Preparation method and application of modified konjac emulsifier

Abstract

The application provides a preparation method of modified konjac emulsifier, relates to the field of konjac processing, and comprises the following steps: step S1, pretreatment, step S2, preparation of a precursor solution, step S3, segmented deacetylation, step S4, preparation of gel particles, step S5, hydrophobic anchor pretreatment, step S6, fractional enzymatic esterification, and step S7, purification and drying. The method realizes multiple modification on konjac glucomannan, obtains modified konjac emulsifier, and effectively solves the problems of strong hydrophilicity and poor emulsification of konjac glucomannan.

Description

Technical Field

[0001] This invention relates to the field of konjac processing technology, specifically to a method for preparing a modified konjac emulsifier and its application. Background Technology

[0002] Excessive fat intake can directly or indirectly lead to obesity and other chronic diseases. Adequate dietary fiber intake is an effective dietary strategy to reduce dietary fat absorption and improve obesity symptoms. In the processing of traditional emulsified meat products such as chicken meatballs, a certain proportion of fat is added to improve the texture, taste, and flavor of the meat products, enhancing their acceptability. Reducing fat content can easily lower the sensory quality of the product. However, long-term consumption of meat products containing animal fat is detrimental to human health (excessive intake of animal fat easily leads to obesity, cardiovascular disease, diabetes, and other metabolic diseases). Replacing animal fat with vegetable oil introduces polyunsaturated fatty acids, which, while improving the nutritional quality of meat products, places higher demands on the emulsifying performance of emulsifiers. Furthermore, commonly used emulsifiers are easily hydrolyzed by digestive enzymes, causing fat release during digestion of the meatballs, making them easily digestible and absorbed by the body. Therefore, there is an urgent need to develop a new type of emulsifier with excellent emulsifying properties and high resistance to digestion. This emulsifier could inhibit fat release in the emulsion system and reduce fat digestion and absorption, providing a solution to the adverse effects of high saturated fatty acid content and easy fat release during digestion in traditional emulsified meat products such as chicken meatballs.

[0003] Konjac glucomannan (KGM) is a highly water-soluble, non-ionic plant polysaccharide and one of the most viscous soluble polysaccharides found in nature, with a content as high as 64% in konjac tubers. As a water-soluble dietary fiber, KGM is difficult for human digestive enzymes to break down and absorb. It reduces food intake by producing a sustained feeling of fullness, slows down fat digestion and absorption, and promotes its excretion, thereby achieving weight loss and lipid reduction. It is currently used in the research and development of various foods with lipid-lowering and weight-loss functions. However, traditional KGM is a strongly hydrophilic polysaccharide with an extreme lack of hydrophobic groups, resulting in very poor oil absorption and low emulsification ability, making it unsuitable for direct use as an oil-water emulsifier. Furthermore, the high viscosity of KGM's hydrosol makes it difficult to mix evenly with the modified material during hydrophobic modification, leading to difficulties in subsequent recovery and drying, and complex byproducts. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention aims to provide a method for preparing a modified konjac emulsifier. This method involves multiple modifications to konjac glucomannan, including deacetylation modification, octenyl succinic anhydride (OSA) esterification modification, polysaccharide composite modification, and enzymatic modification, to obtain a modified konjac emulsifier, effectively solving the problems of strong hydrophilicity and poor emulsification of konjac glucomannan.

[0005] Another objective of this invention is to provide an application of the above-mentioned modified konjac emulsifier in chicken meatballs, for the preparation of vegetable oil-based meatballs, which effectively reduces the amount of starch and animal fat added to the meatballs and reduces the fat digestion and dissolution rate of the meatballs.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for preparing a modified konjac emulsifier, comprising: Step S1, Pretreatment: Prepare the mixed immobilized enzyme in advance; Step S2, Preparation of precursor solution: First, prepare konjac glucomannan ethanol suspension, then prepare xanthan gum aqueous solution, and finally mix and swell to obtain precursor solution; Step S3, Fractional Deacetylation: Add sodium carbonate to the precursor solution, mix and stir, then transfer to a microwave reactor for heating; then add more sodium carbonate and continue microwave heating to obtain a uniform gel; Step S4, Gel Particle Preparation: Pour the gel into a mold and place it on a cold table to pre-freeze and form oriented ice crystals. Then transfer it to an autoclave for CO2 expansion. Afterward, perform freezing and heat preservation, freeze drying, and air jet milling in sequence to obtain gel particles. Step S5, hydrophobic anchoring pretreatment: Immerse the gel particles in stearic acid ethanol solution to obtain hydrophobically anchored gel particles. Step S6, graded enzymatic esterification: First, prepare a pre-dried solvent; then, mix the pre-dried solvent, hydrophobically anchored gel particles, and immobilized enzyme evenly, and add anhydrous magnesium sulfate, mixing thoroughly; then, add octenyl succinic anhydride-ethyl acetate solution dropwise; then, separate the immobilized enzyme, and add octenyl succinic anhydride-ethyl acetate solution, immobilized enzyme, and glycerol to the filtrate, stirring to mix and react. After the reaction is complete, separate and recover the immobilized enzyme again. Step S7, Purification and Drying: First, wash and filter the filtrate with anhydrous ethanol, then freeze-dry it under vacuum to obtain the final product.

[0008] Based on further optimization of the above scheme, in step S1, the preparation method of the mixed immobilized enzyme includes the preparation of a porous microcrystalline cellulose carrier, enzyme loading, and sodium alginate embedding; wherein, the preparation of the porous microcrystalline cellulose carrier specifically involves: first, adding microcrystalline cellulose to an acetate-sodium acetate buffer solution at pH 5±0.1, and adding cellulase (enzyme activity 10000~12000U / g), and stirring at a low speed of 150~170rpm for 29~31min at 44~46℃, with a mass-to-volume ratio of microcrystalline cellulose, cellulase, and acetate-sodium acetate buffer solution of 1g:0.02g:25mL; then... The enzyme was inactivated by treating the sample in a boiling water bath at 100±1℃ for 9–11 min. The precipitate was then collected by filtration and washed 3–4 times with deionized water. Finally, the precipitate was placed under normal pressure and cooled to -40±1℃ at a rate of 0.8–1.2℃ / min for 2–3 h. It was then subjected to constant-temperature sublimation drying at -0.08–-0.09 MPa and -20±1℃ for 10–12 h. Finally, it was heated to 0±0.5℃ at a rate of 0.2–0.3℃ / min under constant-temperature desorption drying conditions for 4–5 h to obtain the porous microcrystalline cellulose carrier.

[0009] Based on further optimization of the above scheme, the enzyme loading is specifically as follows: First, the porous microcrystalline cellulose carrier is added to a sodium acetate buffer solution with a concentration of 0.1 mol / L and a pH of 5.5 ± 0.1, and ultrasonically dispersed at a frequency of 20 kHz and a power of 95–105 W for 9–11 min. The mass-to-volume ratio of the porous microcrystalline cellulose carrier to the sodium acetate buffer solution is 0.2 g: 10 mL. Then, lipase (enzyme activity 10000–12000 U / g) and mannanase (enzyme activity 5000–6000 U / g) are added sequentially, and stirred at 210–230 rpm for 28–32 min at 4 ± 0.5 °C. The mass ratio of the porous microcrystalline cellulose carrier, lipase, and mannanase is 2:1:1, thus completing the enzyme loading.

[0010] Based on further optimization of the above scheme, the sodium alginate encapsulation is specifically as follows: First, a sodium alginate solution with a mass fraction of 0.9%–1.1% (i.e., a mass-to-volume ratio of sodium alginate to water of 0.9–1.1 g: 100 mL) is added to the enzyme-loaded porous microcrystalline cellulose suspension. The volume ratio of the sodium alginate solution to the enzyme-loaded porous microcrystalline cellulose suspension is 2:1. After stirring at 100–120 rpm for 28–32 min at room temperature (i.e., 25±1℃), a calcium chloride solution with a concentration of 0.1 mol / L is added dropwise at a rate of 0.9–1.1 mL / min. The volume ratio of the added mixture to the calcium chloride solution was 1:6-8. After the addition was complete, the mixture was solidified at room temperature for 28-32 minutes, and then the microspheres were collected by filtration. Finally, the filtered microspheres were placed under normal pressure and cooled to -40±1℃ at a rate of 0.8-1.2℃ / min for 2-3 hours for pre-freezing. Then, they were dried by sublimation at -0.08--0.09MPa and -20±1℃ for 8-9 hours. After that, they were heated to 0±0.5℃ at a rate of 0.2-0.3℃ / min under a rate of -0.08--0.09MPa for 3-4 hours for desorption and drying to obtain the mixed immobilized enzyme.

[0011] Based on further optimization of the above scheme, in step S2, the preparation of the konjac glucomannan ethanol suspension is specifically as follows: konjac glucomannan is added to anhydrous ethanol and stirred at 390–410 rpm for 14–16 min, with a mass-to-volume ratio of konjac glucomannan to anhydrous ethanol of 1 g: 25 mL; the preparation of the xanthan gum aqueous solution is specifically as follows: xanthan gum is added to distilled water at 30±1℃ and stirred at 790–810 rpm for 29–31 min, with a mass-to-volume ratio of xanthan gum to distilled water of 0.1 g: 25 mL; the mixing and swelling is specifically as follows: the konjac glucomannan ethanol suspension is added dropwise to the xanthan gum aqueous solution at a rate of 0.9–1.1 mL / min, and stirred at 390–410 rpm in a water bath environment at 29–31℃, and the mixture is kept at this temperature for 0.9–1.1 h after the addition is completed.

[0012] Based on further optimization of the above scheme, step S3 is specifically as follows: First, sodium carbonate is added to the precursor solution, the pH is adjusted to 9.0±0.1, and the mixture is stirred at 280-300 rpm for 9-11 min at -0.08 to -0.09 MPa; then, it is transferred to a microwave reactor and heated at a constant temperature of 59-61°C for 18-22 min with a power of 195-205 W; then, sodium carbonate is added again, the pH is adjusted to 10.0±0.1, and the mixture is heated at a constant temperature of 59-61°C for 18-22 min with a power of 195-205 W; finally, it is cooled to room temperature to obtain a uniform gel.

[0013] Based on further optimization of the above scheme, step S4 specifically involves: First, pouring the uniform gel obtained in step S3 into a mold (e.g., a circular mold with a diameter of 5 cm), and pre-freezing it for 1.9–2.1 h at a rate of 1 °C / min from the bottom on a cold platform at -20 ± 1 °C to form directional ice crystals; then, transferring it to an autoclave, and injecting CO2 into the autoclave at a rate of 0.3–0.4 MPa / min to 11.8–12.2 MPa, maintaining the pressure for 43–47 min, and then releasing it to atmospheric pressure within 4–6 s (generating a mesoporous structure inside the gel); subsequently… The sample was transferred to a freezer at -39 to -41°C and kept at that temperature for 7.9 to 8.1 hours. After the freezer was kept at that temperature, it was first sublimated and dried at -0.08 to -0.09 MPa and -20±1°C for 22 to 26 hours, and then desorbed and dried at -0.08 to -0.09 MPa and 0±0.5°C for 10 to 14 hours. Finally, it was subjected to air jet milling with the chamber temperature below 40°C, ambient humidity RH < 30%, grinding nozzle pressure 0.6 to 0.7 MPa, classifying wheel speed 2200 to 2600 rpm, and feed rate 15 to 20 g / h. After milling, it was passed through a 40 to 60 mesh sieve to obtain the final product.

