Preparation method of composite lipopeptide capable of preventing high-temperature inactivation

Abstract

The invention discloses a preparation method of composite lipopeptide capable of preventing high-temperature inactivation, and belongs to the technical field of bioactive peptide preparation. The preparation method comprises the following steps: mixing and dissolving a modified phospholipid compound, cholesterol and chloroform, and performing rotary evaporation to obtain a lipid film; mixing and shaking the lipid film, the lipopeptide mixture and a phosphate buffer solution, and performing ultrasonic treatment to obtain a composite lipopeptide liposome suspension; and carrying out spray drying on the composite lipopeptide liposome suspension to obtain composite lipopeptide powder. A lipopeptide mixture obtained through fermentation is mixed with a modified phospholipid compound and cholesterol, a high-temperature-resistant composite lipopeptide liposome suspension is constructed, and after high-temperature spray drying, the obtained composite lipopeptide still has good antibacterial activity.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptide preparation, and in particular relates to a method for preparing a composite lipopeptide capable of preventing high temperature inactivation. Background Art

[0002] Antimicrobial peptides are a class of amphiphilic, positively charged small molecule peptides that exist in most organisms and have broad-spectrum antimicrobial activity. According to the structural characteristics of the peptide chain, antimicrobial peptides can be divided into two categories: one is a helical, open linear peptide rich in cysteine; the other is a cyclic peptide that is self-connected into a ring, and these two peptide chain structures form lipopeptides through fatty acids connected to acyl groups. Lipopeptides composed of cyclic peptides and fatty acids have the advantages of a wide antimicrobial spectrum, rapid and powerful effects, and are not easy to develop drug resistance. They have broad application prospects in the fields of medicine, cosmetics, food and agriculture. Bacillus bacteria are widely present in organisms and the environment. They are a class of bacteria with important application value in agriculture, industry, medicine and health. Among them, Bacillus subtilis is a recognized probiotic and has been widely used. In the field of plant disease prevention and control, Bacillus bacteria are the most promising alternative to conventional pesticides. They can promote plant growth, induce plant resistance, inhibit different diseases, and directly antagonize microbial pathogens by competing for space and nutrients. Although the beneficial protective effects of Bacillus involve multiple mechanisms, the production of antimicrobial lipopeptide compounds is one of the most important factors affecting its biocontrol activity.

[0003] Bacillus can produce a variety of antimicrobial substances, mainly including lipopeptide antibiotics such as surfactin, iturin and fengycin. Surfactin, iturin and fengycin are a class of antimicrobial lipopeptides mainly produced by Gram-positive Bacillus through non-ribosomal synthesis pathways. They are generally composed of a β-hydroxy fatty acid and a 7-10 amino acid peptide chain connected by an amide bond. The preparation of lipopeptides is generally obtained by amplifying the culture and fermentation of Bacillus to obtain a fermentation broth, and then separating and purifying the fermentation broth.

[0004] Patent CN116082470A discloses an antibacterial lipopeptide of Bacillus Velezii and its preparation method and application. The invention activates and ferments the lipopeptide-producing Bacillus Velezii, precipitates it by centrifugation, and finally mixes it with an emulsion and spray-dries it to obtain the antibacterial lipopeptide powder.

[0005] Patent CN116496357A discloses an antimicrobial lipopeptide of iturin A and a preparation method thereof. In the invention, Bacillus subtilis is used as a lipopeptide production strain. By optimizing the fermentation medium and fermentation conditions, the content of the antimicrobial lipopeptide produced by the optimized fermentation medium is greatly improved compared with that produced by pure soybean meal fermentation culture.

[0006] Patent CN110790820A discloses a lipopeptide produced by Bacillus strain FJAT-52631 and a preparation method thereof. The invention prepares lipopeptide powder through the steps of activation, fermentation and freeze-drying.

[0007] Due to its structural characteristics, antimicrobial lipopeptides are more resistant to high temperatures than general small molecule peptides. Sometimes, they are placed in a temperature environment of 60℃~100℃ and still maintain good antibacterial activity. Although they cannot be placed in a high temperature environment for a long time, they have good high temperature resistance compared with ordinary oligopeptides. In the actual application of lipopeptides, according to the requirements of the application environment and storage, they need to be dried and solidified into powder for transportation, storage and preparation at any time. Spray drying is a commonly used material drying technology, which is widely used in food, pharmaceutical and chemical industries. Its basic principle is to send liquid materials into the drying chamber for atomization, and then in the process of contact with hot air, the water is rapidly vaporized, so that the liquid material is converted into dry powder, which has the advantages of low cost, rapid drying and suitable for mass production. Freeze drying is also a drying method for treating liquid materials into solid powders, which uses the principle of ice crystal sublimation to achieve drying. Compared with spray drying, the operating cost is high, and the freeze drying time is much longer than spray drying. Therefore, in actual use, the application range of spray drying is wider. Although lipopeptides are resistant to high temperatures, the spray drying temperature is relatively high, usually needing to reach 150°C~180°C, and sometimes even higher drying temperatures are required, which undoubtedly has a great destructive effect on lipopeptides. Although drying can be achieved, the activity is destroyed, resulting in poor antibacterial effect.

