Method for preparing sheep milk cake by using smilax sieboldi coagulase and application thereof

Abstract

The application provides a method for preparing sheep milk cake by using chrysobalanus icaho rennet, which comprises the following steps: extracting rennet from chrysobalanus icaho stems by using resonant sound waves combined with high-osmotic and low-osmotic solution alternation treatment, esterifying terpene alcohol ester by using palmitic acid and alpha-terpineol through lipase catalysis, and fixing the rennet by using a composite carrier composed of sodium alginate and the terpene alcohol ester; obtaining fresh sheep milk, adding calcium chloride and the immobilized chrysobalanus icaho rennet liquid after pasteurization, and obtaining sheep milk cake through milk clotting, whey separation, compression molding and curing. The method uses mild mechanical stimulation of resonant sound waves combined with high-osmotic and low-osmotic solution alternation treatment to significantly promote rennet dissolution and improve extraction efficiency, the immobilized carrier realizes efficient enzyme fixation through hydrophobic interaction, electrostatic attraction and the like, and significantly improves milk clotting efficiency and product stability; the prepared sheep milk cake has good antioxidant activity and ACE inhibition activity, and is beneficial to the development of functional dairy products.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method and application of preparing sheep milk cake using chymosinensis rennet. Background Technology

[0002] Oxidative stress and elevated angiotensin-converting enzyme (ACE) activity are considered closely related to various chronic health risks. Therefore, developing foods with antioxidant and ACE-inhibiting activities has become a research hotspot in the field of functional foods. Dairy products, due to their rich protein resources and balanced amino acid composition, are considered an important source of potential bioactive peptides. Existing research shows that under specific processing or enzymatic conditions, milk proteins can release peptides with antioxidant or ACE-inhibiting activities, thereby endowing dairy products with certain functional properties.

[0003] In existing technologies, the aforementioned antioxidant and ACE-inhibiting activities are mostly found in whey protein, casein hydrolysates, or fermented dairy product systems, with related results primarily existing in the form of protein hydrolysates, peptide powders, or functional ingredients. These technologies typically rely on exogenous proteases or specific fermentation strains, are achieved in liquid or semi-liquid systems, require high control over processing conditions, and have limited integration with traditional solid dairy products. Therefore, how to stably introduce antioxidant and ACE-inhibiting activities into structurally dense, formed dairy products remains an unsolved problem in existing technologies.

[0004] Sheep milk, due to its high total milk solids content and rich protein and fat content, is considered a high-quality raw material for curdled dairy products and has good processing adaptability in products such as milk cake and cheese. However, current sheep milk product development focuses primarily on flavor and nutrition, with relatively insufficient systematic exploration and application of its potential functional activities. Especially in milk cake, a fresh cheese product, current production technology mainly uses acid coagulation, with the process goal focused on achieving milk protein coagulation and shaping, paying less attention to changes in protein structure and the formation of bioactive peptides, making it difficult to stably obtain antioxidant or ACE inhibitory activities in the product.

[0005] In the traditional acid-coagulated milk cake production process, the acid slurry is mostly derived from natural fermentation or reuse, resulting in unstable coagulation conditions and a complex microbial composition. This affects both product quality consistency and limits the precise control of the degree of protein hydrolysis. Under these conditions, milk proteins exist primarily as intact proteins or large molecular aggregates, which is not conducive to the formation of bioactive low-molecular-weight peptides, thus restricting the formation of the milk cake's functional properties. Furthermore, if additional protein hydrolysates or functional ingredients are added to impart antioxidant or ACE-inhibiting activity to the milk cake, it often destroys the original texture and flavor of the milk cake, and exhibits poor dispersibility and stability in the finished product, making practical application difficult.

[0006] To address the shortcomings of traditional acid coagulation processes, some existing technologies attempt to use plant-derived coagulants to replace or assist in the coagulation process. *Cynanchum paniculatum* is a plant used both as food and medicine; its extracts contain enzymes with coagulation activity, exhibiting high temperature and acid / alkali resistance and high coagulation activity. Existing research indicates that *Cynanchum paniculatum* rennet, when acting on milk proteins, has different enzymatic cleavage characteristics compared to traditional animal-derived rennets, which can affect curd structure and flavor formation. It has already found some application in the preparation of cow's milk or buffalo milk curd and cheese. However, existing methods for extracting rennet from *Cynanchum paniculatum* are inefficient and suffer from significant activity loss. Traditional immersion or mechanical crushing methods yield low extraction rates, while high temperatures, strong acids or alkalis, or conventional ultrasonic treatments easily lead to enzyme denaturation and inactivation. Simultaneously, the whey removal process during milk cake preparation causes substantial loss of whey protein, reducing product yield and nutritional value, and making it difficult to retain bioactive peptides derived from whey protein. Furthermore, there is limited research on the application of *Cynanchum paniculatum* rennet in sheep milk systems, and its impact on protein structural changes and potential bioactivity formation during sheep milk cake preparation remains unclear. Therefore, developing a preparation method that can ensure the basic quality and structural stability of sheep milk cake while enabling the product to possess antioxidant and ACE-inhibiting activities remains of significant research and application value. Summary of the Invention

