Fermentation product containing giant salamander peptide as well as preparation method and application of fermentation product

Abstract

The invention relates to the field of biological medicine, and provides a preparation method of a fermentation product containing giant salamander peptide, which comprises the steps of stepped enzymolysis pretreatment, staged inoculated fermentation, synergistic enzymolysis and molecular weight grading purification. The method comprises the following steps: firstly, carrying out staged enzymolysis on giant salamander tissues by utilizing pepsin, trypsin and flavourzyme to obtain a primary peptide solution; then, bacillus subtilis, lactobacillus plantarum and bifidobacterium are adopted for directional fermentation, and flora metabolism is optimized through dissolved oxygen control and pH automatic regulation. Then, compound protease is added for secondary enzymolysis, so that the yield of the small molecule peptide is increased. And finally, carrying out ultrafiltration and nanofiltration graded purification, vacuum concentration, freeze drying and gamma-ray sterilization to obtain the micromolecular giant salamander peptide. The molecular weight of the obtained product is mainly distributed within 0.5-5 kDa (lt), and the proportion of 3 kDa is larger than or equal to 80%), the DPPH free radical scavenging rate is larger than or equal to 85%, the ABTS < + > scavenging rate is larger than or equal to 80%, and the obvious anti-oxidation and auxiliary liver protection effects are achieved.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a fermentation product containing giant salamander peptide, and a preparation method and application thereof. Background Art

[0002] Due to its rich biological activity, giant salamander peptides have broad application prospects in the fields of functional foods, medicines and health products. In the food industry, active peptides have become the research focus of high value-added nutrients due to their antioxidant, immunomodulatory and anti-aging functions. As a small molecule peptide extracted from giant salamander protein, the antioxidant capacity of giant salamander peptides directly affects the stability, nutritional value and bioavailability of the product. In food and biomedical applications, oxidation reactions not only lead to the degradation of active peptides, but also reduce their functionality and even affect the sensory quality of the end product. Therefore, improving the antioxidant properties of giant salamander peptides and keeping them stable during storage and use is the key to optimizing their application value. In addition, fermentation, as an efficient biotransformation method, plays a vital role in improving protein hydrolysis efficiency, optimizing peptide structure and enhancing biological activity. By rationally designing the fermentation process, the functional properties of giant salamander peptides can be effectively improved, such as enhancing antioxidant capacity, improving flavor, and improving solubility. Therefore, developing a preparation method that has both high-efficiency antioxidant properties and optimized fermentation technology can not only enhance the biological activity of giant salamander peptides, but also broaden their application scope in food, health care products and biomedicine, which is of great significance for promoting the industrialization of functional peptide products.

[0003] At present, the preparation and application research of giant salamander peptides has made certain progress, but there are still many shortcomings. For example, the Chinese patent with publication number CN102676622A discloses giant salamander nanopeptides and giant salamander wine, but the method has limitations in antioxidant properties and fails to effectively inhibit the generation of free radicals, resulting in the product being easily degraded during storage, affecting its biological activity. In addition, the improvement of antioxidant capacity not only depends on the screening of specific peptide segments, but is also closely related to the control of the oxidative environment during its preparation process, and the prior art is still insufficient in this regard. On the other hand, the Chinese patent with publication number CN106047968B discloses a giant salamander active peptide and its use, but its fermentation process parameters are not optimized enough, and the synergistic effect of different microbial strains is not fully considered, resulting in a low yield of the target peptide, and some fermentation products have the problem of bitterness or poor flavor. In addition, the dissolved oxygen, pH and metabolite accumulation in the fermentation process are not finely regulated, affecting the quality and stability of the final product. Therefore, in order to address the antioxidant stability and fermentation optimization issues of giant salamander peptides, it is urgent to develop an efficient and controllable preparation method to improve their biological activity and industrial feasibility. Summary of the invention

[0004] (1) Technical issues solved The purpose of the present invention is to provide a fermentation product containing giant salamander peptide and a preparation method and application thereof, so as to solve the problems of insufficient antioxidant and fermentation rate of giant salamander peptide at present.

[0005] (2) Technical solution In order to achieve the above object, the present invention provides the following technical solutions: A method for preparing a fermentation product containing giant salamander peptide comprises the following steps: S1. Stepwise enzymatic pretreatment, using pepsin and trypsin to hydrolyze the giant salamander tissue in stages to obtain primary peptide solution; S2. Inoculation and optimized fermentation in stages, the primary peptide solution is mixed with the carbon and nitrogen source medium, and Bacillus subtilis and Lactobacillus plantarum are inoculated in stages for directed fermentation to obtain the primary fermentation liquid; S3. Synergistic enzymatic hydrolysis, adding composite protease to the fermentation product for staged synergistic enzymatic hydrolysis to increase the yield of small molecule peptides; S4. Molecular weight fractionation ultrafiltration and freeze drying, the enzymatic hydrolysate is subjected to molecular weight fractionation ultrafiltration, vacuum concentration, freeze drying and non-thermal sterilization packaging to obtain small molecule giant salamander peptides; Furthermore, the stepwise enzymatic hydrolysis in step S1 is specifically as follows: the epidermal tissue of the giant salamander is mixed with deionized water in a mass ratio of 1:(8-12), and ultrasonic cleaning is performed at 15-20°C, the ultrasonic frequency is 40-45kHz, and the cleaning time is 15-20min; then, it is placed in a citric acid-disodium hydrogen phosphate buffer system with a pH of 1.5-2.5, and 1.0-2.0 wt.% of EC 3.4.23.1 pepsin is added in sequence, and hydrolysis is performed at a temperature of 37-42°C for 4-6h; then, the system is adjusted to a pH of 7.8-8.2 and 1.0-2.0 wt.% of EC 3.4.21.4 trypsin is added, and hydrolysis is performed at a temperature of 50-55°C for 3-5h; in the final enzymatic hydrolysis stage, the pH is adjusted to 6.0-7.0, and 1.0-2.0 wt.% is continued to be added. Flavourzyme flavor protease is hydrolyzed at 45~50℃ for 2~3h. Finally, the enzymatic hydrolysate is filtered using a microporous filter membrane with a pore size of 0.22~0.45μm to obtain a relatively pure primary peptide solution.

[0006] The present invention adopts the design of step-by-step enzymolysis mainly to enhance the hydrolysis efficiency of giant salamander tissue protein and the biological activity of the obtained peptide segment. By accurately controlling the enzymolysis order, pH conditions and temperature, the synergistic effect of different enzymes is optimized, the degradation of protein is more sufficient, and the generation of non-target products is reduced. First, deionized water is used in combination with ultrasonic cleaning according to a specific mass ratio, under suitable ultrasonic frequency and temperature conditions, it is helpful to destroy the tissue structure, improve the release efficiency of protein, and reduce impurity interference. Subsequently, under a citric acid-sodium hydrogen phosphate buffer system, by adjusting the pH to the optimum range of pepsin, it can act on the specific peptide bond of giant salamander protein efficiently, promote the initial degradation of protein, reduce molecular weight and release some functional peptide segments. On this basis, the system pH is adjusted to an alkaline range suitable for the action of trypsin, so that it further acts on a specific carboxyl terminal peptide bond, enhances the specificity of hydrolysis, and improves the enrichment of small molecule peptides. In the final enzymatic hydrolysis stage, the pH of the system is adjusted to the optimal range of Flavourzyme flavor protease, so that it can exert the synergistic effect of internal and external peptidases, further degrade oligopeptides, improve the flavor and solubility of peptide segments, and optimize their biological activity. Finally, through microporous membrane filtration, incompletely degraded macromolecular proteins and impurities are effectively removed to obtain a relatively pure primary peptide solution. The synergy between the components is reflected in the sequential action of different enzymes under specific pH and temperature conditions, making the degradation of proteins more precise, while avoiding the generation of bitter peptides caused by excessive degradation, and ultimately improving the yield and bioavailability of the target peptide.

