Preparation method of alligator ginseng anti-fatigue active polypeptide liquid

Abstract

This invention relates to the field of bioactive polypeptide extraction technology and discloses a method for preparing crocodile ginseng anti-fatigue active polypeptide liquid, comprising the following steps: ginseng powder is microwave-disrupted and subjected to compound enzymatic hydrolysis; a portion of the extract is extracted as a polysaccharide intermediate liquid; the remaining substrate is inoculated with *Aspergillus oryzae* and *Lactobacillus plantarum* for two-stage variable-temperature and variable-aeration fermentation to obtain ginseng fermentation broth; crocodile meat is steamed, homogenized, and defatted with lipase, then subjected to enzymatic hydrolysis with compound protease, and at the end of the enzymatic hydrolysis, the polysaccharide intermediate liquid is pumped in for mixing to obtain crocodile polypeptide liquid; finally, the ginseng fermentation broth and crocodile polypeptide liquid are mixed, L-ascorbic acid is added for closed fermentation, and the polypeptide liquid is obtained through ultrafiltration, adsorption, and microfiltration. This invention promotes the release of hydrolytic enzymes through variable-temperature fermentation, increasing the content of rare saponins; utilizes the spatial complexation of polysaccharides and polypeptides to mask the bitterness of the polypeptides; and combines defatting treatment with closed aerobic fermentation to block oxidation pathways and remove fishy and muttony odors, thereby improving the system stability and anti-fatigue activity of the polypeptide liquid.

Description

Technical Field

[0001] This invention relates to the field of bioactive polypeptide extraction and processing technology, specifically a method for preparing an anti-fatigue active polypeptide liquid from crocodile ginseng. Background Technology

[0002] Animal-derived peptides are often combined with plant-based active ingredients to prepare anti-fatigue liquid products. When preparing peptides using animal muscle tissues such as crocodile meat, the traditional enzymatic hydrolysis process exposes hydrophobic amino acid residues inside the protein to the aqueous phase, resulting in a noticeable bitter taste in the final peptide product. At the same time, some lipid components such as triglycerides are usually left in the interstitial spaces of animal muscle tissue. These lipids will undergo oxidation reactions during subsequent heating, enzymatic hydrolysis, and storage, thereby producing a fishy and gamey odor and reducing the palatability of the product.

[0003] On the other hand, ginseng, as a commonly used anti-fatigue excipient, contains saponins that are mostly in the form of large molecular glycosides. Among them, Rb series protopanaxadiones, which have relatively low biological activity, account for a large proportion. Conventional water extraction or simple enzymatic hydrolysis processing methods are difficult to break their stable glycosidic bonds, which means that large molecular saponins cannot be effectively deglycosylated and transformed into rare saponins with high anti-fatigue activity, resulting in the problem of low bioavailability of raw materials.

[0004] Furthermore, existing compound polypeptide liquid preparation processes mostly employ the method of directly and physically mixing animal polypeptide liquid with plant extracts. In this conventional mixing process, the free amino groups at the ends of polypeptides and the reducing active substances in ginseng are easily exposed to air and undergo oxidation or structural changes due to oxygen. This simple processing method not only fails to resolve the flavor conflict caused by the bitterness of polypeptides and the oxidation products of lipids, but also leads to problems such as poor stability and low conversion rate of anti-fatigue active ingredients in the final polypeptide liquid system after long-term storage, making it difficult to meet the production requirements of high-quality polypeptide liquid. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing crocodile ginseng anti-fatigue active polypeptide liquid, which solves the problems of existing animal polypeptide extracts having a bitter and fishy taste, low effective conversion rate of ginsenosides, and poor stability caused by easy oxidation of conventional mixing systems.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a crocodile ginseng anti-fatigue active polypeptide liquid, comprising the following steps: S1. After microwave cell wall breaking of ginseng powder, water is added and compound enzymatic hydrolysis is carried out using plant enzyme hydrolysate. Part of the liquid is extracted as polysaccharide intermediate liquid, and the remaining liquid is cooled after enzyme inactivation to obtain ginseng fermentation substrate. S2. Inoculate the ginseng fermentation substrate with Aspergillus oryzae and Lactobacillus plantarum for fermentation. After the first stage of fermentation, raise the temperature and reduce the aeration rate to carry out the second stage of fermentation. After inactivation and filtration, obtain the ginseng fermentation liquid. S3. The crocodile meat is steamed and homogenized with water, then inactivated and cooled after being hydrolyzed with lipase to obtain defatted crocodile slurry. S4. Add a complex protease to the defatted crocodile slurry for enzymatic hydrolysis. Before the end of the enzymatic hydrolysis, pump in a polysaccharide intermediate liquid for mixing. After enzyme inactivation, centrifugation and filtration, obtain crocodile polypeptide solution. S5. Mix ginseng fermentation liquid with crocodile polypeptide liquid, add L-ascorbic acid and ferment in a sealed container. After sterilization, ultrafiltration, activated carbon adsorption and microfiltration, the polypeptide liquid is obtained.

[0007] By adopting the above technical solution, the reaction conditions of each component in the system are changed due to the multi-step reaction control and material flow separation. Therefore, the effects of increasing the content of rare saponins, masking the bitterness of polypeptides and preventing the oxidation of active groups are achieved.

[0008] In the S2 ginseng fermentation process, the first stage provides a suitable temperature and high aeration to promote the biomass accumulation of Aspergillus oryzae and Lactobacillus plantarum. Then, in the second stage, the physical environment is changed by raising the temperature and lowering the aeration. This drastic change in environmental conditions will interfere with the normal metabolic pathway of Aspergillus oryzae, causing changes in the permeability of its cell wall structure and even autolysis, thereby releasing the β-glucosidase that was originally enriched in the fungal cell into the fermentation broth.

[0009] The free β-glucosidase comes into full contact with the ginseng fermentation substrate, specifically catalyzing the enzymatic cleavage of the glycosidic bond at the C-20 position of Rb series protopanaxadiones, removing some sugar groups and generating rare saponins Rg3 and Rh2 with high anti-fatigue activity. At the same time, the lactic acid produced by Lactobacillus plantarum metabolism constructs a slightly acidic liquid environment, maintaining the chemical stability of the free aglycone structure after deglycosylation.

[0010] To address the bitterness of polypeptides, this invention utilizes the intermolecular interaction between polysaccharides and polypeptides to form a spatial encapsulation. When animal muscle proteins are cleaved by complex proteases, hydrophobic amino acid residues that were originally located inside the protein are exposed to the aqueous phase, forming bitter precursors.

[0011] To mask this residual flavor, this method involves extracting a portion of the ginseng polysaccharide intermediate liquid in S1 and pumping it into the blending system at the end of the alligator protein hydrolysis reaction in S4. The hydrophilic ginseng polysaccharide macromolecules form physical entanglement around the free short peptides through hydrogen bonds and van der Waals forces. The free carbonyl groups of the polysaccharide crosslink with the terminal amino groups of the polypeptide, forming a polysaccharide-peptide complex.

[0012] In addition, to address system stability and lipid oxidation issues, during the later stages of fermentation in S5, the system containing live bacteria was placed in a closed space without mechanical ventilation. In this closed environment, Lactobacillus plantarum used the substrate carbon source for anaerobic metabolism, continuously consuming the residual dissolved oxygen in the liquid phase. Combined with the reducing properties of L-ascorbic acid, the dissolved oxygen concentration in the mixed liquid phase rapidly decreased to a low level. The anaerobic environment blocked the oxidation pathway of free amino groups of short peptides and prevented the formation of fishy and muttony odor substances from residual lipids from the early enzymatic hydrolysis.

