Chymosin complex, its extraction method and application

Abstract

The present application is named a kind of composite rennet and its extraction method and application, belong to biological pharmaceutical technical field.The technical problem to be solved is that the existing rennet extraction process has large enzyme activity loss, low product purity and complicated industrial process;Food-grade rennet product has many impurities, and enzyme activity ratio is difficult to control, which cannot better meet the requirements of product quality for medical applications such as chronic gastritis.The technical solution points are an extraction method of a kind of composite rennet, including the following steps: after the fourth lamb stomach is frozen and crushed, add mixed buffer solution for homogenization, then add mixed buffer solution for extraction, adjust pH for activation after the obtained extract is centrifuged and filtered, further centrifuged, take supernatant for ultrafiltration, concentration, drying, and then get.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a compound rennet, its extraction method, and its application. Background Technology

[0002] Rennet is an aspartic protease primarily derived from the fourth stomach (abomasum) of young ruminants. This enzyme is secreted in the gastric mucosa in zymogen form and becomes biologically active after activation in an acidic environment. It can specifically cleave casein to achieve a curdling effect and can also synergistically work with pepsin to hydrolyze proteins. Natural animal-derived rennets, due to their high substrate specificity and few hydrolytic side effects, are widely used not only in the processing of dairy products such as cheese but also have significant applications in the pharmaceutical field. Chronic gastritis is a common digestive system disease. Some patients with chronic gastritis have damaged gastric mucosal glands and insufficient secretion of endogenous digestive enzymes, leading to decreased protein digestion capacity, loss of appetite, and postprandial bloating. Animal gastric extracts containing rennet and pepsin can exogenously supplement digestive enzymes, improve the gastric digestive environment, and alleviate gastric discomfort symptoms, showing clear application prospects in chronic gastritis and related indigestion conditions.

[0003] In existing technologies, animal-derived rennet is mostly extracted from the fourth gastric mucosa of lambs or calves. Conventional processes typically involve tissue homogenization, acid extraction, solid-liquid separation, salting-out precipitation, multi-step purification, and drying to obtain the rennet product. However, existing extraction processes still have many shortcomings. First, rennet is a bioactive protein that is highly sensitive to temperature and pH, and enzyme activity is easily lost during the entire extraction, activation, and purification process. Traditional extraction-salting-out processes often have long extraction cycles, crude process conditions, and the activation process is difficult to control precisely, resulting in a low overall enzyme activity recovery rate and significant batch-to-batch fluctuations in enzyme activity, making it difficult to consistently obtain products with high enzyme activity. Secondly, traditional purification processes are lengthy, requiring multiple salting out and repeated chromatography, with cumbersome operation steps, high reagent consumption, long production time, and high operating costs when scaled up for industrial use. If the purification process is simplified, the product will contain a large number of impurities such as proteins and tissue degradation impurities, resulting in insufficient product purity. These impurities will further affect the safety and stability of subsequent pharmaceutical preparations, making it difficult to simultaneously meet the practical requirements of high enzyme activity, high purity, and process simplification.

[0004] Furthermore, most current research focuses on the application of rennet in food processing. When applied to the pharmaceutical use of rennet for chronic gastritis, the requirements for the activity ratio, impurity control, and enzyme activity retention of the rennet-pepsin complex system are far higher than in the food industry. Rennet obtained through conventional food-grade extraction often suffers from high levels of impurities, uncontrollable ratios of the two enzymes, and significant activity loss. Direct use of rennet in gastric disease-related preparations can lead to unstable efficacy, limiting its effectiveness in pharmaceutical applications.

[0005] Relevant patent documents retrieved: This document, published in China (CN106011115A) on October 12, 2016, discloses a method for preparing and using an extract of effective components from the fourth gastric cavity of lamb. The method involves defatting the fourth gastric cavity of lamb with petroleum ether at low temperature, followed by freezing with liquid nitrogen, grinding and pulverizing, dissolving in phosphate buffer, undergoing low-temperature autolysis, adjusting the pH to 3.3 with hydrochloric acid solution, activating at low temperature, adjusting the pH to 5.6 with sodium oxide solution, filtering, centrifuging, discarding the residue, and desalting the supernatant by dialysis with distilled water. After centrifugation to remove the precipitate (salt-soluble proteins) formed during dialysis, the supernatant is freeze-dried to obtain the extract. Testing showed that the extract obtained in this paper had a protein content of 64.3%, rennet activity of 25182 Ru / g, pepsin activity of 3796.4 u / g, and proteolytic activity of 4641.1 u / g. The bands for rennet and pepsin were clearly visible in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis image. The method yields extracts with high protein purity and strong biological activity, which can be used as raw materials for the preparation of drugs or health products for the treatment or prevention of various stomach diseases.

[0006] This document, published in China (CN112410323A) on February 26, 2021, discloses a method for preparing lamb abomasal rennet standards. The method involves defatting with petroleum ether, ultrasonic-assisted extraction of rennet, desalting, freeze concentration, gradient precipitation with saturated ammonium sulfate, centrifugation, desalting via dialysis bag, and freeze-drying to obtain total lamb abomasal rennet. Two ionic strengths of rennet are then obtained by gradient elution with sodium chloride solution on an anion exchange column, followed by desalting via dialysis bag and freeze-drying to obtain purified rennet. The two ionic strength rennet fractions, B1 and B2, are then separated using HPLC gel column chromatography to obtain rennet active components such as B1-P1, B2-P1, and P2. Since the B2-P2 electrophoresis band contains impurity bands, only B1-P1 and B2-P1 are used as rennet standards. The lamb abomasal chymotrypsin standard obtained by the method described in this literature has high chymotrypsin activity, high purity, and clear electrophoretic bands; the method is mature, simple and easy to operate, and easy to scale up for large-scale preparation; the obtained lamb abomasal chymotrypsin standard can be used in the pharmaceutical and food fields.

