CN116082470A - Bacillus bailii antibacterial lipopeptide and preparation method and application thereof - Google Patents
Bacillus bailii antibacterial lipopeptide and preparation method and application thereof Download PDFInfo
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- 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 food biology, and particularly relates to bacillus bailii antibacterial lipopeptid as well as a preparation method and application thereof. The antibacterial lipopeptide provided by the invention is prepared from the following components in percentage by weight: bacillus bailii Bacillus velezensis BVQ121 of M20211570 is prepared by fermenting, emulsifying to construct an O/W emulsifying system and spray drying. The BVQ strain has good inhibition effect on common food-borne pathogenic bacteria. The invention improves the preparation method when preparing the antibacterial lipopeptide, adopts the means of combining an emulsifying system and a spray drying technology, solves the problem of toxicity of an organic solvent while improving the water solubility of AMPs, and increases the antibacterial activity of AMPs redissolved substances.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to bacillus bailii antibacterial lipopeptid as well as a preparation method and application thereof.
Background
The antibacterial lipopeptid (Antimicrobial peptides, AMPs) has the advantages of wide antibacterial spectrum, rapid action, difficult generation of drug resistance and the like, and has wide application prospect in medicine, cosmetics and food industry. AMPs are small molecular proteins composed of different amino acids, and have the advantages of small molecular weight, low immunogenicity, special mechanism, easy in vivo degradation, difficult drug resistance generation and the like, and the advantages endow the antimicrobial lipopeptides with great application potential in the food industry. The bacillus has the outstanding advantages of multiple types of AMPs, multiple functions, easily available raw materials, short production period, easy large-scale fermentation production and the like; meanwhile, the AMPs have stable structures and still keep activity in high-temperature and acidic environments; in addition, AMPs also have good broad-spectrum antibacterial activity, can inhibit a plurality of food-borne pathogenic bacteria, and also have remarkable inhibition effects on viruses, mycoplasma and the like. More importantly, compared with chemical surfactants, the AMPs have the advantages of low toxicity, easiness in biodegradation, good environmental compatibility and the like, can replace chemical surfactants in the aspects of food raw material safety control, food processing links, food preservation, food activity packaging and the like, and has great application potential.
AMPs have the obvious advantages of low cost, no toxicity, short period and the like, and the acceleration of the research of antimicrobial lipopeptides of microorganisms is necessarily the mainstream. Current research on bacillus bailii AMPs is focused mainly on plant antifungal activity, such as the effect of bacillus bailii D61-a on the bacterial community structure of rice rhizosphere soil; the bacillus belicus explores biocontrol of penicillium citrinum and the like. On the other hand, AMPs are mostly extracted by acid precipitation and other extraction methods, but AMPs after precipitation and drying are difficult to redissolve in water due to the chemical structure of hydrophobic aliphatic hydrocarbon chains, and most AMPs are redissolved by toxic organic solvents such as methanol or acetonitrile, so that the defect is greatly limited in application of AMPs extract in food.
Disclosure of Invention
The invention aims to solve the technical problem of providing the bacillus bailii antibacterial lipopeptide aiming at the defects of the prior art.
The invention also solves the technical problem of providing a preparation method of the antibacterial lipopeptide.
The invention finally solves the technical problem of providing the application of the antibacterial lipopeptide in inhibiting food from being infected by food-borne pathogenic bacteria.
In order to solve the technical problems, the invention adopts the following technical scheme:
the bacillus beleiensis antibacterial lipopeptide is characterized by comprising the following components of bacillus beleiensis Bacillus velezensis BVQ and bacillus beleiensis CCTCC NO: m20211570 is prepared by fermenting, emulsifying to construct an O/W emulsifying system and spray drying.
Wherein the molecular weight of the bacillus belicus antimicrobial lipopeptide is 1-3Kda, and the yield is 1.527+/-0.03 g/L.
The preparation method of the bacillus bailii antibacterial lipopeptide comprises the following steps:
(1) Bacillus beleiensis Bacillus velezensis BVQ, 121 cctccc NO: activating M20211570 strain;
(2) Inoculating the strain activated in the step (1) into a Landy modified culture medium for fermentation culture to obtain a fermentation broth, and centrifuging to retain a supernatant;
(3) Regulating the pH of the supernatant in the step (2), centrifuging at a low temperature after overnight at a low temperature, and discarding the supernatant to collect a precipitate;
(4) Adding emulsion into the precipitate obtained in the step (3), and shaking and uniformly mixing to obtain an O/W emulsion system;
(5) And (3) spray drying the emulsion system obtained in the step (4) to prepare antibacterial lipopeptide powder.
