A method for producing a blood pressure lowering euphausia superba small molecular protein peptide

Abstract

The application discloses a kind of production methods of blood pressure reducing Euphausia superba small molecule protein peptide, and it is related to the technical field of deep processing and comprehensive utilization of marine biological resources.The method comprises the following steps: after frozen Euphausia superba is thawed by low-temperature microwave, low-temperature heat pump drying is carried out, and condensate is collected; the condensate is used as a fermentation substrate, and Aspergillus oryzae and Bacillus subtilis are inoculated for fermentation; after the fermentation liquor is purified by ultrafiltration, it is self-assembled with chitosan oligosaccharide under acidic conditions to form a nano co-assembly, and then dried to obtain a powder.The application significantly improves the content of small molecule active peptide and improves the bitterness by enzyme-coupling fermentation process, and at the same time, the nano delivery system is constructed by self-assembly of chitosan oligosaccharide, which improves the stability and bioavailability of the product, and the whole process is low-temperature processing, safe and environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of deep processing and comprehensive utilization of marine biological resources, specifically a method for producing small molecule protein peptides from Antarctic krill that lower blood pressure. Background Technology

[0002] First, the enzymatic hydrolysis process is limited, resulting in products with limited activity. For example, existing technologies often use a single protease (such as trypsin) or ultrasound-assisted enzymatic hydrolysis, yielding products that are single peptides or mixtures of crude peptides with specific sequences. While these methods can achieve some ACE inhibitory activity, the product lineage is fixed, the active sites are not sufficiently exposed, and residual hydrophobic amino acids can easily lead to bitterness, affecting the sensory quality of the final product.

[0003] Second, there is a lack of formulation and stability design for small molecule peptides. Existing technologies often use enzymatic hydrolysates or lyophilized powders as the final product, without considering the degradation of small molecule peptides in the gastrointestinal environment. Small molecule peptides are easily further hydrolyzed by gastric acid and digestive enzymes, resulting in low oral bioavailability and difficulty in guaranteeing antihypertensive effects. Furthermore, existing methods often use strong acids (such as hydrochloric acid) for pretreatment, which not only poses a risk of reagent residue but may also damage the native protein structure, reducing the yield and activity of the target peptide.

[0004] Third, current technologies do not functionally combine chitin derivatives with antihypertensive peptides. Chitin and chitosan oligosaccharides themselves have certain ACE inhibitory activity and good film-forming properties and biocompatibility. However, in existing antihypertensive peptide preparation processes, chitin is usually treated as a byproduct and is not effectively utilized.

[0005] Therefore, developing a method for producing Antarctic krill small molecule protein peptides that can improve the activity of antihypertensive peptides, reduce the bitterness of the product, and have targeted delivery or sustained-release functions has important application value. Summary of the Invention

[0006] The purpose of this invention is to provide a method for producing small molecule protein peptides from Antarctic krill that lower blood pressure, in order to solve the problems mentioned in the background art.

[0007] This invention provides a method for producing small molecule protein peptides from Antarctic krill that lower blood pressure, comprising the following steps: S1. Low-temperature microwave thawing and partial enzyme inactivation: Frozen Antarctic krill raw materials, which are rapidly frozen on board after being caught and whose core temperature reaches below -20°C, are placed in a microwave thawing device and thawed at low temperature under the conditions of microwave power of 200-500W / kg and frequency of 915MHz or 2450MHz. The temperature of the krill body is controlled not to exceed 60°C during the thawing process, preferably between 40-55°C. The microwave thawing time is set to 5-20 minutes according to the amount of raw materials. In step S1, microwave thawing simultaneously deactivates some of the low-temperature enzymes in Antarctic krill, reducing enzyme activity by 40-60%. It should be noted that the mechanism of microwave thawing is as follows: when microwaves penetrate the frozen krill body, water molecules rotate at high speed in an alternating electric field, generating heat through friction, thus achieving uniform thawing from the inside out. Simultaneously, the thermal and non-thermal effects of microwaves synergistically act on the cryoenzymes (mainly trypsin, carboxypeptidase, and lipase) within the Antarctic krill body, causing partial conformational changes and achieving partial inactivation. Compared to natural thawing, this step reduces enzyme activity, effectively minimizing protein loss due to enzymatic degradation during thawing and subsequent shelling.

[0008] S2. Low-temperature heat pump drying and condensate collection: After thawing in step S1, the whole Antarctic krill was placed in a low-temperature heat pump drying device, with circulating hot air as the drying medium; the drying temperature was controlled at 35-50℃, and the drying time was 4-8 hours. During the drying process, condensed gas and condensate are collected; the condensate is rich in volatile flavor substances, water-soluble proteins and peptides, and free amino acids. Drying endpoint control: On a wet basis, dry until the internal moisture content of the shrimp is 30-50% and the surface moisture content is 5-10%, forming a semi-dry state with dry outside and moist inside; It should be noted that heat pump drying can save 30% to 50% of energy compared to traditional hot air drying. Moreover, under low temperature conditions below 50℃, it effectively avoids thermal denaturation of krill proteins, lipid oxidation, and astaxanthin degradation, thus preserving the natural nutrients and flavor components of Antarctic krill to the maximum extent.

