A temperature-sensitive peppermint essential oil microcapsule and a preparation method thereof

Abstract

This invention provides a thermosensitive peppermint essential oil microcapsule and its preparation method. First, peppermint essential oil is encapsulated in yeast cells to prepare microcapsules, improving the problems of volatility and poor stability of peppermint essential oil. Second, the microcapsules are immersed in Bacillus subtilis fermentation broth, where enzymes such as glucanase and mannoglucase in the fermentation broth enzymatically modify the microcapsules, partially converting polysaccharides on the cell wall into oligosaccharides and hydrolyzing long molecular chains into shorter ones, thus improving the dispersibility of the microcapsules in water. Finally, the enzymatically modified microcapsules are immersed in a polylysine-carboxymethyl chitosan solution for surface modification, ultimately obtaining thermosensitive peppermint essential oil microcapsules, broadening the application of peppermint essential oil in the field of food antibacterial preservation.

Description

Technical Field

[0001] This invention belongs to the field of microcapsule technology, specifically relating to a thermosensitive peppermint essential oil microcapsule and its preparation method. Background Technology

[0002] Peppermint essential oil is a colorless or pale yellow, clear, aromatic volatile oil obtained from the fresh stems and leaves of the peppermint plant (Mentha haplocalyx) through distillation, freezing, and partial defatting. It possesses properties such as dispelling wind and heat, reducing inflammation and pain, clearing the head and eyes, relieving itching, and calming the nerves. It is widely used both domestically and internationally in the food, health product, pharmaceutical, beverage, and daily chemical industries. However, peppermint essential oil is highly volatile, remaining liquid at room temperature. It is unstable when exposed to light and heat, and is not heat-resistant during processing. Its poor processing and storage stability severely limits its development and application.

[0003] Microcapsules are tiny, sealed capsules containing solids, liquids, or gases. The encapsulated substance is called the core material, and the substance that encapsulates the core material to achieve microencapsulation is called the wall material. Microencapsulation of peppermint essential oil can effectively improve its stability, protecting it from environmental influences, reducing loss and degradation during processing and storage, thus extending its shelf life and facilitating transportation.

[0004] Yeast cells are spherical or ellipsoidal single-celled organisms with diameters ranging from a few micrometers to 20 μm. They are safe, non-toxic, widely available, grow and reproduce rapidly, and are easy to cultivate on a large scale. They have the typical structure of natural eukaryotic cells, and their complete cell wall and cell membrane structure have certain strength and permeability, making them ideal microcapsule wall materials. Yeast cells have unique advantages as microcapsule wall materials: (1) No or very few other chemical reagents need to be added during the microcapsule preparation process; only water, yeast cells, and active core materials need to be in high-frequency contact. (2) The loading capacity of the core material can reach more than 70%, which is very suitable for the encapsulation of drugs and food additives. (3) They have a natural double-layer cavity structure, which can avoid the volatilization of volatile substances and the oxidation and deterioration caused by light and oxygen. (4) The obtained microcapsule products are uniform in size, non-toxic, biocompatible, and easily biodegradable. At present, the application scope of yeast microencapsulation technology includes food, medicine, cosmetics, biological pesticides, textiles, nicotine encapsulation in smoking cessation products, and carbonless copy paper, among other fields. However, because yeast cell walls have a special triple helix structure, they are insoluble in water and appear as aggregated granules. They are slightly soluble in dimethyl sulfoxide (DMSO). Due to their insolubility, their dispersibility is reduced, which greatly limits their application. Summary of the Invention

[0005] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention aims to provide a thermosensitive peppermint essential oil microcapsule and its preparation method. First, peppermint essential oil is encapsulated in yeast cells to prepare microcapsules, improving the problems of volatility and poor stability of peppermint essential oil. Second, the microcapsules are immersed in Bacillus subtilis fermentation broth, where enzymes such as glucanase and mannoglucanase in the fermentation broth enzymatically modify the microcapsules, partially converting polysaccharides on the cell wall into oligosaccharides and hydrolyzing long molecular chains into shorter ones, thus improving the dispersibility of the microcapsules in water. Finally, the enzymatically modified microcapsules are immersed in a polylysine-carboxymethyl chitosan solution for surface modification, ultimately obtaining thermosensitive peppermint essential oil microcapsules, thus broadening the application of peppermint essential oil in the field of food antibacterial preservation.

[0006] Technical solution: A thermosensitive peppermint essential oil microcapsule, wherein the thermosensitive peppermint essential oil microcapsule uses brewer's yeast cells as the wall material and peppermint essential oil as the core material, and its surface is modified by immersion in Bacillus subtilis fermentation broth and polylysine-carboxymethyl chitosan solution.

