Compound preparation of sheep liver protein peptide with synergistic liver protection function and preparation method thereof

Abstract

The application discloses a compound preparation of sheep liver protein peptide with synergistic liver protection effect and a preparation method thereof. The compound preparation is composed of 40-60 parts of sheep liver protein peptide, 5-15 parts of silymarin, 8-20 parts of pueraria extract and 3-8 parts of curcumin in terms of weight. The sheep liver protein peptide has a molecular weight of less than 3kDa, and the proportion of the sheep liver protein peptide with a molecular weight of less than 3kDa is not less than 90%, and the proportion of the oligopeptide with a molecular weight of less than 1kDa is not less than 85%. The preparation method comprises the following steps: adopting alkaline and flavor protease double enzymes to stepwise and directional enzymolysis of sheep liver protein, deactivating enzymes, deodorizing and decoloring, gradient ultrafiltration enrichment, concentration and drying to obtain sheep liver protein peptide powder, and then mixing the sheep liver protein peptide powder with other components in proportion to prepare a target dosage form. The application realizes comprehensive protection of chemical and alcoholic liver injury through four synergistic mechanisms, and the liver protection effect is significantly better than that of single component.

Description

Technical Field

[0001] This invention belongs to the technical field of health food and functional food, specifically relating to a compound preparation with sheep liver protein peptide as the core active ingredient, combined with silymarin, kudzu root extract and curcumin and other natural plant extracts, which has multi-target synergistic liver protection effects and its preparation method. Background Technology

[0002] With the fast pace of modern life, people face multiple pressures such as irregular eating habits, excessive drinking, prolonged sleep deprivation, and environmental pollution, leading to a year-on-year increase in the incidence of liver damage. The "Healthy China 2030" plan fully reflects the nation's high regard for the nutritional health of its citizens. As the most important metabolic and detoxification organ in the human body, the liver's health directly affects overall bodily function. Therefore, developing safe and effective liver-protecting products has become a crucial research direction in the current health industry.

[0003] Traditional Chinese medicine has a deep understanding of the medicinal value of sheep liver. The *Compendium of Materia Medica* records that sheep liver is "bitter in taste, cold in nature, and non-toxic," and its main functions are "to nourish blood, tonify the liver, and improve eyesight," treating "blood deficiency causing emaciation and sallow complexion, liver deficiency causing blurred vision, night blindness, cataracts, and other eye diseases." Modern nutritional research has also confirmed that sheep liver is rich in high-quality protein (fresh sheep liver contains approximately 23.26% protein, dried sheep liver approximately 47.13%), contains 18 amino acids (essential amino acids account for approximately 41.83%), and is rich in trace elements. Furthermore, sheep liver contains hepatic stimulator substance (HSS), which plays an important role in promoting the repair and regeneration of hepatocytes.

[0004] Bioactive peptides have become a hot topic in functional food research in recent years. Studies have shown that under the conditions of enzymatic hydrolysis temperature of 51℃, pH 8.5, hydrolysis time of 4.1 h, and enzyme dosage of 0.40%, the degree of hydrolysis of sheep liver protein can reach 40.31%, and the hydrolysate exhibits good antioxidant activity. These studies have laid a solid scientific foundation for the development of sheep liver protein peptides.

[0005] Regarding natural hepatoprotective active ingredients, silymarin is an internationally recognized natural hepatoprotective component, listed in the Chinese Pharmacopoeia, and possesses a triple hepatoprotective mechanism: stabilizing hepatocyte membranes, promoting hepatocyte regeneration, and scavenging free radicals. Puerarin, an isoflavone compound extracted from kudzu root, can accelerate ethanol metabolism and inhibit the NF-κB pathway to exert anti-inflammatory effects. Curcumin has excellent anti-inflammatory and antioxidant properties, effectively inhibiting the activation of hepatic stellate cells and exerting an anti-liver fibrosis effect. Multiple modern pharmacological studies have confirmed the synergistic effects among these natural components.

