Preparation method of a composite lipopeptide for preventing high-temperature inactivation

Abstract

The present invention discloses a preparation method of a composite lipopeptide for preventing high-temperature inactivation, belonging to the technical field of bioactive peptide preparation. The preparation method comprises the following steps: a lipid film is obtained by mixing and dissolving a modified phospholipid compound, cholesterol and chloroform and then performing rotary evaporation; after the lipid film, a lipopeptide mixture and a phosphate buffer solution are mixed and shaken, a composite lipopeptide liposome suspension is obtained through ultrasonic treatment; and the composite lipopeptide liposome suspension is spray-dried to obtain a composite lipopeptide powder. In the present invention, the lipopeptide mixture obtained by fermentation is mixed with the modified phospholipid compound and cholesterol to construct a high-temperature-resistant composite lipopeptide liposome suspension, 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 sp. strain FJAT - 52631 and its preparation method. Through steps such as activation, fermentation, and lyophilization, lipopeptide powder was prepared.

[0007] Due to the structural characteristics of antibacterial lipopeptides, they are more heat - resistant than ordinary small - molecule peptides. Sometimes, when placed in a temperature environment of 60°C - 100°C, they still maintain good antibacterial activity. Although they cannot be placed in a high - temperature environment for a long time, compared with ordinary oligopeptides, they already have good heat - resistant properties. In the practical application of lipopeptides, according to the requirements of the application environment and storage, etc., it is necessary to dry and solidify them into powder form for the purposes of transportation, storage, and preparation at any time. Spray drying is a commonly used material drying technology, widely used in industries such as food, pharmaceuticals, and chemicals. Its basic principle is to atomize liquid materials into the drying chamber and then, during the contact with hot air, the water quickly vaporizes, turning the liquid materials into dry powder, which has advantages such as low cost, rapid drying, and suitability for large - batch production. Freeze - drying is also a drying method for processing liquid materials into solid powder, which realizes drying by using the principle of ice crystal sublimation. Compared with spray drying, the operating cost is high, and the freeze - drying time is much longer than that of spray drying. Therefore, in actual use, the application scope of spray drying is more extensive. Although lipopeptides have heat - resistant properties, due to the relatively high temperature of spray drying, usually reaching 150°C - 180°C, and sometimes even higher drying temperatures, this undoubtedly has a greater destructive effect on lipopeptides. Although drying can be achieved, the activity is damaged, resulting in poor antibacterial effects.

[0008] Therefore, it is of great significance to protect the lipopeptide mixture obtained by fermentation from heat inactivation so that it retains 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 heat - resistant composite lipopeptide liposome suspension. After high - temperature spray drying, the obtained composite lipopeptide still has good antibacterial activity, solving the technical problems proposed in the background art. Specifically, the technical solution of the present invention includes the following content:

[0010] A preparation method of a composite lipopeptide for preventing heat inactivation, the preparation method comprising the following steps:

[0011] The modified phospholipid compound, cholesterol, and chloroform are mixed and dissolved, and then rotary evaporated at 40°C - 45°C for 40 min - 50 min to obtain a lipid film;

[0012] After mixing and shaking the lipid film, lipopeptide mixture and phosphate buffer solution, it is treated with ultrasonic power of 200W - 300W for 2min - 3min to obtain a composite lipopeptide liposome suspension;

[0013] The composite lipopeptide liposome suspension is spray-dried to obtain a composite lipopeptide powder.

[0014] Furthermore, the preparation method of the modified phospholipid compound includes the following steps:

[0015] The terminal amino phospholipid compound, aldehyde cross-linking agent and chloroform are mixed and stirred to form a mixed dispersion;

[0016] After the mixed dispersion is mixed with acetic acid and then treated by microwave, a mixed solution is obtained. The mixed solution is subjected to reduced pressure evaporation and washing and drying to obtain the modified phospholipid compound.

[0017] Furthermore, the terminal amino phospholipid compound includes 1-palmitoyl-2-oleoyl phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0018] Furthermore, the aldehyde cross-linking agent includes glyoxal.