[0013] Preferably, in S1, the step of adding water to ginseng powder after microwave cell wall disruption for compound enzymatic hydrolysis includes: taking 100 parts of ginseng powder and microwave cell wall disruption at 600-700W power for 3-5 minutes; adding 1000-1200 parts of water and mixing evenly; adjusting the system temperature to 45-50℃; adjusting the pH to 4.5-5.0 with citric acid; adding 0.2-0.3 parts of pectinase and 0.3-0.4 parts of cellulase; and stirring at a constant temperature for enzymatic hydrolysis for 2.5-3.0 hours.

[0014] By employing the above technical solution, microwaves are used to induce the vaporization and expansion of intracellular water, thereby disrupting the cell structure. Simultaneously, pectinase and cellulase are used to enzymatically hydrolyze the pectin network and cellulose skeleton in the plant cell wall, releasing intracellular contents and providing a material basis for subsequent substrate transformation.

[0015] Preferably, in step S1, the step of extracting a portion of the liquid as a polysaccharide intermediate solution and cooling the remaining liquid after enzyme inactivation to obtain the ginseng fermentation substrate includes: heating the liquid after adding pectinase and cellulase for enzymatic hydrolysis to 50-55℃, adjusting the pH to 5.5-6.0, adding 0.3-0.5 parts of β-glucanase, and stirring at a constant temperature for 2.0-2.5 hours for enzymatic hydrolysis; extracting 5-10% of the total volume of the system liquid and refrigerating it as a polysaccharide intermediate solution; and inactivating the enzyme in the remaining liquid at 90-95℃ for 10-15 minutes and cooling it to 35-37℃ to obtain the ginseng fermentation substrate.

[0016] By adopting the above technical solution, β-glucanase degrades macromolecular glucan into water-soluble polysaccharides and oligosaccharides with appropriate molecular weights. 5-10% of the total volume of the extraction system is used as an intermediate liquid, which retains this part of plant polysaccharides for subsequent peptide encapsulation, while ensuring that the remaining substrate has sufficient carbon source for microbial fermentation.

[0017] Preferably, in step S2, the step of inoculating the ginseng fermentation substrate with Aspergillus oryzae and Lactobacillus plantarum for fermentation includes: inoculating 2-8 parts of Aspergillus oryzae culture and 5-8 parts of Lactobacillus plantarum culture into the cooled ginseng fermentation substrate; setting the temperature for the first stage fermentation to 32-34℃ and the aeration rate to 1.0-1.2 L / (L·min), and fermenting for 36 h; raising the temperature to 38-40℃ and reducing the aeration rate to 0.2-0.3 L / (L·min) for the second stage fermentation, and fermenting for 36 h.

[0018] By adopting the above technical solution, the normal proliferation of aerobic microorganisms was maintained under the ambient temperature and oxygen-rich conditions in the first stage. On this basis, the second stage of heating and low ventilation changed the metabolic flow of microorganisms, initiated the autolysis release mechanism mentioned above, and increased the extracellular concentration of the target enzyme.

[0019] Preferably, in step S3, the steps of steaming and homogenizing crocodile meat with water, adding lipase for enzymatic hydrolysis, inactivating and cooling to obtain defatted crocodile slurry include: mixing 100 parts of crocodile meat with 300 parts of water, steaming at 105-110℃ for 1.5-2.0h, cooling to 40-45℃ and homogenizing into meat paste; adjusting the pH of the system to 7.0-7.5, maintaining the temperature at 50-55℃, adding 0.3-0.5 parts of lipase, stirring and enzymatically hydrolyzing for 1.5-2.0h, inactivating at 85℃ for 10min, and cooling to 50-55℃ to obtain defatted crocodile slurry.

[0020] By adopting the above technical solution, lipase is used to specifically cleave the triglycerides remaining in the interstitial spaces of muscle tissue, thereby reducing free fatty acids and lipid oxidation precursors that can produce unpleasant flavors, thus cutting off the generation pathway of fishy and muttony odors in subsequent processes from the source.

[0021] Preferably, in step S4, the step of adding a complex protease to the defatted alligator slurry for enzymatic hydrolysis includes: adjusting the pH of the defatted alligator slurry to 7.5-8.0, adding 0.5-1.0 parts of a complex protease, the complex protease being composed of papain and protease in a weight ratio of 1:2 to 1:4, and hydrolyzing at a constant temperature of 50-55°C for a total hydrolysis time of 3.0-3.5 h.

[0022] By adopting the above technical solution, papain performs endo-cleavage to open the protein polypeptide chain, while the protease performs exo-cleavage at both ends of the polypeptide chain. This ratio allows the large protein molecules to be degraded into oligopeptides with concentrated molecular weight, which is beneficial to their solubility and anti-fatigue effects.

[0023] Preferably, in S4, the step of pumping in the polysaccharide intermediate solution for mixing before the end of enzymatic hydrolysis includes: when the complex protease hydrolysis is in the last 30-40 minutes, pumping the polysaccharide intermediate solution into the system, maintaining 50-55°C and pH 7.5-8.0 and continuing to stir until the total hydrolysis time is over.

[0024] By adopting the above technical solution, ginseng polysaccharide is introduced at the moment when free peptides are generated but before hydrophobic aggregation and polymerization occur. This can avoid excessive non-enzymatic browning caused by premature addition of polysaccharide. At the same time, the shear force generated by stirring promotes the uniform distribution of polysaccharide chains around the peptides, achieving effective complexation and encapsulation.

[0025] Preferably, in step S5, the step of mixing ginseng fermentation liquid with crocodile polypeptide liquid and adding L-ascorbic acid for closed fermentation includes: mixing 100 parts of ginseng fermentation liquid, 100-300 parts of crocodile polypeptide liquid and 0.1-0.4 parts of L-ascorbic acid in a closed container, retaining a headspace air layer at the top of the container that accounts for 10-15% of the total volume of the closed container, without mechanical ventilation, and fermenting in a closed container at 30-32°C for 12-16 hours.

[0026] By adopting the above technical solution, the reserved trace air layer is used to maintain the initial physiological metabolism. Subsequently, the dissolved oxygen in the liquid phase is depleted, creating a reducing and sealed environment, thereby achieving deep fusion of the components and regulating the acid-base balance of the final liquid.

[0027] Preferably, in step S5, the ultrafiltration step includes: pumping the feed liquid into an ultrafiltration membrane with a molecular weight cutoff of 10,000-50,000 Da for cross-flow filtration and collecting the permeate.

[0028] By adopting the above technical solution, based on the size of the polysaccharide-peptide complex formed in the early reaction and the saponin molecules after conversion, an ultrafiltration membrane with a molecular weight cutoff of 10,000-50,000 Da is selected. This molecular weight cutoff range of 10,000-50,000 Da allows the target complex active substances to pass through, while effectively retaining residual large undegraded proteins, microbial cell fragments and other suspended particles, avoiding the interception and loss of complexed active ingredients by traditional small-pore ultrafiltration membranes.

[0029] Preferably, in step S5, the steps of activated carbon adsorption and microfiltration to obtain polypeptide solution include: adding 0.1-0.2 parts of powdered activated carbon to the permeate, stirring and adsorbing at 25-30℃ for 30-40 min, filtering by plate and frame filter and filtration through a series of 0.22μm microporous membranes, and collecting the filtrate to obtain polypeptide solution.

[0030] By adopting the above technical solution, activated carbon is used to adsorb residual free pigment molecules and tiny impurity and odor molecules. Then, the detached carbon particles and tiny bacteria are intercepted by a 0.22-micron filter membrane, ensuring the clarity and sterility of the final product of the polypeptide liquid.