[0009] It should be further explained that during the drying process, the evaporator of the heat pump system condenses the water vapor in the hot and humid air discharged from the drying chamber. The condensate and condensate are collected and stored together by a collection device. The condensate is rich in volatile flavor compounds of Antarctic krill (including pyrazines, aldehydes, ketones, and other flavor compounds), water-soluble proteins and peptides, free amino acids, and trace amounts of krill oil carried by the airflow.

[0010] Step S3: Low-temperature drum shell removal and separation: The semi-dried Antarctic krill obtained in step S2 is fed into a low-temperature rotating drum device; the drum rotation speed is 10-30 r / min, the processing temperature is 10-25℃, and the processing time is 3-10 min. Under the action of rotational friction and collision, the shrimp shell, shrimp head, shrimp tail, shrimp viscera and shrimp meat are separated; the molted krill meat and the shell containing chitin are collected separately. The krill meat after being deshelled is further dried at low temperature or directly packaged to obtain the finished product of Antarctic krill jerky. It should be noted that in a semi-dry state, due to the higher internal moisture content and lower surface moisture content of the shrimp meat, there is a significant difference in shrinkage rate and weakened binding force between the shrimp shell and the shrimp meat. Under the rotational friction and collision action of the drum, the shrimp shell, head, tail, and internal organs are easily separated from the shrimp meat. The separated material is graded through the sieve: the harder shell parts, such as the shrimp shell, remain in the drum and are discharged from one end; the shelled shrimp meat (shrimp kernels), due to its softer texture, falls through the sieve into the collection tank.

[0011] S4. Preparation of food-grade chitin: Rinse the shell portion collected in step S3 with clean water and drain for later use; Alkaline deproteinization: Add the shell material to a food-grade sodium hydroxide solution with a mass fraction of 3-8% at a solid-liquid ratio of 1:(5-10)(w / v), stir at 50-70℃ for 1-3 hours, separate the solid and liquid, and collect the solid part; Acid decalcification: The deproteinized solid is added to a food-grade lactic acid solution with a mass fraction of 3-10% at a solid-liquid ratio of 1:(5-8)(w / v), and stirred at 20-30℃ for 2-4 hours to dissolve and remove the minerals; Washing and dehydration: The acid-treated solids are rinsed with pure water until the pH of the washing solution is 6.5-7.5, and then centrifuged to dehydrate until the water content of the solids is 40-60%. Low-temperature drying: The dehydrated solid is placed in a low-temperature heat pump drying device and dried at 40-50℃ until the moisture content is ≤10%, thus obtaining the Antarctic krill-derived chitin product. It should be noted that lactic acid is a food-grade organic acid, which not only has a good decalcification effect but also has high safety, meeting the requirements for food-grade and pharmaceutical-grade applications. At the same time, it avoids the excessive degradation of chitin molecular chains by strong acids such as hydrochloric acid.

[0012] S5. Fermentation preparation of Antarctic krill juice for lowering blood pressure: The condensate collected in step S2 was used as the basic culture medium for fermentation, and glucose or sucrose was added to adjust the carbon-nitrogen ratio to (15-25):1. Inoculate with Aspergillus oryzae spore suspension and Bacillus subtilis culture, and ferment for 24-48 hours at 25-32℃, aeration rate of 0.5-1.5 vvm, and stirring speed of 150-300 r / min. In step S5, the total inoculation amount of Aspergillus oryzae and Bacillus subtilis is 2-10% of the fermentation liquid volume, and the ratio of viable cells of Aspergillus oryzae to Bacillus subtilis is 1:(1-3). After fermentation, heat the fermentation broth to 80-85℃ and maintain for 15-20 minutes to inactivate the enzymes. After cooling, centrifuge to remove the bacterial cells and obtain a clear fermentation broth. It should be noted that during fermentation, the complex enzyme system secreted by *Aspergillus oryzae* and *Bacillus subtilis* hydrolyzes proteins, peptides, and residual phospholipids in the condensate into small peptides, amino acids, and free fatty acids. Simultaneously, various organic acids (citric acid, gluconic acid, etc.) and flavor precursors produced by metabolism impart rich flavor layers to the fermentation broth. *Aspergillus oryzae* secretes prolyl endopeptidase and carboxypeptidase, which can specifically recognize and remove C-terminal proline and hydrophobic amino acid residues, increasing the exposure of ACE-inhibiting peptides. *Bacillus subtilis* secretes neutral protease and aminopeptidase, further degrading medium- and long-chain peptides in the hydrolysate into small active peptides. The synergistic effect of these two enzymes increases the proportion of peptides with a molecular weight <3kDa in the fermentation broth and significantly improves bitterness.

