A method for preparing a bitter gourd seed peptide by enzymatic hydrolysis

Abstract

The application discloses a preparation method of balsam pear seed peptides by enzymolysis, and belongs to the technical field of peptide preparation. The preparation method comprises the following steps: after balsam pear seeds are crushed, water is added for soaking treatment to obtain material A; cellulase and pectinase are added to the material A for pretreatment, enzyme inactivation is performed, and a primary enzymolysis liquid is obtained; compound protease is added to the primary enzymolysis liquid for step-by-step enzymolysis, enzyme inactivation is performed, solid-liquid separation is performed, and an enzymolysis liquid is obtained; the enzymolysis liquid is dried to obtain polypeptide powder; the polypeptide powder is extracted by ethanol, filtered, and dried to obtain the balsam pear seed peptides; and the compound protease in step (3) is bromelain, pepsin and carboxypeptidase B. Compared with the prior art, the balsam pear seed peptides prepared by the application have better alpha-glucosidase inhibiting capacity, are close to chemical hypoglycemic drugs, can be used as natural alpha-glucosidase inhibitor raw materials, and have lower side effects.

Description

Technical Field

[0001] This invention belongs to the field of peptide preparation technology, and relates to a method for preparing bitter melon seed peptides by enzymatic hydrolysis. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia, with type 2 diabetes mellitus (T2DM) being the most common, primarily affecting adults. The core pathogenesis of diabetes is insulin resistance or insufficient insulin secretion, which can lead to serious complications affecting the cardiovascular system, retina, kidneys, and nervous system in the long term. Currently, T2DM is incurable; clinical management primarily involves controlling blood sugar through diet, exercise, medication, or a combination of these methods to manage the condition.

[0003] Alpha-glucosidase is a key intestinal enzyme involved in the final digestion of carbohydrates, playing a crucial role in regulating postprandial blood glucose levels. Clinically used alpha-glucosidase inhibitors primarily work by blocking the cleavage of disaccharides into absorbable monosaccharides, effectively slowing intestinal glucose absorption and inhibiting the postprandial spike in blood glucose, thus playing a role in the prevention and treatment of diabetes. However, commonly used alpha-glucosidase inhibitors (e.g., acarbose, voglibose, miglitol) often cause side effects such as bloating, vomiting, and gastrointestinal disturbances, highlighting the urgent need to develop novel inhibitors with lower adverse reactions. Therefore, current research focuses on the application value of natural products in the adjunctive treatment of type 2 diabetes mellitus (T2DM). Polysaccharides, peptides, and polyphenols extracted from plant, animal, and marine sources have been shown to possess alpha-glucosidase inhibitory activity, showing promise as a replacement for traditional drugs and becoming a future trend in drug development.

[0004] Bitter melon seeds are the kernels of the bitter melon plant (Momordica charantia L.), belonging to the genus Momordica of the Cucurbitaceae family. Bitter melon seeds are rich in active ingredients, such as proteins, polypeptides, and oils, as well as small amounts of flavonoids and saponins. They possess biological activities including lowering blood sugar, anti-tumor activity, lowering blood pressure, lowering blood lipids, and antibacterial activity. The development and utilization of the medicinal value of bitter melon seeds has broad prospects.

[0005] Patent document CN112618406A (published on April 9, 2021) discloses a bitter melon seed peptide powder with free radical scavenging activity. The bitter melon seed peptide powder is obtained by sequentially defatting bitter melon seeds, extracting proteins, enzymatically hydrolyzing proteins, separating and purifying, concentrating, and drying. It was found that bitter melon seed peptides obtained by enzymatic hydrolysis with different biological enzymes all exhibited good antioxidant activity. However, this invention did not investigate the inhibitory activity of the bitter melon seed peptide powder on α-glucosidase.