[0031] This invention provides a method for preparing an anti-fatigue active polypeptide liquid from crocodile ginseng. It has the following beneficial effects: 1. This invention employs a two-stage ginseng fermentation process with varying temperature and aeration to induce Aspergillus oryzae to autolyze and release β-glucosidase. This enzyme can specifically decompose the glycosidic bonds of protoginsenosides, converting macromolecular saponins into rare Rg3 and Rh2 saponins with high anti-fatigue activity, thereby increasing the content of effective components in the polypeptide solution.

[0032] 2. In this invention, a portion of ginseng polysaccharide is extracted as an intermediate liquid and pumped into the enzymatic hydrolysis system at the end of the alligator protein hydrolysis process. This allows the hydrophilic ginseng polysaccharide to physically entangle and cross-link with the free alligator short peptides, blocking the direct contact between the hydrophobic amino acid residues of the peptides and the taste buds, masking the bitterness often found in animal peptides, and improving the overall taste of the product.

[0033] 3. In the early processing of crocodile meat, lipase is added to eliminate lipid oxidation precursors. In the final process, the oxygen-consuming metabolism of Lactobacillus plantarum and L-ascorbic acid are used to construct a closed anaerobic fermentation environment, which cuts off the pathway for residual oil to produce fishy and muttony smells. At the same time, it prevents the oxidation and deterioration of free amino groups of short peptides, and ensures the structural stability and flavor harmony of the polypeptide liquid system. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the dynamic comparison of extracellular β-glucosidase activity according to the present invention; Figure 2 This is a schematic diagram illustrating the dynamic comparison of the total amount of rare saponins in this invention. Figure 3 This is a schematic diagram illustrating the change in the hydrophobicity index of the free polypeptide surface in this invention. Figure 4 This is a schematic diagram showing the distribution of the browning index measurement results of the present invention; Figure 5 This is a schematic diagram illustrating the change in dissolved oxygen concentration in the system of this invention; Figure 6 This is a schematic diagram illustrating the pH value changes in the system of this invention; Figure 7 This is a schematic diagram showing the distribution of bitterness residue in the final product of this invention. Figure 8 This is a schematic diagram showing the distribution of residual fishy and muttony odor in the final product of this invention. Figure 9 This is a schematic diagram showing the distribution of the overall flavor harmony of the final product of this invention; Figure 10 This is a schematic diagram of the polysaccharide retention concentration distribution of the present invention; Figure 11 This is a schematic diagram showing the concentration distribution of the water-soluble short peptides of the present invention. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Please see the appendix Figure 1 - Appendix Figure 11 : The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0037] The ginseng is made from the dried roots of ginseng plants (Araliaceae family) that have been artificially cultivated for 5 years or less, with a moisture content of no more than 15%. The ginseng powder is obtained by ultra-fine grinding and passing through a 200-mesh sieve. The total ginsenoside content in the ginseng powder is no less than 3.5% (based on dry weight), which includes one or more of the original ginsenosides Rb1, Rb2, Rc, and Rd.

[0038] The crocodile meat is made from pure muscle tissue of artificially raised Siamese crocodiles, with all fascia and visible fat removed.

[0039] Pectinase (CAS No. 9032-75-1) is a food-grade enzyme preparation with an enzyme activity greater than or equal to 500 U / mg.

[0040] Cellulase (CAS No. 9012-54-8) is a food-grade enzyme preparation with an enzyme activity greater than or equal to 400 U / mg.

[0041] β-glucanase (CAS No. 9025-70-1) is a food-grade enzyme preparation with an enzyme activity greater than or equal to 500 U / mg.

[0042] Lipase (CAS No. 9001-62-1) is a food-grade enzyme preparation with an enzyme activity greater than or equal to 5000 U / g.

[0043] Papain (CAS No. 9001-73-4) is a food-grade enzyme preparation with an enzyme activity greater than or equal to 8000 U / g.

[0044] The protease (CAS No. 9001-92-7) is a food-grade enzyme preparation with an enzyme activity greater than or equal to 8000 U / g.

[0045] Aspergillus oryzae: ATCC10124.

[0046] Lactobacillus plantarum: ATCC14917.

[0047] Powdered activated carbon (CAS No. 7440-44-0) is a food-grade porous carbon material with a particle size controlled between 200 and 300 mesh.

[0048] The ultrafiltration membrane uses a cross-flow filtration membrane element made of polyethersulfone, with a membrane pore size corresponding to a molecular weight cutoff of 10,000 to 50,000 Da.

[0049] D101 type macroporous adsorption resin: polystyrene type non-polar adsorbent, used for the enrichment and desalination of saponin components; Ginsenoside Rg3 and ginsenoside Rh2: both with a purity of ≥98%, purchased from the China National Institutes for Food and Drug Control, used for plotting the content determination standard curve.

[0050] p-Nitrophenyl-β-D-glucopyranoside (CAS No. 2492-87-7): Purity ≥98%, used for the determination of β-glucosidase activity.

[0051] 8-Aniline-1-naphthalenesulfonic acid: a fluorescent reagent used for the determination of protein hydrophobicity.

[0052] In this embodiment, the unit of measurement for the amount of each material and solvent is uniformly referred to as parts by weight.

[0053] Example 1: This embodiment provides a method for preparing a crocodile ginseng anti-fatigue active polypeptide liquid, including the following steps: S1. Take 100 parts of ginseng powder and microwave it at 650W for 4 minutes. Add 1100 parts of water to the treated ginseng powder and mix well. Adjust the system temperature to 48℃, adjust the pH to 4.8 with 0.1mol / L citric acid, add 0.25 parts of pectinase and 0.35 parts of cellulase, and stir at a constant temperature for 2.8 hours for enzymatic hydrolysis. Then raise the temperature to 53℃, adjust the pH to 5.8, add 0.4 parts of β-glucanase, and continue to stir at a constant temperature for 2.2 hours for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, extract 8% of the total volume of liquid and refrigerate it at 4℃ as ginseng polysaccharide intermediate solution. Keep the remaining liquid at 95℃ for 12 minutes to inactivate the enzyme, and then cool it to 36℃.

[0054] S2. Add 6 parts of Aspergillus oryzae culture and 6 parts of Lactobacillus plantarum culture to the cooled liquid; set the first stage fermentation temperature at 33℃ and the aeration rate at 1.1L / (L·min) for 36h; raise the temperature to 39℃ and reduce the aeration rate to 0.25L / (L·min) for 36h; after fermentation, inactivate the ginseng at 95℃ for 20min and collect the ginseng fermentation liquid by filtration.

[0055] S3. Take 100 parts of crocodile meat and mix with 300 parts of water. Cook at 108℃ for 1.8 hours. After cooling to 45℃, homogenize into meat paste. Adjust the pH of the system to 7.3 and maintain the temperature at 53℃. Add 0.4 parts of lipase and stir to hydrolyze for 1.8 hours. Then inactivate at 85℃ for 10 minutes and cool down to 53℃.

[0056] S4. Adjust the pH of the cooled system to 7.8 using 0.1 mol / L NaOH, add 0.75 parts of a complex protease (composed of papain and protease in a weight ratio of 1:2), and hydrolyze at a constant temperature of 53℃ for a total hydrolysis time of 3.2 h. When the hydrolysis is in its last 35 min, pump the ginseng polysaccharide intermediate solution stored in S1 into the system, maintain 53℃ and pH 7.8 and continue stirring until the total hydrolysis time is over. Then heat to 95℃ to inactivate the enzyme for 15 min, cool and centrifuge at 4800 r / min for 20 min, take the supernatant and filter it through a 0.22 μm filter membrane to collect the crocodile polypeptide solution.

