WO2013071723A1 - 一种抗菌脂肽及其制备方法及在兽医药中的应用 - Google Patents

一种抗菌脂肽及其制备方法及在兽医药中的应用 Download PDF

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WO2013071723A1
WO2013071723A1 PCT/CN2012/072447 CN2012072447W WO2013071723A1 WO 2013071723 A1 WO2013071723 A1 WO 2013071723A1 CN 2012072447 W CN2012072447 W CN 2012072447W WO 2013071723 A1 WO2013071723 A1 WO 2013071723A1
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lipopeptide
antibacterial
fermentation
antibacterial lipopeptide
preparation
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PCT/CN2012/072447
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English (en)
French (fr)
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韩健宝
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南京森楠生物技术研究有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the invention relates to a preparation method of an antibacterial lipopeptide and the application thereof in a veterinary medicine, and belongs to the technical field of biomedicine. Background technique
  • Lipopeptides are mainly derived from some metabolites secreted by bacteria, yeasts, and fungi, and structurally contain hydrophilic peptide bonds and lipophilic aliphatic hydrocarbon chains.
  • the amino acid in the peptide chain can form a cyclic structure in the form of a lactone or amide bond with a hydroxyl group or an amino group of the aliphatic hydrocarbon chain.
  • the number of carbon atoms in the fatty chain portion of the lipopeptide molecule is generally between 12 and 19, and the number of amino acid residues in the peptide chain portion is between 6 and 13.
  • the lipopeptide antibiotic produced by Bacillus subtilis in the natural growth and fermentation culture is its most important antibacterial substance. Lipopeptide antibiotics include iturins, fengycins and surfactins, in which itosine and gentiana have four strong antifungal activities, while surfactants The virus, tumor, and mycoplasma all have a high inhibitory activity of 4 ⁇ .
  • the antibacterial lipopeptide belongs to one of the antibacterial peptide families, and has the characteristics of high antibacterial activity, wide antibacterial spectrum, stable structure, and harmlessness to humans, beasts and animals. Its antibacterial mechanism is different from that of general antibiotics. It forms pores on the cell membrane of microorganisms and causes the contents of the cells to flow out, which leads to bacterial death or sequestration of mono- and divalent cations, thereby inhibiting the activity of various enzymes in the process of bacterial metabolism. It is not easy to produce drug resistance. In addition, because peptides are easily decomposed in nature, they have the potential to be developed as a new, environmentally friendly biopharmaceutical.
  • the invention provides a biological preparation method of an antibacterial lipopeptide, which has high yield and low production cost.
  • the obtained antibacterial lipopeptide has good antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, and has been applied to clinical practice of veterinary medicine, and has achieved good results.
  • One object of the present invention is to provide a method for preparing an antimicrobial lipopeptide.
  • Another object of the present invention is to provide a method for purifying an antimicrobial lipopeptide.
  • the antibacterial lipopeptide prepared by the method of the present invention has achieved good effects in the field of veterinary medicine, and provides a new use of an antibacterial lipopeptide.
  • the preparation method of the antibacterial lipopeptide of the invention comprises: cultivation of seed bacteria, fermentation culture, crude extraction of antibacterial lipopeptides, and purification of antibacterial lipopeptides.
  • the preparation method of the antibacterial lipopeptide of the invention comprises: cultivation of seed bacteria, fermentation culture, crude extraction of antibacterial lipopeptide, purification of antibacterial lipopeptide, identification of antibacterial lipopeptide and evaluation of preparation results.
  • the seed bacteria in the above seed culture is a Gram-positive bacteria, preferably Bacillus subtilis, and more preferably B. subtilis Al (purchased from the General Microorganisms Collection of China).
  • the culture method of the seed bacteria is as follows: the strain is first transferred to the plate medium for cultivation; the activated strain is taken, inoculated in a liquid medium, and the seed bacteria are cultured overnight; the plate medium is LB medium, culture The temperature is 32-42 ° C, and the culture time is 12-48 h; preferably, the culture temperature is 35-40 ° C, and the culture time is 20-36 h; further preferably, the preparation method of the seed bacteria is: A1 strain is transferred to The cells were cultured on an LB plate at 37 ° C for 24 hours; the activated strain was taken with an inoculating loop, inoculated into an Erlenmeyer flask containing 100 ml of LB liquid medium, and cultured overnight as a seed fungus.
  • MnSO 4 2.0-8.0mg CuS0 4 0.1-0.2 mg distilled water 1000 ml
  • the NS-L-I medium can be replaced with a medium in which the glucose content is adjusted, lactose, sodium L-glutamate, trace elements, and the pH of the medium are adjusted.
  • the fermentation conditions are: seed inoculation amount is 1% ⁇ 15%; fermentation temperature is 20 °C ⁇ 37 °C, fermentation time is 24 ⁇ 72h, fermentation speed is 50 ⁇ 300 r / min, fermentation initial pH is pH 5.0 ⁇ 9.0;
  • the inoculum is 8%
  • the fermentation temperature is 29 ° C
  • the fermentation time is 48 hours
  • the fermentation speed is 150 r/min
  • the initial fermentation pH is pH 7.0.
  • the crude extraction process of the above antibacterial lipopeptide is as follows: the fermentation broth is centrifuged under acidic conditions to remove the bacteria; the supernatant is adjusted with an acidic solution to adjust the desired pH, centrifuged, and the precipitate is collected and dried to obtain an antibacterial lipopeptide. Extract.
  • the acidic condition is pH 2.0 ⁇ 5.0; the acidic solution is hydrochloric acid solution; the centrifugal speed is
  • the crude extraction process of the antibacterial lipopeptide is as follows: the prepared fermentation broth is centrifuged at 7000 rpm for 10 min at pH 3.0 to remove the cells, and the supernatant is adjusted to a desired pH with 6 mol/L HCL, and then centrifuged at 7000 rpm. lOmin, collect the precipitate. The precipitate was dried in an oven at 70 ° C for 20 hours to obtain a crude extract of the antibacterial lipopeptide.
  • the purification process of the above antibacterial lipopeptide adopts the acid-alcohol method, and the specific steps are as follows: taking the crude extract of the un-baked antibacterial lipopeptide, adding the alcohol substance, adjusting the pH near neutral with an alkaline solution, and then extracting the alcohol with the alcohol substance The extract is purified by a glucan gel column and concentrated to obtain a purified lipopeptide.
  • the specific steps of the purification process of the preferred antibacterial lipopeptide are as follows: taking the unbaked crude antibacterial lipopeptide extract, plus After adding the alcohol, adjust the pH to 5.0 ⁇ 8.0 with NaOH, extract 1-3 times with alcohol and combine the extract, then pass it through Sephadex LH-20 column, and finally use centrifugal concentrator Concentration to complete drying yields a purified antibacterial lipopeptide.
  • the preparation method of the above antibacterial lipopeptide is evaluated by drying the obtained antibacterial peptide in an oven at 70 ° C, and the yield is required to be 6.0 to 7.5 g/L; preferably 6.8 g/L.
  • the animal pathogens are selected and cultured until the logarithmic phase, and the bacterial suspension is prepared separately, and the viable cell count is greater than
  • the solid agar solid medium is melted and poured into the culture medium. After cooling and solidification, the three sterilized Oxford cups are placed on the solid medium plane equidistantly, and the LB semi-solid medium of the bacteria is separately cultured. Melt, cool to 40 °C ⁇ 50 °C, transfer 1ml of the bacterial suspension to the medium, mix, and then pipet 8ml containing culture medium onto the agar plate. After solidification, the Oxford Cup was pulled out, and 30 ⁇ l of pure antibacterial lipopeptides of different concentrations were added to the oxholes with a pipette, and physiological saline was used as a control.
