A highly branched cyclodextrin complex, and a preparation method and application thereof

Abstract

The present application relates to the technical field of enzyme preparation stabilizer, and provides a high-branched cyclodextrin complex, a low-DE high-branched polysaccharide substance prepared by two-stage enzymatic hydrolysis of starch by starch branching enzyme, which synergistically acts with oligosaccharides and amino acids to protect the three-dimensional structure of enzyme protein in multiple dimensions. The high-branched cyclodextrin complex can significantly improve the tolerance of enzyme preparation to high temperature, freezing, acid and alkali environment, greatly prolong the shelf life of enzyme preparation at room temperature, and solve the industry pain points of existing industrial enzyme preparation, such as easy high-temperature freezing inactivation, fast storage activity attenuation and poor processing tolerance.

Description

Technical Field

[0001] This invention relates to the field of enzyme preparation stabilizer technology, and in particular to a highly branched cyclodextrin complex, its preparation method, and its application. Background Technology

[0002] Enzyme preparations are a class of biocatalysts with highly efficient catalytic activity. Due to their advantages of high efficiency, specificity, and environmental friendliness, they are widely used in food processing, animal feed, detergents, and bio-fermentation. However, natural enzyme proteins are three-dimensional active proteins that are extremely sensitive to the environment. Under conditions of high temperature, low temperature, extreme acids and alkalis, high salt, mechanical shearing, surfactants, and long-term storage at room temperature, they are prone to peptide chain unfolding, protein aggregation, and destruction of active sites, leading to a significant decrease in enzyme activity or even complete inactivation. This severely limits the processing application scenarios and shelf life of enzyme preparations.

[0003] Currently, commonly used enzyme stabilizers in the industry are mostly trehalose, maltodextrin, sorbitol, inorganic salts, and single sugars. These stabilizers have drawbacks such as poor compatibility, insufficient heat resistance, and weak anti-degradation ability. Although trehalose can provide some protection for enzyme proteins, its mechanism of action is singular, only achieving simple physical encapsulation. It cannot simultaneously meet the multiple stability requirements of enzyme preparations, such as high temperature resistance, acid and alkali resistance, antioxidant properties, and resistance to protease degradation, resulting in limited stabilization effects. Ordinary maltodextrin has low branching degree and loose molecular structure, resulting in weak enzyme encapsulation ability, low encapsulation rate, and easy hygroscopic clumping at high temperatures. Sugar excipients are prone to Maillard reactions at high temperatures. Inorganic salts lack polyhydroxyl groups and rely solely on weak charge neutralization. Their freeze-thaw protection ability is far lower than that of sugars and dextrins, and they have significant side effects. The salt addition range is extremely narrow, and even slight over-limitation can cause significant enzyme inactivation. At the same time, high-salt enzyme preparations will increase the sodium and phosphorus content of the final product, which does not meet the standards for low-salt and low-mineral additive products. Based on this, developing a composite enzyme stabilizer with multi-mechanism synergy, broad adaptability, and excellent stabilization effect is of great practical significance for improving the quality of industrial enzyme preparations. Summary of the Invention

[0004] The purpose of this invention is to provide a highly branched cyclodextrin complex, which can significantly improve the stability of industrial enzyme preparations.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a highly branched cyclodextrin complex, comprising the following steps: (1) The waxy starch suspension was mixed with starch branching enzyme for enzymatic hydrolysis, and then filtered, decolorized, deodorized, concentrated and dried to obtain highly branched cyclodextrin. (2) After mixing the highly branched cyclodextrin with water, add oligosaccharides and amino acids, and stir to obtain a mixture; (3) The mixture is obtained by shearing, homogenizing and drying.

[0006] Preferably, the enzymatic hydrolysis in step (1) includes a first enzymatic hydrolysis and a second enzymatic hydrolysis. The first enzymatic hydrolysis process involves mixing a 20-40% (w / v) concentration of waxy starch suspension with 50-150 U / g of dry starch starch branching enzyme and performing a single enzymatic hydrolysis reaction to obtain hydrolysate 1. The second enzymatic hydrolysis process involves spraying and liquefying hydrolysate 1, then instantaneously mixing and keeping it warm with steam, cooling it down, adding 100-500 U / g of dry starch starch branching enzyme, mixing it, and performing a second enzymatic hydrolysis reaction.

[0007] Preferably, the time for one enzymatic hydrolysis reaction is 20-40 min, and the temperature for one enzymatic hydrolysis reaction is 65-75℃.

[0008] Preferably, the secondary enzymatic hydrolysis reaction takes 12-24 hours and is carried out at a temperature of 65-75°C.

[0009] Preferably, the waxy starch suspension has a mass-volume concentration of 20-40% and a pH value of 7.5-8.5.

[0010] Preferably, the decolorization time in step (1) is 25-45 min, the decolorization temperature is 70-80℃, the concentration temperature is 60-70℃, the concentration vacuum degree is -0.08~-0.09MPa, the drying inlet air temperature is 150-170℃, and the drying outlet air temperature is 75-85℃.

[0011] Preferably, the mass ratio of the high branched-chain cyclodextrin, oligosaccharide and amino acid in step (2) is (55-75):(15-25):(5-15), and the mass ratio of the high branched-chain cyclodextrin to water is 1:(1.5-3.5).

[0012] Preferably, the oligosaccharide is any one or more of raffinose, stachyose, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, galactooligosaccharide, manno-oligosaccharide, and inulin.

[0013] Preferably, the amino acid is any one or more of glutamic acid, aspartic acid, methionine, L-cysteine, lysine, glycine, proline, L-alanine, arginine, and methionine.

[0014] Preferably, the shearing time in step (3) is 10-15 min, and the shearing speed is 4000-6000 r / min.