[0057] S5. Mix 100 parts of ginseng fermentation broth and 200 parts of crocodile polypeptide broth in a sealed tank, add 0.2 parts of L-ascorbic acid, and maintain a headspace air layer of 12% of the total volume at the top of the tank. Do not perform mechanical aeration and ferment in a sealed environment at 31°C for 14 hours. Sterilize the liquid at 115°C for 15 minutes and cool it down to 25°C. Pump the liquid into an ultrafiltration membrane with a molecular weight cutoff of 30,000 Da for cross-flow filtration and collect the permeate. Add 0.15 parts of powdered activated carbon to the permeate and stir and adsorb at 25°C for 35 minutes. Filter the solution through a plate and frame filter press and a series of 0.22 μm microporous membranes. Collect the filtrate to obtain the final anti-fatigue active polypeptide broth.

[0058] Example 2: This embodiment provides a method for preparing a crocodile ginseng anti-fatigue active polypeptide liquid, including the following steps: S1. Take 100 parts of ginseng powder and microwave it at 600W for 3 minutes. Add 1000 parts of water to the treated ginseng powder and mix well. Adjust the system temperature to 45℃, adjust the pH to 4.5 with 0.1mol / L citric acid, add 0.2 parts of pectinase and 0.3 parts of cellulase, and stir at a constant temperature for 2.5 hours for enzymatic hydrolysis. Then raise the temperature to 50℃, adjust the pH to 5.5, add 0.3 parts of β-glucanase, and continue to stir at a constant temperature for 2.0 hours for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, extract 5% of the total volume of liquid and refrigerate it at 4℃ as ginseng polysaccharide intermediate solution. Keep the remaining liquid at 90℃ for 15 minutes to inactivate the enzyme, and then cool it to 35℃.

[0059] S2. Add 5 parts of Aspergillus oryzae solution and 5 parts of Lactobacillus plantarum solution to the cooled liquid; set the first stage fermentation temperature at 32℃ and the aeration rate at 1.0L / (L·min) for 36h; raise the temperature to 38℃ and reduce the aeration rate to 0.2L / (L·min) for 36h; after fermentation, inactivate at 95℃ for 20min and collect the ginseng fermentation liquid by filtration.

[0060] S3. Take 100 parts of crocodile meat and mix with 300 parts of water. Cook at 105℃ for 1.5 hours. After cooling to 40℃, homogenize into meat paste. Adjust the pH of the system to 7.0, keep the temperature at 50℃, add 0.3 parts of lipase, stir and hydrolyze for 1.5 hours, then inactivate at 85℃ for 10 minutes, and cool down to 50℃.

[0061] S4. Adjust the pH of the cooled system to 7.5 using 0.1 mol / L NaOH, add 0.5 parts of a complex protease (composed of papain and protease in a weight ratio of 1:2), and hydrolyze at a constant temperature of 50°C for a total hydrolysis time of 3.0 h. When the hydrolysis is in the last 30 min, pump the ginseng polysaccharide intermediate solution stored in S1 into the system, maintain 50°C and pH 7.5 and continue stirring until the total hydrolysis time is over. Then heat to 90°C to inactivate the enzyme for 15 min, cool and centrifuge at 4500 r / min for 25 min, take the supernatant and filter it through a 0.22 μm filter membrane to collect the crocodile polypeptide solution.

[0062] S5. Mix 100 parts of ginseng fermentation broth and 100 parts of crocodile polypeptide broth in a sealed tank, add 0.1 parts of L-ascorbic acid, and maintain a headspace air layer of 10% of the total volume at the top of the tank. Do not perform mechanical aeration, and ferment in a sealed environment at 30°C for 12 hours. Sterilize the liquid at 115°C for 15 minutes, and then cool it to 25°C. Pump the liquid into an ultrafiltration membrane with a molecular weight cutoff of 10000 Da for cross-flow filtration, and collect the permeate. Add 0.1 parts of powdered activated carbon to the permeate, stir and adsorb at 25°C for 30 minutes, filter through a plate and frame filter press and a series of 0.22 μm microporous membranes, and collect the filtrate to obtain the final anti-fatigue active polypeptide broth.

[0063] Example 3: This embodiment provides a method for preparing a crocodile ginseng anti-fatigue active polypeptide liquid, including the following steps: S1. Take 100 parts of ginseng powder and microwave it at 700W for 5 minutes. Add 1200 parts of water to the treated ginseng powder and mix well. Adjust the system temperature to 50℃, adjust the pH to 5.0 with 0.1mol / L citric acid, add 0.3 parts of pectinase and 0.4 parts of cellulase, and stir at a constant temperature for 3.0 hours. Then raise the temperature to 55℃, adjust the pH to 6.0, add 0.5 parts of β-glucanase, and continue to stir at a constant temperature for 2.5 hours. After the enzymatic hydrolysis is completed, extract 10% of the total volume of liquid and refrigerate at 4℃ as ginseng polysaccharide intermediate solution. Keep the remaining liquid at 95℃ for 10 minutes to inactivate the enzyme, and then cool it to 37℃.

[0064] S2. Add 8 parts of Aspergillus oryzae culture and 8 parts of Lactobacillus plantarum culture to the cooled liquid; set the first stage fermentation temperature at 34℃ and the aeration rate at 1.2L / (L·min) for 36h; raise the temperature to 40℃ and reduce the aeration rate to 0.3L / (L·min) for 36h; after fermentation, inactivate the ginseng at 95℃ for 20min, and collect the ginseng fermentation liquid after filtration.

[0065] S3. Take 100 parts of crocodile meat and mix with 300 parts of water. Cook at 110℃ for 2.0h. After cooling to 45℃, homogenize into meat paste. Adjust the pH of the system to 7.5, keep the temperature at 55℃, add 0.5 parts of lipase, stir and hydrolyze for 2.0h, then inactivate at 85℃ for 10min, and cool down to 55℃.

[0066] S4. Adjust the pH of the cooled system to 8.0 using 0.1 mol / L NaOH, add 1.0 part of a complex protease (composed of papain and protease in a weight ratio of 1:2), and hydrolyze at a constant temperature of 55℃ for a total hydrolysis time of 3.5 h. When the hydrolysis is in progress for the last 40 min, pump the ginseng polysaccharide intermediate solution stored in S1 into the system, maintain 55℃ and pH 8.0 and continue stirring until the total hydrolysis time is over. Then heat to 95℃ to inactivate the enzyme for 15 min, cool and centrifuge at 5000 r / min for 20 min, take the supernatant and filter it through a 0.22 μm filter membrane to collect the crocodile polypeptide solution.

[0067] S5. Mix 100 parts of ginseng fermentation broth with 300 parts of crocodile polypeptide broth in a sealed tank, add 0.4 parts of L-ascorbic acid, and maintain a headspace air layer of 15% of the total volume at the top of the tank. Do not perform mechanical aeration and ferment in a sealed environment at 32°C for 16 hours. Sterilize the liquid at 115°C for 20 minutes and cool it to 30°C. Pump the liquid into an ultrafiltration membrane with a molecular weight cutoff of 50,000 Da for cross-flow filtration and collect the permeate. Add 0.2 parts of powdered activated carbon to the permeate and stir and adsorb at 30°C for 40 minutes. Filter the solution through a plate and frame filter press and a series of 0.22 μm microporous membranes. Collect the filtrate to obtain the final anti-fatigue active polypeptide broth.

[0068] Example 4: This embodiment provides a method for preparing a crocodile ginseng anti-fatigue active polypeptide liquid, including the following steps: S1. Take 100 parts of ginseng powder and microwave it at 650W for 4 minutes. Add 1100 parts of water to the treated ginseng powder and mix well. Adjust the system temperature to 48℃, adjust the pH to 4.8 with 0.1mol / L citric acid, add 0.25 parts of pectinase and 0.35 parts of cellulase, and stir at a constant temperature for 2.8 hours for enzymatic hydrolysis. Then raise the temperature to 53℃, adjust the pH to 5.8, add 0.4 parts of β-glucanase, and continue to stir at a constant temperature for 2.2 hours for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, extract 8% of the total volume of liquid and refrigerate it at 4℃ as ginseng polysaccharide intermediate solution. Keep the remaining liquid at 95℃ for 12 minutes to inactivate the enzyme, and then cool it to 36℃.