[0007] This document, published in China (CN103555698A) on February 5, 2014, discloses an extraction process for a compound rennet, belonging to the field of biopharmaceutical technology. The extraction process includes a step of stimulating and activating lamb quadrigeminal fragments with an acid solution. In this process, the various steps and parameters work together synergistically, resulting in an extraction process that is not only short in reaction time and mild in reaction conditions, but also avoids the use of large amounts of organic solvents and environmental pollution, and achieves high yield. The obtained compound rennet exhibits high in vitro rennet activity and high protein activity, opening up new avenues for research on the extraction process of compound rennets.

[0008] This document, published in China (CN112226426A) on January 15, 2021, discloses a method for extracting and separating rennet from lamb abomasum. The method involves pre-extraction treatments such as tearing the abomasum into smaller particles and soaking to disperse the abomasum tissue, disrupt cells, and enhance mixing. Combined with ultrasound-assisted extraction, this significantly improves the rennet extraction rate, greatly shortens the extraction time, and reduces the cost of rennet preparation, providing technical support for the standardized production of lamb stomach extracts by relevant enterprises. Simultaneously, the solubility of rennet in lamb stomach was systematically studied, and the optimal solvent and extraction pH were selected, providing a reliable and feasible method for large-scale rennet extraction. The lamb abomasum rennet obtained by this method has high purity and strong biological activity. Furthermore, the extraction method is simple and feasible, and the entire extraction process is carried out at low temperatures, better preserving the activity of the lamb stomach rennet. The obtained rennet can be applied in cheese production.

[0009] The publication, dated October 26, 1994, is from China and has the publication number CN1093908A. It discloses a stomach medicine (Wei You Le) that is a novel biochemical stomach medicine refined using specific scientific methods to treat digestive tract diseases, particularly stomach ailments. It uses the fourth stomach of lamb as the main raw material, and its production process is simple and reasonable. Mass production has been approved under the document No. (28) of the Ministry of Health. Clinical observation shows that the overall effective rate of this product is 83%. It provides a novel, non-toxic, safe, and reliable biochemical drug with no side effects for treating superficial gastritis, atrophic gastritis, upper abdominal distension, and digestive system diseases such as spitting up, diarrhea, and loss of appetite in children. This has significant social and economic value in fully utilizing the biochemical resources of pastoral areas and in treating common stomach ailments.

[0010] This document, published in China (CN117904243B) on July 23, 2024, discloses an antimicrobial peptide derived from Hu sheep and its application, belonging to the field of biotechnology. The method includes: firstly, defatting Hu sheep milk and then extracting proteins to obtain freeze-dried Hu sheep milk protein powder; secondly, alkali-dissolving the freeze-dried Hu sheep milk protein powder to obtain a Hu sheep milk protein solution; thirdly, enzymatically hydrolyzing the Hu sheep milk protein solution; fourthly, adding an enzymatic catalyst during the enzymatic hydrolysis process to obtain Hu sheep milk protein hydrolysate; fifthly, separating and desalting the Hu sheep milk protein hydrolysate using macroporous nonpolar resin; sixthly, purifying it using gel chromatography; and finally, freeze-drying it to obtain the antimicrobial peptide derived from Hu sheep. This preparation method promotes pepsin activity, increases the degree of hydrolysis, and accelerates the enzymatic hydrolysis rate, thereby improving the antibacterial rate of the protease hydrolysate and increasing the yield of antimicrobial peptides derived from Hu sheep.

[0011] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The pepsin and chymotrypsin activities were low. The relevant evidence is that the yield of CN106011115A (corresponding to Comparative Example 1 of this invention) was only 5.2%, and the pepsin and chymotrypsin activities obtained by using the technical solution in this literature were low, and the pepsin hydrolysis activity was low. Further adjustments and improvements to the technical solution are needed.

[0012] In summary, there is an urgent need to develop an extraction process that can simplify industrial operations, reduce enzyme activity loss, improve the purity of rennet products, and obtain rennet products suitable for pharmaceutical applications such as chronic gastritis.

[0013] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: 1. Large amounts of organic reagents were needed for defatting, but these reagents inactivated the enzymes, leading to decreased enzyme activity. Later, degreasing with organic reagents was discontinued, which improved enzyme activity. 2. Initially, ammonium sulfate was used to precipitate rennet, but it was found to be time-consuming, labor-intensive, and required desalting. Later, ultrafiltration was used, which significantly reduced reagent consumption and labor. Summary of the Invention

[0014] The purpose of this invention is to provide: A method for preparing a compound rennet, and related technologies thereof, to solve technical problems such as improving yield, improving rennet activity, or combinations thereof.

[0015] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0016] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0017] Definitions of standard terms can be found in the references *Food Enzymology (Second Edition)* (Yang Ruijin and Zhao Wei, China Light Industry Press, 2025), *Livestock Physiology* (Fourth Edition, Chen Jie, China Agriculture Press), and *Internal Medicine (9th Edition)* (Ge Junbo and Xu Yongjian, People's Medical Publishing House).