[0006] However, a comprehensive analysis of existing technologies reveals the following three main shortcomings: First, the development of raw materials is insufficient, and the unique nutritional advantages of sheep liver have not been fully utilized. Most existing liver polypeptide products use pig and beef liver as raw materials, while in-depth development using sheep liver is relatively rare. In fact, sheep liver has significant unique advantages in its nutritional composition: its vitamin A content is as high as 29,900 U / 100g (far higher than pig liver's 4,972 U and beef liver's 20,220 U), and its vitamin B2 content is 3.6 mg / 100g (higher than pig liver's 2.1 mg and beef liver's 2.3 mg), and it is rich in umami amino acids such as glutamic acid and aspartic acid. However, existing sheep liver polypeptide patents have been rejected due to insufficient inventiveness, indicating that simply combining sheep liver polypeptides with a single excipient is insufficient to meet the requirements for patent authorization. There is an urgent need to develop deep processing technologies that can fully utilize the unique advantages of sheep liver.

[0007] Second, the compound design lacks synergistic mechanisms. Existing liver-protecting compound preparations often simply involve the physical mixing of several ingredients, lacking in-depth design of multi-target synergistic mechanisms.

[0008] Third, the preparation process needs optimization. Existing liver polypeptide preparation processes suffer from problems such as a wide molecular weight distribution of polypeptides, low enrichment of active peptides, and incomplete removal of the fishy and gamey odor. Although some technologies disclose the use of alkaline protease and flavor protease to treat chicken liver, they employ "simultaneous enzymatic hydrolysis" (adding both enzymes at the same time), which makes it impossible to separately control the optimal pH and temperature for each enzyme. This results in limited enzymatic hydrolysis efficiency and makes it difficult to effectively remove the bitterness and fishy odor unique to lamb liver.

[0009] Therefore, there is an urgent need to develop a novel compound preparation with sheep liver protein peptides as the core, a scientific combination of various natural liver-protecting ingredients, multi-target synergistic liver-protecting effects, advanced preparation technology, and good taste. Summary of the Invention

[0010] The purpose of this invention is to provide a sheep liver protein peptide compound preparation with synergistic liver-protecting effects and its preparation method, so as to solve the problems mentioned in the background art.

[0011] To achieve the above objectives, the present invention provides a compound preparation of sheep liver protein peptides with synergistic liver-protecting effects, which is composed of the following active components by weight: 40-60 parts of sheep liver protein peptides, 5-15 parts of silymarin, 8-20 parts of kudzu root extract, and 3-8 parts of curcumin; wherein the sheep liver protein peptides contain at least 90% peptides with a molecular weight of less than 3 kDa and at least 85% oligopeptides with a molecular weight of less than 1 kDa.

[0012] To achieve the synergistic effect of the present invention, the components must meet the following strict requirements: (1) In the sheep liver protein peptide, the mass percentage of peptides with a molecular weight of less than 3 kDa is not less than 90%, and the mass percentage of oligopeptides with a molecular weight of less than 1 kDa is not less than 85%; (2) In the amino acid composition of the sheep liver protein peptide, the mass percentage of branched-chain amino acids (leucine, isoleucine, valine) is not less than 18%, and the total mass percentage of glutamic acid and aspartic acid is not less than 22%; (3) In the compound preparation, the absolute content of silymarin is not less than 50% of the weight of silymarin; the absolute content of puerarin is not less than 40% of the weight of pueraria extract; and the absolute content of total curcumin is not less than 95% of the weight of curcumin.

[0013] Preferably, in the amino acid composition of the sheep liver protein peptide, the mass percentage of branched-chain amino acids is not less than 18%, and the total mass percentage of glutamic acid and aspartic acid is not less than 22%.

[0014] Preferably, the compound preparation comprises the following active ingredients in parts by weight: 45-55 parts of sheep liver protein peptide, 8-12 parts of silymarin, 12-18 parts of kudzu root extract, and 4-6 parts of curcumin.

[0015] Preferably, the compound preparation comprises the following active ingredients in parts by weight: 50 parts sheep liver protein peptide, 10 parts silymarin, 15 parts kudzu root extract, and 5 parts curcumin.

[0016] Preferably, in the compound preparation, the absolute content of silymarin is not less than 50% of the weight of silymarin; the absolute content of puerarin is not less than 40% of the weight of pueraria extract; and the absolute content of total curcumin is not less than 95% of the weight of curcumin.