[0014] Based on further optimization of the above scheme, step S5 specifically involves: adding the gel particles obtained in step S4 to a 1.5% anhydrous ethanol solution of stearic acid, with a mass-to-volume ratio of gel particles to anhydrous ethanol solution of stearic acid of 1g:10mL; then adding 4.8–5.2% lipase by mass of the gel particles; and refluxing at 39.5–40.5℃ and 140–160 rpm for 1.9–2.1 h (to allow the stearic acid to covalently bind with the hydroxyl groups of konjac glucomannan through esterification); after the reaction is complete, filtering with a 400-mesh filter cloth and washing with anhydrous ethanol at 45±1℃ 3–4 times; after washing, evaporating the ethanol in a fume hood until there is no alcohol odor, thus obtaining the surface-hydrophobically anchored gel particles.

[0015] Based on further optimization of the above scheme, the specific steps for preparing the pre-dried solvent in step S6 are as follows: Ethyl acetate and anhydrous ethanol are mixed at a volume ratio of 3:1, and anhydrous magnesium sulfate is added. The mass-volume ratio of anhydrous magnesium sulfate to the mixture is 0.8-1g:10mL. The bottle is capped with a ground glass stopper and left to stand at room temperature in the dark for 24-26 hours, shaking for 1 minute every 6 hours (to improve the water absorption contact efficiency). Then, the mixture is filtered at normal pressure using a dry sintered sand funnel and qualitative filter paper (all equipment is dried and cooled at 100℃ beforehand to prevent secondary water absorption). The clear filtrate is collected to obtain the pre-dried solvent.

[0016] Based on further optimization of the above scheme, step S6 specifically involves: First, vacuum drying the surface-anchored hydrophobic gel particles at -0.08 to -0.09 MPa and 58 to 62°C for 11.5 to 12.5 hours; then, mixing and stirring the pre-dried solvent, surface-anchored hydrophobic gel particles, and mixed immobilized enzyme at 345 to 355 rpm for 9 to 11 minutes, with a volume-to-mass ratio of 50 mL: 1 g: 0.1 g; next, adding anhydrous magnesium sulfate and continuously stirring at 345 to 355 rpm for 14 to 16 minutes. In the first step, anhydrous magnesium sulfate was mixed with hydrophobically anchored gel particles at a mass ratio of 0.4:1. Then, an octenyl succinic anhydride (OSA)-ethyl acetate solution (0.1 g / mL, i.e., a mass-to-volume ratio of octenyl succinic anhydride to ethyl acetate of 0.1 g:1 mL) was added dropwise at a rate of 0.35–0.45 mL / min, with a mass ratio of octenyl succinic anhydride to hydrophobically anchored gel particles of 0.28:1. The mixture was stirred at 295–305 rpm while being added dropwise. After the addition was complete, the mixture was kept at a constant temperature of 37.5–38.5 °C for 1.9–2.1 h (rapid grafting of O2 onto the gel particle surface). SA hydrophobic groups enhance particle dispersibility in the organic phase; after the reaction, the mixed immobilized enzyme was initially separated and recovered using a 20-mesh standard sieve, yielding a filtrate (because the mixed immobilized enzyme was encapsulated in sodium alginate and calcium chloride to form microspheres, its diameter after freeze-drying was greater than 500 μm, while the gel particles, after air jet milling and passing through a 40-60 mesh sieve, had a particle size less than 500 μm; therefore, the mixed immobilized enzyme was effectively separated by the 20-mesh sieve); then, 0.1 g / mL of octenyl succinic anhydride-ethyl acetate solution and the mixed immobilized enzyme were added to the filtrate. The volume-to-mass ratio of the immobilized enzyme to the hydrophobically anchored gel particles was 0.84 mL:0.05 g:1 g. Simultaneously, 1.9%–2.1% (by weight of the hydrophobically anchored gel particles) of glycerol was added. The mixture was stirred at 345–355 rpm for 9–11 min, then heated to 44.5–45.5 °C and stirred at a constant temperature for 2.9–3.1 h (to promote the diffusion of OSA and enzyme into the gel pores, achieving uniform esterification of internal hydroxyl groups). After the reaction, the immobilized enzyme was first separated and recovered again using a 20-mesh standard sieve, and then the filtrate was filtered under normal pressure using a 200-mesh filter cloth to collect the gel particle filter cake.

[0017] Based on further optimization of the above scheme, step S7 specifically involves: First, adding three times the volume of anhydrous ethanol to the gel particle filter cake from step S6, stirring at 270–290 rpm for 7–9 minutes, and then allowing it to stand for 1.9–2.1 hours; then, washing the precipitate 3–4 times with anhydrous ethanol, filtering it through a 200-mesh filter at normal pressure, and then placing the precipitate in a fume hood (wind speed 0.7–0.8 m / s) at 28–30°C. In an environment with relative humidity RH < 40%, the powder is spread thinly (thickness not greater than 2 mm) for 5.8–6.2 h to obtain precipitated powder. Finally, the precipitated powder is pre-frozen at -40±1℃ for 2–4 h, then sublimated and dried at -0.08–-0.09 MPa and -20±1℃ for 7–9 h. After that, the temperature is raised to 0±0.5℃ at a rate of 0.2–0.3℃ / min and dried at a constant temperature for 3.5–4.5 h. The modified konjac emulsifier is obtained by passing it through a 40-mesh sieve.

[0018] The application of a modified konjac emulsifier in chicken meatball products includes: First, rosemary extract and modified konjac emulsifier were mixed and added to an ethanol-water solution with a volume ratio of 1:1 (i.e., anhydrous ethanol to water volume ratio of 1:1). The mixture was ultrasonically dissolved for 4–6 minutes at a frequency of 20 kHz and a power of 95–105 W. The mass-volume ratio of rosemary extract, modified konjac emulsifier, and ethanol-water solution was 0.01 g:0.22 g:5 mL. The mixture was then spray-dried (inlet air temperature 122–124℃, outlet air temperature 58–62℃, feed rate 3–3.5 mL / min) to obtain emulsifier powder. Then, soybean oil, soy protein isolate, ice water at 2±2℃, and calcium chloride (…) were… CaCl 2) Add to a high-speed disperser and homogenize at 7800-8200 rpm for 2-3 minutes to obtain a pre-emulsion. The mass ratio of soybean oil, soy protein isolate, ice water, and calcium chloride is 20:1:5:0.03. Then, wash the chicken breast (after removing the skin and excess fat) and cut it into even small pieces (approximately 1cm). 3The meat is ground into coarse mince using a meat grinder (3mm perforated plate). The coarse mince is then mixed with ternary compound salt and ice water at 2±2℃ in a chopper at 950-1050 rpm for 1-2 minutes. The mass ratio of coarse mince, ternary compound salt, and ice water is 80:2:7.5. The pH is adjusted to 6.5±0.1 with 0.1mol / L NaOH solution, and chopping continues for 28-32 seconds to obtain minced meat. Next, emulsifier powder and modified konjac emulsifier are added to the pre-emulsion and stirred at 495-505 rpm for 28-32 seconds to form a mixture. The mass ratio of emulsifier powder, modified konjac emulsifier, and pre-emulsion is 0.22:0.38:13. This mixture is then added to the minced meat and stirred at 1450-1050 rpm. Chop the meat mixture at 1550 rpm for 2–3 minutes, adding ice water at 2±2℃ during this time. The mass ratio of the mixture, minced meat, and ice water is 0.44:100:7.5. Then add yeast extract and continue chopping at 1450–1550 rpm for 1–2 minutes (keeping the center temperature of the minced meat no higher than 10℃ throughout the process). The mass ratio of yeast extract to minced meat is 0.01:20. Next, add glucono-delta-lactone to the minced meat mixture and stir at 795–805 rpm for 28–32 seconds. The mass ratio of glucono-delta-lactone to minced meat is 0.03:20. After stirring, immediately transfer the mixture to a refrigerator at 4±0.5℃ and let it stand for 1.9–2.1 hours. Finally, shape the meatballs and preheat them.

[0019] Based on further optimization of the above scheme, the ternary composite salt includes sodium chloride (NaCl), potassium chloride (KCl) and calcium lactate, with a mass ratio of 5:3:2.

[0020] Based on further optimization of the above scheme, the meatball forming and preheating are specifically as follows: the minced meat that has been left to stand in the refrigerator is kneaded into meatballs (by hand or by machine) with a diameter of 3±0.5cm and a weight of 25±5g; then, it is heated in a water bath environment of 42±1℃ for 10 to 12 minutes, and then cooled with ice water to a core temperature of 4±0.5℃.

[0021] Based on further optimization of the above scheme, after the meatballs are formed and preheated, the products are refrigerated. Specifically, the preheated meatballs are cooled to -18±1℃ at a rate of 1 to 1.2℃ / min and stored in an environment of -18±1℃.

[0022] The following are the technical effects of this invention: This invention utilizes a mixed immobilized enzyme preparation method, employing porous microcrystalline cellulose as a high specific surface area carrier. Lipase and mannanase are physically adsorbed and then encapsulated in sodium alginate. This process not only utilizes lipase to catalyze the esterification reaction but also leverages mannanase to hydrolyze the glycosidic bonds of konjac glucomannan, increasing reaction sites. This balances esterification efficiency with molecular chain regulation, preventing excessive polysaccharide degradation. Furthermore, the porous and encapsulated structures enhance the stability of the mixed immobilized enzyme, effectively avoiding problems such as easy inactivation, difficulty in separation and recovery, and enzyme residue in organic phase systems. Through the synergistic effect of intermolecular hydrogen bonds between konjac glucomannan and xanthan gum, a homogeneous and viscosity-controllable complex polysaccharide system is formed, yielding a homogeneous and stable complex polysaccharide precursor solution, providing a uniform reaction matrix for subsequent processing. A staged deacetylation process gently removes acetyl groups from the konjac glucomannan molecule, exposing more hydroxyl reaction sites and increasing the active sites for subsequent esterification reactions. This avoids problems such as violent deacetylation reactions caused by strong bases, severe polysaccharide degradation, and uncontrollable deacetylation. By preparing porous gel particles, the problems of dense konjac glucomannan particle structure, small specific surface area, and high mass transfer resistance are solved. This ensures that the modifying agent can enter the particle interior and the modification is uniform, providing a sufficient reaction interface for subsequent esterification reactions and endowing the particles with the ability to physically adsorb and encapsulate oils, thus providing structural support for the product's anti-digestion properties. Through hydrophobic anchoring pretreatment, lipase catalyzes the esterification reaction between stearic acid and the hydroxyl groups on the surface of the gel particles, forming hydrophobic anchor points on the particle surface. This achieves preliminary hydrophobicization of the particle surface, effectively solving problems such as poor dispersibility, severe agglomeration, and ineffective OSA modification of konjac glucomannan in organic phase systems. By employing a two-step enzymatic esterification strategy, the organic compatibility of the particles is directly enhanced by the surface of OSA, achieving "surface hydrophobicity enhancement." Then, the addition of OSA and mixed immobilized enzymes drives the diffusion of OSA and enzyme molecules into the pores, achieving uniform esterification of hydroxyl groups inside the particles. This results in uniform hydrophobic modification of porous particles from the surface to the interior, significantly improving the uniformity of product substitution. It avoids the problem of uneven modification such as "excessive surface grafting and insufficient internal reaction" during OSA modification, and greatly enhances the emulsifying ability and emulsion stability of the modified emulsifier.