[0008] Therefore, it is of great significance to protect the fermented lipopeptide mixture from high temperature so that it can retain high antibacterial activity during the high-temperature drying process. Summary of the invention

[0009] In view of the deficiencies of the prior art, the present invention mixes the lipopeptide mixture obtained by fermentation with a modified phospholipid compound and cholesterol to construct a high temperature resistant composite lipopeptide liposome suspension. After high temperature spray drying, the obtained composite lipopeptide still has good antibacterial activity, solving the technical problems raised in the background technology. Specifically, the technical solution of the present invention includes the following contents: A method for preparing a composite lipopeptide that prevents high temperature inactivation, the method comprising the following steps: The modified phospholipid compound, cholesterol and chloroform are mixed and dissolved, and then rotary evaporated at 40°C to 45°C for 40min to 50min to obtain a lipid film; The lipid film, lipopeptide mixture and phosphate buffer are mixed and shaken, and then subjected to ultrasonic treatment at 200W-300W power for 2min-3min to obtain a composite lipopeptide liposome suspension; The composite lipopeptide liposome suspension is spray-dried to obtain composite lipopeptide powder.

[0010] Furthermore, the preparation method of the modified phospholipid compound comprises the following steps: The amino-terminated phospholipid compound, the aldehyde-based cross-linking agent and chloroform are mixed and stirred to form a mixed dispersion; The mixed dispersion and acetic acid are mixed and then subjected to microwave treatment to obtain a mixed solution, and the mixed solution is evaporated under reduced pressure and washed and dried to obtain the modified phospholipid compound.

[0011] Furthermore, the amino-terminated phospholipid compound includes 1-palmitoyl-2-oleoylphosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0012] Furthermore, the aldehyde-based cross-linking agent includes glyoxal.

[0013] Furthermore, the mass ratio of the amino-terminated phospholipid compound: the aldehyde cross-linking agent: chloroform is 1: 0.2-0.3: 1000-1500.

[0014] Furthermore, the amount of acetic acid added is 0.4 μL to 0.8 μL per mg of the mixed dispersion.

[0015] Furthermore, the microwave treatment conditions include a temperature of 40°C to 50°C, a power of 200W to 300W, a rotation speed of 50r / min to 100r / min and a treatment time of 10min to 15min.

[0016] Furthermore, the preparation method of the lipopeptide mixture comprises the following steps: After activation, the Bacillus was inoculated into a seed liquid culture medium and cultured at 37°C for 24 hours to obtain a seed liquid; The seed liquid is inoculated into a fermentation medium at an inoculum rate of 5% and cultured at 30±1° C. for 40 hours to obtain a fermentation liquid; The fermentation liquid is acidified to a pH of 2.0 and separated to obtain a precipitate, which is then purified to obtain the lipopeptide mixture.

[0017] Furthermore, the Bacillus is Bacillus subtilis with strain number CICC 10732, Bacillus pumilus with strain number CICC21890 and Bacillus licheniformis with strain number CICC 10103.

[0018] Furthermore, the seed liquid culture medium is composed of 5 g of peptone, 2.5 g of beef extract powder, 2.5 g of yeast extract, 7.5 g of sodium chloride, 5 g of glucose, and distilled water to make up to 1 L.

[0019] Furthermore, the fermentation medium is composed of 20 g of glucose, 5 g of sodium L-glutamate, 0.5 g of magnesium sulfate, 0.5 g of potassium chloride, 1 g of potassium dihydrogen phosphate, 0.15 mg of ferrous sulfate, 0.16 mg of copper sulfate, and distilled water to make up to 1 L.

[0020] Furthermore, the pH of the phosphate buffer is 5.0-5.8.

[0021] Furthermore, the weight ratio of the modified phospholipid compound: cholesterol: chloroform: lipopeptide mixture: phosphate buffer is 10-25: 2.5-10: 1000-2000: 1: 450-1000.