[0019] Furthermore, the mass ratio of the terminal amino phospholipid compound: aldehyde cross-linking agent: chloroform is 1:0.2 - 0.3:1000 - 1500.

[0020] Furthermore, the addition amount of acetic acid is 0.4μL - 0.8μL added per milligram of the mixed dispersion.

[0021] Furthermore, the conditions of the microwave treatment include a temperature of 40°C - 50°C, a power of 200W - 300W, a rotation speed of 50r / min - 100r / min, and a treatment time of 10min - 15min.

[0022] Furthermore, the preparation method of the lipopeptide mixture includes the following steps:

[0023] The Bacillus is activated and inoculated into a seed liquid medium and cultured at 37°C for 24h to obtain a seed solution;

[0024] The seed solution is inoculated into a fermentation medium at an inoculation amount of 5% and cultured at 30±1°C for 40h to obtain a fermentation broth;

[0025] The fermentation broth is adjusted to pH 2.0 and separated to obtain a precipitate, and the precipitate is purified to obtain the lipopeptide mixture.

[0026] Furthermore, the Bacillus strains are Bacillus subtilis with strain number CICC 10732, Bacillus pumilus with strain number CICC 21890, and Bacillus licheniformis with strain number CICC 10103.

[0027] Furthermore, the seed liquid 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 added to make up 1 L.

[0028] Furthermore, the fermentation medium is composed of 20 g of glucose, 5 g of L-sodium 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 added to make up 1 L.

[0029] Furthermore, the pH of the phosphate buffer is 5.0 - 5.8.

[0030] 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.

[0031] Furthermore, the conditions for spray drying include a feed rate of 10 ml - 12 ml / min, an inlet air temperature of 140°C - 150°C, and an outlet air temperature of 50 - 55°C.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] The present invention adopts the idea of liposome encapsulation and utilizes the core structure of liposomes, the phospholipid bilayer. The phospholipid bilayer contains a hydrophobic structural layer and a hydrophilic structural layer, which can achieve the encapsulation of hydrophilic and hydrophobic substances. However, the liposome suspension obtained only by using liposome encapsulation in the prior art is not heat-resistant. By analyzing the raw material phospholipids forming the liposome structure, the terminal amino phospholipid compound containing an amino group is improved. The amino group can covalently crosslink with an aldehyde group to form a heat-resistant imine bond structure, and it is improved with an aldehyde crosslinking agent with a shorter carbon chain, so that the prepared modified phospholipid compound has heat-resistant performance. 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 culture and phosphate buffer and ultrasonically treated to obtain a composite lipopeptide liposome suspension. The composite lipopeptide liposome suspension is prepared by a high-temperature spray drying process to obtain a composite lipopeptide powder with high antibacterial activity. Specific Embodiments

[0034] The technical solutions of the present invention will be clearly and completely described below through embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0035] Unless otherwise specified, the raw materials and reagents used in the present invention below are all commercially available products or can be prepared by known methods.

[0036] Preparation Example 1:

[0037] Preparation of the seed liquid medium specifically includes the following process:

[0038] 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 by moist heat at 121 °C for 15 min, and then cool to room temperature to obtain the seed liquid medium.

[0039] Preparation Example 2:

[0040] Preparation of the fermentation medium specifically includes the following process:

[0041] Weigh 20 g of glucose, 5 g of L-sodium 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 up to 1 L, then sterilize by moist heat at 121 °C for 15 min, and then cool to room temperature to obtain the seed liquid medium.