[0013] S6. Preparation of self-assembled nanocomposites: The clarified fermentation broth obtained in step S5 is concentrated to a solid content of 5-15%, the pH is adjusted to 4.5-5.5 with food-grade acid, chitosan oligosaccharide solution is added, and the mixture is reacted at 20-30℃ and a stirring speed of 100-300 r / min for 1-3 h to obtain a chitosan oligosaccharide-peptide co-assembled suspension. In step S6, the mass ratio of chitosan oligosaccharide to antihypertensive peptide is 1:(3-6); In step S6, the weight-average molecular weight of chitosan oligosaccharide is 1000-3000 Da, and the degree of deacetylation is ≥85%. It should be noted that this invention utilizes the electrostatic interaction between chitosan oligosaccharide and the antihypertensive peptide to spontaneously assemble into a nano-assembly under acidic conditions. The free amino groups on the chitosan oligosaccharide molecule become protonated and positively charged under acidic conditions, forming an ionic complex with the negatively charged carboxyl ionized region of the antihypertensive peptide. This complex assembles into nanoparticles under the synergistic effect of hydrophobic interactions and hydrogen bonding. This assembly has the following advantages: firstly, it protects the stability of the antihypertensive peptide in the acidic environment of the stomach, preventing premature degradation by pepsin; secondly, the chitosan oligosaccharide molecule itself has ACE inhibitory activity, competitively binding to Zn of ACE. 2+ The site and peptide produce synergistic effects; thirdly, the nanoscale particle size facilitates the passage through tight junctions of intestinal epithelial cells, improving oral bioavailability. Compared with single peptide powder, this co-assembly improves peptide retention in simulated gastrointestinal fluid, and increases ACE inhibitory activity by more than 20%.

[0014] S7. Drying and Preparation: The co-assembled suspension obtained in step S6 is subjected to low-temperature vacuum drying or spray drying at 40-50℃ to obtain blood pressure-lowering Antarctic krill small molecule protein peptide powder.

[0015] As a preferred embodiment of the present invention, in step S1, the microwave thawing power is 350W / kg, the frequency is 915MHz, and the thawing time is 12min.

[0016] As a preferred technical solution of the present invention, in step S2, the heat pump drying temperature is preferably 40-45℃ and the drying time is 5-6h.

[0017] As a preferred embodiment of the present invention, in step S5, the spore concentration of the Aspergillus oryzae spore suspension is 10. 6 -10 7 CFU / mL; the viable count of Bacillus subtilis in the bacterial suspension was 10. 7 -10 8 CFU / mL.

[0018] As a preferred embodiment of the present invention, in step S5, the fermentation time is 36 hours.

[0019] As a preferred embodiment of the present invention, in step S6, the concentration of the chitosan oligosaccharide solution is 1-5% (w / v), and the solvent is deionized water.

[0020] As a preferred technical solution of the present invention, in step S7, the drying method is spray drying, the inlet air temperature is 120-140℃, the outlet air temperature is 60-80℃, and the feed flow rate is 5-10mL / min.

[0021] It should be noted that in the technical solution of this invention, step S1 uses microwave thawing instead of traditional running water thawing or natural thawing, which can simultaneously achieve partial inactivation of enzymes, avoid continuous degradation of protein raw materials by proteases during thawing, and ensure the quality of substrates for subsequent enzymatic hydrolysis. The heat pump drying process in step S2 not only achieves energy-saving drying of shrimp, but the condensate produced is rich in water-soluble nutrients, which can be used as the fermentation substrate in step S5, realizing full utilization of resources; steps S3 and S4 respectively yield two high-value-added products: krill jerky and food-grade chitin, which together with the blood pressure-lowering peptide powder obtained in step S7 constitute a multi-product co-production system with a raw material comprehensive utilization rate of ≥95%.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention adopts a combined enzymatic hydrolysis and directional fermentation process. First, the Antarctic krill protein is efficiently degraded into a polypeptide mixture by alkaline protease. Then, the peptide spectrum is trimmed by prolyl endopeptidase and carboxypeptidase secreted by Aspergillus oryzae and Bacillus subtilis. This significantly increases the content of small molecule active peptides with a molecular weight of <3kDa, improves the bitterness, and enhances the ACE inhibitory activity.

[0023] (2) This invention utilizes the electrostatic self-assembly of chitosan oligosaccharide and antihypertensive peptide under acidic conditions to construct a nanoscale co-assembly with protective, sustained-release, and synergistic functions. While providing its own ACE inhibitory activity, chitosan oligosaccharide increases the stability of the antihypertensive peptide in simulated gastrointestinal fluid, improves oral bioavailability, and makes up for the lack of formulation design in the prior art.

[0024] (3) The method of the present invention uses frozen Antarctic krill as raw material and adopts food-grade reagents and mild processing conditions (temperature not exceeding 60°C) throughout the process, which preserves the natural structure and high activity of Antarctic krill protein to the greatest extent. At the same time, it avoids the problems of reagent residue and protein structure damage caused by hydrochloric acid pretreatment in the prior art. The product has high safety and is suitable for large-scale production.

