A licorice extract for resisting heat stress in beef cattle and its preparation method

Abstract

This invention provides a licorice extract for combating heat stress in beef cattle and its preparation method, belonging to the field of feed additives. The preparation method includes: adding a combination enzyme and buffer solution to licorice powder and mixing them evenly to form an enzymatic hydrolysis system; placing the enzymatic hydrolysis system in a high-pressure reactor and performing a pre-activation reaction at low temperature and medium pressure (100 MPa to 300 MPa) and a temperature of 5°C to 50°C, followed by a main enzymatic hydrolysis reaction with increased temperature and pressure; immediately after the main enzymatic hydrolysis reaction, releasing the pressure to atmospheric pressure, boiling to inactivate the enzyme, and centrifuging to collect the supernatant; concentrating and drying the supernatant to obtain licorice extract powder; the combination enzyme includes a basic complex enzyme and a synergistic enzyme, the basic complex enzyme including papain, cellulase, and pectinase; the synergistic enzyme including GUS enzyme and / or acetylated GUS enzyme. This invention, through the synergistic application of the complex enzyme system and high-pressure coupled enzymatic hydrolysis technology, significantly improves the extraction efficiency and bioactivity of licorice active ingredients, effectively enhancing its anti-heat stress performance in beef cattle.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, and specifically relates to a licorice extract for resisting heat stress in beef cattle and its preparation method. Background Technology

[0002] Heat stress is one of the main factors restricting the profitability of beef cattle farming in summer. High temperatures lead to decreased feed intake, increased respiratory rate, aggravated oxidative damage to the body, and impaired intestinal barrier function in beef cattle, ultimately resulting in decreased growth performance and poorer meat quality.

[0003] Licorice is the rhizome of licorice, a perennial plant of the legume family. It is sweet and neutral in nature and enters the twelve meridians. It has the effects of tonifying the middle and replenishing qi, relieving pain, clearing heat and detoxifying, moistening the lungs, relieving cough and resolving phlegm, and harmonizing the properties of other medicines. Modern pharmacology has confirmed that licorice contains glycyrrhizic acid, flavonoids and other effective components, and has pharmacological effects such as inhibiting inflammatory response and protecting the liver. In the existing technology, the preparation of licorice extract mostly adopts hot water extraction or ethanol reflux method, and the target product is mainly glycyrrhizic acid or total flavonoids. However, heat stress in beef cattle is a multi-system syndrome involving oxidative stress, inflammatory response, endocrine disorders and intestinal dysfunction. It is difficult to achieve comprehensive intervention with a single component. The existing extraction methods have the following common technical defects: (1) Low efficiency: Conventional extraction takes several hours and the cell wall is not completely broken, resulting in a limited dissolution rate of active ingredients. (2) Loss of activity: Long-term high-temperature heating easily leads to thermal degradation of flavonoids and polysaccharides.

[0004] To address the aforementioned issues, a stepwise enzymatic hydrolysis and alcohol extraction method can be employed. However, this method suffers from a long process chain, and the resulting licorice extract exhibits low bioavailability in combating heat stress in beef cattle. Therefore, developing a highly efficient, mild method for preparing licorice extract that can simultaneously enrich multiple target active ingredients is of significant value for its application in combating heat stress in beef cattle. Summary of the Invention

[0005] In view of this, the present invention provides a licorice extract for resisting heat stress in beef cattle and a method for preparing the same, aiming to solve at least one technical problem in the background art.

[0006] This invention is implemented as follows: The first aspect of this invention provides a method for preparing a licorice extract that combats heat stress in beef cattle, comprising the following steps: S1. Licorice raw material is pretreated to licorice powder; combined enzyme and buffer solution are added to licorice powder and mixed evenly to form an enzymatic hydrolysis system; S2. The enzymatic hydrolysis system is placed in a high-pressure reactor and subjected to a pre-activation at low temperature and medium pressure, followed by a main enzymatic hydrolysis reaction at high pressure of 100 MPa to 300 MPa and temperature of 5℃ to 50℃. S3. After the main enzymatic hydrolysis reaction is completed, immediately release the pressure to normal pressure, boil to inactivate the enzyme, and centrifuge to collect the supernatant. S4. The supernatant is concentrated and dried to obtain licorice extract powder; The combined enzyme comprises a basic complex enzyme and a synergistic enzyme. The basic complex enzyme comprises papain, cellulase, and pectinase. The synergistic enzyme comprises GUS enzyme and / or acetylated GUS enzyme.

[0007] Furthermore, according to the enzyme activity ratio, papain: cellulase: pectinase = (0.5~1):(1~2):1.

[0008] Furthermore, according to the enzyme activity ratio, the ratio of synergistic enzyme to basic complex enzyme is 1:10~50.