[0007] Further, step S2 includes mixing the primary peptide solution with a composite carbon and nitrogen source culture medium, wherein the culture medium contains the following mass fraction components: glucose 15-25wt.%, maltose 5-10wt.%, peptone 10-20wt.%, yeast extract 10-20wt.%, magnesium sulfate 0.05-0.1wt.%, dipotassium hydrogen phosphate 0.02-0.05wt.%, sodium chloride 0.02-0.05wt.%, EDTA-2Na 0.05-0.10wt.; adjusting the pH value to 7.0-7.4, and then sterilizing at 110-120°C for 15-20min, cooling to 25-30°C, and adding calcium carbonate twice in a gradient manner, adding 1.5-2.0wt.% for the first time at 0h of fermentation, and adding 1.5-2.0wt.% after 12h, and then inoculating CGMCC at 5-8vol%. 1.1086 Bacillus subtilis was pre-cultured at 37-40℃ for 8-12h, during which DO≥30% was maintained by an online dissolved oxygen probe; then CGMCC 1.557 Lactobacillus plantarum was inoculated at 2-4vol%, and oxygen was step-wise reduced to DO 5-10% within 0-2h, and cultured at 37-40℃ with shaking for 32-36h; then Bifidobacterium CICC 6079 was inoculated at 2-3vol%, and high-purity nitrogen was introduced at a flow rate of 2-3L / min. Strict anaerobic fermentation was carried out at 38-40℃ and 50-100rpm for 36-40h, and the pH was maintained at 7.2-7.6 by an automatic alkali replenishment system.

[0008] The present invention adopts a design of phased inoculation and optimized fermentation, which is mainly used to enhance the bioconversion efficiency of giant salamander peptides and the enrichment of functional peptides. Nutrition is provided by a composite carbon and nitrogen source culture medium, calcium carbonate is added in a gradient to control the pH, Bacillus subtilis, Lactobacillus plantarum and Bifidobacterium are inoculated in sequence, dissolved oxygen control and automatic alkali supplementation are combined to achieve bacterial community synergistic metabolism and efficient fermentation, and finally obtain functional peptide segments with optimized biological activity.

[0009] Furthermore, the synergistic enzymatic hydrolysis in step S3 includes the following steps: placing the primary fermentation liquid in a constant temperature water bath and linearly heating it to 70-75°C at a temperature of 1-2°C / min, and maintaining it for 5-10 minutes to complete the thermal inactivation of endogenous enzymes; reducing the material temperature to 38-45°C at a cooling rate of 0.5-1°C / min through an ice water circulation system; accurately weighing pepsin powder ≥2500U / mg, trypsin powder ≥1000BTEE U / g and alkaline protease powder ≥2000U / g, and premixing them in sterile physiological saline at a mass ratio of 1:1:(0.5-1) to prepare a composite enzyme solution, wherein the total amount of enzyme added is 1.0-2.0wt.% of the total amount of substrate; using a step-by-step feeding method, firstly mixing 60vol.% of the composite enzyme solution with the substrate to start primary enzymatic hydrolysis, and hydrolyzing for 2-3h at 38-42°C and pH7.0-7.4; then adding the remaining 40vol.% of the composite enzyme solution and adjusting the pH to 7.6~7.8, switch to 45~50℃ and continue enzymatic hydrolysis for 4~5h.

[0010] The present invention adopts the design of synergistic enzymolysis mainly for enhancing the enzymolysis efficiency of giant salamander peptides and the enrichment of functional peptides. Through precise temperature control, the primary fermentation liquid is first heated to a specific temperature in a linear temperature rise mode to ensure that the endogenous enzyme is fully thermally inactivated to avoid interfering with the formation of the target peptide in the subsequent enzymolysis process. Subsequently, the ice water circulation system is used to accurately cool down the system so that the system quickly reaches a temperature range suitable for the action of exogenous enzymes, providing a stable environment for efficient enzymolysis. In the selection of enzyme preparations, a composite enzyme solution is prepared by pepsin, trypsin and alkaline protease in a specific mass ratio, and premixed uniformly in sterile physiological saline to ensure that each enzyme species works synergistically under appropriate conditions. Using the step-by-step feeding method, 60 vol.% of the composite enzyme solution is first added, and primary enzymolysis is performed under appropriate temperature and pH conditions to promote the moderate loosening of the protein structure in the substrate, which is conducive to the in-depth subsequent enzymolysis. Subsequently, the remaining 40 vol.% of the composite enzyme solution is added, and the pH and temperature are appropriately adjusted to make the enzymolysis conditions more conducive to the release of small molecule peptides. The synergy between the components is reflected in the organic combination of temperature control, endogenous enzyme inactivation, reasonable ratio of complex enzymes and step-by-step feeding strategy, which makes the enzymatic hydrolysis process more precise and controllable, and ultimately improves the yield and biological activity of the target peptide.

[0011] Furthermore, the molecular weight fractionation ultrafiltration in step S4 comprises the following steps: performing 30kDa polyacrylonitrile membrane endotoxin removal, 10kDa / 3kDa composite ultrafiltration system molecular weight fractionation and 1~5kDa nanofiltration membrane fine screening on the enzymatic hydrolyzate in sequence, and controlling the filtration rate to ≤20L / (m²·h) to form a small molecule peptide enrichment solution; Furthermore, the vacuum concentration in step S4 comprises the following steps: using a scraped film evaporator to concentrate the feed liquid to 15-18% of the original volume at a temperature range of 40-45° C. and a vacuum degree of 0.09-0.095 MPa; Furthermore, the freeze drying in step S4 includes the following steps: adding a composite protective agent consisting of 0.2-0.3 wt.% trehalose and 0.1-0.2 wt.% ascorbic acid to the concentrated solution, mixing well, firstly performing a primary drying stage, controlling the system temperature at -50--45°C and maintaining it for 6-8 h, then performing a secondary drying stage, heating to 20-25°C, maintaining it for 4-6 h, and the vacuum degree is less than 5 Pa; Furthermore, the non-thermal sterilization packaging process in step S4 is sterilization by irradiation with γ rays of 50-80 kGy, and adding 50-100 g / kg of silica gel desiccant, and the packaging environment requires a humidity of less than 5% and a temperature of 20-26°C.

[0012] The present invention adopts the design of molecular weight classification ultrafiltration, vacuum concentration, freeze drying and non-thermal sterilization packaging, which is mainly used to enhance the purification efficiency, stability and storage safety of giant salamander peptides. First, endotoxins are removed by 30kDa polyacrylonitrile membrane to ensure the safety of the terminal product, and the enzymatic hydrolysate is molecular weight graded by 10kDa / 3kDa composite ultrafiltration system, combined with 1~5kDa nanofiltration membrane fine screening, effectively enriching small molecule peptides and improving the purity of the target component. In this process, by strictly controlling the permeation rate, the filtration process is ensured to be gentle and controllable, and the loss or denaturation of the target peptide is avoided. Subsequently, a scraped film evaporator is used to perform vacuum concentration under low temperature and suitable vacuum conditions to reduce the degradation of heat-sensitive components, and the feed liquid is concentrated to a suitable range to provide optimized initial conditions for subsequent drying. In the freeze-drying stage, trehalose and ascorbic acid are added as composite protective agents to enhance the antioxidant stability of the product, and under the synergistic effect of primary low-temperature drying and secondary gentle temperature drying, the accurate removal of moisture is ensured, while avoiding the collapse of the material structure, and improving the solubility and biological activity of the terminal product. Finally, gamma ray irradiation sterilization is used to ensure sterility and safety, and silica gel desiccant is added to control the humidity in the package, combined with strict temperature and humidity management to further improve the storage stability of the product. Each process link cooperates with each other, from the enrichment, gentle concentration, stable drying of the target peptide to the final packaging and sterilization, forming an efficient and stable preparation process to ensure the high purity, high activity and long-term storage safety of the giant salamander peptide product.

[0013] The present invention also discloses a fermentation product containing a giant salamander peptide, and the fermentation product containing a giant salamander peptide is prepared by the above method; The fermentation product containing giant salamander peptides comprises active peptide segments with molecular weight distribution in the range of 0.5-5 kDa, of which those less than 3 kDa account for ≥80%; The fermentation product containing giant salamander peptide has a DPPH free radical scavenging rate of ≥85% and ABTS at a test concentration of 0.1 mg / mL. + Free radical scavenging rate ≥ 80%, total antioxidant capacity 12.0~15.0 U / mg; The invention also discloses the use of a fermentation product containing giant salamander peptide in assisting liver protection.