[0069] S2. Add 6 parts of Aspergillus oryzae culture and 6 parts of Lactobacillus plantarum culture to the cooled liquid; set the temperature for the first stage of fermentation at 34℃ and the aeration rate at 1.2L / (L·min), and ferment for 36h; for the second stage of fermentation, raise the temperature to 40℃ and reduce the aeration rate to 0.2L / (L·min), and ferment for 36h; after fermentation, inactivate the ginseng at 95℃ for 20min, and collect the ginseng fermentation liquid by filtration.

[0070] S3. Take 100 parts of crocodile meat and mix with 300 parts of water. Cook at 108℃ for 1.8 hours. After cooling to 45℃, homogenize into meat paste. Adjust the pH of the system to 7.3 and maintain the temperature at 53℃. Add 0.4 parts of lipase and stir to hydrolyze for 1.8 hours. Then inactivate at 85℃ for 10 minutes and cool down to 53℃.

[0071] S4. Adjust the pH of the cooled system to 7.8 using 0.1 mol / L NaOH, add 0.75 parts of a complex protease (composed of papain and protease in a weight ratio of 1:2), and hydrolyze at a constant temperature of 53℃ for a total hydrolysis time of 3.2 h. When the hydrolysis is in its last 35 min, pump the ginseng polysaccharide intermediate solution stored in S1 into the system, maintain 53℃ and pH 7.8 and continue stirring until the total hydrolysis time is over. Then heat to 95℃ to inactivate the enzyme for 15 min, cool and centrifuge at 4800 r / min for 20 min, take the supernatant and filter it through a 0.22 μm filter membrane to collect the crocodile polypeptide solution.

[0072] S5. Mix 100 parts of ginseng fermentation broth and 200 parts of crocodile polypeptide broth in a sealed tank, add 0.2 parts of L-ascorbic acid, and maintain a headspace air layer of 12% of the total volume at the top of the tank. Do not perform mechanical aeration and ferment in a sealed environment at 31°C for 14 hours. Sterilize the liquid at 115°C for 15 minutes and cool it down to 25°C. Pump the liquid into an ultrafiltration membrane with a molecular weight cutoff of 30,000 Da for cross-flow filtration and collect the permeate. Add 0.15 parts of powdered activated carbon to the permeate and stir and adsorb at 25°C for 35 minutes. Filter the solution through a plate and frame filter press and a series of 0.22 μm microporous membranes. Collect the filtrate to obtain the final anti-fatigue active polypeptide broth.

[0073] Example 5: This embodiment provides a method for preparing a crocodile ginseng anti-fatigue active polypeptide liquid, including the following steps: S1. Take 100 parts of ginseng powder and microwave it at 650W for 4 minutes. Add 1100 parts of water to the treated ginseng powder and mix well. Adjust the system temperature to 48℃, adjust the pH to 4.8 with 0.1mol / L citric acid, add 0.25 parts of pectinase and 0.35 parts of cellulase, and stir at a constant temperature for 2.8 hours for enzymatic hydrolysis. Then raise the temperature to 53℃, adjust the pH to 5.8, add 0.4 parts of β-glucanase, and continue to stir at a constant temperature for 2.2 hours for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, extract 10% of the total volume of liquid and refrigerate it at 4℃ as ginseng polysaccharide intermediate solution. Keep the remaining liquid at 95℃ for 12 minutes to inactivate the enzyme, and then cool it to 36℃.

[0074] S2. Add 6 parts of Aspergillus oryzae culture and 2 parts of Lactobacillus plantarum culture to the cooled liquid; set the temperature for the first stage of fermentation at 33℃ and the aeration rate at 1.1L / (L·min), and ferment for 36h; raise the temperature to 39℃ and reduce the aeration rate to 0.25L / (L·min) for the second stage of fermentation, and ferment for 36h; after fermentation, inactivate the ginseng at 95℃ for 20min, and collect the ginseng fermentation liquid by filtration.

[0075] S3. Take 100 parts of crocodile meat and mix with 300 parts of water. Cook at 108℃ for 1.8 hours. After cooling to 45℃, homogenize into meat paste. Adjust the pH of the system to 7.3 and maintain the temperature at 53℃. Add 0.4 parts of lipase and stir to hydrolyze for 1.8 hours. Then inactivate at 85℃ for 10 minutes and cool down to 53℃.

[0076] S4. Adjust the pH of the cooled system to 7.8 using 0.1 mol / L NaOH, add 0.75 parts of a complex protease (composed of papain and protease in a weight ratio of 1:4), and perform enzymatic hydrolysis at 53°C for a total hydrolysis time of 3.2 h. When the hydrolysis is in its last 40 min, pump the ginseng polysaccharide intermediate solution stored in S1 into the system, maintain 53°C and pH 7.8, and continue stirring until the total hydrolysis time is over. Then, heat to 95°C to inactivate the enzyme for 15 min, cool, centrifuge at 4800 r / min for 20 min, and filter the supernatant through a 0.22 μm filter membrane to collect the crocodile polypeptide solution.

[0077] S5. Mix 100 parts of ginseng fermentation broth and 200 parts of crocodile polypeptide broth in a sealed tank, add 0.2 parts of L-ascorbic acid, and maintain a headspace air layer of 12% of the total volume at the top of the tank. Do not perform mechanical aeration and ferment in a sealed environment at 31°C for 14 hours. Sterilize the liquid at 115°C for 15 minutes and cool it down to 25°C. Pump the liquid into an ultrafiltration membrane with a molecular weight cutoff of 30,000 Da for cross-flow filtration and collect the permeate. Add 0.15 parts of powdered activated carbon to the permeate and stir and adsorb at 25°C for 35 minutes. Filter the solution through a plate and frame filter press and a series of 0.22 μm microporous membranes. Collect the filtrate to obtain the final anti-fatigue active polypeptide broth.

[0078] Comparative Example 1: Compared with Example 1, the difference is that in step S2, the fermentation process is maintained at a temperature of 33°C and an aeration rate of 1.1 L / (L·min) for a continuous isothermal aerobic fermentation for 72 hours. All other aspects are the same.

[0079] Comparative Example 2: Compared with Example 1, the difference is that after the enzymatic hydrolysis in step S1, the intermediate liquid of ginseng polysaccharide is not extracted, but all the liquid is directly inactivated at 95°C; when the complex enzymatic hydrolysis in step S4 reaches the last 35 minutes, no liquid is pumped in, and stirring is continued until the total enzymatic hydrolysis time ends. The rest are the same.

[0080] Comparative Example 3: Compared with Example 1, the difference is that in step S5, an open container is used for conventional mechanical aeration fermentation for 14 hours under continuous mechanical stirring and air introduction. All other steps are the same.

[0081] Comparative Example 4: Compared with Example 1, the difference is that in step S5, a conventional ultrafiltration membrane with a molecular weight cutoff of 500 to 1500 Da, which is commonly used for extracting small molecule active peptides, is used for cross-flow filtration; all other aspects are the same.

[0082] Comparative Example 5: Compared with Example 1, the difference is that in the degreasing treatment of crocodile meat in step S3, after homogenization into meat paste, lipase was not added for pre-degradation and deodorization treatment, but directly entered the complex protease hydrolysis process in step S4. The rest are the same.

