CN114806976B - Lactobacillus brevis and preparation method of antibacterial substances thereof - Google Patents
Lactobacillus brevis and preparation method of antibacterial substances thereof Download PDFInfo
- Publication number
- CN114806976B CN114806976B CN202210699465.5A CN202210699465A CN114806976B CN 114806976 B CN114806976 B CN 114806976B CN 202210699465 A CN202210699465 A CN 202210699465A CN 114806976 B CN114806976 B CN 114806976B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- lactobacillus brevis
- strain
- drying
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 37
- 240000001929 Lactobacillus brevis Species 0.000 title claims abstract description 33
- 235000013957 Lactobacillus brevis Nutrition 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000000126 substance Substances 0.000 title abstract description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 54
- 230000004151 fermentation Effects 0.000 claims abstract description 54
- 239000001963 growth medium Substances 0.000 claims abstract description 29
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 12
- 235000013305 food Nutrition 0.000 claims abstract description 12
- 238000001035 drying Methods 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims description 22
- 239000003755 preservative agent Substances 0.000 claims description 17
- 230000002335 preservative effect Effects 0.000 claims description 17
- 239000003480 eluent Substances 0.000 claims description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 10
- 238000001694 spray drying Methods 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 229920002774 Maltodextrin Polymers 0.000 claims description 5
- 239000005913 Maltodextrin Substances 0.000 claims description 5
- 229940035034 maltodextrin Drugs 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 238000005374 membrane filtration Methods 0.000 claims description 5
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 4
- 229920000881 Modified starch Polymers 0.000 claims description 4
- 239000004368 Modified starch Substances 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000000919 ceramic Substances 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 239000003456 ion exchange resin Substances 0.000 claims description 4
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 4
- 235000019426 modified starch Nutrition 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 229920000858 Cyclodextrin Polymers 0.000 claims description 3
- 238000005352 clarification Methods 0.000 claims description 3
- 239000012510 hollow fiber Substances 0.000 claims description 3
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 20
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 11
- 241000588724 Escherichia coli Species 0.000 abstract description 9
- 241000228245 Aspergillus niger Species 0.000 abstract description 7
- 241000233866 Fungi Species 0.000 abstract description 5
- 239000011347 resin Substances 0.000 abstract description 5
- 229920005989 resin Polymers 0.000 abstract description 5
- 241000235342 Saccharomycetes Species 0.000 abstract description 4
- 239000000047 product Substances 0.000 abstract description 4
- 238000001228 spectrum Methods 0.000 abstract description 4
- 230000001954 sterilising effect Effects 0.000 abstract description 3
- 239000012467 final product Substances 0.000 abstract description 2
- 235000021474 generally recognized As safe (food) Nutrition 0.000 abstract description 2
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract 2
- 230000000813 microbial effect Effects 0.000 abstract 1
- 239000006041 probiotic Substances 0.000 abstract 1
- 235000018291 probiotics Nutrition 0.000 abstract 1
- 238000009792 diffusion process Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 7
- 230000002401 inhibitory effect Effects 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000192125 Firmicutes Species 0.000 description 3
- 241000186660 Lactobacillus Species 0.000 description 3
- 230000003385 bacteriostatic effect Effects 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 2
- 101000765308 Aspergillus niger N-(5'-phosphoribosyl)anthranilate isomerase Proteins 0.000 description 2
- 235000002566 Capsicum Nutrition 0.000 description 2
- 241000223218 Fusarium Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 2
- 108010053775 Nisin Proteins 0.000 description 2
- 241000758706 Piperaceae Species 0.000 description 2
- 230000002421 anti-septic effect Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000004309 nisin Substances 0.000 description 2
- 235000010297 nisin Nutrition 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- KIPLYOUQVMMOHB-MXWBXKMOSA-L [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O Chemical compound [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O KIPLYOUQVMMOHB-MXWBXKMOSA-L 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 210000004763 bicuspid Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- CYDMQBQPVICBEU-UHFFFAOYSA-N chlorotetracycline Natural products C1=CC(Cl)=C2C(O)(C)C3CC4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-UHFFFAOYSA-N 0.000 description 1
- 229960004475 chlortetracycline Drugs 0.000 description 1
- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 description 1
- 235000019365 chlortetracycline Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 229940063650 terramycin Drugs 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Nutrition Science (AREA)
- General Chemical & Material Sciences (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (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 accepted GRAS strain. The fermentation product has good antibacterial 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: the strain (1) is inoculated in a fermentation culture medium for fermentation culture; (2) sterilizing after fermentation to obtain clear fermentation liquor; (3) Adsorbing the clarified fermentation broth by anion exchange resin; (4) adding the resin after adsorption into the analysis liquid for analysis; and (5) drying the resolved liquid to obtain a final product. The strain and the obtained product provided by the invention are safe and reliable, have wide bacteriostasis spectrum, and have higher application value in the fields of food, feed, agriculture and the like.