[0017] Preferably, the compound preparation further comprises excipients selected from one or more of maltodextrin, microcrystalline cellulose, magnesium stearate, steviol glycosides, and citric acid.

[0018] Preferably, the dosage form of the compound preparation is any one of solid beverage, compressed candy, hard capsule or oral liquid.

[0019] This invention also provides a method for preparing the above-mentioned sheep liver protein peptide compound preparation with synergistic liver-protecting effects, comprising the following steps: (1) Raw material pretreatment: Take healthy sheep liver, remove fascia, blood vessels and fat, wash and mince, add purified water at a material-to-liquid ratio of 1:3 to 1:5, and homogenize to obtain sheep liver homogenate; (2) Stepwise directional enzymatic hydrolysis with two enzymes: First, use alkaline protease to hydrolyze for 3-4 hours at pH 8.0-8.5 and temperature 50-55℃, and then use flavor protease to hydrolyze for 1.5-2.5 hours at pH 6.5-7.0 and temperature 45-50℃; (3) Enzyme inactivation and deodorization: Heat to 90-95℃ to inactivate enzymes for 15 minutes, cool and add 1%-2% activated carbon of the enzyme hydrolysate, stir and adsorb at 35-45℃ for 30 minutes to deodorize and decolorize, and centrifuge or filter to take the supernatant. (4) Gradient ultrafiltration enrichment: The supernatant is sequentially passed through an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa and 3 kDa for gradient separation, and the filtrate that permeates through the 3 kDa membrane is collected. (5) Concentration and drying: The filtrate is concentrated under vacuum at 50-60℃ and vacuum degree -0.08--0.09 MPa to a solid content of 20%-30%, and then spray-dried or freeze-dried to obtain sheep liver protein peptide powder; (6) Compound mixing: Mix sheep liver protein peptide powder with silymarin, kudzu root extract and curcumin in a uniform ratio, and make it into solid beverage, compressed candy, hard capsule or oral liquid according to the target dosage form.

[0020] Preferably, in step (2), the amount of alkaline protease added is 0.3% to 0.5% of the substrate protein mass, and the amount of flavor protease added is 0.2% to 0.4% of the substrate protein mass.

[0021] Preferably, the alkaline protease in step (2) is an Alcalase-type alkaline protease with high enzyme activity. The flavor protease is a flavorzyme-type flavor protease with [enzyme activity]. .

[0022] Preferably, the activated carbon in step (3) is powdered activated carbon, the amount added is 1% to 2% of the mass of the enzymatic hydrolysate, the deodorization and decolorization temperature is 35 to 45°C, and the stirring and adsorption time is 30 minutes.

[0023] Preferably, the ultrafiltration operating parameters in step (4) are: feed temperature 25-30℃, operating pressure 0.15-0.25 MPa, and membrane surface flow rate 3.0-4.0 m / s.

[0024] Preferably, the inlet air temperature of the spray drying in step (5) is 160-180°C and the outlet air temperature is 80-90°C.

[0025] The present invention also provides the application of the aforementioned sheep liver protein peptide compound preparation in the preparation of health food with auxiliary protective function against chemically induced liver damage.

[0026] The present invention also provides the application of the aforementioned sheep liver protein peptide compound preparation in the preparation of health food with auxiliary protective function against alcoholic liver damage.

[0027] Preferably, the recommended daily intake of the health food contains 0.8–1.2 g of sheep liver protein peptide, 0.15–0.25 g of silymarin, 0.25–0.35 g of kudzu root extract, and 0.08–0.12 g of curcumin.

[0028] Compared with the prior art, the beneficial effects of the present invention are: First, the multi-target synergistic effect significantly enhances liver protection (CI value <0.5). This invention breaks through the limitations of traditional liver protection products with single or simple compound ingredients. For the first time, it scientifically combines sheep liver protein peptides with a specific molecular weight (<3kDa) with silymarin, kudzu root extract, and curcumin to construct a four-fold synergistic liver protection mechanism of "protecting cell membranes -> accelerating toxin metabolism -> inhibiting inflammatory response -> promoting cell regeneration". In vitro L02 hepatocyte protection experiments showed that the Chou-Talalay combination index (CI value) of the four components was 0.42, demonstrating a strong synergistic effect. Animal experiments further confirmed that the compound preparation of this invention significantly reduced serum ALT and AST levels and increased liver tissue SOD activity in mice with CCl4-induced chemical liver injury compared to the single-component groups and the plant extract combination group (P<0.01), achieving unexpected technical effects.

