CN114806976A - Lactobacillus brevis and preparation method of antibacterial substance thereof - Google Patents
Lactobacillus brevis and preparation method of antibacterial substance thereof Download PDFInfo
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- 230000004151 fermentation Effects 0.000 claims abstract description 55
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- 238000001035 drying Methods 0.000 claims abstract description 13
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- 239000001963 growth medium Substances 0.000 claims description 33
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- 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
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3571—Microorganisms; Enzymes
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/121—Brevis
-
- 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
- C12R2001/225—Lactobacillus
- C12R2001/24—Lactobacillus brevis
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- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
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- General Chemical & Material Sciences (AREA)
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- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
Abstract
The invention discloses lactobacillus brevis CGMCC No.19407 and a preparation method of antibacterial substances thereof, and relates to the field of microbial fermentation and extraction. The lactobacillus brevis provided by the invention belongs to probiotics and is a generally recognized GRAS strain. The fermentation product has good bacteriostatic activity on various fungi and bacteria, such as aspergillus niger, saccharomycetes, staphylococcus aureus, escherichia coli and the like. The invention also provides an extraction preparation method of the antibacterial substance, which comprises the following steps: (1) inoculating the strain to a fermentation medium for fermentation culture; (2) after fermentation, sterilizing to obtain clear fermentation liquor; (3) adsorbing the clarified fermentation liquor by anion exchange resin; (4) adding the desorption solution into the resin after adsorption for desorption; (5) drying the desorption solution to obtain a final product. The bacterial strain and the obtained product provided by the invention are safe and reliable, have wide antibacterial spectrum and have higher application value in the fields of food, feed, agriculture and the like.
Description
Technical Field
The invention relates to a Lactobacillus brevis and a preparation method and application of a biological preservative thereof, belonging to the field of microorganisms.
Background
According to the deployment of the national plan for suppressing animal-derived bacteria drug resistance action (2017-2020), from 2020, 13 drug feed additives such as aureomycin and terramycin are abandoned by the ministry of agriculture, and the search for suitable substitute resistance products is a challenge facing the feed industry at present. Meanwhile, with the increasing serious problems of antibiotic abuse, food safety and the like, health and safety become higher requirements of consumers for food selection. How to realize the antibiotic forbidding of feed, the antibiotic reduction of breeding and the antibiotic-free of food becomes an important problem facing the feed and food industries.
Lactic acid bacteria serving as GRAS strains can generate various antibacterial substances, and the application of lactic acid bacteria and metabolites thereof to solve the safety problem of food and feed is a hotspot of current research, such as Nisin, and is widely applied to the food industry. However, Nisin has narrow bacteriostatic spectrum, has good inhibitory effect only on partial gram-positive bacteria, and has no inhibitory effect on fungi such as mould, yeast and the like. The lactobacillus brevis metabolite disclosed by the patent has good bacteriostatic activity on various fungi, gram-negative and gram-positive bacteria, such as aspergillus niger, sachima yeast, fusarium, staphylococcus aureus, escherichia coli and the like, and is wide in bacteriostatic spectrum.
At present, most of lactobacillus bacteriostatic products are mainly live bacterium fermentation liquor or microecological preparations, but live lactobacillus is difficult to be directly added into food and cannot be directly used as a preservative. The lactobacillus fermentation liquor can be directly added into animal feed as feed, but the activity of the viable bacteria microecological preparation is easy to reduce and is not easy to store. The bacteriostatic components of the fermentation liquor are separated and extracted, and the fermentation liquor is convenient to store, transport, add and use.
Disclosure of Invention
The invention provides a preparation method of a lactobacillus brevis and a preservative thereof for solving the technical defects in the prior art, thereby providing a biological preservative with wide bacteriostatic range and solving the safety problem of food and feed.
In order to realize the purpose of the invention, the technical scheme is as follows:
the invention provides a lactobacillus brevis strain with wide bacteriostatic activity, and the preservation number of the strain is CGMCC number 19407.