[0015] The present invention also provides a highly branched cyclodextrin complex prepared by the above preparation method, wherein the highly branched cyclodextrin complex has a cyclic structure in which glucose is bonded to the non-reducing end of dextrin via an α-1,6 glycosidic bond, and the DE value of the highly branched cyclodextrin is 0.8-3.

[0016] The present invention also provides the application of the highly branched cyclodextrin complex in improving the stability of enzyme preparations, wherein the mass ratio of the highly branched cyclodextrin complex to the enzyme preparation to be treated is (2-10):1.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects: 1. The highly branched cyclodextrin prepared by the technical solution of this invention achieves precise targeted encapsulation of enzyme proteins, solving the contradiction of "enzyme protection and activity inhibition". It has the characteristics of hyperbranched multi-cavity structure, large specific surface area and high water solubility. It can selectively encapsulate hydrophobic groups and flexible and volatile regions on the surface of enzyme proteins, stabilize the three-dimensional spatial conformation of enzyme molecules, effectively isolate enzyme molecules from the damage of high temperature, oxygen and acid and alkali ions, achieve the dual effect of high stability and high enzyme activity retention, and significantly improve the storage period and working condition tolerance of enzyme preparations.

[0018] 2. The high-branched cyclodextrin compound prepared by the technical solution of this invention has strong compatibility and forms a ternary synergistic protection system with oligosaccharides and amino acids: Compared with the monosaccharides and disaccharides used in traditional technologies, oligosaccharide (DP3-10) molecules have a large number of continuous hydroxyl groups and medium- to long-chain backbone structures. They also have the dual functions of water substitution effect and high glass transition temperature glass matrix, which can better bind free water, inhibit large ice crystals and ice recrystallization, and have no low-temperature crystallization problem. By isolating the molecular chain, it blocks the hydrophobic aggregation of enzyme proteins. The enzyme activity retention after freeze-thaw is significantly better than that of monosaccharides and disaccharides. It also has strong thermal stability, low reducing group content, and is not easily hydrolyzed and volatilized at high temperatures, forming a continuous heat-insulating glass film, which greatly reduces the enzyme activity loss caused by thermal denaturation and Maillard browning. Meanwhile, amino acids act as molecular chaperones, making it difficult for enzyme proteins to unfold during dehydration. Multiple amino / carboxyl groups build an intermolecular network to lock the protein spatial conformation. They also scavenge thermal oxidative free radicals and protect the metal ionicity of the active center. The three components work in a complementary and non-antagonistic manner, forming multiple synergistic stabilizing effects through a three-layer three-dimensional protective structure. This simultaneously addresses three types of stress: low-temperature freeze-thaw cycles, high-temperature drying, and long-term storage of dry powder. Compared to traditional protective agent formulations, this approach has a significant effect, greatly improving the overall activity retention rate of industrial enzymes such as TG enzymes and PG enzymes throughout the entire process.

[0019] 3. This invention innovatively employs a ternary composite encapsulation stabilization system of highly branched cyclodextrin, functional oligosaccharides, and amino acids to replace the traditional stabilization system of trehalose, maltodextrin, polyols, and inorganic salts. Through a triple synergistic mechanism of "cavity-targeted encapsulation, hydration membrane protection, and conformational rigidity reinforcement," it comprehensively solves industry pain points such as easy denaturation, easy oxidation, poor temperature and acid / alkali resistance, and rapid storage degradation of enzyme preparations, resulting in a low-cost, water-soluble, freeze-resistant, high-temperature resistant, and acid / alkali resistant natural high-efficiency enzyme preparation stabilizer. Attached Figure Description

[0020] Figure 1 This is a comparative schematic diagram of ice crystals of highly branched cyclodextrin, trehalose, and maltodextrin prepared in Example 1 of the present invention. Figure 1 In this text, A represents the ice crystal area of ​​trehalose, B represents the ice crystal area of ​​maltodextrin, and C represents the ice crystal area of ​​the highly branched cyclodextrin prepared in the examples; the scale bar is 100 μm. Detailed Implementation

[0021] This invention provides a method for preparing a highly branched cyclodextrin complex, comprising the following steps: (1) The waxy starch suspension was mixed with starch branching enzyme for enzymatic hydrolysis, and then filtered, decolorized, deodorized, concentrated and dried to obtain highly branched cyclodextrin. (2) After mixing the highly branched cyclodextrin with water, add oligosaccharides and amino acids, and stir to obtain a mixture; (3) The mixture is obtained by shearing, homogenizing and drying.

[0022] In this invention, the preparation process of the waxy starch suspension is as follows: waxy starch is added to water and stirred evenly to obtain a waxy starch suspension. The waxy starch in this invention is preferably any one of waxy corn starch, waxy wheat starch, and waxy cassava starch; the pH value of the waxy starch suspension is preferably 7.5-8.5, more preferably 7.6-8.0, and even more preferably 7.8.

[0023] In this invention, a waxy starch suspension is mixed with starch branching enzyme for enzymatic hydrolysis. The enzymatic hydrolysis includes a first-step hydrolysis and a second-step hydrolysis. The first-step hydrolysis involves mixing a 20-40% (w / v) waxy starch suspension with a starch branching enzyme at a concentration of 50-150 U / g dry starch and performing a single enzymatic hydrolysis reaction to obtain hydrolysate 1. The second-step hydrolysis involves spraying and liquefying hydrolysate 1, then momentarily steam-mixing and maintaining the temperature. After cooling, a second enzymatic hydrolysis reaction is performed by adding a starch branching enzyme at a concentration of 100-500 U / g dry starch. The stepwise enzymatic hydrolysis process described in this invention can further increase the degree of branching, promote intramolecular cyclization, and increase the proportion of highly branched cyclodextrins.

[0024] In this invention, during the first enzymatic hydrolysis step, the mass-volume concentration of the waxy starch suspension is preferably 20-40%, more preferably 25-35%, and even more preferably 30%; the amount of starch branching enzyme added is preferably 50-150 u / g dry starch, more preferably 80-120 u / g dry starch, and even more preferably 100 u / g dry starch. The mass-volume concentration of the enzymatic hydrolysate 1 in this invention is preferably 20-40%, more preferably 25-35%, and even more preferably 30%.