[0007] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention aims to provide a method for preparing sheep milk cake using rennet from *Symplocos buergeriana*. This method involves extracting rennet from the stems of *Symplocos buergeriana* using resonant acoustic waves combined with alternating hypertonic and hypotonic solutions. Palmitic acid and α-terpineol are then esterified using lipase catalysis to generate terpene esters, which are then immobilized using a composite carrier composed of sodium alginate. Fresh sheep milk is pasteurized, and then calcium chloride and the immobilized *Symplocos buergeriana* rennet solution are added. The mixture is then subjected to coagulation, whey removal, pressing, and maturation to obtain sheep milk cake. This method utilizes the gentle mechanical stimulation of resonant acoustic waves combined with alternating hypertonic and hypotonic treatments to significantly promote rennet dissolution and improve extraction efficiency. The immobilization carrier achieves efficient enzyme immobilization through hydrophobic interactions and electrostatic attraction, significantly improving coagulation efficiency and product stability. The prepared sheep milk cake exhibits good antioxidant and ACE inhibitory activity, which is beneficial for the development of functional dairy products.

[0008] Technical solution: A method for preparing sheep milk cake using chymosin from *Cynanchum paniculatum*, comprising the following steps: Step 1. Extraction of chymotrypsin from *Cynanchum paniculatum* The dried stems of *Cynanchum paniculatum* were cut into 5-8 cm segments and crushed. The mixture was then treated with a combination of resonant acoustic wave treatment and alternating hypertonic and hypotonic solutions: Hypertonic solution was added at a ratio of 1 g:(10-15) mL, and the mixture was treated for 10-30 min at a sound wave frequency of 20-40 kHz, an amplitude of 1-5 mm, and a temperature of 20-40℃. The residue was then filtered. Hypotonic solution was added at a ratio of 1 g:(10-15) mL, and the mixture was treated under the same conditions for 2-5 cycles. All filtrates were then combined. The mixture was centrifuged at 4000-8000 r / min for 15-30 min and filtered. The filtrate was then concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa, and purified by gel filtration chromatography to obtain *Cynanchum paniculatum* chymotrypsin. The hypertonic solution was a 5-20% sucrose solution, and the hypotonic solution was a 0.1-1% buffer solution. Step 2. Preparation of immobilized *Cynanchum paniculatum* chymoses Palmitic acid and α-terpineol were esterified at a mass ratio of 1:(1-5) under the catalysis of Novozym 435 lipase, and the terpene ester was obtained after purification; the amount of lipase added was 2-5% based on the total mass of palmitic acid and α-terpineol; the esterification reaction conditions were a reaction temperature of 30-50℃ and a reaction time of 4-8h. Terpene esters and sodium alginate were dispersed in water at a mass ratio of 1:(2-6) and dissolved and stirred at 50-70℃ and 300-600r / min to form a composite carrier matrix. The chymotrypsin solution of *Cynanchum paniculatum* was mixed with the composite carrier matrix at a volume ratio of 1:(1-5) and stirred. Then it was dropped into calcium chloride solution and cured and crosslinked at a treatment temperature of 20-30℃ for 1-3h. After washing with deionized water 3-5 times and drying at 40-60℃, immobilized *Cynanchum paniculatum* chymotrypsin was obtained. Step 3. Preparation of sheep milk cake Fresh sheep milk is pasteurized at 85-100℃ for 5-10 minutes and cooled to 78-82℃. First, 0.01-0.03% calcium chloride is added and stirred evenly. Then, 22-25% immobilized chymotrypsin solution at a concentration of 0.5-1.0g / L is added. After curdling, whey removal, pressing and molding, and maturation, sheep milk cake is obtained. Preferably, the conditions for curdling in step 3 are a curdling temperature of 78-82℃ and a curdling time of 6-12 minutes. Preferably, the pressing conditions in step 3 are a processing pressure of 2-4 kg / cm² and a processing time of 2-2.5 h.

[0009] The sheep milk cake prepared by the above method can be used in the preparation of products that help with anti-oxidation and regulate blood pressure levels. Beneficial effects

[0010] 1. This invention utilizes resonant acoustic waves combined with osmotic pressure difference-osmotic pressure cycling to extract rennet from *Cynanchum paniculatum*. First, a specific frequency of acoustic waves is used to generate mechanical resonance coupling with the fixed mechanical vibration frequency of the *Cynanchum paniculatum* cell wall, causing fatigue fracture of the cell wall. On the one hand, this increases the permeability of the *Cynanchum paniculatum* cell wall to promote the permeation of the solution system and the outward diffusion of rennet molecules. On the other hand, the mild mechanical stimulation of the resonant acoustic waves can activate the stress response of *Cynanchum paniculatum* cells, prompting the release of cell wall relaxation enzymes to further reduce the mechanical strength of the cell wall. This avoids the adverse effects of extreme physicochemical conditions caused by local hot spots and cavitation collapse, thereby protecting the three-dimensional conformation and active center integrity of rennet. Secondly, by alternating treatment with hypertonic and hypotonic solutions, repeated plasmolysis and deplasmolysis processes are induced in *Cynanchum paniculatum* cells. This causes the cell membrane to undergo repeated mechanical stress loading and unloading, resulting in structural fatigue and local rupture of the membrane lipid bilayer. This leads to a reversible increase in membrane permeability and a significant reduction in the resistance to mass exchange driven by osmotic pressure difference. Rennet can then more easily diffuse through cell wall pores into the external solution to accelerate dissolution. At the same time, the acoustic flow effect of resonant sound waves generates macroscopic eddies in the solution system, which promptly breaks the concentration boundary layer at the extraction interface, maintains the driving force of the high concentration gradient, and maximizes the extraction efficiency of *Cynanchum paniculatum* rennet.