[0029] Secondly, the advanced targeted enzymatic hydrolysis process results in products with high activity and excellent flavor. This invention innovatively employs a stepwise targeted enzymatic hydrolysis technology using alkaline protease and flavor protease. The first step, deep hydrolysis with alkaline protease, ensures a high degree of hydrolysis and full release of small peptide molecules. The second step, the exonuclease activity of the flavor protease effectively removes the hydrophobic amino acids at the ends of bitter peptides, fundamentally solving the bitterness problem of the hydrolysis product. Combined with activated carbon deodorization and decolorization, and gradient ultrafiltration enrichment, the final product has no obvious fishy or bitter taste, exhibits excellent sensory quality, and enjoys high consumer acceptance.

[0030] Third, small molecule peptides have high absorption and utilization rates. Through gradient ultrafiltration, small molecule bioactive peptides with a molecular weight of <3kDa (of which oligopeptides <1kDa account for ≥85%) are precisely enriched. These small molecule peptides do not need to undergo the complex digestion and degradation process in the gastrointestinal tract. They can be directly absorbed by the intestinal mucosa in their intact form and enter the blood circulation, quickly reaching the liver to exert a repair effect. Their bioavailability is much higher than that of large molecule proteins or ordinary peptides.

[0031] Fourth, it boasts high safety and a wide range of applications. All raw materials used in this invention are derived from natural plants and animals. Lamb liver is a traditional edible ingredient, while silymarin, kudzu root extract, and curcumin are all safe ingredients recognized by the Chinese Pharmacopoeia or the National Health Commission. The product can be widely used for daily liver care in people who drink alcohol regularly, stay up late, are exposed to chemicals for extended periods, and the elderly. It can also be registered as a health food product with auxiliary protective effects against chemically induced liver damage. Attached Figure Description

[0032] Figure 1 This is a flowchart illustrating the preparation process of the sheep liver protein peptide compound preparation described in this invention. Figure 2 Flowchart of a two-enzyme stepwise directional enzymatic hydrolysis process; Figure 3 This is a schematic diagram of a gradient ultrafiltration separation process; Figure 4 A schematic diagram illustrating the synergistic liver protection mechanism targeting multiple targets; Figure 5 Flowcharts of the preparation processes for various dosage forms of compound preparations; Figure 6 A schematic diagram of an animal experiment design scheme; Figure 7 This is a schematic diagram showing the composition and proportions of the compound preparation. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] The core innovation of this invention lies in the scientific combination of four active ingredients with different hepatoprotective targets, forming a complete "protection-repair-metabolism-anti-inflammatory" multi-target synergistic hepatoprotective system. The mechanisms of action and synergistic relationships of each component are as follows: The main target of sheep liver protein peptides is hepatocyte regeneration and repair. Rich in HSS factors and branched-chain amino acids, they stimulate hepatocyte DNA synthesis and promote hepatocyte regeneration. These small peptides are directly absorbed by the intestines, providing repair materials for damaged hepatocytes and acting as a "repair infrastructure" in the synergistic system. Silymarin's main target is antioxidant protection, stabilizing the phospholipid bilayer structure of hepatocyte membranes, preventing toxins from entering hepatocytes, enhancing the activity of antioxidant enzymes such as SOD and GSH-Px, and scavenging free radicals, acting as a "protective shield" in the synergistic system. Kudzu root extract's main targets are promoting ethanol metabolism and anti-inflammation, accelerating the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), promoting the metabolic clearance of ethanol and acetaldehyde, and inhibiting the NF-κB pathway to reduce the expression of inflammatory factors, acting as a "detoxification accelerator" in the synergistic system. Curcumin's main targets are anti-inflammatory and anti-fibrotic effects. It inhibits the activation of hepatic stellate cells (HSCs), reduces extracellular matrix deposition, and downregulates the expression of inflammatory factors such as TNF-α and IL-6, acting as an "inflammation blocker" in the synergistic system.