[0009] Furthermore, the method for preparing the acetylated GUS enzyme includes: Dissolve β-glucuronidase in phosphate buffer at pH 7.5–8.5 at a concentration of 5 mg / mL–10 mg / mL; Add acetic anhydride to a final concentration of 10mM~50mM, and react at 4℃~10℃ for 30min~90min; Unreacted acetic anhydride was removed by dialysis, and the reaction product was collected to obtain acetylated GUS enzyme.

[0010] Furthermore, the enhancing enzyme includes GUS enzyme and acetylated GUS enzyme, with an enzyme activity ratio of GUS enzyme:acetylated GUS enzyme = 1:0.5~2.

[0011] Furthermore, step S2 specifically includes: The enzymatic hydrolysis system was placed in a high-pressure reactor and pressurized to 100 MPa to 200 MPa at 5℃ to 10℃ and held at that pressure for 1 min to 5 min for low-temperature and high-pressure pre-activation. The pressure is then increased to 200 MPa to 300 MPa and heated to 40°C to 50°C, and held at that pressure for 15 to 30 minutes to carry out the main enzymatic hydrolysis reaction.

[0012] Furthermore, in step S3, the time taken to depressurize to atmospheric pressure is ≤15s.

[0013] Furthermore, in step S1, the solid-liquid ratio of licorice powder to buffer solution is 1 g: (10~20) mL; In step S1, the buffer solution used is either disodium hydrogen phosphate-citrate buffer or citrate-sodium citrate buffer, and the pH of the buffer solution is 4-6.

[0014] Furthermore, the S4 concentrate also includes: Adjust the pH of the concentrate to 8.0~9.0 and keep it at 40℃~50℃ for 10min~20min; Add chitosan solution to the concentrate to make the final concentration of chitosan in the mixture 2.5 mg / mL to 10 mg / mL, and stir until homogeneous; Licorice extract powder was obtained by spray drying.

[0015] A second aspect of the present invention provides a licorice extract for resisting heat stress in beef cattle prepared by the above-described preparation method.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention significantly improves the extraction efficiency and bioactivity of licorice active ingredients by synergistically applying a complex enzyme system (basic enzyme + acetylated GUS enhancer enzyme) and high-pressure coupled enzymatic hydrolysis technology (pre-activation + main enzymatic hydrolysis + instantaneous pressure relief), effectively enhancing its resistance to heat stress in beef cattle.

[0017] 2. The licorice extract prepared by this invention significantly improves the daily weight gain of beef cattle, reduces the diarrhea rate, improves the digestibility of nutrients, enhances antioxidant capacity, improves immune function, and optimizes rumen fermentation function.

[0018] 3. The preparation method of the present invention introduces acetylated GUS enzyme (acetylated β-glucuronidase) as a synergistic enzyme on the basis of a basic complex enzyme composed of papain, cellulase and pectinase, which significantly improves the release efficiency of licorice active ingredients.

[0019] 4. The preparation method of the present invention adopts high-pressure coupled enzymatic hydrolysis technology, which maximizes the enzymatic hydrolysis efficiency and the retention of active ingredients through a three-stage process of low-temperature medium-pressure pre-activation + main enzymatic hydrolysis reaction + instantaneous pressure relief.

[0020] 5. The preparation method of the present invention further employs chitosan-citric acid solution purification technology to improve the purity and bioactivity of the extract. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] A method for preparing a licorice extract that combats heat stress in beef cattle, comprising the following steps: S1. Licorice raw material pretreatment is licorice powder (passed through a 20-40 mesh sieve); combined enzyme and buffer solution are added to licorice powder and mixed evenly to form an enzymatic hydrolysis system; The buffer solution is a disodium hydrogen phosphate-citric acid buffer or a citrate-sodium citrate buffer, with a pH of 4-6. The solid-liquid ratio of licorice powder to buffer solution is 1 g: (10-20) mL. The following examples use 1:15 as an example, but are not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0023] The combined enzyme includes a basic complex enzyme and a synergistic enzyme, with the synergistic enzyme: basic complex enzyme ratio being 1:10~50 according to the enzyme activity ratio. The basic complex enzymes include papain, cellulase, and pectinase; according to the enzyme activity ratio, papain: cellulase: pectinase = (0.5~1):(1~2):1; in specific implementation, licorice is rich in cellulose, which is the main supporting structure of the cell wall. Cellulase can destroy the network structure composed of cellulose, causing cracks in the cell wall, opening channels for other enzymes and solvents, allowing glycyrrhizic acid, flavonoids, etc. encapsulated in the cell to be released; pectinase is used to decompose the pectin substances that connect cells, making the tissue loose and the cells dispersed, reducing the viscosity of the extraction system, promoting solvent penetration and diffusion of active ingredients into the solvent; papain is used to hydrolyze proteins that are bound to target components (such as flavonoids and glycosides) as well as storage proteins in cells.