[0022] Furthermore, the spray drying conditions include a feed rate of 10 ml to 12 ml / min, an inlet air temperature of 140° C. to 150° C., and an outlet air temperature of 50° C. to 55° C.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts the idea of ​​liposome encapsulation, and utilizes the core structure phospholipid bilayer of liposome, which contains a hydrophobic structure layer and a hydrophilic structure layer, so as to realize the encapsulation of hydrophilic and hydrophobic substances. However, since the liposome suspension obtained by only using the liposome encapsulation in the prior art is not resistant to high temperature, the raw material phospholipids forming the liposome structure are analyzed, and the terminal amino phospholipid compound containing the amino structure is improved, and the amino group can be covalently cross-linked with the aldehyde group to form a high temperature resistant imine bond structure, and the aldehyde cross-linking agent with a shorter carbon chain is used for improvement, so that the prepared modified phospholipid compound has high temperature resistance. Subsequently, the modified phospholipid compound and cholesterol are prepared into a lipid film, and then mixed and shaken with the lipopeptide composition obtained by fermentation and culture, and a phosphate buffer solution, and ultrasonically treated to obtain a composite lipopeptide liposome suspension, and the composite lipopeptide liposome suspension is prepared and treated by a high temperature spray drying process to obtain a composite lipopeptide powder with high antibacterial activity. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below through the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available or can be prepared by known methods.

[0026] Preparation Example 1: The preparation of seed liquid culture medium specifically includes the following processes: Weigh 5 g of peptone, 2.5 g of beef extract powder, 2.5 g of yeast extract, 7.5 g of sodium chloride and 5 g of glucose, mix them, add distilled water to make up to 1 L, then sterilize them by wet heat at 121° C. for 15 min, and then cool to room temperature to obtain a seed liquid culture medium.

[0027] Preparation Example 2: The preparation of fermentation medium specifically includes the following processes: Weigh 20 g of glucose, 5 g of sodium L-glutamate, 0.5 g of magnesium sulfate, 0.5 g of potassium chloride, 1 g of potassium dihydrogen phosphate, 0.15 mg of ferrous sulfate and 0.16 mg of copper sulfate, mix them, add distilled water to make the volume to 1 L, then sterilize it with wet heat at 121°C for 15 min, and then cool it to room temperature to obtain a seed liquid culture medium.

[0028] Preparation Example 3: The preparation of the lipopeptide mixture specifically includes the following processes: Bacillus subtilis with strain number CICC 10732, Bacillus pumilus with strain number CICC 21890 and Bacillus licheniformis with strain number CICC 10103 were inoculated in PDA medium and activated in an incubator at 32°C ± 1°C for 24 hours. After activation, a sterilized inoculation loop was used to pick a loop from the PDA medium and inoculated into the seed liquid medium obtained in Preparation Example 1, and then placed in a shaker at 37°C for 24 hours to obtain a seed liquid. The seed liquid was inoculated into the fermentation medium obtained in Preparation Example 2 according to an inoculation amount of 5%, and cultured at a speed of 150r / min for 40 hours in a shaker environment at 30°C ± 1°C. After the culture was completed, the bacteria were removed by centrifugation to obtain a fermentation liquid. After the fermentation liquid was adjusted to pH 2.0 with hydrochloric acid, it was allowed to stand in a refrigerator at 5°C for 24 hours. After standing, the precipitate was separated and collected at a centrifugal speed of 8000r / min. The precipitate was dissolved with methanol, the pH was adjusted to 7, and then placed in a refrigerator at 5°C for 12 hours, and then eluted to obtain a lipopeptide mixture.

[0029] Preparation Example 4: The preparation of modified phospholipid compounds specifically includes the following processes: 10 mg of 1-palmitoyl-2-oleoyl phosphatidylethanolamine was weighed with an electronic analytical balance and placed in a round-bottom flask, and then 10 g of chloroform solution was added to mix so that 1-palmitoyl-2-oleoyl phosphatidylethanolamine was evenly dissolved. 2 mg of glyoxal was weighed with an electronic analytical balance and added to the chloroform solution containing 1-palmitoyl-2-oleoyl phosphatidylethanolamine to mix and disperse evenly to obtain a mixed dispersion, and then 8.8 μL of acetic acid was taken with a pipette and added to the mixed dispersion and placed in a microwave reactor at 40°C, the power was adjusted to 200 W, and the reaction was stirred at a speed of 50 r / min for 10 minutes. After the reaction was completed, the obtained mixed solution was cooled to room temperature, and then the solid was collected by reduced pressure evaporation, rinsed with ethanol, and then rinsed with deionized water, and then dried to obtain modified 1-palmitoyl-2-oleoyl phosphatidylethanolamine.