[0042] Preparation Example 3:

[0043] Preparation of the lipopeptide mixture specifically includes the following process:

[0044] Bacillus subtilis with strain number CICC 10732, Bacillus pumilus with strain number CICC 21890, and Bacillus licheniformis with strain number CICC 10103 were co-inoculated on a slant of PDA medium and activated in an incubator at 32°C ± 1°C for 24 h. After activation, a loopful was picked from the PDA medium with a sterilized inoculation loop and inoculated into the seed liquid medium obtained in Preparation Example 1, and then placed in a shaker at 37°C and cultured for 24 h to obtain a seed solution. The seed solution was inoculated into the fermentation medium obtained in Preparation Example 2 at an inoculation amount of 5%, and cultured in a shaker at 30°C ± 1°C at a rotation speed of 150 r / min for 40 h. After the cultivation, the cells were removed by centrifugation to obtain a fermentation broth. The pH of the fermentation broth was adjusted to 2.0 with hydrochloric acid and then left standing in a refrigerator at 5°C for 24 h. After standing, the precipitate was separated and collected by centrifugation at a speed of 8000 r / min. The precipitate was dissolved in methanol, the pH was adjusted to 7, and then placed in a refrigerator at 5°C for 12 h, and then eluted to obtain a lipopeptide mixture.

[0045] Preparation Example 4:

[0046] Preparation of the modified phospholipid compound specifically includes the following process:

[0047] Weigh 10 mg of 1-palmitoyl-2-oleoyl phosphatidylethanolamine with an electronic analytical balance and place it in a round-bottom flask, then add 10 g of chloroform solution for mixing to dissolve 1-palmitoyl-2-oleoyl phosphatidylethanolamine evenly. Then weigh 2 mg of glyoxal with an electronic analytical balance and add it to the chloroform solution containing 1-palmitoyl-2-oleoyl phosphatidylethanolamine and mix and disperse evenly to obtain a mixed dispersion. Then pipette 8.8 μL of acetic acid into the mixed dispersion and place it in a microwave reactor at 40°C, adjust the power to 200 W, and stir and react at a rotation speed of 50 r / min for 10 min. After the reaction, the obtained mixed solution was cooled to room temperature, then evaporated under reduced pressure to collect the solid, rinsed with ethanol, then rinsed with deionized water, and then dried to obtain modified 1-palmitoyl-2-oleoyl phosphatidylethanolamine.

[0048] Preparation Example 5:

[0049] Preparation of the modified phospholipid compound specifically includes the following process:

[0050] Weigh 10 mg of 1-palmitoyl-2-oleoyl phosphatidylethanolamine with an electronic analytical balance and place it in a round-bottom flask. Then add 11 g of chloroform solution and mix to dissolve 1-palmitoyl-2-oleoyl phosphatidylethanolamine evenly. Weigh 2.2 mg of glyoxal with an electronic analytical balance and add it to the chloroform solution containing 1-palmitoyl-2-oleoyl phosphatidylethanolamine, and mix and disperse evenly to obtain a mixed dispersion. Then use a pipette to aspirate 11.4 μL of acetic acid and add it to the mixed dispersion, and place it in a microwave reactor at 42 °C. Adjust the power to 220 W and stir and react at a speed of 60 r / min for 11 min. After the reaction is completed, cool the obtained mixture to room temperature, then evaporate under reduced pressure to collect the solid, rinse it with ethanol, then rinse it with deionized water, and then dry it to obtain modified 1-palmitoyl-2-oleoyl phosphatidylethanolamine.

[0051] Preparation Example 6:

[0052] The preparation of the modified phospholipid compound specifically includes the following process:

[0053] Weigh 10 mg of 1-palmitoyl-2-oleoyl phosphatidylethanolamine with an electronic analytical balance and place it in a round-bottom flask. Then add 12 g of chloroform solution and mix to dissolve 1-palmitoyl-2-oleoyl phosphatidylethanolamine evenly. Weigh 2.4 mg of glyoxal with an electronic analytical balance and add it to the chloroform solution containing 1-palmitoyl-2-oleoyl phosphatidylethanolamine, and mix and disperse evenly to obtain a mixed dispersion. Then use a pipette to aspirate 14.4 μL of acetic acid and add it to the mixed dispersion, and place it in a microwave reactor at 44 °C. Adjust the power to 240 W and stir and react at a speed of 70 r / min for 12 min. After the reaction is completed, cool the obtained mixture to room temperature, then evaporate under reduced pressure to collect the solid, rinse it with ethanol, then rinse it with deionized water, and then dry it to obtain modified 1-palmitoyl-2-oleoyl phosphatidylethanolamine.

