A marine shrimp peptide-based intestinal flora modulator and a preparation method thereof

Abstract

The present application relates to the field of intestinal barrier function, immune function and intestinal flora regulation, and discloses an intestinal flora regulator based on marine shrimp peptide and a preparation method thereof; the intestinal flora regulator comprises an oligopeptide component, the oligopeptide component is prepared from marine shrimp protein raw materials through enzymolysis, enzyme inactivation, solid-liquid separation and membrane fractionation, the molecular weight of the oligopeptide component is mainly concentrated below 3 kDa, and the oligopeptide component contains short peptides composed of 4 to 8 amino acid residues and proline-rich short peptides. The preparation method comprises marine shrimp raw material pretreatment, protease enzymolysis, enzyme inactivation, solid-liquid separation, membrane fractionation, concentration and drying. The intestinal flora regulator can be used for regulating intestinal flora structure, improving intestinal mucosal barrier function and reducing intestinal permeability. The present application improves the specificity and stability of the obtained active component by controlled enzymolysis and low molecular weight peptide component enrichment of marine shrimp protein.

Description

Technical Field

[0001] This invention relates to the fields of intestinal barrier function, immune function, and intestinal flora regulation, specifically to an intestinal flora regulator based on marine shrimp peptides and its preparation method. Background Technology

[0002] Gut microbiota homeostasis and intestinal mucosal barrier integrity play crucial roles in metabolic balance and gut health. Imbalances in gut microbiota structure, decreased goblet cell secretion, or increased intestinal permeability can easily induce intestinal barrier damage and related metabolic disorders. Therefore, developing products that can simultaneously regulate gut microbiota and the intestinal barrier has become an important research direction in the fields of functional foods and bioactive products.

[0003] In existing technologies, bioactive peptide products are often prepared by enzymatic hydrolysis of protein raw materials, and attempts are made to use them to improve intestinal function. Marine shrimp protein is abundant, providing a basis for the preparation of bioactive peptides. However, existing methods mostly use conventional enzymatic hydrolysis or crude separation to obtain shrimp protein hydrolysates. The peptide composition and molecular weight distribution of the obtained products are relatively dispersed, lacking targeted enrichment and clear definition of low molecular weight short peptide components.

[0004] The existing structure and preparation method make it difficult for the obtained shrimp peptide components to stably act on the process of gut microbiota regulation and mucosal barrier repair after entering the intestine. This can easily lead to problems such as incomplete improvement of gut microbiota, insufficient promotion of mucin secretion, and insignificant decrease in intestinal permeability, making it difficult to simultaneously achieve gut microbiota regulation and intestinal barrier protection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a gut microbiota regulator based on marine shrimp peptides and its preparation method, thereby solving the technical problems existing in the prior art.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: A gut microbiota regulator based on marine shrimp peptides, comprising: The oligopeptide component is obtained from marine shrimp protein raw material through enzymatic hydrolysis, enzyme inactivation, solid-liquid separation and membrane fractionation. In the oligopeptide component, peptides with a molecular weight not greater than 3kDa account for more than 60% of the total mass of the oligopeptide component, peptides with a molecular weight not greater than 1kDa account for more than 40% of the total mass of the oligopeptide component, and the oligopeptide component contains short peptides composed of 4 to 8 amino acid residues.

[0007] Preferably, the marine shrimp protein raw material is derived from the shrimp meat, shrimp kernels, shrimp heads, or by-product protein extracts of marine shrimp.

[0008] Preferably, the oligopeptide component contains proline-rich short peptides.

[0009] Preferably, the gut microbiota regulator is used to regulate the gut microbiota structure, improve the intestinal mucosal barrier function, or reduce intestinal permeability.

[0010] Preferably, the intestinal flora regulator is formulated as a powder, granule, tablet, capsule, oral liquid, or gavage preparation.

[0011] A method for preparing an intestinal flora regulator based on marine shrimp peptides includes the following steps: S1. Pre-treat the marine shrimp raw material to obtain marine shrimp protein raw material liquid; S2. Add protease to the marine shrimp protein raw material solution for enzymatic hydrolysis, so that the protein in the marine shrimp protein raw material solution is broken down into oligopeptide components, and an enzymatic hydrolysate containing oligopeptide components is obtained. S3. The enzyme hydrolysate is subjected to enzyme inactivation treatment and solid-liquid separation to obtain a clear enzymatic hydrolysate. S4. Perform membrane fractionation on the clarified enzymatic hydrolysis product to separate and collect oligopeptide components with a molecular weight not greater than 3kDa. S5. The oligopeptide component is concentrated and dried to obtain the intestinal flora regulator.