Also provides the application of the lactobacillus brevis strain with wide bacteriostatic activity in preparing biological preservatives.
Further provided is a method for producing a biological preservative using the lactobacillus brevis strain. The method specifically comprises the following steps: fermenting and culturing the lactobacillus brevis strain, and drying the fermented culture solution to obtain the biological preservative.
Preferably, the fermentation culture conditions are 30-37 ℃, 50-100 rpm, and the culture is 24-48h, wherein the fermentation culture medium is MRS liquid culture medium.
In one embodiment, the following culturing is performed prior to the fermentation culture:
(1) slant culture: inoculating the lactobacillus brevis strain on a solid slant culture medium under an aseptic condition, and culturing for 24-48h at 30-37 ℃, wherein the solid slant culture medium is a skim milk culture medium;
(2) seed culture: inoculating the lactobacillus brevis cultured in the step (1) to a liquid seed culture medium under an aseptic condition, and standing and culturing at 30-37 ℃ for 12 h; wherein the seed culture medium is a liquid MRS culture medium.
Preferably, the drying is a spray drying process. More specifically, the conditions of spray drying are that the inlet temperature is 120-210 ℃, the outlet temperature is 60-100 ℃, and 5-20% of modified starch, maltodextrin and/or cyclodextrin by weight are added after drying.
In another embodiment, the method comprises the steps of: clarifying the fermentation liquor, and collecting clear liquid; treating the clarified fermentation broth with anion exchange resin; eluting the anion exchange resin by using an analytic solution, and collecting an eluent; drying the eluent to obtain the biological preservative.
Preferably, the clarification method is selected from the group consisting of tube centrifuge centrifugation, disk centrifuge centrifugation, ceramic membrane filtration, hollow fiber membrane filtration.
Further preferably, the anion exchange resin is a strongly basic ion exchange resin or a weakly basic ion exchange resin.
In still another preferred embodiment, the analysis solution is one or a mixture of two of 0.5-5% hydrochloric acid, 0.5-5% sodium chloride and 0.5-5% sodium hydroxide solution.
The eluent drying method is vacuum drying, spray drying or freeze drying.
The invention also provides the biological preservative prepared by the method.
The invention also provides the Lactobacillus brevis strain and application of the biological preservative in food and feed as a preservative.
The invention also provides a preparation method of the broad-spectrum antibacterial substance, which comprises the following steps:
(1) inoculating the strain to a fermentation culture medium for fermentation culture;
(2) clarifying the fermentation liquor, and collecting clear liquid;
(3) treating the clarified fermentation broth with anion exchange resin;
(4) eluting the anion exchange resin by using an analytic solution, and collecting an eluent;
(5) drying the eluent to obtain the final product.
Preferably, the concentration of glucose in the MRS medium in the step 2 is 30 g/L.
Preferably, the culture temperature in the step 3 is 33 ℃.
Preferably, the inlet temperature in the step 4 is 190 ℃ and the outlet temperature is 90 ℃.
Preferably, the additive in step 4 is 10% maltodextrin.
Preferably, the clarification mode in the step 5 is filtering by using a 50-200nm ceramic membrane, and the operating pressure is 0.3 MPa.
Preferably, the anion exchange resin in the step 5 is D301.
Preferably, the drying method in the step 5 is a spray drying method.
The bacteriostatic substance produced by the lactobacillus brevis strain provided by the invention has good bacteriostatic activity on various fungi and bacteria, such as aspergillus niger, fusarium, staphylococcus aureus, escherichia coli and the like, and has wide bacteriostatic spectrum. Meanwhile, the generated antibacterial substance can be effectively extracted and purified by the method disclosed by the patent, and is convenient to add and use. Has wide application value in the fields of food, feed and agriculture.
Drawings
FIG. 1 shows the inhibitory effect of Lactobacillus brevis zczx-1 on Staphylococcus aureus in example two.