Description
Technical Field
The invention relates to a preparation method and application of lactobacillus brevis and a biological preservative thereof, belonging to the field of microorganisms.
Background
According to the deployment of national plan for inhibiting animal-derived bacterial drug-resistant action (2017-2020), since 2020, 13 drug feed additives such as aureomycin and terramycin are abandoned by the department of agriculture, and finding a suitable replacement resistant product is a challenge facing the current feed industry. Meanwhile, along with the increasing serious problems of antibiotics abuse, food safety and the like, health safety becomes a higher requirement of consumers on food selection. How to realize the forbidden resistance of the feed, the reduced resistance of the cultivation and the no resistance of the food become important problems commonly faced by the feed industry and the food industry.
Lactic acid bacteria as GRAS strain can produce various antibacterial substances, and the application of lactic acid bacteria and metabolites thereof to solve the problem of food and feed safety is a hot spot of current research, such as Nisin, and has been widely applied to the food industry. However, nisin has narrower antibacterial spectrum, has better inhibition effect on partial gram-positive bacteria, and has no inhibition effect on fungi such as mould, yeast and the like. The Lactobacillus brevis metabolite has good antibacterial activity on various fungi, gram negative bacteria and gram positive bacteria, such as Aspergillus niger, meiqi yeast, fusarium, staphylococcus aureus, escherichia coli and the like, and has wide antibacterial spectrum.
At present, the lactobacillus antibacterial product mainly comprises a viable bacteria fermentation liquid or a microecological preparation, but the viable bacteria of the lactobacillus are difficult to directly add into food and cannot be directly used as a preservative. The lactobacillus fermentation broth can be directly added into animal feed as feed, but the activity of the live bacteria microecological preparation is easy to reduce and difficult to preserve. The antibacterial components of the fermentation broth are separated and extracted, and the fermentation broth is convenient to preserve, transport and add for use.
Disclosure of Invention
The invention provides a preparation method of lactobacillus brevis and a biological preservative thereof for solving the technical defects in the prior art, thereby providing a biological preservative with wide bacteriostasis and solving the safety problem of food and feed.
In order to achieve the aim of the invention, the technical scheme adopted is as follows:
the invention provides a lactobacillus brevis strain with wide antibacterial activity, and the preservation number of the strain is CGMCC No. 19407.
Also provides the application of the lactobacillus brevis strain with wide antibacterial activity in preparing biological preservative.
Further provided are methods 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 condition is 30-37 ℃,50-100 rpm, and the fermentation culture condition is 24-48h, wherein the fermentation culture medium is MRS liquid culture medium.
In one embodiment, the following cultivation is performed prior to fermentation cultivation:
(1) Slant culture: inoculating the lactobacillus brevis strain on a solid slant culture medium under a sterile condition, and culturing for 24-48 hours at the temperature of 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 a sterile condition, and carrying out stationary culture 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, spray drying is carried out under the condition that the inlet temperature is 120-210 ℃ and the outlet temperature is 60-100 ℃, and modified starch, maltodextrin and/or cyclodextrin accounting for 5-20% of the weight of the modified starch, maltodextrin and/or cyclodextrin are added after drying.