  • the present invention provides an antibacterial lipopeptide which is mainly prepared by the following method:
  • Step 1 cultivating the seed fungus
  • Step 2 fermenting the seed bacteria to prepare a fermentation broth
  • Step 3 centrifuge the fermentation broth at 10 ° C or below and remove the bacterial extract to prepare a crude extract of the antibacterial lipopeptide;
  • Step 4 Purify the crude extract of the antimicrobial lipopeptide.
  • the seed fungus in the above step 1 is a Gram-positive bacteria, preferably Bacillus subtilis, more preferably B. subtilis Al strain (purchased from the General Microbial Culture Collection of China);
  • the culture method of the seed bacteria is: the strain is first transferred to the plate medium for cultivation, and the activated strain is taken and inoculated into the liquid medium;
  • the culture method of the seed bacteria is: the strain is first transferred to the plate medium for cultivation, the activated strain is taken, inoculated in a liquid medium, and cultured for 18-36 hours to obtain a seed fungus;
  • the plate medium is LB medium
  • the culture temperature is 32-42 ° C
  • the culture time is 12-48 h; preferably, the culture temperature is 35-40 ° C, and the culture time is 20-36 h;
  • the method for preparing the seed fungus in the step 1 is further as follows: B.
  • the strain of the A1 strain is transferred to
  • the cells were cultured on an LB plate at 37 ° C for 24 hours; the activated strain was taken with an inoculating loop, inoculated in a container containing 100 ml of LB liquid medium, and cultured for 20 to 48 hours to obtain a seed fungus.
  • NS-LI medium can choose various components such as glucose, lactose, sodium L-glutamate and pH. One or more of the various factors in the value are adjusted.
  • the fermentation conditions in the step 2 are: the inoculum of the seed bacteria is 1% to 15%; the fermentation temperature is 20 ° C ⁇ 37 ° C, the fermentation time is 24 ⁇ 72 h, the fermentation speed is 50 ⁇ 300 r / min, the initial pH of the fermentation The pH is 5.0 ⁇ 9.0; the fermentation conditions in the step 2 are preferably: seed inoculation amount 5-13%, fermentation temperature 25-33 ° C, fermentation time 36-60 hours, fermentation speed 100-200 r/min, The initial pH of the fermentation is pH 6.0-8.0;
  • the fermentation conditions in the step 2 are further preferably: seed inoculum 8%, fermentation temperature 29 ° C, fermentation time 48 hours, fermentation speed 150 r/min, and initial fermentation pH pH 7.0.
  • the supernatant is preferably centrifuged 1-5 times, and the precipitate is collected after centrifugation, and dried to obtain a crude extract of the antibacterial lipopeptide; wherein the centrifugation method is preferably: the supernatant is centrifuged under acidic conditions. Further, in the step 3, it is further preferred that: the prepared fermentation broth is centrifuged at a temperature of 0-5 ° C and a pH of 2.0 to 5.0 to remove the cells, and the supernatant is adjusted to pH 2.0 with an acidic solution. 5.0, centrifugation, collecting the precipitate, drying the precipitate, which is a crude extract of the antibacterial lipopeptide;
  • the acidic solution in the step 3 is a hydrochloric acid solution; the centrifugal speed is 6000-8000 rpm, the centrifugation time is 5-15 min; the drying temperature is 60-80 ° C, and the drying is 10-30 h.
  • the purification process of the above antibacterial lipopeptide adopts an acid-alcohol method; the specific steps are: taking a crude extract of the antibacterial lipopeptide, adding an alcohol (such as decyl alcohol, ethanol, etc.) and adjusting the pH with an alkaline solution; .0 ⁇ 8.0, and then extracted with alcohol, the extract is obtained, the extract is purified, and concentrated, and the purified lipopeptide is obtained.
  • an alcohol such as decyl alcohol, ethanol, etc.
  • the step of purifying the preferred antibacterial lipopeptide in the step 4 is as follows: taking the crude extract of the antibacterial lipopeptide, adding the alcohol substance, adjusting the pH to 7.0 with NaOH, extracting 1-3 times with the alcohol substance and pumping it The extracts were combined, and then passed through a Sephadex LH-20 column, and finally concentrated to a complete dryness by a centrifugal concentrator to obtain a purified lipopeptide.
  • the identification process of the antibacterial lipopeptide is: taking the purified lipopeptide to be dissolved in an appropriate amount of alcohol (mainly sterol, ethanol, etc.), and then taking a certain amount of the antibacterial lipopeptide solution and spotting it on the silica gel plate multiple times. After the sample is air-dried, it is saturated in a chromatography cylinder containing a developing agent for 30 minutes.
  • the preparation method of the above antibacterial lipopeptide is evaluated by drying the obtained antibacterial peptide in an oven at 70 ° C, and the yield is required to be 6.0 to 7.5 g/L; preferably 6.8 g/L.
  • the animal pathogenic bacteria are selected and cultured until the logarithmic phase, and the bacterial suspension is prepared separately, and the viable cell count is greater than 10 6 CFU/ml, and is reserved.
  • the solid agar solid medium is melted and poured into the culture medium. After cooling and solidification, the three sterilized Oxford cups are placed on the solid medium plane equidistantly, and the LB semi-solid medium of the bacteria is separately cultured. Melt, cool to 40 °C ⁇ 50 °C, transfer 1ml of the bacterial suspension to the medium, mix, and then pipet 8ml containing culture medium onto the agar plate.
  • the Oxford Cup was pulled out, and 30 ⁇ l of pure antibacterial lipopeptides of different concentrations were added to the oxholes with a pipette, and physiological saline was used as a control. Each treatment was repeated 3 times, and finally the bacteria were placed in a 37 ° C incubator for 24 hours. After the completion of the culture, the diameter of the culture sub-measure bacteriostatic sputum was taken out.
  • the bacteriostatic diameter is greater than 20mm for high sensitivity, less than 20mm for greater than 14mm for medium sensitivity, and less than 14mm for insensitivity.
  • Bacillus subtilis (B. subtilis Al strain) was prepared.
  • the preparation method of the seed bacteria was as follows: B. TM / ⁇ ⁇ 1 strain was transferred to LB plate and cultured at 37 ° C for 24 h; , inoculated in an Erlenmeyer flask containing 100 ml of LB liquid medium, and cultured overnight as a seed fungus.
  • the seed culture was used for fermentation culture.
  • the medium was: NS-LI medium (glucose 20.0 g, sodium L-glutamate 5.0 g, MgS0 4 0.5 g, KCl 0.5 g, KH 2 P0 4 1 g, FeSO 4 0.15 mg , MnSO 4 5.0 mg, CuSO 4 0.16 mg, distilled water 1 000 ml, pH 7.0).
  • the fermentation conditions were as follows: seed inoculum 8%, fermentation temperature 29 ° C, fermentation time 48 hours, fermentation speed 150 r / min, initial fermentation pH 7.0.
  • the crude extraction process of the above antibacterial lipopeptide is as follows: The prepared fermentation broth is taken at 4 ° C and pH 4
  • the crude antibacterial lipopeptide is added to the alcohol, and the pH is adjusted to 7.0 with NaOH, and then the alcohol is used.
  • the material was extracted twice and the extracts were combined, and then passed through a Sephadex LH-20 column, and finally concentrated to a complete dryness by a centrifugal concentrator to obtain a purified lipopeptide.
  • the purified antibacterial lipopeptide of Example 1 was dissolved in a small amount of alcohol, and a certain amount of the antibacterial lipopeptide solution was spotted on a silica gel plate, and after being air-dried, it was placed in a developing agent.
  • the purified antibacterial lipopeptide obtained by drying in an oven at 70 ° C requires a yield of more than 6.8 g / liter.
  • Example 1 The antibacterial effect evaluation method of the purified antibacterial lipopeptide was as follows:
  • the 10 animal pathogens shown in Table 1 were selected and cultured until the logarithmic phase, and the bacterial suspension was prepared separately, and the viable cell count was greater than 10 6 CFU/ml, and was used.