[0024] In the subsequent enzymatic hydrolysis process, the basic complex enzyme can promote the precipitation of medicinal components in licorice (mainly glycyrrhizic acid, with glycyrrhizin flavonoids and other components as auxiliary components), but the bioavailability needs to be improved, especially for glycyrrhizic acid. To solve this technical problem, a synergist enzyme is added during enzymatic hydrolysis. The synergist enzymes are GUS enzyme (β-glucuronidase) and / or acetylated GUS enzyme, preferably GUS enzyme and acetylated GUS enzyme. According to the enzyme activity ratio, GUS enzyme:acetylated GUS enzyme = 1:0.5~2. The following examples use an approximate ratio of 1:1 for illustration, but are not limited to the listed values. Other unlisted values ​​within the range are also applicable. Both GUS enzyme and acetylated GUS enzyme can promote the conversion of large molecule glycyrrhizic acid into small molecules and improve the utilization rate. Among them, acetylated GUS enzyme is a mutant of GUS enzyme, a functionally enhanced enzyme obtained through structural modification. Its stability, substrate selectivity and catalytic efficiency are all superior to GUS enzyme. Furthermore, GUS enzyme and acetylated GUS enzyme form a synergistic system. When working together, GUS enzyme preferentially acts on the initial high concentration of glycyrrhizic acid (GL) to generate the intermediate product GAMG; acetylated GUS enzyme has a higher affinity for GAMG, deeply hydrolyzing it into highly active glycyrrhetinic acid (GA). Together, they achieve the GL→GAMG→GA conversion chain, significantly increasing the GA content in the extract. Simultaneously, acetylation modification enhances the enzyme's tolerance to acidic pH and high-pressure environments, allowing the two enzymes to fully exert their synergistic effect under the extraction process conditions of this invention.

[0025] The method for preparing the acetylated GUS enzyme includes: dissolving β-glucuronidase in phosphate buffer at pH 7.5-8.5 at a concentration of 5 mg / mL-10 mg / mL; adding acetic anhydride to a final concentration of 10 mM-50 mM; reacting at 4℃-10℃ for 30 min-90 min; removing unreacted acetic anhydride by dialysis; collecting the reaction product to obtain the acetylated GUS enzyme.

[0026] S2. The enzymatic hydrolysis system is placed in a high-pressure reactor and subjected to a pre-activation at low temperature and medium pressure, followed by a main enzymatic hydrolysis reaction at high pressure of 100 MPa to 300 MPa and temperature of 5℃ to 50℃. Enzymatic hydrolysis offers advantages such as mild conditions, high safety, and optimized environment, but it also suffers from drawbacks such as low extraction efficiency and long processing time. These drawbacks are the result of factors such as decreased substrate concentration, enzyme inactivation, and product inhibition. To address these issues, this invention places the enzymatic hydrolysis reaction in a specific environment. Studies have shown that most enzymes exhibit an initial increase followed by a decrease in activity under certain pressure conditions. However, different enzymes have different activity windows corresponding to different conditions. This application utilizes a combined process of low-temperature, medium-high pressure pre-activation followed by high-temperature, high-pressure enzymatic hydrolysis to achieve highly efficient combined enzyme activity.

[0027] Specifically, it includes: S21. The enzymatic hydrolysis system is placed in a high-pressure reactor and pressurized to 100 MPa to 200 MPa at 5℃ to 10℃, and held at that pressure for 1 min to 5 min for pre-activation. The following example uses a pressurization of 150 MPa at 5℃ and a holding pressure for 5 min as an illustration, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0028] Before the main enzymatic hydrolysis reaction, the enzymatic hydrolysis system is pretreated under low temperature and high pressure. High pressure induces reversible conformational changes in enzyme molecules, including exposing the active site, optimizing subunit assembly, and increasing substrate affinity, transforming the enzyme from a low-activity latent state to a high-activity activated state, thereby significantly improving the efficiency of subsequent enzymatic hydrolysis reactions and the conversion rate of the target product. Specifically, this includes: (1) Low temperature and high pressure induce reversible conformational pre-expansion of enzyme molecules, making the active site more accessible to the substrate; (2) At low temperature, the activation volume of the enzyme-substrate complex changes from negative to positive, and applying pressure can reduce the activation energy of the catalytic reaction; (3) The pre-activation stage completes the stable assembly of the enzyme-substrate complex, and the main reaction stage directly enters the efficient catalytic cycle; (4) The multi-enzyme system is activated synchronously to avoid the problem of asynchronous activity of different enzymes in the main reaction stage.

[0029] S22. The pressure is then increased to 200 MPa to 300 MPa (preferably 220 MPa to 280 MPa) and heated to 40°C to 50°C, holding at that pressure for 15 to 30 minutes to carry out the main enzymatic hydrolysis reaction. The following examples illustrate this by increasing the pressure to 250 MPa and heating to 45°C, holding at that pressure for 25 minutes. However, the values ​​listed are not limited to these values, and other unlisted values ​​within the same range are also applicable.

