WO2018035892A1 - 一种乳酸片球菌来源的抗菌六肽及其制备方法 - Google Patents

一种乳酸片球菌来源的抗菌六肽及其制备方法 Download PDF

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WO2018035892A1
WO2018035892A1 PCT/CN2016/098420 CN2016098420W WO2018035892A1 WO 2018035892 A1 WO2018035892 A1 WO 2018035892A1 CN 2016098420 W CN2016098420 W CN 2016098420W WO 2018035892 A1 WO2018035892 A1 WO 2018035892A1
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antimicrobial peptide
antibacterial
lactobacillus
peptide
escherichia coli
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French (fr)
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陆健
蔡国林
王娟
朱德伟
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A23B2/7295—Antibiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A23B2/762—Organic compounds containing nitrogen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B4/00—Preservation of meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A23B4/22—Microorganisms; Enzymes; Antibiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/1526—Amino acids; Peptides; Protein hydrolysates; Nucleic acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/195—Antibiotics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/58—Multistep processes
    • 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
    • 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
    • A23V2400/41—Pediococcus
    • A23V2400/413—Acidilactici
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • the invention relates to an antibacterial hexapeptide derived from Pediococcus lacticis and a preparation method thereof, and belongs to the field of bioengineering.
  • antibiotics are widely used as feed additives in poultry feeds, which play an important role in promoting animal growth and preventing certain diseases, but as antibiotic abuse events occur frequently, people are given antibiotics.
  • the side effects have been further recognized. Abuse of antibiotics not only causes endogenous infections in animals, but also produces drug-resistant strains, and it will reduce the immune function of livestock and poultry. More seriously, antibiotics remaining in livestock and poultry products will flow into the human body through different channels and cause human body damage. harm.
  • the EU Regulatory Commission has decided to ban antibiotic growth promoters in animal farming since January 2006. Beginning in December 2013, the US FDA issued the Veterinary Feed Directive, which requires licensed veterinarians to supervise the use of antibiotics. It is planned to ban the use of preventive antibiotics in livestock feed for three years from 2014. Reduce the problem of antibiotic resistance brought to consumers by food animals. South Korea will also ban the use of feed antibiotics in July 2018.
  • the present invention separates a lactobacillus which inhibits bacteria from a fermentation broth of a lactic acid bacteria capable of inhibiting the growth of Escherichia coli by liquid chromatography-mass spectrometry (LC-). MS) preliminary identification of the molecular weight of the lactomycin was 705.63 Da, the isoelectric point was 3.58, and the amino acid sequence was ENGEEE. The present invention also confirms that the lactomycin has an inhibitory effect on various Gram-positive bacteria by a chemical synthesis method, and names it as Lactobacillus R16.
  • a first object of the present invention is to provide an antimicrobial peptide having an amino acid sequence of: ENGEEE (as shown in SEQ ID NO. 1), designated as lactobacillus R16.
  • the antimicrobial peptide is isolated from a fermentation broth of a lactic acid bacterium capable of inhibiting the growth of Escherichia coli.
  • a second object of the invention is to provide a nucleotide fragment encoding the antimicrobial peptide.
  • a third object of the present invention is to provide a recombinant vector or genetically engineered bacteria capable of expressing the antimicrobial peptide.
  • a plurality of (eg, 20) coding genes of the antimicrobial peptide may be ligated end to end, and a thrombin cleavage site is added between each two genes, and the entire gene is ligated to the vector to obtain a recombinant vector, which is transformed into a host strain for expression.
  • the expression product is digested with thrombin to obtain the antimicrobial peptide.
  • the vector may be pPIC9K.
  • the host strain may be Pichia pastoris GS115.
  • a fourth object of the present invention is to provide a composition containing the antimicrobial peptide.
  • the composition is a feed additive, a feed, a biological preservative.
  • a fifth object of the present invention is to provide the use of the antimicrobial peptide, a nucleotide fragment encoding the antimicrobial peptide, a recombinant vector capable of expressing the antimicrobial peptide, or a genetically engineered bacteria capable of expressing the antimicrobial peptide.
  • the application is as a feed additive or a biological preservative.
  • a sixth object of the present invention is to provide a method for inhibiting bacteria by using the antimicrobial peptide.
  • the bacterium is a Gram-positive or Gram-negative bacterium.
  • the bacteria comprise: Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and the like.