[0006] Patent document CN114107414B (published on March 15, 2024) discloses a method for preparing bitter melon polypeptides by fermentation. The method involves pretreating bitter melon seeds, activating Aspergillus oryzae strains, and expanding the Aspergillus oryzae liquid culture medium. The expanded strains are inoculated into the pretreated bitter melon seed material for fermentation. After fermentation, the fermented material is dried and pulverized. Bitter melon seed polypeptides are extracted with ethanol solution, collected by centrifugation, filtered, and distilled to obtain an aqueous solution of bitter melon polypeptides. The solution volume is adjusted with water, the pH is adjusted with ammonia, centrifuged, and the supernatant is collected. The pH is adjusted with acid, anhydrous ethanol is added, precipitation is initiated, the precipitate is collected, and dried to obtain the bitter melon polypeptides. The prepared low-molecular-weight bitter melon polypeptides have a high reconstitution rate, a good taste without greasiness, and a strong ability to regulate blood sugar. However, this invention did not investigate the inhibitory activity of the bitter melon polypeptide powder on α-glucosidase.

[0007] Therefore, there is an urgent need for a method to prepare bitter melon seed peptides with α-glucosidase inhibitory activity. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a method for preparing bitter melon seed peptides via enzymatic hydrolysis.

[0009] 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.

[0010] 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.

[0011] The definitions of standard chemical terms can be found in the 2025 edition of the Chinese Pharmacopoeia.

[0012] Unless otherwise stated, conventional methods within the scope of the art, such as methods for detecting α-glucosidase inhibitory activity, shall be used.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] As used herein, the term "about" refers to a quantity or range of values ​​that is an approximation within experimental variability (or within statistical experimental error). In this invention, the term "about" shall have the meaning of being within 10% of the specified value or range, preferably within 5%.

[0017] The term "cellulase" used in this article refers to a complex hydrolytic enzyme system composed of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. It can specifically hydrolyze the β-1,4-glucosidic bonds of cellulose in plant cell walls, degrading macromolecular cellulose stepwise into cellobiose and glucose. It is commonly prepared by fermentation using industrial strains (Trichoderma and Aspergillus niger).

[0018] The term "pectinase" used in this article includes three types: pectin esterase, polygalacturonase, and pectin lyase. These enzymes specifically hydrolyze the ester and glycosidic bonds of pectin polysaccharides in plant intercellular matrix, breaking down pectin into small molecule galacturonic acid. Acidic pectinase derived from Aspergillus niger is commonly used in food / biological extraction.

[0019] The term "bromelain" used in this article refers to a thiol-type plant endopeptidase extracted from the stems and fruits of pineapple. Its active center depends on the cysteine ​​thiol group, which preferentially hydrolyzes the carboxyl-terminal peptide bonds of basic amino acids such as lysine and arginine in polypeptide chains, breaking large protein molecules to generate medium and short chain polypeptides.

[0020] The term "chymotrypsin" used in this article refers to a serine-type endopeptidase extracted from porcine / bovine pancreas that specifically breaks the carboxyl side peptide bonds of hydrophobic / aromatic amino acids such as phenylalanine, tyrosine, tryptophan, and leucine on polypeptide chains, cleaving long peptides into medium and short peptides.

[0021] The term "carboxypeptidase B" used in this article comes from the metalloexopeptidase (containing a zinc ion cofactor) of the animal pancreas. It cleaves two basic amino acids, lysine and arginine, sequentially from the C-terminus (carboxyl terminus) of the polypeptide, gradually pruning the terminal residues of the polypeptide and converting medium- to long peptides into small, active peptides.

[0022] The term "solid-liquid separation" as used in this article refers to the conventional operation of separating the solid phase and the liquid phase in a system to obtain a clear liquid phase or solid product, including but not limited to centrifugation, filtration, vacuum filtration, pressure filtration, membrane separation, etc.

[0023] The term "crushing" as used in this article refers to the conventional operation of breaking solid raw materials through mechanical force to reduce their particle size, increase their specific surface area, and improve their extraction and reaction efficiency, including but not limited to shear crushing, grinding crushing, cryogenic crushing, ultrafine crushing, and air jet crushing.

[0024] The term "drying" as used in this article refers to the routine operation of removing free moisture or organic solvents from materials by controlling environmental conditions such as temperature, airflow, and pressure, thereby reducing the moisture content of the materials and maintaining the stability of the physicochemical properties of the formulation, including but not limited to hot air drying, vacuum drying, low-temperature air drying, fluidized bed drying, etc.

[0025] The term "enzyme inactivation" as used in this article refers to conventional operations that alter the spatial structure of an enzyme protein, cause it to lose its catalytic activity, or terminate the enzymatic reaction by changing the temperature, pH, or adding inhibitors. These operations include, but are not limited to, high-temperature inactivation, boiling water bath inactivation, acid-base inactivation, and inhibitor inactivation.

