Purified cow milk antibacterial peptide produced by fermentation method and preparation method thereof

Abstract

The invention discloses a preparation method and application of a cow milk antibacterial peptide with wide inhibitory activity on gram-positive bacteria and gram-negative bacteria by a fermentation method, the cow milk antibacterial peptide is prepared by taking milk protein as a raw material through a microbial fermentation method and a multi-stage membrane separation and purification process, the yield of the cow milk antibacterial peptide prepared by the method can reach 18-30%, and the yield of the cow milk antibacterial peptide prepared by the method can reach 18-30%. And the peptide content in the product is 60-85%. The cow milk antibacterial peptide prepared by the preparation method disclosed by the invention has inhibitory activity on gram-positive bacteria and gram-negative bacteria. The prepared cow milk antibacterial peptide can be used for preparing drugs and / or health care products, food and skin care products for preventing and / or reducing infectious diseases caused by escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, porphyromonas gingivalis and streptococcus mutans, can also be used as a food preservative, an oral bacteriostatic agent, a preservative and the like, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and specifically discloses a bovine milk antimicrobial peptide, a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides are a class of naturally occurring small molecule peptides. As a new type of antibiotic, they have broad-spectrum antimicrobial activity and will not easily cause microorganisms to develop drug resistance. They have broad application prospects in the fields of medicine, food, cosmetics, etc. Antimicrobial peptides are derived from proteins and are widely distributed in nature. As more and more antimicrobial peptides are found in microorganisms, animals and plants, large-scale preparation of antimicrobial peptides has become a practical demand for the application of antimicrobial peptides.

[0003] The existing antimicrobial peptide preparation technology mainly includes two technical routes: artificial synthesis and biological expression. Artificial synthesis is the synthesis of target peptides by chemical synthesis. Antimicrobial peptides with specific sequences and activities are designed and synthesized, but the preparation cost is high, which is suitable for small-scale research and clinical applications, and artificially synthesized cationic antimicrobial peptides may have a greater toxic effect on mammalian cells. Biological expression is an in vivo expression and in vitro expression method using bacteria, yeast, insect cells, mammalian cells, etc. The gene of the antimicrobial peptide is introduced into the host cell through genetic engineering technology to enable it to produce antimicrobial peptides. The specific process includes gene cloning, transfection or transformation, culture optimization, protein expression and purification. The biological expression method can produce antimicrobial peptides with high activity on a large scale, but it requires complex multi-layer processes during the purification process. Therefore, it is of great significance to develop safe, stable and low-cytotoxic antimicrobial peptides.

[0004] As a new type of food raw material, cow's milk has the advantages of high safety and wide adaptability. As a popular food, cow's milk has rich protein and peptide resources. Its quality and safety have long been highly concerned and regulated, which ensures the high quality and safety of cow's milk as a food raw material, taking into account the advantages of antimicrobial peptides and the two characteristics of high-quality and safe protein sources. The enzymes produced by lactic acid bacteria have a strong hydrolysis effect on proteins, which can decompose the proteins in cow's milk into peptides with various biological activities, thereby improving the utilization value of cow's milk protein. In the list of probiotic strains that can be used for food published by the Ministry of Health, Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus paracasei, Lactobacillus helveticus and Bifidobacterium breve are all listed, which can be widely used in dairy fermentation, with low strain cultivation cost, easy control of fermentation process and simple product purification process. Therefore, the large-scale preparation of antimicrobial peptides can be achieved by hydrolyzing cow's milk protein using edible probiotic fermentation method, and its method and products have good application and development prospects in the preparation of microbial infection drugs or food preservatives, antibacterial agents, etc. It can be widely used in medicine, food, cosmetics and other fields to provide people with better antibacterial protection Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing and applying a bovine milk antimicrobial peptide having a significant inhibitory effect on both Gram-negative bacteria and Gram-positive bacteria. The present invention degrades milk protein by a microbial fermentation method, separates and purifies the antimicrobial peptide components therein, and the prepared bovine milk antimicrobial peptide can be used as a food preservative, oral antibacterial agent, preservative, etc. in the preparation of drugs and / or health products for preventing and / or reducing infectious diseases caused by Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, Porphyromonas gingivalis and Streptococcus mutans, or in new foods and skin care products.

[0006] The preparation method of the bovine milk antimicrobial peptide comprises the following steps:

[0007] (1) Culture medium preparation and fermentation culture

[0008] Prepare skim milk powder water solution with milk protein content of 5% to 20% as fermentation medium, mix thoroughly and sterilize at 110℃ for 15min. Inoculate with 2% inoculation amount, the bacterial content in the fermentation liquid is 2×10^ 5 ~5×10^ 8 CFU / mL, ferment at 200 rpm, 30-60°C for 10-60 hours. Sterilize at 121°C for 20 minutes to inactivate the bacteria and end the fermentation.