  • the invention also provides a preparation method of the antibacterial peptide, wherein the fermentation supernatant obtained by fermenting the bacterium of the bacterium of the bacterium is firstly extracted by ultrafiltration, and then subjected to an anion exchange chromatography column Hitrap Q FF and a gel filtration chromatography Superdux Peptide 10 /300GL was separated and finally isolated and purified by reverse phase column Phenomenex Luna/C 18 to obtain an antibacterial hexapeptide.
  • the ultrafiltration is concentrated 8 to 10 times by ultrafiltration (molecular weight cut off of 3000 Da) to obtain a crude extract.
  • the preparation method is specifically:
  • Ultrafiltration crude extraction ultrafiltration (molecular weight cut off of 3000 Da) is concentrated 8 to 10 times to obtain a crude extract;
  • Hitrap Q FF Separation by anion exchange chromatography
  • the antibacterial peptide ENGEEE of the present invention is compared with a database such as the Antimicrobial Peptide Database, and such an antimicrobial peptide is reported for the first time.
  • the present invention utilizes techniques such as ultrafiltration and chromatography to prepare an active peptide lactobacillus R16 which is significantly resistant to Gram-positive bacteria.
  • the preparation process of the invention is simple, the separation effect is good, the operating conditions are mild, fast and efficient, and the product purity is high, and the purity thereof can be more than 95% by MS.
  • the obtained antimicrobial peptide Lactobacillus R16 of the present invention has good pH stability and thermal stability, and the minimum inhibitory concentration against Escherichia coli and Listeria monocytogenes is 6.4 mg ⁇ mL -1 for golden yellow grapes. The minimum inhibitory concentration of cocci was 3.4 mg ⁇ mL -1 .
  • the antibacterial peptide lactobacillus R16 of the present invention has a certain proliferative effect on Saccharomyces cerevisiae and Lactobacillus plantarum, and has little effect on other probiotics.
  • the antibacterial peptide lactobacillus R16 of the present invention has a certain scavenging ability to hydrogen peroxide, hydroxyl radical, DPPH ⁇ free radical and superoxide anion, and the scavenging rates are 25.65%, 70.13%, 79.42% and 10.11%, respectively.
  • the antibacterial peptide Lactobacillus sp. R16 of the present invention can be used as a biological control, and is an effective supplement to the lactic acid bacteria library of the measured amino acid sequence. It can be used as a feed additive and plays an important role in replacing antibiotics and addressing feed safety issues in the future.
  • FIG. 4 Effect of lactobacillus R16 on the growth of probiotics
  • Figure 5 Scavenging ability of bacteriostatic active substances to hydroxyl radicals, DPPH free radicals and superoxide anion radicals.
  • the antibacterial hexapeptide lactobacillus R16 was prepared as follows:
  • the antibacterial hexapeptide is fermented by Pediococcus lactis, and after the fermentation supernatant is centrifuged, the supernatant is collected and subjected to ultrafiltration (molecular weight cut off of 10,000 Da) to be concentrated 8 to 10 times to obtain a crude extract.
  • Hitrap Q FF Separation by anion exchange chromatography
  • Mass spectrometry conditions capillary voltage, 3.88 kV; cone voltage, 20 V; ion source temperature, 120 ° C; desolvation temperature, 300 ° C; flow rate, 1 mL/min; split ratio, 50:1. The results were analyzed with the software MassLynx 4.1.
  • Mass spectrometry identified this antimicrobial peptide to have a molecular weight of 705.17 Da and an amino acid sequence of E-N-G-E-E-E (sequence as shown in SEQ ID NO. 1).
  • Figure 2 shows that the bacteriostatic activity of lactomycin R16 is maximized at pH 3.0 to 5.0.
  • pH 6.0-8.0 the antibacterial activity of lactobacillus R16 gradually decreased, and the decreasing trend was slow, and the residual activity was maintained at about 75%, indicating that the stability of lactobacillus R16 was lowered under this pH condition, but the pH was against lactic acid bacteria.
  • the stability of the prime R16 has little effect.
  • the lactobacillus R16 has a certain thermal stability. After treatment at 40, 50, 60, 70, 80 °C for 30 min, the antibacterial activity of lactobacillus R16 has no significant change, and it is treated at 90 ° C and 100 ° C for 30 min. Lactobacillus The antibacterial activity of R16 only lost 21.95% and 49.47%, respectively. After treatment at 121 ° C for 15 min, the bacteriostatic activity of lactomycin R16 lost 59.98%. In the granulation process at a temperature of 75-85 ° C for a duration of 1 to 2 min, the activity can reach 100% retention.