[0014] The present invention adopts the design of precise enzymatic hydrolysis, molecular weight fractionation purification and optimized fermentation, which is mainly used to enhance the biological activity and functional stability of giant salamander peptides. Through the step-by-step enzymatic hydrolysis strategy, combined with the synergistic effect of pepsin, trypsin and alkaline protease, the efficient degradation of giant salamander tissue protein is achieved, and the enrichment of small molecule peptides is optimized by step-by-step enzyme addition and dynamic pH regulation. Subsequently, the structure of the peptide segment is further modified by composite bacterial fermentation to enhance its antioxidant capacity and functional properties. During the purification process, molecular weight fractionation ultrafiltration technology is used to accurately screen the target peptide segment through 30kDa endotoxin removal and 10kDa / 3kDa composite ultrafiltration system, and further use 1~5kDa nanofiltration membrane to enrich small molecule peptides, so that the proportion of active peptides less than 3kDa in the final product reaches a high level, ensuring its superior bioavailability and functional activity. After that, vacuum concentration and freeze-drying technology are combined to gently dehydrate under low temperature and vacuum environment to avoid degradation of active peptides. Trehalose and ascorbic acid are added as composite protective agents to enhance the stability of the product and ensure that its antioxidant activity is not lost during storage. Finally, the microbial safety of the product is guaranteed through γ-ray irradiation sterilization and strict packaging environment control, and silica gel desiccant is added to maintain a low humidity environment in the package, thereby further improving its shelf life. The fermentation product containing giant salamander peptide prepared by the present invention has a high small molecule peptide content and excellent antioxidant capacity, and has excellent DPPH free radical scavenging and ABTS + It showed excellent effects in free radical scavenging and good total antioxidant capacity, proving its potential in functional applications such as liver protection. A high degree of synergy is formed between the various process links, and the comprehensive strategy of precise enzymatic hydrolysis, optimized fermentation, graded purification and stable drying is used to ensure the high activity, high stability and wide application value of the final product.

[0015] (3) Beneficial technical effects 1. The present invention uses step-by-step enzymatic hydrolysis to precisely control the enzymatic hydrolysis sequence, pH and temperature, making protein degradation efficient and specific, and reducing non-target products. Ultrasonic cleaning improves protein release rate, pepsin, trypsin and flavor protease act in sequence to improve the enrichment and flavor of small molecule peptides, and microporous membrane purifies target peptides to enhance biological activity and utilization.

[0016] 2. The present invention improves the bioconversion efficiency of giant salamander peptides by inoculating and optimizing fermentation in stages, and optimizes the enrichment of functional peptides by combining precise temperature control and synergistic enzymatic hydrolysis. The composite culture medium, gradient pH control and synergistic metabolism of bacterial flora ensure efficient fermentation, and the step-by-step feeding of exogenous enzymes and dynamic pH adjustment enhance the release of small molecule peptides, ultimately improving the purity, biological activity and stability of the target product.

[0017] 3. The present invention optimizes the purification and stability of giant salamander peptides through ultrafiltration classification, vacuum concentration, freeze drying and non-thermal sterilization, accurately controls the filtration rate to prevent the denaturation of active peptides, improves antioxidant properties through low-temperature concentration and composite protective agents, and ensures long-term storage safety through γ-ray sterilization and humidity control. The synergistic effect of various processes ensures high purity, high activity and stability of the product.

[0018] 4. The present invention synergistically improves the biological activity and stability of giant salamander peptides through precise enzymatic hydrolysis, molecular weight fractionation purification and optimized fermentation, optimizes the functional peptide structure through dynamic pH control and composite bacterial fermentation, and ensures product purity, safety and long-term storage through vacuum concentration, freeze drying and gamma-ray sterilization, ultimately significantly improving antioxidant capacity, and has broad prospects in functional applications such as auxiliary liver protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Example 1 of the present invention, the effect of different concentrations of H2O2 on the survival rate of HepG2 cells, H2O2 treatment for 16 hours, different English letters represent significant differences (p<0.05).

[0020] Figure 2 Example 1 of the present invention, the effect of different concentrations of ADFP on the survival rate of HepG2 cells, ADFP treatment for 16 hours, different English letters represent significant differences (p<0.05).

[0021] Figure 3 Example 1 of the present invention, analysis of the survival rate of HepG2 cells protected from H2O2 oxidative damage by different concentrations of ADFP, ADFP treatment for 24 hours, and then 300 μM H2O2 treatment for 16 hours, different letters represent significant differences (p<0.05).

[0022] FIG4 Example 2 of the present invention, effects of different concentrations of ADFP on the cell cycle of HepG2 cells induced by H2O2. The experimental conditions are the same as those in FIG3. Different letters represent significant differences (p<0.05).

[0023] FIG5 Analysis of the effect of different concentrations of ADFP on H2O2-induced apoptosis (AnnexinV / PI) of HepG2 cells in Example 2 of the present invention. The experimental conditions are the same as those in FIG3 . Different letters represent significant differences (p<0.05).

[0024] FIG6 Example 3 of the present invention, the effect of different concentrations of ADFP on the total antioxidant capacity (TAC) of HepG2 cells, the experimental conditions are the same as those in FIG3 , and different English letters represent significant differences (p<0.05).

[0025] FIG. 7 Example 3 of the present invention, the effect of different concentrations of ADFP on the generation of reactive oxygen species (ROS) in HepG2 cells induced by H2O2. The experimental conditions are the same as those in FIG. 3. Different letters represent significant differences (p<0.05).

[0026] FIG8 Example 3 of the present invention, the effect of different concentrations of ADFP on the superoxide dismutase (SOD) activity of HepG2 cells, the experimental conditions are the same as those in FIG3 , different English letters represent significant differences (p<0.05).

[0027] FIG9 Example 3 of the present invention, the effect of different concentrations of ADFP on the activity of glutathione peroxidase (GPx) in HepG2 cells. The experimental conditions are the same as those in FIG3 . Different letters represent significant differences (p<0.05).

[0028] FIG10 Example 3 of the present invention, the effect of different concentrations of ADFP on the activity of glutathione S-transferase (GST) in HepG2 cells. The experimental conditions are the same as those in FIG3 . Different letters represent significant differences (p<0.05). DETAILED DESCRIPTION

[0029] To make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0030] Example 1

[0031] A method for preparing a fermentation product containing giant salamander peptide comprises the following steps: S1. Step-by-step enzymatic pretreatment, using pepsin and trypsin to hydrolyze the giant salamander tissue in stages to obtain primary peptide solution; the giant salamander epidermal tissue and deionized water were mixed in a mass ratio of 1:8, and ultrasonically cleaned at 15°C with an ultrasonic frequency of 40kHz and a cleaning time of 15min; then it was placed in a citric acid-disodium hydrogen phosphate buffer system with a pH of 1.5, and 1.0 wt.% EC 3.4.23.1 pepsin was added in sequence, and hydrolyzed at 37°C for 4h; then the system was adjusted to pH 7.8 and 1.0wt.% EC 3.4.21.4 trypsin was added, and hydrolyzed at 50°C for 3h; in the final enzymatic stage, the pH was adjusted to 6.0, and 1.0 wt.% Flavourzyme flavor protease was added, and hydrolyzed at 45°C for 2h. Finally, the peptide solution was prepared by using a 0.22μm pore size HPLC system. The enzymatic hydrolysate was filtered through a microporous filter membrane to obtain a relatively pure primary peptide solution.

[0032] S2. Inoculation and optimized fermentation in stages: the primary peptide solution was mixed with the carbon and nitrogen source medium, and Bacillus subtilis and Lactobacillus plantarum were inoculated in stages for directional fermentation to obtain the primary fermentation liquid; the primary peptide solution was mixed with the composite carbon and nitrogen source medium, and the medium contained the following mass fraction components: glucose 15wt.%, maltose 5wt.%, peptone 10wt.%, yeast extract 10wt.%, magnesium sulfate 0.05wt.%, potassium dihydrogen phosphate 0.02wt.%, sodium chloride 0.02wt.%, EDTA-2Na 0.05wt.%; the pH value was adjusted to 7.0 and then sterilized at 110℃ for 15min, cooled to 25℃ and calcium carbonate was added twice in a gradient manner, 1.5wt.% was added for the first time at 0h of fermentation and 1.5wt.% was added 12h later, and then CGMCC1.1086 Bacillus subtilis was inoculated at 5vol%, and pre-cultured at 37℃ for 8h, during which DO was maintained ≥30% by an online dissolved oxygen probe; then CGMCC was inoculated at 2vol% 1.557 plant lactobacillus, step-wise oxygen reduction to DO 5% within 0h, and shaking culture at 37℃ for 32h; then inoculated with Bifidobacterium CICC 6079 at 2vol%, and passed high-purity nitrogen at a flow rate of 2L / min. Strict anaerobic fermentation was carried out at 38℃ and 50rpm for 36h, and the pH was maintained at 7.2 by an automatic alkali replenishment system.