[0030] Preparation Example 5: The preparation of modified phospholipid compounds specifically includes the following processes: 10 mg of 1-palmitoyl-2-oleoyl phosphatidylethanolamine was weighed with an electronic analytical balance and placed in a round-bottom flask, and then 11 g of chloroform solution was added to mix so that 1-palmitoyl-2-oleoyl phosphatidylethanolamine was evenly dissolved. 2.2 mg of glyoxal was weighed with an electronic analytical balance and added to the chloroform solution containing 1-palmitoyl-2-oleoyl phosphatidylethanolamine to mix and disperse evenly to obtain a mixed dispersion, and then 11.4 μL of acetic acid was drawn with a pipette and added to the mixed dispersion and placed in a microwave reactor at 42° C., the power was adjusted to 220 W, and the reaction was stirred at a speed of 60 r / min for 11 minutes. After the reaction was completed, the obtained mixed solution was cooled to room temperature, and then the solid was collected by reduced pressure evaporation, rinsed with ethanol, and then rinsed with deionized water, and then dried to obtain modified 1-palmitoyl-2-oleoyl phosphatidylethanolamine.

[0031] Preparation Example 6: The preparation of modified phospholipid compounds specifically includes the following processes: 10 mg of 1-palmitoyl-2-oleoyl phosphatidylethanolamine was weighed with an electronic analytical balance and placed in a round-bottom flask, and then 12 g of chloroform solution was added to mix so that 1-palmitoyl-2-oleoyl phosphatidylethanolamine was evenly dissolved. 2.4 mg of glyoxal was weighed with an electronic analytical balance and added to the chloroform solution containing 1-palmitoyl-2-oleoyl phosphatidylethanolamine to mix and disperse evenly to obtain a mixed dispersion, and then 14.4 μL of acetic acid was drawn with a pipette and added to the mixed dispersion and placed in a microwave reactor at 44°C, the power was adjusted to 240 W, and the reaction was stirred at a speed of 70 r / min for 12 minutes. After the reaction was completed, the obtained mixed solution was cooled to room temperature, and then the solid was collected by reduced pressure evaporation, rinsed with ethanol, and then rinsed with deionized water, and then dried to obtain modified 1-palmitoyl-2-oleoyl phosphatidylethanolamine.

[0032] Preparation Example 7: The preparation of modified phospholipid compounds specifically includes the following processes: 10 mg of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine was weighed with an electronic analytical balance and placed in a round-bottom flask, and then 13 g of chloroform solution was added to mix so that 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine was evenly dissolved. 2.6 mg of glyoxal was weighed with an electronic analytical balance and added to the chloroform solution containing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine to mix and disperse evenly to obtain a mixed dispersion, and then 18 μL of acetic acid was drawn with a pipette and added to the mixed dispersion and placed in a microwave reactor at 46°C, the power was adjusted to 260 W, and the reaction was stirred at a speed of 80 r / min for 13 minutes. After the reaction was completed, the obtained mixed solution was cooled to room temperature, and then the solid was collected by reduced pressure evaporation, rinsed with ethanol, and then rinsed with deionized water, and then dried to obtain modified 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0033] Preparation Example 8: The preparation of modified phospholipid compounds specifically includes the following processes: 10 mg of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine was weighed with an electronic analytical balance and placed in a round-bottom flask, and then 14 g of chloroform solution was added to mix so that 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine was evenly dissolved. 2.8 mg of glyoxal was weighed with an electronic analytical balance and added to the chloroform solution containing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine to mix and disperse evenly to obtain a mixed dispersion, and then 20 μL of acetic acid was drawn with a pipette and added to the mixed dispersion and placed in a microwave reactor at 48°C, the power was adjusted to 280 W, and the reaction was stirred at a speed of 90 r / min for 14 minutes. After the reaction was completed, the obtained mixed solution was cooled to room temperature, and then the solid was collected by reduced pressure evaporation, rinsed with ethanol, and then rinsed with deionized water, and then dried to obtain modified 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0034] Preparation Example 9: The preparation of modified phospholipid compounds specifically includes the following processes: 10 mg of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine was weighed with an electronic analytical balance and placed in a round-bottom flask, and then 15 g of chloroform solution was added to mix so that 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine was evenly dissolved. 3 mg of glyoxal was weighed with an electronic analytical balance and added to the chloroform solution containing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine to mix and disperse evenly to obtain a mixed dispersion, and then 22.4 μL of acetic acid was drawn with a pipette and added to the mixed dispersion and placed in a microwave reactor at 50°C, the power was adjusted to 300 W, and the reaction was stirred at a speed of 100 r / min for 15 minutes. After the reaction was completed, the obtained mixed solution was cooled to room temperature, and then the solid was collected by reduced pressure evaporation, rinsed with ethanol, and then rinsed with deionized water, and then dried to obtain modified 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0035] Preparation Example 10: The preparation of modified phospholipid compounds specifically includes the following processes: The 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine in Preparation Example 9 was replaced with phosphatidylinositol, and the other conditions were consistent with the example.