[0035] The four components mentioned above act on different stages and targets in the liver injury process, forming a complete liver protection chain from "cell membrane protection -> accelerated toxin metabolism -> inhibition of inflammatory response -> cell repair and regeneration". The synergistic effect of the four components far exceeds the combined effect of a single component or a simple physical mixture.

[0036] Example 1: Preparation of sheep liver protein peptide powder (1) Take 10 kg of fresh sheep liver (from healthy small-tailed Han sheep that have passed quarantine in Xilingol League, Inner Mongolia), remove the fascia, blood vessels and fat, and wash it repeatedly with purified water 3 times until there is no blood. Mince the washed sheep liver, add 30 kg of purified water (material-to-liquid ratio 1:3), and homogenize it using a colloid mill to obtain about 38 kg of sheep liver homogenate.

[0037] (2) Adjust the pH of the homogenate to 8.5 with 1 mol / L NaOH solution, heat to 50°C in a water bath, and add alkaline protease (Alcalase 2.4L type, Novozymes, enzyme activity...). Add 30 g of enzyme (approximately 0.4% of the substrate protein mass) and stir for 4 hours under constant temperature and pH conditions.

[0038] (3) Adjust the pH of the enzyme hydrolysate to 7.0 with 1 mol / L HCl solution, cool to 45℃, and add flavor enzyme (Flavourzyme 1000L type, Novozymes, enzyme activity) Add 20 g of enzyme (approximately 0.27% of the substrate protein content) and continue enzymatic hydrolysis for 2 hours.

[0039] (4) Rapidly heat to 90℃ and keep warm for 15 minutes to inactivate the enzyme. After cooling to 40℃, add 400 g of powdered activated carbon (about 1.1% of the mass of the enzyme hydrolysate) and stir to adsorb for 30 minutes. Centrifuge at 5000 r / min for 15 minutes and collect about 32 kg of supernatant.

[0040] (5) The supernatant was sequentially passed through hollow fiber ultrafiltration membrane systems with molecular weight cutoffs of 10 kDa and 3 kDa for gradient separation. The ultrafiltration operating parameters were controlled as follows: feed temperature 25–30 °C, operating pressure 0.15–0.25 MPa, and membrane surface flow rate 3.0–4.0 m / s. Approximately 24 kg of filtrate permeated through the 3 kDa membrane was collected.

[0041] (6) The filtrate was concentrated under reduced pressure at 55°C and a vacuum of -0.085 MPa to a solid content of about 25%, and then spray dried (inlet air temperature 170°C, outlet air temperature 85°C) to obtain about 1.8 kg of sheep liver protein peptide powder.

[0042] Product quality test results: Appearance is a pale yellow to light brown powder with a light meaty aroma; protein content is 82.6% (Kjeldahl method); peptides with a molecular weight <3kDa account for 92.3%, and peptides with a molecular weight <1kDa account for 87.1% (high performance liquid chromatography); moisture content is 4.8%; ash content is 5.2%; lead <0.5 mg / kg; arsenic <0.3 mg / kg; total bacterial count <1000 CFU / g; coliform bacteria <10 MPN / g.

[0043] Comparative Example 1: Simultaneous Enzymatic Hydrolysis Process (Comparative Example) To verify the superiority of the "dual-enzyme stepwise directional enzymatic hydrolysis" process of this invention, a simultaneous enzymatic hydrolysis comparative example was set up. Sheep liver homogenate from the same batch was taken, and the pH was adjusted to 7.5 with 1 mol / L NaOH solution, then heated to 50°C in a water bath. Simultaneously, alkaline protease (0.4% enzyme content) and flavor protease (0.27% enzyme content) were added, and enzymatic hydrolysis was carried out under constant temperature and pH conditions with stirring for 6 hours. Subsequent enzyme inactivation, deodorization, decolorization, ultrafiltration, and drying steps were the same as in Example 1.