[0033] S3. Synergistic enzymatic hydrolysis, adding composite proteases to the fermentation products for phased synergistic enzymatic hydrolysis to improve the yield of small molecule peptides; placing the primary fermentation broth in a constant temperature water bath and linearly heating it to 70°C at 1°C / min, maintaining it for 5 minutes to complete the thermal inactivation of endogenous enzymes; reducing the material temperature to 38°C at a cooling rate of 0.5°C / min through an ice water circulation system; accurately weighing pepsin powder ≥2500U / mg, trypsin powder ≥1000BTEE U / g and alkaline protease powder ≥2000U / g, premixing them in sterile saline at a mass ratio of 1:1:0.5 to prepare a composite enzyme solution, with the total amount of enzyme added being 1.0wt.% of the total amount of substrate; using a step-by-step feeding method, first mixing 60vol.% of the composite enzyme solution with the substrate to start primary enzymatic hydrolysis, and hydrolyzing at 38°C and pH7.0 for 2h; then adding the remaining 40vol.% of the composite enzyme solution and adjusting it to pH 7.6, switching to 45°C to continue enzymatic hydrolysis for 4h.

[0034] S4. Molecular weight fractionation ultrafiltration and freeze drying, the enzymatic hydrolysate is subjected to molecular weight fractionation ultrafiltration, vacuum concentration, freeze drying and non-thermal sterilization packaging to obtain small molecule giant salamander peptides; the fractionation ultrafiltration includes the following steps: the enzymatic hydrolysate is subjected to 30kDa polyacrylonitrile membrane endotoxin removal, 10kDa / 3kDa composite ultrafiltration system molecular weight fractionation and 1kDa nanofiltration membrane fine screening in sequence, and the filtration rate is controlled to be ≤20L / (m²·h) to form a small molecule peptide enrichment solution; the vacuum concentration includes the following steps: the material solution is concentrated to 15% of the original volume by a scraped film evaporator at a temperature range of 40°C and a vacuum degree of 0.09MPa; the freeze drying includes the following steps: a composite protective agent composed of 0.2wt.% trehalose and 0.1wt.% ascorbic acid is added to the concentrate, and after being fully mixed, a primary drying stage is first performed, the system temperature is controlled at -50°C and maintained for 6h, and then a secondary drying stage is performed, the temperature is raised to 20°C, maintained for 4h, and the vacuum degree is less than 5 Pa; The non-thermal sterilization packaging process uses γ-ray 50 kGy irradiation sterilization and adds 50g / kg of silica gel desiccant. The packaging environment requires humidity less than 5% and temperature of 20℃.

[0035] In this embodiment, a fermentation product containing a giant salamander peptide comprises active peptides with a molecular weight distribution in the range of 0.5-5 kDa, of which 81% are less than 3 kDa; the fermentation product containing a giant salamander peptide has a DPPH free radical scavenging rate of 85% at a test concentration of 0.1 mg / mL, and ABTS + The free radical scavenging rate was 80%, and the total antioxidant capacity was 12.0 U / mg.

[0036] Table 1 is an analysis of the amino acid composition of the fermentation product of giant salamander (ADFP) in Example 1. It can be seen from the table that the fermentation product is rich in a variety of essential and non-essential amino acids, among which aspartic acid (Asp 40.96 mg / g) and glutamic acid (Glu 34.26 mg / g) have high contents, indicating that it is rich in acidic amino acids, which helps to improve protein solubility and flavor characteristics. In addition, arginine (Arg 32.02 mg / g) and tyrosine (Tyr 28.56 mg / g) have high contents, which may give the product certain antioxidant activity and functional properties. Branched-chain amino acids (BCAA), such as valine (Val 12.24 mg / g), isoleucine (Ile 8.12 mg / g) and leucine (Leu 7.58 mg / g), although the content is moderate, play an important role in promoting protein synthesis and enhancing nutritional value. The high content of proline (Pro 62.77 mg / g) may be derived from the degradation of collagen in giant salamander tissues, indicating that the product effectively released collagen-related peptides during processing, which helped to improve its biological activity and functional properties. The high content of tryptophan (Trp 9.51 mg / g) may have a positive effect on neuromodulation and anti-oxidation. Overall, the amino acid composition of the fermentation product is relatively balanced, indicating that through reasonable enzymatic hydrolysis and fermentation processes, the bioavailability of protein can be effectively improved, while giving the product good functional properties.

[0037] Table 1 Analysis of amino acid composition of giant salamander fermentation product (ADFP)

[0038] In this example, the human liver cancer HepG2 cell line was used as an in vitro model to evaluate the effect of hydrogen peroxide (H2O2) on cell oxidative damage and survival rate. In the experiment, H2O2 was added at final concentrations of 0, 100, 200, 300, 400 and 500 μM, and cultured for 16 hours to analyze the survival rate of HepG2 cells under different oxidative stresses (Figure 1). The results showed that H2O2 had a concentration-dependent cytotoxicity to HepG2 cells. As the concentration of H2O2 increased, the cell survival rate gradually decreased, and its half inhibitory concentration (IC50) was 2.34. 50) is about 300 μM. Therefore, this study confirmed that HepG2 cells can be used as an evaluation model for oxidative stress damage in vitro, and selected 300 μM H2O2 as the appropriate concentration for inducing oxidative damage. Literature reports that the endogenous antioxidant defense system in the body is difficult to completely resist the oxidative stress induced by exogenous H2O2, which in turn leads to cell apoptosis or necrosis. Although H2O2 itself does not directly participate in the oxidation reaction, it can react with transition metal ions (such as iron and copper) in the body to produce highly active hydroxyl radicals (•OH). Hydroxyl radicals are highly reactive and can quickly attack lipids, proteins and DNA in cells, causing cell damage and exacerbating oxidative stress reactions.

[0039] This study further used HepG2 cell line as an in vitro model to evaluate the cytotoxicity of giant salamander fermentation product (ADFP). In the experiment, ADFP was added to the final concentrations of 0, 0.025, 0.05, 0.1, 0.2 and 0.25 mg / mL, and cultured for 16 hours to analyze the effects of ADFP on HepG2 cells at different concentrations (Figure 2). The results showed that ADFP did not show significant cytotoxicity to HepG2 cells within the concentration range below 0.05 mg / mL. The protective effect of ADFP on HepG2 cells under H2O2-induced oxidative damage was further explored (Figure 3). In the experiment, ADFP was first added to the final concentrations of 0, 0.0125, 0.025, 0.05, 0.1 and 0.2 mg / mL and cultured for 24 hours, followed by the addition of H2O2 (300 μM) and continued to be cultured for 16 hours to determine the cell survival rate. The results showed that after H2O2 treatment alone, the viability of HepG2 cells dropped to 57.1 ± 0.2%. However, the viability of the cell groups that were protected for 24 hours with 0.025 mg / mL and 0.05 mg / mL ADFP before H2O2 treatment increased to 67.4 ± 0.4% and 71.0 ± 0.8%, respectively, and were significantly higher than those in the H2O2 treatment group (p<0.05). Therefore, ADFP at concentrations of 0.025 and 0.05 mg / mL can effectively reduce H2O2-induced oxidative damage in HepG2 cells and show significant protective effects. Based on this result, the subsequent cell cycle analysis and Annexin V / PI cell apoptosis experiments selected 0.025 and 0.05 mg / mL as the experimental concentrations with the best protective effect.

[0040] Example 2

[0041] A method for preparing a fermentation product containing giant salamander peptide comprises the following steps: S1. Step-by-step enzymatic pretreatment, using pepsin and trypsin to hydrolyze the giant salamander tissue in stages to obtain primary peptide solution; the giant salamander epidermal tissue and deionized water were mixed in a mass ratio of 1:9, and ultrasonically cleaned at 17°C with an ultrasonic frequency of 42kHz and a cleaning time of 17min; then it was placed in a citric acid-disodium hydrogen phosphate buffer system with a pH of 1.8, and 1.3 wt.% EC 3.4.23.1 pepsin was added in sequence, and hydrolyzed at 39°C for 5h; then the system was adjusted to pH 7.9 and 1.3wt.% EC 3.4.21.4 trypsin was added, and hydrolyzed at 52°C for 4h; in the final enzymatic stage, the pH was adjusted to 6.3, and 1.3 wt.% Flavourzyme flavor protease was added, and hydrolyzed at 47°C for 2.3h. Finally, the peptide solution was prepared by using a 0.29μm pore size The enzymatic hydrolysate was filtered through a microporous filter membrane to obtain a relatively pure primary peptide solution.