[0036] Preparation Example 11: The preparation of modified phospholipid compounds specifically includes the following processes: The glyoxal in Preparation Example 9 was replaced by n-hexanal, and the other conditions were kept the same as those in the example. Example

[0037] A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: 10 parts by weight of the modified phospholipid compound obtained in Preparation Example 4 and 2.5 parts by weight of cholesterol were placed in a dry rotary evaporation bottle, and 1000 parts by weight of chloroform were added to mix and dissolve evenly, and then rotary evaporated at 40° C. for 40 minutes to obtain a lipid film; 1 part by weight of the lipopeptide mixture and 450 parts by weight of a phosphate buffer solution with a pH of 5.0 were added to the lipid film, and then placed in a shaker at 37±2°C, shaken at a speed of 150 r / min for 50 min, and then placed in an ultrasonic machine, the ultrasonic power was adjusted to 200 W, and ultrasonicated for 2 min to obtain a composite lipopeptide liposome suspension (0.5 mL of the composite lipopeptide liposome suspension was centrifuged at 5000 r / min for 20 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 5 kDa, and the liquid obtained by centrifugation was taken and the encapsulation efficiency of the composite lipopeptide liposome suspension was measured by the BCA method to be 72.05%). Subsequently, the composite lipopeptide suspension was fed at a feed rate of 10 ml / min, the inlet temperature of the small spray dryer was adjusted to 140°C, and the outlet temperature was adjusted to 50°C, and the composite lipopeptide powder was obtained by spray drying. Example

[0038] A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: 13 parts by weight of the modified phospholipid compound obtained in Preparation Example 5 and 4 parts by weight of cholesterol were placed in a dry rotary evaporation bottle, and 1200 parts by weight of chloroform were added to mix and dissolve evenly, and then rotary evaporated at 41° C. for 42 minutes to obtain a lipid film; 1 part by weight of the lipopeptide mixture and 600 parts by weight of a phosphate buffer solution with a pH of 5.0 were added to the lipid film, and then placed in a shaker at 37±2°C, shaken at a speed of 160r / min for 52min, and then placed in an ultrasonic machine, the ultrasonic power was adjusted to 220W, and ultrasonicated for 2min to obtain a composite lipopeptide liposome suspension (0.5mL of the composite lipopeptide liposome suspension was centrifuged at a speed of 5000r / min for 20min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 5kDa, and the liquid obtained by centrifugation was taken and the encapsulation efficiency of the composite lipopeptide liposome suspension was measured by the BCA method to be 73.27%). Subsequently, the composite lipopeptide suspension was fed at a feed rate of 10ml / min, the inlet temperature of the small spray dryer was adjusted to 140°C, and the outlet temperature was adjusted to 50°C, and the composite lipopeptide powder was obtained by spray drying. Example

[0039] A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: 15 parts by weight of the modified phospholipid compound obtained in Preparation Example 6 and 5.5 parts by weight of cholesterol were placed in a dry rotary evaporation bottle, and 1400 parts by weight of chloroform were added to mix and dissolve evenly, and then rotary evaporated at 42° C. for 44 minutes to obtain a lipid film; 1 part by weight of the lipopeptide mixture and 700 parts by weight of a phosphate buffer solution with a pH of 5.0 were added to the lipid film, and then placed in a shaker at 37±2°C, shaken at a speed of 170r / min for 54min, and then placed in an ultrasonic machine, the ultrasonic power was adjusted to 240W, and ultrasonicated for 3min to obtain a composite lipopeptide liposome suspension (0.5mL of the composite lipopeptide liposome suspension was centrifuged at a speed of 5000r / min for 20min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 5kDa, and the liquid obtained by centrifugation was taken and the encapsulation efficiency of the composite lipopeptide liposome suspension was measured by the BCA method to be 74.31%). Subsequently, the composite lipopeptide suspension was fed at a feed rate of 11ml / min, the inlet temperature of the small spray dryer was adjusted to 145°C, and the outlet temperature was adjusted to 55°C, and the composite lipopeptide powder was obtained by spray drying. Example