  • the feed is easy to carry pathogenic microorganisms such as Escherichia coli and Listeria monocytogenes during transportation and storage. These pathogenic microorganisms may cause intestinal infectious diseases of animals as they enter the animal, posing a serious threat to the health of the animals.
  • pathogenic microorganisms such as Escherichia coli and Listeria monocytogenes
  • the inhibitory effect of lactobacillus R16 on common animal pathogenic bacteria is shown in Table 1.
  • lactobacillus R16 showed strong antibacterial activity against Staphylococcus aureus, Escherichia coli K99, Escherichia coli JM109 and Listeria monocytogenes, and the antibacterial effect against Staphylococcus aureus was the strongest.
  • the antibacterial activity was determined by minimum inhibitory concentration (MIC) as follows:
  • the minimum inhibitory concentration of lactobacillus R16 against Escherichia coli, Listeria monocytogenes and Staphylococcus aureus is shown in Table 2. From Table 2, the minimum inhibition of Escherichia coli and Listeria monocytogenes can be seen. The bacterial concentration was 6.4 mg ⁇ mL -1 , and the minimum inhibitory concentration against Staphylococcus aureus was 3.2 mg ⁇ mL -1 .
  • Each probiotic was subjected to secondary activation, and inoculated into MRS medium containing bacteriostatic active substance of 6.4 mg ⁇ mL -1 at a dose of 1%, and the probiotics inoculated in MRS medium were used as control, 37 ° C. Incubate at constant temperature for 24 hours. The plate count was performed by viable count method. The result is shown in Figure 4.
  • microecological preparations are widely used in feeds.
  • compound microecological preparations composed of yeast, lactic acid bacteria and spores are more common, which not only can degrade and soften crude fiber, but also increase protein content. It can also improve the palatability of feed, regulate the intestinal micro-ecological balance of animals, and promote digestion, absorption and growth.
  • the invention selects Saccharomyces cerevisiae, Pediococcus pentosaceus, Enterococcus faecium, plant lactic acid bacteria and Bacillus subtilis as research objects, and studies the influence of lactobacillus R16 on it, and the results are shown in FIG. 4 .
  • the lactobacillus R16 has a certain proliferation effect on Saccharomyces cerevisiae and Lactobacillus plantarum, and the proliferation effect on Saccharomyces cerevisiae is more obvious, and the number of Saccharomyces cerevisiae is increased by 24.50% compared with the control.
  • Lactobacillus R16 has a certain inhibitory effect on Enterococcus faecium and Bacillus subtilis, but the inhibition rate is low. Compared with the control, Enterococcus faecium and Bacillus subtilis have a total bacterial count of 6.92% and 4.03%, respectively, and are growing.
  • the total number of bacteria can reach about 10 8 CFU ⁇ mL -1 .
  • lactomycin R16 had no effect on Pediococcus pentosace.
  • a certain amount of unsaturated fat, fatty acid, fat-soluble vitamin, carotenoid and other fat-soluble substances in the feed are easily oxidized during the storage of the feed, especially fats and fatty acids, which are oxidized and rancid to produce aldehydes and alcohols. ,fat, Different compounds such as acid, which may produce various odors, seriously affect the palatability of the feed. Some compounds have certain toxicity and have adverse effects on animals.
  • the antioxidant capacity of lactobacillus R16 against hydrogen peroxide, hydroxyl radicals, DPPH ⁇ free radicals and superoxide anion is shown in Fig. 5.
  • lactobacillus R16 has a certain ability to scavenge hydrogen peroxide, hydroxyl radicals, DPPH ⁇ free radicals and superoxide anion, among which DPPH ⁇ free radicals have the strongest ability to scavenge, and the clearance rate reaches 79.42. %, followed by hydroxyl radicals, the scavenging rate was 70.13%, and the scavenging ability to superoxide anion and hydrogen peroxide was the worst, and the scavenging rate was only 10.11% and 25.65%.
  • soybean meal was pulverized with a blade powder machine, and 4 portions were added, and each 40 g was added to a 500 mL fermentation flask, and the numbers were group 5, group 6, group 7, group 8, and sterilized at 105 ° C for 10 min.