[0042] S2. Inoculation and optimized fermentation in stages, the primary peptide solution was mixed with the carbon and nitrogen source medium, and Bacillus subtilis and Lactobacillus plantarum were inoculated in stages for directional fermentation to obtain the primary fermentation liquid; the primary peptide solution was mixed with the composite carbon and nitrogen source medium, the medium containing the following mass fraction components: glucose 18wt.%, maltose 6.5wt.%, peptone 13wt.%, yeast extract 13wt.%, magnesium sulfate 0.07wt.%, potassium dihydrogen phosphate 0.03wt.%, sodium chloride 0.03wt.%, EDTA-2Na 0.07wt.; after adjusting the pH value to 7.1, it was sterilized at 113℃ for 17min, cooled to 27℃ and calcium carbonate was added twice in a gradient manner, 1.7wt.% was added for the first time at 0h of fermentation and 1.7wt.% was added 12h later, and then CGMCC1.1086 Bacillus subtilis was inoculated at 6vol%, and pre-cultured at 38℃ for 9h, during which DO was maintained ≥30% by an online dissolved oxygen probe; then CGMCC was inoculated at 3vol% 1.557 plant lactobacillus, step-wise oxygen reduction to DO 7% within 1 hour, and shaking culture at 38℃ for 33 hours; then inoculated with Bifidobacterium CICC 6079 at 2.3vol%, passed high-purity nitrogen at a flow rate of 2.3L / min, strictly anaerobic fermentation at 39℃ and 65rpm for 37 hours, and the pH was maintained at 7.3 by an automatic alkali replenishment system.

[0043] S3. Synergistic enzymatic hydrolysis, adding composite proteases to the fermentation products for phased synergistic enzymatic hydrolysis to improve the yield of small molecule peptides; placing the primary fermentation broth in a constant temperature water bath and linearly heating it to 72°C at 1.3°C / min, maintaining it for 7 minutes to complete the thermal inactivation of endogenous enzymes; cooling the material temperature to 40°C at a cooling rate of 0.7°C / min through an ice water circulation system; accurately weighing pepsin powder ≥2500U / mg, trypsin powder ≥1000BTEE U / g and alkaline protease powder ≥2000U / g, premixing them in sterile saline at a mass ratio of 1:1:0.7 to prepare a composite enzyme solution, with the total amount of enzyme added being 1.3wt.% of the total amount of substrate; using a step-by-step feeding method, first mixing 60vol.% of the composite enzyme solution with the substrate to start primary enzymatic hydrolysis, and hydrolyzing for 2.3h at 39°C and pH7.1; then adding the remaining 40vol.% of the composite enzyme solution and adjusting the pH to 7.7, switch to 47°C and continue enzymatic hydrolysis for 4.3h.

[0044] S4. Molecular weight fractionation ultrafiltration and freeze drying, the enzymatic hydrolysate is subjected to molecular weight fractionation ultrafiltration, vacuum concentration, freeze drying and non-thermal sterilization packaging to obtain small molecule giant salamander peptides; the fractionation ultrafiltration includes the following steps: the enzymatic hydrolysate is subjected to 30kDa polyacrylonitrile membrane endotoxin removal, 10kDa / 3kDa composite ultrafiltration system molecular weight fractionation and 2.2kDa nanofiltration membrane fine screening in sequence, and the permeation rate is controlled to be ≤20L / (m²·h) to form a small molecule peptide enrichment solution; the vacuum concentration includes the following steps: the material solution is concentrated to 16% of the original volume by using a scraped film evaporator at a temperature range of 42°C and a vacuum degree of 0.092MPa; the freeze drying includes the following steps: a composite protective agent composed of 0.23wt.% trehalose and 0.13wt.% ascorbic acid is added to the concentrate, and after being fully mixed, a primary drying stage is first performed, the system temperature is controlled at -48°C and maintained for 7h, and then a secondary drying stage is performed, the temperature is raised to 22°C, maintained for 5h, and the vacuum degree is less than 5 Pa; The non-thermal sterilization packaging process uses γ-ray 59 kGy irradiation sterilization and adds 65g / kg of silica gel desiccant. The packaging environment requires humidity less than 5% and temperature of 22°C.

[0045] In this embodiment, a fermentation product containing a giant salamander peptide comprises active peptides with a molecular weight distribution in the range of 0.5-5 kDa, of which peptides less than 3 kDa account for 81%; at a test concentration of 0.1 mg / mL, the fermentation product containing a giant salamander peptide has a DPPH free radical scavenging rate of 86%, and ABTS + The free radical scavenging rate was 81%, and the total antioxidant capacity was 12.9 U / mg.

[0046] In this study, cell cycle analysis and Annexin V / PI apoptosis detection were used to evaluate the protective effect of different concentrations of giant salamander fermentation products (ADFP) on H2O2-induced oxidative damage in HepG2 cells (Figures 4 and 5). The results of cell cycle analysis showed that the proportion of cells in the Sub-G1 phase in the H2O2-treated group increased significantly, indicating an increase in the apoptosis rate, while ADFP pretreatment (0.025 and 0.05 mg / mL) significantly reduced the proportion of cells in the Sub-G1 phase. In addition, H2O2 treatment caused cell cycle arrest in the G2 / M phase, while ADFP pretreatment alleviated the arrest phenomenon, indicating that ADFP has the effect of reducing cell apoptosis and maintaining the normal progression of the cell cycle. Annexin V / PI double staining analysis showed that the proportion of live cells in the H2O2-treated group was significantly reduced, while the proportion of live cells was significantly increased after ADFP pretreatment (p<0.05). Although ADFP had no obvious trend in the effect on early apoptotic cells, it could significantly reduce the proportion of late apoptotic and necrotic cells induced by H2O2 (p<0.05), further confirming that ADFP has the ability to protect HepG2 cells from oxidative damage. In summary, ADFP at concentrations of 0.025 and 0.05 mg / mL can effectively reduce H2O2-induced oxidative damage, and its mechanism may include reducing the proportion of Sub-G1 cells, alleviating G2 / M cell cycle arrest, and reducing the proportion of late apoptotic and necrotic cells, indicating that ADFP has potential antioxidant protective effects.

[0047] Example 3

[0048] A method for preparing a fermentation product containing giant salamander peptide comprises the following steps: S1. Step-by-step enzymatic pretreatment, using pepsin and trypsin to hydrolyze the giant salamander tissue in stages to obtain primary peptide solution; the giant salamander epidermal tissue and deionized water were mixed in a mass ratio of 1:10, and ultrasonically cleaned at 18°C ​​with an ultrasonic frequency of 43kHz and a cleaning time of 18min; then it was placed in a citric acid-disodium hydrogen phosphate buffer system with a pH of 2.1, and 1.6 wt.% EC 3.4.23.1 pepsin was added in sequence, and hydrolyzed at 40°C for 5h; then the system was adjusted to pH 8.0 and 1.6wt.% EC 3.4.21.4 trypsin was added, and hydrolyzed at 53°C for 4h; the final enzymatic hydrolysis stage was adjusted to pH 6.6, and 1.6 wt.% Flavourzyme flavor protease was added, and hydrolyzed at 48°C for 2.6h. Finally, the peptide solution was prepared by using a 0.36μm pore size HPLC system. The enzymatic hydrolysate was filtered through a microporous filter membrane to obtain a relatively pure primary peptide solution.

[0049] S2. Inoculation and optimized fermentation in stages: the primary peptide solution was mixed with the carbon and nitrogen source medium, and Bacillus subtilis and Lactobacillus plantarum were inoculated in stages for directional fermentation to obtain the primary fermentation liquid; the primary peptide solution was mixed with the composite carbon and nitrogen source medium, and the medium contained the following mass fraction components: glucose 21wt.%, maltose 8wt.%, peptone 16wt.%, yeast extract 16wt.%, magnesium sulfate 0.08wt.%, potassium dihydrogen phosphate 0.04wt.%, sodium chloride 0.04wt.%, EDTA-2Na 0.08wt.%; the pH value was adjusted to 7.2 and then sterilized at 116℃ for 18min, cooled to 28℃ and calcium carbonate was added twice in a gradient manner, 1.8wt.% was added for the first time at 0h of fermentation and 1.8wt.% was added 12h later, and then CGMCC1.1086 Bacillus subtilis was inoculated at 7vol%, and pre-cultured at 39℃ for 10h, during which DO was maintained ≥30% by an online dissolved oxygen probe; then CGMCC was inoculated at 3vol% 1.557 plant lactobacillus, step-wise oxygen reduction to DO 8% within 1 hour, and shaking culture at 39℃ for 34 hours; then inoculated with Bifidobacterium CICC 6079 at 2.6vol%, passed high-purity nitrogen at a flow rate of 2.6L / min, strictly anaerobic fermentation was carried out at 39℃ and 80rpm for 38 hours, and the pH was maintained at 7.4 by an automatic alkali replenishment system.