  • the solid agar solid medium is melted and poured into the culture medium. After cooling and solidification, the three sterilized Oxford cups are placed on the solid medium plane equidistantly, and the LB semi-solid medium of the bacteria is separately cultured. Melt, cool to 40 ° C ⁇ 50 ° C, remove 1 ml of the bacterial suspension into the medium, mix, and then pipet 8 ml of the culture medium to the agar plate.
  • Gram Staphylococcus aureus 20 High-sensitivity Pneumococci 18 Neutral positive
  • test pigs were 28-day-old healthy weaned piglets with a total of 198 heads weighing 7.52 ⁇ 0.65 kg. Test is divided into
  • the strains that replicate the bacterial diseases of pigs are the preserved strains of the center, and the virulence of the animals is restored to prepare a certain concentration of the bacterial suspension.
  • the five bacteria are: Haemophilus parasuis, porcine pleuropneumonia Bacillus, Streptococcus suis, Pasteurella multocida and Porcine Escherichia coli, 5 kinds of bacterial pig diseases were replicated by inoculation of these 5 kinds of bacteria in each infected group by the corresponding inoculation route and the total number of bacteria, and the negative control group Piglets are not inoculated with bacteria and only provide clean drinking water. Daily observations recorded activity and mortality in weaned piglets.
  • the dosage form and dosage Every 400-500 g of pure antibacterial lipopeptide (prepared in Example 7) was uniformly mixed into the 1 p ⁇ basic diet, fed ad libitum, and fed continuously. 5 ⁇ , no antibacterial substances are added to the drinking water. The basal diet and drinking water without any antibacterial substances were fed during the other test periods. 10 days ⁇ , 9 pigs were randomly selected from each group, and the inoculum was separated and detected by PCR. The results are shown in Table 2.
  • the antibacterial lipopeptide prepared according to the method of Example 1 was repeated for the treatment of five bacterial swine diseases of Haemophilus parasuis, Actinobacillus pleuropneumoniae, Streptococcus suis, Mycobacterium septicum and Porcine Escherichia coli. The effect was the same as in Example 7 (Table 2).
  • the test chicken was a 15-day-old healthy white feather broiler with a total of 700 birds.
  • the trials were divided into 4 large groups, of which 3 groups were infected groups, and 3 kinds of bacterial chicken diseases were inoculated separately.
  • Each group was established with drug combination group and positive control group, and the other group was negative control group without infection.
  • a total of 7 groups 100 birds / group, each group of male and female half, each group is kept in isolation.
  • the strains that replicate chicken bacterial diseases are the preserved strains of the center, and the animal is inoculated with the animal to restore its virulence, and a certain concentration of the bacterial suspension is prepared.
  • the three bacteria are: Salmonella, Escherichia coli and Pasteurella. Through the corresponding inoculation routes and the total number of bacteria, three bacterial bacteria were replicated in each of the three infected bacteria, and the negative control chickens were supplied with clean drinking water.
  • Example 1 Purified antibacterial lipopeptide (Example 1) was mixed with water at a dose of 50g/100kg, and free drinking water was used. Continuous drinking water 7 The grain does not contain any antibacterial substances, and other drinking water and basal diets without any antibacterial substances are fed during the other test period. After 10, 20 chickens were randomly selected from each group, and the inoculated bacteria were separated and detected by PCR. The results are shown in Table 3.
  • Example 7 The antibacterial lipopeptide prepared according to the method of Example 1 was repeated for the same test as in Example 7 (Table 3) in the treatment of Salmonella, Escherichia coli and Pasteurella bacterial chicken diseases.
  • Bacillus subtilis (B. subtilis A1 strain) was prepared, and the preparation method of the seed fungus was: B.
  • the strain of TM / ⁇ ⁇ 1 was transferred to LB plate and cultured at 37 ° C for 24 hours; the activated strain was taken with an inoculating loop, and seeded in an Erlenmeyer flask containing 100 ml of LB liquid medium, and cultured overnight as a seed fungus.
  • the seed culture was used for fermentation culture.
  • the medium was: NS-L-I medium (glucose 20.0 g, L-glutamate sodium 5.0 g, MgS0 4 0.5 g, KC1 0.5 g, KH 2 P0 4 1 g, FeS0 4 0.15mg, MnSO 4 5.0 Mg, CuSO 4 0.16 mg, distilled water 1 000 ml, pH 7.0).
  • the fermentation conditions were as follows: seed inoculum 8%, fermentation temperature 29 ° C, fermentation time 48 hours, fermentation speed 150 r / min, initial fermentation pH 7.0.
  • the crude extraction process of the above antibacterial lipopeptide is as follows: the prepared fermentation broth is centrifuged at 7000 rpm for 10 min at 3 ° C and pH 4.5 to remove the cells, and the supernatant is adjusted to pH 4.0 with 6 mol/L HCL. After centrifugation at 7000 rpm for 1 Omin, the precipitate was collected, and the precipitate was dried in a 70 V oven for 20 h to obtain a crude extract of the antibacterial lipopeptide.
  • Bacillus subtilis (B. subtilis Al strain) was prepared.
  • the preparation method of the seed bacteria was as follows: B. subtilis Al strain was transferred to LB plate and cultured at 37 ° C for 24 h; activated strain was inoculated with inoculation loop, inoculated The flask was cultured overnight in an Erlenmeyer flask containing 100 ml of LB liquid medium.
  • the seed culture is used for fermentation culture.
  • the medium includes NS-L-I medium (glucose 20.0 g, L-glutamic acid sodium 5.0 g, MgS04 0.5 g, KCl 0.5 g, KH2P04 1 g, FeS04 0.15 mg, MnS04 5.0 mg , CuS04 0.16 mg, distilled water 1 000 ml, pH 7.0.).
  • the fermentation conditions were as follows: seed vaccination amount 8%, fermentation temperature 29 ° C, fermentation time 48 hours, fermentation speed 150 r / min, initial fermentation pH pH 7.0.
  • the crude extraction process of the above antibacterial lipopeptide is as follows: The prepared fermentation broth is subjected to pH 2.0 to 5.0.
  • the cells were removed, and the supernatant was adjusted to a desired pH with 6 mol/L HCL, and then centrifuged at 7000 rpm for 10 min to collect a precipitate.
  • the precipitate was dried in an oven at 70 ° C for 20 hours to obtain a crude extract of the antibacterial lipopeptide.
  • the crude antibacterial lipopeptide is purified by the acid-alcohol method.
  • the specific steps are as follows: taking the crude extract of the antibacterial lipopeptide which is not dried, adding the alcohol substance, adjusting the pH to 5.0-8.0 with NaOH, and then using the alcohol substance
  • the extract was extracted twice and the extracts were combined, and then passed through a Sephadex LH-20 column, and finally concentrated to a complete dryness by a centrifugal concentrator to obtain a purified lipopeptide.
  • the purified antibacterial lipopeptide obtained by drying in an oven at 70 ° C requires a yield of more than 6.8 g / liter.
  • the 10 animal pathogens in the following table were selected and cultured until the logarithmic phase, and the bacterial suspension was prepared separately, and the viable cell count was greater than 106 CFU/ml, and was used.
  • the solid agar solid medium is melted and poured into the culture medium. After cooling and solidification, the three sterilized Oxford cups are placed on the solid medium plane equidistantly, and the LB semi-solid medium of the bacteria is separately cultured. Melt, cool to 40V ⁇ 50 °C, transfer 1ml of the bacterial suspension to the medium, mix, and then pipet 8ml containing medium to the agar plate.