[0023] This invention solves the problems of KGM's hydrophilicity, difficulty in modification, and difficulty in emulsification through "hydrophobic anchoring and hierarchical enzymatic esterification". The modified KGM has both a hydrophilic polysaccharide backbone and a hydrophobic OSA side chain and stearic acid anchor, combining the interfacial film strength of a high molecular weight emulsifier with the interfacial activity of a low molecular weight emulsifier. At the same time, through the structure of "mixed immobilized enzyme and porous particles", a highly efficient, controllable, and recyclable green modification process is achieved, breaking through the bottleneck of "inconsistency between surface and core" in traditional polysaccharide modification. The product has a uniform degree of substitution, strong batch stability, and mild enzymatic catalysis avoids excessive degradation of polysaccharides. The viscosity and gel properties of the product are well preserved, making it suitable for different food processing scenarios.

[0024] Furthermore, this invention achieves a dual effect of "replacing animal fat and reducing fat digestion and absorption" by applying modified konjac emulsifier to chicken meatballs: on the one hand, it uses vegetable oil to replace animal fat and reduces the content of saturated fatty acids; on the other hand, it inhibits the release of fat through fat digestion by modifying the emulsifier, thereby improving the health attributes of meat products while ensuring the texture, taste and flavor of the product, thus solving the pain point of "lower fat means lower quality" in low-fat meat products. Attached Figure Description

[0025] Figure 1 This is a test diagram of the modified konjac emulsifier in Example 2 of the present invention; wherein, Figure 1 (a) is a diagram showing the phenomenon of testing expansion force. Figure 1 (b) is a schematic diagram for testing emulsification performance. Figure 1 (c) is a schematic diagram of emulsion stability testing after 30 days.

[0026] Figure 2 This is a cross-sectional view of the chicken meatballs before and after freezing and thawing in Example 4 of the present invention; Figure 2 (a) is a schematic diagram of regular chicken meatballs before freezing. Figure 2 (b) is a schematic diagram of the chicken meatballs prepared in Example 4 before freezing. Figure 2 (c) is a schematic diagram of a conventional chicken meatball process involving three freeze-thaw cycles. Figure 2 (d) is a schematic diagram of the chicken meatballs prepared in Example 4 undergoing three freeze-thaw cycles.

[0027] Figure 3 This is a SEM image of the chicken meatballs before and after freeze-thaw in Example 4 of the present invention; Figure 3 (a) is a schematic diagram of regular chicken meatballs before freezing. Figure 3 (b) is a schematic diagram of the chicken meatballs prepared in Example 4 before freezing. Figure 3 (c) is a schematic diagram of a conventional chicken meatball process involving three freeze-thaw cycles. Figure 3 (d) is a schematic diagram of the chicken meatballs prepared in Example 4 undergoing three freeze-thaw cycles. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0029] Example 1: A method for preparing a modified konjac emulsifier, comprising: Step S1, Pretreatment: Pre-preparation of the mixed immobilized enzyme, including the preparation of porous microcrystalline cellulose carrier, enzyme loading, and sodium alginate embedding; The preparation of the porous microcrystalline cellulose carrier specifically involves the following steps: First, microcrystalline cellulose (CAS: 9004-34-6) is added to an acetate-sodium acetate buffer solution at pH 4.9, along with cellulase (enzyme activity 10000–12000 U / g). The mixture is stirred at 150 rpm for 31 minutes at 44°C. The mass-to-volume ratio of microcrystalline cellulose, cellulase, and acetate-sodium acetate buffer solution is 1 g:0.02 g:25 mL. Then, the mixture is boiled at 99°C. The enzyme was inactivated by treating in a water bath for 11 min. Then, the precipitate was collected by filtration and washed three times with deionized water. Finally, the precipitate was placed under normal pressure and cooled to -39℃ at a rate of 0.8℃ / min for 3 h for pre-freezing. Then, it was dried by sublimation at -0.08MPa and -19℃ for 12 h. After that, it was heated to 0.5℃ at a rate of 0.2℃ / min under -0.08MPa and dried by desorption at a rate of 5 h for 5 h to obtain the porous microcrystalline cellulose carrier.

[0030] The enzyme loading process was as follows: First, the porous microcrystalline cellulose carrier was added to a sodium acetate buffer solution with a concentration of 0.1 mol / L and a pH of 5.4. The mixture was then ultrasonically dispersed at a frequency of 20 kHz and a power of 95 W for 11 min. The mass-to-volume ratio of the porous microcrystalline cellulose carrier to the sodium acetate buffer solution was 0.2 g: 10 mL. Then, lipase (enzyme activity 10000-12000 U / g) and mannanase (enzyme activity 5000-6000 U / g) were added sequentially. The mixture was stirred at 210 rpm for 32 min at 3.5 °C. The mass ratio of the porous microcrystalline cellulose carrier, lipase, and mannanase was 2:1:1, thus completing the enzyme loading process.

[0031] The sodium alginate encapsulation process is as follows: First, a 0.9% sodium alginate solution (i.e., a sodium alginate to water mass-to-volume ratio of 0.9 g: 100 mL) is added to the enzyme-loaded porous microcrystalline cellulose suspension. The volume ratio of the sodium alginate solution to the enzyme-loaded porous microcrystalline cellulose suspension is 2:1. After stirring at 100 rpm for 32 min at room temperature (i.e., 25 ± 1 °C), a 0.1 mol / L calcium chloride solution is added dropwise at a rate of 0.9 mL / min. The volume ratio of the added mixture to the calcium chloride solution was 1:6. After the addition was complete, the mixture was solidified at room temperature for 32 min, and then the microspheres were collected by filtration. Finally, the filtered microspheres were placed under normal pressure and cooled to -39℃ at a rate of 0.8℃ / min for 3 h for pre-freezing. Then, they were dried by sublimation at -0.08 MPa and -19℃ for 9 h. After that, they were heated to 0.5℃ at a rate of 0.2℃ / min under -0.08 MPa and dried by desorption at a constant temperature for 4 h to obtain the mixed immobilized enzyme.

[0032] Step S2, Preparation of the precursor solution: First, prepare a konjac glucomannan ethanol suspension, specifically by adding konjac glucomannan to anhydrous ethanol and stirring at 390 rpm for 16 min. The mass-to-volume ratio of konjac glucomannan to anhydrous ethanol is 1 g: 25 mL. Next, prepare a xanthan gum aqueous solution, specifically by adding xanthan gum to distilled water at 29℃ and stirring at 790 rpm for 31 min. The mass-to-volume ratio of xanthan gum to distilled water is 0.1 g: 25 mL. Finally, perform mixing and swelling to obtain the precursor solution, specifically by adding the konjac glucomannan ethanol suspension dropwise to the xanthan gum aqueous solution at a rate of 0.9 mL / min and stirring at 390 rpm in a water bath at 29℃. After the addition is complete, maintain the temperature for 1.1 h.

[0033] Step S3, fractional deacetylation: First, sodium carbonate was added to the precursor solution to adjust the pH to 8.9, and the mixture was stirred at 280 rpm for 11 min at -0.08 MPa. Then, it was transferred to a microwave reactor and heated at 195 W and 59 °C for 22 min. Next, sodium carbonate was added to adjust the pH to 9.9, and the mixture was heated at 195 W and 59 °C for another 22 min. Finally, the mixture was cooled to room temperature to obtain a uniform gel.

[0034] Step S4, Gel Particle Preparation: First, pour the homogeneous gel obtained in Step S3 into a mold (e.g., a circular mold with a diameter of 5 cm), and pre-freeze it for 2.1 h at a rate of 1 °C / min from the bottom on a cold stage at -19 °C to form oriented ice crystals. Then, transfer it to an autoclave, and inject CO2 into the autoclave at a rate of 0.3 MPa / min to 11.8 MPa. After holding the pressure for 47 min, release it to atmospheric pressure within 6 s (to generate media inside the gel). (Porous structure); then, it was transferred to a freezer at -39℃ and kept warm for 8.1h. After the warming period, it was first sublimated and dried at -0.08MPa and -19℃ for 26h, and then desorbed and dried at -0.08MPa and 0.5℃ for 14h. Finally, it was subjected to air jet milling with the chamber temperature less than 40℃, ambient humidity RH < 30%, grinding nozzle pressure 0.6MPa, classifying wheel speed 2200rpm, and feed rate 15g / h. After milling, it was passed through a 60-mesh sieve to obtain the final product.

[0035] Step S5, Hydrophobic Anchoring Pretreatment: The gel particles are soaked in a stearic acid-ethanol solution to obtain hydrophobically anchored gel particles. Specifically, the gel particles obtained in step S4 are added to a 1.5% stearic acid-ethanol solution with a mass-to-volume ratio of 1g:10mL. Then, 4.8% of the gel particle mass of lipase (the same lipase as in step S1, with an enzyme activity of 10000-12000U / g) is added, and the mixture is refluxed at 39.5℃ and 140rpm for 2.1h (to allow the stearic acid to covalently bind with the hydroxyl groups of konjac glucomannan through esterification). After the reaction, the mixture is filtered through a 400-mesh filter cloth and washed three times with anhydrous ethanol at 44℃. After washing, the ethanol is evaporated in a fume hood until there is no alcohol odor, thus obtaining the hydrophobically anchored gel particles.

[0036] Step S6, graded enzymatic esterification: First, prepare a pre-dried solvent. Specifically, mix ethyl acetate and anhydrous ethanol at a volume ratio of 3:1, then add anhydrous magnesium sulfate. The mass-volume ratio of anhydrous magnesium sulfate to the mixture is 0.8 g: 10 mL. Cover the bottle with a ground glass stopper and let it stand at room temperature in the dark for 26 hours, shaking for 1 minute every 6 hours (to improve water absorption contact efficiency). Then, filter under normal pressure using a dry sintered sand funnel and qualitative filter paper (all equipment is dried and cooled at 100℃ beforehand to prevent secondary water absorption). Collect the clear filtrate to obtain the pre-dried solvent.