In another embodiment, the method comprises the steps of: clarifying the fermentation liquor, and collecting clear liquor; treating the clarified broth with an anion exchange resin; eluting the anion exchange resin by using an analytical 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 a further preferred mode, the resolving liquid is one or two of 0.5-5% hydrochloric acid, 0.5-5% sodium chloride or 0.5-5% sodium hydroxide solution.
The eluent is dried by 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 the application of the biological preservative in foods and feeds as the preservative.
The invention also provides a preparation method of the broad-spectrum antibacterial substance, which comprises the following steps:
(1) The strain is inoculated in a fermentation culture medium for fermentation culture;
(2) Clarifying fermentation liquor and collecting clear liquor;
(3) Treating the clarified broth with an anion exchange resin;
(4) Eluting the anion exchange resin by using an analytical solution, and collecting an eluent;
(5) And (5) drying the eluent to obtain a final product.
Preferably, the glucose concentration in the MRS medium in the step 2 is 30g/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 the step 4 is 10% maltodextrin.
Preferably, the clarification mode in the step 5 is a 50-200nm ceramic membrane filtration mode, and the operation pressure is 0.3MPa.
Preferably, the anion exchange resin model number D301 in the step 5.
Preferably, the drying method in the step 5 is a spray drying method.
The antibacterial substance produced by the lactobacillus brevis strain has good antibacterial activity on various fungi and bacteria, such as aspergillus niger, fusarium, staphylococcus aureus, escherichia coli and the like, and has wide antibacterial spectrum. Meanwhile, the produced antibacterial substances can be effectively extracted and purified by adopting the method disclosed by the patent, and are 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 E.coli inhibitory effect of Lactobacillus brevis zczx-1 in example two.
FIG. 3 shows the inhibitory effect of Lactobacillus brevis zczx-1 on Meissimago bikini in example two.
FIG. 4 shows the inhibitory effect of Lactobacillus brevis zczx-1 on Aspergillus niger in example two.
FIG. 5 shows the antibacterial activity of the fermentation broth, the adsorbed fermentation broth, and the eluate of Lactobacillus brevis zczx-1 (the indicator bacteria is Staphylococcus aureus) by plate diffusion method in example III.
FIG. 6 shows the antibacterial activity of fermentation broth, adsorbed fermentation broth, and eluate of Lactobacillus brevis zczx-1 (Aspergillus niger as indicator) by plate diffusion method in example IV.
FIG. 7 shows the detection of bacteriostatic activity (indicator strain A. Niger) by plate diffusion with a refined biological preservative in example IV.
Preservation of organisms
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 1 month and 17 days in 2020, and has the preservation number of CGMCC: CGMCC No. 19407.
Detailed Description
The present invention will be specifically described with reference to the following examples, which are provided to illustrate the present invention but not to limit the scope of the present invention.
Example 1
Crushing high-salt pickled raw peppers, adding the crushed high-salt pickled raw peppers into a sterilized MRS liquid culture medium, carrying out vibration culture at 37 ℃ and 50rpm for 2 hours, taking the cultured MRS culture medium for gradient dilution, coating the MRS culture medium on an MRS solid culture medium, placing the MRS solid culture medium in a 33 ℃ incubator for static culture for 48 hours, after single bacterial colonies grow out, picking white round single bacterial colonies, and inoculating the white round single bacterial colonies into a 2ml EP tube containing 1.5ml of the MRS culture medium for continuous static culture for 48 hours. Centrifuging at 12000rpm after the culture is finished, taking supernatant, detecting the antibacterial activity of the supernatant of the fermentation broth by adopting a flat plate diffusion method, and screening strains with antibacterial activity on four bacteria by adopting escherichia coli, staphylococcus aureus, saccharomycetes and aspergillus niger as indicator bacteria.