[0040] A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: 18 parts by weight of the modified phospholipid compound obtained in Preparation Example 7 and 7 parts by weight of cholesterol were placed in a dry rotary evaporation bottle, and 1600 parts by weight of chloroform were added to mix and dissolve evenly, and then rotary evaporated at 43° C. for 46 minutes to obtain a lipid film; 1 part by weight of the lipopeptide mixture and 800 parts by weight of a phosphate buffer solution with a pH of 5.8 were added to the lipid film, and then placed in a shaker at 37±2°C, shaken at a speed of 180 r / min for 56 min, and then placed in an ultrasonic machine, the ultrasonic power was adjusted to 260 W, and ultrasonicated for 4 min to obtain a composite lipopeptide liposome suspension (0.5 mL of the composite lipopeptide liposome suspension was centrifuged at a speed of 5000 r / min for 20 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 5 kDa, and the liquid obtained by centrifugation was taken and the encapsulation efficiency of the composite lipopeptide liposome suspension was measured by the BCA method to be 76.02%). Subsequently, the composite lipopeptide suspension was fed at a feed rate of 11 ml / min, the inlet temperature of the small spray dryer was adjusted to 145°C, and the outlet temperature was adjusted to 55°C, and the composite lipopeptide powder was obtained by spray drying. Example

[0041] A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: 22 parts by weight of the modified phospholipid compound obtained in Preparation Example 8 and 8.5 parts by weight of cholesterol were placed in a dry rotary evaporation bottle, and 1800 parts by weight of chloroform were added to mix and dissolve evenly, and then rotary evaporated at 44° C. for 48 minutes to obtain a lipid film; 1 part by weight of the lipopeptide mixture and 900 parts by weight of a phosphate buffer solution with a pH of 5.8 were added to the lipid film, and then placed in a shaker at 37±2°C, shaken at a speed of 190 r / min for 58 min, and then placed in an ultrasonic machine, the ultrasonic power was adjusted to 280 W, and ultrasonicated for 4 min to obtain a composite lipopeptide liposome suspension (0.5 mL of the composite lipopeptide liposome suspension was centrifuged at a speed of 5000 r / min for 20 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 5 kDa, and the liquid obtained by centrifugation was taken and the encapsulation efficiency of the composite lipopeptide liposome suspension was measured by the BCA method to be 76.78%). Subsequently, the composite lipopeptide suspension was fed at a feed rate of 12 ml / min, the air inlet temperature of the small spray dryer was adjusted to 150°C, and the air outlet temperature was adjusted to 55°C, and the composite lipopeptide powder was obtained by spray drying. Example

[0042] A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: 25 parts by weight of the modified phospholipid compound obtained in Preparation Example 9 and 10 parts by weight of cholesterol were placed in a dry rotary evaporation bottle, and 2000 parts by weight of chloroform were added to mix and dissolve evenly, and then rotary evaporated at 45° C. for 50 minutes to obtain a lipid film; 1 part by weight of the lipopeptide mixture and 1000 parts by weight of a phosphate buffer solution with a pH of 5.8 were added to the lipid film, and then placed in a shaker at 37±2°C, shaken at a speed of 200 r / min for 60 min, and then placed in an ultrasonic machine, the ultrasonic power was adjusted to 300 W, and ultrasonicated for 5 min to obtain a composite lipopeptide liposome suspension (0.5 mL of the composite lipopeptide liposome suspension was centrifuged at 5000 r / min for 20 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 5 kDa, and the liquid obtained by centrifugation was taken and the encapsulation efficiency of the composite lipopeptide liposome suspension was measured by the BCA method to be 76.85%). Subsequently, the composite lipopeptide suspension was fed at a feed rate of 12 ml / min, the air inlet temperature of the small spray dryer was adjusted to 150°C, and the air outlet temperature was adjusted to 55°C, and the composite lipopeptide powder was obtained by spray drying.

[0043] Comparative Example 1: A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: The modified phospholipid compound in Example 6 was replaced by the modified phospholipid compound obtained in Preparation Example 10, and the other conditions were kept the same as in Example 6. The encapsulation efficiency of the composite lipopeptide liposome suspension was measured to be 75.56%.