FIG. 2 shows the inhibitory effect of Lactobacillus brevis zczx-1 on Escherichia coli in example II.
FIG. 3 shows the inhibitory effect of Lactobacillus brevis zczx-1 on Erichthyosis bifidus in example II.
FIG. 4 shows the inhibitory effect of Lactobacillus brevis zczx-1 on Aspergillus niger in example two.
FIG. 5 shows the bacteriostatic activity of the fermentation broth, the fermentation broth after adsorption and the eluent of Lactobacillus brevis zczx-1 (Staphylococcus aureus as indicator) detected by the plate diffusion method in the third example.
FIG. 6 shows the bacteriostatic activity of the fermentation broth, the fermentation broth after adsorption and the eluate of Lactobacillus brevis zczx-1 (the indicator bacterium is Aspergillus niger) detected by the plate diffusion method in the fourth example.
FIG. 7 shows the bacteriostatic activity of the refined bio-preservative (Aspergillus niger as an indicator) measured by plate diffusion in example four.
Biological preservation
The strain zczx-1 related to the invention is classified and named as: lactobacillus brevis (Lactobacillus brevis) is preserved in China general microbiological culture Collection center (CGMCC) at 17.1.2020, abbreviated as CGMCC, and is deposited at the institute of microbiology of Zhongkoyao institute No. 3, Naciyao No.1 Hospital, North Kyoho, Beijing, with the preservation number: CGMCC number 19407.
Detailed Description
The present invention will be described in detail with reference to the following examples, which are provided for illustration of the present invention and are not intended to limit the scope of the present invention.
Example one
Crushing the high-salt pickled raw peppers, adding the crushed raw peppers into a sterilized MRS liquid culture medium, carrying out vibration culture at 37 ℃ and 50rpm for 2h, taking the cultured MRS culture medium for gradient dilution, coating the diluted MRS culture medium on an MRS solid culture medium, placing the MRS solid culture medium in a 33 ℃ culture box for standing culture for 48h, and after single bacteria grow out, selecting white round single bacteria colony, inoculating the white round single bacteria colony into a 2ml EP tube containing 1.5ml of the MRS culture medium, and continuing the standing culture for 48 h. And centrifuging at 12000rpm after the culture is finished, taking the supernatant, detecting the bacteriostatic activity of the supernatant of the fermentation liquor by adopting a flat plate diffusion method, selecting strains with bacteriostatic activity for four bacteria by adopting escherichia coli, staphylococcus aureus, saccharomycetes and aspergillus niger as indicator bacteria, and identifying and preserving the strains.
Obtaining a bacterial strain zczx-1 with bacteriostatic activity for four indicator bacteria, inoculating the bacterial strain zczx-1 in an MRS liquid culture medium, standing and culturing for 24 hours at 33 ℃, centrifugally collecting thalli, diluting the thalli to a certain concentration by using a buffer solution, inoculating the thalli in a Biolog automatic microorganism identification and analysis system, and identifying the strains by using a GEN three-plate, wherein the identification result is thatLactobacillus brevis(ii) a The strain zczx-1 is screened in low-temperature high-salt pickled vegetables, is low-temperature resistant and high-salt resistant, has good antibacterial effect on gram-positive bacteria such as escherichia coli, staphylococcus aureus, saccharomycetes and mould fungi and fungi such as gram-negative bacteria, saccharomycetes and mould fungi, and has wide antibacterial activity.
Example two
The glycerol tube preserved Lactobacillus brevis zczx-1 is inoculated on a solid skim milk culture medium under an aseptic condition, cultured for 48h at 37 ℃, selected to be larger colony and inoculated on 50ml of liquid MRS culture medium, and cultured for 24h at 33 ℃ to prepare seed liquid. Inoculating the seed solution into a fermentation culture medium under aseptic conditions, wherein the inoculation amount is 5%, the temperature is 37 ℃, the rpm is 50, and culturing is carried out for 48 hours. After fermentation, the bacteriostatic activity of the fermentation liquor is detected by a plate diffusion method, and indicator bacteria such as escherichia coli and staphylococcus aureus are adopted, as shown in fig. 1 and fig. 2.