[0037] The hydrophobically anchored gel particles were vacuum dried at -0.08 MPa and 58 °C for 12.5 h. Then, the pre-dried solvent, the hydrophobically anchored gel particles, and the mixed immobilized enzyme were mixed and stirred at 345 rpm for 11 min. The volume-to-mass ratio of the pre-dried solvent, the hydrophobically anchored gel particles, and the mixed immobilized enzyme was 50 mL: 1 g: 0.1 g.

[0038] Then, anhydrous magnesium sulfate was added, and the mixture was stirred continuously at 345 rpm for 16 minutes. The mass ratio of anhydrous magnesium sulfate to surface hydrophobic anchored gel particles was 0.4:1.

[0039] Subsequently, an octenyl succinic anhydride (OSA)-ethyl acetate solution with a concentration of 0.1 g / mL (i.e., the mass-to-volume ratio of octenyl succinic anhydride to ethyl acetate is 0.1 g: 1 mL) was added dropwise at a rate of 0.35 mL / min. The mass ratio of octenyl succinic anhydride to the hydrophobically anchored gel particles was 0.28:1. The mixture was stirred at a speed of 295 rpm while being added dropwise. After the addition was completed, the mixture was stirred at a constant temperature of 37.5 °C for 2.1 h (the hydrophobic groups of OSA were rapidly grafted onto the surface of the gel particles to improve the dispersibility of the particles in the organic phase). After the reaction was completed, the mixed immobilized enzyme was preliminarily separated and recovered using a 20-mesh standard sieve to obtain the filtrate.

[0040] Next, octenyl succinic anhydride-ethyl acetate solution and mixed immobilized enzyme at a concentration of 0.1 g / mL were added to the filtrate. The volume-to-mass ratio of the added octenyl succinic anhydride-ethyl acetate solution, mixed immobilized enzyme, and surface hydrophobically anchored gel particles was 0.84 mL:0.05 g:1 g. At the same time, 1.9% of the mass of the surface hydrophobically anchored gel particles was added. The mixture was stirred at 345 rpm for 11 min, then heated to 44.5 °C and stirred at a constant temperature for 3.1 h (to promote the diffusion of OSA and enzyme into the gel pores and achieve uniform esterification of internal hydroxyl groups). After the reaction was completed, the mixed immobilized enzyme was separated and recovered again using a 20-mesh standard sieve, and then the filtrate was filtered under normal pressure using a 200-mesh filter cloth to collect the gel particle filter cake.

[0041] Step S7, Purification and Drying: First, add three times the volume of anhydrous ethanol to the gel particle filter cake from step S6, stir at 270 rpm for 9 min, and then let stand for 2.1 h; then, wash the precipitate three times with anhydrous ethanol, filter it through a 200-mesh filter under normal pressure, and then place the precipitate in a fume hood (wind speed 0.7 m / s) at 28℃ and relative humidity RH < 40%, spread it thinly (thickness not greater than 2 mm) for 6.2 h to obtain precipitate powder; finally, pre-freeze the precipitate powder at -39℃ for 4 h, then sublimate and dry it at -0.08 MPa and -19℃ for 9 h, then heat it to 0.5℃ at a rate of 0.2℃ / min and dry it at a constant temperature for 4.5 h, and pass it through a 40-mesh sieve to obtain the modified konjac emulsifier.

[0042] Example 2: A method for preparing a modified konjac emulsifier, comprising: Step S1, Pretreatment: Pre-preparation of the mixed immobilized enzyme, including the preparation of porous microcrystalline cellulose carrier, enzyme loading, and sodium alginate embedding; The preparation of the porous microcrystalline cellulose carrier is as follows: First, microcrystalline cellulose (CAS: 9004-34-6) is added to an acetate-sodium acetate buffer solution at pH 5, and cellulase (enzyme activity 10000~12000U / g) is added. The mixture is stirred at 160 rpm for 30 min at 45℃. The mass-to-volume ratio of microcrystalline cellulose, cellulase, and acetate-sodium acetate buffer solution is 1 g:0.02 g:25 mL. Then, the mixture is heated in a boiling water bath at 100℃. The enzyme was inactivated by processing for 10 min. Then, the precipitate was collected by filtration and washed three times with deionized water. Finally, the precipitate was placed under normal pressure and cooled to -40℃ at a rate of 1℃ / min for 2.5 h. Then, it was dried by sublimation at -0.085 MPa and -20℃ for 11 h. After that, it was heated to 0℃ at -0.085 MPa and a rate of 0.25℃ / min and dried by desorption at a rate of 4.5 h to obtain the porous microcrystalline cellulose carrier.

[0043] The enzyme loading process is as follows: First, the porous microcrystalline cellulose carrier is added to a sodium acetate buffer solution with a concentration of 0.1 mol / L and a pH of 5.5. The mixture is then ultrasonically dispersed at a frequency of 20 kHz and a power of 100 W for 10 min. The mass-to-volume ratio of the porous microcrystalline cellulose carrier to the sodium acetate buffer solution is 0.2 g: 10 mL. Then, lipase (enzyme activity 10000-12000 U / g) and mannanase (enzyme activity 5000-6000 U / g) are added sequentially. The mixture is stirred at 220 rpm for 30 min at 4 °C. The mass ratio of the porous microcrystalline cellulose carrier, lipase, and mannanase is 2:1:1, thus completing the enzyme loading process.

[0044] The sodium alginate encapsulation process is as follows: First, a 1% (by mass) sodium alginate solution (i.e., a mass-to-volume ratio of 1 g sodium alginate to 100 mL water) is added to the enzyme-loaded porous microcrystalline cellulose suspension. The volume ratio of the sodium alginate solution to the enzyme-loaded porous microcrystalline cellulose suspension is 2:1. After stirring at 110 rpm for 30 min at room temperature (i.e., 25 ± 1 °C), a 0.1 mol / L calcium chloride solution is added dropwise at a rate of 1 mL / min. The volume ratio of the microspheres to the calcium chloride solution was 1:7. After the addition was complete, the microspheres were solidified at room temperature for 30 min, and then collected by filtration. Finally, the filtered microspheres were placed under normal pressure and cooled to -40℃ at a rate of 1℃ / min for 2.5 h. Then, they were dried by sublimation at -0.085 MPa and -20℃ for 8.5 h. After that, they were heated to 0℃ at -0.085 MPa and a rate of 0.25℃ / min and dried by desorption at a rate of 3.5 h to obtain the mixed immobilized enzyme.

[0045] Step S2, Preparation of the precursor solution: First, prepare a konjac glucomannan ethanol suspension, specifically by adding konjac glucomannan to anhydrous ethanol and stirring at 400 rpm for 15 min. The mass-to-volume ratio of konjac glucomannan to anhydrous ethanol is 1 g: 25 mL. Next, prepare a xanthan gum aqueous solution, specifically by adding xanthan gum to distilled water at 30 ± 1 °C and stirring at 800 rpm for 30 min. The mass-to-volume ratio of xanthan gum to distilled water is 0.1 g: 25 mL. Finally, perform mixing and swelling to obtain the precursor solution, specifically by adding the konjac glucomannan ethanol suspension dropwise to the xanthan gum aqueous solution at a rate of 1 mL / min, stirring at 400 rpm in a 30 °C water bath, and maintaining the temperature for 1 h after the addition is complete.

[0046] Step S3, fractional deacetylation: First, sodium carbonate was added to the precursor solution to adjust the pH to 9.0, and the mixture was stirred at 290 rpm for 10 min at -0.085 MPa. Then, it was transferred to a microwave reactor and heated at 200 W and 60 °C for 20 min. Next, sodium carbonate was added to adjust the pH to 10.0, and the mixture was heated at 200 W and 60 °C for another 20 min. Finally, the mixture was cooled to room temperature to obtain a uniform gel.

[0047] Step S4, Gel Particle Preparation: First, pour the homogeneous gel obtained in Step S3 into a mold (e.g., a circular mold with a diameter of 5 cm), and pre-freeze it for 2 hours at a rate of 1°C / min from the bottom on a cold stage at -20°C to form directional ice crystals. Then, transfer it to an autoclave, and inject CO2 into the autoclave at a rate of 0.35 MPa / min to 12 MPa. After holding the pressure for 45 minutes, release it to atmospheric pressure within 5 seconds (to generate mesoporous structures inside the gel). (The structure is then transferred to a -40℃ freezer for 8 hours. After the freezer is heated, it is first sublimated and dried at -0.085MPa and -20℃ for 24 hours, and then desorbed and dried at -0.085MPa and 0℃ for 12 hours. Finally, it is subjected to air jet milling with the chamber temperature less than 40℃, ambient humidity RH < 30%, grinding nozzle pressure 0.65MPa, classifying wheel speed 2400rpm, and feed rate 17.5g / h. After milling, it is passed through a 50-mesh sieve to obtain the final product.)

[0048] Step S5, Hydrophobic Anchoring Pretreatment: The gel particles are soaked in a stearic acid-ethanol solution to obtain hydrophobically anchored gel particles. Specifically, the gel particles obtained in step S4 are added to a 1.5% stearic acid-ethanol solution with a mass-to-volume ratio of 1g:10mL. Then, 5% of the mass of the gel particles (the same lipase as in step S1, with an enzyme activity of 10000-12000U / g) is added, and the mixture is refluxed at 40℃ and 150rpm for 2 hours (to allow the stearic acid to covalently bind with the hydroxyl groups of konjac glucomannan through esterification). After the reaction, the mixture is filtered through a 400-mesh filter cloth and washed three times with anhydrous ethanol at 45℃. After washing, the ethanol is evaporated in a fume hood until there is no alcohol odor, thus obtaining the hydrophobically anchored gel particles.

[0049] Step S6, graded enzymatic esterification: First, prepare a pre-dried solvent. Specifically, mix ethyl acetate and anhydrous ethanol at a volume ratio of 3:1, then add anhydrous magnesium sulfate. The mass-volume ratio of anhydrous magnesium sulfate to the mixture is 0.9 g: 10 mL. Cover the bottle with a ground glass stopper and let it stand at room temperature in the dark for 25 hours, shaking for 1 minute every 6 hours (to improve water absorption contact efficiency). Then, filter under normal pressure using a dry sintered sand funnel and qualitative filter paper (all equipment is dried and cooled at 100℃ beforehand to prevent secondary water absorption). Collect the clear filtrate to obtain the pre-dried solvent.

[0050] The hydrophobically anchored gel particles were vacuum dried at -0.085 MPa and 60 °C for 12 h. Then, the pre-dried solvent, the hydrophobically anchored gel particles, and the mixed immobilized enzyme were mixed and stirred at 350 rpm for 10 min. The volume-to-mass ratio of the pre-dried solvent, the hydrophobically anchored gel particles, and the mixed immobilized enzyme was 50 mL: 1 g: 0.1 g.

[0051] Then, anhydrous magnesium sulfate was added, and the mixture was stirred continuously at 350 rpm for 15 minutes. The mass ratio of anhydrous magnesium sulfate to surface hydrophobic anchored gel particles was 0.4:1.