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Abstract

提供了一种抗菌脂肽的制备方法及其在兽医药中的应用，通过抗菌脂肽制备工艺的优化，抗菌脂肽产量达到6.8g/L。获得的抗菌脂肽对革兰氏阳性菌如葡萄球菌和链球菌效果更佳。在兽医临床试验中，发现其对副嗜血杆菌、胸膜肺炎放线杆菌、链球菌、多杀性巴氏杆菌、肺炎双球菌等猪病原菌，以及沙门氏菌、大肠杆菌、巴氏杆菌等鸡病原菌均有良好的抗菌效果。

Description

一种抗菌脂肽及其制备方法及在兽医药中的应用 技术领域
本发明涉及一种抗菌脂肽的制备方法及其在兽医药中的应用，属于生物医 药技术领域。 背景技术
近年来，研究人员发现某些细菌分泌的产物也具有抗菌活性， 目前研究较 多的主要有两种（阳离子抗菌肽和脂肽）。 脂肽主要来源于一些由细菌、 酵母 菌、 真菌分泌的代谢产物， 结构上含亲水的肽键和亲油的脂肪烃链两部分。 其 中肽链上的氨基酸能以内酯或酰胺键形式与脂肪烃链的 ½、羟基或氨基结合 形成环状结构。 脂肽分子中脂肪链部分的碳原子数一般在 12 ~ 19之间， 肽链 部分的氨基酸残基数在 6 ~ 13之间。枯草芽孢杆菌在自然生长和发酵培养后期 产生的脂肽类抗生素是其最重要的抗菌物质。 脂肽类抗生素包括伊枯草菌素 ( iturins ) 、 泛革素 ( fengycins )和表面活性素 ( surfactin ) , 其中伊枯草菌素 和泛革素具有 4艮强的抗真菌活性， 而表面活性素对病毒、 肿瘤、 支原体都有 4艮 高的抑制活性。
抗菌脂肽属于抗菌肽家族中的一种， 具有抗菌活性高、 抗菌谱广、 结构稳 定， 对人、 兽、 畜无害等特性。 其抗菌机理和一般抗生素有所不同， 它是在微 生物的细胞膜上形成孔洞而使细胞内容物流出从而导致细菌死亡或螯合一、二 价阳离子从而抑制了细菌代谢过程中多种酶类活性， 不易产生耐药性。 此外， 由于是肽类物质在自然界中容易分解，极具潜力开发为一种新型、环境友好的 生物药物。
迄今已有近千种天然抗菌肽被分离出来，但是天然抗菌肽的分离纯化过程 烦瑣且费用较高， 另外多数抗菌肽的活性较弱难以达到应用的水平，有些抗菌 肽还具有副作用。 当前制备抗菌脂肽的方法主要有生物材料提取法，化学合成 法和基因工程表达等方法。天然抗菌脂肽在生物材料中含量低，分离也较烦瑣， 采用这种方法获得的样品难以满足应用需求。化学合成虽可以完成，但成本较 高。 因此， 研发新型、 广谱、 易于大量制备与纯化而又有较高活性的抗菌脂肽 将有利于降低成本， 并为开发代替抗生素的抗菌药物提供更大的发挥空间。 本发明提供了一种抗菌脂肽的生物制备方法， 产量高， 生产成本低。 所得 抗菌脂肽对革兰氏阳性菌和革兰氏阴性菌均有较好的抗菌活性，应用于兽医药 临床实践中， 取得了良好的效果。 发明内容 本发明的目的之一是提供一种抗菌脂肽的制备方法。
本发明的另外一种目的是提供一种抗菌脂肽的纯化方法。
本发明的目的还在于提供一种抗菌脂肽。
本发明方法制备的抗菌脂肽应用于兽医药领域取得了良好的效果，提供了 一种抗菌脂肽的新用途。
本发明所述的抗菌脂肽制备方法为： 种子菌的培养、 发酵培养、 抗菌脂肽 的粗提、 抗菌脂肽的纯化。
本发明所述的抗菌脂肽制备方法为： 种子菌的培养、 发酵培养、 抗菌脂肽 的粗提、 抗菌脂肽的纯化、 抗菌脂肽的鉴定与制备结果评价。
上述种子菌培养中的种子菌为革兰氏阳性菌，优选为枯草芽孢杆菌， 更优 选枯草芽孢杆菌 （B. subtilis Al株， 购于中国普通微生物菌种保藏中心） 。
其中， 种子菌的培养方法为： 菌种先转接至平板培养基上培养； 取活化菌 种，接种于液体培养基中，培养过夜得种子菌； 所述平板培养基为 LB培养基， 培养温度为 32-42 °C , 培养时间为 12-48h; 优选的， 培养温度为 35-40 °C , 培 养时间为 20-36h; 进一步优选种子菌的制备方法为： A1株菌种转接 至 LB平板上， 37°C培养 24h; 用接种环取活化菌种， 接种于含有 100ml LB 液体培养基的锥形瓶中， 培养过夜作为种子菌。
上述的发酵培养中的培养基包括 NS-L-I培养基， 所述培养基为 pH= 6.5-7.5 条件下配方为：
葡萄糖 10.0-30.0 g L-谷氨酸钠 2.0-8.0g MgS04 0.2-0.8 g KC1 0.2-0.8 g KH2PO 20-60 g FeS04 0.10-0.20mg
MnSO42.0-8.0mg CuS04 0.1-0.2 mg 蒸馏水 1000 ml 优选为： 葡萄糖 15.0-25.0 g L-谷氨酸钠 4.0-6.0g MgSO.
0.4-0.6 g
KC1 0.4-0.6 g KH2PO 35-45 g FeS04 0.12-0.18mg MnSO44.0-6.0mg CuS04 0.12-0.18
pH 6.8-7.2;
进一步优选为：葡萄糖 20.0 g L-谷氨酸钠 5.0g MgS04 0.5 g KC1 0.5
KH2P04 1 g FeSO4 0.15mg MnS04 5.0 mg CuS04 0.16 mg 蒸馏 水 1 000 ml
pH 7.0 o
其中， NS-L-I培养基可用其中葡萄糖含量调整， 乳糖， L-谷氨酸钠， 微量 元素等及培养基 pH值进行调整后的培养基取代。
其中， 发酵条件为： 种子菌接种量为 1%~15%； 发酵温度为 20°C ~ 37°C , 发酵时间 24 ~ 72h, 发酵转速 50 ~ 300 r/min, 发酵初始 pH为 pH 5.0~9.0；
进一步优选为： 种子菌接种量 8%, 发酵温度 29°C , 发酵时间 48小时， 发 酵转速 150 r/min, 发酵初始 pH为 pH 7.0。
上述抗菌脂肽的粗提过程为： 取发酵菌液在酸性条件下离心， 去除菌体； 取上清液用酸性溶液分别调节所需 pH, 离心， 收集沉淀， 干燥， 即得抗菌脂 肽粗提物。
所述酸性条件为 pH2.0~5.0; 酸性溶液为盐酸溶液； 离心转速为
6000-8000rpm, 离心时间为 5-15min; 干燥温度 60-80°C , 干燥 10-30h。
进一步优选抗菌脂肽的粗提过程为：取制备的发酵菌液在 pH3.0条件下经 7000rpm离心 lOmin, 去除菌体， 取上清用 6mol/L HCL分别调节所需 pH, 再 以 7000rpm离心 lOmin, 收集沉淀。 将沉淀在 70°C的烘箱中干燥 20h, 即得抗 菌脂肽粗提物。
上述抗菌脂肽的纯化过程采用酸-醇法， 具体步骤为： 取未烘干的抗菌脂 肽粗提物， 加入醇类物质后用碱性溶液调节 pH近中性， 再用醇类物质抽提， 抽提液过葡聚糖凝胶柱纯化， 浓缩， 即得抗菌脂肽的纯化物。
优选的抗菌脂肽的纯化过程具体步骤为： 取未烘干的抗菌脂肽粗提物，加 入醇类物质后用 NaOH调节 pH至 5.0~8.0,再用醇类物质抽提 1-3次并将其抽 提液合并， 然后将其过 Sephadex LH-20层析柱， 最后用离心浓缩仪浓缩至完 全干燥， 即得抗菌脂肽的纯化物。
上述抗菌脂肽的鉴定过程为： 采用薄层色谱法，展开剂的组成为氯仿： 曱 醇：水 = 60~70:20~30: 1~8 (体积比） ， 展开前用展开剂预饱和， 展开后用茚 三酮-丙酮溶液喷雾显色。