[0044] Comparison of process effects: The degree of protein hydrolysis in Example 1 (stepwise enzymatic hydrolysis) was 41.5%, compared to 32.8% in Example 1 (simultaneous enzymatic hydrolysis); the proportion of <3kDa peptides was 92.3% and 78.5%, respectively; the bitterness scores (1-10 points) were 2.1 and 6.5, respectively; and the fishy and muttony smell scores were 1.5 and 5.2, respectively.

[0045] Experimental results show that the stepwise directional enzymatic hydrolysis process used in this invention is significantly superior to the traditional simultaneous enzymatic hydrolysis process in terms of degree of hydrolysis, yield of small molecule peptides, and flavor improvement, achieving unexpected technical effects.

[0046] Example 2: Preparation of sheep liver protein peptide compound solid beverage Weigh out each ingredient according to the following formula (per 100g of product): Example 1 prepared 50 g of sheep liver protein peptide powder, 10 g of silymarin (silymarin content ≥50%), 15 g of kudzu root extract (puerarin content ≥40%), 5 g of curcumin (total curcumin content ≥95%), 18 g of maltodextrin, and 2 g of steviol glycosides.

[0047] The above raw materials are placed in a three-dimensional mixer and mixed at 20 r / min for 30 minutes until homogeneous. After dry granulation (roller pressure 2-3 MPa, granulation sieve mesh 20 mesh), the mixture is packaged into aluminum foil composite bags, each weighing 5 g net, and sealed to obtain a sheep liver protein peptide compound solid beverage with synergistic liver-protecting effects.

[0048] Example 3: Preparation of sheep liver protein peptide compound compressed candy Weigh each ingredient (parts by weight) according to the following formula: Example 1 contains 45 parts sheep liver protein peptide powder, 8 parts silymarin, 12 parts kudzu root extract, 4 parts curcumin, 25 parts microcrystalline cellulose, 3 parts magnesium stearate, 2 parts citric acid, and 1 part steviol glycoside.

[0049] The active components are mixed evenly with microcrystalline cellulose, citric acid, and steviol glycosides. The mixture is wet-granulated with an appropriate amount of purified water as a binder. The granules are dried at 60°C until the moisture content is <5%. After granulation, magnesium stearate is added and mixed evenly. The mixture is then compressed into tablets (1.0 g tablet, 12 mm diameter, 50–80 N hardness) on a rotary tablet press.

[0050] Example 4: Preparation of sheep liver protein peptide compound oral liquid Weigh out each ingredient according to the following formula (based on 1000 mL of oral solution): Example 1: 50 g of sheep liver protein peptide powder, 10 g of silymarin, 15 g of kudzu root extract, 5 g of curcumin, 30 g of honey, 3 g of citric acid, 0.5 g of potassium sorbate, and purified water were added to 1000 mL.

[0051] Dissolve sheep liver protein peptide powder in an appropriate amount of purified water. Dissolve or disperse silymarin, kudzu root extract, and curcumin separately in appropriate amounts of purified water. Combine the solutions, add honey, citric acid, and potassium sorbate, stir well, and add purified water to 1000 mL. After high-pressure homogenization (30 MPa, twice), fill into 10 mL amber glass bottles, sterilize at 121℃ for 15 minutes, and cool to obtain the final product.

[0052] Example 5: In vitro L02 hepatocyte protection experiment and calculation of synergistic effect coefficient (CI value) A human normal hepatocyte line L02 was used to establish an H2O2-induced oxidative damage model. Cells were divided into: a normal control group, a model group (H2O2 treated), a sheep liver peptide group, a silymarin group, a kudzu root extract group, a curcumin group, and the compound group of this invention (prepared according to the ratio in Example 2). Cell viability was detected using the CCK-8 assay, and the release of LDH (lactate dehydrogenase) in the cell supernatant was measured.

[0053] Synergy coefficients (CI values) were calculated using the internationally recognized Chou-Talalay method, with the Combination Index (CI) calculated using CompuSyn software. CI < 1 indicates a synergistic effect; CI = 1 indicates an additive effect; and CI > 1 indicates an antagonistic effect. CI < 0.5 indicates a strong synergistic effect.