  • the inhibitory effect of lactobacillus R16 on Escherichia coli in soybean meal was studied by adding Escherichia coli to the sterilized soybean meal.
  • the results showed that the number of E. coli was increased from 1380 ⁇ 100g -1 before fermentation to 4 ⁇ 10 6 ⁇ 100g -1 after fermentation, and the number of E. coli was added after adding lactobacillus R16. From 1380 ⁇ 100g -1 before fermentation to 123 ⁇ 100g -1 , it shows that Lactobacillus R16 can effectively reduce E. coli in soybean meal while reducing the number of E. coli, and its removal rate reaches 33.44%. .
  • the present invention changes the ratio of water to water of 1:0.8 to 1:0.12 on the basis of the original experiment, and the other experimental conditions remain unchanged, and the soybean meal fermentation experiment is carried out. It showed that the removal rate of Escherichia coli in soybean meal by lactobacillus R16 increased from 33.44% to 56.64%, indicating that under industrial conditions, lactomycin R16 can fully act on Escherichia coli.
  • Lactobacillus is currently the only bacteriocin that is allowed to be used in food preservation, and can be added to foods such as milk and meat products to inhibit the growth of bacteria and extend shelf life.

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Abstract

一种乳酸片球菌来源的抗菌六肽及其制备方法，属于生物工程领域。所述抗菌肽乳酸菌素R16的氨基酸序列为ENGEEE，其pH稳定性和热稳定性好，对大肠杆菌、单增李斯特菌、金黄色葡萄球菌均有抑制作用，能够有效降低豆粕中大肠杆菌的数量。抗菌肽乳酸菌素R16对于酿酒酵母和植物乳杆菌具有一定的增殖作用，对过氧化氢、羟自由基、DPPH•自由基和超氧阴离子具有一定的清除能力。抗菌肽乳酸菌素R16可作为生物防治使用，可作为饲料添加剂使用，在将来替代抗生素和解决饲料安全问题中发挥重要作用。

Description

一种乳酸片球菌来源的抗菌六肽及其制备方法 技术领域
本发明涉及一种乳酸片球菌来源的抗菌六肽及其制备方法，属于生物工程领域。
背景技术
目前，亚治疗剂量的抗生素作为饲料添加剂被广泛应用于禽畜饲料中，其在促进动物生长、预防某些疾病发生方面发挥着重要作用，但是，随着抗生素滥用事件频繁发生，让人们对抗生素的副作用有了进一步的认识。滥用抗生素不仅会引起动物内源性感染，产生耐药菌株，而且会使畜禽免疫功能下降，更严重的是，残留于畜禽产品中的抗生素会通过不同渠道而流入人体，并对人体造成危害。