[0083] Comparative Example 6: Compared with Example 1, the differences are as follows: in step S1, the ginseng polysaccharide intermediate liquid is not extracted; in step S2, aerobic fermentation at a constant temperature of 33°C is used throughout; in step S4, the ginseng polysaccharide intermediate liquid is not pumped in; in step S5, the two sets of filtrates are mixed and then not subjected to closed post-fermentation, and an ultrafiltration membrane with a molecular weight cutoff of 1000 Da is used for separation and purification. All other aspects are the same.

[0084] Test Example 1: The test subjects were the liquid from Example 1 at the end of the first stage of fermentation (36 hours) and the liquid from Example 1 at the end of the second stage of fermentation (72 hours). To eliminate the interference of the cumulative fermentation time on the experimental results, the liquids from Comparative Example 1 and Comparative Example 6 at the end of the total fermentation (36 hours and 72 hours) were extracted simultaneously for comparison and measurement.

[0085] Take 50 mL of fermentation liquid at each corresponding time point, centrifuge at 8000 r / min for 15 min at 4℃, and collect the supernatant as the enzyme solution and saponin extract to be tested.

[0086] Extracellular β-glucosidase activity was determined using the p-nitrobenzene-β-D-glucopyranoside method: p-nitrobenzene-β-D-glucopyranoside (pNPG) powder was prepared into a 10 mmol / L pNPG substrate solution using 0.05 mol / L, pH 5.0 sodium acetate buffer. The enzyme solution to be tested was mixed with the pNPG substrate solution at a volume ratio of 1:1, and the reaction was carried out at 45 °C for 30 min. The reaction was then terminated by adding 0.2 mol / L sodium carbonate solution, and the absorbance was measured at a wavelength of 400 nm. The enzyme activity units of the system were calculated using a standard curve.

[0087] The remaining supernatant was slowly passed through a D101 macroporous adsorption resin column. Water-soluble impurities were first removed by elution with pure water, followed by elution of the saponin component with 70% methanol solution. The methanol eluent was collected and concentrated to dryness under reduced pressure. After being brought to a final volume with methanol, it was filtered through a 0.22 μm microporous membrane.

[0088] The total amounts of saponins Rg3 and Rh2 were determined by high performance liquid chromatography (HPLC). A C18 column was used, and gradient elution was performed using an acetonitrile-water mobile phase. The detection wavelength was set to 203 nm. The peak areas of the target chromatograms were recorded, and the total concentration was calculated based on the working curve of a reference standard with known concentrations.

[0089] The test results are shown in Table 1: Table 1: Results of extracellular β-glucosidase activity and saponin content under different fermentation conditions

[0090] Please see Figure 1 and Figure 2 : From Table 1 and Figure 1 and Figure 2 It can be seen that the fermentation system exhibits different biochemical metabolic characteristics before and after the change of environmental conditions. In Example 1, the system was in an aerobic isothermal environment at 36 h, and the extracellular β-glucosidase activity was 12.38 U / mL. At this time, the saponins in the system had not yet undergone obvious deglycosylation reaction, and the saponin content was measured to be 0.36 mg / mL. Compared with the data of Comparative Example 1 at 36 h, it can be found that its various indicators are similar to those of Example 1. This indicates that in the first fermentation stage, the metabolism of microbial cells is mainly used for biomass accumulation, and the amount of enzymes released into the extracellular space is relatively small.

[0091] When fermentation entered the second stage, the ambient temperature in Example 1 increased to 39°C and the aeration rate decreased. Sampling and testing after 72 hours showed that the extracellular enzyme activity increased to 89.15 U / mL and the total saponin content increased to 4.27 mg / mL. When observing the centrifuged precipitate in the laboratory, cell rupture was found. Based on this, it was inferred that the change in fermentation conditions affected the normal metabolism of Aspergillus oryzae, causing its cells to undergo autolysis. Intracellular β-glucosidase entered the surrounding liquid phase, and the free enzyme came into contact with the ginseng substrate, breaking the glycosidic bond at the C-20 position of the Rb series saponins, and increasing the concentration of products such as Rg3 and Rh2.

[0092] Comparative Example 1 was kept in a constant temperature and aerobic state throughout the fermentation cycle. The enzyme activity and saponin yield at 72h showed a slow increase over time, failing to reach the conversion level of Example 1. This indicates that without changing the physical conditions, the amount of enzymes naturally secreted by microorganisms into the extracellular space is insufficient to drive a large-scale conversion of saponins. Comparative Example 6, due to the lack of relevant pretreatment and dynamic control of fermentation parameters, had low measured values ​​throughout the monitoring period.

[0093] Data comparisons from different systems confirm that altering fermentation conditions can promote the release of enzymes within fungi. Combined with the slightly acidic environment produced by lactobacillus metabolism, this helps stabilize the structure of deglycosylated aglycones, thereby increasing the saponin content in the extract.

[0094] Test Example 2: The test subjects were the enzymatic hydrolysis system liquids of Examples 1 and 5 before the ginseng polysaccharide intermediate liquid was pumped in step S4, and the final product liquids of these two groups at the end of the total enzymatic hydrolysis. The process flow of Comparative Example 2 did not introduce polysaccharide intermediate liquid, so its final state liquid at the end of the total enzymatic hydrolysis was extracted as a reference.

[0095] Take 10 mL of the reaction solution at each corresponding time point, centrifuge at 5000 r / min for 10 min at room temperature to allow large protein particles to precipitate, aspirate the supernatant and filter it through a 0.45 μm filter membrane, and collect the filtrate for later use.

[0096] The obtained filtrate was diluted with 0.01 mol / L phosphate buffer (pH 7.4) to prepare a series of test solutions with protein concentrations of 0.05, 0.1, 0.2, 0.3, and 0.5 mg / mL. 100 μL of 800 μmol / L 8-aniline-1-naphthalenesulfonic acid fluorescent probe solution was added to 4 mL of the above test solutions. The reaction was carried out in the dark for 15 min. The fluorescence intensity of the system was measured using a fluorescence spectrophotometer at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. The slope of the regression curve was calculated by linear fitting to obtain the surface hydrophobicity index of the sample.

[0097] The clarified filtrate was diluted 5 times with ultrapure water at a volume ratio of 1:4. Using ultrapure water as a reference, the sample was placed in a UV-Vis spectrophotometer, and the absorbance value at a wavelength of 420 nm was measured as the browning index. Each sample was scanned three times in parallel and the arithmetic mean was taken.

[0098] The test results are shown in Table 2: Table 2: Results of surface hydrophobicity index and browning index at different system stages

[0099] Please see Figure 3 and Figure 4 : Based on the data in Table 2 and Figure 3 and Figure 4 It can be seen that crocodile protein exposes some hydrophobic structures during the enzymatic hydrolysis process. In Example 1, the surface hydrophobicity index before polysaccharide intervention was 3184.6. Under the action of protease, hydrophobic residues were released into the aqueous phase. In actual sensory tests, it was found that these small molecule peptides rich in hydrophobic ends have a bitter taste.

[0100] When ginseng polysaccharide intermediate liquid was added to the fermentation system and stirred, the hydrophobicity index of Example 1 decreased to 1342.1, and the browning index increased from 0.125 to 0.493. This phenomenon indicates that the hydrophilic ginseng polysaccharide formed a wrapping structure around the polypeptide through physical entanglement and hydrogen bonding. The free carbonyl group of the polysaccharide and the amino group of the polypeptide underwent a cross-linking reaction, which increased the stability of the structure and reduced the contact between the hydrophobic groups and the outside world in space.