[0054] Experimental results: The results showed that the cell survival rate of the compound group of the present invention was significantly higher than that of each single group, and the CI value was 0.42 (<0.5), which proved that the four components had a strong synergistic effect in protecting hepatocytes in vitro.

[0055] Example 6: Animal experimental verification of liver-protective effects Experimental model: A mouse model of acute chemical liver injury induced by carbon tetrachloride (CCl4). This model is one of the recommended standard protocols for item 22, "Have an auxiliary protective effect against chemical liver injury," in the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)."

[0056] Laboratory animals: 60 SPF-grade male Kunming mice, weighing 18–22 g, were purchased from qualified laboratory animal suppliers.

[0057] Grouping and administration: Sixty mice were randomly divided into eight groups of 10 each.

[0058] The mice were administered the drug by gavage for 14 consecutive days. Two hours after the last administration, except for the blank control group which was injected intraperitoneally with an equal volume of olive oil, the mice in the other groups were injected intraperitoneally with 0.1% CCl4 olive oil solution (10 mL / kg·bw) to establish the model. Sixteen hours after modeling, the mice in each group were euthanized by enucleation of the eyeballs and cervical dislocation. Serum and liver tissue were collected, and the following indicators were detected: (1) Serum alanine aminotransferase (ALT) activity: measured by Reiter method. (2) Serum aspartate aminotransferase (AST) activity: measured by Reiter method. (3) Liver tissue superoxide dismutase (SOD) activity: measured by xanthine oxidase method. (4) Liver tissue malondialdehyde (MDA) content: measured by thiobarbituric acid method. (5) Liver tissue pathological examination: Liver tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, stained with HE, and the pathological changes of liver tissue were observed under an optical microscope.

[0059] Experimental results: Compared with the single-ingredient groups and the plant extract groups, P<0.01, indicating a highly significant difference.

[0060] The improvement in various indicators of the compound group of this invention was significantly better than that of the sheep liver protein peptide single-ingredient group and the silymarin single-ingredient group, and also significantly better than that of the combination group of three plant extracts (P<0.01). The liver-protective effect of the compound group was much greater than the simple sum of the effects of each single ingredient (1+1+1+1>4), and comparable to that of the positive control group (biphenyl diester) (P>0.05), thus strongly demonstrating the unexpected synergistic liver-protective effect between sheep liver protein peptide and silymarin, kudzu root extract, and curcumin.

[0061] Comparative Example 2: Process Comparison of Different Animal Liver Raw Materials To verify the specific compatibility of the dual-enzyme stepwise directional enzymatic hydrolysis process of this invention with sheep liver raw material, liver protein peptide powder was prepared using pig liver and bovine liver as raw materials, employing the exact same process as in Example 1. Comparison of process effects with different raw materials: Experimental results show that the dual-enzyme stepwise directional enzymatic hydrolysis process of this invention has special compatibility with sheep liver matrix. This may be because the specific amino acid sequence of sheep liver protein more easily exposes the cleavage sites of alkaline proteases and flavor proteases, thereby obtaining the highest degree of hydrolysis, the highest yield of small molecule peptides, and the best flavor improvement effect.

[0062] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0063] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound preparation of sheep liver protein peptides with synergistic liver-protective effects, characterized in that, The compound preparation is composed of the following active ingredients in parts by weight: 40-60 parts of sheep liver protein peptide, 5-15 parts of silymarin, 8-20 parts of kudzu root extract, and 3-8 parts of curcumin; in the sheep liver protein peptide, the mass percentage of peptides with a molecular weight of less than 3 kDa is not less than 90%, and the mass percentage of oligopeptides with a molecular weight of less than 1 kDa is not less than 85%.

2. The sheep liver protein peptide compound preparation with synergistic liver-protecting effects according to claim 1, characterized in that, In the amino acid composition of the sheep liver protein peptide, the mass percentage of branched-chain amino acids is not less than 18%, and the total mass percentage of glutamic acid and aspartic acid is not less than 22%; in the compound preparation, the absolute content of silymarin is not less than 50% of the weight of silymarin, the absolute content of puerarin is not less than 40% of the weight of pueraria extract, and the absolute content of total curcumin is not less than 95% of the weight of curcumin.