[0050] S3. Synergistic enzymatic hydrolysis, adding composite proteases to the fermentation products for phased synergistic enzymatic hydrolysis to improve the yield of small molecule peptides; placing the primary fermentation broth in a constant temperature water bath and linearly heating it to 73°C at 1.6°C / min, maintaining it for 8 minutes to complete the thermal inactivation of endogenous enzymes; cooling the material temperature to 42°C at a cooling rate of 0.8°C / min through an ice water circulation system; accurately weighing pepsin powder ≥2500U / mg, trypsin powder ≥1000BTEE U / g and alkaline protease powder ≥2000U / g, premixing them in sterile saline at a mass ratio of 1:1:0.8 to prepare a composite enzyme solution, with the total amount of enzyme added being 1.6wt.% of the total amount of substrate; using a step-by-step feeding method, first mixing 60vol.% of the composite enzyme solution with the substrate to start primary enzymatic hydrolysis, and hydrolyzing for 2.6h at 40°C and pH7.2; then adding the remaining 40 vol.% of the composite enzyme solution and adjusting the pH to 7.7, switch to 48°C and continue enzymatic hydrolysis for 4.6h.

[0051] S4. Molecular weight fractionation ultrafiltration and freeze drying, the enzymatic hydrolysate is subjected to molecular weight fractionation ultrafiltration, vacuum concentration, freeze drying and non-thermal sterilization packaging to obtain small molecule giant salamander peptides; the fractionation ultrafiltration includes the following steps: the enzymatic hydrolysate is subjected to 30kDa polyacrylonitrile membrane endotoxin removal, 10kDa / 3kDa composite ultrafiltration system molecular weight fractionation and 3.4kDa nanofiltration membrane fine screening in sequence, and the filtration rate is controlled to be ≤20L / (m²·h) to form a small molecule peptide enrichment solution; the vacuum concentration includes the following steps: the material solution is concentrated to 17% of the original volume by a scraped film evaporator at a temperature range of 43°C and a vacuum degree of 0.093MPa; the freeze drying includes the following steps: a composite protective agent composed of 0.26wt.% trehalose and 0.16wt.% ascorbic acid is added to the concentrate, and after being fully mixed, a primary drying stage is first performed, the system temperature is controlled at -47°C and maintained for 7h, and then a secondary drying stage is performed, the temperature is raised to 23°C, maintained for 5h, and the vacuum degree is less than 5 Pa; The non-thermal sterilization packaging process uses γ-ray 68 kGy irradiation sterilization and adds 80g / kg of silica gel desiccant. The packaging environment requires humidity less than 5% and temperature of 24°C.

[0052] In this embodiment, a fermentation product containing a giant salamander peptide comprises active peptides with a molecular weight distribution in the range of 0.5-5 kDa, of which 82% are less than 3 kDa; the fermentation product containing a giant salamander peptide has a DPPH free radical scavenging rate of 87% at a test concentration of 0.1 mg / mL, and ABTS +·The free radical scavenging rate was 82%, and the total antioxidant capacity was 13.8 U / mg. This example further analyzed the effects of different concentrations of ADFP (0.025 and 0.05 mg / mL) on H2O2-induced oxidative stress damage in HepG2 cells, specifically evaluating the changes in total antioxidant capacity (TAC), reactive oxygen species (ROS) generation, and antioxidant enzyme (SOD, GPx, GST) activity (Figures 6–10). The experimental results showed that after H2O2 treatment, TAC activity decreased significantly (p<0.05), while ADFP pretreatment significantly increased TAC activity (Figure 6), indicating that ADFP reduces oxidative damage by enhancing total antioxidant capacity. At the same time, H2O2 significantly increased ROS generation (p<0.05), while ADFP pretreatment significantly reduced ROS levels (Figure 7), indicating that it can effectively inhibit the accumulation of free radicals induced by oxidative stress. In addition, the analysis of antioxidant enzyme activity showed that H2O2 treatment led to a significant decrease in the activities of SOD, GPx and GST (p<0.05), while ADFP pretreatment significantly increased the activities of these antioxidant enzymes (Figures 8–10), further confirming that ADFP can enhance the antioxidant defense capacity of HepG2 cells and reduce oxidative stress damage by regulating the antioxidant enzyme system. In summary, ADFP at concentrations of 0.025 and 0.05 mg / mL can effectively alleviate H2O2-induced oxidative damage by increasing the activities of TAC, SOD, GPx and GST and reducing ROS generation, showing good antioxidant protection.

[0053] Example 4

[0054] A method for preparing a fermentation product containing giant salamander peptide comprises the following steps: S1. Step-by-step enzymatic pretreatment, using pepsin and trypsin to hydrolyze the giant salamander tissue in stages to obtain primary peptide solution; the giant salamander epidermal tissue and deionized water were mixed in a mass ratio of 1:12, and ultrasonic cleaning was performed at 20°C with an ultrasonic frequency of 45kHz and a cleaning time of 20min; it was then placed in a citric acid-disodium hydrogen phosphate buffer system with a pH of 2.5, and 2.0 wt.% EC 3.4.23.1 pepsin was added in sequence, and hydrolysis was carried out at a temperature of 42°C for 6h; the system was then adjusted to pH 8.2 and 2.0wt.% EC 3.4.21.4 trypsin was added, and hydrolysis was carried out at a temperature of 55°C for 5h; the final enzymatic hydrolysis stage was adjusted to pH 7.0, and 2.0 wt.% Flavourzyme flavor protease was added, and hydrolysis was carried out at 50°C for 3h. Finally, the peptide solution was prepared using a 0.45μm pore size HPLC system. The enzymatic hydrolysate was filtered through a microporous filter membrane to obtain a relatively pure primary peptide solution.

[0055] S2. Inoculation and optimized fermentation in stages, the primary peptide solution was mixed with the carbon and nitrogen source medium, and Bacillus subtilis and Lactobacillus plantarum were inoculated in stages for directional fermentation to obtain the primary fermentation liquid; the primary peptide solution was mixed with the composite carbon and nitrogen source medium, the medium containing the following mass fraction components: glucose 25wt.%, maltose 10wt.%, peptone 20wt.%, yeast extract 20wt.%, magnesium sulfate 0.1wt.%, dipotassium hydrogen phosphate 0.05wt.%, sodium chloride 0.05wt.%, EDTA-2Na 0.10wt.; the pH value was adjusted to 7.4 and then sterilized at 120℃ for 20min, cooled to 30℃ and calcium carbonate was added twice in a gradient manner, 2.0wt.% was added for the first time at 0h of fermentation and 2.0wt.% was added 12h later, and then CGMCC 1.1086 Bacillus subtilis was inoculated at 8vol%, and pre-cultured at 40℃ for 12h, during which DO was maintained ≥30% by an online dissolved oxygen probe; then CGMCC was inoculated at 4vol% 1.557 plant lactobacillus, step-wise oxygen reduction to DO 10% within 2 hours, and shaking culture at 40℃ for 36 hours; then inoculated with Bifidobacterium CICC 6079 at 3vol%, passed high-purity nitrogen at a flow rate of 3L / min, strictly anaerobic fermentation was carried out at 40℃ and 100rpm for 40 hours, and the pH was maintained at 7.6 by an automatic alkali replenishment system.