[0052] Subsequently, an octenyl succinic anhydride (OSA)-ethyl acetate solution with a concentration of 0.1 g / mL (i.e., the mass-to-volume ratio of octenyl succinic anhydride to ethyl acetate is 0.1 g: 1 mL) was added dropwise at a rate of 0.4 mL / min. The mass ratio of octenyl succinic anhydride to the hydrophobically anchored gel particles was 0.28:1. The mixture was stirred at 300 rpm while being added dropwise. After the addition was completed, the mixture was stirred at a constant temperature of 38 °C for 2 h (the hydrophobic groups of OSA were rapidly grafted onto the surface of the gel particles, improving the dispersibility of the particles in the organic phase). After the reaction was completed, the mixed immobilized enzyme was preliminarily separated and recovered using a 20-mesh standard sieve to obtain the filtrate.

[0053] Next, octenyl succinic anhydride-ethyl acetate solution with a concentration of 0.1 g / mL and mixed immobilized enzyme were added to the filtrate. The volume-to-mass ratio of the added octenyl succinic anhydride-ethyl acetate solution, mixed immobilized enzyme and surface hydrophobically anchored gel particles was 0.84 mL:0.05 g:1 g. At the same time, 2% of the mass of surface hydrophobically anchored gel particles of glycerol was added. The mixture was stirred at 350 rpm for 10 min, and then heated to 45 °C and stirred at a constant temperature for 3 h (to promote the diffusion of OSA and enzyme into the gel pores and achieve uniform esterification of internal hydroxyl groups). After the reaction was completed, the mixed immobilized enzyme was separated and recovered again using a 20-mesh standard sieve, and then the filtrate was filtered under normal pressure using a 200-mesh filter cloth to collect the gel particle filter cake.

[0054] Step S7, Purification and Drying: First, add three times the volume of anhydrous ethanol to the gel particle filter cake from step S6, stir at 280 rpm for 8 min, and then let stand for 2 h; then, wash the precipitate three times with anhydrous ethanol, filter it through a 200-mesh filter under normal pressure, and then place the precipitate in a fume hood (wind speed 0.75 m / s) at 29℃ and relative humidity RH < 40%, spread it thinly (thickness not greater than 2 mm) for 6 h to obtain precipitate powder; finally, pre-freeze the precipitate powder at -40℃ for 4 h, then sublimate and dry it at -0.085 MPa and -20℃ for 8 h, then heat it to 0℃ at a rate of 0.25℃ / min and dry it at a constant temperature for 4 h, and pass it through a 40-mesh sieve to obtain the modified konjac emulsifier.

[0055] Example 3: A method for preparing a modified konjac emulsifier, comprising: Step S1, Pretreatment: Pre-preparation of the mixed immobilized enzyme, including the preparation of porous microcrystalline cellulose carrier, enzyme loading, and sodium alginate embedding; The preparation of the porous microcrystalline cellulose carrier specifically involves the following steps: First, microcrystalline cellulose (CAS: 9004-34-6) is added to an acetate-sodium acetate buffer solution at pH 5.1, along with cellulase (enzyme activity 10000–12000 U / g). The mixture is stirred at 170 rpm for 29 minutes at 46°C. The mass-to-volume ratio of microcrystalline cellulose, cellulase, and acetate-sodium acetate buffer solution is 1 g:0.02 g:25 mL. Then, the mixture is boiled at 101°C. The enzyme was inactivated by treating the sample in a water bath for 9 minutes. After that, the precipitate was collected by filtration and washed four times with deionized water. Finally, the precipitate was placed under normal pressure and cooled to -41°C at a rate of 1.2°C / min for 2 hours. Then, it was dried by sublimation at -0.09 MPa and -21°C for 10 hours. After that, it was heated to -0.5°C at a rate of 0.3°C / min under -0.09 MPa and dried by desorption at a rate of 4 hours to obtain the porous microcrystalline cellulose carrier.

[0056] The enzyme loading process was as follows: First, the porous microcrystalline cellulose carrier was added to a sodium acetate buffer solution with a concentration of 0.1 mol / L and a pH of 5.6, and ultrasonically dispersed at a frequency of 20 kHz and a power of 105 W for 9 min. The mass-to-volume ratio of the porous microcrystalline cellulose carrier to the sodium acetate buffer solution was 0.2 g: 10 mL. Then, lipase (enzyme activity 10000-12000 U / g) and mannanase (enzyme activity 5000-6000 U / g) were added sequentially, and the mixture was stirred at 230 rpm for 28 min at 4.5 °C. The mass ratio of the porous microcrystalline cellulose carrier, lipase, and mannanase was 2:1:1, thus completing the enzyme loading.

[0057] The sodium alginate encapsulation process is as follows: First, a 1.1% (mass-volume ratio of sodium alginate to water of 0.9–1.1 g: 100 mL) sodium alginate solution is added to the enzyme-loaded porous microcrystalline cellulose suspension. The volume ratio of sodium alginate solution to the enzyme-loaded porous microcrystalline cellulose suspension is 2:1. After stirring at 120 rpm for 28 min at room temperature (25 ± 1 °C), the solution is added dropwise at a rate of 1.1 mL / min to a 0.1 mol / L calcium chloride solution. The volume ratio of the mixed solution to calcium chloride solution was 1:8. After the addition was complete, the mixture was solidified at room temperature for 28 min, and then the microspheres were collected by filtration. Finally, the filtered microspheres were placed under normal pressure and cooled to -41℃ at a rate of 1.2℃ / min for 2 h for pre-freezing. Then, they were dried by sublimation at -0.09 MPa and -21℃ for 8 h. After that, they were heated to -0.5℃ at a rate of 0.3℃ / min under -0.09 MPa for 3 h for desorption and drying, thus obtaining the mixed immobilized enzyme.

[0058] Step S2, Preparation of the precursor solution: First, prepare a konjac glucomannan ethanol suspension, specifically by adding konjac glucomannan to anhydrous ethanol and stirring at 410 rpm for 14 min. The mass-to-volume ratio of konjac glucomannan to anhydrous ethanol is 1 g: 25 mL. Then, prepare a xanthan gum aqueous solution, specifically by adding xanthan gum to distilled water at 31℃ and stirring at 810 rpm for 29 min. The mass-to-volume ratio of xanthan gum to distilled water is 0.1 g: 25 mL. Finally, perform mixing and swelling to obtain the precursor solution, specifically by adding the konjac glucomannan ethanol suspension dropwise to the xanthan gum aqueous solution at a rate of 1.1 mL / min and stirring at 410 rpm in a 31℃ water bath environment. After the addition is completed, maintain the temperature for 0.9 h.

[0059] Step S3, Fractional Deacetylation: First, sodium carbonate was added to the precursor solution to adjust the pH to 9.1, and the mixture was stirred at 300 rpm for 9 min at -0.09 MPa. Then, it was transferred to a microwave reactor and heated at 205 W and 61 °C for 18 min. Next, sodium carbonate was added to adjust the pH to 10.1, and the mixture was heated at 205 W and 61 °C for another 18 min. Finally, the mixture was cooled to room temperature to obtain a uniform gel.

[0060] Step S4, Gel Particle Preparation: First, pour the homogeneous gel obtained in Step S3 into a mold (e.g., a circular mold with a diameter of 5 cm), and pre-freeze it for 1.9 h at a rate of 1 °C / min from the bottom on a cold stage at -21 °C to form oriented ice crystals. Then, transfer it to an autoclave, and inject CO2 into the autoclave at a rate of 0.4 MPa / min to 12.2 MPa. After holding the pressure for 43 min, release it to atmospheric pressure within 4 s (to generate...) (Mesoporous structure); then, it was transferred to a freezer at -41℃ and kept warm for 7.9h. After the warming period, it was first sublimated and dried at -0.09MPa and -21℃ for 22h, and then desorbed and dried at -0.09MPa and 0℃ for 10h. Finally, it was subjected to air jet milling with the chamber temperature less than 40℃, ambient humidity RH < 30%, grinding nozzle pressure 0.7MPa, classifying wheel speed 2600rpm, and feed rate 20g / h. After milling, it was passed through a 40-mesh sieve to obtain the final product.

[0061] Step S5, Hydrophobic Anchoring Pretreatment: The gel particles are soaked in a stearic acid-ethanol solution to obtain hydrophobically anchored gel particles. Specifically, the gel particles obtained in step S4 are added to a 1.5% stearic acid-ethanol solution with a mass-to-volume ratio of 1g:10mL. Then, 5.2% of the mass of the gel particles (the same lipase as in step S1, with an enzyme activity of 10000-12000U / g) is added, and the mixture is refluxed at 40.5℃ and 160rpm for 1.9h (to allow the stearic acid to covalently bind with the hydroxyl groups of konjac glucomannan through esterification). After the reaction, the mixture is filtered through a 400-mesh filter cloth and washed four times with anhydrous ethanol at 46℃. After washing, the ethanol is evaporated in a fume hood until there is no alcohol odor, thus obtaining the hydrophobically anchored gel particles.

[0062] Step S6, graded enzymatic esterification: First, prepare a pre-dried solvent. Specifically, mix ethyl acetate and anhydrous ethanol at a volume ratio of 3:1, then add anhydrous magnesium sulfate. The mass-volume ratio of anhydrous magnesium sulfate to the mixture is 1g:10mL. Cover the bottle with a ground glass stopper and let it stand at room temperature in the dark for 24 hours, shaking for 1 minute every 6 hours (to improve water absorption contact efficiency). Then, filter under normal pressure using a dry sintered sand funnel and qualitative filter paper (all equipment is dried and cooled at 100℃ beforehand to prevent secondary water absorption). Collect the clear filtrate to obtain the pre-dried solvent.

[0063] The hydrophobically anchored gel particles were vacuum dried at -0.09 MPa and 62 °C for 11.5 h. Then, the pre-dried solvent, the hydrophobically anchored gel particles, and the mixed immobilized enzyme were mixed and stirred at 355 rpm for 9 min. The volume-to-mass ratio of the pre-dried solvent, the hydrophobically anchored gel particles, and the mixed immobilized enzyme was 50 mL: 1 g: 0.1 g.

[0064] Then, anhydrous magnesium sulfate was added, and the mixture was stirred continuously at 355 rpm for 14 minutes. The mass ratio of anhydrous magnesium sulfate to surface hydrophobic anchored gel particles was 0.4:1.

[0065] Subsequently, an octenyl succinic anhydride (OSA)-ethyl acetate solution with a concentration of 0.1 g / mL (i.e., the mass-to-volume ratio of octenyl succinic anhydride to ethyl acetate is 0.1 g: 1 mL) was added dropwise at a rate of 0.45 mL / min. The mass ratio of octenyl succinic anhydride to the hydrophobically anchored gel particles was 0.28:1. The mixture was stirred at 305 rpm while being added dropwise. After the addition was completed, the mixture was stirred at a constant temperature of 38.5 °C for 1.9 h (the hydrophobic groups of OSA were rapidly grafted onto the surface of the gel particles to improve the dispersibility of the particles in the organic phase). After the reaction was completed, the mixed immobilized enzyme was preliminarily separated and recovered using a 20-mesh standard sieve to obtain the filtrate.