优选抗菌脂肽的鉴定过程为： 取抗菌脂肽纯化物溶解在少量醇类中，再取 一定量的该抗菌脂肽溶液分多次点样于硅胶板上，待点样点风干后，放入盛有 展开剂的层析缸中饱和 30min, 展开剂的组成为氯仿： 曱醇：水 = 65:25:4 (体 积比） ， 然后， 待展开剂到达距硅胶板上层边缘 2cm左右时取出， 待有机溶 剂充分挥发后， 再采用含有 0.5 %茚三酮的丙酮溶液喷雾显色。
上述抗菌脂肽的的制备结果评价方法为： 70°C的烘箱中干燥所得纯化的抗 菌脂肽， 产量需为 6.0~7.5g/L; 优选为 6.8 g/L。
上述提纯的抗菌脂肽的抗菌效果评价方法如下：
选用动物病原菌， 将其培养至对数期后， 分别制备成菌悬液， 活菌数大于
106CFU/ml, 备用。 将素琼脂固体培养基融化后倒入培养亚， 待冷却凝固后， 等距平稳地将 3个已经灭菌的牛津杯放在固体培养基平面上，再将分别培养细 菌的 LB半固体培养基融化，冷却至 40 °C ~ 50 °C ,移取 1ml菌悬液到培养基中， 混匀， 然后用移液管移取 8ml含菌培养基至素琼脂平板上。 待凝固后， 拔出牛 津杯， 用移液枪取 30μ1不同浓度的纯抗菌脂肽加入牛津孔内， 以生理盐水作 对照。 每个处理重复 3次， 最后细菌放置于 37°C培养箱培养 24 h。 培养完成 后， 取出培养亚测量抑菌圏的直径大小。 抑菌圏直径大于 20mm为高敏， 小于 20 mm大于 14 mm为中敏， 小于 14 mm为不敏感。 本发明提供一种抗菌脂肽， 主要由下述方法制备：
步骤 1 : 培养种子菌；
步骤 2: 取种子菌发酵培养， 制备发酵菌液；
步骤 3: 将发酵菌液在 10°C以下及酸性条件下离心，去除菌体制备抗菌脂 肽粗提物； 步骤 4: 将抗菌脂肽粗提物纯化。
上述步骤 1中的种子菌为革兰氏阳性菌， 优选为枯草芽孢杆菌， 更优选枯 草芽孢杆菌 （B. subtilis Al株， 购于中国普通微生物菌种保藏中心） ；
其中步骤 1中， 种子菌的培养方法为： 菌种先转接至平板培养基上培养， 取活化菌种， 接种于液体培养基中；
其中步骤 1中， 种子菌的培养方法为： 菌种先转接至平板培养基上培养， 取活化菌种， 接种于液体培养基中， 培养 18-36小时， 得种子菌；
所述步骤 1中平板培养基为 LB培养基， 培养温度为 32-42°C , 培养时间为 12-48h; 优选的， 培养温度为 35-40 °C , 培养时间为 20-36h;
所述步骤 1中进一步优选种子菌的制备方法为： B. A1株菌种转接至
LB平板上， 37°C培养 24h; 用接种环取活化菌种， 接种于含有 100ml LB液体培 养基的容器中， 培养 20-48小时， 得种子菌。
所述步骤 2中，发酵培养中的培养基包括 NS-L-I培养基，所述培养基为 pH= 6.5-7.5条件下按如下比例关系调配：
葡萄糖 10.0-30.0 g L-谷氨酸钠 2.0-8.0g MgS04 0.2-0.8 g
KC1 0.2-0.8 g KH2PO 20-60 g FeS04 0.10-0.20mg
MnS04 2.0-8.0mg CuS04 0.1-0.2 mg 蒸馏水 1000 ml
pH 6.5-7.5;
优选为：葡萄糖 15.0-25.0 g L-谷氨酸钠 4.0-6.0g MgS04 0.4-0.6 g
KC1 0.4-0.6 g KH2PO 35-45 g FeS04 0.12-0.18mg
MnSO44.0-6.0mg CuS04 0.12-0.18 mg 蒸馏水 1000 ml pH 6.8-7.2;
进一步优选为：葡萄糖 20.0 g L-谷氨酸钠 5.0g MgS04 0.5 g KC1 0.5 g
KH2P04 1 g FeSO4 0.15mg MnS04 5.0 mg CuS04 0.16 mg 蒸馏 水 1 000 ml
pH 7.0 o
其中， NS-L-I培养基可选择其中葡萄糖、 乳糖， L-谷氨酸钠等各组分及 pH 值中各个因素的一个或多个进行调整。
所述步骤 2中的发酵条件为：种子菌接种量为 1%~15%；发酵温度为 20°C ~ 37 °C , 发酵时间 24 ~ 72h, 发酵转速 50 ~ 300 r/min, 发酵初始 pH为 pH 5.0~9.0； 所述步骤 2中的发酵条件优选为： 种子菌接种量 5-13%,发酵温度 25-33°C , 发酵时间 36-60小时， 发酵转速 100-200 r/min, 发酵初始 pH为 pH 6.0-8.0;
所述步骤 2中的发酵条件进一步优选为：种子菌接种量 8%,发酵温度 29°C , 发酵时间 48小时， 发酵转速 150 r/min, 发酵初始 pH为 pH 7.0。
所述步骤 3中，去除菌体后，取上清液优选离心 1-5次， 离心后收集沉淀， 干燥， 得抗菌脂肽粗提物； 其中离心方法优选： 上清液在酸性条件下离心； 所述步骤 3中， 进一步优选为： 取制备的发酵菌液在温度为 0-5°C、 pH2.0~5.0条件下离心， 去除菌体， 取上清用酸性溶液调节 pH2.0~5.0, 离心， 收集沉淀， 将沉淀干燥， 即为抗菌脂肽粗提物；
所述步骤 3中酸性溶液为盐酸溶液； 离心转速为 6000-8000rpm, 离心时 间为 5-15min; 干燥温度 60-80 °C , 干燥 10-30h。
所述步骤 4中： 上述抗菌脂肽的纯化过程采用酸-醇法； 具体步骤为： 取 抗菌脂肽粗提物， 加入醇类 （如曱醇、 乙醇等）物质后用碱性溶液调节 pH5.0~8.0, 再用醇类物质抽提， 得抽提液， 抽提液纯化， 浓缩， 即得抗菌脂 肽的纯化物。
所述步骤 4优选的抗菌脂肽的纯化过程步骤为： 取抗菌脂肽粗提物，加入 醇类物质后用 NaOH调节 pH至 7.0, 再用醇类物质抽提 1-3次并将其抽提液 合并， 然后将其过 Sephadex LH-20层析柱， 最后用离心浓缩仪浓缩至完全干 燥， 即得抗菌脂肽的纯化物。
上述抗菌脂肽还可以包括进一步的鉴定过程， 鉴定方法包括如下步骤： 采用薄层色谱法，展开剂的组成为氯仿： 曱醇：水 = 60~70:20~30:1~8 (体 积比） ， 展开前用展开剂预饱和， 展开后用茚三酮-丙酮溶液喷雾显色。
优选抗菌脂肽的鉴定过程为： 取抗菌脂肽纯化物溶解在适量醇类（主指曱 醇、 乙醇等）中， 再取一定量的该抗菌脂肽溶液分多次点样于硅胶板上， 待点 样点风干后， 放入盛有展开剂的层析缸中饱和 30min, 展开剂的组成为氯仿： 曱醇：水 = 65:25:4 (体积比） ， 然后， 待展开剂到达距硅胶板上层边缘 2cm 左右时取出， 待有机溶剂充分挥发后， 再采用含有 0.5 %茚三酮的丙酮溶液喷 雾显色。
上述抗菌脂肽的的制备结果评价方法为： 70°C的烘箱中干燥所得纯化的抗 菌脂肽， 产量需为 6.0~7.5g/L; 优选为 6.8 g/L。
上述提纯的抗菌脂肽的抗菌效果评价方法如下：