[0054] Preparation Example 7:

[0055] The preparation of the modified phospholipid compound specifically includes the following process:

[0056] Weigh 10 mg of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine with an electronic analytical balance and place it in a round-bottom flask. Then add 13 g of chloroform solution for mixing to dissolve 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine evenly. Weigh 2.6 mg of glyoxal with an electronic analytical balance and add it to the chloroform solution containing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine for mixing and dispersing evenly to obtain a mixed dispersion. Then use a pipette to aspirate 18 μL of acetic acid and add it to the mixed dispersion, and place it in a microwave reactor at 46 °C. Adjust the power to 260 W and stir and react at a speed of 80 r / min for 13 min. After the reaction, cool the obtained mixture to room temperature, then evaporate under reduced pressure to collect the solid, rinse it with ethanol, then rinse it with deionized water, and then dry it to obtain modified 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0057] Preparation Example 8:

[0058] The preparation of the modified phospholipid compound specifically includes the following process:

[0059] Weigh 10 mg of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine with an electronic analytical balance and place it in a round-bottom flask. Then add 14 g of chloroform solution for mixing to dissolve 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine evenly. Weigh 2.8 mg of glyoxal with an electronic analytical balance and add it to the chloroform solution containing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine for mixing and dispersing evenly to obtain a mixed dispersion. Then use a pipette to aspirate 20 μL of acetic acid and add it to the mixed dispersion, and place it in a microwave reactor at 48 °C. Adjust the power to 280 W and stir and react at a speed of 90 r / min for 14 min. After the reaction, cool the obtained mixture to room temperature, then evaporate under reduced pressure to collect the solid, rinse it with ethanol, then rinse it with deionized water, and then dry it to obtain modified 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0060] Preparation Example 9:

[0061] The preparation of the modified phospholipid compound specifically includes the following process:

[0062] Weigh 10 mg of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine using an electronic analytical balance and place it in a round-bottom flask. Then add 15 g of chloroform solution for mixing to dissolve 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine evenly. Weigh 3 mg of glyoxal using the electronic analytical balance and add it to the chloroform solution containing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine for mixing and dispersing evenly to obtain a mixed dispersion. Then use a pipette to aspirate 22.4 μL of acetic acid and add it to the mixed dispersion, and place it in a microwave reactor at 50 °C. Adjust the power to 300 W and stir and react at a speed of 100 r / min for 15 min. After the reaction is completed, cool the obtained mixed solution to room temperature, then evaporate under reduced pressure to collect the solid, wash it with ethanol, then wash it with deionized water, and then dry it to obtain modified 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0063] Preparation Example 10:

[0064] The preparation of the modified phospholipid compound specifically includes the following process:

[0065] Replace 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine in Preparation Example 9 with phosphatidylinositol, and keep the other conditions the same as those in Preparation Example 9.

[0066] Preparation Example 11:

[0067] The preparation of the modified phospholipid compound specifically includes the following process:

[0068] Replace glyoxal in Preparation Example 9 with n-hexanal, and keep the other conditions the same as those in Preparation Example 9. Example 1

[0069] The preparation of a composite lipopeptide for preventing high-temperature inactivation specifically includes the following process:

[0070] Take 10 parts by weight of the modified phospholipid compound obtained in Preparation Example 4 and 2.5 parts by weight of cholesterol and place them in a dry rotary evaporation flask. Then add 1000 parts by weight of chloroform for mixing and dissolving evenly. Subsequently, perform rotary evaporation at 40 °C for 40 min to obtain a lipid film;