[0025] In this invention, during the second enzymatic hydrolysis process, the amount of starch branching enzyme added is preferably 100-500 u / g dry starch, more preferably 200-400 u / g dry starch, and even more preferably 300 u / g dry starch.

[0026] In this invention, the time for the first enzymatic hydrolysis reaction is preferably 20-40 min, more preferably 25-35 min, and even more preferably 30 min; the temperature for the first enzymatic hydrolysis reaction is preferably 65-75℃, more preferably 68-72℃, and even more preferably 70℃.

[0027] In this invention, after the enzymatic hydrolysate 1 is sprayed and liquefied, it is instantaneously mixed with steam and kept warm. The spraying temperature is preferably 110-118℃, more preferably 112-117℃, and even more preferably 115℃; the keeping temperature is preferably 95-99℃, more preferably 96-98℃, and even more preferably 97℃; the keeping time is preferably 60-90 min, more preferably 65-85 min, and even more preferably 70 min.

[0028] In this invention, after the heat preservation is completed, flash evaporation and cooling are performed. Preferably, the temperature is reduced to the discharge temperature of 65-75°C, more preferably 68-72°C, and even more preferably 70°C.

[0029] In this invention, after flash evaporation and cooling, starch branching enzyme is added for a secondary enzymatic hydrolysis reaction. The reaction time is preferably 12-24 h, more preferably 14-20 h, and even more preferably 16 h. The reaction temperature is preferably 65-75 °C, more preferably 68-72 °C, and even more preferably 70 °C.

[0030] In this invention, after the secondary enzymatic hydrolysis reaction is completed, the temperature is preferably adjusted to 95°C for enzyme inactivation, and the inactivation time is preferably 25-32 min, more preferably 26-31 min, and even more preferably 30 min.

[0031] In this invention, after the enzyme is inactivated, the enzymatic hydrolysate is filtered to remove insoluble precipitates, yielding the supernatant. Preferably, the filtration process uses a diatomaceous earth filter, with a filter mesh size of 300-500 mesh, more preferably 350-450 mesh, and even more preferably 400 mesh.

[0032] In this invention, the supernatant is decolorized, preferably by adding activated carbon. The amount of activated carbon added is preferably 0.2-0.6%, more preferably 0.3-0.5%, and even more preferably 0.4%. The decolorization temperature is preferably 70-80℃, more preferably 72-78℃, and even more preferably 75℃. The decolorization time is preferably 25-45 min, more preferably 30-40 min, and even more preferably 35 min.

[0033] In this invention, after decolorization, an ion exchange is performed to obtain an ion exchange solution. Preferably, the decolorized solution is filtered through a plate and frame filter before entering the ion exchange column to remove inorganic salt ions, residual proteins, etc. from the feed solution. The feed temperature is preferably 50-55℃, and the feed flow rate is preferably 7-10 m³ / h. 3 The pH value is preferably 4.0-6.0, and the conductivity is preferably ≤20μS / cm.

[0034] In this invention, the ion exchange liquid is passed through a deodorization column and deodorized by a macroporous adsorption resin, adsorbing aldehydes and heterocyclic small molecule odor substances. The deodorization treatment temperature of this invention is preferably 45-55℃, more preferably 48-52℃, and even more preferably 50℃.

[0035] In this invention, after deodorization, evaporation and concentration are performed until the solid content is 50-70%. Preferably, this invention employs a five-effect falling film evaporation concentration method. The concentration temperature is preferably 60-70℃, more preferably 62-68℃, and even more preferably 65℃. The vacuum degree of the concentration is preferably -0.08~-0.09MPa, more preferably -0.083~-0.087MPa, and even more preferably -0.085MPa.

[0036] In this invention, the concentrated solution is dried to obtain highly branched cyclodextrin. The drying method is preferably spray drying, with the inlet air temperature preferably at 150-170°C, more preferably at 155-165°C, and even more preferably at 160°C; the outlet air temperature is preferably at 75-85°C, more preferably at 78-82°C, and even more preferably at 80°C.

[0037] In this invention, the starch branching enzyme (glycogen branching enzyme, GBE, EC2.4.1.18) is a commercially available food-grade enzyme solution, which is a starch branching enzyme AaGBE derived from Aquifex aeolicus VF5, with an enzyme activity of 20,000 U / mL.

[0038] In this invention, highly branched cyclodextrin is mixed with water and stirred until homogeneous. The stirring temperature is preferably 40-60°C, more preferably 45-55°C, and even more preferably 50°C. The stirring time is preferably 10-20 min, more preferably 12-18 min, and even more preferably 15 min. The stirring speed is preferably 400-600 rpm, more preferably 450-550 rpm, and even more preferably 500 rpm. Then, oligosaccharides and amino acids are added and stirring continues to obtain a mixture. The stirring temperature in this invention is preferably 40-50°C, more preferably 42-48°C, and even more preferably 45°C. The stirring time is preferably 20-30 min, more preferably 22-28 min, and even more preferably 25 min.

[0039] In this invention, the preferred mass ratio of the highly branched cyclodextrin, oligosaccharide, and amino acid is (55-75):(15-25):(5-15), more preferably (60-70):(18-20):(8-12), and even more preferably 69.1:19.7:11.2; the preferred mass ratio of the highly branched cyclodextrin to water is 1:(1.5-3.5), more preferably 1:(2-3), and even more preferably 1:2.5.