[0009] (2) Antimicrobial peptide purification

[0010] The fermentation broth is centrifuged at 3000-8000 rpm and 4°C for 20 minutes to remove impurities and insoluble substances in the fermentation broth, and the supernatant after centrifugation is retained. The fermentation supernatant is diluted with pure water to 3-8 times the volume, separated by an ultrafiltration membrane with a molecular weight cutoff of 3-30 kDa, and the ultrafiltrate is collected. The fermentation ultrafiltrate is then separated by a nanofiltration membrane with a molecular weight cutoff of <1 kDa, concentrated to the initial volume of the fermentation broth, and the nanofiltration retentate is collected.

[0011] (3) Collect the nanofiltration retentate and dry it to obtain the milk antimicrobial peptides, which can be stored in a freezer at -20°C.

[0012] The method of the present invention uses milk protein as raw material, and prepares bovine milk antimicrobial peptides through microbial fermentation and multi-stage membrane separation and purification technology. The yield of bovine milk antimicrobial peptides prepared by the method can reach 10-30%, and the peptide content in the product is 60-85%.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The bovine milk antimicrobial peptide prepared according to the method has good microbial growth inhibition activity, and has significant inhibitory effects on both Gram-positive bacteria and Gram-negative bacteria. The IC50 values ​​for Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, Porphyromonas gingivalis and Streptococcus mutans are all lower than 40 mg / mL. It has good solubility and high purity, and is a green, safe and efficient broad-spectrum antimicrobial peptide.

[0015] 2. The present invention discloses a bovine milk antimicrobial peptide and a preparation method thereof, the method comprising milk protein fermentation and collection and purification of the antimicrobial peptide. The method is simple and feasible, and the reaction conditions are mild.

[0016] 3. The present invention uses edible probiotics to ferment milk protein to produce antimicrobial peptides. Compared with other strains, the invention has the advantages of low cost, greenness, safety, etc. The strains used are all on the list of probiotic strains that can be used in food published by the Ministry of Health, can be widely used in dairy fermentation, have low strain cultivation costs, and the fermentation process is easy to control, which is suitable for large-scale preparation.

[0017] 4. Good application prospects. The milk antimicrobial peptide prepared according to the method of the present invention has green and safe raw materials and products, is more convenient to eat, and is easily absorbed by the human body. Therefore, it has broad application prospects as food, medicine, and functional food. It can be used as a drug and / or health product to prevent and / or reduce infectious diseases caused by microorganisms, or used in new foods and skin care products as a food preservative, oral antibacterial agent, preservative, etc. DETAILED DESCRIPTION

[0018] The milk antimicrobial peptide can be prepared according to the following example method and the peptide content and antibacterial activity can be determined.

[0019] Example 1: Production of milk antimicrobial peptides by fermentation with Lactobacillus bulgaricus

[0020] (1) Culture medium preparation and fermentation culture

[0021] A skimmed milk powder aqueous solution with a milk protein content of 8% by mass was prepared as a fermentation medium, which was fully mixed and sterilized at 110° C. for 15 min.

[0022] The Lactobacillus bulgaricus (CICC 20247) strain was stored at -80°C by glycerol preservation. Before the experiment, 200 μL of the bacterial solution was inoculated into 10 mL of the sterile skim milk powder medium prepared in the above step (inoculation volume 2% (V / V)), and cultured in air at 37°C for 8 h to resuscitate the strain. The above resuscitation operation was repeated 2-3 times (here 2 times) to restore the strain to vitality.

[0023] The inoculation volume (V / V) of the bacteria (revitalized strain) was 2% in the fermentation medium, and the bacterial content in the fermentation broth was 1×10^ 7 CFU / mL, fermented in air at 200 rpm and 37°C for 22 h. Sterilized at 121°C for 20 min to inactivate the bacteria and end the fermentation.

[0024] (2) Antimicrobial peptide purification

[0025] The fermentation broth was centrifuged at 8000 rpm and 4°C for 20 min to remove impurities and insoluble substances in the fermentation broth, and the supernatant after centrifugation was retained. The fermentation supernatant was diluted with pure water to 6 times the original supernatant volume, separated by an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the ultrafiltrate (ultrafiltration membrane permeate) was collected. The fermentation ultrafiltrate was then separated by a nanofiltration membrane with a molecular weight cutoff of 150 Da, concentrated to the initial volume of the fermentation broth, and the nanofiltration retentate was collected.