[0026] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides: a method for preparing bitter melon seed peptides by enzymatic hydrolysis, comprising the following steps: (1) After the bitter gourd seeds are crushed, they are soaked in water to obtain material A; (2) Add cellulase and pectinase to material A for pretreatment to inactivate the enzymes and obtain primary enzymatic hydrolysate; (3) Add a complex protease to the primary enzymatic hydrolysate for stepwise enzymatic hydrolysis, inactivate the enzyme, separate the solid and liquid, and obtain the enzymatic hydrolysate; (4) The enzymatic hydrolysate is dried to obtain polypeptide powder; (5) The polypeptide powder is extracted with ethanol, filtered, and dried to obtain the final product.

[0027] In some implementations, the mass ratio of bitter gourd seeds to water in step (1) is 1:6-12. For example, the mass ratio of bitter gourd seeds to water can be selected as 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, or a range derived therefrom.

[0028] In some implementations, the soaking in step (1) is: soaking at 45-55°C for 6-12 hours; for example, the soaking temperature can be selected as 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, or a range derived therefrom; the soaking time can be selected as 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or a range derived therefrom.

[0029] In some embodiments, the amount of cellulase used in step (2) is 0.01%-0.05% of the weight of bitter gourd seeds; for example, it can be selected as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, or a range derived therefrom.

[0030] In some embodiments, the amount of pectinase used in step (2) is 0.005%-0.01% of the weight of bitter gourd seeds; for example, it can be selected as 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, or a range derived therefrom.

[0031] In some implementations, the pretreatment in step (2) is: enzymatic hydrolysis at 45-55°C for 30-60 min, pH The range is 4.5-5; for example, the enzymatic hydrolysis temperature can be selected as 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, or a range derived therefrom; the enzymatic hydrolysis time can be selected as 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min, 49min, 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min, 60min, or a range derived therefrom; the enzymatic hydrolysis pH can be selected as 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or a range derived therefrom.

[0032] In some embodiments, the amount of the complex protease used in step (3) is 0.03%-0.12% of the weight of bitter gourd seeds; it can be selected as 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, or a range derived therefrom.

[0033] In some embodiments, the complex protease in step (3) is bromelain, chymotrypsin, and carboxypeptidase B; preferably, the mass ratio of bromelain, chymotrypsin, and carboxypeptidase B is 1-5:1:0.2-0.5; for example, the mass ratio of bromelain, chymotrypsin, and carboxypeptidase B can be 1:1:0.2, 1:1:0.3, 1:1:0.4, 1:1:0.5, 2:1:0.2, 2:1:0.3, 2:1:0.4, 2:1:0.5, 3:1:0.2, 3:1:0.3, 3:1:0.4, 3:1:0.5, 4:1:0.2, 4:1:0.3, 4:1:0.4, 4:1:0.5, 5:1:0.2, 5:1:0.3, 5:1:0.4, 5:1:0.5, or a range derived therefrom.

[0034] In some implementations, the stepwise enzymatic hydrolysis in step (3) is as follows: add bromelain and chymotrypsin, and perform a first enzymatic hydrolysis at 45-50℃ for 60-90 min, with a pH of 6.5-7.5; then add carboxypeptidase B, and perform a second enzymatic hydrolysis at 30-37℃ for 30-60 min, with a pH of 7.5-8.5.

[0035] For example, the temperature for a single enzymatic hydrolysis can be selected as 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, or a range derived therefrom; the time for a single enzymatic hydrolysis can be selected as 60min, 61min, 62min, 63min, 64min, 65min, 66min, 67min, 68min, 69min, 70min, 71min, 72min, 73min, 74min, 75min, 76min, 77min, 78min, 79min, 80min, 81min, 82min, 83min, 84min, 85min, 86min, 87min, 88min, 89min, 90min, or a range derived therefrom; and the pH for a single enzymatic hydrolysis can be selected as 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or a range derived therefrom.

[0036] For example, the temperature for the secondary enzymatic hydrolysis can be selected as 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, or a range derived therefrom; the time for the secondary enzymatic hydrolysis can be selected as 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min, 49min, 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min, 60min, or a range derived therefrom; and the pH for the primary enzymatic hydrolysis can be selected as 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or a range derived therefrom.