Obtaining a strain zczx-1 with bacteriostatic activity on four indicator bacteria, inoculating into MRS liquid culture medium, standing at 33 ℃ for 24 hours, centrifugally collecting thalli, diluting to a certain concentration with buffer solution, inoculating into a Biolog automatic microorganism identification analysis system, and using GEN three templates for strain identification, wherein the identification result is thatLactobacillus brevisThe method comprises the steps of carrying out a first treatment on the surface of the The strain zczx-1 is screened in low-temperature high-salt pickled vegetables, has low temperature resistance and high salt resistance, and the strain and fermentation liquor thereof have good antibacterial effect on gram-positive bacteria such as escherichia coli, staphylococcus aureus, saccharomycetes, mould and the like, and fungi such as gram-negative bacteria, saccharomycetes, mould and the like, and have wide antibacterial activity.
Example two
The lactobacillus brevis zczx-1 preserved in the glycerol tube is inoculated on a solid skim milk culture medium under the aseptic condition, cultured for 48 hours at 37 ℃, and the larger bacterial colony is picked and inoculated on 50ml of liquid MRS culture medium, and cultured for 24 hours at 33 ℃ to prepare seed liquid. The seed solution was inoculated in a fermentation medium under aseptic conditions at an inoculum size of 5%,37℃and 50rpm, and cultured at 48h. After fermentation, the antibacterial activity of the fermentation broth is detected by a plate diffusion method, and the indicator bacteria are escherichia coli and staphylococcus aureus, which are shown in fig. 1 and 2.
10g of maltodextrin was added to 100ml of the fermentation broth, and spray-drying was performed at an inlet temperature of 190℃and an outlet temperature of 90℃to prepare a microecological preparation. 5g of the microecological preparation is taken and added with 50ml of sterile water for re-dissolution, activity is detected by a plate diffusion method, and the indicator bacteria are composed of Meiqi yeast and Aspergillus niger, and are shown in figures 3 and 4. The special bicuspid mermaid yeast is a direct pathogenic bacterium causing aquatic milk diseases in the crab culture process, and the strain has the characteristics of low temperature resistance and salt resistance, can grow in sodium chloride solution with the concentration of more than 10%, and has higher application value in seawater aquaculture.
Example III
The lactobacillus brevis zczx-1 preserved in the glycerol tube is inoculated on a solid skim milk culture medium under the aseptic condition, cultured for 48 hours at 37 ℃, and the larger bacterial colony is picked and inoculated on 50ml of liquid MRS culture medium, and cultured for 24 hours at 37 ℃ to prepare seed liquid. The seed solution was inoculated in a fermentation medium under aseptic conditions at an inoculum size of 5%,37℃and 50rpm, and cultured at 48h. After fermentation, the antibacterial activity of the fermentation broth is detected by a flat plate diffusion method, and the indicator bacteria are escherichia coli and staphylococcus aureus, so that the activity is good.
Taking 1000ml of fermentation liquor, filtering and sterilizing by adopting a hollow fiber membrane to obtain the sterile fermentation liquor. 100ml of the sterile fermentation broth was taken and added to 10g of 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. And adding 100ml of hydrochloric acid solution with the concentration of 1mol/L into the washed resin, vibrating at 50rpm for 2 hours for eluting, adjusting the pH of the eluent to 4-5 after the eluting is finished, detecting the antibacterial activity of the fermentation liquor, the adsorbed fermentation liquor and the eluent by a flat-plate diffusion method, wherein staphylococcus aureus is adopted as the indicator bacteria, and pH=4 hydrochloric acid is used as a control. The results show that the antibacterial effect of the fermentation liquor and the eluent is not different, and the diameter of the antibacterial ring can reach 1.2-1.5cm. The adsorbed fermentation broth and ph=4 hydrochloric acid had no bacteriostatic activity, see fig. 5.
Example IV
The lactobacillus brevis zczx-1 preserved in the glycerol tube is inoculated on a solid skim milk culture medium under the aseptic condition, cultured for 48 hours at the temperature of 33 ℃, and the larger bacterial colony is picked and inoculated on 50ml of liquid MRS culture medium, and cultured for 24 hours at the temperature of 33 ℃ to prepare seed liquid. The seed solution was inoculated in a fermentation medium under aseptic conditions at an inoculum size of 8%,33℃and 50rpm, and cultured at 24 h. After fermentation, the antibacterial activity of the fermentation broth is detected by a flat plate diffusion method, and the indicator bacteria are escherichia coli and staphylococcus aureus, so that the activity is good.