[0011] 2. This invention utilizes palmitic acid and α-terpineol to generate terpene esters via lipase catalysis, which are then combined with sodium alginate to form a composite carrier for the immobilization of *Cynanchum paniculatum* chymoses. Firstly, the terpene esters generated by the ester bond between palmitic acid and α-terpineol possess both the long-chain hydrophobic properties of palmitic acid and the monoterpene cyclic structure of α-terpineol. The carbonyl and hydroxyl groups in the terpene ester molecule form a hydrogen bond network with the carboxyl groups of sodium alginate. Simultaneously, the hydrophobic long chain of the terpene ester interacts with the hydrophobic pockets on the surface of *Cynanchum paniculatum*, achieving initial immobilization of the chymoses. Secondly, the negatively charged carboxyl groups of sodium alginate bind to the positively charged amino acid residues on the surface of the chymoses through electrostatic attraction. Furthermore, the monoterpene cyclic structure of the terpene ester interacts with the aromatic amino acids in the enzyme molecule through π-π stacking, achieving efficient immobilization of *Cynanchum paniculatum* chymoses.

[0012] 3. The immobilized *Cynanchum paniculatum* chymoses used in this invention act on sheep milk. The terpene ester-sodium alginate composite matrix in the immobilized carrier has a porous structure and abundant surface active sites, which can adsorb casein molecules in sheep milk through electrostatic attraction, hydrogen bonding, and hydrophobic interactions, causing them to accumulate around the immobilized *Cynanchum paniculatum* chymoses, significantly improving its contact efficiency with κ-casein and accelerating the coagulation process of sheep milk. The palmitic acid long chain introduced by the immobilized material specifically binds to β-lactoglobulin in whey protein through hydrophobic interactions to avoid loss during whey discharge. The monoterpene ring structure of the terpene ester interacts with the aromatic amino acid residues in β-lactoglobulin through π-π stacking, further stabilizing the whey protein and the immobilized carrier to form a complex. This effectively integrates whey protein, which originally does not participate in coagulation, into the casein gel network to form a casein-whey protein cross-linked composite coagulation structure, thereby achieving the purpose of efficient coagulation and improving the stability of the coagulation structure.

[0013] 4. The sheep milk cake prepared by this invention exhibits good antioxidant and ACE inhibitory activities. Its DPPH radical scavenging rate, ABTS radical scavenging rate, hydroxyl radical scavenging rate, and ACE inhibition rate all reach good levels. These results indicate that the sheep milk cake can form and retain functionally active peptides while maintaining a good structure and flavor. Compared with traditional acid-coagulated sheep milk cake, the sheep milk cake of this invention has significant advantages in antioxidant and ACE inhibitory activities, and its process is highly controllable, providing a technical solution for the functional development and industrialization of sheep milk cake products. Detailed Implementation

[0014] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: Example 1

[0015] A method for preparing immobilized *Cynanchum paniculatum* chymoses includes the following steps: Step 1. Extraction of chymotrypsin from *Cynanchum paniculatum* The dried stems of *Cynanchum paniculatum* were cut into 5cm segments and crushed. 100.0g of the crushed stems were added to 1L of 5% sucrose solution and treated with resonant sound waves at 20℃, 20kHz frequency, and 1mm amplitude for 10min. After filtration, the residue and filtrate were collected and set aside. The residue was added to 1L of 0.1% phosphate buffer and treated with resonant sound waves at 20℃, 20kHz frequency, and 1mm amplitude for 10min. The hypertonic-hypotonic alternating treatment cycle was repeated twice, and all filtrates were combined to obtain the crude enzyme solution. The crude enzyme solution was centrifuged at 4000r / min for 15min and filtered. Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10-30kDa to concentrate the solution, and then purified by gel filtration chromatography to obtain the *Cynanchum paniculatum* chymotrypsin extract. Step 2. Preparation of immobilized *Cynanchum paniculatum* chymoses Weigh 10.0g of palmitic acid and 15.6g of α-terpineol, mix and dissolve in 200mL of ethanol, add 0.51g of Novozym435 lipase, react at 30℃ for 4h, and then obtain terpene ester by vacuum distillation and drying. Weigh 5.0 g of terpene ester and 10.0 g of sodium alginate and disperse them in 500 mL of deionized water. Stir and dissolve them at 50 °C and 300 r / min to form a composite carrier matrix. Mix 85 mL of *Cynanchum paniculatum* chymotrypsin solution with 85 mL of the composite carrier matrix and stir slowly at 4 °C for 30 min to mix evenly. Then, add 1 L of 2% calcium chloride solution dropwise and cure and crosslink at room temperature for 1 h. Wash three times with 200 mL of water and vacuum dry at 40 °C to obtain immobilized *Cynanchum paniculatum* chymotrypsin. Example 2