[0040] In this invention, the oligosaccharide is any one or more of raffinose, stachyose, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, galactooligosaccharide, manno-oligosaccharide, and inulin. The oligosaccharide of this invention has high water solubility and good molecular flexibility. Constructing a dense hydration protective film can form a continuous and dense hydration protective layer on the enzyme protein surface, locking in the water bound to the enzyme molecules, effectively inhibiting protein dehydration and denaturation caused by moisture loss during storage and transportation. Simultaneously, it can buffer the damage to the enzyme's spatial structure caused by mechanical shear forces such as industrial stirring and pumping, improve the homogeneity of the enzyme system, and prevent protein aggregation and precipitation.

[0041] In this invention, the amino acid is any one or more selected from glutamic acid, aspartic acid, methionine, L-cysteine, lysine, glycine, proline, L-alanine, arginine, and methionine. The amino acid described in this invention is a naturally compatible small molecule substance for enzyme proteins, which can bind to the enzyme protein surface through hydrogen bonds and electrostatic interactions, repairing enzyme molecular structural defects, increasing the rigidity of the protein backbone, significantly enhancing the structural stability of the enzyme protein under extreme acid-base and high-low temperature environments, neutralizing local acid-base fluctuations in the system, scavenging free radicals, and inhibiting enzyme oxidative inactivation.

[0042] In this invention, the mixture is subjected to medium-speed shearing to obtain a pre-emulsion. The shearing time is preferably 5-15 min, more preferably 8-12 min, and even more preferably 10 min. The shearing speed is preferably 4000-6000 r / min, more preferably 4500-5500 r / min, and even more preferably 5000 r / min.

[0043] In this invention, the pre-emulsion is homogenized to obtain the final emulsion. The low pressure of the homogenization is preferably 20-30 bar, more preferably 22-28 bar, and even more preferably 25 bar. The high pressure of the homogenization is preferably 150-200 bar, more preferably 170-80 bar, and even more preferably 175 bar.

[0044] In this invention, the final emulsion is dried, preferably by spray drying or freeze drying. When spray drying is preferred, the inlet air temperature is preferably 145-165°C, more preferably 150-160°C, and even more preferably 155°C; the outlet air temperature is preferably 65-75°C, more preferably 68-72°C, and even more preferably 70°C. When freeze drying is preferred, the drying temperature is preferably -40 to -60°C, more preferably -45 to -55°C, and even more preferably -50°C; the drying time is preferably 20-33 hours, more preferably 25-32 hours, and even more preferably 30 hours.

[0045] The present invention also provides a highly branched cyclodextrin complex prepared by the aforementioned method, wherein the highly branched cyclodextrin complex has a cyclic structure in which glucose is bonded to the non-reducing end of dextrin via an α-1,6 glycosidic bond, and the DE (Dextrose Equivalent, glucose value) of the highly branched cyclodextrin is preferably 0.8-3, more preferably 1.5-2.3, and even more preferably 1.9; the molecular weight of the highly branched cyclodextrin is preferably 70-100 KD, and the mass content of branched starch in the highly branched cyclodextrin is preferably ≥90%.

[0046] The present invention also provides the application of the highly branched cyclodextrin complex in improving the stability of enzyme preparations.

[0047] In this invention, the mass ratio of the highly branched cyclodextrin complex to the enzyme preparation to be treated is preferably (2-10):1, more preferably (4-8):1, and even more preferably 6:1.

[0048] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] Weigh 135g of waxy corn starch and add it to 450ml of water to prepare a 30% (w / v) suspension. Adjust the pH to 7.8, add 0.675g of starch branching enzyme (100u / g dry starch), and perform a first enzymatic hydrolysis reaction at 70℃ for 30min. Then, perform spray liquefaction at a spray temperature of 115℃ and maintain the temperature in a laminar flow tank at 97℃ for 70min. After that, flash evaporate to cool the material, and discharge it to 70℃ into a saccharification tank. Add 2.025g of starch branching enzyme (AaGBE) (300u / g dry starch) for a second enzymatic hydrolysis reaction and perform enzymatic hydrolysis at 70℃ for 16h. Then, adjust the temperature to 95℃ and maintain it for 30min to inactivate the enzyme. The enzymatic hydrolysate was filtered through a diatomaceous earth filter (400 mesh) to remove insoluble precipitates, yielding a supernatant. The supernatant was then decolorized with 0.4% activated carbon at 75°C for 35 min, followed by plate and frame filtration and ion exchange into an ion exchange column to obtain the ion exchange solution. The feed temperature was 52°C, and the feed flow rate was 8 m³ / s. 3 The pH value was 5.0, and the conductivity was 12 μS / cm. The ion exchange liquid was passed through a deodorization column for deodorization using macroporous adsorption resin at a deodorization temperature of 50℃. Then, it was concentrated by five-effect falling film evaporation at 65℃ and -0.085MPa to a solid content of 60%. Then, it was spray dried at an inlet air temperature of 160℃ and an outlet air temperature of 80℃ to obtain highly branched cyclodextrin with a DE value of 1.9.

[0051] 207.3 g of highly branched-chain cyclodextrin was added to 518.25 g of water (the mass of water was 2.5 times the mass of the highly branched-chain cyclodextrin), and stirred at 50°C for 15 min at 500 rpm. Then, 59.1 g of fructooligosaccharides and 33.6 g of aspartic acid were added, and stirring continued at 45°C for 25 min to obtain a mixture. The mixture was sheared at 5000 rpm for 10 min to obtain a pre-emulsion. The pre-emulsion was homogenized to obtain a final emulsion at a low pressure of 25 bar and a high pressure of 175 bar. The final emulsion was spray-dried at an inlet air temperature of 155°C and an outlet air temperature of 70°C to obtain the dry basis of the highly branched-chain cyclodextrin complex.

[0052] The above-mentioned complex components were dissolved in 450 ml of deionized water to prepare a 40 wt% protective solution. 250 ml of protein glutaminase (PG) enzyme solution (20 wt% solids, 500 U / mL enzyme activity) was added. The mixture was stirred in a water bath at 50°C for 15 min. The resulting mixture was sheared at medium speed for 10 min at a speed of 5000 rpm. The homogenization pressure was 25 bar for low pressure and 175 bar for high pressure. The mixture was homogenized twice and then spray-dried in a spray drying tower with an inlet air temperature of 155°C and an outlet air temperature of 70°C.