[0056] S3. Synergistic enzymatic hydrolysis, adding composite proteases to the fermentation products for phased synergistic enzymatic hydrolysis to improve the yield of small molecule peptides; placing the primary fermentation broth in a constant temperature water bath and linearly heating it to 75°C at 2°C / min, maintaining it for 10 min to complete the thermal inactivation of endogenous enzymes; cooling the material temperature to 45°C at a cooling rate of 1°C / min through an ice water circulation system; accurately weighing pepsin powder ≥2500U / mg, trypsin powder ≥1000BTEE U / g and alkaline protease powder ≥2000U / g, premixing them in sterile saline at a mass ratio of 1:1:1 to prepare a composite enzyme solution, with the total amount of enzyme added being 2.0wt.% of the total amount of substrate; using a step-by-step feeding method, first mixing 60vol.% of the composite enzyme solution with the substrate to start primary enzymatic hydrolysis, and hydrolyzing at 42°C and pH7.4 for 3h; then adding the remaining 40vol.% of the composite enzyme solution and adjusting it to pH 7.8, switching to 50°C to continue enzymatic hydrolysis for 5h.

[0057] S4. Molecular weight fractionation ultrafiltration and freeze drying, the enzymatic hydrolysate is subjected to molecular weight fractionation ultrafiltration, vacuum concentration, freeze drying and non-thermal sterilization packaging to obtain small molecule giant salamander peptides; the fractionation ultrafiltration includes the following steps: the enzymatic hydrolysate is subjected to 30kDa polyacrylonitrile membrane endotoxin removal, 10kDa / 3kDa composite ultrafiltration system molecular weight fractionation and 5kDa nanofiltration membrane fine screening in sequence, and the permeation rate is controlled to be ≤20L / (m²·h) to form a small molecule peptide enrichment solution; the vacuum concentration includes the following steps: the material solution is concentrated to 18% of the original volume by using a scraped film evaporator at a temperature range of 45°C and a vacuum degree of 0.095MPa; the freeze drying includes the following steps: a composite protective agent composed of 0.3wt.% trehalose and 0.2wt.% ascorbic acid is added to the concentrate, and after being fully mixed, a primary drying stage is first performed, the system temperature is controlled at -45°C and maintained for 8h, and then a secondary drying stage is performed, the temperature is raised to 25°C, maintained for 6h, and the vacuum degree is less than 5 Pa; The non-thermal sterilization packaging process uses γ-ray 80 kGy irradiation sterilization and adds 100g / kg of silica gel desiccant. The packaging environment requires humidity less than 5% and temperature of 26°C.

[0058] In this embodiment, a fermentation product containing a giant salamander peptide comprises active peptides with a molecular weight distribution in the range of 0.5-5 kDa, of which 83% are less than 3 kDa; the fermentation product containing a giant salamander peptide has a DPPH free radical scavenging rate of 86% at a test concentration of 0.1 mg / mL, and ABTS + The free radical scavenging rate was 83%, and the total antioxidant capacity was 15.0 U / mg.

[0059] Comparative Example 1 The same as Example 1, except that: Flavourzyme flavor protease was not added in the final enzymatic hydrolysis stage of step S1 Comparative Example 2 The same as Example 1, except that: the ultrasonic cleaning frequency in step S1 is fixed to 20kHz (originally 40-45kHz) Comparative Example 3 The same as Example 1, except that: the pepsin hydrolysis temperature in step S1 is fixed at 45°C (originally 37-42°C) Comparative Example 4 The same as Example 1, except that: in step S2, the potassium hydrogen phosphate in the culture medium is replaced with an equal amount of potassium chloride. Comparative Example 5 The same as Example 1, except that calcium carbonate was not added in batches in step S2 (3-4% of the total amount was added only at 0h). Comparative Example 6 Basically the same as Example 1, except that: Step S2: maintain DO≥20% after inoculation of Lactobacillus plantarum (originally step-down oxygen to 5-10%) Comparative Example 7 The same as Example 1, except that the ratio of the complex enzyme in step S3 is fixed to 1:1:0.2 (originally 1:1:(0.5-1)) Comparative Example 8 The same as Example 1, except that the pH of the primary enzymatic hydrolysis in step S3 was fixed at 6.8 (originally 7.0-7.4) Comparative Example 9 It is basically the same as Example 1, except that: In step S4, 30 kDa polyacrylonitrile membrane was not used for endotoxin removal.

[0060] Comparative Example 10 It is basically the same as Example 1, except that: In step S4, the nanofiltration membrane was replaced with a 10 kDa cut-off membrane (originally 1-5 kDa).

[0061] Comparative Example 11 The method is basically the same as Example 1, except that no trehalose and ascorbic acid are added in the freeze-drying step S4.

[0062] Comparative Example 12 It is basically the same as Example 1, except that the primary drying temperature in step S4 is set to -30°C (originally -50~-45°C) Comparative Example 13 The same as Example 1, except that: the radiation sterilization dose in step S4 is reduced to 20 kGy (originally 50-80 kGy) Comparative Example 14 The same as Example 1, except that: Step S4 encapsulation does not use silica gel desiccant Comparative Example 15 The method is basically the same as Example 1, except that: Bifidobacterium CICC 6079 is not inoculated in step S2. Performance Test: The antioxidant capacity was determined (DPPH free radical scavenging rate). The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging experiment was used to determine the scavenging rate of fermentation products at different concentrations on DPPH free radicals. Ascorbic acid was used as a positive control to calculate the IC50 value (half inhibitory concentration).

[0063] Antioxidant capacity assay (ABTS + · Free radical scavenging rate), using ABTS +· (2,2'-Bis(2-phenylthiazoline-6-sulfonic acid diammonium salt) free radical solution, determination of sample to ABTS + The free radical scavenging ability was measured at 734 nm and the antioxidant capacity (TEAC value) was calculated using Trolox as the standard.

[0064] Total antioxidant capacity (FRAP test): The FRAP (ferric ion reducing antioxidant capacity) test was used to determine the ability of the fermentation product to reduce Fe 3+ Reduction to Fe 2+ The antioxidant capacity was calculated by the change of absorbance at 593 nm and compared with the FeSO4 standard curve.

[0065] Analysis of small molecule peptide content (HPLC): High performance liquid chromatography (HPLC) was used with a C18 reverse phase column and acetonitrile-water gradient elution. The ultraviolet absorption peak at 220 nm was detected and the proportion of small molecule peptides (<3 kDa) was calculated.

[0066] The properties of the fermentation products containing giant salamander peptides of Examples 1 to 4 and Comparative Examples 1 to 15 are summarized in Table 2.

[0067] Table 2 Performance of the fermentation products containing giant salamander peptides in Examples 1 to 4 and Comparative Examples 1 to 15

[0068] The experimental results show that the proportion and antioxidant capacity of small molecule peptides are significantly affected by multiple process parameters. Among them, the addition of flavor protease in the final enzymatic hydrolysis stage can effectively increase the enrichment of small molecule peptides and enhance the DPPH and ABTS +Free radical scavenging ability, and the abnormal increase of pepsin hydrolysis temperature will lead to a decrease in enzyme activity, inhibit the full degradation of protein, and reduce the antioxidant capacity. In addition, too low an ultrasonic cleaning frequency affects protein release, resulting in a decrease in primary enzymatic hydrolysis efficiency, which in turn affects the proportion of small molecule peptides in the final product. During the fermentation stage, the replacement of phosphate in the culture medium may cause precipitation, inhibit bacterial metabolism, and thus affect the synthesis of antioxidant peptides, while the non-stepwise addition of calcium carbonate will lead to increased pH fluctuations, affect the survival rate of bifidobacteria, and reduce fermentation efficiency. Oxygen control is essential for the metabolism of Lactobacillus plantarum. Excessive dissolved oxygen levels will lead to acetic acid accumulation, inhibit the modification of functional peptides, and reduce antioxidant properties. Adjustment of the proportion of composite proteases also affects the efficiency of protein hydrolysis. When the alkaline protease content is insufficient, the yield of small molecule peptides decreases significantly. The deviation of enzymatic hydrolysis pH will inhibit trypsin activity and reduce the generation of antioxidant peptides. In the separation and purification stage, the expansion of the interception range of the nanofiltration membrane may lead to the loss of the target small molecule peptide, thereby significantly reducing the antioxidant capacity. Although the failure to remove endotoxins does not affect the antioxidant properties, it will affect the safety of the product. During the freeze-drying process, the lack of appropriate protective agents (such as trehalose and ascorbic acid) will aggravate the oxidative degradation of active peptides and reduce the antioxidant capacity, while unreasonable primary drying temperature may cause structural collapse and affect the solubility of the final product. In addition, insufficient irradiation sterilization dose may lead to excessive microorganisms, and the failure to add silica gel desiccant during packaging will lead to increased humidity during storage and accelerate the attenuation of active ingredients. In summary, reasonable enzymatic hydrolysis, fermentation, purification and drying processes are crucial to improving the enrichment and antioxidant capacity of small molecule peptides, and optimizing key parameters can significantly improve product performance.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that all equivalent structural changes made under the concept of the present invention and using the contents of the present invention specification and drawings should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a fermentation product containing giant salamander peptide, characterized in that: The following steps are involved: S1. Stepwise enzymatic pretreatment, using pepsin and trypsin to hydrolyze the giant salamander tissue in stages to obtain primary peptide solution; S2. Inoculation and optimized fermentation in stages, the primary peptide solution is mixed with the carbon and nitrogen source medium, and Bacillus subtilis and Lactobacillus plantarum are inoculated in stages for directed fermentation to obtain the primary fermentation liquid; S3. Synergistic enzymatic hydrolysis, adding composite protease to the fermentation product for staged synergistic enzymatic hydrolysis to increase the yield of small molecule peptides; S4. Molecular weight fractionation ultrafiltration and freeze drying: The enzymatic hydrolysate is subjected to molecular weight fractionation ultrafiltration, vacuum concentration, freeze drying and non-thermal sterilization packaging to obtain small molecule giant salamander peptides.

2. The method for preparing a fermentation product containing a giant salamander peptide according to claim 1, characterized in that: The stepwise enzymatic hydrolysis in step S1 is specifically as follows: the epidermal tissue of the giant salamander is mixed with deionized water in a mass ratio of 1:(8-12), and ultrasonic cleaning is performed at 15-20°C, the ultrasonic frequency is 40-45kHz, and the cleaning time is 15-20min; then, it is placed in a citric acid-sodium hydrogen phosphate buffer system with a pH of 1.5-2.5, 1.0-2.0 wt.% of EC 3.4.23.1 pepsin is added in sequence, and hydrolysis is performed at a temperature of 37-42°C for 4-6h; then, the system is adjusted to a pH of 7.8-8.2 and 1.0-2.0 wt.% of EC3.4.21.4 trypsin is added, and hydrolysis is performed at a temperature of 50-55°C for 3-5h; in the final enzymatic hydrolysis stage, the pH is adjusted to 6.0-7.0, and 1.0-2.0 wt.% of EC3.4.21.4 trypsin is added. Flavourzyme flavor protease is hydrolyzed at 45~50℃ for 2~3h. Finally, the enzymatic hydrolysate is filtered using a microporous filter membrane with a pore size of 0.22~0.45μm to obtain a relatively pure primary peptide solution.

3. The method for preparing a fermentation product containing a giant salamander peptide according to claim 1, characterized in that: The step S2 comprises mixing the primary peptide solution with a composite carbon and nitrogen source culture medium, wherein the culture medium comprises the following components by mass fraction: 15-25wt.% glucose, 5-10wt.% maltose, 10-20wt.% peptone, 10-20wt.% yeast extract, 0.05-0.1wt.% magnesium sulfate, 0.02-0.05wt.% dipotassium hydrogen phosphate, 0.02-0.05wt.% sodium chloride, and 0.05-0.10wt.% EDTA-2Na; adjusting the pH value to 7.0-7.4, sterilizing by high pressure at 110-120°C for 15-20min, cooling to 25-30°C, and adding calcium carbonate twice in a gradient manner, adding 1.5-2.0wt.% for the first time at 0h of fermentation, and adding 1.5-2.0wt.% after 12h, and then inoculating CGMCC at 5-8vol%. 1.1086 Bacillus subtilis was pre-cultured at 37-40℃ for 8-12h, during which DO≥30% was maintained by an online dissolved oxygen probe; then CGMCC 1.557 Lactobacillus plantarum was inoculated at 2-4vol%, and oxygen was step-wise reduced to DO 5-10% within 0-2h, and cultured at 37-40℃ with shaking for 32-36h; then Bifidobacterium CICC 6079 was inoculated at 2-3vol%, and high-purity nitrogen was introduced at a flow rate of 2-3L / min. Strict anaerobic fermentation was carried out at 38-40℃ and 50-100rpm for 36-40h, and the pH was maintained at 7.2-7.6 by an automatic alkali replenishment system.

4. The method for preparing a fermentation product containing a giant salamander peptide according to claim 1, characterized in that: The synergistic enzymatic hydrolysis in step S3 comprises the following steps: placing the primary fermentation liquid in a constant temperature water bath and linearly heating it to 70-75°C at 1-2°C / min, maintaining it for 5-10 minutes to complete the thermal inactivation of endogenous enzymes; reducing the material temperature to 38-45°C at a cooling rate of 0.5-1°C / min through an ice water circulation system; accurately weighing pepsin powder ≥2500U / mg, trypsin powder ≥1000BTEE U / g and alkaline protease powder ≥2000U / g, and premixing them in sterile physiological saline at a mass ratio of 1:1:(0.5-1) to prepare a composite enzyme solution, wherein the total amount of enzyme added is 1.0-2.0wt.% of the total amount of substrate; using a step-by-step feeding method, firstly mixing 60vol.% of the composite enzyme solution with the substrate to start primary enzymatic hydrolysis, and hydrolyzing for 2-3h at 38-42°C and pH7.0-7.4; then adding the remaining 40vol.% of the composite enzyme solution and adjusting the pH to 1:1:(0.5-1). 7.6~7.8, switch to 45~50℃ and continue enzymatic hydrolysis for 4~5h.

5. The method for preparing a fermentation product containing a giant salamander peptide according to claim 1, characterized in that: The preparation method of the modified polyurethane emulsion is as follows: by weight, 20 to 50 parts of fluorine-containing polyol, 24 to 75 parts of isocyanate and 80 to 120 parts of N, N-dimethylformamide are added to a reaction kettle, reacted at 60 to 80° C. for 2 to 4 hours to obtain a prepolymer, and then 5 to 10 parts of a chain extender are added to the prepolymer, stirred evenly, and reacted at 100 to 140° C. for 1 to 2 hours to obtain a modified polyurethane emulsion.

6. The method for preparing a fermentation product containing a giant salamander peptide according to claim 1, characterized in that: The molecular weight fractionation ultrafiltration in step S4 comprises the following steps: performing 30kDa polyacrylonitrile membrane endotoxin removal, 10kDa / 3kDa composite ultrafiltration system molecular weight fractionation and 1~5kDa nanofiltration membrane fine screening on the enzymatic hydrolyzate in sequence, and controlling the filtration rate to be ≤20L / (m²·h) to form a small molecule peptide enrichment solution.

7. The method for preparing a fermentation product containing a giant salamander peptide according to claim 1, characterized in that: The vacuum concentration in step S4 comprises the following steps: using a scraped film evaporator to concentrate the feed liquid to 15-18% of the original volume at a temperature range of 40-45° C. and a vacuum degree of 0.09-0.095 MPa; The freeze drying in step S4 comprises the following steps: adding a composite protective agent consisting of 0.2-0.3 wt.% trehalose and 0.1-0.2 wt.% ascorbic acid to the concentrated solution, mixing thoroughly and evenly, firstly performing a primary drying stage, controlling the system temperature at -50--45°C and maintaining it for 6-8 hours, then performing a secondary drying stage, heating to 20-25°C, maintaining it for 4-6 hours, and the vacuum degree is less than 5 Pa; The non-thermal sterilization packaging process in step S4 is to use γ-ray 50-80 kGy irradiation sterilization and add 50-100 g / kg of silica gel desiccant. The packaging environment requires humidity less than 5% and temperature 20-26°C.

8. A fermentation product containing giant salamander peptide, characterized in that: The high-strength hot-dip galvanized steel is prepared by the preparation method described in any one of claims 1 to 7; The fermentation product containing giant salamander peptides comprises active peptide segments with molecular weight distribution in the range of 0.5-5 kDa, of which those less than 3 kDa account for ≥80%.

9. A fermentation product containing a giant salamander peptide as claimed in claim 8, characterized in that: The fermentation product containing giant salamander peptide has a DPPH free radical scavenging rate of ≥85% and ABTS at a test concentration of 0.1 mg / mL. + The free radical scavenging rate is ≥80%, and the total antioxidant capacity is 12.0~15.0 U / mg.

10. Use of a fermentation product containing a giant salamander peptide prepared by the preparation method according to any one of claims 1 to 7, or a fermentation product containing a giant salamander peptide according to any one of claims 8 to 9 in assisting liver protection.

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