[0066] Next, 0.1 g / mL of octenyl succinic anhydride-ethyl acetate solution and mixed immobilized enzyme were added to the filtrate. The volume-to-mass ratio of the added octenyl succinic anhydride-ethyl acetate solution, mixed immobilized enzyme, and surface hydrophobically anchored gel particles was 0.84 mL:0.05 g:1 g. At the same time, 2.1% of the mass of the surface hydrophobically anchored gel particles was added. The mixture was stirred at 355 rpm for 9 min, then heated to 45.5 °C and stirred at a constant temperature for 2.9 h (to promote the diffusion of OSA and enzyme into the gel pores and achieve uniform esterification of internal hydroxyl groups). After the reaction was completed, the mixed immobilized enzyme was separated and recovered again using a 20-mesh standard sieve, and then the filtrate was filtered under normal pressure using a 200-mesh filter cloth to collect the gel particle filter cake.

[0067] Step S7, Purification and Drying: First, add three times the volume of anhydrous ethanol to the gel particle filter cake from step S6, stir at 290 rpm for 7 min, and then let stand for 1.9 h; then, wash the precipitate four times with anhydrous ethanol, filter it through a 200-mesh filter under normal pressure, and then place the precipitate in a fume hood (wind speed 0.8 m / s) at 30℃ and relative humidity RH < 40%, spread it thinly (thickness not greater than 2 mm) for 5.8 h to obtain precipitate powder; finally, pre-freeze the precipitate powder at -41℃ for 2 h, then sublimate and dry it at -0.09 MPa and -21℃ for 7 h, then heat it to -0.5℃ at a rate of 0.3℃ / min and dry it at a constant temperature for 3.5 h, and pass it through a 40-mesh sieve to obtain the modified konjac emulsifier.

[0068] Example 4: The application of a modified konjac emulsifier in chicken meatball products includes: First, rosemary extract (CAS: 84604-14-8) and modified konjac emulsifier (prepared in any one of Examples 1 to 3) were mixed and added to an ethanol-water solution with a volume ratio of 1:1 (i.e., the volume ratio of anhydrous ethanol to water was 1:1). The mixture was ultrasonically dissolved for 4 to 6 minutes (preferably 5 minutes) at a frequency of 20 kHz and a power of 95 to 105 W (preferably 100 W). The mass-volume ratio of rosemary extract, modified konjac emulsifier, and ethanol-water solution was 0.01 g: 0.22 g: 5 mL. The mixture was then spray-dried (inlet air temperature 122 to 124 °C, outlet air temperature 58 to 62 °C, feed rate 3 to 3.5 mL / min; preferably, inlet air temperature 123 °C, outlet air temperature 60 °C, feed rate 3.2 mL / min) to obtain emulsifier powder.

[0069] Then, add soybean oil (CAS: 8001-22-7), soy protein isolate (CAS: 9010-10-0), ice water at 2±2℃, and calcium chloride ( CaCl 2) Add to a high-speed disperser and homogenize at a speed of 7800-8200 rpm (preferably 8000 rpm) for 2-3 minutes (preferably 2.5 minutes) to obtain a pre-emulsion. The mass ratio of soybean oil, soy protein isolate, ice water and calcium chloride is 20:1:5:0.03.

[0070] Next, wash the chicken breast (after removing the skin and excess fat) and cut it into even small pieces (about 1cm). 3 The meat is ground into coarse mince using a meat grinder (3mm perforated plate). The coarse mince is then mixed with ternary compound salt and ice water at 2±2℃ in a chopper at 950-1050rpm (preferably 1000rpm) for 1-2 minutes (preferably 1.5 minutes). The mass ratio of coarse mince, ternary compound salt, and ice water is 80:2:7.5. The ternary compound salt includes sodium chloride (NaCl), potassium chloride (KCl), and calcium lactate, with a mass ratio of 5:3:2. The pH is adjusted to 6.5±0.1 (preferably pH 6.5) using a 0.1mol / L NaOH solution, and the mixture is further chopped for 28-32 seconds (preferably 30 seconds) to obtain the meat mince.

[0071] Next, the emulsifier powder and modified konjac emulsifier are added to the pre-emulsion and stirred at 495-505 rpm (preferably 500 rpm) for 28-32 seconds (preferably 30 seconds) to form a mixture. The mass ratio of emulsifier powder, modified konjac emulsifier, and pre-emulsion is 0.22:0.38:13. The mixture is then added to the minced meat and chopped at 1450-1550 rpm (preferably 1500 rpm) for 2-3 minutes (preferably 2.5 minutes), during which ice water at 2±2°C is added. The mass ratio of mixture, minced meat, and ice water is 0.44:100:7.5. Finally, yeast extract (CAS: 8013-01-2), continue chopping at a speed of 1450-1550 rpm (preferably 1500 rpm) for 1-2 minutes (preferably 1.5 minutes, with the center temperature of the minced meat controlled to be no more than 10℃ throughout the process), and the mass ratio of yeast extract to minced meat is 0.01:20.

[0072] Next, glucono-δ-lactone is added to the minced meat mixture and stirred at a speed of 795-805 rpm (preferably 800 rpm) for 28-32 s (preferably 30 s). The mass ratio of glucono-δ-lactone to minced meat is 0.03:20. After stirring, the mixture is immediately transferred to a refrigerator at 4±0.5℃ (preferably 4℃) and left to stand for 1.9-2.1 h (preferably 2 h).

[0073] Finally, the meatballs are shaped and preheated. Specifically, the ground meat that has been left to stand in the refrigerator is kneaded into meatballs (by hand or by machine) with a diameter of 3±0.5cm and a weight of 25±5g. Then, the meatballs are heated in a water bath at 42±1℃ (preferably 42℃) for 10 to 12 minutes (preferably 11 minutes), and then cooled with ice water to a core temperature of 4±0.5℃ (preferably 4℃).

[0074] After the meatballs are formed and preheated, they are refrigerated. Specifically, the preheated meatballs are cooled to -18±1℃ (preferably -18℃) at a rate of 1-1.2℃ / min (preferably 1.1℃ / min) and stored in an environment of -18±1℃ (preferably -18℃).

[0075] The conventional method for preparing chicken meatballs is as follows: First, wash the chicken breast (after removing the skin and excess fat) and cut it into even small pieces (about 1cm). 3The meat was ground into coarse mince using a meat grinder (3mm perforated plate). The coarse mince was then mixed with ternary compound salt and ice water at 2±2℃ in a chopper at 1000rpm for 1.5min. The mass ratio of coarse mince, ternary compound salt, and ice water was 80:2:7.5. The ternary compound salt consisted of sodium chloride (NaCl), potassium chloride (KCl), and calcium lactate, with a mass ratio of 5:3:2. The pH was adjusted to 6.5 with a 0.1mol / L NaOH solution, and the mixture was continued to be chopped for 30s to obtain the meat mince. Next, glyceryl monostearate (GMS) was added to the minced meat and chopped at 1500 rpm for 2.5 minutes, with ice water at 2±2°C added during the process. The mass ratio of glyceryl monostearate, minced meat, and ice water was 0.44:100:7.5. Then, yeast extract was added, and chopping was continued at 1500 rpm for another 1.5 minutes, with the mass ratio of yeast extract to minced meat being 0.01:20. Next, glucono-delta-lactone was added to the minced meat mixture and stirred at 800 rpm for 30 seconds, with the mass ratio of glucono-delta-lactone to minced meat being 0.03:20. After stirring, the mixture was immediately transferred to a refrigerator at 4°C and allowed to stand for 2 hours. Finally, the meatballs were shaped and preheated: the refrigerated minced meat was kneaded (by hand or machine) into meatballs with a diameter of 3±0.5 cm and a weight of 25±5 g; then, it was heated in a water bath at 42°C for 11 minutes, and then cooled with ice water to a core temperature of 4°C. After the meatballs are shaped and preheated, they are refrigerated. Specifically, the preheated meatballs are cooled to -18°C at a rate of 1.1°C / min and then stored in an environment of -18°C.

[0076] Figure 2 and Figure 3 The image shows cross-sectional views of chicken meatballs before and after freeze-thaw cycles; by Figure 2 , Figure 3 It can be seen that before freezing and thawing, the meatballs in Example 4 have a dense and uniform structure, a smooth surface, and low porosity. After freezing and thawing, they can still maintain a dense structure and a smooth surface. The test results show that the emulsifier of the present invention has excellent emulsifying ability and freeze-thaw stability. This is due to the introduction of the OSA hydrophobic group in the emulsifier molecule to improve the emulsifying properties of konjac gel, which can effectively bind water migration, reduce ice crystal size, and significantly reduce the damage of ice crystals to the muscle protein network during freezing and thawing, ultimately making the chicken meatballs exhibit good freeze-thaw stability.

[0077] Comparative Example 1: A method for preparing an emulsifier, comprising: Step S1, Pretreatment: Pre-preparation of the mixed immobilized enzyme, including preparation of microcrystalline cellulose carrier, enzyme loading, and sodium alginate embedding; The preparation of the porous microcrystalline cellulose carrier is as follows: First, microcrystalline cellulose (CAS: 9004-34-6) is added to an acetate-sodium acetate buffer solution at pH 5 and stirred at 160 rpm for 30 min at 45 °C. The mass-volume ratio of microcrystalline cellulose to acetate-sodium acetate buffer solution is 1 g: 25 mL. Then, the precipitate is collected by filtration and washed three times with deionized water. Finally, the precipitate is placed under normal pressure and cooled to -40 °C at a rate of 1 °C / min for 2.5 h. Then, it is dried by sublimation at -0.085 MPa and -20 °C for 11 h. Finally, it is heated to 0 °C at -0.085 MPa and a rate of 0.25 °C / min and dried by desorption at a rate of 4.5 h to obtain the microcrystalline cellulose carrier.

[0078] The enzyme loading was the same as step S1 in Example 2, and the sodium alginate encapsulation was the same as step S1 in Example 2.

[0079] Step S2, Preparation of precursor solution: Same as step S2 in Example 2.

[0080] Step S3, segmented deacetylation: consistent with step S3 in Example 2.

[0081] Step S4, Gel Particle Preparation: Same as Step S4 in Example 2.

[0082] Step S5, hydrophobic anchoring pretreatment: consistent with step S5 in Example 2.

[0083] Step S6, graded enzymatic esterification: consistent with step S6 in Example 2.

[0084] Step S7, purification and drying: consistent with step S7 in Example 2.

[0085] Comparative Example 2: A method for preparing an emulsifier, comprising: Step S1, Pretreatment: Pre-preparation of the mixed immobilized enzyme, including the preparation of porous microcrystalline cellulose carrier, enzyme loading, and sodium alginate embedding; The preparation of the porous microcrystalline cellulose carrier is consistent with step S1 in Example 2.