选用动物病原菌， 将其培养至对数期后， 分别制备成菌悬液， 活菌数大于 106CFU/ml, 备用。 将素琼脂固体培养基融化后倒入培养亚， 待冷却凝固后， 等距平稳地将 3个已经灭菌的牛津杯放在固体培养基平面上，再将分别培养细 菌的 LB半固体培养基融化，冷却至 40 °C ~ 50 °C ,移取 1ml菌悬液到培养基中， 混匀， 然后用移液管移取 8ml含菌培养基至素琼脂平板上。 待凝固后， 拔出牛 津杯， 用移液枪取 30μ1不同浓度的纯抗菌脂肽加入牛津孔内， 以生理盐水作 对照。每个处理重复 3次，最后细菌放置于 37°C培养箱培养 24h。培养完成后， 取出培养亚测量抑菌圏的直径大小。 抑菌圏直径大于 20mm为高敏， 小于 20 mm大于 14 mm为中敏， 小于 14 mm为不敏感。
具体实施方式
实施例 1 抗菌脂肽的制备方法
取枯草芽孢杆菌 （B. subtilis Al株）进行制备， 种子菌的制备方法为： B. ™ /ώ Α1株菌种转接至 LB平板上， 37°C培养 24h; 用接种环取活化菌种， 接 种于含有 100ml LB液体培养基的锥形瓶中， 培养过夜作为种子菌。
取种子菌进行发酵培养， 培养基为： NS-L-I培养基（葡萄糖 20.0 g, L-谷 氨酸钠 5.0g, MgS04 0.5 g, KCl 0.5 g, KH2P04 1 g, FeSO4 0.15mg, MnSO45.0 mg, CuSO4 0.16 mg, 蒸馏水 1 000 ml, pH 7.0 ) 。 发酵条件为： 种子菌接种量 8%, 发酵温度 29°C , 发酵时间 48小时， 发酵转速 150 r/min, 发酵初始 pH为 pH 7.0。
上述抗菌脂肽的粗提过程为： 取制备的发酵菌液在 4°C及 pH4条件下经
7000rpm离心 lOmin, 去除菌体， 取上清液用 6mol/L HCL调节 pH4.0, 再以 7000rpm离心 1 Omin , 收集沉淀， 将沉淀在 70 V的烘箱中干燥 20h , 即得抗菌 脂肽粗提物。
将抗菌脂肽粗提物， 加入醇类物质后用 NaOH调节 pH至 7.0, 再用醇类 物质抽提 2次并将其抽提液合并， 然后将其过 Sephadex LH-20层析柱， 最后 用离心浓缩仪浓缩至完全干燥， 即得抗菌脂肽的纯化物。
实施例 2将纯化后的抗菌脂肽进行鉴定
取实施例 1抗菌脂肽纯化物溶解在少量醇类中，再取一定量的该抗菌脂肽 溶液分多次点样于硅胶板上，待点样点风干后，放入盛有展开剂的层析缸中饱 和 30min, 展开剂的组成为氯仿： 曱醇：水 = 65:25:4 (体积比） ， 然后， 待展 开剂到达距硅胶板上层边缘 2cm左右时取出， 待有机溶剂充分挥发后， 再采 用含有 0.5 %茚三酮的丙酮溶液喷雾显色。
抗菌脂肽的制备结果评价： 70°C的烘箱中干燥所得纯化的抗菌脂肽，产量 需大于 6.8克 /升。
实施例 3 抗菌效果评价
实施例 1提纯的抗菌脂肽的抗菌效果评价方法如下：
选用如表 1所示的 10种动物病原菌， 将其培养至对数期后， 分别制备成 菌悬液， 活菌数大于 106CFU/ml, 备用。 将素琼脂固体培养基融化后倒入培养 亚，待冷却凝固后， 等距平稳地将 3个已经灭菌的牛津杯放在固体培养基平面 上， 再将分别培养细菌的 LB半固体培养基融化， 冷却至 40°C ~ 50°C , 移取 lml菌悬液到培养基中， 混匀， 然后用移液管移取 8ml含菌培养基至素琼脂平 板上。 待凝固后， 拔出牛津杯， 用移液枪取 30μ1不同浓度的实施例 1纯抗菌 脂肽加入牛津孔内， 以生理盐水作对照。 每个处理重复 3次， 最后细菌放置于 37°C培养箱培养 24 h。 培养完成后， 取出培养亚测量抑菌圏的直径大小。 结 果见表 1。
纯抗菌脂肽的抗菌谱 抑菌圈直径
动物病原细菌 敏感度
( mm)
革兰 金黄色葡萄球菌 20 高敏 氏 肺炎双球菌 18 中敏 阳性
猪链球菌 20 高敏 菌
大肠杆菌 E. Col i 22 高敏 革兰 猪副嗜血杆菌 21 高敏 氏 猪多杀性巴氏杆菌 21 高敏 阴性 猪胸膜肺炎放线杆菌 21 高敏 菌 德氏卑沙门氏菌 25 高敏 铜绿假单胞菌 19 中敏 注： 判断标准： 抑菌圏直径大于 20mm为高敏， 小于 20 mm大于 14 mm为中敏， 小 于 14 mm为不敏感。 实施例 4 抗菌脂肽对断奶仔猪 5种细菌病的治疗作用
试验猪为 28日龄健康断奶仔猪， 共 198头， 体重 7.52±0.65kg。 试验分为
6大组， 其中 5大组为感染组， 分别接种复制 5种细菌病， 每组设立药物组合 组和阳性对照组， 另一大组为阴性对照组， 共 1 1个小组， 18头 /小组， 各小组 公母各半， 各组按常规隔离饲养。
复制猪细菌病的菌种为本中心保存菌种，通过本动物接种恢复其毒力后制 备成一定浓度的菌悬液， 这 5种细菌分别是： 猪副嗜血杆菌、 猪胸膜肺炎放线 杆菌、猪链球菌、猪多杀性巴氏杆菌和猪大肠杆菌， 通过相应的接种途径和细 菌总数， 分别对各个感染组接种这 5种细菌而复制出 5种细菌性猪病， 阴性对 照组仔猪不接种细菌， 只供给洁净饮水。每日观察记录对断奶仔猪的活动情况 和病死情况。
接种感染 2天后对药物组合组开始投药， 投药方式和剂量： 每 400-500g 纯抗菌脂肽（实施例 7制备）均匀拌入 1 p屯基础日粮中， 自由采食， 连续饲喂 5 Λ, 饮水中不添加任何抗菌类物质。 其它试验期间内饲喂不含任何抗菌类物 质的基础日粮和饮水。 10天^ , 每小组随机抽取 9头猪， 采用 PCR法分别对 其接种菌进行分离和检测。 结果见表 2。
Figure imgf000011_0001
按实施例 1方法制备的抗菌脂肽重复该试验，在治疗猪副嗜血杆菌、猪胸 膜肺炎放线杆菌、猪链球菌、猪多杀性巴氏杆菌和猪大肠杆菌 5种细菌性猪病 效果上与实施例 7 (表 2 )相同。
实施例 5 抗菌脂肽对白羽肉鸡 3种细菌病的治疗作用
试验鸡为 15 日龄健康白羽肉鸡， 共 700羽。 试验分为 4个大组， 其中 3 大组为感染组， 分别接种复制 3种细菌性鸡病，每组设立药物组合组和阳性对 照组， 另一组为不感染不用药的阴性对照组， 共 7个小组， 100羽 /组， 各组鸡 公母各半， 各组按常规隔离饲养。 复制鸡细菌病的菌种为本中心保存菌种，通过本动物接种本动物恢复其毒 力后制备成一定浓度的菌悬液， 这 3种细菌分别是： 沙门氏菌、 大肠杆菌和巴 氏杆菌，通过相应的接种途径和细菌总数， 分别对各个感染组接种这 3种细菌 而复制出 3个细菌性鸡病， 阴性对照组鸡只供给洁净饮水。
鸡接种感染后，每日观察记录各组鸡的活动情况和病死情况。感染 2天后， 对感染的药物组合组开始投药，投药方式和剂量： 将纯化抗菌脂肽（实施例 1 ) 按照 50g/100kg的剂量兌水混匀， 采取自由饮水方式， 连续饮水 7 ， 基础日 粮中不含任何抗菌类物质，其它试验期间内饲喂不含任何抗菌类物质的饮水和 基础日粮。 10 后， 每小组随机抽取 20只鸡， 采用 PCR法分别对其接种菌 进行分离和检测。 结果见表 3。
Figure imgf000012_0001
按实施例 1方法制备的抗菌脂肽重复该试验，在治疗沙门氏菌、 大肠杆菌 和巴氏杆菌细菌性鸡病效果上与实施例 7 (表 3 )相同。
实施例 6 抗菌脂肽的制备方法
取枯草芽孢杆菌 （B. subtilis A1株）进行制备， 种子菌的制备方法为： B.