[0030] The main enzymatic hydrolysis step is the core execution unit of enzymatic hydrolysis. 200-300 MPa is the peak activity window for complex enzymes such as cellulase, pectinase, and GUS enzyme, optimizing catalytic efficiency and maximizing enzyme activity. In addition, high pressure lowers the activation energy barrier by changing the activation volume, while medium temperature of 40-50℃ provides molecular kinetic energy, doubly promoting the reaction rate. The enzymatic hydrolysis and the mechanical disruption under high pressure are carried out simultaneously, achieving deep disintegration of licorice cells. While releasing glycyrrhizic acid, GUS enzyme / acetylated GUS enzyme immediately converts it into highly active glycyrrhetinic acid, avoiding the accumulation of intermediate products.

[0031] S3. After the main enzymatic hydrolysis reaction is completed, immediately release the pressure to normal pressure, boil to inactivate the enzyme, and centrifuge to collect the supernatant. In practice, after enzymatic hydrolysis, the pressure is rapidly released to atmospheric pressure (≤15s, preferably ≤10s). This sudden pressure change drives multiple physical effects: the cavitation effect generated by the sudden release of dissolved gases secondary breaks down cell residues; the impact effect created by the osmotic pressure difference between the inside and outside of cells forcibly elutes retained components; and the explosive disintegration caused by the instantaneous vaporization and expansion of the solvent that has penetrated into the cells completely disintegrates the partially degraded cell walls. These three factors work synergistically to further improve the yield of the target components and significantly improve the clarity of the extract without increasing additional energy consumption or time, while ensuring no thermal damage throughout the process and fully protecting the heat-sensitive active ingredients.

[0032] S4. The supernatant is concentrated and dried to obtain licorice extract powder; In practice, licorice extract obtained directly from the supernatant exhibits advantages such as anti-inflammatory, antibacterial, and growth-regulating effects, demonstrating good efficacy against heat stress in beef cattle. However, research has revealed that heat stress in beef cattle is a multi-system syndrome involving inflammation, oxidative damage, and immunosuppression. This licorice extract is relatively weak in terms of antioxidant and intestinal protection; furthermore, it is not adequately adapted to rumination. When directly added to feed, some effective components (such as flavonoids) are easily degraded by microorganisms in the rumen, resulting in unstable effective doses reaching the small intestine. Therefore, its efficacy against heat stress in beef cattle can be further enhanced.

[0033] To further enhance the anti-heat stress efficacy of licorice extract in beef cattle, the present invention further optimizes step S4, which is specifically optimized as follows: S40. The supernatant is concentrated under reduced pressure to obtain a concentrated solution; S41. Adjust the pH of the concentrate to 8.0-9.0 and incubate at 40℃-50℃ for 10-20 minutes. This step is a pH-driven conversion, which expands the conjugated system of the compound, enhances the hydrogen donor capacity of the phenolic hydroxyl group, and significantly improves the antioxidant activity compared to before conversion. Simultaneously, the alkaline environment creates optimal conditions for subsequent chitosan self-assembly. The following example uses sodium bicarbonate to adjust the pH of the concentrate to 8.5 and incubate at 45℃ for 15 minutes as an illustration, but is not limited to the listed values; other unlisted values ​​within the range are also applicable.

[0034] S42. Add chitosan solution to the concentrate to make the final concentration of chitosan in the mixture 2.5 mg / mL~10 mg / mL, and stir evenly; wherein the chitosan solution is a chitosan-citric acid solution formed by dissolving chitosan in citric acid; under alkaline conditions, chitosan undergoes deprotonation, and hydrophobic interactions drive its self-assembly to form regular nanoparticles. The chitosan nano-encapsulation formed by chitosan self-assembly has pH-responsive release characteristics, is stable in the neutral environment of the rumen, and is released in the acidic environment of the abomasum, realizing the rumen-specific targeted delivery unique to ruminants, thereby simultaneously solving the stability and delivery problems of licorice extract and effectively improving bioavailability.

[0035] S43. Licorice extract powder is obtained by spray drying.

[0036] Example 1 A method for preparing a licorice extract that combats heat stress in beef cattle, comprising the following steps: S1. Licorice raw material is dried, crushed, and passed through a 20-mesh sieve to obtain licorice powder. 1500 mL of disodium hydrogen phosphate-citric acid buffer (pH=5) is added to 100 g of licorice powder, along with a basic complex enzyme consisting of 0.03 g of papain (800,000 U / g), 0.45 g of cellulase (100,000 U / g), and 0.62 g of pectinase (50,000 U / g), and an enhancing enzyme—acetylated GUS enzyme (acetylated β-glucuronidase, 180,000 U / g)—mixed thoroughly to form an enzymatic hydrolysis system (enzyme activity ratio of enhancing enzyme to basic complex enzyme ≈ 1:10). The preparation steps for acetylated GUS enzyme are as follows: β-glucuronidase is dissolved in a phosphate buffer at pH=8 to a concentration of 7 mg / mL; acetic anhydride is added to a final concentration of 30 mM, and the reaction is carried out at 4℃~10℃ for 60 min; unreacted acetic anhydride is removed by dialysis, and the reaction product is collected to obtain acetylated GUS enzyme.