[0086] The enzyme loading process is as follows: First, the porous microcrystalline cellulose carrier is added to a sodium acetate buffer solution with a concentration of 0.1 mol / L and a pH of 5.5, and ultrasonically dispersed at a frequency of 20 kHz and a power of 100 W for 10 min. The mass-to-volume ratio of the porous microcrystalline cellulose carrier to the sodium acetate buffer solution is 0.2 g: 10 mL. Then, mannanase (enzyme activity 5000-6000 U / g) is added, and the mixture is stirred at 220 rpm for 30 min at 4 °C. The mass ratio of the porous microcrystalline cellulose carrier to the mannanase is 2:1, thus completing the enzyme loading.

[0087] Sodium alginate encapsulation is consistent with step S1 in Example 2.

[0088] Step S2, Preparation of precursor solution: Same as step S2 in Example 2.

[0089] Step S3, segmented deacetylation: consistent with step S3 in Example 2.

[0090] Step S4, Gel Particle Preparation: Same as Step S4 in Example 2.

[0091] Step S5, hydrophobic anchoring pretreatment: consistent with step S5 in Example 2.

[0092] Step S6, graded enzymatic esterification: consistent with step S6 in Example 2.

[0093] Step S7, purification and drying: consistent with step S7 in Example 2.

[0094] Comparative Example 3: A method for preparing an emulsifier, comprising: Step S1, Pretreatment: Pre-preparation of the mixed immobilized enzyme, including the preparation of porous microcrystalline cellulose carrier, enzyme loading, and sodium alginate embedding; The preparation of the porous microcrystalline cellulose carrier is consistent with step S1 in Example 2.

[0095] The enzyme loading process is as follows: First, the porous microcrystalline cellulose carrier is added to a sodium acetate buffer solution with a concentration of 0.1 mol / L and a pH of 5.5, and ultrasonically dispersed at a frequency of 20 kHz and a power of 100 W for 10 min. The mass-to-volume ratio of the porous microcrystalline cellulose carrier to the sodium acetate buffer solution is 0.2 g: 10 mL. Then, lipase (enzyme activity 10000~12000 U / g) is added, and the mixture is stirred at 220 rpm at 4 °C for 30 min. The mass ratio of the porous microcrystalline cellulose carrier to the lipase is 2:1, thus completing the enzyme loading.

[0096] Sodium alginate encapsulation is consistent with step S1 in Example 2.

[0097] Step S2, Preparation of precursor solution: Same as step S2 in Example 2.

[0098] Step S3, segmented deacetylation: consistent with step S3 in Example 2.

[0099] Step S4, Gel Particle Preparation: Same as Step S4 in Example 2.

[0100] Step S5, hydrophobic anchoring pretreatment: consistent with step S5 in Example 2.

[0101] Step S6, graded enzymatic esterification: consistent with step S6 in Example 2.

[0102] Step S7, purification and drying: consistent with step S7 in Example 2.

[0103] Comparative Example 4: A method for preparing an emulsifier, comprising: Step S1, Pre-processing: Same as step S1 in Example 2.

[0104] Step S2, Preparation of precursor solution: Prepare konjac glucomannan ethanol suspension by adding konjac glucomannan to anhydrous ethanol and stirring at 400 rpm for 15 min. The mass-to-volume ratio of konjac glucomannan to anhydrous ethanol is 1 g: 25 mL, thus obtaining the precursor solution.

[0105] Step S3, segmented deacetylation: consistent with step S3 in Example 2.

[0106] Step S4, Gel Particle Preparation: Same as Step S4 in Example 2.

[0107] Step S5, hydrophobic anchoring pretreatment: consistent with step S5 in Example 2.

[0108] Step S6, graded enzymatic esterification: consistent with step S6 in Example 2.

[0109] Step S7, purification and drying: consistent with step S7 in Example 2.

[0110] Comparative Example 5: A method for preparing an emulsifier, comprising: Step S1, Pre-processing: Same as step S1 in Example 2.

[0111] Step S2, Preparation of precursor solution: Same as step S2 in Example 2.

[0112] Step S3, Gel Particle Preparation: Same as step S4 in Example 2.

[0113] Step S4, hydrophobic anchoring pretreatment: consistent with step S5 in Example 2.

[0114] Step S5, graded enzymatic esterification: consistent with step S6 in Example 2.

[0115] Step S6, purification and drying: consistent with step S7 in Example 2.

[0116] Comparative Example 6: A method for preparing an emulsifier, comprising: Step S1, Pre-processing: Same as step S1 in Example 2.

[0117] Step S2, Preparation of precursor solution: Same as step S2 in Example 2.

[0118] Step S3, segmented deacetylation: consistent with step S3 in Example 2.

[0119] Step S4, Gel Particle Preparation: Same as Step S4 in Example 2.

[0120] Step S5, graded enzymatic esterification: First, prepare a pre-dried solvent, consistent with step S6 in the example.

[0121] The gel particles obtained in step S4 were vacuum dried at -0.085 MPa and 60°C for 12 h. Then, the pre-dried solvent, gel particles, and mixed immobilized enzyme were mixed and stirred at 350 rpm for 10 min. The volume-to-mass ratio of the pre-dried solvent, gel particles, and mixed immobilized enzyme was 50 mL: 1 g: 0.1 g.

[0122] The subsequent steps are the same as step S6 in Example 2. Step S7, purification and drying: consistent with step S7 in Example 2.

[0123] Comparative Example 7: A method for preparing an emulsifier, comprising: Step S1, Pre-processing: Same as step S1 in Example 2.

[0124] Step S2, Preparation of precursor solution: Same as step S2 in Example 2.

[0125] Step S3, segmented deacetylation: consistent with step S3 in Example 2.

[0126] Step S4, Gel Particle Preparation: Same as Step S4 in Example 2.

[0127] Step S5, hydrophobic anchoring pretreatment: consistent with step S5 in Example 2.

[0128] Step S6, Enzymatic Esterification: First, prepare a pre-dried solvent. Specifically, mix ethyl acetate and anhydrous ethanol at a volume ratio of 3:1, then add anhydrous magnesium sulfate. The mass-volume ratio of anhydrous magnesium sulfate to the mixture is 0.9 g: 10 mL. Cover the bottle with a ground glass stopper and let it stand at room temperature in the dark for 25 hours, shaking for 1 minute every 6 hours (to improve water absorption contact efficiency). Then, filter under normal pressure using a dry sintered sand funnel and qualitative filter paper (all equipment is dried and cooled at 100℃ beforehand to prevent secondary water absorption). Collect the clear filtrate to obtain the pre-dried solvent.

[0129] The hydrophobically anchored gel particles were vacuum dried at -0.085 MPa and 60 °C for 12 h. Then, the pre-dried solvent, the hydrophobically anchored gel particles, and the mixed immobilized enzyme were mixed and stirred at 350 rpm for 10 min. The volume-to-mass ratio of the pre-dried solvent, the hydrophobically anchored gel particles, and the mixed immobilized enzyme was 50 mL: 1 g: 0.1 g.

[0130] Then, anhydrous magnesium sulfate was added, and the mixture was stirred continuously at 350 rpm for 15 minutes. The mass ratio of anhydrous magnesium sulfate to surface hydrophobic anchored gel particles was 0.4:1.

[0131] Subsequently, an octenyl succinic anhydride (OSA)-ethyl acetate solution with a concentration of 0.1 g / mL (i.e., the mass-to-volume ratio of octenyl succinic anhydride to ethyl acetate is 0.1 g: 1 mL) was added dropwise at a rate of 0.4 mL / min. The mass ratio of octenyl succinic anhydride to the hydrophobically anchored gel particles was 0.28:1. The mixture was stirred at 300 rpm while being added dropwise. After the addition was completed, the mixture was stirred at a constant temperature of 38 °C for 2 h (the hydrophobic groups of OSA were rapidly grafted onto the surface of the gel particles, improving the dispersibility of the particles in the organic phase). After the reaction was completed, the mixed immobilized enzyme was preliminarily separated and recovered using a 20-mesh standard sieve to obtain the filtrate.

[0132] Step S7, purification and drying: consistent with step S7 in Example 2.

[0133] The swelling force, apparent viscosity, emulsifying ability, and emulsification stability of the emulsifiers prepared in Examples 1 to 3 and Comparative Examples 1 to 7 were tested respectively. Expansion force test: Accurately weigh a dry sample powder with a mass of m (usually 1g) and record its initial volume V0; pour the sample into a graduated cylinder and add 50mL of distilled water, gently shake to ensure full contact between the sample and water, and let the graduated cylinder stand at room temperature until the sample is completely swollen and its volume no longer changes (e.g., after 24 hours). Read the final volume V1 occupied by the swollen sample in the graduated cylinder to obtain the expansion force. ; Apparent viscosity test: Apparent viscosity was determined at a concentration of 1% (w / v) using a rotational viscometer. The test method is in accordance with GB / T 15357-2014 "Surfactants and Detergents - Determination of viscosity and flow properties of liquid products by rotational viscometer" or GB / T 5561-1994 "Surfactants - Method for determination of viscosity and flow properties by rotational viscometer". Emulsifying ability and emulsion stability testing: A sample with a mass of m (usually 0.2 g) was dispersed in 50 mL of distilled water and stirred in a 60℃ water bath at 200 rpm for 30 min. After cooling to room temperature, 50 g of soybean oil was slowly added, and the mixture was homogenized at 6000 rpm for 1 min using a high-speed disperser. Then, it was centrifuged at 3000 rpm for 10 min, and the total liquid height and emulsion layer height were recorded. The emulsion was then stored at room temperature for 30 days and centrifuged at 3000 rpm for 10 min, and the total liquid height and the height of the remaining emulsion layer were recorded. ; ; The test results are shown in Table 1 below (ten sets of data were tested for each test group and the average value was taken. Each test group was tested under the same temperature and humidity conditions). Table 1. Comparison of emulsifier performance:

[0134] As shown in the table above, the modified konjac emulsifier prepared using the scheme in the examples exhibits significantly better swelling power, apparent viscosity, emulsifying ability, and emulsifying stability than the comparative examples due to the multiple synergistic effects of "porous carrier + dual enzyme immobilization + xanthan gum synergy + deacetylation + directional pore formation + hydrophobic anchoring + graded esterification".