[0016] A method for preparing immobilized *Cynanchum paniculatum* chymoses includes the following steps: Step 1. Extraction of chymotrypsin from *Cynanchum paniculatum* The dried stems of *Cynanchum paniculatum* were cut into 6cm segments and crushed. 200.0g of the crushed stems were added to 2.4L of 10% sucrose solution and treated with resonant sound waves at 30℃, 30kHz frequency, and 3mm amplitude for 15min. After filtration, the residue and filtrate were collected and set aside. The residue was added to 2.4L of 0.3% phosphate buffer and treated with resonant sound waves at 30℃, 30kHz frequency, and 3mm amplitude for 15min. The hypertonic-hypotonic alternating treatment cycle was repeated 3 times, and all filtrates were combined to obtain the crude enzyme solution. The crude enzyme solution was centrifuged at 6000r / min for 10min and filtered. Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10-30kDa to concentrate the solution, and then purified by gel filtration chromatography to obtain the *Cynanchum paniculatum* chymotrypsin extract. Step 2. Preparation of immobilized *Cynanchum paniculatum* chymoses Weigh 15.0g of palmitic acid and 46.8g of α-terpineol, mix and dissolve in 400mL of ethanol, add 2.16g of Novozym435 lipase, react at 40℃ for 6h, and then obtain terpene alcohol ester by vacuum distillation and drying. 10.0 g of terpene ester and 40.0 g of sodium alginate were weighed and dispersed in 500 mL of deionized water. The mixture was stirred and dissolved at 50 °C and 300 r / min to form a composite carrier matrix. 175 mL of *Cynanchum paniculatum* chymotrypsin solution was mixed with 525 mL of the composite carrier matrix and stirred slowly at 4 °C for 45 min to achieve homogeneity. Then, 2 L of 3% calcium chloride solution was added dropwise and the mixture was cured and crosslinked at room temperature for 2 h. The mixture was washed 4 times with 300 mL of water and vacuum dried at 50 °C to obtain immobilized *Cynanchum paniculatum* chymotrypsin. Example 3

[0017] A method for preparing immobilized *Cynanchum paniculatum* chymoses includes the following steps: Step 1. Extraction of chymotrypsin from *Cynanchum paniculatum* The dried stems of *Cynanchum paniculatum* were cut into 5cm segments and crushed. 300.0g of the crushed stems were added to 4.5L of 20% sucrose solution and treated with resonant sound waves at 40℃, 40kHz frequency, and 5mm amplitude for 30min. After filtration, the residue and filtrate were collected and set aside. The residue was added to 4.5L of 1.0% phosphate buffer and treated with resonant sound waves at 40℃, 40kHz frequency, and 5mm amplitude for 30min. The hypertonic-hypotonic alternating treatment cycle was repeated 3 times, and all filtrates were combined to obtain the crude enzyme solution. The crude enzyme solution was centrifuged at 6000r / min for 10min and filtered. Ultrafiltration concentration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10-30kDa, and then purified by gel filtration chromatography to obtain the *Cynanchum paniculatum* chymotrypsin extract. Step 2. Preparation of immobilized *Cynanchum paniculatum* chymoses Weigh 20.0g of palmitic acid and 93.6g of α-terpineol, mix and dissolve in 500mL of ethanol, add 5.68g of Novozym435 lipase, react at 50℃ for 5h, and then obtain terpene ester by vacuum distillation and drying; 12g of terpene ester and 72g of sodium alginate were weighed and dispersed in 1500mL of deionized water. The mixture was stirred and dissolved at 70℃ and 600r / min to form a composite carrier matrix. 240mL of *Cynanchum paniculatum* chymoses solution was mixed with 1200mL of the composite carrier matrix and stirred slowly at 4℃ for 60min to achieve homogeneity. Then, 3L of 2.5% calcium chloride solution was added dropwise and the mixture was cured and crosslinked at room temperature for 3h. The mixture was washed three times with 400mL of water and vacuum dried at 40℃ to obtain immobilized *Cynanchum paniculatum* chymoses. Example 4

[0018] A method for preparing immobilized *Cynanchum paniculatum* chymoses includes the following steps: Step 1. Extraction of chymotrypsin from *Cynanchum paniculatum* The dried stems of *Cynanchum paniculatum* were cut into 5cm segments and crushed. 150.0g of the crushed stems were added to 1.65L of 10% sucrose solution and treated with resonant sound waves at 25℃, 25kHz frequency, and 2mm amplitude for 12min. After filtration, the residue and filtrate were collected and reserved. The residue was added to 1.65L of 0.2% phosphate buffer and treated with resonant sound waves at 25℃, 25kHz frequency, and 2mm amplitude for 12min. The hypertonic-hypotonic alternating treatment cycle was repeated 4 times, and all filtrates were combined to obtain the crude enzyme solution. The crude enzyme solution was centrifuged at 5000r / min for 10min and filtered. Ultrafiltration concentration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10-30kDa, and then purified by gel filtration chromatography to obtain the *Cynanchum paniculatum* chymotrypsin extract. Step 2. Preparation of immobilized *Cynanchum paniculatum* chymoses Weigh 12.0g of palmitic acid and 28.1g of α-terpineol, mix and dissolve in 200mL of ethanol, add 1.0g of Novozym 435 lipase, react at 35℃ for 5h, and then obtain terpene alcohol ester by vacuum distillation and drying; 8g of terpene ester and 24g of sodium alginate were weighed and dispersed in 800mL of deionized water. The mixture was stirred and dissolved at 55℃ and 350r / min to form a composite carrier matrix. 135mL of *Cynanchum paniculatum* chymoses solution was mixed with 270mL of the composite carrier matrix and stirred slowly at 4℃ for 35min to achieve homogeneity. Then, 1.5L of 2.5% calcium chloride solution was added dropwise and the mixture was cured and crosslinked at room temperature for 1.5h. The mixture was washed three times with 300mL of water and vacuum dried at 45℃ to obtain immobilized *Cynanchum paniculatum* chymoses. Example 5