[0037] In some embodiments, the ethanol extraction in step (5) is performed by using an ethanol solution with a volume fraction of 80-90%; for example, the volume fraction of the ethanol solution may be selected as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or a range derived therefrom.

[0038] Preferably, the ethanol solution is 5-10 times the mass of the polypeptide powder; for example, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or a range derived therefrom.

[0039] Preferably, the extraction is a soaking extraction at room temperature for 18-36 hours; for example, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, or a range derived therefrom.

[0040] Preferably, the ethanol extraction is performed by adding an 80-90% ethanol solution to the polypeptide powder and soaking for 18-36 hours, followed by filtration to obtain the filtrate.

[0041] In some implementations, the filtration in step (5) is to pass the filtrate through a 0.2-0.5 μm filter membrane.

[0042] The beneficial effects of this invention are as follows: This invention provides a method for preparing bitter melon seed peptides by enzymatic hydrolysis. Using bitter melon seeds as raw material, the seeds are crushed, soaked, and pretreated with a combination of cellulase and pectinase to break down the cell walls and release the protein. Then, the seeds are subjected to stepwise controllable enzymatic hydrolysis with bromelain, chymotrypsin and carboxypeptidase B, followed by ethanol purification, membrane filtration and drying to obtain high-purity bitter melon seed peptides.

[0043] Compared with the prior art, the present invention has at least the following beneficial effects: (1) High cell wall disruption efficiency: The synergistic and mild pretreatment of cellulase and pectinase can efficiently degrade the cell wall structure of bitter gourd seeds, significantly improve the protein dissolution rate, and provide sufficient substrate for subsequent enzymatic hydrolysis.

[0044] (2) The enzymatic hydrolysis process is precise and controllable: stepwise enzymatic hydrolysis using bromelain, chymotrypsin and carboxypeptidase B can directionally break peptide bonds, optimize the molecular weight distribution of peptide segments, and improve the yield of small molecule active peptides.

[0045] (3) High product purity and few impurities: Combining ethanol extraction and microporous membrane filtration, polysaccharides, lipids, pigments and macromolecular proteins are effectively removed, improving the purity and stability of bitter melon seed peptides.

[0046] (3) Significant α-glucosidase inhibitory activity: The obtained bitter melon seed peptide can effectively inhibit α-glucosidase activity, block the breakdown of carbohydrates into absorbable monosaccharides, and significantly reduce postprandial blood glucose peak. It can be used as a safe and efficient α-glucosidase inhibitor. Detailed Implementation

[0047] 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.

[0048] The present invention will be further described below by way of specific embodiments. All instruments, devices, equipment, reagents, and products used in the embodiments of the present invention, unless otherwise specified, are obtained through conventional commercial channels. For example, cellulase, pectinase, bromelain, and chymotrypsin are all food-grade raw materials with an enzyme activity of 100,000; carboxypeptidase B was purchased from Shanghai Guchen Biotechnology Co., Ltd., catalog number GC1742.

[0049] Example 1 A method for preparing bitter melon seed peptides by enzymatic hydrolysis The steps are as follows: (1) Bitter gourd seeds are crushed and passed through a 60-mesh sieve to obtain bitter gourd seed powder. Add water to the bitter gourd seed powder in a volume of 10 times the weight of the bitter gourd seed powder, and soak at 50°C for 8 hours to obtain material A.

[0050] (2) Add cellulase (0.02% of the weight of bitter gourd seeds) and pectinase (0.008% of the weight of bitter gourd seeds) to material A for pretreatment (enzymatic hydrolysis at 50℃ for 40 min, pH 4.5-5) to inactivate the enzymes and obtain the primary enzymatic hydrolysate.

[0051] (3) Add 0.1% of the mass of bitter gourd seeds of compound protease (the mass ratio of bromelain, chymotrypsin and carboxypeptidase B is 2:1:0.3) to the primary enzymatic hydrolysate for stepwise enzymatic hydrolysis, inactivate the enzyme, and centrifuge at 3000 rpm for 10 min to obtain the enzymatic hydrolysate. The stepwise enzymatic hydrolysis is specifically as follows: add bromelain and chymotrypsin, and perform a first enzymatic hydrolysis at 45°C for 60 minutes, with a pH of 6.5-7.5; then add carboxypeptidase B, and perform a second enzymatic hydrolysis at 35°C for 30 minutes, with a pH of 7.5-8.5.