[0026] (3) Collect the nanofiltration retentate and dry it to obtain the milk antimicrobial peptides, which can be stored in a freezer at -20°C.

[0027] Example 2: Fermentation of milk antimicrobial peptides by Streptococcus thermophilus and Lactobacillus paracasei

[0028] (1) Culture medium preparation and fermentation culture

[0029] A skim milk powder aqueous solution with a milk protein mass content of 12% was prepared as a fermentation medium, and the mixture was fully mixed and sterilized at 110°C for 15 min. The thermophilic Streptococcus (CICC 20374) and Lactobacillus paracasei (CICC 22165) strains were stored at -80°C by glycerol preservation method, and 200 μL of bacterial solution was inoculated into 10 mL of the sterile skim milk powder medium prepared in the above step (inoculation volume 2% (V / V)) before the experiment, and cultured at 42°C for 8 h to resuscitate the strains, and the above resuscitation operation was repeated 2-3 times (here 2 times) to restore the strains to vitality.

[0030] The inoculation volume was 2% and the bacterial content in the fermentation medium was 2×10^ 7 CFU / mL, ferment at 200 rpm and 42°C for 16 h. Sterilize at 121°C for 20 min to inactivate the bacteria and end the fermentation.

[0031] (2) Antimicrobial peptide purification

[0032] The fermentation broth was centrifuged at 6000 rpm and 4°C for 20 min to remove impurities and insoluble substances in the fermentation broth, and the supernatant after centrifugation was retained. The fermentation supernatant was diluted with pure water to 4 times the original supernatant volume, separated by an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the ultrafiltrate was collected. The fermentation ultrafiltrate was then separated by a nanofiltration membrane with a molecular weight cutoff of 1 kDa, concentrated to the initial volume of the fermentation broth, and the nanofiltration retentate was collected.

[0033] (3) Collect the nanofiltration retentate and dry it to obtain the milk antimicrobial peptides, which can be stored in a freezer at -20°C.

[0034] Example 3: Production of milk antimicrobial peptides by fermentation of Lactobacillus bulgaricus, Lactobacillus helveticus and Bifidobacterium breve

[0035] (1) Culture medium preparation and fermentation culture

[0036] A skim milk powder aqueous solution with a milk protein mass content of 12% was prepared as a fermentation medium, and the mixture was fully mixed and sterilized at 110°C for 15 min. Lactobacillus bulgaricus (CICC 20247), Lactobacillus helveticus (CICC 20243) and Bifidobacterium breve (CICC 6185) strains were stored at -80°C by glycerol preservation method, and 200 μL of bacterial solution was inoculated into 10 mL of the sterile skim milk powder medium prepared in the above step (inoculation amount 2% (V / V)) before the experiment, and cultured for 8 h at 35°C in an anaerobic environment to resuscitate the strains, and the above resuscitation operation was repeated 2-3 times (here 2 times) to restore the strains to vitality.

[0037] The inoculation volume was 2% and the bacterial content in the fermentation medium was 1×10^ 8 CFU / mL, fermented for 30h in an anaerobic environment at 200rpm and 35℃. Sterilized at 121℃ for 20min to inactivate the bacteria and end the fermentation.

[0038] (2) Antimicrobial peptide purification

[0039] The fermentation broth was centrifuged at 6000 rpm and 4°C for 20 min to remove impurities and insoluble substances in the fermentation broth, and the supernatant after centrifugation was retained. The fermentation supernatant was diluted with pure water to 4 times the original supernatant volume, separated by an ultrafiltration membrane with a molecular weight cutoff of 5k Da, and the ultrafiltrate was collected. The fermentation ultrafiltrate was then separated by a nanofiltration membrane with a molecular weight cutoff of 150 Da, concentrated to the initial volume of the fermentation broth, and the nanofiltration retentate was collected.

[0040] (3) Collect the nanofiltration retentate and dry it to obtain the milk antimicrobial peptides, which can be stored in a freezer at -20°C.

[0041] Example 4: Product yield and peptide content determination

[0042] (1) Product yield determination:

[0043] The balance was calibrated to ensure accurate mass measurement. The starting mass of the raw material (skim milk powder) was weighed in a dry environment. The experimental conditions determined in Examples 1, 2, and 3 were followed. The mass of the product was weighed and recorded in a dry environment.