[0101] In Example 5, the proportion of protease was relatively high, and the surface hydrophobicity index of the system before complexation reached 4756.8. At the end of the reaction, a large proportion of polysaccharide solution was introduced to participate in the reaction. The polysaccharide combined with the free short peptide, causing the final hydrophobicity to drop to 1198.5 and the browning index to rise to 0.821.

[0102] Comparative Example 2 did not introduce ginseng polysaccharides throughout the process. At the reaction endpoint, the hydrophobicity index was 3291.3. The system exhibited free polypeptide dispersion characteristics and no obvious non-enzymatic browning was observed. This indicates that simple proteolysis cannot change the problem of polypeptide hydrophobic exposure of the raw materials themselves. Encapsulation with plant polysaccharides can adjust the physicochemical properties of the polypeptide solution.

[0103] Test Example 3: The test subjects were the mixed liquid from the fermentation stage after step S5 of Example 1 and the mixed liquid from the corresponding stage of Comparative Example 3.

[0104] Calibrated dissolved oxygen and pH glass electrodes were installed on the closed fermenter of Example 1 and the open fermenter of Comparative Example 3, respectively, with the probes fixed at a depth of about one-third below the liquid surface.

[0105] Set and maintain the temperature of the two sets of fermentation equipment at 31℃, and connect the data transmission interface of the monitoring instrument to the host computer system.

[0106] Fermentation start-up is recorded as 0h, and the monitoring cycle is set to 14h. The instrument records the dissolved oxygen concentration and pH of the liquid phase system.

[0107] The values ​​at six time points—0h, 2h, 4h, 8h, 12h, and 14h—were extracted and organized.

[0108] The test results are shown in Table 3: Table 3: Dynamic monitoring results of dissolved oxygen concentration and pH value under different fermentation modes

[0109] Please see Figure 5 and Figure 6 : Based on the data in Table 3 and Figure 5 and Figure 6It can be seen that the closed system of Example 1 and the open ventilation system of Comparative Example 3 show different physicochemical properties. After the liquid containing Lactobacillus plantarum was introduced into Example 1, the dissolved oxygen concentration dropped to 1.75 mg / L in the first 4 hours and to 0.24 mg / L in 8 hours. Lactobacillus plantarum metabolized in the closed tank using the carbon source substrate of the system, consuming the free oxygen in the liquid phase.

[0110] When the oxygen concentration approaches the lower limit of the equipment, fermentation enters a low-oxygen-consumption state due to the lack of fresh air supply. This state helps reduce the oxidation of components caused by free oxygen in the liquid phase.

[0111] Comparative Example 3 adopted an open-ventilation operation, and the dissolved oxygen in the liquid phase was maintained in the range of 6.75 to 7.21 mg / L for 14 hours. At the same time, the pH value of Example 1 decreased from 6.45 to 4.58 with oxygen consumption. During the transition from microaerobic to anaerobic environment, Lactobacillus carried out lactic acid fermentation, and the lactic acid produced neutralized the alkaline substances left by the previous enzymatic hydrolysis, making the liquid phase a slightly acidic environment.

[0112] The pH of Comparative Example 3 varied between 6.48 and 5.95, indicating that the acid-producing pathway of Lactobacillus was affected when more oxygen was present.

[0113] This comparative test shows that, combined with the antioxidant buffering effect of L-ascorbic acid in the liquid phase, the combination of a closed environment and microbial strains can maximize the regulation of oxygen content and pH of the polypeptide liquid, preventing the active ingredients from being oxidized and destroyed during post-fermentation.

[0114] Test Example 4: The test subjects were the polypeptide solutions prepared in Examples 1 to 5, and the products of Comparative Examples 2, 5 and 6.

[0115] A sensory evaluation team was established by selecting 10 evaluators who had passed the screening and training according to the GB / T 16291.1 standard.

[0116] 50 mL of each group of peptide solution was extracted and dispensed into tasting cups at room temperature (25°C). The containers were blind-coded with three digits.

[0117] Three evaluation dimensions were set: bitterness residue, fishy and muttony residue, and overall flavor harmony. A scale of 0 to 10 was used for scoring. The lower the score for bitterness and fishy and muttony, the weaker the flavor. The higher the score for overall flavor harmony, the better the taste acceptance.

[0118] The evaluators evaluated the samples in a random sequence, and rinsed their mouths with purified water and rested during the test intervals. The highest and lowest scores were removed from the score sheet and the average score was calculated.

[0119] The test results are shown in Table 4: Table 4: Comprehensive evaluation results of sensory quality of polypeptide solutions under different process conditions

[0120] Please see Figures 7 to 9 : From Table 4 and Figures 7-9 It can be seen that there are differences in the sensory dimensions of each group of samples. The bitterness and fishy smell residue of Examples 1 to 5 are below 2.5, and the overall flavor harmony reaches above 8.0. The hydrophobic amino acids and residual lipids exposed after enzymatic hydrolysis of animal muscle protein usually produce off-flavors. The sensory data are improved after the process.

[0121] In Comparative Example 2, the mixing step of the ginseng polysaccharide intermediate liquid was removed from the process, and its bitterness residue score was 8.2. The evaluation record showed that the sample had a relatively obvious bitterness, which indicates that the combination of plant polysaccharides and peptides has a certain effect on masking the bitterness produced by hydrophobic groups.

[0122] Comparative Example 5, which did not introduce lipase for defatting, had a fishy and muttony odor score of 8.5. The lipids remaining in the muscle tissue oxidize during processing, forming substances with a fishy odor. The pretreatment with lipase helps reduce the formation of fishy odor precursors. Comparative Example 6, as a traditional enzymatic hydrolysis product without relevant pretreatment, had a low overall flavor harmony. The test results show that the combination of defatting and polysaccharide encapsulation can improve the flavor performance of the polypeptide liquid.

[0123] Test Example 5: The test subjects were peptide solutions produced by the process of Examples 1 to 5, Comparative Example 4 and Comparative Example 6.

[0124] Take 5 mL of the final product peptide solution from each group and place it in a centrifuge tube. Add 4 times the volume of anhydrous ethanol and mix well. Let it stand overnight at 4°C. Centrifuge at 10000 r / min for 15 min, discard the supernatant, and reconstitute the precipitate with ultrapure water to the initial volume.

[0125] The total polysaccharide content in the complex solution was determined by the phenol-sulfuric acid colorimetric method. 1.0 mL of 5% phenol solution was added to 1.0 mL of the dilute solution, followed by the rapid addition of 5.0 mL of concentrated sulfuric acid (98% by mass). After shaking well, the solution was placed in a boiling water bath for 15 min and then cooled. The absorbance was measured at a wavelength of 490 nm, and the polysaccharide concentration was calculated using the standard curve.

[0126] Take another 10 mL of polypeptide solution and put it into an Erlenmeyer flask. Add an equal volume of 10% trichloroacetic acid solution and mix well. Let it stand for 30 min to precipitate the undigested protein. Centrifuge to obtain the supernatant.

[0127] The supernatant was digested and titrated using a Kjeldahl nitrogen analyzer to determine the total nitrogen content. After deducting the background value of free amino acids, the retention concentration of water-soluble short peptides was calculated.

[0128] The test results are shown in Table 5: Table 5: Results of Retention Concentration of Polysaccharides and Short Peptides in Final Products under Different Separation Processes

[0129] Please see Figure 10 and Figure 11 : From Table 5 and Figure 10 and Figure 11 It can be seen that the polysaccharide retention concentration in the final products of Examples 1 to 5 is in the range of 10.87 to 14.12 mg / mL, and the water-soluble short peptide retention concentration is between 4.65 and 5.34 g / 100 mL. The volume of the polysaccharide-peptide complex formed in the pre-process is increased, and the pore size used allows the complex of this size to pass through, so that the target component is retained in the liquid product.