[0037] S2. The enzymatic hydrolysis system is placed in a high-pressure reactor and pressurized to 150 MPa at 5°C and held for 5 minutes for pre-activation; then pressurized to 250 MPa and heated to 45°C and held for 25 minutes for the main enzymatic hydrolysis reaction. S3. After the main enzymatic hydrolysis reaction is completed, immediately release the pressure to normal pressure within 10 seconds, boil for about 5 minutes to inactivate the enzyme, and centrifuge to collect the supernatant. S4. The supernatant was concentrated under reduced pressure to 1 / 10 of its original volume at about 50°C, and then freeze-dried to obtain licorice extract powder.

[0038] Example 2 The difference between this embodiment and Example 1 is that the acetylated GUS enzyme in S1 is replaced with a GUS enzyme of equal activity (β-glucuronidase, 200,000 U / g, 0.025g); all other conditions and steps are the same as in Example 1.

[0039] Example 3 The difference between this embodiment and Example 1 is that the acetylated GUS enzyme in S1 is replaced with a mixed enzyme of GUS enzyme and acetylated GUS enzyme, wherein GUS enzyme (200,000 U / g) is 0.013 g and acetylated GUS enzyme (180,000 U / g) is 0.014 g; other conditions and steps are the same as in Example 1.

[0040] Example 4 The difference between this embodiment and Example 1 is that step S4 is modified as follows: the supernatant is concentrated under reduced pressure at approximately 50°C to obtain a concentrated solution; the pH of the concentrated solution is adjusted to 8.5 and kept at 45°C for 15 minutes; a chitosan-citric acid solution is added to the concentrated solution to achieve a final chitosan concentration of 5 mg / mL in the mixture, and the solution is stirred until homogeneous; finally, the solution is spray-dried to obtain licorice extract powder. All other conditions and steps in this embodiment are the same as in Example 1.

[0041] Example 5 The only difference between this embodiment and Example 1 is that the activity of the combined enzyme in S1 is reduced; all other steps and conditions are the same as in Example 1.

[0042] S1 specifically involves: drying and crushing licorice raw materials, and passing them through a 20-mesh sieve to obtain licorice powder; adding 1500mL of disodium hydrogen phosphate-citric acid buffer (pH=5) to 100g of licorice powder, and adding a basic complex enzyme consisting of 0.02g of papain (800,000 U / g), 0.32g of cellulase (100,000 U / g), and 0.64g of pectinase (50,000 U / g), as well as 0.022g of acetylated GUS enzyme (acetylated β-glucuronidase, 180,000 U / g), and mixing them evenly to form an enzymatic hydrolysis system.

[0043] Example 6 The only difference between this embodiment and Example 1 is that the activity of the combined enzyme in S1 is increased; all other steps and conditions are the same as in Example 1.

[0044] S1 specifically involves: drying and crushing licorice raw materials, and passing them through a 20-mesh sieve to obtain licorice powder; adding 1500mL of disodium hydrogen phosphate-citric acid buffer (pH=5) to 100g of licorice powder, and adding a basic complex enzyme consisting of 0.038g of papain (800,000 U / g), 0.6g of cellulase (100,000 U / g), and 0.6g of pectinase (50,000 U / g), as well as 0.033g of acetylated GUS enzyme (acetylated β-glucuronidase, 180,000 U / g), and mixing evenly to form an enzymatic hydrolysis system.

[0045] Example 7 The difference between this embodiment and Example 1 is only that the activity of the synergist-acetylated GUS enzyme in S1 is increased, so that the activity ratio of the synergist to the basic complex enzyme is ≈1:10. All other steps and conditions are the same as in Example 1.

[0046] S1 specifically involves: drying and crushing licorice raw materials, and passing them through a 20-mesh sieve to obtain licorice powder; adding 1500mL of disodium hydrogen phosphate-citric acid buffer (pH=5) to 100g of licorice powder, and adding a basic complex enzyme consisting of 0.03g of papain (800,000 U / g), 0.45g of cellulase (100,000 U / g), and 0.62g of pectinase (50,000 U / g), as well as 0.056g of acetylated GUS enzyme (acetylated β-glucuronidase, 180,000 U / g), and mixing them evenly to form an enzymatic hydrolysis system.