Wherein, in the step (2), the inoculation amount is 1-3% v/v. Preferably, the inoculum size is 1.5% v/v.
Wherein, in the step (2), the fermentation culture is carried out under the following conditions: fermenting at 37deg.C for 24-48h in shaking table 100-300 r/min. Preferred conditions are: fermenting at 37deg.C and shaking table 200r/min for 48 hr.
In the step (2), the formula of the Landy modified culture medium is as follows: glucose 20.0g/L, sodium glutamate 5.0g/L, yeast extract 1.0g/L, mgSO4 0.5g/L, KCl 0.5g/L, KH2PO4 1.0g/L, feSO40.15mg/L, mnSO4 0.5mg/L, cuSO4 0.16mg/L.
Wherein, in the step (3), the pH is adjusted to 2.0-4.0; the low temperature is 4 ℃; the centrifugation is performed under the conditions of 6000-9000r/min for 5-10min. The preferred pH is 4.0, and the centrifugation conditions are 9000r/min and centrifugation at 4℃for 10min.
Wherein, in the step (4), the emulsion is added according to the concentration ratio that the lipopeptide content reaches 10-20 mg/mL. The preferred addition amounts are: the lipopeptides were added in such a concentration ratio that the lipopeptides content was 10mg/mL and 20mg/mL, respectively.
Wherein in the step (4), the emulsion is a mixed solution of glycerol and water, wherein the volume of water is 4-6 times of that of the glycerol. Preferably, the volume of water is 4 times that of glycerol.
In the step (5), the spray drying is performed under the following process conditions: and (3) using a low-temperature spray dryer to continuously spray-dry at 80 ℃, wherein the feeding flow is 40-60mL/min, the feeding temperature is 20 ℃, and the outlet temperature is 60 ℃.
The application of the bacillus belicus antimicrobial lipopeptid in inhibiting food from being infected by food-borne pathogenic bacteria is also within the scope of the invention.
Wherein the food-borne pathogenic bacteria are any one of Edwardsiella ictaluri, escherichia coli and Salmonella. Salmonella is preferred.
Specifically, the O/W emulsion system prepared by using the bacillus bailii antibacterial lipopeptid is uniform in white turbid liquid, and the bacillus bailii antibacterial lipopeptid is proved to have good effect when being used as an emulsifier, and can be used as a food emulsifier.
Specifically, bacillus belgium BVQ121 fermentation liquor has obvious antibacterial effect on proliferation of pathogenic bacteria (Edwardsiella ictaluri, escherichia coli and salmonella), the comparison of the diameters of antibacterial rings shows that the antibacterial effect of the fermentation liquor is positively correlated with the dosage, the inhibition effect of the fermentation liquor on salmonella at the same dosage is most obvious, the diameter of the antibacterial ring reaches 26.50mm at most when 100 mu L of fermentation liquor is added, and the antibacterial rings of escherichia coli and Edwardsiella ictaluri are 24.5mm and 18.0mm respectively
Specifically, the salmonella is continuously adopted as an indicator bacterium to carry out an evaluation experiment, and the comparison of the average diameter of the inhibition zone can find that the glycerol emulsion added with the lipopeptide has obvious inhibition effect, and the inhibition effect of 20mg/mL of the bacillus subtilis lipopeptide emulsion is better than that of 10mg/mL of the bacillus subtilis lipopeptide emulsion. The diameter of a bacteriostasis circle added with a 100 mu LO/W emulsification system (lipopeptide/oil phase=20 mg/mL) is the highest, the diameter reaches (17.85+/-2.33) mm, the conventional lipopeptide re-dissolved by methanol is usually 14.20+/-1.65 mm, and compared with the methanol re-dissolution, the bacteriostasis effect of the lipopeptide re-dissolved substance emulsified by glycerol is improved by about 25%. These data demonstrate that bacillus bailii surfactants as emulsifiers are used in emulsifying systems to enhance their bacteriostatic effects.