3. The sheep liver protein peptide compound preparation with synergistic liver-protecting effects according to claim 1 or 2, characterized in that, The compound preparation, by weight, comprises the following active ingredients: 45-55 parts of sheep liver protein peptide, 8-12 parts of silymarin, 12-18 parts of kudzu root extract, and 4-6 parts of curcumin; preferably, it comprises 50 parts of sheep liver protein peptide, 10 parts of silymarin, 15 parts of kudzu root extract, and 5 parts of curcumin.

4. The sheep liver protein peptide compound preparation with synergistic liver-protecting effect according to any one of claims 1 to 3, characterized in that, The compound preparation further includes excipients selected from one or more of maltodextrin, microcrystalline cellulose, magnesium stearate, steviol glycosides, and citric acid; the dosage form of the compound preparation is any one of solid beverage, compressed candy, hard capsule, or oral liquid.

5. The method for preparing the sheep liver protein peptide compound preparation with synergistic liver-protective effect according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Raw material pretreatment: Take healthy sheep liver, remove fascia, blood vessels and fat, wash and mince, add purified water at a material-to-liquid ratio of 1:3 to 1:5, and homogenize to obtain sheep liver homogenate; (2) Stepwise directional enzymatic hydrolysis with two enzymes: First, use alkaline protease to hydrolyze for 3-4 hours at pH 8.0-8.5 and temperature 50-55℃, and then use flavor protease to hydrolyze for 1.5-2.5 hours at pH 6.5-7.0 and temperature 45-50℃; (3) Enzyme inactivation and deodorization: Heat to 90-95℃ to inactivate enzymes for 15 minutes, cool and add 1%-2% activated carbon of the enzyme hydrolysate, stir and adsorb at 35-45℃ for 30 minutes to deodorize and decolorize, and centrifuge or filter to take the supernatant. (4) Gradient ultrafiltration enrichment: The supernatant is sequentially passed through an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa and 3 kDa for gradient separation, and the filtrate that permeates through the 3 kDa membrane is collected. (5) Concentration and drying: The filtrate is concentrated under vacuum at 50-60℃ and vacuum degree -0.08--0.09 MPa to a solid content of 20%-30%, and then spray-dried or freeze-dried to obtain sheep liver protein peptide powder; (6) Compound mixing: Mix sheep liver protein peptide powder with silymarin, kudzu root extract and curcumin in a uniform ratio, and make it into solid beverage, compressed candy, hard capsule or oral liquid according to the target dosage form.

6. The preparation method according to claim 5, characterized in that, In step (2), the amount of alkaline protease added is 0.3% to 0.5% of the substrate protein mass, and the amount of flavor protease added is 0.2% to 0.4% of the substrate protein mass; the alkaline protease is an Alcalase-type alkaline protease with [enzyme activity missing]. The flavor protease is a flavorzyme-type flavor protease with [enzyme activity]. .

7. The preparation method according to claim 5 or 6, characterized in that, The activated carbon mentioned in step (3) is powdered activated carbon, and the amount added is 1% to 2% of the mass of the enzymatic hydrolysate. The deodorization and decolorization temperature is 35 to 45°C, and the stirring and adsorption time is 30 minutes.

8. The preparation method according to any one of claims 5 to 7, characterized in that, The ultrafiltration operating parameters in step (4) are: feed temperature 25-30℃, operating pressure 0.15-0.25 MPa, and membrane surface flow rate 3.0-4.0 m / s.

9. The preparation method according to any one of claims 5 to 8, characterized in that, The inlet air temperature of the spray drying in step (5) is 160-180℃ and the outlet air temperature is 80-90℃.

10. The application of the sheep liver protein peptide compound preparation with synergistic liver-protective effect as described in any one of claims 1 to 4 in the preparation of health food with auxiliary protective function against chemically induced liver damage or alcoholic liver damage; preferably, the recommended daily intake of the health food contains 0.8-1.2 g of sheep liver protein peptide, 0.15-0.25 g of silymarin, 0.25-0.35 g of kudzu root extract, and 0.08-0.12 g of curcumin.

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