[0044] Comparative Example 2: A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: The modified phospholipid compound in Example 6 was replaced with the modified phospholipid compound obtained in Preparation Example 11, and the other conditions were kept consistent with Example 6. It was found that it was difficult to form a lipid film during the rotary evaporation treatment, and the preparation failed. Analysis of the reasons: Although n-hexanal contains aldehyde groups, it can introduce high-temperature resistant imine bonds through covalent cross-linking of aldehyde groups and amino groups. However, since n-hexanal contains a relatively long hydrophobic carbon chain, after being introduced into the structure of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, it may further lead to an increase in the hydrophobicity of the modified phospholipid compound prepared by this comparative example, thereby hindering the embedding of cholesterol into the phospholipid molecules, making it difficult to form a film.

[0045] Comparative Example 3: A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: The modified phospholipid compound in Example 6 was replaced with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and the other conditions were kept consistent with Example 6. The encapsulation efficiency of the composite lipopeptide liposome suspension was measured to be 76.59%.

[0046] Comparative Example 4: A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: 20 parts by weight of β-cyclodextrin were added to 1500 parts by weight of phosphate buffer with a pH of 5.8, heated and stirred until completely dissolved, and then 5 parts by weight of lipopeptide mixture and 7 parts by weight of Tween 20 were added, and then stirred and allowed to stand for 5 hours at room temperature to obtain a suspension, which was placed in a -20°C refrigerator for 24 hours, and then taken out and dried in a -50°C freeze dryer for 24 hours to obtain a composite lipopeptide powder. 1g of the composite lipopeptide powder was dissolved in 300ml of anhydrous ethanol, and after 400W ultrasonic treatment for 10 minutes, the encapsulation efficiency of the composite lipopeptide was 18.75% by high performance liquid chromatography.

[0047] Comparative Example 5: A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: The air inlet temperature in Example 6 was lowered to 100°C, and the other conditions were kept consistent with Example 6. It was found that the composite lipopeptide powder obtained by drying had a more serious wall sticking phenomenon. This may be due to the low temperature, which caused the water to evaporate slowly, resulting in the spray-dried powder having a higher moisture content, which was prone to wall sticking and was not conducive to production.

[0048] Comparative Example 6: A preparation method of a composite lipopeptide that can prevent high temperature inactivation, specifically comprising the following processes: The inlet air temperature in Example 6 was raised to 200°C, and the other conditions were kept the same as those in Example 6. The composite lipopeptide powder obtained in Examples 1 to 6 and Comparative Examples 1 to 6 was dissolved in a phosphate buffer solution with a pH of 5.0 to a concentration of 50 mg / ml, and then mixed and solidified with the PDA culture medium raw material to obtain a PDA culture medium containing composite lipopeptides (composite lipopeptide concentration of 50 mg / L), and then the apple ring rot fungus cake and tomato gray mold fungus cake with a diameter of 10 mm were inoculated on the culture medium. The blank group did not use composite lipopeptides, and the control group directly used the lipopeptide composition to prepare the PDA culture medium (lipopeptide composition concentration of 50 mg / L). Then it was placed in an incubator at 25°C for 24 hours, and the antibacterial rate was calculated by counting the ratio of the colony diameter of the culture medium containing composite lipopeptides to the colony diameter difference on the blank group culture medium. The results are shown in Table 1 below.

[0049] Table 1 Antibacterial effect of composite lipopeptide Sources Inhibition rate of apple ring rot pathogen (%) Tomato gray mold inhibition rate (%) Example 1 80.41 74.86 Example 2 81.03 75.77 Example 3 82.49 77.81 Example 4 84.75 78.69 Example 5 85.51 79.98 Example 6 86.06 80.47 Comparative Example 1 5.62 3.89 Comparative Example 3 5.97 3.95 Comparative Example 4 18.59 15.07 Comparative Example 6 8.45 5.62 Control group 93.75 88.52 The following conclusions can be drawn from the above antibacterial test results: (1) It can be found from Examples 1 to 6 that the present invention constructs a high-temperature resistant composite lipopeptide liposome suspension by modifying phospholipids to be resistant to high temperatures, and then mixing the fermented lipopeptide mixture with the modified phospholipid compound and cholesterol. After high-temperature spray drying, the obtained composite lipopeptide still has good antibacterial activity.

[0050] (2) It can be found from Comparative Example 1 that since phosphatidylinositol does not contain an amino group in its structure, glyoxal cannot construct a high-temperature resistant imine bond on phosphatidylinositol through covalent cross-linking, so that the modified phospholipid compound prepared in this comparative example cannot be modified to be resistant to high temperature, which in turn leads to low antibacterial properties and activity of the composite lipopeptide obtained by high-temperature spray drying.