[0019] A method for preparing immobilized *Cynanchum paniculatum* chymoses includes the following steps: Step 1. Extraction of chymotrypsin from *Cynanchum paniculatum* The dried stems of *Cynanchum paniculatum* were cut into 8cm segments and crushed. 250.0g of the crushed stems were added to 3.25L of 15% sucrose solution and treated with resonant sound waves at 35℃, 35kHz frequency, and 4mm amplitude for 25min. After filtration, the residue and filtrate were collected and set aside. The residue was added to 3.25L of 0.5% phosphate buffer and treated with resonant sound waves at 35℃, 35kHz frequency, and 4mm amplitude for 25min. The hypertonic-hypotonic alternating treatment cycle was repeated 3 times, and all filtrates were combined to obtain the crude enzyme solution. The crude enzyme solution was centrifuged at 4000r / min for 15min and filtered. Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10-30kDa to concentrate the solution, and then purified by gel filtration chromatography to obtain the *Cynanchum paniculatum* chymotrypsin extract. Step 2. Preparation of immobilized *Cynanchum paniculatum* chymoses Weigh 18.0g of palmitic acid and 70.2g of α-terpineol, mix and dissolve in 300mL of ethanol, add 4.0g of Novozym 435 lipase, react at 30℃ for 4h, and then obtain terpene alcohol ester by vacuum distillation and drying. 10.0 g of terpene ester and 50.0 g of sodium alginate were weighed and dispersed in 1200 mL of deionized water. The mixture was stirred and dissolved at 65 °C and 500 r / min to form a composite carrier matrix. 210 mL of *Cynanchum paniculatum* chymotrypsin solution was mixed with 840 mL of the composite carrier matrix and stirred slowly at 4 °C for 30 min to achieve homogeneity. Then, 2.5 L of 3.5% calcium chloride solution was added dropwise and the mixture was cured and crosslinked at room temperature for 2.5 h. The mixture was washed four times with 350 mL of water and then vacuum dried at 55 °C to obtain immobilized *Cynanchum paniculatum* chymotrypsin. Example 6

[0020] A method for preparing immobilized *Cynanchum paniculatum* chymoses includes the following steps: Step 1. Extraction of chymotrypsin from *Cynanchum paniculatum* The dried stems of *Cynanchum paniculatum* were cut into 5cm segments and crushed. 180.0g of the crushed stems were added to 2.52L of 10% sucrose solution and treated with resonant sound waves at 28℃, 30kHz frequency, and 3mm amplitude for 20min. After filtration, the residue and filtrate were collected and reserved. The residue was added to 2.52L of 0.4% phosphate buffer and treated with resonant sound waves at 28℃, 30kHz frequency, and 3mm amplitude for 20min. The hypertonic-hypotonic alternating treatment cycle was repeated 4 times, and all filtrates were combined to obtain the crude enzyme solution. The crude enzyme solution was centrifuged at 5500r / min for 6min and filtered. Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10-30kDa to concentrate the solution, and then purified by gel filtration chromatography to obtain the *Cynanchum paniculatum* chymotrypsin extract. Step 2. Preparation of immobilized *Cynanchum paniculatum* chymoses Weigh 4.0g of palmitic acid and 16.0g of α-terpineol, mix and dissolve in 500mL of ethanol, add 0.84g of Novozym 435 lipase, react at 38℃ for 5h, and then obtain terpene alcohol ester by vacuum distillation and drying; 9g of terpene ester and 31.5g of sodium alginate were weighed and dispersed in 900mL of deionized water. The mixture was stirred and dissolved at 55℃ and 400r / min to form a composite carrier matrix. 155mL of *Cynanchum paniculatum* chymoses solution was mixed with 400mL of the composite carrier matrix and stirred slowly at 4℃ for 40min to achieve homogeneity. Then, 1.8L of 2.5% calcium chloride solution was added dropwise and the mixture was cured and crosslinked at room temperature for 2h. The mixture was washed three times with 300mL of water and vacuum dried at 50℃ to obtain immobilized *Cynanchum paniculatum* chymoses. Comparative Example 1

[0021] The difference between this comparative example and Example 3 is that resonant acoustic wave processing is not used; the remaining operations are the same as in Example 3. Comparative Example 2

[0022] The difference between this comparative example and Example 3 is that the high-osmotic-low-osmotic alternating treatment is not used; the remaining operations are the same as in Example 3. Comparative Example 3

[0023] The difference between this comparative example and Example 3 is that no terpene esters are added; the remaining operations are the same as in Example 3.