[0047] Example 8 The difference between this embodiment and Example 1 is only that the activity of the synergist-acetylated GUS enzyme in S1 is reduced, so that the activity ratio of the synergist to the basic complex enzyme is ≈1:50. All other steps and conditions are the same as in Example 1.

[0048] S1 specifically involves: drying and crushing licorice raw materials, and passing them through a 20-mesh sieve to obtain licorice powder; adding 1500mL of disodium hydrogen phosphate-citric acid buffer (pH=5) to 100g of licorice powder, and adding a basic complex enzyme consisting of 0.03g of papain (800,000 U / g), 0.45g of cellulase (100,000 U / g), and 0.62g of pectinase (50,000 U / g), as well as 0.012g of acetylated GUS enzyme (acetylated β-glucuronidase, 180,000 U / g), and mixing them evenly to form an enzymatic hydrolysis system.

[0049] Comparative Example 1 The difference between this comparative example and Example 1 is that the synergist in S1 is deleted, while the other conditions and steps are the same as in Example 1.

[0050] Comparative Example 2 The difference between this comparative example and Example 1 is that the pre-activation step in S2 is deleted, while the other conditions and steps are the same as in Example 1.

[0051] Comparative Example 3 The difference between this comparative example and Example 1 is that the pressure in the enzymatic hydrolysis main reaction step in S2 is increased to 350 MPa, while the other conditions and steps are the same as in Example 1.

[0052] Comparative Example 4 The difference between this comparative example and Example 1 is that the pressure in the enzymatic hydrolysis main reaction step in S2 is reduced to 150 MPa, while the other conditions and steps are the same as in Example 1.

[0053] Comparative Example 5 The difference between this comparative example and Example 1 is that the time taken to depressurize to atmospheric pressure in step S3 is 1 minute, while the other conditions and steps are the same as in Example 1.

[0054] Example 9 In this embodiment, the licorice extracts prepared in Examples 1 to 7 and Comparative Examples 1 to 5 were used as additives and added to the concentrated feed in proportion (components are shown in Table 1 below) for heat stress resistance testing. A blank control group and a conventional glycyrrhizic acid group were used as additives for comparison.

[0055] Bulls weighing 300-400 kg and aged 10-12 months (with a THI index greater than 74) were selected as experimental beef cattle. They were randomly divided into groups, with 3 replicates per treatment group and 5 cattle per replicate. The experiment lasted for 40 days (based on the measured temperature and humidity, the THI index was calculated to be above 74, indicating that the experimental beef cattle were under heat stress).

[0056] The feeding and management procedures followed the standard requirements of the experimental cattle farm. Feeding was conducted twice daily at 8:00 and 16:00. The dosage of feed additives was 2 kg / ton of concentrate, and the feed intake was 1.5% of the experimental beef cattle's body weight. The roughage was wheat straw, and cattle had free access to feed and water. Water troughs were cleaned regularly, and the normal immunization program was carried out.

[0057] Table 1

[0058] Note: The premix provides the following per kilogram of concentrate: VA 30,000,000 IU, VD3 1,000,000 IU, VE 80,000 IU, VK3 10g, VB1 10g, VB2 25g, Cu 0.04g, Fe 0.17g, Zn 0.088g, Mn 0.064g, Co 0.02g, I 0.03g.

[0059] (1) Effects on the production performance of heat-stressed beef cattle The production performance of each group of beef cattle was tested, and the indicators included: daily weight gain and diarrhea rate. Daily weight gain = (final weight - initial weight) / number of days in the experiment. The feces of each cow are observed at a fixed time each day (e.g., 8:00-9:00 AM). Diarrhea is usually graded based on the characteristics of the feces. The commonly used scoring criteria are as follows: (1) 0 points, normal, formed, hard or soft strips; (2) 1 point, mild diarrhea, loose, slightly formed feces; (3) 2 points, moderate diarrhea, loose, unformed feces, but without water separation; (4) 3 points, severe diarrhea, watery feces, or with mucus or blood. Individuals with a score ≥2 points are considered to have diarrhea.

[0060] Diarrhea rate (%) = (Number of cattle with diarrhea during the trial period × Number of days with diarrhea) ÷ (Total number of cattle in the trial × Total number of days in the trial) × 100%.

[0061] Table 2

[0062] (2) Effects on nutrient digestibility in heat-stressed beef cattle After the feeding trial, five cattle were randomly selected from each group for a digestive and metabolic experiment. At the start of the experiment, feces were collected continuously for three days, mixed thoroughly, and then sampled at a rate of 5%. Feces from the three consecutive days were sampled and mixed using a weighted average method. The feces were divided into two portions: one portion was dried at 65°C, and its initial moisture content was determined to prepare an air-dried sample for the determination of other components; the other portion was dried at 65°C using 10% sulfuric acid (20 mL sulfuric acid per 100 g feces sample), then pulverized and passed through a 40-mesh sieve for storage and nitrogen analysis. The determination methods were based on the book "Feed Analysis and Feed Quality Testing Technology". The contents of crude protein (CP), crude fat (EE), neutral detergent fiber (NDF), and acid detergent fiber (ADF) in the diet and feces were determined. The apparent digestibility of nutrients in the experimental cattle was determined using an endogenous indicator (acid-insoluble ash method, AIA). The results are shown in Table 3.