[0018] Unless otherwise specified, conventional methods within the scope of the art, such as enzyme activity assays (rennet activity assay, pepsin activity assay), protein content assays (Coomassie brilliant blue assay), and rat chronic gastritis animal modeling, shall be used.

[0019] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0020] The “range” disclosed in this document takes the form of a lower limit and an upper limit. It can be one or more lower limits and one or more upper limits, respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this way are inclusive and composable; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is also expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if the minimum range values ​​are listed as 1 and 2, and if the maximum ranges are listed as 3, 4, and 5, then the following ranges are all expected: 1-2, 1-4, 1-5, 2-3, 2-4, and 2-5.

[0021] In this invention, unless otherwise specified, the numerical range "ab" represents a shortened representation of any combination of real numbers from a to b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed in this document, and "0-5" is simply a shortened representation of these numerical combinations.

[0022] The term “lamb’s fourth stomach” used in this article refers to the abomasum of lambs, which is the stomach among the four stomach chambers of ruminants that is actually responsible for secreting gastric juice and digesting proteins, and is the main secretory organ of rennet.

[0023] The term "rennet" used in this article refers to an aspartic protease mainly derived from the abomasum of ruminants, which can specifically cleave casein, causing milk and other dairy systems to coagulate, and is widely used in cheese preparation.

[0024] The term "homogenization" as used in this article refers to a sample processing operation that uses mechanical shearing, grinding, or other methods to break up tissues or cells, making the sample a relatively homogeneous suspension.

[0025] The term "activation" used in this article refers to the process of converting a proenzyme or a low-activity enzyme into a state with catalytic activity, which can generally be achieved through pH adjustment, changes in the ionic environment, or removal of inhibitory peptides.

[0026] The term "psepsis" used in this article refers to aspartic protease, which is mainly secreted by the chief cells of the gastric mucosa. It is activated under acidic conditions and can catalyze the hydrolysis of proteins into polypeptides. Its optimal pH is slightly acidic.

[0027] The term "enzyme activity" used in this article refers to the catalytic activity of an enzyme, which characterizes the ability of an enzyme to catalyze a reaction of a substrate per unit time, and is usually quantitatively characterized in units of enzyme activity.

[0028] The term "chronic gastritis" used in this article refers to chronic inflammatory lesions of the gastric mucosa caused by various etiologies. Pathologically, it can be seen as infiltration of lymphocytes and plasma cells in the gastric mucosa, and may be accompanied by changes such as glandular atrophy and intestinal metaplasia. It is a common digestive system disease in clinical practice.

[0029] The term "solid-to-liquid ratio" used in this article refers to the ratio of the mass of the solid raw material to the volume of the extraction solvent liquid, usually expressed in g / mL or g / L, and is an important process parameter in the extraction process.

[0030] In a first aspect, the present invention provides: a method for extracting a complex rennet, comprising the steps of: (1) After freezing and crushing the fourth stomach of the lamb, a mixed buffer solution was added to homogenize the fourth stomach of the lamb to obtain the homogenized fourth stomach of the lamb. (2) The homogenized lamb fourth stomach obtained in step (1) was added to a mixed buffer solution for extraction to obtain the extract; (3) After centrifugation and filtration, the extract obtained in step (2) is activated by adjusting the pH to obtain the activated extract. (4) Centrifuge the activated extract obtained in step (3), take the supernatant and perform ultrafiltration, concentration and drying to obtain the final product.

[0031] Wherein, the freezing temperature in step (1) is -24~-15℃; Preferably, the freezing temperature in step (1) is -20°C.

[0032] The size of the pulverized material in step (1) is 1-5 mm. 1-5mm 1-5mm; Preferably, the size of the pulverized material in step (1) is 3mm. 3mm 3mm.

[0033] Wherein, the mixed buffer solution in steps (1) and (2) is a sodium chloride-sodium acetate mixture; Preferably, the mass percentage of sodium chloride in the mixed buffer solution in steps (1) and (2) is 5%-12%, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or any range derived thereof; More preferably, the mass percentage of sodium chloride in the mixed buffer solution in steps (1) and (2) is 10%; Preferably, the mass percentage of sodium acetate in the mixed buffer solution in steps (1) and (2) is 1%-3%, for example, 1%, 1.5%, 2%, 2.5%, 3%, or any range derived therefrom; More preferably, the mass percentage of sodium acetate in the mixed buffer solution in steps (1) and (2) is 2%.

[0034] In step (1), the ratio of feed to liquid in the lamb's fourth stomach (g:mL) is 1:0.5-1.5, for example, 1:0.5, 1:0.7, 1:0.9, 1:1.0, 1:1.2, 1:1.3, 1:1.5, or any range derived therefrom; Preferably, the ratio of lamb's fourth stomach to mixed buffer solution (g:mL) in step (1) is 1:1.

[0035] In step (2), the ratio of the homogenized lamb fourth stomach to the mixed buffer solution (g:mL) is 1:5-10, for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any range derived therefrom. Preferably, in the extraction step (2), the ratio of the homogenized lamb fourth stomach to the mixed buffer solution (g:mL) is 1:8.

[0036] The extraction time in step (2) is 10-24h, for example 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, or any range derived therefrom; Preferably, the extraction time in step (2) is 20 hours.