[0052] (4) Spray drying of enzymatic hydrolysate, with inlet air temperature of 120℃ and outlet air temperature of 75℃, to obtain polypeptide powder; (5) Add 7 times the volume fraction of 85% ethanol solution to the polypeptide powder and soak for 24 hours. Filter through a 0.45μm PTFE membrane and spray dry the filtrate. The inlet air temperature is 120℃ and the outlet air temperature is 75℃ to obtain the product.

[0053] Example 2 A method for preparing bitter melon seed peptides by enzymatic hydrolysis The steps are as follows: (1) Bitter gourd seeds are crushed and passed through a 60-mesh sieve to obtain bitter gourd seed powder. Add water to the bitter gourd seed powder in a volume of 6 times the weight of the bitter gourd seed powder, and soak at 55°C for 6 hours to obtain material A.

[0054] (2) Add cellulase (0.01% of the weight of bitter gourd seeds) and pectinase (0.01% of the weight of bitter gourd seeds) to material A for pretreatment (enzymatic hydrolysis at 45℃ for 60 min, pH 4.5-5) to inactivate the enzymes and obtain the primary enzymatic hydrolysate.

[0055] (3) Add 0.12% of the mass of bitter gourd seeds of compound protease (the mass ratio of bromelain, chymotrypsin and carboxypeptidase B is 5:1:0.5) to the primary enzymatic hydrolysate for stepwise enzymatic hydrolysis, inactivate the enzyme, and centrifuge at 3000 rpm for 10 min to obtain the enzymatic hydrolysate. The stepwise enzymatic hydrolysis specifically involves: adding bromelain and chymotrypsin, performing a first enzymatic hydrolysis at 45°C for 90 minutes, with a pH of 6.5-7.5; then adding carboxypeptidase B, performing a second enzymatic hydrolysis at 30°C for 60 minutes, with a pH of 7.5-8.5.

[0056] (4) Spray drying of enzymatic hydrolysate, with inlet air temperature of 120℃ and outlet air temperature of 75℃, to obtain polypeptide powder; (5) Add 10 times the volume of 90% ethanol solution to the polypeptide powder and soak for 18 hours. Filter through a 0.45μm PTFE membrane and spray dry the filtrate. The inlet air temperature is 120℃ and the outlet air temperature is 75℃ to obtain the product.

[0057] Example 3 A method for preparing bitter melon seed peptides by enzymatic hydrolysis The steps are as follows: (1) Bitter gourd seeds are crushed and passed through a 60-mesh sieve to obtain bitter gourd seed powder. Add water to the bitter gourd seed powder in a volume of 12 times the weight of the bitter gourd seed powder, and soak at 45°C for 12 hours to obtain material A.

[0058] (2) Add cellulase (0.05% of the weight of bitter gourd seeds) and pectinase (0.005% of the weight of bitter gourd seeds) to material A for pretreatment (enzymatic hydrolysis at 55℃ for 30 min, pH 4.5-5) to inactivate the enzymes and obtain the primary enzymatic hydrolysate.

[0059] (3) Add 0.03% of the mass of bitter gourd seeds of compound protease (the mass ratio of bromelain, chymotrypsin and carboxypeptidase B is 1:1:0.2) to the primary enzymatic hydrolysate for stepwise enzymatic hydrolysis, inactivate the enzyme, and centrifuge at 3000 rpm for 10 min to obtain the enzymatic hydrolysate. The stepwise enzymatic hydrolysis is specifically as follows: add bromelain and chymotrypsin, and perform a first enzymatic hydrolysis at 50°C for 60 minutes, with a pH of 6.5-7.5; then add carboxypeptidase B, and perform a second enzymatic hydrolysis at 37°C for 30 minutes, with a pH of 7.5-8.5.