And filtering and sterilizing 5000ml of fermentation liquor by adopting a 100nm ceramic membrane to obtain a sterile fermentation liquor. 500ml of the sterile fermentation broth was taken and added with 50g of D380 anion exchange resin, vibrated at 50rpm for 2h and filtered after the end. The resin was washed with 100ml deionized water and filtered. Adding 200ml sodium hydroxide solution with concentration of 1mol/L into the washed resin, vibrating at 50rpm for 2 hours to perform elution, adjusting the pH of the eluent to 4-5 after the elution is finished, and detecting the antibacterial activity of the fermentation liquor, the adsorbed fermentation liquor and the eluent by a flat-plate diffusion method, wherein the indicator bacteria are aspergillus niger. The results show that the antibacterial effects of the fermentation liquor and the eluent are not different, the antibacterial effects are good, and the diameter of the antibacterial ring can reach 1.8-2.0cm, as shown in figure 6. The fermentation liquor after adsorption has no antibacterial activity.
30ml of the eluate was taken and 15% modified starch was added. Spray drying at 180deg.C at 85deg.C at outlet to obtain refined biological antiseptic, adding 50ml water into 1g solid antiseptic after spray drying, and detecting activity by plate diffusion method, wherein the bacteria is Aspergillus niger, and the bacteriostasis zone of the re-dissolved fermentation liquid can reach 2.5cm, while starch solution with the same mass concentration has no bacteriostasis activity, as shown in figure 7.
Claims (14)
1. A Lactobacillus brevis strain with wide antibacterial activity has a preservation number of CGMCC No. 19407.
2. Use of a strain of lactobacillus brevis with broad antibacterial activity as claimed in claim 1 in the preparation of a biological preservative.
3. A method for producing a biological preservative using the lactobacillus brevis strain of claim 1.
4. A method according to claim 3, comprising 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 4, wherein the fermentation culture conditions are 30-37 ℃,50-100 rpm, and 24-48 h; the fermentation medium is MRS liquid medium.
6. The method according to claim 5, wherein the following cultivation is performed before the fermentation cultivation:
(1) Slant culture: inoculating the lactobacillus brevis strain on a solid slant culture medium under a sterile condition, and culturing for 24-48 hours at the temperature of 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 a sterile condition, and carrying out stationary culture 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 process.
8. The method according to claim 7, wherein the spray drying is carried out at an inlet temperature of 120-210 ℃ and an outlet temperature of 60-100 ℃, and wherein after drying 5-20% by weight of modified starch, maltodextrin and/or cyclodextrin is added.
9. The method according to claim 4, comprising the steps of: clarifying the fermentation liquor, and collecting clear liquor; treating the clarified broth with an anion exchange resin; eluting the anion exchange resin by using an analytical solution, and collecting an eluent; drying the eluent to obtain the biological preservative.
10. The method of claim 9, wherein: the clarification method is selected from tube centrifuge centrifugation, disk centrifuge centrifugation, ceramic membrane filtration, and hollow fiber membrane filtration.
11. The method of claim 9, wherein: the anion exchange resin is a strong basic ion exchange resin or a weak basic ion exchange resin.
12. The method of claim 9, wherein: the resolving liquid is one or two 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 is dried by vacuum drying, spray drying or freeze drying.