10g of maltodextrin was added to 100ml of the fermentation broth, and spray-drying was carried out at an inlet temperature of 190 ℃ and an outlet temperature of 90 ℃ to prepare a microecological preparation. Taking 5g of the microecological preparation, adding 50ml of sterile water for redissolving, detecting the activity by a flat plate diffusion method, and adopting Ericaria and Aspergillus niger as indicator bacteria, and taking figures 3 and 4. The special Erjiangqi yeast is a direct pathogenic bacterium causing the occurrence of the milk disease of the aquatic products in the crab cultivation process, and the bacterial strain has the characteristics of low temperature resistance and salt tolerance, can grow in a sodium chloride solution with the concentration of more than 10 percent, and has higher application value in seawater aquaculture.
EXAMPLE III
The glycerol tube preserved Lactobacillus brevis zczx-1 is inoculated on a solid skim milk culture medium under an aseptic condition, cultured for 48h at 37 ℃, a larger colony is selected to be inoculated on 50ml of liquid MRS culture medium, and cultured for 24h at 37 ℃ to prepare a seed solution. Inoculating the seed liquid into a fermentation culture medium under aseptic conditions, wherein the inoculation amount is 5%, the temperature is 37 ℃, the rpm is 50, and the culture is carried out for 48 hours. After fermentation, the antibacterial activity of the fermentation liquor is detected by a flat plate diffusion method, and the indicator bacteria adopt escherichia coli and staphylococcus aureus and have good activity.
Taking 1000ml of fermentation liquor, and filtering and sterilizing by adopting a hollow fiber membrane to obtain sterile fermentation liquor. 100ml of sterile fermentation liquor is added into 10g D301 anion exchange resin, vibrated at 50rpm for 2h, and filtered after the end. The resin was washed with an equal volume of deionized water and filtered. Adding 100ml of hydrochloric acid solution with the concentration of 1mol/L into the washed resin, vibrating at 50rpm for 2h for elution, adjusting the pH of the eluent to 4-5 after the elution is finished, detecting the bacteriostatic activity of the fermentation liquid, the fermentation liquid after adsorption and the eluent by using a flat plate diffusion method, using staphylococcus aureus as an indicator bacterium, and using hydrochloric acid with the pH =4 as a contrast. The results show that the antibacterial effects of the fermentation liquor and the eluent are not different, and the diameter of the antibacterial zone can reach 1.2-1.5 cm. The fermentation broth after adsorption and pH =4 hydrochloric acid had no bacteriostatic activity, see fig. 5.
Example four
The glycerol tube preserved Lactobacillus brevis zczx-1 is inoculated on a solid skim milk culture medium under an aseptic condition, cultured for 48h at 33 ℃, selected to be larger colony and inoculated on 50ml of liquid MRS culture medium, and cultured for 24h at 33 ℃ to prepare seed liquid. Inoculating the seed liquid into a fermentation culture medium under aseptic conditions, wherein the inoculation amount is 8%, the temperature is 33 ℃, the rpm is 50, and culturing is carried out for 24 hours. After fermentation, the antibacterial activity of the fermentation liquor is detected by a flat plate diffusion method, and the indicator bacteria adopt escherichia coli and staphylococcus aureus and have good activity.