[0071] Add 1 part by weight of the lipopeptide mixture and 450 parts by weight of phosphate buffer solution with a pH of 5.0 to the lipid film, then place it in a shaker at 37±2°C and shake at a speed of 150 r / min for 50 min. Then put it into an ultrasonic machine, adjust the ultrasonic power to 200 W, and ultrasonicate for 2 min to obtain a composite lipopeptide liposome suspension (take 0.5 mL of the composite lipopeptide liposome suspension and centrifuge it at a speed of 5000 r / min for 20 min using an ultrafiltration centrifugal tube with a molecular weight cut-off of 5 kDa. Absorb the liquid obtained by centrifugal separation and measure the encapsulation efficiency of the composite lipopeptide liposome suspension by the BCA method, which is 72.05%). Subsequently, adjust the inlet air temperature of the small spray dryer to 140°C and the outlet air temperature to 50°C, and obtain the composite lipopeptide powder by spray drying at a feeding rate of 10 ml / min. Example 2

[0072] Preparation of a composite lipopeptide for preventing high-temperature inactivation, specifically including the following process:

[0073] Take 13 parts by weight of the modified phospholipid compound obtained in Preparation Example 5 and 4 parts by weight of cholesterol and place them in a dry rotary evaporation flask. Then add 1200 parts by weight of chloroform and mix and dissolve evenly. Subsequently, perform rotary evaporation at 41°C for 42 min to obtain a lipid film;

[0074] Add 1 part by weight of the lipopeptide mixture and 600 parts by weight of phosphate buffer solution with a pH of 5.0 to the lipid film, then place it in a shaker at 37±2°C and shake at a speed of 160 r / min for 52 min. Then put it into an ultrasonic machine, adjust the ultrasonic power to 220 W, and ultrasonicate for 2 min to obtain a composite lipopeptide liposome suspension (take 0.5 mL of the composite lipopeptide liposome suspension and centrifuge it at a speed of 5000 r / min for 20 min using an ultrafiltration centrifugal tube with a molecular weight cut-off of 5 kDa. Absorb the liquid obtained by centrifugal separation and measure the encapsulation efficiency of the composite lipopeptide liposome suspension by the BCA method, which is 73.27%). Subsequently, adjust the inlet air temperature of the small spray dryer to 140°C and the outlet air temperature to 50°C, and obtain the composite lipopeptide powder by spray drying at a feeding rate of 10 ml / min. Example 3

[0075] Preparation of a composite lipopeptide for preventing high-temperature inactivation, specifically including the following process:

[0076] Take 15 parts by weight of the modified phospholipid compound obtained in Preparation Example 6 and 5.5 parts by weight of cholesterol and place them in a dry rotary evaporation flask. Then add 1400 parts by weight of chloroform and mix and dissolve evenly. Subsequently, perform rotary evaporation at 42°C for 44 min to obtain a lipid film;

[0077] 1 part by weight of the lipopeptide mixture and 700 parts by weight of phosphate buffer solution with a pH of 5.0 were added to the lipid film, and then it was placed in a shaker at 37 ± 2 °C and shaken at a speed of 170 r / min for 54 min. Then it was put into an ultrasonic machine, the ultrasonic power was adjusted to 240 W, and ultrasonic treatment was carried out for 3 min to obtain a composite lipopeptide liposome suspension (0.5 mL of the composite lipopeptide liposome suspension was centrifuged and separated at a speed of 5000 r / min for 20 min using an ultrafiltration centrifugal tube with a molecular weight cut-off of 5 kDa, and the liquid obtained by centrifugal separation was aspirated 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 rate of 11 ml / min, the inlet air temperature of the small spray dryer was adjusted to 145 °C, and the outlet air temperature was adjusted to 55 °C, and the composite lipopeptide powder was obtained by spray drying. Example 4

[0078] Preparation of a composite lipopeptide for preventing high-temperature inactivation, specifically including the following process:

[0079] 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 flask, and then 1600 parts by weight of chloroform was added and mixed and dissolved evenly. Subsequently, rotary evaporation was carried out at 43 °C for 46 min to obtain a lipid film;