[0063] Table 3

[0064] (3) Effects on blood biochemical parameters of heat-stressed beef cattle On the last day of the feeding trial, 20 mL of blood was collected from the jugular vein on an empty stomach. Two tubes were collected using anticoagulant tubing and centrifuged at 3500 rpm for 10 min to separate the serum, which was then placed into 0.5 EP tubes for later testing. Blood biochemical indicators, including serum antioxidant and serum immune indicators, are shown in Tables 4 and 5 below.

[0065] Serum antioxidant markers: The levels of superoxide dismutase (SOD), glutathione reductase (GSH-Px), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) in serum were measured using a kit method. Serum immune markers: The concentrations of interleukin-4 (IL-4), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA); the concentrations of immunoglobulin M (IgM) and immunoglobulin G (IgG) were measured by immunoturbidimetric assay.

[0066] Table 4

[0067] Table 5

[0068] (4) Effects on rumen fermentation function in heat-stressed beef cattle Rumen fluid collection and measurement: On the last day of the experiment, rumen fluid was collected promptly through the mouth using a specialized rumen fluid collection device. Sufficient rumen fluid was collected from each cow and poured into a 1000mL beaker, mixed thoroughly, and filtered through four layers of gauze into another 500mL beaker. 50mL of the filtered rumen fluid was placed in a 100mL beaker, mixed thoroughly, and dispensed into 2mL yellow-capped enzyme-free cryovials (6 tubes, pre-labeled), and stored in liquid nitrogen. The pH of the remaining rumen fluid from step one was measured three times, and the average value was taken. 80mL of the filtered rumen fluid was mixed with 20mL of 25% metaphosphate, dispensed into three 10mL LEP tubes (9-10mL per tube), and stored in liquid nitrogen for VFA determination. The filtered rumen fluid was then aliquoted into four 10 mL EP tubes for measuring ammonia nitrogen (NH3-N) content and total microbial protein (MCP), and stored in liquid nitrogen. NH3-N was determined using the phenol-sodium hypochlorite colorimetric method, MCP concentration was determined using a kit provided by Nanjing Jiancheng Biotechnology Co., Ltd., and VFA concentration was determined using a high-performance gas chromatograph, as shown in Table 6.

[0069] Table 6

[0070] The data in Tables 2 to 6 show that the licorice extract prepared in the embodiments of the present invention has a good promoting effect on the production performance, apparent nutrient digestibility, serum antioxidant and immune properties, and rumen fermentation capacity of heat-stressed beef cattle, thus achieving anti-heat stress.

[0071] During the trial period, the control group without any additives showed the weakest production performance, apparent nutrient digestibility, serum antioxidant and immune properties, and rumen fermentation capacity in heat-stressed beef cattle. The conventional glycyrrhizic acid group, the embodiments of the present invention, and the comparative examples all showed significant improvements in production performance, apparent nutrient digestibility, serum antioxidant and immune properties, and rumen fermentation capacity in heat-stressed beef cattle. This indicates that both licorice extract and glycyrrhizic acid reagent can effectively improve heat stress resistance.

[0072] A comparison of Examples 1 to 3 shows that, in terms of heat stress resistance, Example 3 > Example 1 > Example 2, indicating that the synergistic effect of the combined enzymes on the performance of licorice extract is: GUS enzyme + acetylated GUS enzyme > acetylated GUS enzyme > GUS enzyme. Comparative Example 1, which removed the synergistic enzyme, showed a significantly reduced heat stress resistance compared to Example 1, comparable to the conventional glycyrrhizic acid group. This is because, although the active ingredients in licorice can be effectively extracted without the synergistic enzyme, its bioavailability is far less than that of Example 1.

[0073] Comparing Example 1 and Example 4, it can be seen that Example 4 further optimizes the heat stress resistance by adding pH conversion and chitosan coating after enzymatic extraction.

[0074] A comparison of Examples 1 with Examples 5 to 8 shows that increasing the enzyme activity of the combined enzyme or the synergist within a certain range can enhance the anti-heat stress effect.

[0075] Comparing Comparative Example 2 with Example 1, it can be seen that the removal of the pre-activation step in enzymatic hydrolysis reduces the production performance of heat-stressed beef cattle. The reason is that without pre-activation, some enzymes in the combined enzyme cannot fully exert their enzymatic hydrolysis performance within a limited time, resulting in a reduced extraction rate of effective components, and thus limiting the heat stress resistance effect of beef cattle.

