A fermentation method for producing purified milk antimicrobial peptides and its preparation method

Abstract

The present invention discloses a method for preparing a bovine milk antimicrobial peptide having broad inhibitory activity against Gram-positive and Gram-negative bacteria by a fermentation method and its application. The bovine milk antimicrobial peptide is prepared using milk protein as raw material through a microbial fermentation method and a multi-stage membrane separation and purification process. The yield of the bovine milk antimicrobial peptide prepared by this method can reach 18-30%, and the peptide content in the product is 60-85%. The bovine milk antimicrobial peptide prepared by the present invention has inhibitory activity against Gram-positive and Gram-negative bacteria. The prepared bovine milk antimicrobial peptide can be used to prepare drugs and / or health products, foods, and skin care products for preventing and / or reducing infectious diseases caused by Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Porphyromonas gingivalis, and Streptococcus mutans. It can also be used as a food preservative, oral antibacterial agent, preservative, etc., and has good application prospects.

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 class of antibiotics, they possess broad-spectrum antimicrobial activity and are unlikely to induce drug resistance in microorganisms. They hold broad application prospects in the fields of medicine, food, and cosmetics. Derived from proteins, antimicrobial peptides are widely distributed in nature. As more and more antimicrobial peptides are discovered in microorganisms, animals, and plants, large-scale production of antimicrobial peptides has become a practical need for their application.

[0003] Existing technologies for producing antimicrobial peptides primarily rely on artificial synthesis and biological expression. Artificial synthesis involves chemically synthesizing the target peptide. Antimicrobial peptides with specific sequences and specific activities are designed and synthesized, but production costs are high, making them suitable for small-scale research and clinical applications. Furthermore, artificially synthesized cationic antimicrobial peptides may have significant toxicity to mammalian cells. Biological expression involves in vivo and in vitro expression using bacteria, yeast, insect cells, and mammalian cells. Genetic engineering techniques are used to introduce the antimicrobial peptide gene into host cells, enabling production of the peptide. The specific process involves gene cloning, transfection or transformation, culture optimization, protein expression, and purification. While biological expression allows for large-scale production of highly active antimicrobial peptides, it requires complex, multi-step purification. Therefore, the development of safe, stable, and low-cytotoxic antimicrobial peptides is of great importance.

[0004] As a novel food ingredient, cow's milk offers advantages such as high safety and wide adaptability. As a universally popular food, cow's milk is rich in protein and peptide resources, and its quality and safety have long been the focus of high attention and regulation. This ensures the high quality and safety of cow's milk as a food ingredient, combining the advantages of antimicrobial peptides with the potential to be a high-quality and safe protein source. Enzymes produced by lactic acid bacteria have a strong hydrolytic effect on proteins, breaking them down into peptides with diverse biological activities, thereby increasing the value of milk proteins. The Ministry of Health's list of probiotic strains for food use includes Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus paracasei, Lactobacillus helveticus, and Bifidobacterium breve. These strains are widely used in dairy fermentation, offering low cultivation costs, easy-to-control fermentation processes, and simple product purification. Therefore, the use of edible probiotic fermentation to hydrolyze cow's milk protein for large-scale production of antimicrobial peptides holds great promise for the development of drugs for microbial infections, food preservatives, and antimicrobial agents. 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 present invention aims to provide a method for preparing and applying a bovine milk antimicrobial peptide having a significant inhibitory effect on both Gram-negative and Gram-positive bacteria. The present invention degrades milk protein through a microbial fermentation method, and separates and purifies the antimicrobial peptide component. 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 novel 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 a skim milk powder aqueous solution with a milk protein content of 5% to 20% as the fermentation medium, mix thoroughly and sterilize at 110℃ for 15 minutes. Inoculate with 2% of the inoculum, and the bacterial content in the fermentation liquid is 2×10^ 5 ~5×10^ 8 CFU / mL, ferment at 200 rpm and 30-60°C for 10-60 h. Sterilize at 121°C for 20 min to inactivate the bacteria and terminate the fermentation.