[0037] In some specific embodiments, step (2) further includes a step of refrigerating the obtained extract after the extraction process; Preferably, the refrigeration temperature is 0-5°C, for example, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, or any range derived therefrom; More preferably, the refrigeration temperature is 4°C.

[0038] Preferably, the refrigeration treatment is an overnight refrigeration treatment.

[0039] Wherein, the centrifugation speed in step (3) is 2000-4000 rpm, for example 2000 rpm, 2400 rpm, 2800 rpm, 3000 rpm, 3200 rpm, 3600 rpm, 4000 rpm, or any range derived therefrom; Preferably, the centrifugation speed in step (3) is 3000 rpm.

[0040] The centrifugation time in step (3) is 5-40 min, for example 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, or any range derived therefrom; Preferably, the centrifugation time in step (3) is 30 minutes.

[0041] The filtration in step (3) involves filtering the supernatant obtained after centrifugation to remove lumpy impurities and grease.

[0042] Among them, the pH adjustment in step (3) is divided into primary pH adjustment and secondary pH adjustment, and the activation step is between primary pH adjustment and secondary pH adjustment; Preferably, the range of the pH adjustment is 2.5-4.5, for example, 2.5, 2.8, 3.0, 3.2, 3.5, 3.8, 4.0, 4.2, 4.5, or any range derived therefrom; More preferably, the initial pH adjustment is 3.0.

[0043] In some specific embodiments, the solvent for adjusting the pH in the first step is HCl; Specifically, the concentration of the HCl is 4-6 mol / L; Furthermore, the concentration of the HCl is 5 mol / L.

[0044] Preferably, the activation duration is 15-30 minutes, for example, 15 minutes, 18 minutes, 20 minutes, 22 minutes, 25 minutes, 28 minutes, 30 minutes, or any range derived therefrom; More preferably, the activation duration is 20 minutes.

[0045] Preferably, the range of the secondary pH adjustment is 5.0-5.5, for example 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or any range derived therefrom; More preferably, the secondary pH adjustment is 5.3.

[0046] Wherein, the centrifugation speed in step (4) is 2000-4000 rpm, for example 2000 rpm, 2400 rpm, 2800 rpm, 3000 rpm, 3200 rpm, 3600 rpm, 4000 rpm, or any range derived therefrom; Preferably, the centrifugation speed in step (4) is 3000 rpm.

[0047] The centrifugation time in step (4) is 5-40 min, for example 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, or any range derived therefrom; Preferably, the centrifugation time in step (4) is 30 minutes.

[0048] Wherein, the pore size of the ultrafiltration in step (4) is 1000-10000 Da, for example 1000 Da, 1500 Da, 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da, 5000 Da, 5500 Da, 6000 Da, 6500 Da, 7000 Da, 7500 Da, 8000 Da, 8500 Da, 9000 Da, 9500 Da, 10000 Da, or any range derived therefrom; Preferably, the molecular weight obtained by ultrafiltration in step (4) is 5000 Da.

[0049] Wherein, the concentration described in step (4) is to concentrate to 1 / 8-1 / 15 of the original volume, for example 1 / 8, 1 / 9, 1 / 10, 1 / 11, 1 / 12, 1 / 13, 1 / 14, 1 / 15, or any range derived thereof; Preferably, the concentration in step (4) is to concentrate to 1 / 10 of the original volume.

[0050] Wherein, the drying in step (4) is freeze drying; Preferably, the drying temperature in step (4) is -60 to -40°C, for example -60°C, -55°C, -50°C, -48°C, -45°C, -42°C, -40°C, or any range derived therefrom; More preferably, the drying temperature in step (4) is -50°C.

[0051] In some specific embodiments, the extraction method of the compound rennet includes the following steps: (1) Take the frozen lamb fourth stomach, cut it into small pieces, add a 1:1 (relative to the wet weight of the fourth stomach) mixture of 10% sodium chloride and 2% sodium acetate buffer solution and homogenize it. (2) Add 8 times the volume (relative to the wet weight of the fourth stomach) of 10% sodium chloride and 2% sodium acetate mixed buffer solution and stir for 20 hours. Place in a refrigerator at 4 degrees Celsius overnight. (3) Centrifuge at 3000 rpm for 30 min at room temperature. After centrifugation, filter the supernatant with gauze to remove lumpy impurities and grease. (4) Adjust the pH to 3.0 with 5 mol HCl, activate for 15~30 min, and then adjust the pH back to 5.3; (5) The material is centrifuged at 3000 rpm for 30 min at room temperature. The supernatant is then ultrafiltered using an ultrafiltration membrane and concentrated to 1 / 10 of the original volume. The solution is then freeze-dried to obtain the final product. (6) The product obtained in step (5) is tested according to the pharmacopoeia standards.

[0052] In some specific embodiments, the compound rennet obtained based on the above-described extraction method for compound rennet has a rennet activity ≥6000 Ru / g and a pepsin activity ≥2000 IU / g.

[0053] Preferably, the rennet activity can reach 6800 Ru / g; Preferably, the pepsin activity can reach 2200 IU / g.

[0054] Secondly, the present invention provides: a compound rennet, wherein the rennet activity is ≥6000 Ru / g and the pepsin activity is ≥2000 iu / g.

[0055] In some specific embodiments, the rennet activity can reach 6800 Ru / g; In some specific embodiments, the pepsin activity can reach 2200 iu / g.

[0056] Thirdly, the present invention provides the application of the extraction method or the compound rennet in the preparation of products related to the treatment and / or prevention of gastric diseases.