[0080] 1 part by weight of the lipopeptide mixture and 800 parts by weight of phosphate buffer solution with a pH of 5.8 were added to the lipid film, and then it was placed in a shaker at 37 ± 2 °C and shaken at a speed of 180 r / min for 56 min. Then it was put into an ultrasonic machine, the ultrasonic power was adjusted to 260 W, and ultrasonic treatment was carried out for 4 min to obtain a composite lipopeptide liposome suspension (0.5 mL of the composite lipopeptide liposome suspension was centrifuged and separated at a speed of 5000 r / min for 20 min using an ultrafiltration centrifugal tube with a molecular weight cut-off of 5 kDa, and the liquid obtained by centrifugal separation was aspirated 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 rate of 11 ml / min, the inlet air temperature of the small spray dryer was adjusted to 145 °C, and the outlet air temperature was adjusted to 55 °C, and the composite lipopeptide powder was obtained by spray drying. Example 5

[0081] Preparation of a composite lipopeptide for preventing high-temperature inactivation, specifically including the following process:

[0082] 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 flask, and then 1800 parts by weight of chloroform was added and mixed and dissolved evenly. Subsequently, rotary evaporation was carried out at 44 °C for 48 min to obtain a lipid film;

[0083] Add 1 part by weight of the lipopeptide mixture and 900 parts by weight of phosphate buffer solution with a pH of 5.8 to the lipid film, then place it in a shaker at 37 ± 2 °C and shake it at a speed of 190 r / min for 58 min. Then put it into an ultrasonic machine, adjust the ultrasonic power to 280 W, and ultrasonicate for 4 min to obtain a composite lipopeptide liposome suspension (take 0.5 mL of the composite lipopeptide liposome suspension and centrifuge it at a speed of 5000 r / min for 20 min using an ultrafiltration centrifugal tube with a molecular weight cut-off of 5 kDa. Absorb the liquid obtained by centrifugation and measure the encapsulation efficiency of the composite lipopeptide liposome suspension by the BCA method, which is 76.78%). Subsequently, adjust the inlet air temperature of the small spray dryer to 150 °C and the outlet air temperature to 55 °C, and obtain the composite lipopeptide powder by spray drying at a feeding rate of 12 ml / min. Example 6

[0084] Preparation of a composite lipopeptide for preventing high-temperature inactivation, specifically including the following process:

[0085] Take 25 parts by weight of the modified phospholipid compound obtained in Preparation Example 9 and 10 parts by weight of cholesterol and place them in a dry rotary evaporation flask. Then add 2000 parts by weight of chloroform and mix and dissolve evenly. Subsequently, perform rotary evaporation at 45 °C for 50 min to obtain a lipid film;

[0086] Add 1 part by weight of the lipopeptide mixture and 1000 parts by weight of phosphate buffer solution with a pH of 5.8 to the lipid film, then place it in a shaker at 37 ± 2 °C and shake it at a speed of 200 r / min for 60 min. Then put it into an ultrasonic machine, adjust the ultrasonic power to 300 W, and ultrasonicate for 5 min to obtain a composite lipopeptide liposome suspension (take 0.5 mL of the composite lipopeptide liposome suspension and centrifuge it at a speed of 5000 r / min for 20 min using an ultrafiltration centrifugal tube with a molecular weight cut-off of 5 kDa. Absorb the liquid obtained by centrifugation and measure the encapsulation efficiency of the composite lipopeptide liposome suspension by the BCA method, which is 76.85%). Subsequently, adjust the inlet air temperature of the small spray dryer to 150 °C and the outlet air temperature to 55 °C, and obtain the composite lipopeptide powder by spray drying at a feeding rate of 12 ml / min.

[0087] Comparative Example 1:

[0088] Preparation of a composite lipopeptide for preventing high-temperature inactivation, specifically including the following process:

[0089] Replace the modified phospholipid compound in Example 6 with the modified phospholipid compound obtained in Preparation Example 10, and keep the other conditions the same as in Example 6. The encapsulation efficiency of the composite lipopeptide liposome suspension is measured to be 75.56%.