[0024] Performance testing Extraction rate of chymotrypsin from *Cynanchum paniculatum* First, the stem powder of *Cynanchum paniculatum* was extracted using ultrasonic cell disruption. Ten times its mass of 0.05 mol / L phosphate buffer (pH 7.0) was added, and the mixture was sonicated at 300 W for 10 min. Subsequently, the mixture was centrifuged at 8000 rpm for 15 min at 4°C, and the supernatant was collected. The total protein content (A) was determined using the Coomassie Brilliant Blue G-250 method. 总 Then, the protein content (A1) in the *Cynanchum paniculatum* chymoses solution was measured. The extraction rate was calculated using the formula: *Cynanchum paniculatum* chymoses extraction rate (%) = A1 / A 总 ×100%, of which the protein assay used bovine serum albumin as the standard, and the absorbance was measured at a wavelength of 595 nm.

[0025] (2) Chymotrypsin activity of *Cynanchum paniculatum* Prepare 10% skim milk by dissolving skim milk powder in 0.01 mol / L CaCl2 solution. Take 1 mL of skim milk, preheat it in an 85℃ water bath for 5 min, then add 0.1 mL of enzyme solution. Accurately record the coagulation time, ensuring the effective coagulation time is within 40 min. The calculation formula is as follows: Rennet activity (U / mL) = (2400 × V) / (t × v), where V represents the volume of skim milk, v represents the volume of enzyme solution, and t represents the coagulation time.

[0026] (3) Immobilization efficiency of *Cynanchum paniculatum* chymoses The enzyme immobilization ability of the carrier is evaluated by comparing the changes in enzyme activity before and after immobilization. The volume of enzyme solution added before immobilization is accurately recorded, and the activity of the enzyme solution is determined by the method described in (2) as the total enzyme activity (U). 总 After the immobilization reaction was completed, all supernatant and washing solution were collected, combined, and the residual enzyme activity (U) was measured. 余 The formula for calculating immobilization efficiency is: Immobilization efficiency (%) = (U 总 -U 余 ) / U 总 ×100%.

[0027] Table 1. Extraction rate, enzyme activity, and immobilization efficiency of *Cynanchum paniculatum* chymoses from Examples 1-6 and Comparative Examples 1-3.

[0028] As shown in Table 1, the extraction rate, enzyme activity, and immobilization efficiency of *Cynanchum paniculatum* chymoses in Examples 1-6 were all superior to those in Comparative Examples 1-2. In particular, Example 3 showed the best extraction and immobilization effect of *Cynanchum paniculatum* chymoses. This indicates that the use of resonant acoustic wave treatment combined with alternating high-osmotic and low-osmotic cycles effectively broke the cell wall of *Cynanchum paniculatum* and fully released intracellular enzyme proteins to improve the extraction rate of chymoses. At the same time, the terpene ester-sodium alginate composite carrier achieved efficient immobilization of *Cynanchum paniculatum* chymoses through hydrophobic interactions and electrostatic adsorption. Example 7

[0029] A method for preparing sheep milk cake using chymosin from *Symplocos edulis* includes the following steps: S1. Pasteurize 1000mL of fresh sheep milk at 90℃ for 8min, then cool it to 80℃ and add 0.2g of calcium chloride to the sheep milk and stir well. Then add 230mL of immobilized ginseng rennet solution with a concentration of 0.75g / L and coagulate at 80℃ for 8min. After the coagulated milk is completely formed, drain the whey and set it. S2. Place the shaped curd into a mold at 3kg / cm 2 Pressed under pressure for 2.2 hours, then cut and vacuum-packed to obtain the finished sheep milk cake. Example 8

[0030] A method for preparing sheep milk cake using chymosin from *Symplocos edulis* includes the following steps: S1. Pasteurize 1000mL of fresh sheep milk at 95℃ for 5min, then cool it to 80℃ and add 0.3g of calcium chloride to the sheep milk and stir well. Then add 250mL of immobilized ginseng rennet solution with a concentration of 1.0g / L and coagulate at 82℃ for 6min. After the coagulation is completely formed, drain the whey and set the shape. S2. Place the shaped curd into a mold at 4kg / cm². 2 Pressed under pressure for 2 hours, then cut and vacuum-packed to obtain the finished sheep milk cake. Example 9

[0031] A method for preparing sheep milk cake using chymosin from *Symplocos edulis* includes the following steps: S1. Pasteurize 1000mL of fresh sheep milk at 85℃ for 10min, then cool it to 78℃ and add 0.1g of calcium chloride to the sheep milk and stir well. Then add 220mL of immobilized ginseng rennet solution with a concentration of 0.5g / L and coagulate at 78℃ for 12min. After the coagulated milk is completely formed, drain the whey and set it. S2. Place the set curd into a mold at 2kg / cm 2 Pressed under pressure for 2.5 hours, then cut and vacuum-packed to obtain the finished sheep milk cake. Example 10