™ /ώ Α1株菌种转接至 LB平板上， 37°C培养 24h; 用接种环取活化菌种， 接 种于含有 100ml LB液体培养基的锥形瓶中， 培养过夜作为种子菌。
取种子菌进行发酵培养， 培养基为： NS-L- I培养基（葡萄糖 20.0 g , L- 谷氨酸钠 5.0g , MgS04 0.5 g , KC1 0.5 g, KH2P04 1 g, FeS04 0.15mg, MnSO4 5.0 mg, CuSO4 0.16 mg, 蒸馏水 1 000 ml, pH 7.0 ) 。 发酵条件为： 种子菌接种量 8%, 发酵温度 29°C , 发酵时间 48小时， 发酵转速 150 r/min, 发酵初始 pH为 pH 7.0。
上述抗菌脂肽的粗提过程为：取制备的发酵菌液在 3°C及 pH4.5条件下经 7000rpm离心 lOmin, 去除菌体， 取上清液用 6mol/L HCL调节 pH4.0, 再以 7000rpm离心 1 Omin , 收集沉淀， 将沉淀在 70 V的烘箱中干燥 20h , 即得抗菌 脂肽粗提物。
取抗菌脂肽粗提物， 加入乙醇后用 NaOH调节 pH至 7.0, 再乙醇类物质 抽提 2次并将其抽提液合并， 然后将其过 Sephadex LH-20层析柱， 最后用离 心浓缩仪浓缩至完全干燥， 即得抗菌脂肽的纯化物。
实施例 7抗菌脂肽的制备方法
取枯草芽孢杆菌 （B. subtilis Al株）进行制备， 种子菌的制备方法为： B. subtilis Al株菌种转接至 LB平板上， 37°C培养 24h; 用接种环取活化菌种， 接 种于含有 100ml LB液体培养基的锥形瓶中， 培养过夜作为种子菌。
取种子菌进行发酵培养， 培养基包括 NS-L- I培养基（葡萄糖 20.0 g, L- 谷氨酸钠 5.0g, MgS04 0.5 g, KCl 0.5 g, KH2P04 1 g, FeS04 0.15mg, MnS04 5.0 mg, CuS04 0.16 mg, 蒸馏水 1 000 ml, pH 7.0。 ) 。 发酵条件为： 种子菌 接种量 8%, 发酵温度 29°C , 发酵时间 48小时， 发酵转速 150 r/min, 发酵初始 pH为 pH 7.0。
上述抗菌脂肽的粗提过程为： 取制备的发酵菌液在 pH2.0~5.0条件下经
7000rpm离心 lOmin, 去除菌体， 取上清用 6mol/L HCL分别调节所需 pH, 再 以 7000rpm离心 lOmin, 收集沉淀。 将沉淀在 70°C的烘箱中干燥 20h, 即得抗 菌脂肽粗提物。
将抗菌脂肽粗提物采用酸-醇法进行纯化， 具体步骤为： 取未烘干的抗菌 脂肽粗提物， 加入醇类物质后用 NaOH调节 pH至 5.0~8.0, 再用醇类物质抽 提 2次并将其抽提液合并， 然后将其过 Sephadex LH-20层析柱， 最后用离心 浓缩仪浓缩至完全干燥， 即得抗菌脂肽的纯化物。
将纯化后的抗菌脂肽进行鉴定： 取抗菌脂肽纯化物溶解在少量醇类中， 再取一定量的该抗菌脂肽溶液分多次点样于硅胶板上， 待点样点风干后， 放 入盛有展开剂的层析缸中饱和 30min, 展开剂的组成为氯仿： 曱醇：水= 65:25:4 (体积比）， 然后，待展开剂到达距硅胶板上层边缘 2cm左右时取出， 待有机溶剂充分挥发后， 再采用含有 0.5 %茚三酮的丙酮溶液喷雾显色。
抗菌脂肽的制备结果评价： 70°C的烘箱中干燥所得纯化的抗菌脂肽，产量 需大于 6.8克 /升。
上述提纯的抗菌脂肽的抗菌效果评价方法如下：
选用如下表的 10种动物病原菌， 将其培养至对数期后， 分别制备成菌悬 液， 活菌数大于 106CFU/ml, 备用。 将素琼脂固体培养基融化后倒入培养亚， 待冷却凝固后， 等距平稳地将 3个已经灭菌的牛津杯放在固体培养基平面上， 再将分别培养细菌的 LB半固体培养基融化， 冷却至 40V ~ 50 °C , 移取 1ml 菌悬液到培养基中，混匀，然后用移液管移取 8ml含菌培养基至素琼脂平板上。 待凝固后， 拔出牛津杯， 用移液枪取 30μ1不同浓度的纯抗菌脂肽加入牛津孔 内， 以生理盐水作对照。 每个处理重复 3次， 最后细菌放置于 37°C培养箱培 养 24 h。 培养完成后， 取出培养亚测量抑菌圏的直径大小， 结果与实施例 3 表 1相同。

Claims

权 利 要 求 书
1、 一种抗菌脂肽的制备方法， 其特征在于： 该方法包括种子菌的培养、 发酵培养、 抗菌脂肽的粗提、 抗菌脂肽的纯化、 抗菌脂肽的鉴定与制备结果评 价。
2、 根据权利要求 1所述的制备方法， 其特征在于： 上述种子菌培养中的种 子菌包括枯草芽孢杆菌（B. subtilis Al株，购于中国普通微生物菌种保藏中心）。
3、 根据权利要求 2所述的制备方法， 其特征在于： 种子菌的制备方法为： ™ fo Al株菌种转接至 LB平板上， 37°C培养 24h; 用接种环取活化菌种， 接种于含有 100ml LB液体培养基的锥形瓶中， 培养过夜作为种子菌。
4、 根据权利要求 1所述的制备方法， 其特征在于： 上述的发酵培养中的培 养基包括 NS-L- I培养基（葡萄糖 20.0 g, L-谷氨酸钠 5.0g, MgSO40.5 g, KC1 0.5 g, KH2P04 1 g, FeS04 0.15mg, MnSO45.0 mg, CuSO40.16 mg, 蒸馏水 1 000 ml, pH 7.0 ) 。
5、 根据权利要求 4所述的制备方法， 其特征在于： NS-L-I培养基可用葡萄 糖含量调整， 乳糖， L-谷氨酸钠， 微量元素及培养基 pH值进行调整后的培养 基取代。
6、 根据权利要求 1所述的制备方法， 其特征在于： 种子菌接种量是
1%~15%, 发酵温度是 20°C ~ 37°C , 发酵时间为 24 ~ 72小时， 发酵转速为 50 ~ 300转 /分钟， 发酵初始 pH为 pH 5.0~9.0。