欧盟监管委员会决定，自2006年1月起在动物养殖业中禁用抗生素生长促进剂。从2013年12月开始，美国FDA发布了《兽医饲料指令》，要求有执照的兽医监督抗生素的使用，计划从2014年起，用3年时间禁止在牲畜饲料中使用预防性抗生素，最大限度地减少食用牲畜带给消费者抗生素耐药性问题。韩国也将在2018年7月全面禁止饲用抗生素的使用。
随着细菌对抗生素耐药性和抗生素的残留问题日益严重，研究和开发绿色饲料添加剂产品已经成为世界性的研究课题，大量研究表明，中草药提取物、微生物制剂、酶制剂、益生元、抗菌肽、酸化剂等新型饲料添加剂能有效减少或者替代饲用抗生素的使用。其中，抗菌肽由于具有较好的抗菌和免疫调节的活性，且不易残留、没有副作用、没有抗药性，对环境不产生不良影响等优点，成为了抗生素潜在的有效替代物。
发明内容
为了解决上述问题，本发明从能够抑制大肠杆菌生长的乳酸菌的发酵液中分离得到一种对细菌具有抑制作用的乳酸菌素，通过液相色谱-质谱联用技术(liquid chromatography-mass spectrometry，LC-MS)初步鉴定该乳酸菌素的分子量大小为705.63Da，等电点为3.58，氨基酸序列为ENGEEE。本发明还通过化学合成的方法确认该乳酸菌素对多种革兰氏阳性菌具有抑制作用，将其命名为乳酸菌素R16。
本发明的第一个目的是提供一种抗菌肽，所述抗菌肽的氨基酸序列为：ENGEEE(如SEQ ID NO.1所示)，命名为乳酸菌素R16。
所述抗菌肽，是从能够抑制大肠杆菌生长的乳酸菌的发酵液中分离得到的。
本发明的第二个目的是提供编码所述抗菌肽的核苷酸片段。
本发明的第三个目的是提供一种能够表达所述抗菌肽的重组载体或者基因工程菌。比如， 可以将若干(如20)个该抗菌肽的编码基因首尾连接，在每两段基因中间添加凝血酶酶切位点，整段基因与载体连接得到重组载体，转化入宿主菌表达。表达产物经凝血酶酶切即可得到该抗菌肽。
在本发明的一种实施方式中，所述载体可以是pPIC9K。
在本发明的一种实施方式中，所述宿主菌可以是毕赤酵母GS115。
本发明的第四个目的是提供含有所述抗菌肽的组合物。
在本发明的一种实施方式中，所述组合物为饲料添加剂、饲料、生物防腐剂。
本发明的第五个目的是提供所述抗菌肽、编码所述抗菌肽的核苷酸片段、能够表达所述抗菌肽的重组载体或者能够表达所述抗菌肽的基因工程菌的应用。
在本发明的一种实施方式中，所述应用，是作为饲料添加剂或者生物防腐剂。
本发明的第六个目的是提供一种抑制细菌的方法，是使用所述的抗菌肽。
在本发明的一种实施方式中，所述细菌为革兰氏阳性菌或革兰氏阴性菌。
在本发明的一种实施方式中，所述细菌包括：大肠杆菌、单增李斯特菌、金黄色葡萄球菌等。
本发明还提供一种所述抗菌肽的制备方法，是将乳酸片球菌发酵得到的发酵上清液先由超滤进行粗提，然后经阴离子交换色谱柱Hitrap Q FF和凝胶过滤色谱Superdux Peptide10/300GL分离，最后用反相柱Phenomenex Luna/C18分离纯化得到抗菌六肽。
在本发明的一种实施方式中，所述超滤是超滤(截留分子量3000Da)浓缩8～10倍，得到粗提液。
在本发明的一种实施方式中，所述制备方法具体是：
(1)超滤粗提：超滤(截留分子量3000Da)浓缩8～10倍，得到粗提液；
(2)经阴离子交换色谱Hitrap Q FF的分离：粗提液上样于Hitrap Q FF色谱柱分离得到2个洗脱峰，收集对大肠杆菌有抑菌作用的活性峰L2，透析后冷冻干燥；
(3)经凝胶过滤色谱Superdux Peptide 10/300GL的分离：将阴离子交换色谱的洗脱峰L2上样于Superdux Peptide 10/300GL凝胶过滤层析柱，分离得到2个洗脱峰，收集对大肠杆菌有抑菌作用的活性峰N2，透析后冷冻干燥；
(4)反相C18柱分离：将上一步冷冻干燥的样品用5％的乙腈(含0.1％TFA，下同)溶解，并经5％的乙腈平衡的Phenomenex Luna/C18柱，用浓度为5-50％的线性梯度递增的乙腈洗脱，收集活性洗脱峰Ⅱ，并经旋转蒸发和冷冻干燥后，得到最终产品。
本发明的有益效果：