[0009] (2) Antimicrobial peptide purification

[0010] The fermentation broth was centrifuged at 3000-8000 rpm and 4°C for 20 minutes to remove impurities and insoluble matter. The supernatant was retained. The fermentation supernatant was diluted to 3-8 times its volume with pure water and separated through an ultrafiltration membrane with a molecular weight cutoff of 3-30 kDa. The ultrafiltrate was collected. The fermentation ultrafiltrate was further separated through a nanofiltration membrane with a molecular weight cutoff of <1 kDa and concentrated to the original volume of the fermentation broth. The nanofiltration retentate was collected.

[0011] (3) Collect the nanofiltration retentate and dry it to obtain the milk antimicrobial peptides. Store it 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 this 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 this method exhibits excellent microbial growth inhibition activity, showing significant inhibitory effects against both Gram-positive and Gram-negative bacteria. IC50 values ​​against Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, Porphyromonas gingivalis, and Streptococcus mutans were all below 40 mg / mL. With excellent solubility and high purity, this peptide is a green, safe, and highly effective broad-spectrum antimicrobial peptide.

[0015] 2. The present invention discloses a bovine milk antimicrobial peptide and a preparation method thereof, which comprises 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. This invention utilizes edible probiotics to ferment milk protein to produce antimicrobial peptides. Compared to other bacterial strains, this invention offers advantages such as low cost, environmental friendliness, and safety. The strains used are all on the Ministry of Health's list of probiotic strains approved for food use and are widely applicable in dairy fermentation. The low cost of cultivation and the easily controllable fermentation process make this method suitable for large-scale production.

[0017] 4. Promising application prospects. The bovine milk antimicrobial peptides prepared according to the method of the present invention are green, safe, and convenient to consume, and are easily absorbed by the human body. Therefore, they have broad application prospects as foods, medicines, and functional foods. They can be used as drugs and / or health supplements to prevent and / or reduce the risk of microbial infections, or in novel foods and skincare products as food preservatives, oral antibacterial agents, and preservatives. DETAILED DESCRIPTION

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

[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 then sterilized at 110° C. for 15 minutes.

[0022] Lactobacillus bulgaricus (CICC 20247) was stored at -80°C using glycerol. 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)). The culture was incubated in air at 37°C for 8 h to resuscitate the strain. The resuscitation operation was repeated 2-3 times (here twice) to restore the strain to viability.

[0023] The bacteria (revitalized strain) were inoculated into the fermentation medium at a 2% inoculum volume (V / V), 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 terminate the fermentation.

[0024] (2) Antimicrobial peptide purification

[0025] The fermentation broth was centrifuged at 8000 rpm and 4°C for 20 minutes to remove impurities and insoluble matter, retaining the supernatant. The fermentation supernatant was diluted with pure water to 6 times the original supernatant volume and separated through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The ultrafiltrate (ultrafiltration membrane permeate) was collected. The fermentation ultrafiltrate was further separated through a nanofiltration membrane with a molecular weight cutoff of 150 Da and concentrated to the original volume of the fermentation broth. The nanofiltration retentate was collected.

[0026] (3) Collect the nanofiltration retentate and dry it to obtain the milk antimicrobial peptides. Store it 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 solution with a milk protein content of 12% by weight was prepared as the fermentation medium. After thorough mixing, it was sterilized at 110°C for 15 minutes. Streptococcus thermophilus (CICC 20374) and Lactobacillus paracasei (CICC 22165) strains were stored at -80°C using glycerol. 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)). The culture was incubated at 42°C for 8 hours to allow the strains to recover. This recovery process was repeated 2-3 times (here, twice) to restore the strains' vitality.

[0030] The bacteria were inoculated into the fermentation medium at a 2% inoculum level, and the bacterial content in the fermentation liquid 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 then terminate the fermentation.