7、 根据权利要求 1所述的制备方法， 其特征在于： 抗菌脂肽粗提物的制 备过程是发酵菌液在 pH2.0~5.0条件下经 7000rpm离心 10分钟， 去除菌体， 取上清用 6mol/L HCL分别调节所需 pH, 再以 7000rpm离心 10分钟， 收集沉 淀， 将沉淀在 70°C的烘箱中干燥 20小时， 即得。
8、 根据权利要求 1所述的制备方法， 其特征在于： 抗菌脂肽的纯化过程 采用酸-醇法， 具体步骤是取未烘干的抗菌脂肽粗提物， 加入醇类物质后用 NaOH调节 pH至 5.0~8.0, 再用醇类物质抽提 2次并将其抽提液合并， 然后将 其过 Sephadex LH-20层析柱， 最后用离心浓缩仪浓缩至完全干燥， 即得抗菌 脂肽的纯化物。
9、 根据权利要求 1所述的制备方法， 其特征在于： 抗菌脂肽的鉴定方法 为取抗菌脂肽纯化物溶解在少量醇类中，再取一定量的该抗菌脂肽溶液分多次 点样于硅胶板上，待点样点风干后，放入盛有展开剂的层析缸中饱和 30分钟， 展开剂的组成为氯仿： 曱醇：水 = 65:25:4 (体积比）， 然后， 待展开剂到达距 硅胶板上层边缘 2厘米时取出， 待有机溶剂充分挥发后， 再采用含有 0.5 %茚 三酮的丙酮溶液喷雾显色。
10、 根据权利要求 1所述的制备方法， 其特征在于： 所得纯化的抗菌脂肽， 含量大于 6.8克 /升。
11、根据权利要求 1获得的抗菌脂肽在制备抑制革兰氏阳性菌和革兰氏阴 性菌药物中的应用。
12、根据权利要求 1获得的抗菌脂肽在制备对治疗断奶仔猪和白羽肉鸡细 菌病药物中的应用。
13、 一种抗菌脂肽， 其特征在于该抗菌脂肽的制备方法包括如下步骤： 步骤 1 : 培养种子菌；
步骤 2: 种子菌发酵培养， 制备发酵菌液；
步骤 3: 将发酵菌液在 10°C以下及酸性条件下离心，去除菌体制备抗菌脂 肽粗提物；
步骤 4: 将抗菌脂肽粗提物纯化。
14、 如权利要求 13所述的抗菌脂肽， 其特征在于： 步骤 1中的种子菌为 革兰氏阳性菌。
15、 如权利要求 14所述的抗菌脂肽， 其特征在于： 步骤 1中的种子菌为 枯草芽孢杆菌 B. subtilis Al。
16、 如权利要求 13所述的抗菌脂肽， 其特征在于： 步骤 1种子菌的培养方 法为： 菌种先转接至平板培养基上培养， 取活化菌种， 接种于液体培养基中。
17、 如权利要求 13所述的抗菌脂肽， 其特征在于： 步骤 2中， 发酵培养包 括 NS-L-I培养基， 所述培养基为 pH= 6.5-7.5条件下按如下比例关系调配：
葡萄糖 10.0-30.0 g L-谷氨酸钠 2.0-8.0g MgS04 0.2-0.8 g KC1 0.2-0.8 g KH2PO 20-60 g FeS04 0.10-0.20mg
MnSO42.0-8.0mg CuS04 0.1-0.2 mg 蒸馏水 1000 ml。
18、 如权利要求 17所述的抗菌脂肽， 其特征在于： 发酵条件为： 种子菌接 种量为 1%~15%, 发酵温度为 20°C ~ 37°C , 发酵时间 24 ~ 72h, 发酵转速 50 ~ 300 r/min, 发酵初始 pH为 pH 5.0~9.0。
19、 如权利要求 13所述的抗菌脂肽， 其特征在于： 步骤 3将发酵菌液在温 度为 0-5°C、 pH2.0~5.0条件下离心。
20、 如权利要求 13所述的抗菌脂肽， 其特征在于： 步骤 3去除菌体后， 取 上清液离心 1-5次， 离心后收集沉淀， 干燥， 得抗菌脂肽粗提物。
21、 如权利要求 13所述的抗菌脂肽， 其特征在于： 步骤 4为取抗菌脂肽 粗提物， 加入醇类物质后用碱性溶液调节 pH5.0~8.0, 再用醇类物质抽提， 得 抽提液， 抽提液纯化， 浓缩。
22、如权利要求 13-21任一所述的抗菌脂肽在制备治疗动物细菌病药物中 的应用。
23、 如权利要求 22所述的应用， 其特征在于所述的动物为猪或鸡。
24、 一种抗菌脂肽的制备方法， 其特征在于该方法包括如下步骤： 步骤 1 : 培养种子菌；
步骤 2: 取种子菌发酵培养， 制备发酵菌液；
步骤 3: 将发酵菌液在 10°C以下及酸性条件下离心，去除菌体制备抗菌脂 肽粗提物；
步骤 4: 将抗菌脂肽粗提物纯化。
25、 如权利要求 24所述的方法， 其特征在于： 步骤 1中的种子菌为革兰 氏阳性菌。
26、 如权利要求 25所述的方法， 其特征在于： 步骤 1中的种子菌为枯草 芽孢杆菌 B. subtilis Ah
27、 如权利要求 26所述的方法， 其特征在于： 步骤 1种子菌的培养方法为： 菌种先转接至平板培养基上培养， 取活化菌种， 接种于液体培养基中。
28、如权利要求 26所述的方法，其特征在于：步骤 2中，发酵培养包括 NS-L-I 培养基， 所述培养基为 pH= 6.5-7.5条件下按如下比例关系调配：
葡萄糖 10.0-30.0 g L-谷氨酸钠 2.0-8.0g MgS04 0.2-0.8 g KC1 0.2-0.8 g KH2PO 20-60 g FeS04 0.10-0.20mg MnSO42.0-8.0mg CuS04 0.1-0.2 mg 蒸馏水 1000 ml。
29、 如权利要 28所述的方法， 其特征在于： 发酵条件为： 种子菌接种量为 1%~15%,发酵温度为 20°C ~ 37°C ,发酵时间 24 ~ 72h,发酵转速 50 ~ 300 r/min, 发酵初始 pH为 pH 5.0~9.0。
30、 如权利要求 24所述的方法， 其特征在于步骤 3 , 将发酵菌液在温度为 0-5 °C、 pH2.0~5.0条件下离心。
31、 如权利要求 24所述的方法， 其特征在于： 步骤 3去除菌体后， 取上清 液离心 1-5次， 离心后收集沉淀， 干燥， 得抗菌脂肽粗提物。
32、 如权利要求 24所述的方法， 其特征在于： 步骤 4为取抗菌脂肽粗提 物， 加入醇类物质后用碱性溶液调节 pH5.0~8.0, 再用醇类物质抽提， 得抽提 液， 抽提液纯化， 浓缩。
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