(1)本发明的抗菌肽ENGEEE，与抗菌肽数据库(TheAntimicrobial Peptide Database)等数据库比对，此种抗菌肽是首次被报道。
(2)本发明利用超滤和色谱等技术，制备对革兰氏阳性菌具有明显抗性的活性肽乳酸菌素R16。本发明的制备工艺简单、分离效果好、操作条件温和、快速高效、产品纯度高，经MS鉴定其纯度可达95％以上。
(3)本发明中的获得的抗菌肽乳酸菌素R16的pH稳定性和热稳定性好，对大肠杆菌和单增李斯特菌的最小抑菌浓度为6.4mg·mL-1，对金黄色葡萄球菌的最小抑菌浓度为3.4mg·mL-1。
(4)本发明的抗菌肽乳酸菌素R16对于酿酒酵母和植物乳杆菌具有一定的增殖作用，对其他对益生菌的影响很小。
(5)本发明的抗菌肽乳酸菌素R16对过氧化氢、羟自由基、DPPH·自由基和超氧阴离子具有一定的清除能力，清除率分别为25.65％、70.13％、79.42％和10.11％。
(6)本发明的抗菌肽乳酸菌素R16可作为生物防治使用，是对已测氨基酸序列的乳酸菌素库的有效补充。可作为饲料添加剂使用，在将来替代抗生素和解决饲料安全问题中发挥重要作用。
附图说明
图1：六肽的质谱鉴定图；
图2：不同pH值对乳酸菌素R16的影响；
图3：温度对乳酸菌素R16的影响；
图4：乳酸菌素R16对益生菌生长的影响；
图5：抑菌活性物质对羟自由基、DPPH自由基和超氧阴离子自由基的清除能力。
具体实施方式
本发明所涉及的各种缓冲液配方均可以在实验手册中查到。
实施例1：乳酸菌素R16的制备
按照下面的方法制备抗菌六肽乳酸菌素R16：
1.抗菌六肽经乳酸片球菌发酵而来，发酵上清液经离心之后，收集上清液超滤(截留分子量10000Da)浓缩8～10倍，得到粗提液。
2.经阴离子交换色谱Hitrap Q FF的分离：粗提液上样于Hitrap Q FF色谱柱分离得到2个洗脱峰，收集对大肠杆菌有抑菌作用的活性峰，并将收集的活性峰L2经Mill-Q水充分透析后冷冻干燥。
3.经凝胶过滤色谱Superdux Peptide 10/300GL的分离：将阴离子交换色谱的洗脱峰L2上样于Superdux Peptide 10/300GL凝胶过滤层析柱，分离得到2个洗脱峰，收集对大肠杆菌有抑菌作用的活性峰，并将收集的活性峰N2经Mill-Q水充分透析后冷冻干燥。
4.反相C18柱的分离：将上一步冷冻干燥的样品用5％的乙腈(含0.1％TFA，下同)溶解，并经5％的乙腈平衡的C18柱，用浓度为5-50％的线性梯度递增的乙腈洗脱，收集活性洗脱峰H3，并经旋转蒸发和冷冻干燥后，最终得到产品。
5.LC-MS的鉴定：将上述步骤得到的具有抑菌活性的冷冻干燥样品，用Mill-Q水溶解后经LC-MS鉴定，流动相为A(乙腈:水:甲酸＝30:970:1(V:V:V))和B(乙腈:水:甲酸＝700:300:1(V:V:V))；洗脱程序为0-10min，100％A，0％B；20min，70％A，30％B；30min，0％A，100％B；35min，100％A，0％B；flow rate，1mL/min；柱温30℃。质谱条件：毛细管电压，3.88kV；圆锥体电压，20V；离子源温度，120℃；去溶温度，300℃；流速，1mL/min；分流比，50:1。结果用软件MassLynx 4.1分析。
质谱鉴定此抗菌肽的分子量为705.17Da，其氨基酸序列为E-N-G-E-E-E(序列如SEQ ID NO.1所示)。
经与抗菌肽数据库(TheAntimicrobial Peptide Database)等数据库比对，此种抗菌肽并未见报道。
实施例2：乳酸菌素R16的生物学特性
(1)乳酸菌素R16在pH中的稳定性
取10份浓度为100mg·mL-1纯化过后的活性物质乳酸菌素R16，用1.0mol·L-1NaOH和1.0mol·L-1HCl将其pH分别调成1.0～10.0，置于37℃的水浴锅处理2h，后将其pH调至4.5[62]，做抑菌实验，观察其对大肠杆菌的抑制情况。结果如图2所示。
图2显示，在pH 3.0～5.0时，乳酸菌素R16的抑菌活性达到最大。在pH 6.0～8.0时，乳酸菌素R16的抑菌活性逐步降低，且降低趋势缓慢，残留活性维持在75％左右，说明乳酸菌素R16在此pH条件下稳定性有一定降低，但此pH对乳酸菌素R16的稳定性影响不大。
