/or spores of the bacillus;
wherein the fermentation strain is obtained by seed culture of corresponding strains;
the corresponding strain of the fermentative species is a strain having at least 90% homology to the 16S rDNA of the strain of Bacillus of any one of claims 1-3; preferably the corresponding strain of the species fermentum is a strain having at least 95% homology to the 16S rDNA of the strain of bacillus according to any one of claims 1-3; further preferred is a strain of the bacillus according to any of claims 1-3, wherein the corresponding strain of the species zymogen is a strain of bacillus according to any of the claims.
7. The method according to claim 6, wherein the fermentation medium comprises the following components in 1L of water, based on 1L of water:
5-10g of beef extract; preferably 8 to 10 g;
5-10g of peptone; preferably 8 to 10 g; and
3-8g of NaCl; preferably 4-6 g;
preferably, the pH value of the fermentation medium is 7-8;
further preferably, in step B, the temperature of the fermentation culture is 18-40 ℃, preferably 36-38 ℃.
8. The production method according to claim 6 or 7, characterized by further comprising:
step K, carrying out cell disruption treatment on the cell suspension of the bacillus under a low-temperature condition to obtain a cell-free disruption solution of the bacillus;
step L, carrying out centrifugal separation on cell-free broken liquid of the bacillus, and taking supernatant cell-free extracting solution as crude enzyme of the bacillus;
wherein the low temperature is 0-4 ℃.
9. Use of the acetaldehyde-degrading Bacillus belgii preparation of claim 4 or 5 or the acetaldehyde-degrading Bacillus belgii preparation prepared by the preparation method of any one of claims 6 to 8 in the preparation of an anti-hangover medicament, comprising:
d, cleaning the bacterial cells and/or spores of the bacillus by using normal saline to obtain pure bacterial cells and/or spores of the bacillus;
step E, in a physiological saline solution system, under the low temperature condition, breaking pure bacterial cells of the bacillus by adopting ultrasonic waves, centrifuging, and taking supernate to obtain cell-free extracting solution as a crude enzyme pure product of the bacillus;
step F, one or more of bacterial cells, spores and crude enzymes of the bacillus are frozen and dried, and the frozen and dried bacillus belius preparation is diluted to prepare the medicament for relieving alcoholism;
wherein the low temperature is 0-4 ℃.
10. Use according to claim 9,
in the step F, diluting the freeze-dried Bacillus belgii preparation by using normal saline to prepare an anti-inebriation medicament and prepare a liquid anti-inebriation medicament;
or, in the step F, diluting the freeze-dried Bacillus belgii preparation by using edible starch to prepare a solid anti-alcohol agent;
preferably, the anti-alcoholism medicament is an oral preparation.
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| CN107058168A (en) * | 2017-01-21 | 2017-08-18 | 淮海工学院 | A kind of bacillus megaterium and its method and product for producing Pullulanase |
| KR101780229B1 (en) * | 2016-09-05 | 2017-09-21 | 조선대학교산학협력단 | Extremely alkaline mannanase from Bacillus subtilis subsp. inaquosorum CSB31 isolated from fermented food Kimchi and the use thereof |
| CN113073058A (en) * | 2021-03-17 | 2021-07-06 | 中国农业大学 | Bacillus subtilis mafic-Y7 with soybean antigen protein degradation activity and application thereof |
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| KR101780229B1 (en) * | 2016-09-05 | 2017-09-21 | 조선대학교산학협력단 | Extremely alkaline mannanase from Bacillus subtilis subsp. inaquosorum CSB31 isolated from fermented food Kimchi and the use thereof |
| CN107058168A (en) * | 2017-01-21 | 2017-08-18 | 淮海工学院 | A kind of bacillus megaterium and its method and product for producing Pullulanase |
| CN113073058A (en) * | 2021-03-17 | 2021-07-06 | 中国农业大学 | Bacillus subtilis mafic-Y7 with soybean antigen protein degradation activity and application thereof |
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