[0053] Example 2

[0054] Weigh 135g of waxy corn starch and add it to 450ml of water to prepare a 30% (w / v) suspension. Adjust the pH to 7.8, add 0.675g of starch branching enzyme (100u / g dry starch), and perform a first enzymatic hydrolysis reaction at 70℃ for 30min. Then, perform spray liquefaction at a spray temperature of 115℃ and maintain the temperature in a laminar flow tank at 97℃ for 70min. After that, flash evaporate to cool the material, and discharge it to 70℃ into a saccharification tank. Add 2.025g of starch branching enzyme (AaGBE) (300u / g dry starch) for a second enzymatic hydrolysis reaction and perform enzymatic hydrolysis at 70℃ for 16h. Then, adjust the temperature to 95℃ and maintain it for 30min to inactivate the enzyme. The enzymatic hydrolysate was filtered through a diatomaceous earth filter (400 mesh) to remove insoluble precipitates, yielding a supernatant. The supernatant was then decolorized with 0.4% activated carbon at 75°C for 35 min, followed by plate and frame filtration and ion exchange into an ion exchange column to obtain the ion exchange solution. The feed temperature was 52°C, and the feed flow rate was 8 m³ / s. 3 The pH value was 5.0, and the conductivity was 12 μS / cm. The ion exchange liquid was passed through a deodorization column for deodorization using macroporous adsorption resin at a deodorization temperature of 50℃. Then, it was concentrated by five-effect falling film evaporation at 65℃ and -0.085MPa to a solid content of 60%. Then, it was spray dried at an inlet air temperature of 160℃ and an outlet air temperature of 80℃ to obtain highly branched cyclodextrin with a DE value of 1.9.

[0055] 207.3 g of highly branched-chain cyclodextrin was added to 518.25 g of water (the mass of water was 2.5 times the mass of the highly branched-chain cyclodextrin), and stirred at 50°C for 15 min at 500 rpm. Then, 59.1 g of fructooligosaccharides and 33.6 g of aspartic acid were added, and stirring continued at 45°C for 25 min to obtain a mixture. The mixture was sheared at 5000 rpm for 10 min to obtain a pre-emulsion. The pre-emulsion was homogenized to obtain a final emulsion at a low pressure of 25 bar and a high pressure of 175 bar. The final emulsion was spray-dried at an inlet air temperature of 155°C and an outlet air temperature of 70°C to obtain the dry basis of the highly branched-chain cyclodextrin complex.

[0056] The above-mentioned complex components were dissolved in 500 ml of deionized water to prepare a 40 wt% protective solution. 250 ml of protein glutaminase (PG) enzyme solution (20 wt% solids, 500 U / mL enzyme activity) was added. The mixture was stirred in a water bath at 50 °C for 15 min. The resulting mixture was sheared at medium speed for 10 min at a speed of 5000 rpm. The homogenization pressure was 25 bar at low pressure and 175 bar at high pressure. The mixture was homogenized twice and then dried in a freeze dryer at -50 °C for 30 h.

[0057] Example 3

[0058] Weigh 135g of waxy corn starch and add it to 450ml of water to prepare a 30% (w / v) suspension. Adjust the pH to 7.8, add 0.675g of starch branching enzyme (100u / g dry starch), and perform a first enzymatic hydrolysis reaction at 70℃ for 30min. Then, perform spray liquefaction at a spray temperature of 115℃ and maintain the temperature in a laminar flow tank at 97℃ for 70min. After that, flash evaporate to cool the material, and discharge it to 70℃ into a saccharification tank. Add 2.025g of starch branching enzyme (AaGBE) (300u / g dry starch) for a second enzymatic hydrolysis reaction and perform enzymatic hydrolysis at 70℃ for 16h. Then, adjust the temperature to 95℃ and maintain it for 30min to inactivate the enzyme. The enzymatic hydrolysate was filtered through a diatomaceous earth filter (400 mesh) to remove insoluble precipitates, yielding a supernatant. The supernatant was then decolorized with 0.4% activated carbon at 75°C for 35 min, followed by plate and frame filtration and ion exchange into an ion exchange column to obtain the ion exchange solution. The feed temperature was 52°C, and the feed flow rate was 8 m³ / s. 3 The pH value was 5.0, and the conductivity was 12 μS / cm. The ion exchange liquid was passed through a deodorization column for deodorization using macroporous adsorption resin at a deodorization temperature of 50℃. Then, it was concentrated by five-effect falling film evaporation at 65℃ and -0.085MPa to a solid content of 60%. Then, it was spray dried at an inlet air temperature of 160℃ and an outlet air temperature of 80℃ to obtain highly branched cyclodextrin with a DE value of 1.9.

[0059] 207.3 g of highly branched cyclodextrin was added to 518.25 g of water (the mass of water was 2.5 times the mass of the highly branched cyclodextrin), and stirred at 50°C for 15 min at 500 rpm. Then, 59.1 g of galacto-oligosaccharides and 33.6 g of glycine were added, and stirring continued at 45°C for 25 min to obtain a mixture. The mixture was sheared at 5000 rpm for 10 min to obtain a pre-emulsion. The pre-emulsion was homogenized to obtain a final emulsion at a low pressure of 25 bar and a high pressure of 175 bar. The final emulsion was spray-dried at an inlet air temperature of 155°C and an outlet air temperature of 70°C to obtain the dry basis of the highly branched cyclodextrin complex.