[0025] (4) This invention uses an integrated processing flow of microwave thawing, heat pump drying, drum shell removal, chitin extraction, fermentation and self-assembly to add Antarctic krill small molecule protein peptide products with blood pressure lowering function, while retaining two traditional products: krill jerky and food-grade chitin. This achieves high-value utilization of all components of raw materials and significantly improves the added value of the industry. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Preparation Example 1 Preparation of Aspergillus oryzae spore suspension: Aspergillus oryzae standard strains were inoculated onto PDA slant culture medium and activated at 28℃ for 72 h. Spores were eluted with sterile physiological saline, filtered through sterile gauze, and the spore precipitate was collected by centrifugation, resuspended in sterile physiological saline, and the spore concentration was adjusted to 5 × 10⁻⁶. 6 CFU / mL was used to obtain a suspension of Aspergillus oryzae spores.

[0028] Preparation Example 2 Preparation of Bacillus subtilis bacterial culture: The Bacillus subtilis standard strain was inoculated into LB liquid medium and cultured at 30℃ with shaking for 24 h. The bacterial pellet was collected by centrifugation at 8000 r / min for 10 min, washed twice with sterile physiological saline, and resuspended. The viable cell concentration was adjusted to 1×10⁻⁶. 6 CFU / mL was used to obtain Bacillus subtilis bacterial suspension.

[0029] Preparation Example 3 Preparation of chitosan oligosaccharide solution: Dissolve chitosan oligosaccharide powder in deionized water to prepare a 3% (w / v) chitosan oligosaccharide solution. Adjust the pH to 5 with food-grade citric acid, and then filter through a 0.22μm microporous membrane for sterilization. Example

[0030] A method for producing blood pressure-lowering Antarctic krill small molecule protein peptides includes the following steps: S1. Low-temperature microwave thawing and partial enzyme inactivation: 100 kg of frozen Antarctic krill (core temperature -20℃) were placed in batches in a microwave thawing apparatus. The microwave frequency was 915 MHz, and the power was set to 350 W / kg. During thawing, the surface temperature of the krill was monitored in real time using an infrared thermometer, and the maximum temperature was controlled to not exceed 55℃. The thawing time was 12 minutes, until the krill were completely thawed, soft to the touch, and free of ice crystals. Shrimp samples were taken before and after thawing, and protease activity was determined using the Folin-phenol method. The results showed that the total protease activity of the krill decreased by approximately 46% after microwave thawing compared to before thawing, indicating that microwave treatment effectively achieved partial enzyme inactivation. S2. Low-temperature heat pump drying and condensate collection: Thawed Antarctic krill were evenly spread in the tray of a low-temperature heat pump dryer, with a layer thickness of approximately 2-3 cm. The drying temperature was set to 45℃, and the drying medium was circulating air. After 5.5 hours of drying, samples were taken for testing: the internal moisture content of the krill was 42%, and the surface moisture content was 8%, meeting the requirement of a semi-dry state with dry exterior and moist interior. During the drying process, the condensate precipitated on the evaporator side was collected by a condensate collector, with a total of approximately 12.5 L of condensate collected. The total solids content in the condensate was approximately 4.2% (w / v), the total nitrogen content was 1.8 g / L, and the free amino acid content was 3.5 g / L. S3. Low-temperature drum desquamation and separation: The semi-dried Antarctic krill was fed into a low-temperature rotary drum deshelling device. The drum speed was set to 20 rpm, and the temperature was controlled by circulating 10-15℃ cold water through a cooling jacket. The processing time was 6 minutes. Approximately 41.2 kg of deshelled krill jerky and approximately 28.5 kg of shells (including shells, heads, and tails) were collected. The deshelled krill jerky was further dried at a low temperature until the moisture content was ≤12%, resulting in the finished Antarctic krill jerky product. S4. Preparation of food-grade chitin: Take 28.5 kg of the shell parts collected in step S3, rinse with clean water to remove any residual shrimp meat on the surface, and drain. Alkaline deproteinization: The shell was added to 300L of a 5% (w / w) food-grade sodium hydroxide solution (solid-liquid ratio approximately 1:10), and stirred in a 60℃ water bath for 2 hours. After the reaction was complete, the solid and liquid fractions were separated by a plate centrifuge (3000 rpm, 10 min), and the solid fraction was collected. Acid decalcification: The deproteinized solid is added to 200L of food-grade lactic acid solution with a mass fraction of 6% (solid-liquid ratio of about 1:8) and stirred for 3 hours at room temperature (25℃). Washing and dehydration: The acid-treated solids were repeatedly rinsed with pure water until the pH of the washing solution reached 7. They were then centrifuged in a plate centrifuge at 1500 rpm for 10 minutes to dehydrate until the water content of the solids was approximately 50%. Low-temperature drying: The dehydrated solids were placed in a low-temperature heat pump drying device and dried at 45℃ until the moisture content was ≤10%. Approximately 3.1 kg of Antarctic krill-derived chitin was finally obtained, with a yield of 10.9% (based on the wet weight of the shells). Testing showed that the chitin purity was 91.5%, ash content was 0.8%, and protein residue was 0.7%, meeting the quality standards for food-grade chitin. S5. Fermentation preparation of Antarctic krill juice for lowering blood pressure: Take 12.5 L of the condensate collected in step S2, allow it to stand and separate into layers, remove a small amount of oil phase from the surface, and take approximately 11.8 L of the aqueous phase. Filter the condensate through a 0.22 μm microporous membrane for sterilization. The total nitrogen content of the condensate was measured to be 1.8 g / L, and glucose was added to adjust the carbon-to-nitrogen ratio to approximately 20:1. The fermentation substrate was placed in a 20L fermenter, sterilized at 121℃ for 20 min, cooled to 30℃, and then inoculated with Aspergillus oryzae spore suspension prepared in Preparation Example 1 and Bacillus subtilis bacterial culture prepared in Preparation Example 2. The total inoculum volume was 5% of the fermentation liquid volume, with a viable cell ratio of Aspergillus oryzae to Bacillus subtilis of 1:2. Fermentation conditions: temperature 30℃, aeration rate 1.0 vvm, stirring speed 200 rpm, fermentation time 36 h. After fermentation, the temperature was raised to 85℃ and held for 15 minutes to inactivate the enzymes. After cooling, the cells were removed by centrifugation, yielding approximately 10.2 L of clear fermentation broth. S6. Preparation of self-assembled nanocomposites: The clarified fermentation broth obtained in step S5 was concentrated by rotary evaporation at 50°C to a solid content of 10%. The pH was adjusted to 4.8 with food-grade citric acid. The chitosan oligosaccharide solution prepared in Preparation Example 3 was slowly added while stirring at 200 r / min. The mass ratio of chitosan oligosaccharide to antihypertensive peptide was 1:4. The reaction was continued for 2 h to obtain a chitosan oligosaccharide-peptide co-assembly suspension. The average particle size of the co-assembly was 215 nm and the Zeta potential was +18.5 mV, as determined by dynamic light scattering. S7. Drying and Preparation: The co-assembled suspension obtained in step S6 was subjected to low-temperature vacuum drying at 50°C (vacuum degree -0.08MPa) until the moisture content was ≤5%, yielding approximately 2.1 kg of blood pressure-lowering Antarctic krill small molecule protein peptide powder.