(2)乳酸菌素R16的热稳定性
取8份浓度为100mg·mL-1纯化过后的活性物质乳酸菌素R16，分别置于40、50、60、70、80、90、100℃中处理30min，121℃高压灭菌15min，做抑菌实验，观察其对大肠杆菌的抑制情况。结果如图3所示。
从图3可以看出，乳酸菌素R16具有一定的热稳定性，在40、50、60、70、80℃处理30min，乳酸菌素R16的抑菌活性无明显变化，在90℃和100℃处理30min后，乳酸菌素 R16的抑菌活性仅仅分别损失了21.95％和49.47％。121℃处理15min，乳酸菌素R16的抑菌活性损失了59.98％。在温度为75～85℃，持续时间为1～2min的制粒过程中，其活性能够达到100％的保留。
(3)乳酸菌素R16的抑菌谱
取8份浓度为100mg·mL-1纯化过后的活性物质乳酸菌素R16，运用牛津杯法，检测其对大肠杆菌、单增李斯特菌、金黄色葡萄球菌的抑制作用。
饲料在运输、储存过程中容易携带大肠杆菌、单增李斯特菌等病原微生物，这些病原微生物随着饲料进入动物体内，会引起动物肠道传染病，对动物的健康构成严重威胁。乳酸菌素R16对常见动物致病菌的抑制作用如表1所示。
表1 乳酸菌素R16的抑菌谱
Figure PCTCN2016098420-appb-000001
注：“+++”为强抑制；“++”为较强抑制；“+”为抑制；“-”为无抑制
从表1可知，乳酸菌素R16对金黄色葡萄球菌、大肠杆菌K99，大肠杆菌JM109和单增李斯特菌表现出较强的抑菌活性，其中对金黄色葡萄球菌的抑菌效果最强。
(4)乳酸菌素R16的最低抑菌浓度
采用最小抑菌浓度法(minimum inhibitory concentration，MIC)检测抗菌活性，操作如下：
(a)将菌种按1％接种量接种于LB液体培养基中，37℃，200r·min-1摇床培养过夜。
(b)将上述培养液按1％接种量接种于20mL LB液体培养基中，37℃，200r·min-1摇床培养至OD600＝0.5左右。
(c)将OD600＝0.5的菌悬液按1‰接种量接种于10mL LB液体培养基中，震荡摇匀，使细菌密度在1×105～5×105CFU·mL-1，测定MIC。
(d)将抑菌活性物质进行梯度稀释，使其终浓度为256、128、64、32、16、8、4、2和1mg·mL-1。
(e)向无菌组织培养板中加入450μL制备好的菌悬液，再分别加入50μL相应浓度的 抑菌活性物质稀释液，待测抑菌活性物质的浓度分别为25.6、12.8、6.4、3.2、1.6、0.8、0.4、0.2和0.1mg·mL-1。以500μL菌悬液为阳性对照，以500μL LB液体培养基为阴性对照，37℃恒温培养箱培养18～24h。
(f)观察各个孔底部是否有细菌沉淀，无肉眼可见细菌沉淀的最小浓度为该抑菌活性物质的MIC。
乳酸菌素R16对大肠杆菌，单增李斯特菌和金黄色葡萄球菌的最小抑菌浓度如表2所示，从表2可以看出抑菌活性物质对大肠杆菌和单增李斯特菌的最小抑菌浓度为6.4mg·mL-1，对金黄色葡萄球菌的最小抑菌浓度为3.2mg·mL-1。
表2 乳酸菌素R16对菌种的最低抑菌浓度
Figure PCTCN2016098420-appb-000002
(5)乳酸菌素R16对益生菌的影响
将各益生菌进行二级活化，按1％的接种量接种于含有抑菌活性物质为6.4mg·mL-1的MRS培养基中，以接种于MRS培养基中的益生菌为对照，37℃恒温培养24h。采用活菌计数法进行涂板计数。结果如图4所示。
微生态制剂作为绿色饲料添加剂的一种，被广泛应用于饲料中，其中以酵母菌，乳酸菌和芽孢菌构成的复合型微生态制剂更为常见，其不仅能够降解和软化粗纤维，提高蛋白含量，还能够改善饲料的适口性，调节动物肠道微生态平衡，促进消化吸收和生长。本发明选取酿酒酵母、戊糖片球菌、屎肠球菌，植物乳酸菌和枯草芽孢杆菌作为研究对象，研究了乳酸菌素R16对其的影响，结果见图4所示。