[0060] (4) Spray drying of enzymatic hydrolysate, with inlet air temperature of 120℃ and outlet air temperature of 75℃, to obtain polypeptide powder; (5) Add 5 times the volume fraction of 80% ethanol solution to the polypeptide powder and soak for 36 hours. Filter through a 0.45μm PTFE membrane and spray dry the filtrate. The inlet air temperature is 120℃ and the outlet air temperature is 75℃ to obtain the product.

[0061] Comparative Example 1 The difference between this comparative example and Example 1 is that there is no pretreatment step.

[0062] Specifically, a method for preparing bitter melon seed peptides via enzymatic hydrolysis. The steps are as follows: (1) Bitter gourd seeds are crushed and passed through a 60-mesh sieve to obtain bitter gourd seed powder. Add water to the bitter gourd seed powder in a volume of 10 times the weight of the bitter gourd seed powder, and soak at 50°C for 8 hours to obtain material A.

[0063] (2) Add 0.2% of the mass of bitter gourd seeds of compound protease (the mass ratio of bromelain, chymotrypsin and carboxypeptidase B is 2:1:0.3) to material A for stepwise enzymatic hydrolysis, inactivate the enzyme, and centrifuge at 3000 rpm for 10 min to obtain the enzymatic hydrolysate. The stepwise enzymatic hydrolysis is specifically as follows: add bromelain and chymotrypsin, and perform a first enzymatic hydrolysis at 45°C for 60 minutes, with a pH of 6.5-7.5; then add carboxypeptidase B, and perform a second enzymatic hydrolysis at 35°C for 30 minutes, with a pH of 7.5-8.5.

[0064] (3) Spray drying of enzymatic hydrolysate, with inlet air temperature of 120℃ and outlet air temperature of 75℃, to obtain polypeptide powder; (4) Add 7 times the volume fraction of 85% ethanol solution to the polypeptide powder and soak for 24 hours. Filter through a 0.45μm PTFE membrane and spray dry the filtrate. The inlet air temperature is 120℃ and the outlet air temperature is 75℃ to obtain the product.

[0065] Comparative Example 2 The difference between this comparative example and Example 1 is that the complex protease is different.

[0066] Specifically, a method for preparing bitter melon seed peptides via enzymatic hydrolysis. The steps are as follows: (1) Bitter gourd seeds are crushed and passed through a 60-mesh sieve to obtain bitter gourd seed powder. Add water to the bitter gourd seed powder in a volume of 10 times the weight of the bitter gourd seed powder, and soak at 50°C for 8 hours to obtain material A.

[0067] (2) Add cellulase (0.02% of the weight of bitter gourd seeds) and pectinase (0.008% of the weight of bitter gourd seeds) to material A for pretreatment (enzymatic hydrolysis at 50℃ for 40 min, pH 4.5-5) to inactivate the enzymes and obtain the primary enzymatic hydrolysate.

[0068] (3) Add 0.1% of the mass of bitter gourd seeds of compound protease (the mass ratio of chymotrypsin and carboxypeptidase B is 1:0.3) to the primary enzymatic hydrolysate for stepwise enzymatic hydrolysis, inactivate the enzyme, and centrifuge at 3000 rpm for 10 min to obtain the enzymatic hydrolysate. The stepwise enzymatic hydrolysis is specifically performed as follows: add chymotrypsin and perform a first enzymatic hydrolysis at 45°C for 60 minutes, with a pH of 6.5-7.5; then add carboxypeptidase B and perform a second enzymatic hydrolysis at 35°C for 30 minutes, with a pH of 7.5-8.5.

[0069] (4) Spray drying of enzymatic hydrolysate, with inlet air temperature of 120℃ and outlet air temperature of 75℃, to obtain polypeptide powder; (5) Add 7 times the volume fraction of 85% ethanol solution to the polypeptide powder and soak for 24 hours. Filter through a 0.45μm PTFE membrane and spray dry the filtrate. The inlet air temperature is 120℃ and the outlet air temperature is 75℃ to obtain the product.

[0070] Comparative Example 3 The difference between this comparative example and Example 1 is that the complex protease is different.

[0071] Specifically, a method for preparing bitter melon seed peptides via enzymatic hydrolysis. The steps are as follows: (1) Bitter gourd seeds are crushed and passed through a 60-mesh sieve to obtain bitter gourd seed powder. Add water to the bitter gourd seed powder in a volume of 10 times the weight of the bitter gourd seed powder, and soak at 50°C for 8 hours to obtain material A.

[0072] (2) Add cellulase (0.02% of the weight of bitter gourd seeds) and pectinase (0.008% of the weight of bitter gourd seeds) to material A for pretreatment (enzymatic hydrolysis at 50℃ for 40 min, pH 4.5-5) to inactivate the enzymes and obtain the primary enzymatic hydrolysate.

[0073] (3) Add 0.1% of the mass of bitter gourd seeds of compound protease (the mass ratio of bromelain and chymotrypsin is 2:1) to the primary enzymatic hydrolysate for enzymatic hydrolysis, inactivate the enzyme, and centrifuge at 3000 rpm for 10 min to obtain the enzymatic hydrolysate. The enzymatic hydrolysis specifically involves adding bromelain and chymotrypsin, and performing a single enzymatic hydrolysis at 45°C for 60 minutes, with a pH of 6.5-7.5.

[0074] (4) Spray drying of enzymatic hydrolysate, with inlet air temperature of 120℃ and outlet air temperature of 75℃, to obtain polypeptide powder; (5) Add 7 times the volume fraction of 85% ethanol solution to the polypeptide powder and soak for 24 hours. Filter through a 0.45μm PTFE membrane and spray dry the filtrate. The inlet air temperature is 120℃ and the outlet air temperature is 75℃ to obtain the product.

[0075] Comparative Example 4 The difference between this comparative example and Example 1 is that the complex protease is different.

[0076] Specifically, a method for preparing bitter melon seed peptides via enzymatic hydrolysis. The steps are as follows: (1) Bitter gourd seeds are crushed and passed through a 60-mesh sieve to obtain bitter gourd seed powder. Add water to the bitter gourd seed powder in a volume of 10 times the weight of the bitter gourd seed powder, and soak at 50°C for 8 hours to obtain material A.

[0077] (2) Add cellulase (0.02% of the weight of bitter gourd seeds) and pectinase (0.008% of the weight of bitter gourd seeds) to material A for pretreatment (enzymatic hydrolysis at 50℃ for 40 min, pH 4.5-5) to inactivate the enzymes and obtain the primary enzymatic hydrolysate.

[0078] (3) Add 0.1% of the mass of bitter gourd seeds of compound protease (the mass ratio of bromelain and carboxypeptidase B is 2:0.3) to the primary enzymatic hydrolysate for stepwise enzymatic hydrolysis, inactivate the enzyme, and centrifuge at 3000 rpm for 10 min to obtain the enzymatic hydrolysate. The stepwise enzymatic hydrolysis is specifically as follows: add bromelain and hydrolyze once at 45°C for 60 minutes, with a pH of 6.5-7.5; then add carboxypeptidase B and hydrolyze a second time at 35°C for 30 minutes, with a pH of 7.5-8.5.

[0079] (4) Spray drying of enzymatic hydrolysate, with inlet air temperature of 120℃ and outlet air temperature of 75℃, to obtain polypeptide powder; (5) Add 7 times the volume fraction of 85% ethanol solution to the polypeptide powder and soak for 24 hours. Filter through a 0.45μm PTFE membrane and spray dry the filtrate. The inlet air temperature is 120℃ and the outlet air temperature is 75℃ to obtain the product.

[0080] Comparative Example 5 Bitter melon polypeptide powder prepared in Example 1 of CN114107414B.

[0081] Comparative Example 6 Bitter melon seed peptide powder prepared in Example 1 of CN112618406A.

[0082] Detection example α-glucosidase inhibitory activity assay, refer to (1) Solution preparation: Prepare the solution using 0.1 mol / L PBS (pH 7.0).

[0083] (2) Group settings 4-Nitrophenyl-α-D-glucopyranoside (p-NPG) was used as the substrate, and acarbose was used as the positive control. The experiment was carried out in 96-well plates. Blank group, control group, positive control group and sample group were set up as shown in Table 1. Each group had 3 replicates.

[0084] Table 1

[0085] After adding each component solution, quickly shake to mix, and immediately place in 37℃ for 10 min. At the same time, add 20 μL of p-NPG (2.0 mmol / L) to each group and continue shaking to mix, and react at 37℃ for 15 min. Add 100 μL of Na2CO3 aqueous solution (0.2 mol / L) to each group to terminate the reaction, and measure the absorbance value at 405 nm in an ELISA reader.