[0007] Furthermore, the preparation method of the polylysine-carboxymethyl chitosan solution is as follows: S1. Weigh chitosan and soak it in polyethylene glycol solution. Stir until uniform and swell. After swelling, freeze at -20℃ for 15 hours, restore to room temperature, and filter to obtain pretreated chitosan. S2. Place the pretreated chitosan in a 40% sodium hydroxide solution and alkalize it at 25-65℃ for 4-8 hours, stirring at 100-180r / min until homogeneous to obtain alkalized chitosan. S3. Alkalized chitosan was mixed and stirred with 70% ethanol solution, chloroacetic acid was added, and carboxylation reaction was carried out at 40℃ for 4h. After neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20min, and drying at 60℃ for 20h, carboxymethyl chitosan was obtained. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of (1-3):2, and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution.

[0008] Furthermore, in step S1, the mass-to-volume ratio of chitosan to polyethylene glycol solution is 1g:(4-5)mL, and the polyethylene glycol solution is composed of polyethylene glycol 800, water, and nanocellulose in a mass-to-volume ratio of 1g:(2-8)mL:(0.2-0.7)mL.

[0009] Furthermore, in step S2, the mass-to-volume ratio of pretreated chitosan to sodium hydroxide solution is 1 g: (5-10) mL.

[0010] Furthermore, in step S3, the mass-to-volume ratio of alkalized chitosan: ethanol solution: chloroacetic acid is 1g: (4-10)mL: (3-8)mL.

[0011] The above-mentioned method for preparing thermosensitive peppermint essential oil microcapsules includes the following steps: Step 1. Mix peppermint essential oil with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min, pour out, vacuum filter, wash, and freeze dry under vacuum to obtain peppermint essential oil microcapsules; Step 2. Immerse the peppermint essential oil microcapsules in Bacillus subtilis fermentation broth and freeze-dry at -40℃ for 20 hours to obtain modified peppermint essential oil microcapsules; Step 3. Immerse the modified peppermint oil microcapsules again in a polylysine-carboxymethyl chitosan solution and freeze-dry at -40°C for 20 hours to obtain thermosensitive peppermint oil microcapsules.

[0012] Furthermore, in step 1, the ratio of peppermint essential oil to the core wall of the activated brewer's yeast cells is (1-5):1.

[0013] Furthermore, in step 2, the mass-to-volume ratio of peppermint oil microcapsules to Bacillus subtilis fermentation broth is 1g:(10-30)mL.

[0014] Furthermore, in step 3, the mass-to-volume ratio of the modified peppermint oil microcapsules to the polylysine-carboxymethyl chitosan solution is 1 g: (5-20) mL.

[0015] The application of the above-mentioned thermosensitive peppermint essential oil microcapsules in food antibacterial preservation.

[0016] Beneficial effects: This invention uses peppermint essential oil as the core material and yeast cells as the wall material to prepare microcapsules. The microencapsulation of peppermint essential oil can effectively prevent the volatilization of the essential oil and improve the stability of peppermint essential oil, making it immune to environmental influences, thereby reducing the loss of peppermint essential oil during processing and storage. In this invention, peppermint essential oil microcapsules are soaked in Bacillus subtilis fermentation broth. Enzymes such as glucanase and mannoglucase in the fermentation broth modify the surface of the microcapsules by enzymatic modification, causing the polysaccharides on the cell wall to be partially converted into oligosaccharides, and the long molecular chains to be hydrolyzed into short molecular chains, thereby improving the solubility of the microcapsules and their dispersibility in water. This invention involves immersing enzymatically modified peppermint oil microcapsules in a polylysine-carboxymethyl chitosan solution. The amino groups of the polylysine side chains and the carboxyl groups of the carboxymethyl chitosan side chains form a network structure through electrostatic interactions. The carboxyl groups on the carboxymethyl chitosan undergo a condensation reaction with the hydroxyl groups on the yeast cell wall, causing the network structure to adhere to the surface of the microcapsules, thereby increasing the encapsulation rate of peppermint oil. At the same time, carboxymethyl groups are introduced onto the surface of the microcapsules, achieving a surface modification effect and improving the solubility of the microcapsules. The carboxymethyl chitosan used in this invention is a temperature-sensitive material. The prepared polylysine-carboxymethyl chitosan network is attached to the surface of peppermint oil microcapsules, thus preparing temperature-sensitive peppermint oil microcapsules. When the temperature rises, it accelerates the growth of microorganisms in the environment and increases the rate of spoilage of fruits, vegetables and other foods. However, the temperature rise will increase the pore size of the network on the surface of the temperature-sensitive peppermint oil microcapsules, and release a large amount of peppermint oil inside, which will play an antibacterial and preservation role and improve the storage quality. Attached Figure Description Figure 1 The graph shows the peppermint oil release rate of the microcapsules prepared in Example 5; Figure 2 The graph shows the peppermint oil release rate of the microcapsules prepared for Comparative Example 2. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments: Activation of Saccharomyces cerevisiae: Inoculate freeze-dried Saccharomyces cerevisiae powder into YEPD liquid medium (1% yeast extract, 2% peptone, 2% glucose, deionized water as solvent, sterilized), activate and culture in a shaker at 30℃ and 200 r / min for 20 h, collect the cells by centrifugation at 5000 r / min for 10 min, wash 3 times with deionized water, resuspend for later use, which is the activated Saccharomyces cerevisiae cells.