Specifically, the retention time of the antibacterial peptide of the strain is between 7 and 20 minutes according to the peak retention time of the crude lipopeptide sample extracted from BVQ121 on HPLC and the comparison of the standard substances by utilizing high performance liquid chromatography. The retention time of the antibacterial lipopeptide sample in 7-10 minutes, 12-16 minutes and 16-20 minutes is consistent with that of a standard product, and the antibacterial lipopeptide is mainly composed of homologs of Surfacin, and the BVQ fermentation product component is proved, wherein the homologs of Surfacin have proven antibacterial effects on several food-borne pathogenic bacteria.
Specifically, by MALDI-TOF-MS mass spectrometry, referring to a reference and comparing with a chart, the antibacterial peptide obtained by the invention has the main components of C13-C16Iturin, C12-C15 Surfactin and C15-C23 Fengycin and partial homologs of three antibacterial peptides, and each component has corresponding application in food processing links.
The bacillus beliae producing the antibacterial lipopeptide is classified and named as bacillus beliae Bacillus velezensis, and has a strain number of BVQ and is preserved in China Center for Type Culture Collection (CCTCC) on the 12 th month 8 of 2021, wherein the preservation number is: m20211570.
The beneficial effects are that:
(1) The BVQ strain shows good inhibition effect on common food-borne pathogenic bacteria, expands the bacteriostasis spectrum, and has a certain research value.
(2) The invention improves the preparation method when extracting the antibacterial lipopeptide, adopts a means of combining an emulsifying system and a spray drying technology, improves the water solubility of AMPs, solves the toxicity problem of an organic solvent, and increases the antibacterial activity of AMPs redissolved matters.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
Fig. 1 is a diagram for identifying the form of bacillus belicus provided by the invention.
Fig. 2 is a graph showing the salmonella inhibition effect of the bacillus behenicus high-density fermentation broth. And (3) injection: A. edwardsiella ictaluri, escherichia coli, salmonella, and Salmonella; the left is 50 mu L of Bacillus bailii fermentation broth, and the right is 100 mu L of Bacillus bailii fermentation broth.
Fig. 3 is a diagram of an emulsifying system for extracting antibacterial peptide from bacillus behenicus high-density fermentation broth and an antibacterial result of an emulsifier. And (3) injection: 1 is a glycerin/water emulsion, 2 is an O/W emulsion system (lipopeptide/oil phase=10 mg/mL), and 3 is an O/W emulsion system (lipopeptide/oil phase=20 mg/mL). A is blank, B is glycerol/water emulsion, C is O/W emulsion system (lipopeptide/oil phase=10 mg/mL), D is O/W emulsion system (lipopeptide/oil phase=20 mg/mL).
FIG. 4 is a high performance liquid chromatogram of the antibacterial peptide extracted from the bacillus subtilis high-density fermentation broth;
FIG. 5 is a mass spectrum of the antibacterial peptide extracted from the bacillus subtilis high-density fermentation broth;
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
Example 1 identification of species
Bacillus belgium of the patent is separated from fermented soybean residues prepared by Nanjing industrial university laboratories of Nanjing, jiangsu, 10 months and 30 days, molecular biology methods are adopted to identify bacillus belgium strains, the 16S rRNA sequence of the bacillus belgium is shown as SEQ ID NO.1, the bacillus belgium is aligned in a GenBank nucleic acid database, the bacillus belgium is identified as bacillus belgium through sequence alignment, the bacillus belgium is named as bacillus belgium BVQ, and the bacillus belgium is registered and preserved in China center for type culture collection (CCTCC NO) at 12 months and 8 days of 2021: m20211570.
The bacillus beijerinus BVQ colony presents milky white color, has smooth surface, and is mostly round (figure 1); high temperature resistance, acid resistance, digestion resistance, activity after 1h of high temperature treatment at 110 ℃, activity under the condition of ph=2.0, activity after simulated gastric digestion.
EXAMPLE 2 bacteriostatic Effect of fermentation and fermentation broth
1. Flat plate preparation
Taking 10.0g of peptone, 10.0g of NaCl, 10.0g of agar, 5.0g of yeast powder, and distilled water to a volume of 1.0L, uniformly stirring, and then placing into a sterilizing pot at 121 ℃ for sterilization for 20min. Taking out the plate after the plate is cooled to about 70 ℃, pouring the plate, and putting the plate into an ultra-clean bench for cooling and solidifying.