[0060] The above-mentioned complex components were dissolved in 500 ml of deionized water to prepare a 40 wt% protective solution. 250 ml of protein glutaminase (PG) enzyme solution (20 wt% solids, 500 U / mL enzyme activity) was added. The mixture was stirred in a water bath at 50 °C for 15 min. The resulting mixture was sheared at medium speed for 10 min at a speed of 5000 rpm. The homogenization pressure was 25 bar for low pressure and 175 bar for high pressure. The mixture was homogenized twice and then spray-dried in a spray drying tower with an inlet air temperature of 155 °C and an outlet air temperature of 70 °C.

[0061] Example 4

[0062] Weigh 135g of waxy corn starch and add it to 450ml of water to prepare a 30% (w / v) suspension. Adjust the pH to 7.8, add 0.675g of starch branching enzyme (100u / g dry starch), and perform a first enzymatic hydrolysis reaction at 70℃ for 30min. Then, perform spray liquefaction at a spray temperature of 115℃ and maintain the temperature in a laminar flow tank at 97℃ for 70min. After that, flash evaporate to cool the material, and discharge it to 70℃ into a saccharification tank. Add 2.025g of starch branching enzyme (AaGBE) (300u / g dry starch) for a second enzymatic hydrolysis reaction and perform enzymatic hydrolysis at 70℃ for 16h. Then, adjust the temperature to 95℃ and maintain it for 30min to inactivate the enzyme. The enzymatic hydrolysate was filtered through a diatomaceous earth filter (400 mesh) to remove insoluble precipitates, yielding a supernatant. The supernatant was then decolorized with 0.4% activated carbon at 75°C for 35 min, followed by plate and frame filtration and ion exchange into an ion exchange column to obtain the ion exchange solution. The feed temperature was 52°C, and the feed flow rate was 8 m³ / s. 3 The pH value was 5.0, and the conductivity was 12 μS / cm. The ion exchange liquid was passed through a deodorization column for deodorization using macroporous adsorption resin at a deodorization temperature of 50℃. Then, it was concentrated by five-effect falling film evaporation at 65℃ and -0.085MPa to a solid content of 60%. Then, it was spray dried at an inlet air temperature of 160℃ and an outlet air temperature of 80℃ to obtain highly branched cyclodextrin with a DE value of 1.9.

[0063] 207.3 g of highly branched-chain cyclodextrin was added to 518.25 g of water (the mass of water was 2.5 times the mass of the highly branched-chain cyclodextrin), and stirred at 50°C for 15 min at 500 rpm. Then, 59.1 g of mannosaccharide and 33.6 g of lysine were added, and stirring continued at 45°C for 25 min to obtain a mixture. The mixture was sheared at 5000 rpm for 10 min to obtain a pre-emulsion. The pre-emulsion was homogenized to obtain a final emulsion at a low pressure of 25 bar and a high pressure of 175 bar. The final emulsion was spray-dried at an inlet air temperature of 155°C and an outlet air temperature of 70°C to obtain the dry basis of the highly branched-chain cyclodextrin complex.

[0064] The above-mentioned complex components were dissolved in 500 ml of deionized water to prepare a 40 wt% protective solution. 250 ml of protein glutaminase (PG) enzyme solution (20 wt% solids, 500 U / mL enzyme activity) was added. The mixture was stirred in a water bath at 50°C for 15 min. The resulting mixture was sheared at medium speed for 10 min at a speed of 5000 rpm. The homogenization pressure was 25 bar for low pressure and 175 bar for high pressure. The mixture was homogenized twice and then spray-dried in a spray drying tower with an inlet air temperature of 155°C and an outlet air temperature of 70°C.

[0065] Compare with Example 1

[0066] 207.3 g of highly branched cyclodextrin was added to 518.25 g of water (the mass of water was 2.5 times the mass of the highly branched cyclodextrin), and stirred at 50°C for 15 min at 500 rpm until homogeneous. Then, 59.1 g of fructooligosaccharides was added and stirring continued at 45°C for 25 min to obtain a mixture. The mixture was sheared at 5000 rpm for 10 min to obtain a pre-emulsion. The pre-emulsion was homogenized to obtain a final emulsion at a low pressure of 25 bar and a high pressure of 175 bar. The final emulsion was spray-dried at an inlet air temperature of 155°C and an outlet air temperature of 70°C to obtain the complex component.

[0067] The above-mentioned complex components were dissolved in 500 ml of deionized water to prepare a 40 wt% protective solution. 250 ml of protein glutaminase (PG) enzyme solution (20 wt% solids, 500 U / mL enzyme activity) was added. The mixture was stirred in a water bath at 50°C for 15 min. The resulting mixture was sheared at medium speed for 10 min at a speed of 5000 rpm. The homogenization pressure was 25 bar for low pressure and 175 bar for high pressure. The mixture was homogenized twice and then spray-dried in a spray drying tower with an inlet air temperature of 155°C and an outlet air temperature of 70°C.

[0068] Compare with Example 2

[0069] 207.3 g of highly branched cyclodextrin was added to 518.25 g of water (the mass of water was 2.5 times the mass of the highly branched cyclodextrin), and stirred at 50°C for 15 min at 500 rpm. Then, 33.6 g of aspartic acid was added and stirring continued at 45°C for 25 min to obtain a mixture. The mixture was sheared at 5000 rpm for 10 min to obtain a pre-emulsion. The pre-emulsion was homogenized to obtain a final emulsion at a low pressure of 25 bar and a high pressure of 175 bar. The final emulsion was spray-dried at an inlet air temperature of 155°C and an outlet air temperature of 70°C to obtain the complex component.

[0070] The above-mentioned complex components were dissolved in 500 ml of deionized water to prepare a 40 wt% protective solution. 250 ml of protein glutaminase (PG) enzyme solution (20 wt% solids, 500 U / mL enzyme activity) was added. The mixture was stirred in a water bath at 50°C for 15 min. The resulting mixture was sheared at medium speed for 10 min at a speed of 5000 rpm. The homogenization pressure was 25 bar for low pressure and 175 bar for high pressure. The mixture was homogenized twice and then spray-dried in a spray drying tower with an inlet air temperature of 155°C and an outlet air temperature of 70°C.