[0032] A method for preparing sheep milk cake using chymosin from *Symplocos edulis* includes the following steps: S1. Pasteurize 1000mL of fresh sheep milk at 92℃ for 6min, then cool it to 80℃ and add 0.25g of calcium chloride to the sheep milk and stir well. Then add 240mL of immobilized ginseng rennet solution with a concentration of 0.8g / L and coagulate at 81℃ for 10min. After the coagulation is completely formed, drain the whey and set the shape. S2. Place the shaped curd into a mold at 3kg / cm 2 Pressed under pressure for 2 hours, then cut and vacuum-packed to obtain the finished sheep milk cake. Comparative Example 4

[0033] The difference between this comparative example and Example 7 is that the immobilized *Cynanchum paniculatum* chymoses prepared in Comparative Example 1 was used. The remaining operations were performed in accordance with Example 7. Comparative Example 5

[0034] The difference between this comparative example and Example 7 is that the immobilized *Cynanchum paniculatum* chymoses prepared in Comparative Example 2 was used. The remaining operations were performed in accordance with Example 7. Comparative Example 6

[0035] The difference between this comparative example and Example 7 is that the immobilized *Cynanchum paniculatum* chymoses prepared in Comparative Example 3 was used. The remaining operations were performed in accordance with Example 7. Comparative Example 7

[0036] The difference between this comparative example and Example 7 is that it uses *Cynanchum paniculatum* chymoses that have not undergone immobilization. The remaining operations are the same as in Example 7. Comparative Example 8

[0037] The difference between this comparative example and Example 7 is that lactic acid bacteria are used to ferment the sour liquid; the remaining operations are the same as in Example 7. Performance testing

[0038] (1) Whey protein content in whey samples The whey protein content was determined by centrifuging the liquid sample collected during the whey drainage process at 4℃ and 8000r / min for 15min to remove fat and precipitate, and then the whey protein content was determined by the Coomassie Brilliant Blue G-250 method.

[0039] (2) DPPH free radical scavenging rate Accurately weigh DPPH standard and prepare a 0.1 mmol / L DPPH working solution with anhydrous ethanol, then store it protected from light. Mix 2 mL of 1.0 mg / mL milk cake extract with 2 mL of DPPH working solution and react at room temperature in the dark for 30 min. Then measure the absorbance at 517 nm (A). 样品 Anhydrous ethanol was used instead of DPPH working solution to determine the background absorbance of the sample (A). 本底 The blank absorbance (A) was determined using distilled water instead of the sample. 空白 The formula for calculating DPPH free radical scavenging rate is: DPPH free radical scavenging rate (%) = [1 - (A 样品 -A 本底 ) / A 空白 ] × 100%.

[0040] (3) ABTS+ free radical scavenging rate ABTS was reacted with potassium persulfate to generate an ABTS cationic radical stock solution, which was diluted with phosphate buffer (pH 7.4) to a working solution with an absorbance of 0.70 at 734 nm. 0.1 mL of a milk cake extract with a protein concentration of 1.0 mg / mL was mixed with 3.9 mL of the ABTS working solution and reacted at room temperature in the dark for 6 min. The absorbance was then measured at 734 nm. 样品 ); ); Using phosphate buffer instead of ABTS working solution to determine background (A 本底 ), and use distilled water instead of the sample to determine the blank (A) 空白 The formula for calculating the ABTS+ free radical scavenging rate is: ABTS+ free radical scavenging rate (%) = [1-(A...] 样品 -A 本底 ) / A 空白 ] × 100%.

[0041] (4) Hydroxyl radical scavenging rate The Fenton reaction was used to determine hydroxyl radicals. The reaction system contained 1.0 mL of 9 mmol / L FeSO4 solution, 1.0 mL of 9 mmol / L salicylic acid-ethanol solution, 1.0 mL of milk cake extract with a protein concentration of 1.0 mg / mL, and 1.0 mL of 8.8 mmol / L H2O2 solution. The reaction was carried out in a water bath at 37 °C for 30 min, and the absorbance was measured at 510 nm. 样品 Background measurements were taken using distilled water instead of H2O2 (A). 本底 ), and use distilled water instead of the sample to determine the blank (A) 空白 The formula for calculating the hydroxyl radical scavenging rate is: Hydroxyl radical scavenging rate (%) = [1 - (A...] 样品 -A 本底 ) / A 空白 ] × 100%.

[0042] (5) ACE inhibition rate Mix 50 μL of milk cake extract (1.0 mg / mL protein concentration) with 50 μL of ACE enzyme solution (0.05 U / mL), preheat at 37 °C for 5 min, add 150 μL of 5 mmol / L substrate HHL solution, react at 37 °C for 30 min, and terminate the reaction by adding 250 μL of 1 mol / L HCl. Then, extract hippuric acid with 1.5 mL of ethyl acetate, centrifuge, and collect the upper organic phase. Measure the absorbance at 228 nm (A). 样品 Background measurements were performed using distilled water instead of ACE enzyme solution (A). 本底 ), and use distilled water instead of the sample to determine the blank (A) 空白 The formula for calculating the ACE inhibition rate is: ACE inhibition rate (%) = [1 - (A... 样品 -A 本底 ) / (A 空白 -A 本底 )] × 100%.