[0031] In this embodiment, some of the raw materials used are the same as those obtained in Preparation Examples 1-3, and the other examples are the same. Example

[0032] A method for producing small molecule protein peptides from Antarctic krill that lower blood pressure is basically the same as in Example 1, except that: In step S5, the ratio of viable Aspergillus oryzae to Bacillus subtilis is 1:1, and the total inoculum size is 3% of the fermentation broth volume; in step S6, the mass ratio of chitosan oligosaccharide to antihypertensive peptide is 1:6. The remaining steps and parameters are the same as in Example 1. Example

[0033] A method for producing small molecule protein peptides from Antarctic krill that lower blood pressure is basically the same as in Example 1, except that: In step S6, the mass ratio of chitosan oligosaccharide to antihypertensive peptide is 1:3, and the concentration of the chitosan oligosaccharide solution is 1% (w / v). The remaining steps and parameters are the same as in Example 1.

[0034] Comparative Example 1 The difference between this comparative example and Example 1 is that in step S5, Aspergillus oryzae and Bacillus subtilis are not added for fermentation. Instead, the condensate is directly concentrated before subsequent steps. All other steps and parameters are the same as in Example 1.

[0035] Comparative Example 2 The difference between this comparative example and Example 1 is that, in step S6, chitosan oligosaccharide solution is not added; instead, an equal volume of deionized water is added to the fermentation concentrate instead of chitosan oligosaccharide solution. The remaining steps and parameters are the same as in Example 1.

[0036] Comparative Example 3 The difference between this comparative example and Example 1 is that in step S6, the pH is not adjusted to 4.8, but the fermentation concentrate is kept neutral and directly added to the chitosan oligosaccharide solution. The remaining steps and parameters are the same as in Example 1.

[0037] Comparative Example 4 The difference between this comparative example and Example 1 is that natural thawing (thawing at 4°C for 12 hours) is used instead of microwave thawing in step S1, while the other steps and parameters are the same as in Example 1.

[0038] Comparative Example 5 The difference between this comparative example and Example 1 is that in step S2, conventional hot air drying (75°C, drying for 8 hours) is used instead of low-temperature heat pump drying. The remaining steps and parameters are the same as in Example 1.

[0039] test: I. Assay for ACE inhibitory activity Using FAPGG as a substrate, the ACE inhibitory activity of the antihypertensive peptide powders prepared in each example and comparative example was determined using a full-wavelength microplate reader (340 nm). The samples were dissolved in deionized water and diluted to different concentrations, and the half-maximal inhibitory concentration (IC50) was determined. 50 (value), repeat the measurement 3 times.