[0057] The stomach ailments mentioned include, but are not limited to, chronic gastritis, abdominal distension, and diarrhea; The products mentioned include: pharmaceuticals and food. Preferably, the product is a drug.

[0058] The phrase "the ratio of the lamb's fourth stomach to the mixed buffer solution in step (1) is 1:0.5-1.5" is a generalization derived from the foregoing explanation and / or the phrases "the ratio of the added feed to the buffer solution is 1:1" in Example 1, "the ratio of the added feed to the buffer solution is 1:0.5" in Example 2, and "the ratio of the added feed to the buffer solution is 1:1.5" in Example 3. Therefore, those skilled in the art can reasonably infer that "the ratio of the lamb's fourth stomach to the mixed buffer solution in step (1) is 1:0.5-1.5", its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of this invention.

[0059] The statement that "the mass percentage of sodium chloride in the mixed buffer solution described in steps (1) and (2) is 5%-12% and the mass percentage of sodium acetate is 1%-3%" is a generalization derived from the foregoing explanation and / or the "10% sodium chloride and 2% sodium acetate mixed buffer solution" in Example 1, the "12% sodium chloride and 1% sodium acetate mixed buffer solution" in Example 2, and the "5% sodium chloride and 3% sodium acetate mixed buffer solution" in Example 3. Therefore, those skilled in the art can reasonably infer that "the mass percentage of sodium chloride in the mixed buffer solution described in steps (1) and (2) is 5%-12% and the mass percentage of sodium acetate is 1%-3%", its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of this invention.

[0060] The statement "In step (2), the ratio of the homogenized lamb fourth stomach to the mixed buffer solution is 1:5-10" is a generalization derived from the foregoing explanation and / or the statements in Example 1 ("adding 8 times the volume of the lamb fourth stomach"), Example 2 ("adding 5 times the volume of the lamb fourth stomach"), and Example 3 ("adding 10 times the volume of the lamb fourth stomach"), etc., in Example 1. Therefore, those skilled in the art can reasonably infer that the statement "In step (2), the ratio of the homogenized lamb fourth stomach to the mixed buffer solution is 1:5-10," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of this invention.

[0061] The statement "the amount of HCl used is 4-6 mol / L" is a generalization derived from the foregoing explanation and / or "5 mol HCl" in Example 1, "4 mol HCl" in Example 2, and "6 mol HCl" in Example 3. Therefore, those skilled in the art can reasonably infer that "the amount of HCl used is 4-6 mol / L," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of this invention.

[0062] The phrase "the range of pH adjustment at one time is 2.5-4.5" is a generalization derived from the foregoing explanation and / or the phrases "adjusting pH to 3.0 for one pH adjustment" in Example 1, "adjusting pH to 4.5 for one pH adjustment" in Example 2, and "adjusting pH to 2.5 for one pH adjustment" in Example 3, etc. Therefore, those skilled in the art can reasonably infer that "the range of pH adjustment at one time is 2.5-4.5," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0063] The phrase "the range of secondary pH adjustment is 5.0-5.5" is a generalization derived from the foregoing explanation and / or from "secondary pH adjustment back to 5.3" in Example 1, "secondary pH adjustment back to 5.5" in Example 2, and "secondary pH adjustment back to 5.0" in Example 3. Therefore, those skilled in the art can reasonably infer that "the range of secondary pH adjustment is 5.0-5.5," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0064] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: (1) High enzyme activity and higher purity: The product obtained by the method of the present invention has a rennet activity ≥6000 Ru / g and a pepsin activity ≥2000 iu / g; (2) Compared with traditional processes: the process is simple, the extraction buffer is different, no organic reagents are needed for defatting, and no heating is required to reduce energy consumption; (3) Compared with the prior art, the present invention has better technical effects in terms of pepsin activity, chymotrypsin activity and protein hydrolysis activity.

[0065] According to experimental tests, the present invention increases the pepsin activity from 695 IU / g in the prior art to over 1800 IU / g.

[0066] According to experimental tests, the present invention increases the activity of rennet from 805 Ru / g in the prior art to over 5200 Ru / g.

[0067] According to experimental tests, this invention increases protein hydrolysis activity from 820 U / g in the prior art to over 1900 U / g. Furthermore, based on the present invention: (1) Based on the comparison of Examples 1-3 and Comparative Examples 2-4, the present invention achieves new technical effects by using a combination of specific pH, specific types of buffer systems, and specific buffer concentrations: under simplified process conditions that do not require degreasing with organic solvents or long-term dialysis, the product achieves a suitable industrial yield while obtaining high pepsin activity, rennet coagulation activity, and protein hydrolysis activity. The product impurity level is controllable, meeting the quality requirements for pharmaceutical raw materials for chronic gastritis. The combined technical effect is superior to the sum of the effects of each individual technique. Deviating from the pH conditions, changing the type of buffer solution, or exceeding the specified buffer solution concentration alone cannot simultaneously achieve the comprehensive technical effects of yield, multi-enzyme activity, and process scalability.