[0031] (2) Antimicrobial peptide purification

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

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

[0034] Example 3: Production of milk antimicrobial peptides by fermentation with 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 content of 12% was prepared as the fermentation medium. After thorough mixing, the mixture was sterilized at 110°C for 15 minutes. Lactobacillus bulgaricus (CICC 20247), Lactobacillus helveticus (CICC 20243), and Bifidobacterium breve (CICC 6185) strains were stored at -80°C using glycerol. 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)). The culture was incubated at 35°C in an anaerobic environment for 8 hours to allow the strains to recover. This recovery process was repeated 2-3 times (here, twice) to restore the strains' vitality.

[0037] The bacteria were inoculated into the fermentation medium at a rate of 2%, and the bacterial content in the fermentation liquid was 1×10^ 8 CFU / mL, and fermented in an anaerobic environment at 200 rpm and 35°C for 30 h. Sterilize at 121°C for 20 min to inactivate the bacteria and terminate the fermentation.

[0038] (2) Antimicrobial peptide purification

[0039] The fermentation broth was centrifuged at 6000 rpm and 4°C for 20 minutes to remove impurities and insoluble matter, retaining the supernatant. The fermentation supernatant was diluted with pure water to 4 times the original supernatant volume and separated through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The ultrafiltrate was then separated through a nanofiltration membrane with a molecular weight cutoff of 150 Da and concentrated to the original volume of the fermentation broth. The nanofiltration retentate was then collected.

[0040] (3) Collect the nanofiltration retentate and dry it to obtain the milk antimicrobial peptides. Store it 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 ranged from 15 to 35 g, and the yields of the three bovine milk antimicrobial peptide products were 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, and 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 and clear, 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 digested digestion tube into the Kjeldahl nitrogen analyzer. Add 1 to 2 drops (2 drops in this case) of a mixed indicator (a 1 g / L methyl red ethanol solution and a 1 g / L bromocresol green ethanol solution in a 1:5 ratio, V / V) to the receiving flask. Insert the condenser into the flask. Before using the Kjeldahl nitrogen analyzer, add water, boric acid solution (20 g / L), and sodium hydroxide solution (400 g / L). Turn on the instrument and automatically complete the addition (10 mL of water, 25 mL of boric acid solution, and 40 mL of sodium hydroxide solution), distillation (5 minutes), and rinsing (10 mL of water). After completion, remove the receiving flask and titrate with a standard hydrochloric acid solution until the solution turns light gray-red. Perform a reagent blank test at the same time.

[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 titrant consumed by the reagent blank, in milliliters (mL);

[0058] c: concentration of hydrochloric acid standard 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: Nitrogen conversion factor for protein. The nitrogen conversion factor 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 range from 67% to 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 determination

[0067] (1) Strains and recovery

[0068] Information on the strains and corresponding culture media used in the experiments is shown in Table 3. All strains were stored at −80°C using glycerol stock. Before the experiment, 200 μL of bacterial suspension was inoculated into 10 mL of the corresponding liquid culture medium (2% (v / v)) and incubated at 37°C for 12 h to allow the strains to recover. This recovery procedure was repeated 2–3 times (twice in this case) to restore the strains to viability.

[0069] Table 3 Strain and culture medium information

[0070]

[0071]

[0072] (2) Preparation of bacterial suspension

[0073] 1 mL of the revived bacterial suspension was transferred to 10 mL of the corresponding liquid culture medium and cultured in air at 37° and 120 rpm for 12 h. The OD600 was measured 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 suspension and 100 μL of mixed peptide solutions of varying concentrations were added to a 96-well plate, resulting in final peptide concentrations of 52.1, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 mg / mL, respectively. The 96-well plate was incubated at 37°C for 18 hours. The absorbance at 600 nm was measured using a full-wavelength scanning multi-function reader to determine the half-inhibitory concentration (50%) of the mixed peptide against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0076] (4) Experimental results

[0077] Table 4 shows the results of antibacterial experiments against six bacterial species. The table lists the half-inhibitory concentrations (CIs) of the bovine milk antimicrobial peptide against the six pathogenic bacteria. The results indicate that the bovine milk antimicrobial peptide prepared by this method exhibits inhibitory activity against both Gram-negative and Gram-positive bacteria. IC50 values ​​for Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, Porphyromonas gingivalis, and Streptococcus mutans are all below 40 mg / mL. The peptide exhibits a particularly strong inhibitory effect against the growth of Escherichia coli.