[0040] II. Sensory Evaluation of Bitterness A blind testing panel of 10 people prepared the antihypertensive peptide powders of each example and comparative example into a 1% (w / v) solution with deionized water, using distilled water as a control. The bitterness was evaluated using a 9-point scoring method (1 point indicates no bitterness, and 9 points indicate extremely strong bitterness), and the average value was taken.

[0041] III. Simulated Gastrointestinal Fluid Stability Test Following the preparation methods for artificial gastric and intestinal fluids in the 2020 edition of the Chinese Pharmacopoeia, the antihypertensive peptide powders prepared in each example and comparative example were added to artificial gastric fluid (pH 1.5, containing pepsin), incubated at 37°C for 2 hours, then the pH was adjusted to 6.8, and artificial intestinal fluid (containing trypsin) was added for further incubation for 4 hours. The peptide content in the samples before and after incubation was measured (using the Folin-phenol method), and the peptide retention rate (%) was calculated.

[0042] IV. Uric Acid Lowering Function Test The blood pressure-lowering Antarctic krill small molecule protein peptide powder prepared in Example 1 was mixed with deionized water to prepare a 10 mg / mL solution. The solution was then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was collected to obtain a uric acid-lowering active peptide component with a molecular weight of <3 kDa.

[0043] (1) Laboratory animals and grouping Sixty male Kunming mice (weighing 18-22g) were selected and randomly divided into 6 groups of 10 mice each after one week of acclimatization: Blank control group: administered normal saline by gavage; Model group: Inosine (500 mg / kg) + potassium oxonate (100 mg / kg) administered by gavage; Positive control group: administered hypoxanthine (500 mg / kg) + potassium oxonate (100 mg / kg) + allopurinol (10 mg / kg) by gavage; Low-dose group: oral administration of hypoxanthine (500 mg / kg) + potassium oxonate (100 mg / kg) + peptide component from Example 1 (200 mg / kg); Medium-dose group: oral administration of hypoxanthine (500 mg / kg) + potassium oxonate (100 mg / kg) + peptide component from Example 1 (500 mg / kg); High-dose group: oral administration of hypoxanthine (500 mg / kg) + potassium oxonate (100 mg / kg) + peptide component from Example 1 (1000 mg / kg).

[0044] (2) Experimental methods The patients were administered the medication by gavage for 7 consecutive days. After the last administration, the patients were fasted for 12 hours. Blood was collected from the orbital rim to separate serum and measure serum uric acid levels. Liver tissue was collected to measure xanthine oxidase (XOD) activity.

[0045] V. Summary of Results Table 1: ACE inhibitory activity and bitterness evaluation results of different formulations Example 1 0.086±0.004 2.3±0.4 Example 2 0.092±0.005 2.6±0.5 Example 3 0.095±0.006 2.5±0.4 Comparative Example 1 0.162±0.009 6.8±0.6 Comparative Example 2 0.114±0.007 2.8±0.5 Comparative Example 3 0.131±0.008 3.2±0.6 Comparative Example 4 0.155±0.010 6.2±0.5 Comparative Example 5 0.148±0.009 5.8±0.6 Table 2: Results of stability tests on simulated gastrointestinal fluids treated with different formulations Example 1 92.6±2.3 88.4±2.6 Example 2 90.8±2.5 86.2±2.9 Example 3 89.5±2.8 84.7±3.1 Comparative Example 1 68.3±3.2 62.5±3.5 Comparative Example 2 65.7±3.5 58.9±3.8 Comparative Example 3 72.4±3.1 65.3±3.4 Comparative Example 4 70.2±3.3 64.1±3.6 Comparative Example 5 71.5±3.0 65.8±3.3 Table 3: Effects of the blood pressure-lowering Antarctic krill small molecule protein peptides prepared in Example 1 on serum uric acid and liver XOD activity in hyperuricemic mice. Blank control group 132.5±12.3 18.6±2.1 Model group 286.7±25.8 45.8±4.3 Positive control group 168.2±18.6 27.3±3.2 low-dose group 241.3±22.5 40.2±3.9 medium dose group 204.6±20.8 35.5±3.8 High-dose group 174.3±16.2 31.3±3.1 VI. Discussion of Results As shown in Table 1, the ACE inhibitory activity of the blood pressure-lowering Antarctic krill small molecule protein peptide powder prepared in Examples 1-3 of this invention is significantly better than that of the comparative examples. The IC50 of Example 1... 50 The value was 0.086 mg / mL, which was approximately 47% lower than that of Comparative Example 1 (concentrated with condensate only, 0.162 mg / mL), indicating that the directed fermentation of Aspergillus oryzae and Bacillus subtilis significantly improved the activity of the antihypertensive peptide, increasing the proportion of small molecule peptides with greater ACE inhibitory activity in the product. The IC50 values ​​of Example 1 and Comparative Example 2 were compared. 50 The concentration of the antihypertensive peptide (ACE) was 0.114 mg / mL. The addition of chitosan oligosaccharide increased the ACE inhibitory activity of the antihypertensive peptide by approximately 25%, confirming the synergistic effect between the ACE inhibitory activity of chitosan oligosaccharide and the antihypertensive peptide. Comparative Example 3 (unadjusted pH) IC50 value (0.114 mg / mL) 50 The value (0.131 mg / mL) was between that of Example 1 and Comparative Example 2, indicating that the self-assembly effect was better under acidic conditions (pH 4.8) than under neutral conditions. This is presumably because pH 4.8 is closer to the pKa value of chitosan oligosaccharide, which is conducive to the formation of stable ionic complexes. The IC50 values ​​of Comparative Examples 4 and 5... 50 The values ​​were 0.155 mg / mL and 0.148 mg / mL, respectively, both significantly higher than those in Example 1, indicating that microwave thawing and low-temperature heat pump drying play an important role in protecting protein activity and improving the efficiency of subsequent enzymatic hydrolysis.