[0043] Table 2. Whey protein content, antioxidant activity, and ACE inhibition rate of whey feed liquids from Examples 7-10 and Comparative Examples 4-8.

[0044] As shown in Table 2, the whey content of the whey discharge liquid in Examples 7-10 was significantly lower than that in Comparative Examples 4-8. This indicates that the terpene ester-sodium alginate composite carrier in the immobilized *Cynanchum paniculatum* rennet effectively adsorbs casein through electrostatic attraction, hydrogen bonding, and hydrophobic interactions. Simultaneously, the palmitic acid long chain specifically binds to β-lactoglobulin, and the terpene ester monoterpene ring stabilizes whey protein through π-π stacking, efficiently integrating whey protein that originally did not participate in curdling into the casein gel network, forming a composite curd structure, thereby significantly reducing protein loss during whey discharge. Furthermore, the carboxyperoxidase activity and ACE inhibition rate of Examples 7-10 were significantly better than those of Comparative Examples 4-8, indicating that the sheep milk cake described in this invention produces more active peptides during curdling with the immobilized *Cynanchum paniculatum* rennet, and the casein-whey protein cross-linked composite curd structure ensures that active peptides are not lost, thus exhibiting excellent antioxidant and blood pressure regulating effects.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing sheep milk cake using *Cynanchum paniculatum* chymoses, characterized in that, Includes the following steps: Step 1. Extraction of chymotrypsin from *Cynanchum paniculatum* The dried stems of *Cynanchum paniculatum* were cut into small sections and crushed. The mixture was then treated with resonant sound waves in combination with alternating high-tonic and low-tonic solutions. After centrifugation, filtration, ultrafiltration concentration, and purification, *Cynanchum paniculatum* chymotrypsin was obtained. Step 2. Preparation of immobilized *Cynanchum paniculatum* chymoses Palmitic acid and α-terpineol were esterified under the catalysis of lipase, and the terpene ester was purified to obtain terpene ester. The terpene ester was dissolved and stirred with sodium alginate to form a composite carrier matrix. The chymotrypsin solution obtained in step 1 was mixed and stirred with the composite carrier matrix, and then dripped into calcium chloride solution to solidify and crosslink. After washing and drying, immobilized chymotrypsin was obtained. Step 3. Preparation of sheep milk cake After sterilizing sheep milk, immobilized chymosin var. chinensis rennet solution is added, followed by curdling, whey removal, pressing, and maturation to obtain sheep milk cake.

2. The method for preparing sheep milk cake using *Cynanchum paniculatum* chymoses according to claim 1, characterized in that: The conditions for resonant acoustic wave treatment in step 1 are: acoustic wave frequency 20-40 kHz, acoustic wave amplitude 1-5 mm, treatment temperature 20-40℃, and treatment time 10-30 min; the conditions for alternating treatment with hypertonic and hypotonic solutions are: hypertonic solution is 5-20% sucrose solution, hypotonic solution is 0.1-1% buffer salt solution, the number of cycles is 2-5, and the single treatment time is 10-30 min.

3. The method for preparing sheep milk cake using *Cynanchum paniculatum* chymoses according to claim 1, characterized in that: In step 2, the mass ratio of palmitic acid to α-terpineol is 1:(1-5); the amount of lipase added is 2-5%, based on the total mass of palmitic acid and α-terpineol; the esterification reaction conditions are a reaction temperature of 30-50℃ and a reaction time of 4-8h.

4. The method for preparing sheep milk cake using *Cynanchum paniculatum* chymoses according to claim 1, characterized in that: In step 2, the mass ratio of terpene ester to sodium alginate is 1:(2-6); the volume ratio of *Cynanchum paniculatum* chymotrypsin solution to composite carrier matrix is ​​1:(1-5); the concentration of calcium chloride solution is 0.1-0.5 mol / L; and the curing and crosslinking conditions are a treatment temperature of 20-30℃ and a treatment time of 1-3 h.

5. The method for preparing sheep milk cake using *Cynanchum paniculatum* chymoses according to claim 1, characterized in that: In step 3, sheep milk is sterilized using a high-temperature short-time sterilization method, with a sterilization temperature of 85-100℃ and a sterilization time of 5-10 minutes.

6. The method for preparing sheep milk cake using *Cynanchum paniculatum* chymoses according to claim 1, characterized in that: In step 3, the amount of immobilized *Cynanchum paniculatum* chymoses solution added is 22-25%, and the concentration of the immobilized *Cynanchum paniculatum* chymoses solution is 0.5-1.0 g / L; the coagulation conditions are a coagulation temperature of 78-82℃ and a coagulation time of 6-12 min.

7. The method for preparing sheep milk cake using *Cynanchum paniculatum* chymoses according to claim 1, characterized in that: The conditions for pressing and molding in step 3 are a processing pressure of 2-4 kg / cm² and a processing time of 2-2.5 h.

8. Sheep milk cake prepared by the method according to any one of claims 1-7.

9. The use of the sheep milk cake according to claim 8 in the preparation of products that contribute to antioxidant activity and regulate blood pressure levels.

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