Taking 5000ml of fermentation liquor, and filtering and sterilizing by adopting a 100nm ceramic membrane to obtain sterile fermentation liquor. 500ml of sterile fermentation liquor is added into 50g D380 anion exchange resin, vibrated at 50rpm for 2h, and filtered after the end. The resin was washed with 100ml of deionized water and filtered. Adding 200ml of 1mol/L sodium hydroxide solution into the washed resin, vibrating at 50rpm for 2h for elution, adjusting the pH of the eluent to 4-5 after the elution is finished, and detecting the bacteriostatic activity of the fermentation liquor, the fermentation liquor after adsorption and the eluent by using a flat plate diffusion method, wherein the indicator bacteria adopt Aspergillus niger. The results show that the fermentation liquor and the eluent have no difference in bacteriostasis effect, and are both good, and the diameter of the bacteriostasis zone can reach 1.8-2.0cm, as shown in figure 6. The fermentation liquor after adsorption has no bacteriostatic activity.
30ml of eluent is added with 15 percent of modified starch. Setting the inlet temperature at 180 ℃ and the outlet temperature at 85 ℃, carrying out spray drying to prepare a refined biological preservative, adding 50ml of water into 1g of the solid preservative after the spray drying is finished, redissolving, detecting the activity by a flat plate diffusion method, wherein aspergillus niger is adopted as an indicator bacterium, the bacteriostatic circle of redissolution fermentation liquor can reach 2.5cm, and the starch solution with the same mass concentration has no bacteriostatic activity, which is shown in figure 7.
Claims (15)
1. A Lactobacillus brevis strain with wide antibacterial activity has a preservation number of CGMCC number 19407.
2. Use of a strain of lactobacillus brevis with broad bacteriostatic activity according to claim 1 for the preparation of a biological preservative.
3. A method for producing a biological preservative using a strain of lactobacillus brevis as claimed in claim 1.
4. A method according to claim 3, characterized by the steps of: fermenting and culturing the lactobacillus brevis strain, and drying the fermented culture solution to obtain the biological preservative.
5. The method according to claim 3, wherein the fermentation culture conditions are 30-37 ℃ and 50-100 rpm, and the culture is carried out for 24-48h, wherein the fermentation culture medium is MRS liquid culture medium.
6. The method according to claim 5, wherein the following culturing is carried out before the fermentation culturing:
(1) slant culture: inoculating the Lactobacillus brevis strain on a solid slant culture medium under an aseptic condition, and culturing for 24-48h at 30-37 ℃, wherein the solid slant culture medium is a skim milk culture medium;
(2) seed culture: inoculating the lactobacillus brevis cultured in the step (1) to a liquid seed culture medium under an aseptic condition, and standing and culturing at 30-37 ℃ for 12 h; wherein the seed culture medium is a liquid MRS culture medium.
7. The method of claim 4, wherein the drying is a spray drying method.
8. The method as claimed in claim 6, wherein the spray drying is carried out at an inlet temperature of 120 ℃ and an outlet temperature of 60 ℃ to 100 ℃ and wherein 5% to 20% by weight of modified starch, maltodextrin and/or cyclodextrin is added after drying.
9. The method according to claim 4, characterized in that it comprises the following steps: clarifying the fermentation liquor, and collecting clear liquid; treating the clarified fermentation broth with anion exchange resin; eluting the anion exchange resin by using an analytic solution, and collecting an eluent; drying the eluent to obtain the biological preservative.
10. The method of claim 9, wherein: the clarifying method is selected from tube centrifuge centrifugation, disc centrifuge centrifugation, ceramic membrane filtration and hollow fiber membrane filtration.
11. The method of claim 9, wherein: the anion exchange resin is strong-base ion exchange resin or weak-base ion exchange resin.
12. The method of claim 9, wherein: the resolving liquid is one or a mixture of 0.5-5% hydrochloric acid, 0.5-5% sodium chloride or 0.5-5% sodium hydroxide solution.
13. The method of claim 9, wherein: the eluent drying method is vacuum drying, spray drying or freeze drying.
14. A biological preservative prepared by the method of any one of claims 3 to 13.
15. A strain of lactobacillus brevis according to claim 1, use of a biological preservative according to claim 14 as a preservative in food, feed.
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