[0130] Comparative Example 4 used a conventional small-pore ultrafiltration membrane for purification. The polysaccharide concentration was 3.21 mg / mL and the peptide concentration was 2.87 g / 100 mL. During the filtration operation, the equipment working pressure rose rapidly. After disassembling the membrane module, it was found that there were intercepted substances attached to the surface of the retrieval side. This indicates that the larger polysaccharide-peptide complex was intercepted, resulting in a decrease in the concentration of substances in the filtrate.

[0131] Comparative Example 6 did not employ a complex construction process. Its polysaccharide concentration was 1.05 mg / mL, and its short peptide concentration was low. Due to the lack of encapsulation and protection by hydrophilic polysaccharides, a large number of free short peptides with exposed hydrophobic groups would undergo strong hydrophobic adsorption on the membrane surface when they came into contact with the polyethersulfone ultrafiltration membrane, resulting in severe membrane fouling and retention loss. Therefore, the concentration of short peptides in the filtrate was low.

[0132] Experimental data show that the separation pore size must be matched with the size of the polymer in the feed solution to ensure the retention rate of active substances in the final product.

[0133] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a crocodile ginseng anti-fatigue active polypeptide liquid, characterized in that, The method includes the following steps: S1. After microwave cell wall breaking of ginseng powder, water is added and compound enzymatic hydrolysis is carried out using plant enzyme hydrolysate. Part of the liquid is extracted as polysaccharide intermediate liquid, and the remaining liquid is cooled after enzyme inactivation to obtain ginseng fermentation substrate. S2. Inoculate the ginseng fermentation substrate with Aspergillus oryzae and Lactobacillus plantarum for fermentation. After the first stage of fermentation, raise the temperature and reduce the aeration rate to carry out the second stage of fermentation. After inactivation and filtration, obtain ginseng fermentation liquid. S3. The crocodile meat is steamed and homogenized with water, then inactivated and cooled after being hydrolyzed with lipase to obtain defatted crocodile slurry. S4. Add a complex protease to the defatted crocodile slurry for enzymatic hydrolysis, and pump in the polysaccharide intermediate liquid before the end of enzymatic hydrolysis for mixing. After enzyme inactivation, centrifugation and filtration, crocodile polypeptide liquid is obtained. S5. The ginseng fermentation liquid is mixed with the crocodile polypeptide liquid, L-ascorbic acid is added and fermented in a sealed manner, and then sterilized, ultrafiltered, adsorbed by activated carbon and filtered through micropores to obtain the polypeptide liquid.

2. The method for preparing the anti-fatigue active polypeptide liquid of crocodile ginseng according to claim 1, characterized in that, In S1, the step of adding water to the ginseng powder after microwave cell wall disruption for compound enzymatic hydrolysis includes: Take 100 parts of ginseng powder and microwave it at 600-700W for 3-5 minutes. Add 1000-1200 parts of water and mix well. Adjust the system temperature to 45-50℃, adjust the pH to 4.5-5.0 with citric acid, add 0.2-0.3 parts of pectinase and 0.3-0.4 parts of cellulase, and stir at a constant temperature for 2.5-3.0 hours for enzymatic hydrolysis.

3. The method for preparing the anti-fatigue active polypeptide liquid of crocodile ginseng according to claim 2, characterized in that, In S1, the step of extracting a portion of the liquid as a polysaccharide intermediate and then cooling the remaining liquid after enzyme inactivation to obtain the ginseng fermentation substrate includes: Heating the liquid after enzymatic hydrolysis to 50-55℃, adjusting the pH to 5.5-6.0, adding 0.3-0.5 parts of β-glucanase, and stirring at a constant temperature for 2.0-2.5 hours; extracting 5-10% of the total volume of the liquid and refrigerating it as the polysaccharide intermediate liquid; keeping the remaining liquid at 90-95℃ for 10-15 minutes to inactivate the enzyme, and cooling it to 35-37℃ to obtain the ginseng fermentation substrate.

4. The method for preparing the anti-fatigue active polypeptide liquid of crocodile ginseng according to claim 1, characterized in that, In S2, the step of inoculating the ginseng fermentation substrate with Aspergillus oryzae and Lactobacillus plantarum for fermentation includes: Add 5-8 parts of Aspergillus oryzae solution and 2-8 parts of Lactobacillus plantarum solution to the cooled ginseng fermentation substrate; set the first stage fermentation temperature at 32-34℃ and the aeration rate at 1.0-1.2L / (L·min) for 36h; raise the temperature to 38-40℃ and reduce the aeration rate to 0.2-0.3L / (L·min) for 36h.

5. The method for preparing the anti-fatigue active polypeptide liquid of crocodile ginseng according to claim 1, characterized in that, In step S3, the process of steaming and homogenizing the crocodile meat with water, then adding lipase for enzymatic hydrolysis, inactivating the lipase, and cooling to obtain defatted crocodile slurry includes: Mix 100 parts of crocodile meat with 300 parts of water, steam at 105-110℃ for 1.5-2.0 hours, cool to 40-45℃ and homogenize into a meat paste; adjust the pH of the system to 7.0-7.5, maintain the temperature at 50-55℃, add 0.3-0.5 parts of lipase, stir and enzymatically hydrolyze for 1.5-2.0 hours, inactivate at 85℃ for 10 minutes, and cool to 50-55℃ to obtain defatted crocodile slurry.

6. The method for preparing the anti-fatigue active polypeptide liquid of crocodile ginseng according to claim 1, characterized in that, In step S4, the step of adding a complex protease to the defatted alligator slurry for enzymatic hydrolysis includes: Adjust the pH of the defatted crocodile slurry to 7.5-8.0, add 0.5-1.0 parts of a complex protease, which is composed of papain and protease in a weight ratio of 1:2 to 1:4, and perform enzymatic hydrolysis at a constant temperature of 50-55℃ for a total hydrolysis time of 3.0-3.5h.

7. The method for preparing the anti-fatigue active polypeptide liquid of crocodile ginseng according to claim 1, characterized in that, In S4, the step of pumping in the polysaccharide intermediate solution for blending before the end of enzymatic hydrolysis includes: When the complex protease hydrolysis is in its final 30-40 minutes, the polysaccharide intermediate solution is pumped into the system, and the mixture is stirred at 50-55°C and pH 7.5-8.0 until the total hydrolysis time is completed.

8. The method for preparing the anti-fatigue active polypeptide liquid of crocodile ginseng according to claim 1, characterized in that, In S5, the step of mixing the ginseng fermentation liquid with the crocodile polypeptide liquid and adding L-ascorbic acid for closed fermentation includes: Mix 100 parts of ginseng fermentation liquid, 100-300 parts of crocodile polypeptide liquid and 0.1-0.4 parts of L-ascorbic acid in a sealed container. The top of the container is reserved with a headspace air layer accounting for 10-15% of the total volume of the sealed container. No mechanical ventilation is performed. Ferment in a sealed container at 30-32°C for 12-16 hours.

9. The method for preparing the anti-fatigue active polypeptide liquid of crocodile ginseng according to claim 1, characterized in that, In S5, the ultrafiltration step includes: The feed solution is pumped into an ultrafiltration membrane with a molecular weight cutoff of 10,000-50,000 Da for cross-flow filtration, and the permeate is collected.

10. The method for preparing the anti-fatigue active polypeptide liquid of crocodile ginseng according to claim 1, characterized in that, In step S5, the activated carbon adsorption and microporous filtration process to obtain the polypeptide solution includes: Add 0.1-0.2 parts of powdered activated carbon to the permeate, stir and adsorb at 25-30℃ for 30-40 min, filter by plate and frame filter and filter through a series of 0.22μm microporous membranes, and collect the filtrate to obtain the polypeptide solution.

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