[0068] (2) Based on the comparison of Examples 1-3 and Comparative Examples 2-4, the present invention purposefully selects a narrow range not mentioned in the prior art from the wide range of pH, buffer salt types and buffer concentration parameters disclosed in the prior art: a specific pH range, a specific buffer salt system and a specific concentration range of the buffer salt, and achieves unexpected technical effects. Under the premise of simplifying the process, eliminating the risk of organic solvent residue and shortening the production cycle, it can synergistically ensure the dissolution and activity retention of rennet and pepsin, and obtain high rennet activity, pepsin activity and protein hydrolysis activity at an industrially achievable yield level. Attached Figure Description

[0069] Figure 1 The image shows the SDS-PAGE electrophoresis diagram of the lyophilized rennet obtained in Example 1; where M in the diagram is the marker. Detailed Implementation

[0070] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0071] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0072] Specific raw material information is shown in Table 1: Table 1. Raw Material Information

[0073] Example 1 A method for extracting a complex rennet, specifically comprising: (1) Take the frozen lamb fourth stomach, cut it into small pieces, add a 10% sodium chloride and 2% sodium acetate mixed buffer solution with a feed-to-liquid ratio of 1:1 (relative to the wet weight of the fourth stomach) to homogenize, and obtain the homogenized lamb fourth stomach. (2) Add 8 times the volume (relative to the wet weight of the fourth stomach) of the lamb after homogenization in step (1) to the fourth stomach and stir for 20 hours to obtain the extract. Place it in a refrigerator at 4 degrees overnight. (3) Centrifuge the extract obtained in step (2) at room temperature at 3000 rpm for 30 min. After centrifugation, filter the supernatant with gauze to remove blocky impurities and oil. (4) Adjust the pH to 3.0 with 5 mol HCl for the first pH adjustment, activate for 20 min, and then adjust the pH back to 5.3 for the second time; (5) The material is centrifuged at 3000 rpm for 30 min at room temperature. The supernatant is then ultrafiltered using an ultrafiltration membrane and concentrated to 1 / 10 of the original volume. Finally, it is freeze-dried.

[0074] The SDS-PAGE electrophoresis image of the lyophilized rennet obtained in Example 1 is shown below. Figure 1 .

[0075] Example 2 A method for extracting a complex rennet, specifically comprising: (1) Take the frozen lamb fourth stomach, cut it into small pieces, add a mixture of 12% sodium chloride and 1% sodium acetate in a feed-to-liquid ratio of 1:0.5 (relative to the wet weight of the fourth stomach) to homogenize it, and obtain the homogenized lamb fourth stomach. (2) Add 5 times the volume (relative to the wet weight of the fourth stomach) of the lamb after homogenization in step (1) to the fourth stomach and stir for 10 hours to obtain the extract. Place it in a refrigerator at 4 degrees overnight. (3) Centrifuge the extract obtained in step (2) at room temperature at 2000 rpm for 30 min. After centrifugation, filter the supernatant with gauze to remove blocky impurities and oil. (4) Adjust the pH to 4.5 with 4 mol HCl for the first pH adjustment, activate for 20 min, and then adjust the pH back to 5.5 for the second time; (5) The material is centrifuged at 3000 rpm for 30 min at room temperature. The supernatant is then ultrafiltered using an ultrafiltration membrane and concentrated to 1 / 8 of the original volume. Finally, it is freeze-dried.

[0076] Example 3 A method for extracting a complex rennet, specifically comprising: (1) Take the frozen lamb fourth stomach, cut it into small pieces, add 5% sodium chloride and 3% sodium acetate mixed buffer solution with a feed-to-liquid ratio of 1:1.5 (relative to the wet weight of the fourth stomach) to homogenize, and obtain the homogenized lamb fourth stomach. (2) Add 10 times the volume (relative to the wet weight of the fourth stomach) of the lamb after homogenization in step (1) to a mixed buffer of 5% sodium chloride and 3% sodium acetate and stir for 24 hours to obtain the extract. Place it in a refrigerator at 4 degrees overnight. (3) Centrifuge the extract obtained in step (2) at room temperature at 4000 rpm for 30 min. After centrifugation, filter the supernatant with gauze to remove blocky impurities and oil. (4) Adjust the pH to 2.5 with 6 mol HCl for the first pH adjustment, activate for 20 min, and then adjust the pH back to 5.0 for the second time; (5) The material is centrifuged at 3000 rpm for 30 min at room temperature. The supernatant is then ultrafiltered using an ultrafiltration membrane and concentrated to 1 / 15 of the original volume. Finally, it is freeze-dried to obtain the final product.

[0077] Comparative Example 1 The rennet prepared according to the technical solution described in Example 6 of Patent Document CN106011115A.

[0078] Comparative Example 2 Compared with Example 1, the only difference is that the pH for the first adjustment is changed to 5.0 and the pH for the second adjustment is changed to 4.5. The other conditions are basically the same as those in Example 1.

[0079] Comparative Example 3 Compared with Example 1, the only difference is that the mixing buffer in steps (1) and (2) is replaced with 0.1 mol / L phosphate buffer, and the other conditions are basically the same as in Example 1.

[0080] Comparative Example 4 Compared with Example 1, the only difference is that the mass percentage of sodium chloride in the mixed buffer solution in steps (1) and (2) is replaced with 15%, and the mass percentage of sodium acetate is replaced with 5%. The other conditions are basically the same as in Example 1.

[0081] Detection Example 1 1. Detection method 1-1 Detection method for pepsin enzyme activity: Refer to the potency determination method for pepsin under the "Pepsin" section of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part II (UV-Vis spectrophotometry, General Chapter 0401). (1) Reagent type: Preparation of hydrochloric acid solution: Take 65 mL of 1 mol / L hydrochloric acid solution and add water to 1000 mL.

[0082] Preparation of reference solution: Accurately weigh an appropriate amount of tyrosine dried to constant weight at 105℃, and add the above hydrochloric acid solution to prepare a solution containing 0.5 mg per 1 mL.

[0083] Preparation of the test solution: Take an appropriate amount of this product, accurately weigh it, and prepare a solution containing about 0.2-0.4 units per 1 mL using the above hydrochloric acid solution.

[0084] (2) Measurement steps: Take 6 test tubes. Accurately add 1 mL of the reference solution to 3 tubes and 1 mL of the test solution to the other 3 tubes. Place them in a water bath at 37℃±0.5℃ and incubate for 5 minutes. Accurately add 5 mL of hemoglobin test solution preheated to 37℃±0.5℃, shake well, and accurately time the reaction. Incubate for 10 minutes in a water bath at 37℃±0.5℃. Immediately add 5 mL of 5% trichloroacetic acid solution, shake well, filter, and collect the filtrate for later use.

[0085] Take two more test tubes and accurately add 5 mL of hemoglobin test solution to each. Incubate in a water bath at 37℃±0.5℃ for 10 minutes, and then accurately add 5 mL of 5% trichloroacetic acid solution. Add 1 mL of the test solution to one tube and 1 mL of the above hydrochloric acid solution to the other tube. Shake well, filter, and take the filtrate as the test blank and the control blank, respectively.

[0086] Measure the absorbance at a wavelength of 275 nm using spectrophotometry (Appendix IVA) and calculate the average absorbance of the reference standard. Average absorbance of the test sample Calculate using the following formula:

[0087] In the formula: : Average absorbance of the reference standard; : Average absorbance of the test sample; Ws: Amount of tyrosine (μg) in 1 mL of reference solution; W: Sample size (g); n: Dilution factor of the test sample; 181.19: Molar mass of tyrosine.

[0088] Unit definition: Under the above conditions, the amount of enzyme that can catalyze the hydrolysis of hemoglobin to produce 1 μmol of tyrosine per minute is defined as 1 unit of protease activity (U).

[0089] 1-2 Methods for detecting rennet activity: The procedure for determining the potency of rennet should be performed according to the national drug standard for compound rennet (WS1-XG-2002). The specific steps are as follows: (1) Reagent preparation: ① Calcium chloride-sodium acetate solution: Take 2.05g of sodium acetate and 1.11g of calcium chloride, dissolve them in water and prepare a 1000 mL solution, and adjust the pH to 6.3±0.1.

[0090] ②Substrate solution: Take 12g of whole milk powder, add an appropriate amount of the above calcium chloride-sodium acetate solution, grind evenly, transfer to a 100 mL volumetric flask, add calcium chloride-sodium acetate solution to make up to the mark, and shake well; the substrate solution should be freshly prepared before use.

[0091] ③ pH 6.3 phosphate buffer: Dissolve 7.8 g of sodium dihydrogen phosphate and 2.7 g of disodium hydrogen phosphate in water to prepare 1000 mL, and adjust the pH to 6.3±0.05.

[0092] (2) Preparation of standard solutions Accurately weigh the rennet standard, dissolve and dilute it with pH 6.3 phosphate buffer to prepare a standard solution containing 0.4-0.6 rennet activity units (RU) per 1 mL; keep the solution below 20°C and use it within 2 hours after preparation.

[0093] (3) Preparation of test solution Accurately weigh approximately 0.1 g of the sample to be tested, place it in a mortar, add a small amount of pH 6.3 phosphate buffer and grind to disperse evenly, transfer to a 100 mL volumetric flask, dilute to the mark with the phosphate buffer, and shake well; keep the test solution below 20°C and prepare fresh before use.

[0094] (4) Measurement steps Accurately pipette 10 mL of substrate solution into a test tube and incubate in a constant temperature water bath at 30 ± 0.5 °C for 10 min. Accurately add 1 mL of standard solution, shake well, and immediately start timing. Use a glass rod to continuously dip into the solution in the tube and let it flow down the test tube wall to form a uniform thin layer. When visible particles appear in the thin layer, the curdling endpoint is reached. Record the time from the addition of solution to the appearance of curd particles. Perform three parallel determinations on the same solution. The relative standard deviation (RSD) of the three determinations should not exceed 5%. Take the average of the three determinations as the average curdling time Ts (in seconds) of the standard solution.

[0095] Take 1 mL of the test solution and follow the same procedure as above to obtain the average curdling time Tx (unit: s).

[0096] Note: The best reproducibility is achieved when the curdling time is controlled within the range of 180-240 s; if it exceeds this time range, the dilution factor of the test solution needs to be adjusted and the test repeated.

[0097] (5) Calculation formula

[0098] In the formula: S: Rennet standard, activity indicated in RU / mg; Ts: Average coagulation time of the standard solution, in seconds; Tx: Average coagulation time of the test solution, in seconds; Ws: Mass of rennet standard in 1 mL of standard solution, in g; W: Sample mass of the test sample, in grams; n: Dilution factor of the test sample.

[0099] 1-3 Calculation method of yield Yield = Weight of compound rennet lyophilized powder (g) / Wet weight of lamb's fourth ventricle (g) 100%; 1-4 Detection of protein hydrolysis activity: The Folin-phenol method, referring to SB / T 10317-1999 Protease Activity Assay, is as follows: Accurately weigh 20 mg of the sample to be tested, accurate to 0.0002 g, dissolve in a small amount of distilled water, mix well, centrifuge at 10000 rpm for 2 minutes, pour off the supernatant, dissolve the sediment in a small amount of water, shake well, centrifuge, repeat this process 3 times, transfer the supernatant to a 10 mL volumetric flask, dilute to volume with water, and shake well; take 4 test tubes, add 1 mL of enzyme solution to each, take 1 tube as a blank, add 5 mL of 0.44 g / L trichloroacetic acid solution, and use the other 3 tubes as test tubes, each adding... Add 5 mL of casein, shake well, and incubate in a 35°C water bath for 10 minutes. Remove the test tube, add 5 mL of 0.44 g / L trichloroacetic acid to each of the three test tubes, and add 5 mL of casein to the blank tube. Continue incubation for 10 minutes, filter the precipitate, and take 2 mL of the supernatant from each tube. Add 5 mL of 0.55 mol / L sodium carbonate and 1 mL of Folin-phenol reagent, and develop the color in a 35°C water bath for 20 minutes. Then, measure the absorbance at 660 nm using a UV spectrophotometer. Calculate the protein hydrolysis activity using the following formula: ; In the formula: A represents the average absorbance value at 660 nm; K represents the amount of tyrosine (1 g / mL) with an absorbance of 1 in the standard curve; 4 represents the total volume of the enzyme-catalyzed reaction (mL); 10 represents the reaction time (minutes); n is the dilution factor of the enzyme solution.

[0100] The enzyme activity test results are as follows: Table 2.

[0101] Verification of technical effectiveness and / or analysis of technical problem solving Existing rennet extraction processes suffer from drawbacks such as significant enzyme activity loss, insufficient product purity, complex processes requiring organic solvent defatting, and high energy consumption, resulting in products that are difficult to meet the needs of pharmaceutical applications. This invention optimizes the extraction buffer system, eliminating the organic defatting step and employing mild extraction conditions to achieve the preparation of a complex enzyme extract. This invention significantly improves pepsin activity, rennet activity, and proteolytic activity while maintaining industrial-scale yield; simultaneously, the process eliminates the need for organic solvent defatting and heating, simplifying the process, reducing energy consumption, and producing a product suitable for pharmaceutical applications related to chronic gastritis.

[0102] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for extracting a compound rennet, characterized in that, Including the following steps: (1) After freezing and crushing the fourth stomach of the lamb, a mixed buffer solution was added to homogenize the fourth stomach of the lamb to obtain the homogenized fourth stomach of the lamb. (2) The homogenized lamb fourth stomach obtained in step (1) was added to a mixed buffer solution for extraction to obtain the extract; (3) After centrifugation and filtration, the extract obtained in step (2) is activated by adjusting the pH to obtain the activated extract. (4) Centrifuge the activated extract obtained in step (3), take the supernatant and perform ultrafiltration, concentration and drying to obtain the final product; The mixed buffer solution used in steps (1) and (2) is a sodium chloride-sodium acetate mixture.

2. The extraction method according to claim 1, characterized in that, The freezing temperature in step (1) is -24~-15℃; The size of the pulverized material in step (1) is 1-5mm. 1-5mm 1-5mm; In steps (1) and (2), the mass percentage of sodium chloride in the mixed buffer solution is 5%-12%, and the mass percentage of sodium acetate is 1%-3%.

3. The extraction method according to claim 2, characterized in that, The freezing temperature in step (1) is -20℃; the mass percentage of sodium chloride in the mixed buffer solution in steps (1) and (2) is 5%-10%, and the mass percentage of sodium acetate is 2%.

4. The extraction method according to claim 1, characterized in that, In step (1), the ratio of the lamb's fourth stomach to the mixed buffer solution is 1g:0.5-1.5mL; In step (2), the ratio of the homogenized lamb fourth stomach to the mixed buffer solution is 1g:5-10mL; the extraction time in step (2) is 10-24h. Step (2) after extraction further includes the step of refrigerating the obtained extract; the refrigeration temperature is 0-5℃.

5. The extraction method according to claim 1, characterized in that, Step (3) involves adjusting the pH, which is divided into primary pH adjustment and secondary pH adjustment. The activation step is between the primary pH adjustment and the secondary pH adjustment. The range of the first pH adjustment is 2.5-4.5; the activation duration is 15-30 min; and the range of the second pH adjustment is 5.0-5.

5.

6. The extraction method according to claim 1, characterized in that, In steps (3) and (4), the centrifugation speed is 2000-4000 rpm and the duration is 5-40 min; The pore size of the ultrafiltration in step (4) is 1000-10000 Da; The concentration described in step (4) is to concentrate to 1 / 8 to 1 / 15 of the original volume; The drying in step (4) is freeze drying; the drying temperature in step (4) is -60~-40℃.

7. The extraction method according to claim 1, characterized in that, The compound rennet obtained by this extraction method has a rennet activity ≥6000 Ru / g and a pepsin activity ≥2000 IU / g.

8. A compound rennet, characterized in that, The compound rennet is prepared by the extraction method described in any one of claims 1-7, and its rennet activity is ≥6000 Ru / g, and its pepsin activity is ≥2000 IU / g.

9. The use of the extraction method according to any one of claims 1-7 or the compound rennet according to claim 8 in the preparation of products related to the treatment and / or prevention of gastric diseases.

10. The application according to claim 9, characterized in that, The stomach ailments include chronic gastritis, bloating, and diarrhea.

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