Claims

1. A method for preparing a modified konjac emulsifier, characterized in that: include: Step S1, Pretreatment: Prepare the mixed immobilized enzyme in advance; The preparation method of the mixed immobilized enzyme includes the preparation of a porous microcrystalline cellulose carrier, enzyme loading, and sodium alginate embedding. Specifically, the preparation of the porous microcrystalline cellulose carrier involves: first, adding microcrystalline cellulose to an acetate-sodium acetate buffer solution at pH 5±0.1, and then adding cellulase. The mixture is stirred at a low speed of 150-170 rpm for 29-31 min at 44-46℃. The mass-to-volume ratio of microcrystalline cellulose, cellulase, and acetate-sodium acetate buffer solution is 1 g:0.02 g:25 mL, and the cellulase activity is 10000-12000 U / g. Then, the mixture is heated at 100±1℃. The enzyme was inactivated by treating the sample in a boiling water bath for 9–11 min. The precipitate was then collected by filtration and washed 3–4 times with deionized water. Finally, the precipitate was placed under normal pressure and cooled to -40±1℃ at a rate of 0.8–1.2℃ / min for 2–3 h. It was then subjected to constant-temperature sublimation drying at -0.08–-0.09 MPa and -20±1℃ for 10–12 h. Finally, it was heated to 0±0.5℃ at a rate of 0.2–0.3℃ / min under constant-temperature desorption drying for 4–5 h to obtain the porous microcrystalline cellulose carrier. The enzyme loading process is as follows: First, the porous microcrystalline cellulose carrier is added to a sodium acetate buffer solution with a concentration of 0.1 mol / L and a pH of 5.5 ± 0.

1. The mixture is then ultrasonically dispersed at a frequency of 20 kHz and a power of 95–105 W for 9–11 min. The mass-to-volume ratio of the porous microcrystalline cellulose carrier to the sodium acetate buffer solution is 0.2 g: 10 mL. Next, lipase and mannanase are added sequentially, and the mixture is stirred at 210–230 rpm for 28–32 min at 4 ± 0.5 °C. The mass ratio of the porous microcrystalline cellulose carrier, lipase, and mannanase is 2:1:1, thus completing the enzyme loading. The lipase activity is 10000–12000 U / g, and the mannanase activity is 5000–6000 U / g. Step S2, Preparation of precursor solution: First, prepare konjac glucomannan ethanol suspension, then prepare xanthan gum aqueous solution, and finally mix and swell to obtain precursor solution; Step S3, Fractional Deacetylation: Add sodium carbonate to the precursor solution, mix and stir, then transfer to a microwave reactor for heating; then add more sodium carbonate and continue microwave heating to obtain a uniform gel; Step S4, Gel Particle Preparation: Pour the gel into a mold and place it on a cold table to pre-freeze and form oriented ice crystals. Then transfer it to an autoclave for CO2 expansion. Afterward, perform freezing and heat preservation, freeze drying, and air jet milling in sequence to obtain gel particles. Step S5, Hydrophobic Anchoring Pretreatment: The gel particles are soaked in a stearic acid ethanol solution to obtain hydrophobically anchored gel particles. Specifically, the gel particles obtained in step S4 are added to a 1.5% stearic acid anhydrous ethanol solution with a mass-to-volume ratio of 1g:10mL. Then, 4.8-5.2% of lipase by weight of the gel particles is added, and the mixture is refluxed at 39.5-40.5℃ and 140-160rpm for 1.9-2.1h. After the reaction, the mixture is filtered through a 400-mesh filter cloth and washed 3-4 times with anhydrous ethanol at 45±1℃. After washing, the ethanol is evaporated in a fume hood until there is no alcohol odor, thus obtaining the hydrophobically anchored gel particles. Step S6, graded enzymatic esterification: First, prepare a pre-dried solvent; then, mix the pre-dried solvent, hydrophobically anchored gel particles, and mixed immobilized enzyme evenly, and add anhydrous magnesium sulfate, mixing thoroughly; then, add octenyl succinic anhydride-ethyl acetate solution dropwise; then, separate the mixed immobilized enzyme, and add octenyl succinic anhydride-ethyl acetate solution, mixed immobilized enzyme, and glycerol to the filtrate, stirring and mixing to react. After the reaction is complete, first use a 20-mesh standard sieve to separate and recover the mixed immobilized enzyme again, and then use a 200-mesh filter cloth to filter the filtrate under normal pressure, collecting the gel particle filter cake; Step S7, Purification and Drying: First, wash and filter the gel particle filter cake with anhydrous ethanol to obtain a precipitate, and then freeze-dry it under vacuum to obtain the final product.

2. The method for preparing a modified konjac emulsifier according to claim 1, characterized in that: The sodium alginate encapsulation process is as follows: First, a sodium alginate solution with a mass fraction of 0.9%–1.1% is added to the enzyme-loaded porous microcrystalline cellulose suspension, with a volume ratio of sodium alginate solution to enzyme-loaded porous microcrystalline cellulose suspension of 2:

1. After stirring at 100–120 rpm for 28–32 min at room temperature, a calcium chloride solution with a concentration of 0.1 mol / L is added dropwise at a rate of 0.9–1.1 mL / min, with a volume ratio of the added mixture to the calcium chloride solution of 1:6–8. The addition is then completed. Afterward, the microspheres were solidified at room temperature for 28–32 min, and then collected by filtration. Finally, the filtered microspheres were placed under normal pressure and cooled to -40±1℃ at a rate of 0.8–1.2℃ / min for 2–3 h for pre-freezing. Then, they were subjected to constant temperature sublimation drying at -0.08–-0.09 MPa and -20±1℃ for 8–9 h. Afterward, they were heated to 0±0.5℃ at a rate of 0.2–0.3℃ / min under a rate of -0.08–-0.09 MPa for 3–4 h for constant temperature desorption and drying to obtain the mixed immobilized enzyme.

3. The preparation method of the modified konjac emulsifier according to claim 1, characterized in that: In step S2, the preparation of the konjac glucomannan ethanol suspension specifically involves: adding konjac glucomannan to anhydrous ethanol and stirring at 390–410 rpm for 14–16 min, with a mass-to-volume ratio of konjac glucomannan to anhydrous ethanol of 1 g: 25 mL; the preparation of the xanthan gum aqueous solution specifically involves: adding xanthan gum to distilled water at 30 ± 1 °C and stirring at 790–810 rpm for 29–31 min, with a mass-to-volume ratio of xanthan gum to distilled water of 0.1 g: 25 mL; and the mixing and swelling specifically involves: adding the konjac glucomannan ethanol suspension dropwise to the xanthan gum aqueous solution at a rate of 0.9–1.1 mL / min, stirring at 390–410 rpm in a water bath at 29–31 °C, and maintaining the temperature for 0.9–1.1 h after the addition is complete.

4. The preparation method of the modified konjac emulsifier according to claim 3, characterized in that: Step S3 specifically involves: First, adding sodium carbonate to the precursor solution, adjusting the pH to 9.0±0.1, and stirring at 280-300 rpm for 9-11 minutes at -0.08 to -0.09 MPa; then, transferring it to a microwave reactor and heating at a power of 195-205 W and a temperature of 59-61°C for 18-22 minutes; next, adding sodium carbonate, adjusting the pH to 10.0±0.1, and continuing to heat at a power of 195-205 W and a temperature of 59-61°C for 18-22 minutes; finally, cooling to room temperature to obtain a uniform gel.

5. The method for preparing a modified konjac emulsifier according to claim 4, characterized in that: Step S4 specifically involves: First, pouring the uniform gel obtained in step S3 into a mold, and pre-freezing it for 1.9–2.1 hours at a rate of 1°C / min from the bottom on a cold platform at -20±1°C to form directional ice crystals; then, transferring it to an autoclave, and injecting CO2 into the autoclave at a rate of 0.3–0.4 MPa / min to 11.8–12.2 MPa, maintaining the pressure for 43–47 minutes, and then releasing it to atmospheric pressure within 4–6 seconds; subsequently, transferring it to a freezer at -39–-41°C for 7 hours. After heat preservation for 9 to 8.1 hours, the material is first sublimated and dried at -0.08 to -0.09 MPa and -20±1℃ for 22 to 26 hours, and then desorption and dried at -0.08 to -0.09 MPa and 0±0.5℃ for 10 to 14 hours. Finally, it is subjected to air jet milling with the chamber temperature less than 40℃, ambient humidity RH < 30%, grinding nozzle pressure 0.6 to 0.7 MPa, classifying wheel speed 2200 to 2600 rpm, and feed rate 15 to 20 g / h. After milling, it is passed through a 40 to 60 mesh sieve to obtain the final product.

6. The method for preparing a modified konjac emulsifier according to claim 5, characterized in that: In step S6, the specific steps for preparing the pre-dried solvent are as follows: Ethyl acetate and anhydrous ethanol are mixed at a volume ratio of 3:1, and anhydrous magnesium sulfate is added. The mass-volume ratio of anhydrous magnesium sulfate to the mixture is 0.8-1 g: 10 mL. The bottle is capped with a ground glass stopper and left to stand at room temperature in the dark for 24-26 hours, shaking for 1 minute every 6 hours. After that, the mixture is filtered under normal pressure using a dry sintered sand funnel and qualitative filter paper. The clear filtrate is collected to obtain the pre-dried solvent.

7. The method for preparing a modified konjac emulsifier according to claim 6, characterized in that: Step S6 specifically involves: First, vacuum drying the surface-anchored hydrophobic gel particles at -0.08 to -0.09 MPa and 58 to 62°C for 11.5 to 12.5 hours. Then, mixing and stirring the pre-dried solvent, surface-anchored hydrophobic gel particles, and mixed immobilized enzyme at 345 to 355 rpm for 9 to 11 minutes. The volume-to-mass ratio of the pre-dried solvent, surface-anchored hydrophobic gel particles, and mixed immobilized enzyme is 50 mL: 1 g: 0.

1. g; then, add anhydrous magnesium sulfate, and stir continuously at 345-355 rpm for 14-16 min, with the mass ratio of anhydrous magnesium sulfate to surface hydrophobically anchored gel particles being 0.4:1; then, add a 0.1 g / mL octenyl succinic anhydride-ethyl acetate solution at a rate of 0.35-0.45 mL / min, with the mass ratio of octenyl succinic anhydride to surface hydrophobically anchored gel particles being 0.28:1, while adding dropwise at 295- The mixture was stirred at 305 rpm. After the addition was complete, it was kept at 37.5–38.5 °C and stirred for 1.9–2.1 h. After the reaction was completed, the mixed immobilized enzyme was initially separated and recovered using a 20-mesh standard sieve to obtain a filtrate. Then, 0.1 g / mL of octenyl succinic anhydride-ethyl acetate solution and the mixed immobilized enzyme were added to the filtrate. The volume mass of the added octenyl succinic anhydride-ethyl acetate solution, the mixed immobilized enzyme, and the surface hydrophobically anchored gel particles was specified. The ratio was 0.84 mL: 0.05 g: 1 g. Glycerol of 1.9% to 2.1% of the mass of hydrophobically anchored gel particles was added simultaneously. The mixture was stirred at 345 to 355 rpm for 9 to 11 minutes, then heated to 44.5 to 45.5 °C and stirred at a constant temperature for 2.9 to 3.1 hours. After the reaction was completed, the mixed immobilized enzyme was first separated and recovered again using a 20-mesh standard sieve, and then the filtrate was filtered under normal pressure using a 200-mesh filter cloth to collect the gel particle filter cake.

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