[0071] Compare with Example 3

[0072] 59.1 g of fructooligosaccharides and 33.6 g of aspartic acid were added to 518.25 g of water and mixed at 45°C for 25 min to obtain a mixture. The mixture was then sheared at 5000 rpm for 10 min to obtain a pre-emulsion. The pre-emulsion was homogenized to obtain a final emulsion at a low pressure of 25 bar and a high pressure of 175 bar. The final emulsion was then spray-dried at an inlet air temperature of 155°C and an outlet air temperature of 70°C to obtain the complex component.

[0073] The above-mentioned complex components were dissolved in 500 ml of deionized water to prepare a 40 wt% protective solution. 250 ml of protein glutaminase (PG) enzyme solution (20 wt% solids, 500 U / mL enzyme activity) was added. The mixture was stirred in a water bath at 50°C for 15 min. The resulting mixture was sheared at medium speed for 10 min at a speed of 5000 rpm. The homogenization pressure was 25 bar for low pressure and 175 bar for high pressure. The mixture was homogenized twice and then spray-dried in a spray drying tower with an inlet air temperature of 155°C and an outlet air temperature of 70°C.

[0074] Compare with Example 4

[0075] 207.3g of ordinary maltodextrin (DE15) was added to 518.25g of water (the mass of water was 2.5 times the mass of maltodextrin), and stirred at 50℃ for 15 minutes at 500 rpm. Then, 59.1g of fructooligosaccharides and 33.6g of aspartic acid were added, and stirring continued at 45℃ for 25 minutes to obtain a mixture. The mixture was sheared at 5000 rpm for 10 minutes to obtain a pre-emulsion. The pre-emulsion was homogenized to obtain a final emulsion at a low pressure of 25 bar and a high pressure of 175 bar. The final emulsion was spray-dried at an inlet air temperature of 155℃ and an outlet air temperature of 70℃ to obtain the complex component.

[0076] The above-mentioned complex components were dissolved in 500 ml of deionized water to prepare a 40 wt% protective solution. 250 ml of protein glutaminase (PG) enzyme solution (20 wt% solids, 500 U / mL enzyme activity) was added. The mixture was stirred in a water bath at 50°C for 15 min. The resulting mixture was sheared at medium speed for 10 min at a speed of 5000 rpm. The homogenization pressure was 25 bar for low pressure and 175 bar for high pressure. The mixture was homogenized twice and then spray-dried in a spray drying tower with an inlet air temperature of 155°C and an outlet air temperature of 70°C.

[0077] Compare with Example 5

[0078] 207.3 g of ordinary maltodextrin (DE15) was added to 518.25 g of water (the mass of water was 2.5 times the mass of maltodextrin), and stirred at 50°C for 15 min at 500 rpm until homogeneous. Then, 42.5 g of trehalose was added and stirring continued at 45°C for 25 min to obtain a mixture. The mixture was sheared at 5000 rpm for 10 min to obtain a pre-emulsion. The pre-emulsion was homogenized to obtain a final emulsion at a low pressure of 25 bar and a high pressure of 175 bar. The final emulsion was spray-dried at an inlet air temperature of 155°C and an outlet air temperature of 70°C to obtain the complex component.

[0079] The above-mentioned complex components were dissolved in 500 ml of deionized water to prepare a 40 wt% protective solution. 250 ml of protein glutaminase (PG) enzyme solution (20 wt% solids, 500 U / mL enzyme activity) was added. The mixture was stirred in a water bath at 50°C for 15 min. The resulting mixture was sheared at medium speed for 10 min at a speed of 5000 rpm. The homogenization pressure was 25 bar for low pressure and 175 bar for high pressure. The mixture was homogenized twice and then spray-dried in a spray drying tower with an inlet air temperature of 155°C and an outlet air temperature of 70°C.

[0080] Experimental Example 1 (one)

[0082] Measurement method: The present invention uses the "sucrose sandwich method" to determine the ice crystal area of ​​trehalose, maltodextrin and the highly branched cyclodextrin prepared in Example 1.

[0083] A 1 mg / mL polysaccharide solution and a 46% sucrose solution were mixed in a 1:1 (v / v) ratio, with a loading volume of 1 μL. The temperature control program for the hot and cold stages was set as follows: first, the temperature was lowered to 0°C at a rate of 10°C / min; then, it was lowered to -40°C at a rate of 80°C / min; finally, it was raised to -6°C at a rate of 80°C / min, and held at this temperature for 60 min. Microscopic images of ice crystals were acquired at 0 min, 15 min, 30 min, and 60 min. The average area of ​​the ice crystals at 60 min was calculated using ImageJ software. The polysaccharide adsorbs on the surface of the ice crystals, blocking the spontaneous process of Ostwald ripening—the dissolution of small ice crystals and the engulfment and growth of large ice crystals—is called polysaccharide recrystallization inhibition activity. The average area of ​​the ice crystals is a direct quantitative measure of polysaccharide recrystallization inhibition activity (IRI activity). The stronger the inhibition activity, the more effectively recrystallization is blocked, and the smaller the ice crystal area after annealing. Under the same conditions, the smaller the ice crystal size and the smaller the average area, the higher the recrystallization inhibition activity of the sample and the better the freeze-thaw stability of the product.

[0084] The results are as follows Figure 1 As shown in Figure A, the ice crystal area of ​​trehalose is 12.53 μm. 2 , Figure 1 The ice crystal area of ​​the maltodextrin shown in Figure B is 12.57 μm. 2 , Figure 1 The ice crystal area of ​​the highly branched cyclodextrin prepared in Example 1, shown in Figure C, is 10.23 μm. 2 The smallest ice crystal area indicates that the high-branched cyclodextrin prepared in this invention has the highest recrystallization inhibition activity and the best freeze-thaw resistance. (two)

[0086] The spray-dried samples of Examples 1-4 and Control Examples 1-5 were dissolved in deionized water until the concentration was the same, and then the enzyme activity was measured and the enzyme activity retention rate was calculated.