[0090] Comparative Example 2:

[0091] Preparation of a composite lipopeptide against high-temperature inactivation, specifically including the following process:

[0092] Replace the modified phospholipid compound in Example 6 with the modified phospholipid compound obtained in Preparation Example 11, and keep the other conditions the same as those in Example 6. It is found that during the rotary evaporation treatment, it is difficult to form a lipid film and the preparation fails. Analysis of the reason: Although n-hexanal contains an aldehyde group and can introduce heat-resistant imine bonds through the covalent cross-linking of the aldehyde group and amino group, due to the long hydrophobic carbon chain in n-hexanal, after being introduced into the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine structure, it may further lead to an increase in the hydrophobicity of the modified phospholipid compound prepared in this comparative example, thus hindering the embedding of cholesterol into phospholipid molecules and making it difficult for the film to form.

[0093] Comparative Example 3:

[0094] Preparation of a composite lipopeptide against high-temperature inactivation, specifically including the following process:

[0095] Replace the modified phospholipid compound in Example 6 with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and keep the other conditions the same as those in Example 6. The encapsulation efficiency of the composite lipopeptide liposome suspension is measured to be 76.59%.

[0096] Comparative Example 4:

[0097] Preparation of a composite lipopeptide against high-temperature inactivation, specifically including the following process:

[0098] Add 20 parts by weight of β-cyclodextrin to 1500 parts by weight of phosphate buffer solution with a pH of 5.8, heat and stir until completely dissolved, then add 5 parts by weight of the lipopeptide mixture and 7 parts by weight of Tween 20, and then place it in a room temperature environment, stir and let it stand for 5 h to obtain a suspension. Freeze the suspension in a -20°C refrigerator for 24 hours, take it out and dry it in a -50°C freeze dryer for 24 hours to obtain the composite lipopeptide powder. Take 1 g of the composite lipopeptide powder and dissolve it in 300 ml of absolute ethanol. After ultrasonic treatment at 400 W for 10 min, detect the encapsulation efficiency of the composite lipopeptide by high performance liquid chromatography, which is 18.75%.

[0099] Comparative Example 5:

[0100] Preparation of a composite lipopeptide against high-temperature inactivation, specifically including the following process:

[0101] Lower the inlet air temperature in Example 6 to 100°C, and keep the other conditions the same as those in Example 6. It is found that the dried composite lipopeptide powder has a relatively serious wall sticking phenomenon. This may be due to the lower temperature, resulting in slower evaporation of moisture, making the powder obtained by spray drying have a higher moisture content and being prone to wall sticking, which is not conducive to production.

[0102] Comparative Example 6:

[0103] Preparation of a composite lipopeptide for preventing high-temperature inactivation, which specifically includes the following process:

[0104] Adjust the inlet air temperature in Example 6 to 200 °C, and keep the other conditions the same as those in Example 6. Dissolve the composite lipopeptide powders obtained in Examples 1-6 and Comparative Examples 1-6 in a phosphate buffer solution with a pH of 5.0 to a concentration of 50 mg / ml, and then mix and solidify with the PDA medium raw materials to obtain a PDA medium containing the composite lipopeptide (the composite lipopeptide concentration is 50 mg / L). Subsequently, inoculate apple ring rot pathogen and tomato gray mold pathogen disks with a diameter of 10 mm on the medium. The blank group does not use the composite lipopeptide, and the control group directly uses the lipopeptide composition to prepare the PDA medium (the lipopeptide composition concentration is 50 mg / L). Then place it in an incubator at 25 °C for 24 h, and calculate the antibacterial rate by statistically analyzing the ratio of the colony diameter of the medium containing the composite lipopeptide to the colony diameter on the blank medium. The results are shown in Table 1 below.