[0051] (3) It can be found from Comparative Example 3 that although 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine can participate in the formation of a liposome structure encapsulating the composite lipopeptide, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is not resistant to high temperatures and is easily broken or even oxidatively degraded in the high temperature environment of spray drying, thereby losing its protective effect on the lipopeptide mixture, resulting in low antibacterial activity of the composite lipopeptide prepared by this comparative example.

[0052] (4) It can be found from Comparative Example 4 that the encapsulation effect of β-cyclodextrin is based on the hydrophobic effect of the hydrophobic inner cavity of the structure, which can achieve the encapsulation of hydrophobic substances. Although the lipopeptide mixture has hydrophilic and hydrophobic structures, due to the strong hydrophilicity of the lipopeptide, less of the lipopeptide mixture enters the hydrophobic inner cavity of cyclodextrin, and the hydrophilic part of the lipopeptide mixture cannot be encapsulated, resulting in low antibacterial activity.

[0053] (5) It can be found from Comparative Example 6 that although further increasing the inlet air temperature is beneficial to shortening the spray drying time, the antibacterial activity of the composite lipopeptide powder obtained by drying is relatively low. This may be because the composite lipopeptide liposome suspension obtained by the present invention, which is constructed by improving the high temperature resistance of phospholipids, can only have a high temperature protection effect within a certain temperature range, and has a more serious inactivation phenomenon when exceeding a certain temperature.

[0054] The above-described embodiments provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected.

Claims

1. A method for preparing a composite lipopeptide that prevents high temperature inactivation, characterized in that: The preparation method comprises the following steps: The modified phospholipid compound, cholesterol and chloroform are mixed and dissolved, and then rotary evaporated at 40°C to 45°C for 40min to 50min to obtain a lipid film; The lipid film, lipopeptide mixture and phosphate buffer are mixed and shaken, and then subjected to ultrasonic treatment at 200W-300W power for 2min-3min to obtain a composite lipopeptide liposome suspension; The composite lipopeptide liposome suspension is spray-dried to obtain the composite lipopeptide.

2. The method for preparing a composite lipopeptide that prevents high temperature inactivation according to claim 1, characterized in that: The preparation method of the modified phospholipid compound comprises the following steps: The amino-terminated phospholipid compound, the aldehyde-based cross-linking agent and chloroform are mixed and stirred to form a mixed dispersion; The mixed dispersion and acetic acid are mixed and then subjected to microwave treatment to obtain a mixed solution, and the mixed solution is evaporated under reduced pressure and washed and dried to obtain the modified phospholipid compound.

3. The method for preparing a composite lipopeptide that prevents high temperature inactivation according to claim 2, characterized in that: The amino-terminated phospholipid compound includes 1-palmitoyl-2-oleoylphosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

4. The method for preparing a composite lipopeptide that prevents high temperature inactivation according to claim 2, characterized in that: The aldehyde-based cross-linking agent includes glyoxal.

5. The method for preparing a composite lipopeptide that prevents high temperature inactivation according to claim 2, characterized in that: The mass ratio of the amino-terminated phospholipid compound: the aldehyde cross-linking agent: chloroform is 1: 0.2-0.3: 1000-1500.

6. The method for preparing a composite lipopeptide that prevents high temperature inactivation according to claim 2, characterized in that: The amount of acetic acid added is 0.4 μL to 0.8 μL per mg of the mixed dispersion.

7. The method for preparing a composite lipopeptide that prevents high temperature inactivation according to claim 2, characterized in that: The microwave treatment conditions include a temperature of 40° C. to 50° C., a power of 200 W to 300 W, a rotation speed of 50 r / min to 100 r / min, and a treatment time of 10 min to 15 min.

8. The method for preparing a composite lipopeptide that prevents high temperature inactivation according to claim 1, characterized in that: The preparation method of the lipopeptide mixture comprises the following steps: After activation, the Bacillus was inoculated into a seed liquid culture medium and cultured at 37°C for 24 hours to obtain a seed liquid; The seed liquid is inoculated into a fermentation medium at an inoculum rate of 5% and cultured at 30±1° C. for 40 hours to obtain a fermentation liquid; The fermentation liquid is acidified to a pH of 2.0 and separated to obtain a precipitate, which is then purified to obtain the lipopeptide mixture.

9. The method for preparing a composite lipopeptide that prevents high temperature inactivation according to claim 1, characterized in that: The pH of the phosphate buffer is 5.0-5.

8.

10. The method for preparing a composite lipopeptide that prevents high temperature inactivation according to claim 1, characterized in that: The weight ratio of the modified phospholipid compound: cholesterol: chloroform: lipopeptide mixture: phosphate buffer is 10-25: 2.5-10: 1000-2000: 1: 450-1000.