[0044] The yield calculation formula is as follows:

[0045]

[0046] The yield determination results of the three bovine milk antimicrobial peptide products are shown in Table 1. The mass of the product obtained per liter of fermentation broth ranges from 15 to 35 g, and the yields of the three bovine milk antimicrobial peptide products are approximately 18% to 30%.

[0047] Table 1 Yield results of milk antimicrobial peptides

[0048]

[0049] (2) Peptide content determination:

[0050] Accurately weigh 0.2g of sample (accurate to 0.001g), add 5mL of water to fully dissolve, then add 5mL of trichloroacetic acid solution (300g / L), mix and let stand for 5min, filter to remove the precipitate, transfer the filtrate as the sample test solution to a dry digestion tube, add 0.2g of copper sulfate, 3g of potassium sulfate and 10mL of concentrated sulfuric acid (mass concentration 98%), mix and place in a graphite digestion instrument, place a small funnel at the mouth of the digestion tube, turn on the instrument, and when the digestion furnace temperature reaches 420℃, continue digestion for 1h until the liquid is blue-green, clear and transparent, end heating, cool to room temperature, and obtain the test solution. At the same time, perform a reagent blank test (except that the sample is not included, the rest of the process is exactly the same as the aforementioned sample digestion process) to obtain a reagent blank.

[0051] Place the digestion tube after digestion into the Kjeldahl nitrogen analyzer, add 1-2 drops (2 drops in this case) of mixed indicator (1g / L methyl red ethanol solution and 1g / L bromocresol green ethanol solution mixed in a ratio of 1:5, V / V) into the receiving bottle, insert the condenser into the bottle, add water, boric acid solution (20g / L) and sodium hydroxide solution (400g / L) before using the Kjeldahl nitrogen analyzer, turn on the instrument to automatically complete the process of adding liquid (10mL water, 25mL boric acid solution and 40mL sodium hydroxide solution), distillation (5min) and elution (10mL water), take out the receiving bottle after completion, and titrate with hydrochloric acid standard titration solution to light gray-red as the end point. At the same time, perform a reagent blank test.

[0052] The peptide content in the sample was calculated according to formula (1):

[0053]

[0054] Where:

[0055] X: peptide content in the sample, in grams per hundred grams (g / 100g);

[0056] V1: The volume of hydrochloric acid standard titrant consumed by the test solution, in milliliters (mL);

[0057] V2: The volume of hydrochloric acid standard titration solution consumed by the reagent blank, in milliliters (mL);

[0058] c: concentration of standard hydrochloric acid titration solution, in moles per liter (mol / L);

[0059] 0.0140: The mass of nitrogen equivalent to 1.0 mL of hydrochloric acid [c(HCl) = 1.000 mol / L] standard titration solution, in grams (g);

[0060] m: mass of the sample, in grams (g);

[0061] F: The conversion factor of nitrogen into protein, the conversion factor of nitrogen in various foods, is taken as 6.25;

[0062] 100: Conversion factor.

[0063] Table 2 lists the peptide content determination results of three bovine milk antimicrobial peptide products. The peptide contents of the three bovine milk antimicrobial peptide products are between 67% and 85%. The antimicrobial peptide products prepared and separated by this method have high purity.

[0064] Table 2 Peptide content results of milk antimicrobial peptides

[0065]

[0066] Example 5: Antibacterial activity assay

[0067] (1) Strains and recovery

[0068] The strains and corresponding culture media used in the experiment are shown in Table 3. The strains were stored at -80°C by glycerol preservation. Before the experiment, 200 μL of bacterial solution was inoculated into 10 mL of the corresponding liquid culture medium (2% (V / V)), and cultured at 37°C for 12 h to revive the strains. The above revival operation was repeated 2-3 times (here 2 times) to restore the vitality of the strains.

[0069] Table 3 Strain and culture medium information

[0070]

[0071]

[0072] (2) Preparation of bacterial suspension

[0073] Take 1 mL of the revived bacterial suspension and transfer it back into 10 mL of the corresponding liquid culture medium. Culture it in air at 37° and 120 r / min for 12 h. Measure OD600 with a UV spectrophotometer to calculate the bacterial concentration.