14. Use of the lactobacillus brevis strain as claimed in claim 1 as a preservative in food and feed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210699465.5A CN114806976B (en) | 2022-06-20 | 2022-06-20 | Lactobacillus brevis and preparation method of antibacterial substances thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210699465.5A CN114806976B (en) | 2022-06-20 | 2022-06-20 | Lactobacillus brevis and preparation method of antibacterial substances thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114806976A CN114806976A (en) | 2022-07-29 |
| CN114806976B true CN114806976B (en) | 2023-10-03 |
Family
ID=82520732
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210699465.5A Active CN114806976B (en) | 2022-06-20 | 2022-06-20 | Lactobacillus brevis and preparation method of antibacterial substances thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114806976B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117481326A (en) * | 2023-10-23 | 2024-02-02 | 浙江溢滔食品技术有限公司 | Preparation method of solid seasoning with antibacterial and fresh-keeping functions |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020013670A (en) * | 2000-08-14 | 2002-02-21 | 백남수, 권오훈 | Probiotic preparations for aquacultured fish and its production method |
| CN102605029A (en) * | 2012-03-01 | 2012-07-25 | 中国农业大学 | Lactobacillus brevis bacteriocin and preparation method thereof |
| CN102660477A (en) * | 2012-05-10 | 2012-09-12 | 北京市农林科学院 | Lactobacillus brevis, freeze-dried powder of Lactobacillus brevis and application of freeze-dried powder |
| KR20150032379A (en) * | 2013-09-16 | 2015-03-26 | (사)광주명품김치산업화사업단 | Lactobacillus brevis 4-12 having GABA production capacity and methods for preparing Kimchi comprising the same |
| CN109645403A (en) * | 2018-11-30 | 2019-04-19 | 四川东坡中国泡菜产业技术研究院 | The Lactobacillus brevis of low temperature resistant antiphagin and its application in pickles |
| KR20190061122A (en) * | 2017-11-27 | 2019-06-05 | 한국식품연구원 | Natural bacteriocin produced by Lactobacillus brevis and it's gene sequences |
| KR102065580B1 (en) * | 2018-11-12 | 2020-01-13 | 재단법인 발효미생물산업진흥원 | Lactobacillus brevis SCML 432 strain having antimicrobial activity and probiotics properties and uses thereof |
| CN111778191A (en) * | 2020-07-17 | 2020-10-16 | 鲁东大学 | Lactobacillus brevis strain and culture method and application thereof |
| CN112458002A (en) * | 2020-10-22 | 2021-03-09 | 宁波大学 | Lactobacillus acidophilus strain for reducing urine blood, screening method thereof and application of lactobacillus acidophilus strain in preparing functional yoghourt |
| CN113215067A (en) * | 2021-06-29 | 2021-08-06 | 浙江师范大学 | VBNC (viable but non-viable) state lactobacillus brevis CSHRR5-3 strain and application thereof |
| CN113444664A (en) * | 2021-07-01 | 2021-09-28 | 陕西广播电视大学（陕西工商职业学院） | Lactobacillus brevis capable of producing gamma-aminobutyric acid and application thereof |
-
2022
- 2022-06-20 CN CN202210699465.5A patent/CN114806976B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020013670A (en) * | 2000-08-14 | 2002-02-21 | 백남수, 권오훈 | Probiotic preparations for aquacultured fish and its production method |
| CN102605029A (en) * | 2012-03-01 | 2012-07-25 | 中国农业大学 | Lactobacillus brevis bacteriocin and preparation method thereof |
| CN102660477A (en) * | 2012-05-10 | 2012-09-12 | 北京市农林科学院 | Lactobacillus brevis, freeze-dried powder of Lactobacillus brevis and application of freeze-dried powder |
| KR20150032379A (en) * | 2013-09-16 | 2015-03-26 | (사)광주명품김치산업화사업단 | Lactobacillus brevis 4-12 having GABA production capacity and methods for preparing Kimchi comprising the same |
| KR20190061122A (en) * | 2017-11-27 | 2019-06-05 | 한국식품연구원 | Natural bacteriocin produced by Lactobacillus brevis and it's gene sequences |
| KR102065580B1 (en) * | 2018-11-12 | 2020-01-13 | 재단법인 발효미생물산업진흥원 | Lactobacillus brevis SCML 432 strain having antimicrobial activity and probiotics properties and uses thereof |
| CN109645403A (en) * | 2018-11-30 | 2019-04-19 | 四川东坡中国泡菜产业技术研究院 | The Lactobacillus brevis of low temperature resistant antiphagin and its application in pickles |