[0076] Comparing Comparative Examples 3 and 4 with Example 1, it can be seen that excessively high or low pressure during the enzymatic hydrolysis reaction leads to a decrease in the content of active ingredients in licorice extract and insufficient improvement in the heat stress resistance of beef cattle. The reasons are as follows: excessively high pressure causes the structure of some enzyme proteins to be destroyed and inactivated, resulting in a decrease in enzymatic hydrolysis efficiency; low pressure is insufficient to fully activate the enzyme system, and the main enzymatic hydrolysis time is too short, resulting in incomplete enzymatic hydrolysis and insufficient release of effective ingredients.

[0077] Comparing Comparative Example 5 with Example 1, it can be seen that the slow depressurization resulted in a lower content of active ingredients in the licorice extract compared to Example 1, but it was still better than other comparative examples. The enzymatic hydrolysis reaction decomposed and released most of the active ingredients, and the rapid depressurization was an auxiliary method that allowed the residual ingredients to be extracted.

[0078] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing a licorice extract that combats heat stress in beef cattle, characterized in that, The preparation method includes the following steps: S1. Licorice raw material is pretreated to licorice powder; combined enzyme and buffer solution are added to licorice powder and mixed evenly to form an enzymatic hydrolysis system; S2. The enzymatic hydrolysis system is placed in a high-pressure reactor and subjected to a pre-activation at low temperature and medium pressure, followed by a main enzymatic hydrolysis reaction at high pressure of 100 MPa to 300 MPa and temperature of 5℃ to 50℃. S3. After the main enzymatic hydrolysis reaction is completed, immediately release the pressure to normal pressure, boil to inactivate the enzyme, and centrifuge to collect the supernatant. S4. The supernatant is concentrated and dried to obtain licorice extract powder; The combined enzyme comprises a basic complex enzyme and a synergistic enzyme. The basic complex enzyme comprises papain, cellulase, and pectinase. The synergistic enzyme comprises GUS enzyme and / or acetylated GUS enzyme.

2. The method for preparing a licorice extract for resisting heat stress in beef cattle according to claim 1, characterized in that, According to the enzyme activity ratio, papain: cellulase: pectinase = (0.5~1):(1~2):

1.

3. The method for preparing a licorice extract for resisting heat stress in beef cattle according to claim 2, characterized in that, According to the enzyme activity ratio, the ratio of synergistic enzyme to basic complex enzyme is 1:10~50.

4. The method for preparing a licorice extract for resisting heat stress in beef cattle according to claim 1, characterized in that, The method for preparing the acetylated GUS enzyme includes: Dissolve β-glucuronidase in phosphate buffer at pH 7.5–8.5 at a concentration of 5 mg / mL–10 mg / mL; Add acetic anhydride to a final concentration of 10mM~50mM, and react at 4℃~10℃ for 30min~90min; Unreacted acetic anhydride was removed by dialysis, and the reaction product was collected to obtain acetylated GUS enzyme.

5. The method for preparing a licorice extract for resisting heat stress in beef cattle according to claim 4, characterized in that, The enhancing enzymes include GUS enzyme and acetylated GUS enzyme, with an enzyme activity ratio of GUS enzyme:acetylated GUS enzyme = 1:0.5~2.

6. The method for preparing a licorice extract for resisting heat stress in beef cattle according to claim 1, characterized in that, The steps in S2 specifically include: The enzymatic hydrolysis system was placed in a high-pressure reactor and pressurized to 100 MPa to 200 MPa at 5℃ to 10℃ and held at that pressure for 1 min to 5 min for low-temperature and high-pressure pre-activation. The pressure is then increased to 200 MPa to 300 MPa and heated to 40°C to 50°C, and held at that pressure for 15 to 30 minutes to carry out the main enzymatic hydrolysis reaction.

7. The method for preparing a licorice extract for resisting heat stress in beef cattle according to claim 1, characterized in that, In step S3, the time to depressurize to atmospheric pressure is ≤15s.

8. The method for preparing a licorice extract for resisting heat stress in beef cattle according to claim 1, characterized in that, In step S1, the solid-liquid ratio of licorice powder and buffer solution is 1g:(10~20)mL; In step S1, the buffer solution used is either disodium hydrogen phosphate-citrate buffer or citrate-sodium citrate buffer, and the pH of the buffer solution is 4-6.

9. A method for preparing a licorice extract for resisting heat stress in beef cattle according to claim 1, characterized in that, The S4 concentrate also includes: Adjust the pH of the concentrate to 8.0~9.0 and keep it at 40℃~50℃ for 10min~20min; Add chitosan solution to the concentrate to make the final concentration of chitosan in the mixture 2.5 mg / mL to 10 mg / mL, and stir until homogeneous; Licorice extract powder was obtained by spray drying.

10. The licorice extract for resisting heat stress in beef cattle prepared by the method according to any one of claims 1 to 9.

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