[0074] (3) Antibacterial experiment

[0075] The above bacterial suspension was diluted to 5×10^ 5 CFU / mL. 100 μL of bacterial solution and 100 μL of mixed peptide solution of different concentrations were added to a 96-well plate, so that the final concentrations of the mixed peptides were 52.1, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 mg / mL, respectively. After the 96-well plate was cultured at 37°C for 18 hours, the absorbance value at 600 nm was measured using a full-wavelength scanning multifunctional reader to determine the half-inhibitory concentration of the mixed peptides against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0076] (4) Experimental results

[0077] The results of the antibacterial experiments on 6 kinds of bacteria are shown in Table 4, which lists the half-inhibitory concentrations of the growth of the bovine milk antimicrobial peptides on the 6 kinds of pathogenic bacteria. The results show that the bovine milk antimicrobial peptides prepared by this method have inhibitory activity against both Gram-negative and Gram-positive bacteria, and the IC50 values ​​for Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, Porphyromonas gingivalis and Streptococcus mutans are all lower than 40 mg / mL. Among them, the growth of Escherichia coli has a prominent inhibitory effect.

[0078] Table 4 IC50 antibacterial results of milk antimicrobial peptides Unit: mg / mL

[0079]

[0080] Comparative Example 1: Production of milk antimicrobial peptides by fermentation with Lactobacillus bulgaricus

[0081] Step (1) medium preparation and fermentation culture conditions are exactly the same as those in Example 1. The difference from Example 1 is step (2) of antimicrobial peptide purification. The fermentation broth is centrifuged at 8000 rpm and 4°C for 20 min to remove impurities and insoluble substances in the fermentation broth, and the supernatant after centrifugation is dried and stored. The bovine milk peptide product obtained by this technical solution only shows low inhibitory activity against Escherichia coli (IC50=42.35 mg / mL) and does not show growth inhibitory activity against the other five microorganisms. The yield is 35% and the product peptide content is 43%.

[0082] Comparative Example 2: Fermentation of milk antimicrobial peptides by Streptococcus thermophilus and Lactobacillus paracasei

[0083] The difference from Example 2 is that the milk protein content in the skimmed milk powder medium in the medium preparation and fermentation culture conditions in step (1) is 5%, and the other conditions are the same. The half-inhibitory concentration IC50 value of the bovine milk peptide product obtained by this technical solution for 6 kinds of microorganisms is 11.67-32.96 mg / mL, and the yield is 7%.

[0084] By comparing the technical effects of the embodiments and comparative examples, the bovine milk antimicrobial peptide preparation technology applied for in the present invention has significant advantages in antibacterial effect, product yield and peptide content. Only under the technical scheme described in the present application can a bovine milk antimicrobial peptide product with guaranteed antibacterial effect, high yield and high product peptide content be obtained.

Claims

1. A method for preparing milk antimicrobial peptides by fermentation, characterized in that: After fermentation, centrifugation, ultrafiltration separation, nanofiltration concentration and drying, the milk antimicrobial peptides are finally obtained; The specific steps include: (1) A skimmed milk powder aqueous solution with a milk protein content of 5% to 30% was used as the fermentation medium, and the inoculum size was 2×10^ 5 ~5×10^ 8 CFU / mL inoculated bacteria, fermented at 30-60℃ for 10-60h; (2) centrifuging the fermentation broth at 3000-8000 rpm and retaining the supernatant; (3) diluting the fermentation supernatant with pure water to 3-8 times the volume of the supernatant, separating through an ultrafiltration membrane, and collecting the ultrafiltrate; (4) the fermentation ultrafiltrate is separated by a nanofiltration membrane, concentrated to the initial volume of the fermentation broth, and the nanofiltration retentate is collected; (5) The retentate is dried to obtain bovine milk antimicrobial peptide dry powder.

2. The method according to claim 1, characterized in that: The bacterial species used are one or a combination of two or more of Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus paracasei, Lactobacillus helveticus and Bifidobacterium breve.

3. The method according to claim 1, characterized in that: The ultrafiltration membrane in step (3) has a molecular weight cut-off of 3 to 30 kDa.

4. The method according to claim 1, characterized in that: The molecular weight cut-off of the nanofiltration membrane in step (4) is ≤1 kDa.

5. A bovine milk antimicrobial peptide prepared by fermentation according to any one of claims 1 to 4.

6. A use of the antimicrobial peptide according to claim 5, characterized in that: The invention is used as a bacterial growth inhibitor or bactericide. The antimicrobial peptide has growth inhibition activity on one or more of Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, Porphyromonas gingivalis and Streptococcus mutans.

7. The bovine milk antimicrobial peptide according to claim 5 is used in the preparation of medicines and / or health products or skin care products for preventing and / or reducing infectious diseases caused by one or more of Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, Porphyromonas gingivalis and Streptococcus mutans, or can also be used as a food preservative, an oral antibacterial agent, or a preservative.

8. The use according to claim 7, characterized in that: The anti-infection drug uses the milk antimicrobial peptide as an active ingredient, and pharmaceutically acceptable carriers or auxiliary materials can be added therein.

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