2. BVQ121 activation of cells
LB liquid medium: 10.0g of peptone, 10.0g of NaCl, 5.0g of yeast powder, and distilled water to a volume of 1.0L, placing into a sterilizing pot at 121 ℃ for sterilizing for 20min after stirring uniformly. After the strain is cooled, the strain is inoculated into LB liquid culture medium by an inoculating loop, the activation condition is that the temperature is 37 ℃, the shaking table is 200r/min, and the activation time is 24 hours.
3. BVQ121 fermentation broth treatment
Landy modified medium: glucose 20.0g, sodium glutamate 5.0g, yeast extract 1.0g, mgSO 4 0.5g，KCl 0.5g，KH 2 PO 4 1.0g，FeSO 4 0.15mg，MnSO 4 0.5mg，CuSO 4 0.16mg of distilled water is added to a constant volume of 1.0L, and the mixture is placed into a sterilizing pot at 115 ℃ for sterilization for 25min after being stirred uniformly. Inoculating 1.5% v/v of activated bacteria liquid for fermentation after cooling, wherein the fermentation conditions are as follows: the temperature is 37 ℃, the shaking table is 200r/min, and the fermentation time is 48h. After fermentation, 9000r/min and centrifugation are carried out at 4 ℃ for 10min to remove bacterial precipitate, and the supernatant is reserved for standby.
4. Oxford cup bacteriostasis zone experiment
The prepared plates were taken out, 50. Mu.L of different indicator bacteria solutions (E.coli, salmonella, edwardsiella ictaluri) were added thereto, respectively, and the plates were uniformly coated with a disposable coating rod, one plate was coated with each indicator bacteria. Two sterilized oxford cups are taken out by forceps and placed in two areas of a flat plate so as to ensure that the results of the bacteria inhibition zones are not overlapped with each other. A set of blank experiments was set up and 50 and 100. Mu.L of treated broth were added dropwise to the two areas of the remaining three plates, respectively, using a pipette. Covering the culture dish, placing the culture dish into an incubator at 37 ℃ corresponding to the indicator bacteria for culturing for 12 hours, taking out the culture dish, opening the culture dish for observation and measuring the size (mm) of the bacteriostasis zone, photographing the record, repeating the experiment for three times, and taking an average value.
Results: as can be seen from FIG. 2, the Bacillus belicus BVQ and the fermentation broth have obvious antibacterial effect on the proliferation of several pathogenic bacteria, and the comparison of the diameters of the antibacterial circle shows that the antibacterial effect of the fermentation broth is positively correlated with the dosage, the inhibitory effect of the fermentation broth on salmonella at the same dosage is most obvious, the diameter of the antibacterial circle reaches 26.50mm when 100 mu L of the fermentation broth is added, and the antibacterial circles on escherichia coli and Edwardsiella ictaluri are 24.5mm and 18.0mm respectively.
Example 3 production technique of lipopeptid in fermentation liquor and evaluation of bacteriostatic effect of product
Inoculating the activated bacterial liquid according to the inoculum size of 1.5% v/v in a sterilized Landy culture medium, and culturing at 37 ℃ for 48 hours to prepare a fermentation broth. And (3) centrifuging the fermented liquid 9000r/min at 4 ℃ for 5min, and taking the supernatant for later use.
The supernatant was pH-adjusted to 4.0 with HCl, precipitated overnight at low temperature, 9000r/min and centrifuged at 4℃for 10min. Centrifuging to remove supernatant, collecting yellow precipitate, adding the obtained precipitate into the prepared emulsion system for dissolution, and spray drying.
The preparation method of the O/W emulsion system comprises the following steps: fully mixing glycerol and water (the volume of water is 4 times of that of the glycerol) to prepare emulsion, respectively constructing a mixed solution of lipopeptide and the emulsion according to the proportion of lipopeptide content of 10mg/mL and 20mg/mL, adding a rotor to shake and uniformly mixing, obtaining a uniformly mixed emulsion system after 24 hours, and preserving for later use.
Wherein, the conditions of the spray drying process are as follows: a low temperature spray dryer was used to continuously spray dry at 80℃with a feed flow of 10%, a feed temperature of 20℃and an outlet temperature of 60 ℃.