[0018] Preparation of Bacillus subtilis fermentation broth: (1) Seed culture: Activated Bacillus subtilis was inoculated into LB liquid medium (1% tryptone, 0.5% yeast extract, 1% NaCl, sterilized), and cultured on a shaker at 35℃ and 200 r / min for 12 h, with OD600 controlled at 0.8-1.2; (2) Fermentation culture: 3% (v / v) inoculum was transferred to fermentation medium (1% glucose, 0.5% yeast extract, 1% peptone, 0.5% NaCl, pH 7.0-7.2, sterilized), and fermented at 35℃ and 200 r / min for 30 h; (3) Post-treatment: The fermentation broth was centrifuged at 5000 r / min for 10 min, and the supernatant was taken as Bacillus subtilis fermentation broth, which was stored at 4℃ for later use.

[0019] Example 1 The preparation steps of a thermosensitive peppermint essential oil microcapsule are as follows: S1. Weigh 1 kg of chitosan and soak it in 5 L of polyethylene glycol solution. The polyethylene glycol solution is composed of polyethylene glycol 800, water and nanocellulose in a mass-volume ratio of 1:5:0.5. Stir evenly, swell, freeze at -20℃ for 15 h, restore to room temperature, and filter to obtain pretreated chitosan. S2. Pretreated chitosan was placed in a 40% sodium hydroxide solution at a mass-volume ratio of 1:5 and alkalized at 40°C for 8 hours. The mixture was stirred at 150 r / min until homogeneous to obtain alkalized chitosan. S3. Alkaline chitosan was mixed and stirred with 70% ethanol solution. After adding chloroacetic acid, the mass-volume ratio of alkaline chitosan: ethanol solution: chloroacetic acid was 1:5:8. The carboxylation reaction was carried out at 40℃ for 4 hours, followed by neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20 minutes, and drying at 60℃ for 20 hours to obtain carboxymethyl chitosan. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of 1:2 and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution. S5. Mix peppermint essential oil with a core-to-wall ratio of 1:1 with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min and pour out, vacuum filter to recover the unencapsulated oil, wash off the peppermint oil remaining on the surface of the yeast cells with ethanol, and freeze dry under vacuum for 24 h to obtain peppermint essential oil microcapsules. S6. The peppermint essential oil microcapsules were immersed in Bacillus subtilis fermentation broth at a mass-to-volume ratio of 1g:10mL and freeze-dried at -40℃ for 20h to obtain modified peppermint essential oil microcapsules. S7. The modified peppermint oil microcapsules were immersed again in a polylysine-carboxymethyl chitosan solution at a mass-to-volume ratio of 1g:5mL, and then freeze-dried at -40℃ for 20h to obtain thermosensitive peppermint oil microcapsules.

[0020] Example 2 The preparation steps of a thermosensitive peppermint essential oil microcapsule are as follows: S1. Weigh 1 kg of chitosan and soak it in 5 L of polyethylene glycol solution. The polyethylene glycol solution is composed of polyethylene glycol 800, water and nanocellulose in a mass-volume ratio of 1:5:0.5. Stir evenly, swell, freeze at -20℃ for 15 h, restore to room temperature, and filter to obtain pretreated chitosan. S2. Pretreated chitosan was placed in a 40% sodium hydroxide solution at a mass-volume ratio of 1:5 and alkalized at 40°C for 6 hours. The mixture was stirred at 150 r / min until homogeneous to obtain alkalized chitosan. S3. Alkaline chitosan was mixed and stirred with 70% ethanol solution. After adding chloroacetic acid, the mass-volume ratio of alkaline chitosan: ethanol solution: chloroacetic acid was 1:5:8. The carboxylation reaction was carried out at 40℃ for 4 hours, followed by neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20 minutes, and drying at 60℃ for 20 hours to obtain carboxymethyl chitosan. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of 1:1 and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution. S5. Mix peppermint essential oil with a core-to-wall ratio of 1:1 with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min and pour out, vacuum filter to recover the unencapsulated oil, wash off the peppermint oil remaining on the surface of the yeast cells with ethanol, and freeze dry under vacuum for 24 h to obtain peppermint essential oil microcapsules. S6. Peppermint essential oil microcapsules were immersed in Bacillus subtilis fermentation broth at a mass-to-volume ratio of 1g:10mL and freeze-dried at -40℃ for 20h to obtain modified peppermint essential oil microcapsules. S7. The modified peppermint oil microcapsules were immersed again in a polylysine-carboxymethyl chitosan solution at a mass-to-volume ratio of 1g:5mL, and then freeze-dried at -40℃ for 20h to obtain thermosensitive peppermint oil microcapsules.

[0021] Example 3 The preparation steps of a thermosensitive peppermint essential oil microcapsule are as follows: S1. Weigh 1 kg of chitosan and soak it in 5 L of polyethylene glycol solution. The polyethylene glycol solution is composed of polyethylene glycol 800, water and nanocellulose in a mass-volume ratio of 1:5:0.5. Stir evenly, swell, freeze at -20℃ for 15 h, restore to room temperature, and filter to obtain pretreated chitosan. S2. Pretreated chitosan was placed in a 40% sodium hydroxide solution at a mass-volume ratio of 1:5 and alkalized at 40°C for 4 hours. The mixture was stirred at 150 r / min until homogeneous to obtain alkalized chitosan. S3. Alkaline chitosan was mixed and stirred with 70% ethanol solution. After adding chloroacetic acid, the mass-volume ratio of alkaline chitosan: ethanol solution: chloroacetic acid was 1:5:8. The carboxylation reaction was carried out at 40℃ for 4 hours, followed by neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20 minutes, and drying at 60℃ for 20 hours to obtain carboxymethyl chitosan. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of 3:2 and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution. S5. Mix peppermint essential oil with a core-to-wall ratio of 1:1 with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min and pour out, vacuum filter to recover the unencapsulated oil, wash off the peppermint oil remaining on the surface of the yeast cells with ethanol, and freeze dry under vacuum for 24 h to obtain peppermint essential oil microcapsules. S6. The peppermint essential oil microcapsules were immersed in Bacillus subtilis fermentation broth at a mass-to-volume ratio of 1g:15mL and freeze-dried at -40℃ for 20h to obtain modified peppermint essential oil microcapsules. S7. The modified peppermint oil microcapsules were immersed again in a polylysine-carboxymethyl chitosan solution at a mass-to-volume ratio of 1g:10mL, and then freeze-dried at -40℃ for 20h to obtain thermosensitive peppermint oil microcapsules.

[0022] Example 4 The preparation steps of a thermosensitive peppermint essential oil microcapsule are as follows: S1. Weigh 1 kg of chitosan and soak it in 5 L of polyethylene glycol solution. The polyethylene glycol solution is composed of polyethylene glycol 800, water and nanocellulose in a mass-volume ratio of 1:5:0.5. Stir evenly, swell, freeze at -20℃ for 15 h, restore to room temperature, and filter to obtain pretreated chitosan. S2. Pretreated chitosan was placed in a 40% sodium hydroxide solution at a mass-volume ratio of 1:10 and alkalized at 40°C for 8 hours. The mixture was stirred at 150 r / min until homogeneous to obtain alkalized chitosan. S3. Alkaline chitosan was mixed and stirred with 70% ethanol solution. After adding chloroacetic acid, the mass-volume ratio of alkaline chitosan: ethanol solution: chloroacetic acid was 1:5:8. The carboxylation reaction was carried out at 40℃ for 4 hours, followed by neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20 minutes, and drying at 60℃ for 20 hours to obtain carboxymethyl chitosan. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of 3:2 and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution. S5. Mix peppermint essential oil with a core-to-wall ratio of 2:1 with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min and pour out, vacuum filter to recover the unencapsulated oil, wash off the peppermint oil remaining on the surface of the yeast cells with ethanol, and freeze dry under vacuum for 24 h to obtain peppermint essential oil microcapsules. S6. The peppermint oil microcapsules were immersed in Bacillus subtilis fermentation broth at a mass-to-volume ratio of 1g:20mL and freeze-dried at -40℃ for 20h to obtain modified peppermint oil microcapsules. S7. The modified peppermint oil microcapsules were immersed again in a polylysine-carboxymethyl chitosan solution at a mass-to-volume ratio of 1g:10mL, and then freeze-dried at -40℃ for 20h to obtain thermosensitive peppermint oil microcapsules.

[0023] Example 5 The preparation steps of a thermosensitive peppermint essential oil microcapsule are as follows: S1. Weigh 1 kg of chitosan and soak it in 5 L of polyethylene glycol solution. The polyethylene glycol solution is composed of polyethylene glycol 800, water and nanocellulose in a mass-volume ratio of 1:5:0.5. Stir evenly, swell, freeze at -20℃ for 15 h, restore to room temperature, and filter to obtain pretreated chitosan. S2. Pretreated chitosan was placed in a 40% sodium hydroxide solution at a mass-volume ratio of 1:10 and alkalized at 40°C for 8 hours. The mixture was stirred at 150 r / min until homogeneous to obtain alkalized chitosan. S3. Alkaline chitosan was mixed and stirred with 70% ethanol solution. After adding chloroacetic acid, the mass-volume ratio of alkaline chitosan: ethanol solution: chloroacetic acid was 1:5:8. The carboxylation reaction was carried out at 40℃ for 4 hours, followed by neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20 minutes, and drying at 60℃ for 20 hours to obtain carboxymethyl chitosan. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of 3:2 and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution. S5. Mix peppermint essential oil with a core-to-wall ratio of 3:1 with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min and pour out, vacuum filter to recover the unencapsulated oil, wash away the peppermint oil remaining on the surface of the yeast cells with ethanol, and freeze dry under vacuum for 24 h to obtain peppermint essential oil microcapsules. S6. The peppermint oil microcapsules were immersed in Bacillus subtilis fermentation broth at a mass-to-volume ratio of 1g:20mL and freeze-dried at -40℃ for 20h to obtain modified peppermint oil microcapsules. S7. The modified peppermint oil microcapsules were immersed again in a polylysine-carboxymethyl chitosan solution at a mass-to-volume ratio of 1g:20mL, and then freeze-dried at -40℃ for 20h to obtain thermosensitive peppermint oil microcapsules.

[0024] Example 6 The preparation steps of a thermosensitive peppermint essential oil microcapsule are as follows: S1. Weigh 1 kg of chitosan and soak it in 5 L of polyethylene glycol solution. The polyethylene glycol solution is composed of polyethylene glycol 800, water and nanocellulose in a mass-volume ratio of 1:5:0.5. Stir evenly, swell, freeze at -20℃ for 15 h, restore to room temperature, and filter to obtain pretreated chitosan. S2. Pretreated chitosan was placed in a 40% sodium hydroxide solution at a mass-volume ratio of 1:10 and alkalized at 40°C for 6 hours. The mixture was stirred at 150 r / min until homogeneous to obtain alkalized chitosan. S3. Alkaline chitosan was mixed and stirred with 70% ethanol solution. After adding chloroacetic acid, the mass-volume ratio of alkaline chitosan: ethanol solution: chloroacetic acid was 1:5:8. The carboxylation reaction was carried out at 40℃ for 4 hours, followed by neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20 minutes, and drying at 60℃ for 20 hours to obtain carboxymethyl chitosan. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of 3:2 and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution. S5. Mix peppermint essential oil with a core-to-wall ratio of 4:1 with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min and pour out, vacuum filter to recover the unencapsulated oil, wash off the peppermint oil remaining on the surface of the yeast cells with ethanol, and freeze dry under vacuum for 24 h to obtain peppermint essential oil microcapsules. S6. The peppermint essential oil microcapsules were immersed in Bacillus subtilis fermentation broth at a mass-to-volume ratio of 1g:30mL and freeze-dried at -40℃ for 20h to obtain modified peppermint essential oil microcapsules. S7. The modified peppermint oil microcapsules were immersed again in a polylysine-carboxymethyl chitosan solution at a mass-to-volume ratio of 1g:20mL, and then freeze-dried at -40℃ for 20h to obtain thermosensitive peppermint oil microcapsules.

[0025] Example 7 The preparation steps of a thermosensitive peppermint essential oil microcapsule are as follows: S1. Weigh 1 kg of chitosan and soak it in 5 L of polyethylene glycol solution. The polyethylene glycol solution is composed of polyethylene glycol 800, water and nanocellulose in a mass-volume ratio of 1:5:0.5. Stir evenly, swell, freeze at -20℃ for 15 h, restore to room temperature, and filter to obtain pretreated chitosan. S2. Pretreated chitosan was placed in a 40% sodium hydroxide solution at a mass-volume ratio of 1:10 and alkalized at 40°C for 8 hours. The mixture was stirred at 150 r / min until homogeneous to obtain alkalized chitosan. S3. Alkaline chitosan was mixed and stirred with 70% ethanol solution. After adding chloroacetic acid, the mass-volume ratio of alkaline chitosan: ethanol solution: chloroacetic acid was 1:5:8. The carboxylation reaction was carried out at 40℃ for 4 hours, followed by neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20 minutes, and drying at 60℃ for 20 hours to obtain carboxymethyl chitosan. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of 3:2 and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution. S5. Mix peppermint essential oil with a core-to-wall ratio of 5:1 with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min and pour out, vacuum filter to recover the unencapsulated oil, wash off the peppermint oil remaining on the surface of the yeast cells with ethanol, and freeze dry under vacuum for 24 h to obtain peppermint essential oil microcapsules. S6. The peppermint essential oil microcapsules were immersed in Bacillus subtilis fermentation broth at a mass-to-volume ratio of 1g:25mL and freeze-dried at -40℃ for 20h to obtain modified peppermint essential oil microcapsules. S7. The modified peppermint oil microcapsules were immersed again in a polylysine-carboxymethyl chitosan solution at a mass-to-volume ratio of 1g:20mL, and then freeze-dried at -40℃ for 20h to obtain thermosensitive peppermint oil microcapsules.

[0026] Comparative Example 1 The difference between this comparative example and Example 5 is that the peppermint oil microcapsules were not immersed in the Bacillus subtilis fermentation broth, as detailed below: The preparation steps of a thermosensitive peppermint essential oil microcapsule are as follows: S1. Weigh 1 kg of chitosan and soak it in 5 L of polyethylene glycol solution. The polyethylene glycol solution is composed of polyethylene glycol 800, water and nanocellulose in a mass-volume ratio of 1:5:0.5. Stir evenly, swell, freeze at -20℃ for 15 h, restore to room temperature, and filter to obtain pretreated chitosan. S2. Pretreated chitosan was placed in a 40% sodium hydroxide solution at a mass-volume ratio of 1:10 and alkalized at 40°C for 8 hours. The mixture was stirred at 150 r / min until homogeneous to obtain alkalized chitosan. S3. Alkaline chitosan was mixed and stirred with 70% ethanol solution. After adding chloroacetic acid, the mass-volume ratio of alkaline chitosan: ethanol solution: chloroacetic acid was 1:5:8. The carboxylation reaction was carried out at 40℃ for 4 hours, followed by neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20 minutes, and drying at 60℃ for 20 hours to obtain carboxymethyl chitosan. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of 3:2 and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution. S5. Mix peppermint essential oil with a core-to-wall ratio of 3:1 with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min and pour out, vacuum filter to recover the unencapsulated oil, wash away the peppermint oil remaining on the surface of the yeast cells with ethanol, and freeze dry under vacuum for 24 h to obtain peppermint essential oil microcapsules. S6. Immerse peppermint essential oil microcapsules in a polylysine-carboxymethyl chitosan solution at a mass-to-volume ratio of 1g:20mL, and freeze-dry at -40℃ for 20h to obtain thermosensitive peppermint essential oil microcapsules.

[0027] Comparative Example 2 The difference between this comparative example and Example 5 is that the peppermint oil microcapsules were not immersed in the polylysine-carboxymethyl chitosan solution, as detailed below: The preparation steps of a peppermint essential oil microcapsule are as follows: S1. Weigh 1 kg of chitosan and soak it in 5 L of polyethylene glycol solution. The polyethylene glycol solution is composed of polyethylene glycol 800, water and nanocellulose in a mass-volume ratio of 1:5:0.5. Stir evenly, swell, freeze at -20℃ for 15 h, restore to room temperature, and filter to obtain pretreated chitosan. S2. Pretreated chitosan was placed in a 40% sodium hydroxide solution at a mass-volume ratio of 1:10 and alkalized at 40°C for 8 hours. The mixture was stirred at 150 r / min until homogeneous to obtain alkalized chitosan. S3. Alkaline chitosan was mixed and stirred with 70% ethanol solution. After adding chloroacetic acid, the mass-volume ratio of alkaline chitosan: ethanol solution: chloroacetic acid was 1:5:8. The carboxylation reaction was carried out at 40℃ for 4 hours, followed by neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20 minutes, and drying at 60℃ for 20 hours to obtain carboxymethyl chitosan. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of 3:2 and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution. S5. Mix peppermint essential oil with a core-to-wall ratio of 3:1 with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min and pour out, vacuum filter to recover the unencapsulated oil, wash away the peppermint oil remaining on the surface of the yeast cells with ethanol, and freeze dry under vacuum for 24 h to obtain peppermint essential oil microcapsules. S6. The peppermint essential oil microcapsules were immersed in Bacillus subtilis fermentation broth at a mass-to-volume ratio of 1g:20mL and freeze-dried at -40℃ for 20h to obtain modified peppermint essential oil microcapsules.

[0028] Comparative Example 3 The difference between this comparative example and Example 5 is that the peppermint oil microcapsules were not immersed in the Bacillus subtilis fermentation broth and polylysine-carboxymethyl chitosan solution, as detailed below: The preparation steps of a thermosensitive peppermint essential oil microcapsule are as follows: S1. Weigh 1 kg of chitosan and soak it in 5 L of polyethylene glycol solution. The polyethylene glycol solution is composed of polyethylene glycol 800, water and nanocellulose in a mass-volume ratio of 1:5:0.5. Stir evenly, swell, freeze at -20℃ for 15 h, restore to room temperature, and filter to obtain pretreated chitosan. S2. Pretreated chitosan was placed in a 40% sodium hydroxide solution at a mass-volume ratio of 1:10 and alkalized at 40°C for 8 hours. The mixture was stirred at 150 r / min until homogeneous to obtain alkalized chitosan. S3. Alkaline chitosan was mixed and stirred with 70% ethanol solution. After adding chloroacetic acid, the mass-volume ratio of alkaline chitosan: ethanol solution: chloroacetic acid was 1:5:8. The carboxylation reaction was carried out at 40℃ for 4 hours, followed by neutralization with hydrochloric acid, washing with alcohol, dehydration centrifugation at 5000r / min for 20 minutes, and drying at 60℃ for 20 hours to obtain carboxymethyl chitosan. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of 3:2 and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution. S5. Mix peppermint essential oil with a core-to-wall ratio of 3:1 with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge at 3000 r / min for 10 min and pour out, vacuum filter to recover the unencapsulated oil, wash away the peppermint oil remaining on the surface of the yeast cells with ethanol, and freeze dry under vacuum for 24 h to obtain peppermint essential oil microcapsules.

[0029] Performance testing: (1) Encapsulation rate of peppermint essential oil microcapsules Accurately weigh 0.2 g of the microcapsules prepared in Examples 1-7 and Comparative Examples 1-3, add 20 mL of anhydrous ethanol, and extract ultrasonically at 60℃ for 80 min. After filtration, the concentration of peppermint essential oil is determined by ultraviolet spectrophotometry at a wavelength of 204 nm. The microencapsulation effect is measured by the encapsulation efficiency, and the calculation formula is as follows: Encapsulation rate (%) = (Mass of peppermint essential oil in microcapsules / Initial total mass of peppermint essential oil) × 100% Table 1. Encapsulation efficiency of microcapsules prepared in Examples 1-7 and Comparative Examples 1-3

[0030] As shown in Table 1, the encapsulation efficiency of the peppermint oil microcapsules prepared in Examples 1-7 was 72.11-77.44%. In Examples 1-3, changing the core-to-wall ratio did not significantly alter the encapsulation efficiency. In Examples 4-7, the encapsulation efficiency first increased and then slightly decreased with increasing core-to-wall ratio. The maximum encapsulation efficiency of 77.44% was achieved when the core-to-wall ratio was 3:1 (Example 5), possibly because the peppermint oil diffused freely into the solution system outside the microcapsules. In Comparative Example 1, the peppermint oil microcapsules were not immersed in Bacillus subtilis fermentation broth, and the encapsulation efficiency did not change significantly. In Comparative Example 2, the peppermint oil microcapsules were not immersed in polylysine-carboxymethyl chitosan solution, preventing the formation of a network structure on the microcapsule surface, thus resulting in a decreased encapsulation efficiency. In Comparative Example 3, the peppermint oil microcapsules were not immersed in either Bacillus subtilis fermentation broth or polylysine-carboxymethyl chitosan solution, and the encapsulation efficiency also decreased.

[0031] (2) Solubility of peppermint essential oil microcapsules The moisture content of the microcapsules was measured using a moisture analyzer at room temperature.

[0032] The thermosensitive peppermint oil microcapsules prepared in the examples and comparative examples were added to distilled water and centrifuged. The supernatant was discarded, and the precipitate was transferred to a known mass container and dried at 105°C to constant weight. The solubility of the microcapsules was calculated using the following formula:

[0033] In the formula: m is the mass of the microcapsule / g; m1 is the mass of the container / g; m2 is the mass of the insoluble matter in the container and microcapsules / g; ω is the moisture content of the microcapsule / % by mass. Table 2. Solubility of microcapsules prepared in Examples 1-7 and Comparative Examples 1-3

[0034] As shown in Table 2, the solubility of peppermint oil microcapsules prepared in Examples 1-7 was 89.22-94.14%, while the solubility of microcapsules in Comparative Examples 1-3 all decreased significantly. This indicates that immersing the microcapsules in Bacillus subtilis fermentation broth for enzymatic modification and simultaneously attaching carboxymethyl groups to the surface of the microcapsules can improve the solubility of the microcapsules, thereby further improving their dispersibility in aqueous solution.

[0035] (3) Preservation effect Fresh strawberries were used as the test fruit. The experiment was divided into 3 groups with 20 strawberries in each group, and the average value of the experimental results was taken. The temperature-sensitive peppermint essential oil microcapsules prepared in Example 5 and Comparative Example 2 were dissolved in water to make a microcapsule preservation solution. Fresh strawberries were soaked in the microcapsule preservation solution and stored in storage boxes at different temperatures (15℃, 30℃, 40℃) for 30 days. The peppermint essential oil release rate was recorded.

[0036] like Figure 1 As shown, the release rate of peppermint essential oil gradually increases with increasing temperature. This indicates that the polylysine-carboxymethyl chitosan network modified on the surface of the peppermint essential oil microcapsules in this invention is temperature-sensitive. When the temperature rises, it accelerates the growth of microorganisms in the environment, increasing the rate of spoilage of fresh strawberries. However, rising temperature also increases the pore size of the network on the surface of the temperature-sensitive peppermint essential oil microcapsules, leading to a large release of peppermint essential oil from within, thus achieving an antibacterial and preservative effect and improving storage quality. Figure 2 The microcapsules prepared without immersing the peppermint essential oil microcapsules in a polylysine-carboxymethyl chitosan solution (Comparative Example 2) show that the release rate of peppermint essential oil did not increase significantly when the temperature rose. This may be because the wall material of the microcapsules is not affected by temperature, and therefore the difference in the release rate of peppermint essential oil is not significant.