[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) culture medium preparation and fermentation conditions were identical to those in Example 1. The difference from Example 1 was in step (2) antimicrobial peptide purification: the fermentation broth was centrifuged at 8000 rpm and 4°C for 20 minutes to remove impurities and insoluble matter from the fermentation broth. The supernatant was retained after centrifugation and dried for storage. The bovine milk peptide product obtained by this technical solution exhibited only low inhibitory activity against Escherichia coli (IC50 = 42.35 mg / mL) and showed no growth inhibitory activity against the other five microorganisms. The yield was 35%, and the product peptide content was 43%.

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

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

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

Claims

1. An application of a milk antimicrobial peptide prepared by a fermentation method, characterized in that: After fermentation, centrifugation, ultrafiltration separation, nanofiltration concentration and drying, the milk antimicrobial peptides are finally obtained; The specific steps include: (1) Culture medium preparation and fermentation culture A skim milk powder aqueous solution with a milk protein concentration of 12% was prepared as the fermentation medium. The mixture was thoroughly mixed and sterilized at 110°C for 15 min. Lactobacillus bulgaricus, Lactobacillus helveticus, and Bifidobacterium breve strains were stored at -80°C using glycerol. 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 steps at a 2% (v / v) inoculation volume. The culture was incubated in an anaerobic environment at 35°C for 8 h to allow the strains to recover. This recovery procedure was repeated twice. The bacteria were inoculated into the fermentation medium at a rate of 2%, and the bacterial content in the fermentation liquid was 1×10^ 8 CFU / mL, fermented in an anaerobic environment at 200 rpm and 35 °C for 30 h; sterilized at 121 °C for 20 min to inactivate the bacteria and terminate the fermentation; (2) Antimicrobial peptide purification The fermentation broth was centrifuged at 6000 rpm and 4°C for 20 min to remove impurities and insoluble matter from the fermentation broth, and the supernatant was retained. The fermentation supernatant was diluted with pure water to 4 times the original supernatant volume and separated by ultrafiltration membrane with a molecular weight cutoff of 5 kDa, and the ultrafiltrate was collected. The fermentation ultrafiltrate was further separated by nanofiltration membrane with a molecular weight cutoff of 150 Da and concentrated to the initial volume of the fermentation broth, and the nanofiltration retentate was collected. (3) Collect the nanofiltration retentate and dry it to obtain milk antimicrobial peptides; store it in a freezer at -20°C; The milk antimicrobial peptide prepared by the fermentation method is used to prepare a bacterial growth inhibitor or bactericide, and the antimicrobial peptide has growth inhibitory activity against one or more of Staphylococcus aureus, Escherichia coli, Propionibacterium acnes, Pseudomonas aeruginosa, Porphyromonas gingivalis and Streptococcus mutans.

2. A use of the bovine milk antimicrobial peptide of claim 1 in the preparation of a microbial growth inhibitor, wherein the growth inhibitor is an anti-infective drug, a skin care product, an oral antibacterial agent, a food preservative, or a preservative; and the microorganism is Staphylococcus aureus, Escherichia coli, Propionibacterium acnes, Pseudomonas aeruginosa, Porphyromonas gingivalis, and Streptococcus mutans.

3. The use according to claim 2, characterized in that: The anti-infection medicine uses the milk antimicrobial peptide as an active ingredient, and pharmaceutically acceptable carriers or auxiliary materials are added therein.

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