The pre-prepared plate was divided into four areas and 50 μl of salmonella suspension was used as indicator bacteria and was applied dropwise to the PDA plate, which was uniformly coated with a coating bar. Taking out the sterilized oxford cups, placing the oxford cups in four areas on a flat plate, setting LB culture medium added with 100 mu L as a blank control group, respectively dripping the prepared lipopeptide-free glycerol emulsion and an O/W emulsion system added with 10mg/mL and 20mg/mL lipopeptid into the three oxford cups, and sequentially adding 100 mu L of sample volumes. Then, the plate is placed in a 37 ℃ incubator for culturing for 12 hours, and then the size of the inhibition zone is observed and measured, and the plate is repeated three times to obtain an average value.
The inventor consults related documents to find that the antibacterial lipopeptide extracted from bacillus can bear the high temperature of 120 ℃ for 2-3 hours without obviously reducing the antibacterial effect, so that the final spray drying process in the lipopeptide production technology of the invention can not influence the antibacterial performance of the product.
Results: the preparation method comprises the steps of fermenting and culturing the Bellis strain, extracting the antibacterial lipopeptide by adopting an acid precipitation method, wherein the yield is 1.527+/-0.03 g/L, and the antibacterial effect is obvious. And adding the obtained precipitate into a prepared emulsifying system for dissolution, and then carrying out a spray drying process, wherein the molecular weight is 1-3Kda. The O/W emulsion system prepared from glycerol is shown in FIG. 3. The emulsion is a white turbid liquid uniformly, and the bacillus berryis lipopeptide has proved to achieve good effect when being used as an emulsifier. The salmonella is continuously adopted as indicator bacteria for evaluation experiments, and the experimental results are shown in figure 3, and the comparison of the average diameter of the inhibition zones can show that the glycerol emulsion added with the lipopeptid has obvious inhibition effect, and the inhibition effect of 20mg/mL bacillus berensis lipopeptid emulsion is better than that of 10mg/mL bacillus berensis lipopeptid emulsion. The diameter of a bacteriostasis circle added with a 100 mu L O/W emulsifying system (lipopeptide/oil phase=20 mg/mL) is highest, the diameter reaches (17.85+/-2.33) mm, the conventional lipopeptide re-dissolved by methanol is usually 14.20+/-1.65 mm, compared with methanol re-dissolution, the bacteriostasis effect of the lipopeptide re-dissolved substance after glycerol emulsification is improved by about 25%, and the toxicity problem of an organic solvent is solved while the water solubility of AMPs is improved. These data demonstrate that bacillus subtilis lipopeptid can be used as an emulsifier to improve the bacteriostatic effect in O/W emulsion systems.
EXAMPLE 4 identification of the composition of the lipopeptides of fermentation broths
1. High performance liquid chromatography
Analysis was performed using Agilent high performance liquid chromatography 1260 and ZORBAX Eclipse XDB-C18 columns (Analytical C18,5um, 150X 4.6 mm). Both the sample and the standard are dissolved in a 20% acetonitrile aqueous solution, mobile phase A is acetonitrile (containing 0.1% trifluoroacetic acid), mobile phase B is ultrapure water (containing 0.1% trifluoroacetic acid), mobile phase A: the proportion of B is 8:2, the flow rate is 1mL/min, the detection wavelength is 214nm, the sample concentration is 5mg/mL, and the sample injection volume is 60 mu L.
The analysis is shown in FIG. 4.
Results: the retention time of the antibacterial peptide of the strain was between 7 and 20 minutes, based on the peak-off retention time on HPLC of the crude lipopeptide sample extracted from BVQ121 compared to the standard. The retention time of the antibacterial lipopeptide sample in 7-10 minutes, 12-16 minutes and 16-20 minutes is consistent with that of a standard product, and the antibacterial lipopeptide is mainly composed of homologs of Surfacin, and the BVQ fermentation product component is proved, wherein the homologs of Surfacin have proven antibacterial effects on several food-borne pathogenic bacteria.