[0087] Retention rate of enzyme activity after drying = Total PG enzyme activity (U) of all powder after drying / Total PG enzyme activity (U) of spray-fed feed solution × 100%

[0088] The results showed that the enzyme activity retention rate of the sample prepared in Example 1 was 96.01%, the enzyme activity retention rate of the sample prepared in Example 2 was 96.87%, the enzyme activity retention rate of the sample prepared in Example 3 was 95.79%, the enzyme activity retention rate of the sample prepared in Example 4 was 95.65%, the enzyme activity retention rate of the sample prepared in Control Example 1 was 89.57%, the enzyme activity retention rate of the sample prepared in Control Example 2 was 88.49%, the enzyme activity retention rate of the sample prepared in Control Example 3 was 80.36%, the enzyme activity retention rate of the sample prepared in Control Example 4 was 79.12%, and the enzyme activity retention rate of the sample prepared in Control Example 5 was 76.24%. (three)

[0090] The spray-dried samples of Examples 1-4 and Control Examples 1-5 were subjected to accelerated testing at 40°C (accelerated conditions according to the ICH standard). The samples of each treatment group were placed in a constant temperature environment of 40°C and taken out at 30d, 60d and 90d respectively. After being dissolved in deionized water to the same concentration, enzyme activity was measured and the enzyme activity retention rate was calculated.

[0091] Table 1. Enzyme activity retention rates of different treatment groups in the accelerated experiment at 40℃.

[0092] As shown above, the high-branched cyclodextrin composition, due to the addition of a certain amount of high-branched cyclodextrin as a coating agent, encapsulates substances in a long chain with a helical structure, thus playing a certain barrier role. At the same time, it combines with oligosaccharides and amino acids to better protect enzyme molecules, resulting in a significantly higher enzyme activity retention rate for enzyme preparations encapsulated in the high-branched cyclodextrin composition compared to other groups.

[0093] In summary, the technical solution of this invention utilizes a low-DE value, highly branched polysaccharide obtained by two-stage enzymatic hydrolysis of starch with starch branching enzyme. This polysaccharide, along with oligosaccharides and amino acids, works synergistically to protect the three-dimensional structure of the enzyme protein in multiple dimensions. The highly branched cyclodextrin complex described in this invention can significantly improve the enzyme preparation's tolerance to high temperatures, freezing, and acid / alkali environments, while also greatly extending the shelf life of the enzyme preparation at room temperature. This solves the industry pain points of existing industrial enzyme preparations, such as easy high-temperature freezing inactivation, rapid activity decline during storage, and poor processing tolerance.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a highly branched cyclodextrin complex, characterized in that, Includes the following steps: (1) The waxy starch suspension was mixed with starch branching enzyme for enzymatic hydrolysis, and then filtered, decolorized, deodorized, concentrated and dried to obtain highly branched cyclodextrin. (2) After mixing the highly branched cyclodextrin with water, add oligosaccharides and amino acids, and stir to obtain a mixture; (3) The mixture is obtained by shearing, homogenizing and drying.

2. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis in step (1) includes a first enzymatic hydrolysis and a second enzymatic hydrolysis. The first enzymatic hydrolysis process is to mix a 20-40% (w / v) waxy starch suspension with a starch branching enzyme of 50-150 U / g dry starch and carry out a first enzymatic hydrolysis reaction to obtain hydrolysate 1. The second enzymatic hydrolysis process is to spray and liquefy hydrolysate 1, then mix it with steam for instantaneous heat preservation, cool it down, add a starch branching enzyme of 100-500 U / g dry starch and mix it to carry out a second enzymatic hydrolysis reaction.

3. The preparation method according to claim 1, characterized in that, The time for one enzymatic hydrolysis reaction is 20-40 min, and the temperature for one enzymatic hydrolysis reaction is 65-75℃; The time for the secondary enzymatic hydrolysis reaction is 12h-24h, and the temperature for the secondary enzymatic hydrolysis reaction is 65-75℃.

4. The preparation method according to claim 1, characterized in that, The waxy starch suspension has a mass-volume concentration of 20-40% and a pH value of 7.5-8.

5.

5. The preparation method according to claim 1, characterized in that, The decolorization time in step (1) is 25-45 min, and the decolorization temperature is 70-80℃; The concentration temperature is 60-70℃, and the concentration vacuum degree is -0.08~-0.09MPa; The inlet air temperature of the dryer is 150-170℃, and the outlet air temperature of the dryer is 75-85℃.

6. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of the high branched-chain cyclodextrin, oligosaccharide and amino acid is (55-75):(15-25):(5-15), and the mass ratio of the high branched-chain cyclodextrin to water is 1:(1.5-3.5).

7. The preparation method according to claim 6, characterized in that, The oligosaccharide is any one or more of raffinose, stachyose, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, galactooligosaccharide, manno-oligosaccharide, and inulin; The amino acid is any one or more of glutamic acid, aspartic acid, methionine, L-cysteine, lysine, glycine, proline, L-alanine, arginine, and methionine.

8. The preparation method according to claim 1, characterized in that, The shearing time in step (3) is 10-15 min, and the shearing speed is 4000-6000 r / min.

9. The highly branched cyclodextrin complex prepared by the method according to any one of claims 1-8, characterized in that, The highly branched cyclodextrin complex has a cyclic structure in which glucose is bonded to the non-reducing end of dextrin via an α-1,6 glycosidic bond, and the DE value of the highly branched cyclodextrin is 0.8-3.

10. The application of the highly branched cyclodextrin complex of claim 9 in improving the stability of enzyme preparations, characterized in that, The mass ratio of the highly branched cyclodextrin complex to the enzyme preparation to be treated is (2-10):1.

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