[0046] The bitterness evaluation results showed that the bitterness scores of Examples 1-3 were significantly lower than those of Comparative Example 1 (concentrated condensate only), indicating that the fermentation treatment by Aspergillus oryzae and Bacillus subtilis effectively reduced the bitterness of the peptide products by removing the C-terminal hydrophobic amino acid residues. The bitterness scores of Comparative Examples 2 and 3 were between those of Examples 1 and Comparative Example 1, indicating that chitosan oligosaccharide has a certain masking effect on bitterness, but fermentation treatment plays a major role in the debittering process.

[0047] Table 2 shows that the peptide retention rates in simulated gastrointestinal fluid for Examples 1-3 were significantly higher than those for Comparative Examples 1-5, indicating that the chitosan oligosaccharide-peptide co-assembly has a good protective effect on the antihypertensive peptide. The peptide retention rate of Example 1 after incubation in simulated gastric fluid for 2 hours was 92.6%, while that of the unencapsulated Comparative Example 1 was only 68.3%, indicating that the co-assembly effectively prevented the degradation of the antihypertensive peptide by pepsin. The retention rate of Example 1 in simulated intestinal fluid (88.4%) was also better than that of Comparative Example 1 (62.5%), further verifying the protective effect of the co-assembly on the antihypertensive peptide throughout the digestive tract. The retention rate of Comparative Example 3 was slightly higher than that of Comparative Example 1 but lower than that of Example 1, indicating that even under incompletely optimized pH conditions, chitosan oligosaccharide and the antihypertensive peptide can still form a partially protective structure, but sufficient self-assembly (pH 4.8) can provide better protection.

[0048] Table 3 shows that the Antarctic krill small molecule protein peptide component with a molecular weight <3kDa prepared in Example 1 has significant uric acid-lowering activity. Compared with the model group, the serum uric acid levels in the medium- and high-dose groups decreased by 28.6% and 39.2%, respectively (P<0.01), and the liver XOD activity decreased by 22.4% and 31.7%, respectively (P<0.05), indicating that this peptide component can reduce uric acid production by inhibiting XOD activity, thereby exerting a uric acid-lowering effect.

[0049] In summary, this invention significantly improves the bioactivity and bitterness of antihypertensive peptides through microwave thawing, low-temperature heat pump drying, and enzymatic hydrolysis-fermentation coupling processes. Furthermore, the nano-assemblies formed by the self-assembly of chitosan oligosaccharides significantly enhance the stability of the antihypertensive peptides in the gastrointestinal tract, achieving a dual improvement in both bioactivity and delivery performance. Simultaneously, the co-production process of this invention, while preparing antihypertensive peptides, also yields two high-value-added products: krill jerky and food-grade chitin, realizing the high-value utilization of all components of the raw materials.

[0050] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0051] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the scope defined by the invention, and all such modifications and additions should fall within the protection scope of the present invention.