[0086] The formula for calculating the inhibition rate of bitter melon seed peptides against α-glucosidase is as follows: Inhibition rate (%) = (A1-A2) × / (A1-A0) In the formula, A0 is the absorbance of the blank group, A1 is the absorbance of the positive control group, and A2 is the absorbance of the sample group.

[0087] The results are shown in Table 2.

[0088] Table 2

[0089] Verification of technical effectiveness and / or analysis of technical problem solving 1. The bitter melon seed peptides prepared in Examples 1-3 of this invention have an α-glucosidase inhibition rate of 87%-92%. The existing technologies for preparing bitter melon polypeptides or bitter melon seed peptides, such as Comparative Examples 5 and 6, have an α-glucosidase inhibition rate of no more than 60%. Compared with the existing technologies, the bitter melon seed peptides prepared in this invention have a better α-glucosidase inhibition ability.

[0090] 2. Comparison of Comparative Examples 1-4 shows that in this invention, the lack of cellulase and pectinase pretreatment, as well as any one of the three proteases, significantly reduces the α-glucosidase inhibitory ability of the prepared product. Only with cellulase and pectinase pretreatment and the combined action of the three proteases can bitter melon seed peptides with high α-glucosidase inhibitory ability be prepared.

[0091] 3. The bitter melon seed peptide prepared by this invention has an activity very close to that of chemical hypoglycemic drugs and can be used as a raw material for natural α-glucosidase inhibitors with fewer side effects.

[0092] 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 preparing bitter melon seed peptides via enzymatic hydrolysis, characterized in that, Includes the following steps: (1) After the bitter gourd seeds are crushed, they are soaked in water to obtain material A; (2) Add cellulase and pectinase to material A for pretreatment to inactivate the enzymes and obtain primary enzymatic hydrolysate; (3) Add a complex protease to the primary enzymatic hydrolysate for stepwise enzymatic hydrolysis, inactivate the enzyme, separate the solid and liquid, and obtain the enzymatic hydrolysate; (4) The enzymatic hydrolysate is dried to obtain polypeptide powder; (5) The polypeptide powder is extracted with ethanol, filtered, and dried to obtain the final product; The complex protease mentioned in step (3) is bromelain, chymotrypsin and carboxypeptidase B.

2. The preparation method according to claim 1, characterized in that, The mass ratio of bitter gourd seeds to water in step (1) is 1:6-12; The soaking in step (1) is: soaking at 45-55℃ for 6-12 hours.

3. The preparation method according to claim 1, characterized in that, The amount of cellulase used in step (2) is 0.01%-0.05% of the weight of bitter gourd seeds; The amount of pectinase used in step (2) is 0.005%-0.01% of the weight of bitter gourd seeds.

4. The preparation method according to claim 1, characterized in that, The pretreatment described in step (2) is: enzymatic hydrolysis at 45-55℃ for 30-60 min, with a pH of 4.5-5.

5. The preparation method according to claim 1, characterized in that, The amount of the complex protease used in step (3) is 0.03%-0.12% of the weight of bitter gourd seeds.

6. The preparation method according to claim 1, characterized in that, The mass ratio of bromelain, chymotrypsin and carboxypeptidase B in step (3) is 1-5:1:0.2-0.

5.

7. The preparation method according to claim 1, characterized in that, The stepwise enzymatic hydrolysis described in step (3) is as follows: add bromelain and chymotrypsin, and perform a first enzymatic hydrolysis at 45-50℃ for 60-90 min, with a pH of 6.5-7.5; then add carboxypeptidase B, and perform a second enzymatic hydrolysis at 30-37℃ for 30-60 min, with a pH of 7.5-8.

5.

8. The preparation method according to claim 1, characterized in that, The ethanol extraction in step (5) is performed using an 80-90% ethanol solution.

9. The preparation method according to claim 1, characterized in that, The ethanol solution mentioned in step (5) is 5-10 times the mass of the polypeptide powder; The extraction process involves soaking at room temperature for 18-36 hours.

10. The preparation method according to claim 1, characterized in that, The filtration in step (5) involves passing the liquid phase through a 0.2-0.5 μm filter membrane.

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