由图4可知，乳酸菌素R16对酿酒酵母和植物乳杆菌有一定的增殖作用，其中对酿酒酵母的增殖作用更为明显，酿酒酵母的数量与对照相比，增加了24.50％。乳酸菌素R16对屎肠球菌和枯草芽孢杆菌有一定的抑制作用，但抑制幅度较低，屎肠球菌和枯草芽孢杆菌与对照相比，其细菌总数分别减少了6.92％和4.03％，且在生长24h之后，其细菌总数都能够达到108CFU·mL-1左右。与对照相比，戊糖片球菌的细菌总数无明显的变化，可见乳酸菌素R16对戊糖片球菌无影响。
(6)乳酸菌素R16的抗氧化能力
饲料中存在一定量的不饱和脂肪、脂肪酸、脂溶性维生素、类胡萝卜素及其他脂溶性物质，在饲料存放过程中极易被氧化，特别是脂肪和脂肪酸，其会被氧化酸败产生醛、醇、脂、 酸等不同化合物，这些物质可能对产生各种异味，严重影响饲料的适口性，有些化合物本身具有一定的毒性，对动物会产生不良影响。乳酸菌素R16对过氧化氢、羟自由基，DPPH·自由基和超氧阴离子的抗氧化能力如图5所示。
从图5可见，乳酸菌素R16对过氧化氢、羟自由基，DPPH·自由基和超氧阴离子都有一定的清除能力，其中对DPPH·自由基的清除的能力最强，清除率达到了79.42％，其次是羟自由基，清除率为70.13％，对超氧阴离子和过氧化氢的清除能力最差，清除率仅为10.11％和25.65％。
实施例3：乳酸菌素R16的应用
应用方法：
(A)将豆粕用刀片粉粹机进行粉粹，取4份，每份40g加入500mL发酵瓶中，编号为组5，组6，组7，组8，于105℃灭菌10min。
(B)发酵条件见表3。
表3 含乳酸菌素R16的豆粕初始发酵条件
Figure PCTCN2016098420-appb-000003
(C)采用饲料中大肠菌群的测定国标测定大肠杆菌的数量。
通过向灭菌过后的豆粕中添加大肠杆菌，研究了乳酸菌素R16对豆粕中大肠杆菌的抑制效果。结果显示，在没有添加乳酸菌素R16时，大肠杆菌的数量由发酵前1380个·100g-1增加到了发酵后4×106个·100g-1，在添加了乳酸菌素R16之后，大肠杆菌的数量由发酵前1380个·100g-1降低到了123个·100g-1，说明，乳酸菌素R16在有效降低大肠杆菌数量的同时，能够大幅度的去除豆粕中的大肠杆菌，其去除率达到了33.44％。
考虑到工业乳酸菌发酵豆粕与实验室有一定区别，本发明在原有的实验基础上，将1:0.8的料水比改为1:0.12，其他实验条件保持不变，进行了豆粕发酵实验，结果显示乳酸菌素R16对豆粕中大肠杆菌的去除率由33.44％增加到了56.64％，这说明工业条件下，乳酸菌素R16更能够充分作用于大肠杆菌。
实施例4：乳酸菌素R16的应用
乳酸菌素作为目前被唯一允许使用在食品防腐的细菌素，可以被添加到牛奶、肉制品等食品中抑制杂菌生长，延长货架期等应用。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2016098420-appb-000004

Claims (10)

  1. 一种抗菌肽，其特征在于，所述抗菌肽的氨基酸序列为如SEQ ID NO.1所示。
  2. 一种组合物，其特征在于，所述组合物含有权利要求1所述的抗菌肽。
  3. 根据权利要求2所述的组合物，其特征在于，所述组合物为饲料添加剂、饲料或者生物防腐剂。
  4. 编码权利要求1所述的抗菌肽的核苷酸片段。
  5. 一种能够表达权利要求1所述的抗菌肽的重组载体或者基因工程菌。
  6. 权利要求1所述的抗菌肽、权利要求4所述的核苷酸片段、权利要求5所述的重组载体，或者是权利要求5所述的基因工程菌的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述应用是作为饲料添加剂或者生物防腐剂。
  8. 一种抑制细菌的方法，其特征在于，所述方法是使用权利要求1所述的抗菌肽。
  9. 根据权利要求8所述的方法，其特征在于，所述细菌包括大肠杆菌、单增李斯特菌、金黄色葡萄球菌。
  10. 一种权利要求1所述的抗菌肽的制备方法，其特征在于，所述方法是将乳酸片球菌发酵得到的发酵上清液先由超滤进行粗提，然后经阴离子交换色谱柱Hitrap Q FF和凝胶过滤色谱Superdux Peptide 10/300GL分离，最后用反相柱Phenomenex Luna/C18分离纯化得到抗菌六肽。
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