Claims

1. A method for producing small molecule protein peptides from Antarctic krill that lower blood pressure, characterized in that: Includes the following steps: S1. Low-temperature microwave thawing and partial enzyme inactivation: Frozen Antarctic krill raw materials, which are rapidly frozen on board after being caught and whose core temperature reaches below -20°C, are placed in a microwave thawing device and thawed at low temperature under the conditions of microwave power of 200-500W / kg and frequency of 915MHz or 2450MHz; the temperature of the shrimp body is controlled not to exceed 60°C during the thawing process; the microwave thawing time is set to 5-20 minutes according to the amount of raw materials. S2. Low-temperature heat pump drying and condensate collection: After thawing in step S1, the whole Antarctic krill was placed in a low-temperature heat pump drying device, with circulating hot air as the drying medium; the drying temperature was controlled at 35-50℃, and the drying time was 4-8 hours; during the drying process, condensed gas and condensate were collected. Step S3: Low-temperature drum shell removal and separation: The semi-dried Antarctic krill obtained in step S2 is fed into a low-temperature rotary drum device; the drum rotation speed is 10-30 r / min, the processing temperature is 10-25℃, and the processing time is 3-10 min; under the action of rotational friction and collision, the shrimp shell, shrimp head, shrimp tail, shrimp viscera and shrimp meat are separated; the deshelled krill meat and the shell containing chitin are collected separately; the deshelled krill meat is further dried at low temperature or directly packaged to obtain the finished Antarctic krill meat jerky; S4. Preparation of food-grade chitin: Rinse the shell portion collected in step S3 with clean water and drain for later use; Alkaline deproteinization: Add the shell material to a food-grade sodium hydroxide solution with a mass fraction of 3-8% at a solid-liquid ratio of 1:(5-10)(w / v), stir at 50-70℃ for 1-3 hours, separate the solid and liquid, and collect the solid part; Acid decalcification: The deproteinized solid is added to a food-grade lactic acid solution with a mass fraction of 3-10% at a solid-liquid ratio of 1:(5-8)(w / v), and stirred at 20-30℃ for 2-4 hours to dissolve and remove the minerals; Washing and dehydration: The acid-treated solids are rinsed with pure water until the pH of the washing solution is 6.5-7.5, and then centrifuged to dehydrate until the water content of the solids is 40-60%. Low-temperature drying: The dehydrated solid is placed in a low-temperature heat pump drying device and dried at 40-50℃ until the moisture content is ≤10%, thus obtaining the Antarctic krill-derived chitin product. S5. Fermentation preparation of Antarctic krill juice for lowering blood pressure: The condensate collected in step S2 was used as the basic culture medium for fermentation, and glucose or sucrose was added to adjust the carbon-nitrogen ratio to (15-25):

1. Inoculate with Aspergillus oryzae spore suspension and Bacillus subtilis culture, and ferment for 24-48 hours at 25-32℃, aeration rate of 0.5-1.5 vvm, and stirring speed of 150-300 r / min. After fermentation, heat the fermentation broth to 80-85℃ and maintain for 15-20 minutes to inactivate the enzymes. After cooling, centrifuge to remove the bacterial cells and obtain a clear fermentation broth. S6. Preparation of self-assembled nanocomposites: The clarified fermentation broth obtained in step S5 is concentrated to a solid content of 5-15%, the pH is adjusted to 4.5-5.5 with food-grade acid, chitosan oligosaccharide solution is added, and the mixture is reacted at 20-30℃ and a stirring speed of 100-300 r / min for 1-3 h to obtain a chitosan oligosaccharide-peptide co-assembled suspension. In step S6, the mass ratio of chitosan oligosaccharide to antihypertensive peptide is 1:(3-6); S7. Drying and Preparation: The co-assembled suspension obtained in step S6 is subjected to low-temperature vacuum drying or spray drying at 40-50℃ to obtain blood pressure-lowering Antarctic krill small molecule protein peptide powder.

2. The method for producing a blood pressure-lowering Antarctic krill small molecule protein peptide according to claim 1, characterized in that: In step S1, the microwave thawing power is 350W / kg, the frequency is 915MHz, and the thawing time is 12min.

3. The method for producing a blood pressure-lowering Antarctic krill small molecule protein peptide according to claim 1, characterized in that: In step S1, the shrimp temperature is controlled between 40-55℃ during the thawing process.

4. The method for producing a blood pressure-lowering Antarctic krill small molecule protein peptide according to claim 1, characterized in that: In step S2, the drying endpoint is controlled as follows: on a wet basis, the shrimp is dried until the internal moisture content is 30-50% and the surface moisture content is 5-10%, forming a semi-dry state with dry outside and moist inside.

5. The method for producing a blood pressure-lowering Antarctic krill small molecule protein peptide according to claim 1, characterized in that: In step S2, the heat pump drying temperature is 40-45℃ and the drying time is 5-6 hours.

6. The method for producing a blood pressure-lowering Antarctic krill small molecule protein peptide according to claim 1, characterized in that: In step S5, the spore concentration of the Aspergillus oryzae spore suspension is 10. 6 -10 7 CFU / mL; the viable count of Bacillus subtilis in the bacterial suspension was 10. 7 -10 8 CFU / mL.

7. The method for producing a blood pressure-lowering Antarctic krill small molecule protein peptide according to claim 1, characterized in that: In step S5, the total inoculation amount of Aspergillus oryzae and Bacillus subtilis is 2-10% of the fermentation liquid volume, and the ratio of viable Aspergillus oryzae to Bacillus subtilis is 1:(1-3).

8. The method for producing a blood pressure-lowering Antarctic krill small molecule protein peptide according to claim 1, characterized in that: In step S6, the concentration of the chitosan oligosaccharide solution is 1-5% (w / v), and the solvent is deionized water.

9. The method for producing a blood pressure-lowering Antarctic krill small molecule protein peptide according to claim 1, characterized in that: In step S6, the weight-average molecular weight of chitosan oligosaccharide is 1000-3000 Da, and the degree of deacetylation is ≥85%.

10. The method for producing a blood pressure-lowering Antarctic krill small molecule protein peptide according to claim 1, characterized in that: In step S7, the drying method is spray drying, with an inlet air temperature of 120-140℃, an outlet air temperature of 60-80℃, and a feed flow rate of 5-10mL / min.