| CN111778191A (en) * | 2020-07-17 | 2020-10-16 | 鲁东大学 | Lactobacillus brevis strain and culture method and application thereof |
| CN113061553A (en) * | 2020-07-17 | 2021-07-02 | 鲁东大学 | Lactobacillus brevis strain and culture method and application thereof |
| CN112458002A (en) * | 2020-10-22 | 2021-03-09 | 宁波大学 | Lactobacillus acidophilus strain for reducing urine blood, screening method thereof and application of lactobacillus acidophilus strain in preparing functional yoghourt |
| CN113215067A (en) * | 2021-06-29 | 2021-08-06 | 浙江师范大学 | VBNC (viable but non-viable) state lactobacillus brevis CSHRR5-3 strain and application thereof |
| CN113444664A (en) * | 2021-07-01 | 2021-09-28 | 陕西广播电视大学（陕西工商职业学院） | Lactobacillus brevis capable of producing gamma-aminobutyric acid and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Antimicrobial compounds produced by probiotic Lactobacillus brevis isolated from dairy products;Abeer Ahmed Rushdy et al.;《Ann Microbiol》;第63卷;第81-90页 * |
| Probiotic characterization of Lactobacillus brevis KU15153 showing antimicrobial and antioxidant effect isolated from kimchi;Hye Ji Jang et al.;《Food Sci Biotechnol》;第28卷(第5期);第1521-1528页 * |
| Probiotic Properties of Lactobacillus brevis KU200019 and Synergistic Activity with Fructooligosaccharides in Antagonistic Activity against Foodborne Pathogens;Kariyawasam Majuwana Gamage Menaka Menike Kariyawasam et al.;《Food Sci. Anim. Resour.》;第40卷(第2期);第297-310页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114806976A (en) | 2022-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108611299B (en) | Lactobacillus plantarum for producing antibacterial peptide and application thereof | |
| KR101349692B1 (en) | The Alcohol resistant strain of lactic acid bacteria, Pediococcus acidilactici and its use | |
| CN105176874A (en) | Bacillus coagulans fm 603 and application thereof | |
| CN112391325B (en) | Lactobacillus paracasei and application thereof | |
| CN105175518A (en) | Bacteriocin generated by bacillus coagulans FM603 and preparing method thereof | |
| CN114806976B (en) | Lactobacillus brevis and preparation method of antibacterial substances thereof | |
| CN106635916B (en) | Acetobacter orientalis YZD-09 and application thereof | |
| CN111909878A (en) | Lactobacillus casei with function of removing patulin | |
| CN112980735A (en) | Clostridium butyricum, microbial inoculum, application of clostridium butyricum and microbial inoculum and preparation method of microbial inoculum | |
| CN113980853B (en) | Lactic acid-producing lactococcus garvieae WBT0008 and application thereof | |
| CN113293110B (en) | Preparation method of antibacterial lipopeptid compound | |
| CN112746044B (en) | Lactobacillus plantarum mutant strain and application thereof in alfalfa silage | |
| CN111733117B (en) | Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof | |
| CN116478862B (en) | Strain, preparation and method for degrading aflatoxin | |
| CN110452857B (en) | Lactobacillus plantarum for producing non-protein micromolecule antibacterial metabolites and application thereof | |
| CN112175871A (en) | Marigold flower and fresh flower compound leavening agent and application thereof | |
| CN109468285B (en) | Lactobacillus plantarum GS high-enzyme activity fermentation method | |
| CN106167777A (en) | A kind of cultural method of the bacillus amyloliquefaciens producing Substance | |
| CN105104712A (en) | Microbiological feed additive and preparation method thereof | |
| CN106636262B (en) | Method for improving yield of nisin by controlling pH value of fermentation culture system | |
| CN112094777B (en) | Lactobacillus plantarum and application thereof in Laoshan milk goat feed | |
| CN110564620A (en) | Method for culturing polyphenol oxidase produced by sinapine degrading bacteria | |
| CN114081098B (en) | Method for preparing composite immunopotentiator from Subtilosin A and dandelion root polysaccharide and application thereof | |
| CN114426939B (en) | Lactobacillus rhamnosus with ochratoxin A removal effect | |
| CN117343876B (en) | Antibacterial and anti-inflammatory bacillus belicus and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |