Enzyme-responsive intelligent antibacterial gelatin microgel and preparation method thereof

Abstract

The present application relates to the field of food preservation technology, and particularly relates to an enzyme-responsive intelligent antibacterial gelatin microgel and a preparation method thereof. The method comprises the following steps: (1) preparation of a sugar glycosidase-containing aqueous phase; (2) preparation of a plant essential oil-containing oil phase; and (3) preparation of the enzyme-responsive intelligent antibacterial gelatin microgel. The enzyme-responsive intelligent antibacterial gelatin microgel loaded with plant essential oil or key active components thereof and prepared by the method can effectively protect the active components of the essential oil and intelligently respond to controllable release, can be used as a new antibacterial and fresh-keeping material, can inhibit the formation of bacterial biofilm, and can effectively prolong the shelf life of fresh soybean milk.

Description

Technical Field

[0001] The present invention relates to the technical field of food preservation, and in particular to an enzyme-responsive intelligent antibacterial gelatin microgel and a preparation method thereof. Background Art

[0002] Foodborne microbial contamination is a major factor affecting food safety and nutritional quality. In the natural environment, most microorganisms can secrete components such as exopolysaccharides, proteins, and e-DNA into the extracellular environment under suitable conditions, and organically wrap the bacterial cells to form a highly organized three-dimensional polymer structure, namely a biofilm. Due to the buffering and protective effects of the biofilm matrix molecules, the bacteria encapsulated in the biofilm show strong tolerance to adverse environmental factors such as disinfectants, fungicides, and preservatives. The resistance of biofilm-bound bacteria is hundreds to thousands of times that of planktonic bacteria. Therefore, once spoilage bacteria or pathogens in the food industry form biofilms, they will be difficult to completely remove and kill through conventional disinfection methods, often resulting in serious economic losses to production, and may significantly shorten the shelf life of food in circulation, bringing food microbial safety risks and nutrient loss. Therefore, preventing and controlling the formation of microbial biofilms is of great significance to the safe production, circulation, and consumption of food.

[0003] Soy products are nutritious and rich in protein. They are extremely susceptible to contamination from various microorganisms in raw materials, packaging materials, and the production and processing environment. This can lead to spoilage, such as rancidity, flatulence, protein coagulation and degradation, and even the production of toxic biogenic amines and bacterial toxins. Thermotolerant Bacillus is one of the dominant spoilage bacteria that causes soy milk to spoil. It has the characteristic of rapidly forming biofilms, which easily form in large numbers on the surfaces of production equipment and utensils, and within food matrices. These biofilms are often difficult to completely remove and kill, and are a major threat to the processing and safe storage of soy products. Traditional sterilization methods for soy products primarily rely on high-temperature sterilization, but the sterilization intensity is often too high, destroying the nutritional active ingredients and reducing sensory quality.

[0004] In recent years, with increasing public awareness of food safety and nutritional health, the development and application of new food preservation control technologies have garnered significant industry attention. A growing number of studies have demonstrated that active ingredients in plant essential oils exhibit excellent inhibitory and scavenging activity against the biofilm formation of food spoilage bacteria and pathogens, demonstrating their potential as green, safe, and highly effective natural food preservatives. For example, Sichuan pepper essential oil, a natural plant essential oil extracted from Sichuan pepper peel, contains active molecules such as linalool and limonene, demonstrating excellent biofilm inhibition and scavenging activity. However, essential oil components are mostly volatile, poorly water-soluble small molecules, exhibiting sensitivity and instability to environmental factors such as light, oxygen, and temperature. In practical applications, the sustained and stable antibacterial effects have yet to meet production requirements. Furthermore, essential oil components have a distinctive aromatic odor and a low sensory threshold. Excessive use can negatively impact food flavor, while insufficient use can lead to unsatisfactory preservation and antiseptic effects. In order to overcome these shortcomings, it is urgent to protect the pepper essential oil or its main active components through embedding, encapsulation and other technologies to enhance the stability and durability of the pepper essential oil's antibacterial and anti-biofilm properties.

[0005] At present, microencapsulation and pickering emulsion encapsulation are mainly used to protect the active components of plant essential oils, delay their release, and enhance the stability and durability of the antibacterial effect. However, the related products obtained by these technologies still have some problems and defects in product storage and application in real food systems. The release rate of the carrier of the embedded active molecules in the environment is uncontrollable. Some of them begin to release in a crash-like manner after entering the food, causing the effective concentration of the antibacterial components in the food system to drop below the effective antibacterial concentration in a relatively short period of time, losing the antibacterial and antimicrobial functions, and the preservation effect is poor. Although some products prepared with embedded carriers have good stability during storage, when rapid release is required in food applications, such as when the amount of spoilage bacteria in the food matrix is ​​large, it is difficult to achieve rapid release and achieve the ideal antibacterial, antimicrobial and preservation effects.

[0006] Therefore, the development of an antibacterial material loaded with plant essential oils that can achieve sustained and controlled release and is safe for consumption is of great significance and practical value for promoting the application of plant essential oil preservatives in complex food systems. Summary of the Invention

[0007] The present invention aims to address at least one of the technical problems existing in the above-mentioned prior art. It provides an enzyme-responsive intelligent antibacterial gelatin microgel. This enzyme-responsive intelligent antibacterial gelatin microgel is a material loaded with plant essential oil or its key active components, enabling effective protection of the active components and intelligent, responsive, and controlled release.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned enzyme-responsive intelligent antibacterial gelatin microgel, so as to facilitate and better prepare the enzyme-responsive intelligent antibacterial gelatin microgel.

[0009] In order to achieve the above invention objectives, the specific technical solutions adopted by the present invention are:

[0010] A method for preparing enzyme-responsive intelligent antibacterial gelatin microgel comprises the following steps:

[0011] (1) Preparation of the aqueous phase containing glycosidase: gelatin is added to ultrapure water, stirred in a water bath to obtain a uniform and transparent gelatin solution, β-glucosidase is then added to the solution, mixed uniformly to obtain the aqueous phase, and the pH value of the aqueous phase is adjusted;

[0012] (2) Preparation of an oil phase containing plant essential oil: adding plant essential oil or a single component of plant essential oil to plant oil and mixing uniformly to obtain an oil phase;

[0013] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase containing glycosidase prepared in step (1) and the oil phase containing plant essential oil prepared in step (2) are slowly mixed, an emulsifier is added for emulsification, and the mixture is immediately placed in an ice water bath. TG enzyme is then added for cross-linking treatment, and finally centrifugation and washing with ultrapure water are performed to obtain enzyme-responsive intelligent antibacterial gelatin microgel.

[0014] As a better embodiment of the present application, in step (1), the mass fraction of the gelatin solution is 12.5-20% (specifically 12.5%, 13%, 13.5%, 14%, 15.5%, 16%, 16.5%, 17%, 18.5%, 19%, 19.5%, 20%, etc.); the temperature of the water bath stirring is 45-55°C (specifically 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, etc.).

[0015] As a preferred embodiment of the present application, the activity unit of β-glucosidase in step (1) is 0.5-1.0 U / mL (specifically 0.5 U / mL, 0.6 U / mL, 0.7 U / mL, 0.8 U / mL, 0.9 U / mL, 1.0 U / mL, etc.); the volume ratio of gelatin solution to β-glucosidase solution is 50:1-25:1 (specifically 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, etc.); the pH value of the aqueous phase is 3.9-5.1 (specifically 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, etc.).

[0016] As a preferred embodiment of the present application, in step (2), the vegetable oil is soybean oil; the plant essential oil or the single component of the plant essential oil is pepper essential oil, linalool or limonene; the volume ratio of the vegetable oil, the plant essential oil or the single component of the plant essential oil to the emulsifier is 460:40:1 to 300:200:5 (specifically 460:40:1, 450:50:1, 440:60:1, 430:70:1, 460:7 ...30:70:1, 460:70:1, 430:70:1, 430:70:1, 430:70:1, 430:70:1, 430:70:1, 430:70:1 :1, 420:80:1, 410:90:1, 410:100:1, 400:110:1, 390:120:1, 380:130:1, 370:140:1, 360:150:1, 350:160:1, 340:170:1, 330:180:1, 320:190:1, 310:200:2, 300:200:5, etc.).

[0017] As a preferred embodiment of the present application, in step (3), the volume ratio of the water phase to the oil phase is 1:1 to 9:1.

[0018] As a preferred embodiment of the present application, in step (3), the emulsifier is Tween-20; the emulsification temperature is 45-55°C (specifically 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, etc.), and the emulsification speed is 200-1000r / min (specifically 200r / min, 300r / min, 400r / min, 500r / min, 600r / min, 700r / min, 800r / min, 900r / min, 1000r / min, etc.).

[0019] As a preferred embodiment of the present application, in step (3), the amount of emulsifier Tween-20 added is 0.5-1.0 mL / 100 mL (specifically 0.5 mL / 100 mL, 0.6 mL / 100 mL, 0.7 mL / 100 mL, 0.8 mL / 100 mL, 0.9 mL / 100 mL, 1.0 mL / 100 mL, etc.), and the emulsification time is 10-30 min (specifically 10 min, 15 min, 20 min, 25 min, 30 min, etc.).

[0020] As a preferred embodiment of the present application, the ice water bath time in step (3) is 10-20 min; the amount of TG enzyme added is 5%-10% of the amount of gelatin added (specifically 5%, 6%, 7%, 8%, 9%, 10%, etc.).

[0021] As a better embodiment of the present application, the cross-linking conditions in step (3) are: cross-linking at 20-35°C (specifically 20°C, 25°C, 30°C, 35°C, etc.) for 30-60 min (specifically 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.).

[0022] The third invention object of the present invention is to protect the application of enzyme-responsive intelligent antibacterial gelatin microgel prepared by any of the above methods or combinations of method steps in the preservation of fresh soy milk.

[0023] The specific application method is:

[0024] The enzyme-responsive smart antibacterial gelatin microgel prepared by the above method is directly added to fresh soy milk (cooled to room temperature), stirred and dispersed evenly, filled and sealed, and stored at room temperature of 25°C or 4°C. Based on the volume of fresh soy milk, the addition amount of the enzyme-responsive smart antibacterial gelatin microgel is 5% to 20% (specifically, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc.).

[0025] Comparative experiments show that the addition of the enzyme-responsive intelligent antibacterial gelatin microgel provided by the present invention can significantly inhibit the growth of the total colony count in fresh soy milk, delay the formation time of biofilm at the gas-liquid interface of soy milk, inhibit the flocculation instability, oxidative browning and rancidity of the soy milk slurry, and extend the storage shelf life of the fresh soy milk at 25°C and 4°C by 1 day and 4 days, respectively.

[0026] Enzyme-responsive intelligent antibacterial gelatin microgels can efficiently encapsulate and protect plant essential oils and their active molecules, enhancing the chemical stability and sustained release ability of the active molecules; they have good enzyme response characteristics, realizing the controllable intelligent release of active molecules; through the antibacterial active components of essential oils, they cooperate with glycosidases that have the ability to hydrolyze the extracellular polysaccharides of bacterial biofilm matrix to enhance the antibacterial and anti-biofilm effects.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention utilizes the strong ability of food spoilage bacteria such as Bacillus to produce extracellular proteases. Using gelatin with a certain degree of cross-linking as the shell matrix molecule, plant essential oils or their components, in conjunction with glycoside hydrolases, a protease-responsive composite antibacterial gelatin microgel is prepared. When extracellular proteases secreted by contaminating microorganisms are present in the food matrix, the microgel shell protein matrix is ​​hydrolyzed, causing the gelatin microgel morphology to change and gradually releasing the essential oil active molecules and glycoside hydrolases embedded in the microgel. The response release rate can be precisely regulated by the hydrolytic activity of the extracellular proteases produced by the contaminating microorganisms. Among them, Sichuan pepper essential oil contains active components such as linalool and limonene, which can exert direct antibacterial and antimicrobial activity. At the same time, β-glucosidase can target and hydrolyze extracellular polysaccharide molecules in the matrix components of the biofilm of putrefactive bacteria, delaying the formation and accumulation of biofilms. It can also effectively destroy the formed biofilms and disperse the bacteria in the biofilms, transforming them into free bacteria, enhancing the accessibility of essential oil components to the surface of bacteria, improving the bactericidal effect, and ultimately realizing the real-time intelligent and controllable release of active small molecules and extracellular matrix hydrolases, enhancing the synergistic antibacterial and anti-biofilm effects.

[0029] (2) The preparation method of the present invention has a simple process, low requirements on equipment, and the raw materials are all industrialized and easy to obtain. The cost is low and it is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The particle size distribution and microscope image of Example 10 of the present invention are shown.

[0031] Figure 2 This is a laser confocal scanning microscope image of Example 10 of the present invention.

[0032] Figure 3-1 and Figure 3-2 All of them are enzyme response characteristic result diagrams of Example 10 of the present invention.

[0033] Figure 4-1 and Figure 4-2 All of them are graphs showing the cumulative release of linalool and β-glucosidase according to Example 10 of the present invention.

[0034] Figure 5-1 and Figure 5-2 All of them are antibacterial and anti-biofilm curves of Example 10 of the present invention and Comparative Examples 1-4.

[0035] Figure 6 1 is a diagram showing the preservation effects of Example 10 of the present invention and Comparative Examples 1-4 on fresh soy milk.

[0036] Figure 7 This is a graph showing the changes in the total number of colonies in fresh soy milk in Example 10 of the present invention and Comparative Examples 1-4.

[0037] Figure 8 This is a graph showing the test results of the hydrolysis and dispersion ability of α-amylase, β-glucosidase, cellulase and pectinase on Bacillus amyloliquefaciens biofilm.

[0038] Figure 9 The results show the effects of different gelatin concentrations on microgel particle size distribution (A), enzyme activity recovery rate and linalool encapsulation efficiency (B).

[0039] Figure 10 The results show the effect of different emulsification speeds on microgel particle size distribution (A), enzyme activity recovery rate and linalool encapsulation efficiency (B).

[0040] Figure 11 The results show the effects of different pH values ​​on microgel particle size distribution (A), enzyme activity recovery rate and linalool encapsulation efficiency (B).

[0041] Figure 12 The results show the effect of different water-oil ratios on microgel particle size distribution (A), enzyme activity recovery rate and linalool encapsulation efficiency (B). DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0043] It should be noted that, unless otherwise specified, the raw materials used in the present invention are the raw materials commonly used in the art, and the test / test methods are the methods conventionally used in the art.

[0044] In this application, unless otherwise specified, all ratios refer to mass ratios; % refers to mass %.

[0045] Example 1

[0046] (1) Preparation of the aqueous phase containing glycosidase: 5.625 g of gelatin was added to 44 mL of ultrapure water and stirred in a water bath at 45°C to obtain a uniform gelatin solution. 1 mL of β-glucosidase with an enzyme activity unit of 0.5 U / mL was then added and mixed to obtain the aqueous phase. The pH value of the aqueous phase was adjusted to 5.1.

[0047] (2) Preparation of the oil phase containing plant essential oil: Add 1.2 mL of linalool to 13.8 mL of soybean oil and mix well to obtain the oil phase.

[0048] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 3:1, and then 0.30 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 600 r / min for 15 min, and then immediately placed in an ice water bath for 20 min. Then, 10% of the amount of TG enzyme added to the gelatin was added and cross-linked at 20°C for 60 min. The mixture was centrifuged and washed three times with ultrapure water to obtain enzyme-responsive intelligent antibacterial gelatin microgel.

[0049] Example 2

[0050] (1) Preparation of aqueous phase: Add 6.25 g of gelatin to 49 mL of ultrapure water and stir in a 46°C water bath to obtain a uniform gelatin solution. Then add 1 mL of β-glucosidase with an enzyme activity unit of 0.6 U / mL and mix well to obtain the aqueous phase. The pH value of the aqueous phase is adjusted to 4.8.

[0051] (2) Preparation of oil phase: Add 0.8 mL of linalool to 9.2 mL of soybean oil and mix well to obtain the oil phase.

[0052] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 5:1, and then 0.50 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 800 r / min for 25 min, and then immediately placed in an ice water bath for 20 min. TG enzyme at 8% of the gelatin addition was added and cross-linked at 25°C for 40 min. The enzyme-responsive intelligent antibacterial gelatin microgel was obtained by centrifugation and washing three times with ultrapure water.

[0053] Example 3

[0054] (1) Preparation of aqueous phase: Add 6.5625 g of gelatin to 51.5 mL of ultrapure water and stir in a 47°C water bath to obtain a uniform gelatin solution. Then add 1 mL of β-glucosidase with an enzyme activity unit of 0.7 U / mL and mix well to obtain the aqueous phase. The pH value of the aqueous phase was adjusted to 4.5.

[0055] (2) Preparation of oil phase: Add 0.6 mL of linalool to 6.9 mL of soybean oil and mix well to obtain the oil phase.

[0056] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 7:1, and then 0.40 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 1000 r / min for 30 min, and then immediately placed in an ice water bath for 20 min. Then, 9% of the amount of TG enzyme added to the gelatin was added and cross-linked at 30°C for 50 min. The enzyme-responsive intelligent antibacterial gelatin microgel was obtained by centrifugation and washing three times with ultrapure water.

[0057] Example 4

[0058] (1) Preparation of aqueous phase: Add 8.53125 g of gelatin to 51.5 mL of ultrapure water and stir in a 48°C water bath to obtain a uniform gelatin solution. Then add 1 mL of β-glucosidase with an enzyme activity unit of 0.5 U / mL and mix well to obtain the aqueous phase. The pH value of the aqueous phase was adjusted to 3.9.

[0059] (2) Preparation of oil phase: Add 0.5 mL of linalool to 6.9 mL of soybean oil and mix well to obtain the oil phase.

[0060] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 7:1, and then 0.60 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 800 r / min for 10 min, and then immediately placed in an ice water bath for 20 min. Then, 5% of the amount of TG enzyme added to the gelatin was added and cross-linked at 35°C for 30 min. The enzyme-responsive intelligent antibacterial gelatin microgel was obtained by centrifugation and washing three times with ultrapure water.

[0061] Example 5

[0062] (1) Preparation of aqueous phase: Add 7.3125 g of gelatin to 44 mL of ultrapure water and stir in a 49°C water bath to obtain a uniform gelatin solution. Then add 1 mL of β-glucosidase with an enzyme activity unit of 0.9 U / mL and mix well to obtain the aqueous phase. The pH value of the aqueous phase was adjusted to 4.2.

[0063] (2) Preparation of oil phase: Add 1.1 mL of linalool to 13.8 mL of soybean oil and mix well to obtain the oil phase.

[0064] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 3:1, and then 0.35 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 1000 r / min for 10 min, and then immediately placed in an ice water bath for 20 min. Then, 10% of the TG enzyme added to the gelatin was added and cross-linked at 25°C for 40 min. The enzyme-responsive intelligent antibacterial gelatin microgel was obtained by centrifugation and washing three times with ultrapure water.

[0065] Example 6

[0066] (1) Preparation of aqueous phase: Add 8.125 g of gelatin to 49 mL of ultrapure water, stir in a 51°C water bath to obtain a uniform gelatin solution, then add 1 mL of β-glucosidase with an enzyme activity unit of 1.0 U / mL, mix well to obtain the aqueous phase, and adjust the pH value of the aqueous phase to 4.5.

[0067] (2) Preparation of oil phase: Add 0.8 mL of linalool to 9.2 mL of soybean oil and mix well to obtain the oil phase.

[0068] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 5:1, and then 0.45 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 600 r / min for 25 min, and then immediately placed in an ice water bath for 20 min. Then, 7% of the amount of TG enzyme added to the gelatin was added and cross-linked at 30°C for 50 min. The enzyme-responsive intelligent antibacterial gelatin microgel was obtained by centrifugation and washing three times with ultrapure water.

[0069] Example 7

[0070] (1) Preparation of aqueous phase: Add 10 g of gelatin to 49 mL of ultrapure water and stir in a 52°C water bath to obtain a uniform gelatin solution. Then add 1 mL of β-glucosidase with an enzyme activity unit of 1.0 U / mL and mix well to obtain the aqueous phase. The pH value of the aqueous phase was adjusted to 3.9.

[0071] (2) Preparation of oil phase: Add 0.9 mL of linalool to 9.2 mL of soybean oil and mix well to obtain the oil phase.

[0072] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 5:1, and then 0.55 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 1000 r / min for 15 min, and then immediately placed in an ice water bath for 20 min. TG enzyme at 8% of the gelatin addition was added and cross-linked at 25°C for 40 min. The enzyme-responsive intelligent antibacterial gelatin microgel was obtained by centrifugation and washing three times with ultrapure water.

[0073] Example 8

[0074] (1) Preparation of aqueous phase: Add 10.5 g of gelatin to 51.5 mL of ultrapure water and stir in a 53°C water bath to obtain a uniform gelatin solution. Then add 1 mL of β-glucosidase with an enzyme activity unit of 0.6 U / mL and mix well to obtain the aqueous phase. The pH value of the aqueous phase was adjusted to 4.2.

[0075] (2) Preparation of oil phase: Add 0.7 mL of linalool to 6.9 mL of soybean oil and mix well to obtain the oil phase.

[0076] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 7:1, and then 0.50 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 600 r / min for 25 min, and then immediately placed in an ice water bath for 20 min. Then, 10% of the amount of TG enzyme added to the gelatin was added and cross-linked at 25°C for 50 min. The enzyme-responsive intelligent antibacterial gelatin microgel was obtained by centrifugation and washing three times with ultrapure water.

[0077] Example 9

[0078] (1) Preparation of aqueous phase: Add 9 g of gelatin to 44 mL of ultrapure water and stir in a 55°C water bath to obtain a uniform gelatin solution. Then add 1 mL of β-glucosidase with an enzyme activity unit of 0.5 U / mL and mix well to obtain the aqueous phase. The pH value of the aqueous phase was adjusted to 4.7.

[0079] (2) Preparation of oil phase: Add 1.2 mL of linalool to 13.8 mL of soybean oil and mix well to obtain the oil phase.

[0080] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 3:1, and then 0.60 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 800 r / min for 20 min, and then immediately placed in an ice water bath for 20 min. TG enzyme of 6% of the gelatin addition was added and cross-linked at 35°C for 45 min. The enzyme-responsive intelligent antibacterial gelatin microgel was obtained by centrifugation and washing three times with ultrapure water.

[0081] Example 10

[0082] (1) Preparation of aqueous phase: Add 8.125 g of gelatin to 49 mL of ultrapure water, stir in a 50°C water bath to obtain a uniform gelatin solution, then add 1 mL of β-glucosidase with an enzyme activity unit of 0.5 U / mL, mix well to obtain the aqueous phase, and adjust the pH value of the aqueous phase to 4.5.

[0083] (2) Preparation of oil phase: Add 0.8 mL of linalool to 9.2 mL of soybean oil and mix well to obtain the oil phase.

[0084] (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 5:1, and then 0.50 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 800 r / min for 30 min, and then immediately placed in an ice water bath for 20 min. Then, 10% of the amount of TG enzyme added to the gelatin was added and cross-linked at 20°C for 60 min. The enzyme-responsive intelligent antibacterial gelatin microgel was obtained by centrifugation and washing three times with ultrapure water.

[0085] Comparative Example 1

[0086] (1) Preparation of aqueous phase: Add 8.125 g of gelatin to 50 mL of ultrapure water, stir in a 50°C water bath to obtain a uniform gelatin solution, and adjust the pH value of the aqueous phase to 4.5.

[0087] (2) Preparation of oil phase: 10 mL of soybean oil is the oil phase.

[0088] (3) Preparation of blank gelatin microgels: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 5:1, and then 0.50 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 800 r / min for 30 min, and then immediately placed in an ice water bath for 20 min. TG enzyme at 10% of the amount of gelatin added was added and cross-linked at 20°C for 60 min. The blank gelatin microgels were then washed three times by centrifugation with ultrapure water to obtain the blank gelatin microgels.

[0089] Comparative Example 2

[0090] (1) Preparation of aqueous phase: Add 8.125 g of gelatin to 50 mL of ultrapure water, stir in a 50°C water bath to obtain a uniform gelatin solution, and adjust the pH value of the aqueous phase to 4.5.

[0091] (2) Preparation of oil phase: 10 mL of soybean oil is the oil phase.

[0092] (3) Preparation of blank gelatin microgels: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 5:1, and then 0.50 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 800 r / min for 30 min, and then immediately placed in an ice water bath for 20 min. Then, 10% of the amount of TGase added to the gelatin was added and cross-linked at 20°C for 60 min. The blank gelatin microgels were then centrifuged and washed three times with ultrapure water to obtain blank gelatin microgels.

[0093] (4) Preparation of a mixture of blank gelatin microgel, β-glucosidase, and linalool: 1 mL of β-glucosidase with an enzyme activity unit of 0.5 U / mL was added to the blank gelatin microgel prepared in step (3) and mixed evenly.

[0094] Comparative Example 3

[0095] (1) Preparation of aqueous phase: Add 8.125 g of gelatin to 50 mL of ultrapure water, stir in a 50°C water bath to obtain a uniform gelatin solution, and adjust the pH value of the aqueous phase to 4.5.

[0096] (2) Preparation of oil phase: 10 mL of soybean oil is the oil phase.

[0097] (3) Preparation of blank gelatin microgels: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 5:1, and then 0.50 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 800 r / min for 30 min, and then immediately placed in an ice water bath for 20 min. TG enzyme at 10% of the amount of gelatin added was added and cross-linked at 20°C for 60 min. The blank gelatin microgels were then washed three times by centrifugation with ultrapure water to obtain the blank gelatin microgels.

[0098] (4) Preparation of a mixture of blank gelatin microgel, β-glucosidase, and linalool: 0.8 mL of linalool was added to the blank gelatin microgel prepared in step (3) and mixed evenly.

[0099] Comparative Example 4

[0100] (1) Preparation of aqueous phase: Add 8.125 g of gelatin to 50 mL of ultrapure water, stir in a 50°C water bath to obtain a uniform gelatin solution, and adjust the pH value of the aqueous phase to 4.5.

[0101] (2) Preparation of oil phase: 10 mL of soybean oil is the oil phase.

[0102] (3) Preparation of blank gelatin microgels: The aqueous phase prepared in step (1) and the oil phase prepared in step (2) were slowly mixed in a ratio of 5:1, and then 0.5 mL of Tween-20 was added. The mixture was emulsified at 50°C and a speed of 800 r / min for 30 min, and then immediately placed in an ice water bath for 20 min. Then, 10% of the amount of TGase added to the gelatin was added and cross-linked at 20°C for 60 min. The blank gelatin microgels were then washed three times by centrifugation with ultrapure water to obtain the blank gelatin microgels.

[0103] (4) Preparation of a mixture of blank gelatin microgel, β-glucosidase, and linalool: 1 mL of β-glucosidase with an enzyme activity unit of 0.5 U / mL and 0.8 mL of linalool were added to the blank gelatin microgel prepared in step (3) and mixed evenly.

[0104] Experimental Example 1

[0105] The enzyme-responsive intelligent antibacterial gelatin microgels obtained in Examples 1-10 were measured for their average particle size and particle size distribution using a Rise-2006 laser particle size analyzer at a speed of 200 r / min and a light shielding ratio greater than 1.0. 100 mg of the enzyme-responsive intelligent antibacterial gelatin microgel was dispersed in sterile distilled water to an appropriate concentration. A 20 μL sample was then placed on a glass slide without a coverslip to prevent droplet compression. The sample was observed under a 40x optical microscope objective and photographed.

[0106] Weigh 1 g of the enzyme-responsive intelligent antibacterial gelatin microgel prepared in Examples 1-10 and add it to a centrifuge tube containing 9 mL of 95% ethanol. Ultrasonic treatment was performed in a 50°C water bath for 1 h to completely extract linalool from the gelatin microgel. The insoluble matter was centrifuged at 4000 r / min for 10 min to remove the insoluble precipitate. The supernatant was aspirated and the sample absorbance was measured at a wavelength of 276 nm. The free concentration of linalool was obtained according to the standard curve, and the linalool encapsulation efficiency was calculated according to the formula.

[0107]

[0108] Where C0 represents the initial concentration, mg / mL; C1 represents the free phase concentration, mg / mL.

[0109] The activity of β-glucosidase was determined by 3,5-dinitrosalicylic acid method (DNS). 1.0 mL of β-glucosidase solution with an enzyme activity unit of 0.5 U / mL or 0.5 g of enzyme-responsive intelligent antibacterial gelatin microgel obtained in Example 1-10 was placed in a test tube, 1 mL of PBS buffer solution with pH 5 was added and preheated in a 50°C water bath for 5 min, and then 1 mL of 1 mg / mL salicin solution preheated at 50°C was added; the free enzyme and enzyme-responsive intelligent antibacterial gelatin microgel were reacted in a 50°C water bath for 30 min. After the reaction, 1.5 mL of DNS reagent was added, mixed and immediately boiled in water for 5 min to inactivate the enzyme. 1 mL of supernatant was immediately taken, 1 mL of distilled water and 1.5 mL of DNS reagent were added, boiled in water for 5 min, cooled to room temperature in an ice water bath, and finally diluted to 25 mL, and the absorbance OD was measured. 540nm The same amount of heat-inactivated free enzyme was used as a blank control. Enzyme activity is defined as the amount of enzyme required to hydrolyze 1 μmol of glucose per minute under the above conditions. The β-glucosidase activity recovery rate was calculated according to the formula.

[0110]

[0111] Where C0 represents the total activity of free enzyme, U; C1 represents the total activity of immobilized enzyme, U.

[0112] Taking Example 10 as an example, the results obtained are: particle size 1.153 μm, β-glucosidase enzyme activity recovery rate 73.97%, and linalool encapsulation efficiency 77.51%. Particle size distribution results and microscope images take Example 10 as an example. Figure 1 As shown, the results of other embodiments are similar to those of embodiment 10 and are not described in detail here.

[0113] The results obtained fully meet the requirements of active molecule carriers. The β-glucosidase enzyme activity recovery rate and linalool encapsulation efficiency exceed 70%, indicating that the waste of active substances can be minimized during the microgel preparation process; the microgel particle size is normally distributed, the particle size distribution range is narrow, the dispersion is uniform, and the microgel is round.

[0114] Experimental Example 2

[0115] The enzyme-responsive intelligent antibacterial gelatin microgels obtained in Examples 1-10 were observed using a FV 1200 laser confocal scanning microscope. A mixed fluorescent dye solution was prepared using 0.1 wt.% Nile blue A and 0.1 wt.% Nile red, which was stored in the dark at room temperature. Nile blue A was dissolved in ultrapure water, and Nile red was dissolved in isopropanol. 40 μL of the mixed dye was added to 1 g of antibacterial gelatin microgel, and 10 μL of the dyed antibacterial gelatin microgel was placed on a glass slide and gently covered with a coverslip. Nile red was used to stain the oil phase, and Nile blue A was used to stain gelatin. Nile red and Nile blue A fluorescent dyes were excited by 488 nm argon laser and 633 nm He-Ne laser, respectively.

[0116] The laser confocal scanning microscope image is taken as an example of Example 10. Figure 2 The results of other embodiments are similar to those of embodiment 10, and are not described in detail here.

[0117] Figure 2 -A is gelatin excited at 633 nm; Figure 2 -B is the oil phase excited at 488 nm; Figure 2 -C is the superposition of A and B. Figure 2 It can be seen that the enzyme-responsive intelligent antibacterial gelatin microgel is spherical, gelatin (red) is distributed throughout the microgel, and the oil phase (green) is distributed inside the microgel. This is mainly because gelatin aggregates to form protein gel during the curing process, and eventually forms a huge three-dimensional network microstructure, which contains a large number of pores and voids. The oil phase fills these gelatin pores, and finally forms a stable O / W gelatin microgel.

[0118] Experimental Example 3

[0119] Preparation of bacterial suspension: Inoculate Bacillus amyloliquefaciens DY1a into LB medium and culture at 37°C, 160 rpm for 24 hours to obtain an activated bacterial suspension. Then centrifuge the bacterial suspension at 4000 rpm for 10 minutes at 4°C. Wash the bacterial pellet three times with sterile phosphate buffer (pH 7.4) and adjust the bacterial OD with sterile PBS buffer. 600nm to 0.10(~5×10 8 CFU / mL).

[0120] Preparation of extracellular protease at different concentrations: The bacterial suspension and LB liquid culture medium were inoculated into a test tube at a ratio of 1:3, cultured at 37°C, 160 r / min, and shaken for 48 h. The supernatant was centrifuged at 4000 r / min for 10 min, and the supernatant was filtered and sterilized with a 0.22 μm microporous filter membrane to obtain a 1.25 U / mL crude extracellular protease solution. The 1.25 U / mL crude extracellular protease solution was diluted with sterile distilled water to prepare extracellular protease solutions with different concentration gradients (0, 0.25, 0.5, 0.75, 1.0, and 1.25 U / mL).

[0121] Effect of different extracellular protease concentrations on enzyme response effect: Weigh the enzyme-responsive intelligent antibacterial gelatin microgel obtained in Example 1-10 with a mass of B and add extracellular protease solutions with different concentration gradients into a beaker at a mass ratio of 1:10. In a 37°C water bath, enzymatically hydrolyze for 5 hours, centrifuge at 4000 r / min for 2 minutes, discard the supernatant, record the mass of the unhydrolyzed gelatin microgel as A, and calculate the hydrolysis rate of the gelatin microgel according to the formula.

[0122] Effect of different hydrolysis times on enzyme response effect: Weigh the enzyme-responsive intelligent antibacterial gelatin microgel obtained in Example 1-10 with a mass of B and 1.25U / mL extracellular protease solution in a mass ratio of 1:10 and add them to a beaker. In a 37°C water bath, they were enzymatically hydrolyzed for a certain time (0, 1, 2, 3, 4 and 5h). The sample was centrifuged at 4000r / min for 2min, and the supernatant was discarded. The mass of the gelatin microgel that was not enzymatically hydrolyzed was recorded as A, and the hydrolysis rate of the gelatin microgel was calculated according to the formula.

[0123]

[0124] The enzyme response effect diagram takes Example 10 as an example. Figure 3-1 and Figure 3-2 The results of other embodiments are similar to those of embodiment 10, and are not described in detail here.

[0125] Depend on Figure 3-1 and Figure 3-2It can be seen that the hydrolysis rate of the enzyme-responsive smart antimicrobial gelatin microgel increases with increasing extracellular enzyme activity and prolonged enzymatic hydrolysis time, indicating that the synthesized enzyme-responsive smart antimicrobial gelatin microgel can be hydrolyzed under extracellular protease conditions. The hydrolysis rate after 5 hours of enzymatic hydrolysis was 56.00%, demonstrating that the enzyme-responsive smart antimicrobial gelatin microgel has good enzyme responsiveness. More importantly, when the extracellular protease activity was 0 U / mL, the enzyme-responsive smart antimicrobial gelatin microgel was not decomposed. However, when extracellular protease was added to the environment, the gelatin matrix was hydrolyzed, the microgel morphology changed, and the active molecules embedded in the microgel were released into the application environment. This demonstrates the intelligent release property of the enzyme-responsive smart antimicrobial gelatin microgel. The active molecules embedded in the gelatin microgel are only released when microorganisms secrete extracellular proteases.

[0126] Experimental Example 4

[0127] Weigh 2 g of the enzyme-responsive intelligent antibacterial gelatin microgel obtained in Example 1-10 and mix it with the crude extracellular protease solution in a mass ratio of 1:10. The mixture was enzymatically hydrolyzed at 37 ° C for 10 h. Samples were taken at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 10 h of enzymatic hydrolysis, respectively, and centrifuged at 4000 r / min for 10 min. The supernatant was taken and the hydrolase activity and linalool concentration were determined by the methods of Experimental Example 3 and Experimental Example 2, respectively.

[0128] The cumulative release of linalool and β-glucosidase is shown in Example 10. Figure 4-1 and Figure 4-2 The results of other embodiments are similar to those of embodiment 10, and are not described in detail here.

[0129] Depend on Figure 4-1 and Figure 4-2 It can be seen that the release behaviors of linalool and β-glucosidase differed over the first two hours as the extracellular protease activity increased. This is mainly because in the initial stage, the protease mainly acts on the outermost gelatin matrix, and the diffusion rate of linalool from the in situ to the microgel surface is slow, while the enzyme loaded on the gelatin matrix is ​​gradually released. With the increase of extracellular enzyme activity and the extension of enzymatic hydrolysis time, the cumulative release rate of linalool and enzyme in the antibacterial gelatin microgel showed an upward trend, indicating that the synthesized antibacterial gelatin microgel can be hydrolyzed under extracellular protease conditions. After 4 hours of enzymatic hydrolysis at 1.2 U / mL of extracellular hydrolase, the release rate of linalool and enzyme reached more than 50%, demonstrating that the antibacterial gelatin microgel has a strong sustained-release and controlled-release effect.

[0130] Experimental Example 5

[0131] Bacterial growth curve determination: Bacterial suspension was prepared according to the method in Example 4, and 0.7 mL of bacterial suspension was inoculated into 350 mL of LB liquid medium to make the bacterial suspension concentration 106 CFU / mL, 15g of enzyme-responsive intelligent antibacterial gelatin microgels obtained in Examples 1-10 and Comparative Examples 1-2 were added, cultured at 37°C and 160r / min, and samples were taken at 0, 2, 4, 6, 9, 12, 15, 18, 21, and 24h, respectively, and the OD values ​​were determined. 600nm The inhibition growth curve was drawn.

[0132] Biofilm inhibition curve determination: The bacterial suspension was prepared according to the method in Example 4, and 1 mL of the bacterial suspension was inoculated into 4 mL of TSBS medium to make the bacterial suspension concentration 10 6 CFU / mL, 3 g of the enzyme-responsive intelligent antibacterial gelatin microgel obtained in Examples 1-10 and Comparative Examples 1-2 was added, and the mixture was cultured at 37°C. Samples were taken at 12, 18, 22, 26, 30, 34, 38, 42, 44, 48, 52, 56, and 60 h, respectively. The amount of biofilm formation was determined according to the method of 2.2.2.3, and a curve of the ability to inhibit biofilm formation was drawn.

[0133] The antibacterial growth curve and the anti-biofilm curve are shown in Example 10 and Comparative Examples 1-2. Figure 5-1 and Figure 5-2 The results of other embodiments are similar to those of embodiment 10, and are not described in detail here.

[0134] Depend on Figure 5-1 and Figure 5-2 As can be seen, Examples 1-10 of the present invention all exhibited long-lasting antibacterial and anti-biofilm effects, while Comparative Examples 1 and 2 exhibited poor antibacterial and anti-biofilm effects. This suggests that the enzyme-responsive intelligent antibacterial gelatin microgels prolonged the duration over which the active molecules exerted their antibacterial and anti-biofilm effects. This is primarily due to the absence of active molecules in Comparative Example 1, and the rapid volatilization loss of the unencapsulated active molecules in Comparative Example 2 during the incubation process due to instability. In contrast, the active molecules in Example 10, due to their encapsulation, exhibited reduced volatility and were released slowly, achieving a longer-lasting antibacterial and anti-biofilm effect than the active molecules alone.

[0135] Experimental Example 6

[0136] To prepare fresh soymilk, select plump, pest-free, and brightly colored soybeans. Wash with drinking water and soak overnight in clean water at a ratio of 1:3 soybeans to water at room temperature. Peel and rinse the soybeans, then grind and boil the slurry in an electric soymilk maker at a ratio of 1:10 soybeans to water to produce fresh soymilk. Filter through three layers of gauze to remove dregs and other impurities. Once the soymilk cools to 60°C, package it in 50mL Erlenmeyer flasks, seal with parafilm, and sterilize in a 90°C waterbath for 30 minutes before cooling to room temperature.

[0137] 10 g of the enzyme-responsive intelligent antibacterial gelatin microgel obtained in Examples 1-10 and the control sample prepared in Comparative Examples 1-2 were added to 25 mL of fresh soy milk, sealed, and stored at 25° C. The appearance changes of the fresh soy milk were observed by taking photos every 24 hours.

[0138] The preservation effect of fresh soy milk is shown in Example 10 and Comparative Examples 1-2. Figure 6 The results of other embodiments are similar to those of embodiment 10, and are not described in detail here.

[0139] Depend on Figure 6 As can be seen, Examples 1-10 of the present invention all have a long-lasting fresh-keeping effect on fresh soy milk, while Comparative Examples 1 and 2 have poor preservation effects. As storage time increases, Example 10 consistently outperforms Comparative Examples 1 and 2. Comparative Example 1 began to produce an ammonia odor and biofilm on the second day, and showed stratification. As the number of days increased, the biofilm gradually thickened and completely deteriorated. Comparative Example 2 only began to produce a visible biofilm and stratification on the fourth day, while Example 10 began to produce a thinner biofilm on the fifth day. This is because Comparative Example 1 does not contain antibacterial and anti-biofilm ingredients, allowing bacteria to rapidly multiply in the soy milk, causing it to deteriorate and smell in a very short time. In contrast, Comparative Example 2, due to its antibacterial effect, deteriorated more slowly than the control group in the early stages of storage. However, due to the volatility of linalool at room temperature, it deteriorated more rapidly in the later stages. Example 10 showed a good preservation effect, which was mainly attributed to the slow release of linalool in the early stage. The low amount of biofilm generated in the end was also related to the decomposition of the biofilm by the embedded β-glucosidase in Example 10, which proved that the enzyme-responsive intelligent antibacterial gelatin microgel had a good soy milk preservation effect.

[0140] Experimental Example 7

[0141] 10 g of the enzyme-responsive intelligent antibacterial gelatin microgels obtained in Examples 1-10 and Comparative Examples 1-2 were added to 25 mL of fresh soy milk. The milk was sealed and stored at 4°C and 25°C, respectively. Samples were taken every 24 hours to determine the total bacterial count in the fresh soy milk. The total bacterial count in the fresh soy milk was determined using the plate count method described in GB 4789.2-2022, "Microbiological Examination of Food: Determination of Total Aerobic Count."

[0142] The total bacterial count of fresh soy milk is changed by taking Example 10 and Comparative Example 1-2 as examples. Figure 7 The results of other embodiments are similar to those of embodiment 10, and are not described in detail here.

[0143] Soy milk is rich in nutrients and provides a good environment for the growth of microorganisms. Therefore, the spoilage of soy milk can be determined by detecting changes in the total colony count. According to GB 7101-2022 "National Food Safety Standard for Beverages", the total colony count should not exceed 100 CFU / mL. Figure 7 It can be seen that the total colony count increases with the increase of storage time, but the total colony count in Example 10 is always lower than that in Comparative Examples 1 and 2. Example 10 can store fresh soy milk at 25°C for more than 1 day, while Comparative Examples 1 and 2 far exceed the limit standard on the first day, indicating that the enzyme-responsive intelligent antibacterial gelatin microgel effectively slows down the spoilage of soy milk.

[0144] The enzyme-responsive intelligent antibacterial gelatin microgel prepared in the embodiment of the present invention is safe and non-toxic, has high encapsulation efficiency and antibacterial and anti-biofilm activity. The enzyme-responsive intelligent antibacterial gelatin microgel is used to preserve fresh soy milk, and the storage time at 25°C exceeds 1 day, which proves that it has a good preservation effect, effectively prevents the fresh soy milk from deteriorating, and extends the storage shelf life.

[0145] Experimental Example 8: Screening of glycoside hydrolases for hydrolyzing biofilms

[0146] The bacterial biofilm formed by static culture at 37°C for 24 hours was collected, and the free bacteria attached to its surface were cleaned. 2 mL of 2 U / mL α-amylase, β-glucosidase, cellulase and pectinase were added to the test tube respectively, and the reaction was hydrolyzed for 6 hours under the optimal reaction temperature. The biofilm treated with glycoside hydrolase was filtered through a cell sieve with a pore size of 40 μm, and the filtrate was centrifuged at 10,000 r / min for 10 minutes. The supernatant was taken and the total sugar content was determined by the phenol-sulfuric acid method. The high or low free total sugar content reflects the effect of different glycoside hydrolases. The results are shown in Figure 8 As can be seen from the figure, the total sugar content of the supernatant after treatment with β-glucosidase is significantly higher than that of other candidate glycoside hydrolases, and it has the strongest ability to hydrolyze the Bacillus amyloliquefaciens biofilm in the present invention.

[0147] Experimental Example 9: Single Factor Experiment Results

[0148] 9.1 Effect of different gelatin concentrations on microgels

[0149] Effects of different gelatin concentrations on microgel particle size, linalool encapsulation efficiency and enzyme activity recovery rate Figure 9 shown. Figure 9 In A, as the gelatin concentration increases, the particle size of the microgel becomes larger. This may be because the high concentration of gelatin solution increases the viscosity of the system, making it difficult to be emulsified into small droplets during the emulsification process. On the other hand, the higher the gelatin concentration, the greater the proportion of its molecules in the emulsion and the chance of mutual collision, making it easier to form microgels with larger particle sizes. Figure 9 In B, within a certain range, as the gelatin concentration increases, the linalool encapsulation efficiency increases. However, when the gelatin concentration exceeds 16.25%, the encapsulation efficiency decreases due to the low ball formation rate in the system and the gelatin's tendency to clump. Regarding enzyme activity recovery, as the gelatin concentration increases, the thicker the microgel wall material, the more enzyme it can load, and the higher the enzyme activity recovery rate. However, when the gelatin concentration reaches 16.25%, its enzyme activity reaches saturation, and further increasing the gelatin concentration does not significantly increase the enzyme activity recovery rate. Therefore, based on the microgel particle size, linalool encapsulation efficiency, and enzyme activity recovery rate, the gelatin concentration levels for preparing microgels were determined to be 12.5%, 16.25%, and 20%.

[0150] 9.2 Effect of different emulsification speeds on microgels

[0151] Effects of different emulsification speeds on microgel particle size, linalool encapsulation efficiency and enzyme activity recovery rate Figure 10 As shown in the figure. When the emulsification speed is too low, the shear force generated by the rotor is too small to disperse the oil phase more evenly in the liquid. The formed emulsion is also unstable, and the droplets easily settle or contact each other, resulting in adhesion of the resulting microgels, resulting in large spherical particle size. As the emulsification speed increases, the linalool encapsulation efficiency and enzyme activity recovery rate both show a trend of first increasing and then decreasing. This is mainly because increasing the emulsification speed can increase the shear force applied to various parts of the emulsion, which improves the dispersion of the enzyme and linalool in the system, forming more uniform droplets. These droplets are stabilized by the centrifugal force of stirring and are less likely to aggregate. As a result, the resulting microgels are evenly distributed and have a high encapsulation efficiency. However, when the emulsification speed exceeds 800 r / min, the emulsion system will form turbulence, which in turn causes uneven forces on the droplets. Collision between droplets causes deformation and damage, exacerbating the collision and agglomeration of the dispersed microgels, resulting in adhesion and even demulsification, thereby reducing the linalool encapsulation efficiency and enzyme activity recovery rate. Therefore, considering the particle size of microgel, linalool encapsulation efficiency and enzyme activity recovery rate, the emulsification speed levels for preparing microgel were determined to be 600 r / min, 800 r / min and 1000 r / min.

[0152] 9.3 Effect of Different pH Values ​​on Microgels

[0153] Effects of different pH values ​​on microgel particle size, linalool encapsulation efficiency and enzyme activity recovery rate Figure 11As shown. When the pH value of the system is between 4 and 5, the pH value in the system is close to the isoelectric point of gelatin, the net charge in the solution is close to zero, and the tendency of the particles to aggregate with each other and reduce the free energy of the system is equal to the tendency of the strong Brownian motion between the particles to prevent them from settling under the action of gravity, thereby making the microgel particle size distribution uniform and the particle size change little. With the increase of pH, the linalool encapsulation efficiency and enzyme activity recovery rate show a trend of first increasing and then decreasing. When pH = 4.5, the linalool encapsulation efficiency and enzyme activity recovery rate are the highest. This may be because the number of microgels formed at this pH is the largest, so the enzyme activity recovery rate and linalool encapsulation rate show a peak. Therefore, based on the particle size of the microgel, the linalool encapsulation efficiency and the enzyme activity recovery rate, the pH levels for preparing microgels were determined to be 3.9, 4.2 and 4.5.

[0154] 9.4 Effects of Different Water-Oil Ratios on Microgels

[0155] The water-oil ratio affects the difficulty of emulsification, which in turn leads to differences in the morphology and properties of microgels. The effects of different water-oil ratios on microgel particle size, linalool encapsulation efficiency, and enzyme activity recovery are shown in Figure 2. Figure 12 As shown in the figure, a higher water-to-oil ratio facilitates the dispersion of the aqueous phase into smaller droplets, resulting in smaller microgel particle size. At a water-to-oil ratio of 1:1, the oil phase accounts for too large a proportion, while the water phase accounts for too small a proportion, leading to poor solution fluidity, adhesion between microgels, and increased particle size. Furthermore, when the water-to-oil ratio is low, the aqueous phase is insufficient to encapsulate the oil phase, resulting in a decrease in ball formation, enzyme activity recovery, and encapsulation efficiency, resulting in a waste of resources. Increasing the water-to-oil ratio improves the emulsification degree of the emulsion, allowing for uniform dispersion of the droplets, enhanced microgel fluidity, and rounded microgels, increasing ball formation and encapsulation efficiency. However, when the water-to-oil ratio is too high, more empty capsules are formed, and the encapsulation efficiency decreases. Therefore, based on the microgel particle size, linalool encapsulation efficiency, and enzyme activity recovery, the water-to-oil ratios for preparing microgels were determined to be 3:1, 5:1, and 7:1.

[0156] Finally, it should be noted that the above embodiments do not limit the present invention in any form. Those skilled in the art will appreciate that modifications and improvements can be made based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are intended to fall within the scope of protection claimed in the present invention.

Claims

1. Application of enzyme-responsive intelligent antibacterial gelatin microgel in fresh soy milk preservation, characterized in that: The preparation method of enzyme-responsive intelligent antibacterial gelatin microgel comprises the following steps: (1) Preparation of aqueous phase containing glycosidase: Add gelatin to ultrapure water and stir in a water bath to obtain a uniform and transparent gelatin solution. Then add β-glucosidase to the solution and mix well to obtain an aqueous phase. The pH value of the aqueous phase is adjusted to 3.9-5.1; the activity unit of β-glucosidase is 0.5-1.0 U / mL. (2) Preparation of an oil phase containing plant essential oil: adding plant essential oil or a single component of plant essential oil to plant oil and mixing them evenly to obtain an oil phase; the plant oil is soybean oil; the plant essential oil or the single component of plant essential oil is Sichuan pepper essential oil, linalool or limonene; (3) Preparation of enzyme-responsive intelligent antibacterial gelatin microgel: slowly mix the aqueous phase containing glycosidase prepared in step (1) and the oil phase containing plant essential oil prepared in step (2), add an emulsifier for emulsification, immediately place in an ice water bath, then add TG enzyme for cross-linking treatment, and finally centrifuge and wash with ultrapure water to obtain enzyme-responsive intelligent antibacterial gelatin microgel; the cross-linking condition is cross-linking at 20-35°C for 30-60 min; The enzyme-responsive intelligent antibacterial gelatin microgel is used to preserve fresh soy milk. The specific application method is as follows: The enzyme-responsive intelligent antibacterial gelatin microgel prepared by the above method is directly added to the fresh soy milk cooled to room temperature, stirred and dispersed evenly, filled and sealed, and stored at room temperature of 25°C or 4°C; based on the volume of the fresh soy milk, the addition amount of the enzyme-responsive intelligent antibacterial gelatin microgel is 5%~20%.

2. The use according to claim 1, characterized in that In step (1), the mass fraction of the gelatin solution is 12.5-20%; and the temperature of the water bath stirring is 45-95°C.

3. The use according to claim 1, characterized in that In step (1), the volume ratio of the gelatin solution to the β-glucosidase solution is 50:1 to 25:

1.

4. The use according to claim 1, wherein In step (2), the volume ratio of the vegetable oil, the plant essential oil or a single component of the plant essential oil to the emulsifier is 460:40:1 to 300:200:

5.

5. The use according to claim 1, characterized in that In step (3), the volume ratio of the water phase to the oil phase is 1:1 to 9:

1.

6. The use according to claim 1, wherein In step (3), the emulsifier is Tween-20; the emulsification temperature is 45-55°C, and the emulsification speed is 200-1000 r / min.

7. The use according to claim 1, wherein In step (3), the amount of emulsifier Tween-20 added is 0.5~1.0 mL / 100 mL, and the emulsification time is 10~30 min.

8. The use according to claim 1, wherein The ice water bath time in step (3) is 10 to 20 minutes; the amount of TG enzyme added is 5% to 10% of the amount of gelatin added.

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