2. MALDI-TOF-MS mass spectrometry
Mass spectrometry detection conditions: 10mg of antibacterial peptide, methanol solvent, positive ion detection and DHB matrix. MALDI instrument mass spectrometry was performed in a reflective positive ion mode using a UltrafleXtreme MALDI-TOF mass spectrometer (Bruker dalton). The ions generated by intelligent beam laser irradiation obtain mass spectrum in the mass range of 100-1700 Da, the irradiation frequency is 1000Hz, PIE is 120ns, and the lens voltage is 7kV. The first ion source voltage is 20kV and the second ion source voltage is 17.65kV. Each spectrum was produced by an average of 1000 laser shots and the laser irradiance was set to 50-55%. All mass spectra were processed using Flexanalysis software (version: 3.3, bruker).
The analysis is shown in FIG. 5.
Results: the reference and the comparison chart are consulted, and the main components of the antibacterial peptide obtained by the invention are C13-C16Iturin, C12-C15 Surfactin and C15-C23 Fengycin and partial homologs of the three antibacterial peptides, and each component has corresponding application in food processing links.
The invention provides bacillus bailii antibacterial lipopeptid, a preparation method and an application thought and method thereof, and particularly the method and the method for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by those skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (10)
1. The bacillus beleiensis antibacterial lipopeptide is characterized by comprising the following components of bacillus beleiensis Bacillus velezensis BVQ and bacillus beleiensis CCTCC NO: m20211570 is prepared by fermenting, emulsifying, constructing an O/W emulsifying system and spray drying, wherein the molecular weight of the bacillus belicus antimicrobial lipopeptide is 1-3Kda.
2. The method for preparing the bacillus bailii antibacterial lipopeptide according to claim 1, which is characterized by comprising the following steps:
(1) Bacillus beziacillus Bacillus velezensis CCTCC NO: activating M20211570 BVQ121 strain;
(2) Inoculating the strain activated in the step (1) into a Landy modified culture medium for fermentation culture to obtain a fermentation broth, and centrifuging to retain a supernatant;
(3) Regulating the pH of the supernatant in the step (2), centrifuging at a low temperature after overnight at a low temperature, and discarding the supernatant to collect a precipitate;
(4) Adding emulsion into the precipitate obtained in the step (3), and shaking and uniformly mixing to obtain an O/W emulsion system;
(5) And (3) spray drying the emulsion system obtained in the step (4) to prepare antibacterial lipopeptide powder.
3. The method of claim 2, wherein in step (2), the inoculation is performed in an amount of 1-3% v/v; the conditions of the fermentation culture are as follows: fermenting at 37deg.C for 24-48 hr in shaking table 100-300 r/min; the formula of the Landy improved culture medium is as follows: glucose 20.0g/L, sodium glutamate 5.0g/L, yeast extract 1.0g/L, mgSO 4 0.5g/L，KCl 0.5g/L，KH 2 PO 4 1.0g/L，FeSO 4 0.15mg/L，MnSO 4 0.5mg/L，CuSO 4 0.16mg/L。
4. The method according to claim 2, wherein in the step (3), the pH is adjusted to 2.0 to 4.0; the low temperature is 4 ℃; the centrifugation is performed under the conditions of 6000-9000r/min for 5-10min.
5. The method according to claim 2, wherein in the step (4), the emulsion is added in such an amount that the concentration ratio of the lipopeptide content reaches 10 to 20 mg/mL.
6. The method according to claim 2 or 5, wherein in the step (4), the emulsion is a mixed solution of glycerin and water, wherein the volume of water is 4 to 6 times that of glycerin.
7. The method according to claim 2, wherein in step (5), the spray drying is performed under the following process conditions: and (3) using a low-temperature spray dryer to continuously spray-dry at 80 ℃, wherein the feeding flow is 40-60mL/min, the feeding temperature is 20 ℃, and the outlet temperature is 60 ℃.
8. Use of the bacillus bailii antimicrobial lipopeptide of claim 1 for inhibiting food infestation by food-borne pathogenic bacteria.
9. The use according to claim 8, wherein the food-borne pathogenic bacteria is any of edwardsiella ictaluri, escherichia coli, salmonella.
10. The bacillus beliae producing the antibacterial lipopeptid is characterized in that the bacillus beliae is classified and named as bacillus beliae Bacillus velezensis, the strain number is BVQ, and the bacillus beliae is preserved in China Center for Type Culture Collection (CCTCC) on the 12 th month 8 of 2021, and the preservation number is CCTCC NO: m20211570.
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