[0105]

[0106] The following conclusions can be drawn from the above antibacterial test results:

[0107] (1) It can be found from Examples 1-6 that the present invention modifies phospholipids to be heat-resistant, and then mixes the lipopeptide mixture obtained by fermentation with the modified phospholipid compound and cholesterol to construct a heat-resistant composite lipopeptide liposome suspension. After high-temperature spray drying, the obtained composite lipopeptide still has good antibacterial activity.

[0108] (2) It can be found from Comparative Example 1 that since phosphatidylinositol does not contain an amino group in its structure, glyoxal cannot covalently crosslink to construct a heat-resistant imine bond on phosphatidylinositol, resulting in the inability of the modified phospholipid compound prepared in this comparative example to be heat-resistant modified, and further leading to low antibacterial performance and low activity of the composite lipopeptide obtained by high-temperature spray drying.

[0109] (3) It can be found from Comparative Example 3 that although 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine can participate in the formation of the liposome structure encapsulating the composite lipopeptide, due to its heat intolerance, it is extremely easy to rupture or even oxidize and degrade in the high-temperature environment of spray drying, thus losing the protective effect on the lipopeptide mixture, resulting in low antibacterial activity of the composite lipopeptide prepared by this comparative example.

[0110] (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 in the structure to achieve the encapsulation of hydrophobic substances. Although the lipopeptide mixture has hydrophilic and hydrophobic structures, due to the strong hydrophilicity of the lipopeptide, less 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.

[0111] (5)It can be found from Comparative Example 6 that further increasing the inlet air temperature, although this is beneficial to shortening the spray-drying time, the antibacterial activity of the obtained composite lipopeptide powder is relatively low. This may be because the composite lipopeptide liposome suspension constructed by improving the high-temperature resistance performance of phospholipids in the present invention can only have a high-temperature protection effect within a certain temperature range, and has a relatively serious inactivation phenomenon when the temperature exceeds a certain value.

[0112] The above-described embodiments have detailed the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

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 40 min to 50 min to obtain a lipid film; The lipid film, lipopeptide mixture and phosphate buffer are mixed and shaken, and then treated with ultrasonic power of 200 W to 300 W for 2 min to 3 min to obtain a composite lipopeptide liposome suspension; The composite lipopeptide liposome suspension is spray-dried to obtain the composite lipopeptide; The preparation method of the modified phospholipid compound comprises the following steps: The amino-terminal phospholipid compound, aldehyde cross-linking agent and chloroform are mixed and stirred to form a mixed dispersion; The mixed dispersion and acetic acid are mixed and then treated by microwave to obtain a mixed solution, and the mixed solution is subjected to reduced pressure evaporation and washing and drying to obtain the modified phospholipid compound; The amino-terminal phospholipid compound is 1-palmitoyl-2-oleoyl phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; The aldehyde cross-linking agent is glyoxal.

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

3. The preparation method of a composite lipopeptide for preventing high-temperature inactivation according to claim 1, characterized in that, The addition amount of the acetic acid is 0.4 μL to 0.8 μL added per milligram of the mixed dispersion.

4. The preparation method of a composite lipopeptide for preventing high-temperature inactivation according to claim 1, characterized in that, The conditions of the microwave treatment 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.

5. The preparation method of a composite lipopeptide for preventing high-temperature inactivation according to claim 1, characterized in that, The preparation method of the lipopeptide mixture comprises the following steps: The bacillus is activated and inoculated into a seed liquid medium and cultured at 37 °C for 24 h to obtain a seed solution; The seed solution is inoculated into a fermentation medium according to an inoculation amount of 5% and cultured at 30 ± 1 °C for 40 h to obtain a fermentation broth; The fermentation broth is adjusted to an acidity with a pH of 2.0 and separated to obtain a precipitate, and the precipitate is purified to obtain the lipopeptide mixture.

6. The preparation method of a composite lipopeptide for preventing high-temperature inactivation according to claim 1, characterized in that, The pH of the phosphate buffer is 5.0 to 5.

8.

7. The preparation method of a composite lipopeptide for preventing 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 to 25: 2.5 to 10: 1000 to 2000: 1: 450 to 1000.

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