[0037] Strawberries soaked in the microcapsule preservation solution at the above different temperatures were stored for one week, and the rate of spoiled fruit was calculated using the following formula: Damaged fruit rate (%) = Damaged fruit / Total number of fruits × 100% Table 3. Fruit spoilage rate of strawberries preserved by thermosensitive peppermint oil microcapsules prepared in Example 5 and Comparative Example 2.

[0038] As shown in Table 3, the present invention soaks peppermint oil microcapsules in a polylysine-carboxymethyl chitosan solution, forming a temperature-sensitive network on the surface of the microcapsules. When the temperature rises, a large amount of peppermint oil can be released, thereby achieving a better antibacterial effect. After 7 days of storage, the highest rate of spoiled fruit is 12%. In contrast, Comparative Example 2 did not coat the surface of the peppermint oil microcapsules with a polylysine-carboxymethyl chitosan network. The wall material of the microcapsules themselves is not sensitive to temperature, so it cannot release an appropriate amount of peppermint oil according to changes in ambient temperature. After 7 days of storage, the rate of spoiled strawberries is as high as 62%.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A thermosensitive peppermint essential oil microcapsule, characterized in that: The thermosensitive peppermint oil microcapsules use brewer's yeast cells as the wall material and peppermint oil as the core material, and their surface is modified by immersion in Bacillus subtilis fermentation broth and polylysine-carboxymethyl chitosan solution.

2. The thermosensitive peppermint essential oil microcapsule according to claim 1, characterized in that, The preparation method of the polylysine-carboxymethyl chitosan solution is as follows: S1. Weigh chitosan and soak it in polyethylene glycol solution. Stir until uniform, swell, freeze, restore to room temperature, and filter to obtain pretreated chitosan. S2. Place the pretreated chitosan in a 40% sodium hydroxide solution and alkalize it at 25-65℃ for 4-8 hours, stirring at 100-180r / min until homogeneous to obtain alkalized chitosan. S3. Alkalized chitosan was mixed and stirred with 70% ethanol solution, chloroacetic acid was added, and carboxylation reaction was carried out at 40℃ for 4 hours. After neutralization, alcohol washing, dehydration centrifugation and drying, carboxymethyl chitosan was obtained. S4. Dissolve polylysine and carboxymethyl chitosan in water at a mass ratio of (1-3):2, and stir thoroughly until completely dissolved to obtain a polylysine-carboxymethyl chitosan solution.

3. The thermosensitive peppermint essential oil microcapsule according to claim 2, characterized in that, In step S1, the mass-to-volume ratio of chitosan to polyethylene glycol solution is 1g:(4-5)mL. The polyethylene glycol solution is composed of polyethylene glycol 800, water, and nanocellulose in a mass-to-volume ratio of 1g:(2-8)mL:(0.2-0.7)mL.

4. The thermosensitive peppermint essential oil microcapsule according to claim 2, characterized in that, In step S2, the mass-to-volume ratio of pretreated chitosan to sodium hydroxide solution is 1 g: (5-10) mL.

5. The thermosensitive peppermint essential oil microcapsule according to claim 2, characterized in that, In step S3, the mass-volume ratio of alkalized chitosan: ethanol solution: chloroacetic acid is 1g: (4-10)mL: (3-8)mL.

6. The method for preparing thermosensitive peppermint essential oil microcapsules according to claim 1, characterized in that, Includes the following steps: Step 1. Mix peppermint essential oil with activated brewer's yeast cells, add water, place in a constant temperature magnetic stirrer and stir, centrifuge and pour out, vacuum filter, wash, vacuum freeze dry to obtain peppermint essential oil microcapsules; Step 2. Immerse the peppermint essential oil microcapsules in Bacillus subtilis fermentation broth and freeze-dry to obtain modified peppermint essential oil microcapsules; Step 3. Immerse the modified peppermint oil microcapsules again in a polylysine-carboxymethyl chitosan solution and freeze-dry to obtain thermosensitive peppermint oil microcapsules.

7. The method for preparing thermosensitive peppermint essential oil microcapsules according to claim 6, characterized in that, In step 1, the ratio of peppermint essential oil to the core wall of activated brewer's yeast cells is (1-5):

1.

8. The method for preparing thermosensitive peppermint essential oil microcapsules according to claim 6, characterized in that, In step 2, the mass-to-volume ratio of peppermint oil microcapsules to Bacillus subtilis fermentation broth is 1g:(10-30)mL.

9. The method for preparing thermosensitive peppermint essential oil microcapsules according to claim 6, characterized in that, In step 3, the mass-to-volume ratio of the modified peppermint oil microcapsules to the polylysine-carboxymethyl chitosan solution is 1 g: (5-20) mL.

10. The application of the thermosensitive peppermint essential oil microcapsules according to claim 1 in food antibacterial preservation.

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