[0012] Preferably, the pretreatment includes washing, impurity removal, crushing, and homogenization.

[0013] Preferably, the protease is an alkaline protease, trypsin, papain, pepsin, or neutral protease.

[0014] Preferably, alkaline protease is used for enzymatic hydrolysis in step S2, and the enzymatic hydrolysis conditions are: material-to-liquid ratio of 1:2, hydrolysis temperature of 55°C, pH value of 8.0, enzyme addition amount of 7000 U / g, and hydrolysis time of 6 h.

[0015] Preferably, the solid-liquid separation is performed by centrifugal separation or filtration separation; The membrane grading is ultrafiltration grading; The drying process employs either freeze drying or spray drying.

[0016] In summary, the present invention has the following main beneficial effects: This application obtains marine shrimp peptide components, primarily low-molecular-weight short peptides, from marine shrimp protein raw materials through controlled enzymatic hydrolysis, solid-liquid separation, and membrane fractionation. This achieves the effect of transforming ordinary shrimp protein raw materials into intestinal flora regulators with clearly defined component characteristics and a stable preparation pathway. This method goes beyond simple enzymatic hydrolysis of the raw materials; it optimizes enzyme selection, controls hydrolysis conditions, and combines fractional enrichment to give the resulting components more clearly defined technical characteristics in terms of molecular weight distribution and short peptide composition. This distinguishes it from existing ordinary shrimp protein hydrolysates and improves product specificity and reproducibility.

[0017] By acting on the synergistic regulatory process among marine shrimp peptide components and gut microbiota structure, intestinal mucosal barrier, and intestinal permeability, this invention achieves the effect of not only regulating the microbiota but also simultaneously improving intestinal barrier function. Specifically, through the regulation of gut microbiota homeostasis by the marine shrimp peptide components, it promotes the restoration of beneficial bacteria structure and improves the intestinal environment; by improving goblet cell status and mucin secretion processes, it enhances the stability of the intestinal mucosal barrier; and by reducing abnormal changes related to intestinal permeability, it reduces the transport of enterogenic harmful substances into the body and improves the protective capacity of the intestinal barrier. Therefore, this application does not stop at improving a single indicator but establishes a continuous and complete chain of action around microbiota regulation and barrier protection.

[0018] By correlating the preparation characteristics of marine shrimp peptide components with functional verification at the cellular and animal levels, the technical solution, product components, and functional realization pathways mutually support each other. On the one hand, by clearly defining the source of raw materials, enzymatic hydrolysis methods, and fractionation steps, those skilled in the art can stably prepare the target components accordingly. On the other hand, by verifying the intestinal flora, mucosal tissue state, and permeability indicators, the product's application is established on a concrete implementation basis. Therefore, this application can form a complete technical logic from preparation to application in the specification, which not only helps to demonstrate the substantial features of this application compared to the prior art but also enhances the clarity, supportability, and feasibility of the technical solution. Attached Figure Description

[0019] Figure 1 This refers to the change in mouse body weight during the modeling period of this invention.

[0020] Figure 2 This refers to the total food intake of each mouse during the administration period of this invention.

[0021] Figure 3 This is PAS staining of LS174t cells MUC2 according to the present invention.

[0022] Figure 4 This invention relates to the effect of gut microbiota metabolites on the secretion of MUC2 in LS174t cells. Detailed Implementation

[0023] The technical solutions of 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.

[0024] Example 1 refer to Figure 1 This invention relates to a gut microbiota regulator based on marine shrimp peptides, with the preferred feed being the meat of the Chinese mantis shrimp (Sinapis sinensis). First, the fresh Chinese mantis shrimp meat is cleaned to remove impurities and non-target tissues, then crushed and homogenized to obtain a marine shrimp protein raw material liquid. The disclosure clearly states that the Chinese mantis shrimp is used as the experimental raw material, and its meat is enzymatically hydrolyzed to prepare SCPs.

[0025] Alkaline protease was added to the marine shrimp protein raw material solution for enzymatic hydrolysis. The preferred hydrolysis conditions were: a material-to-liquid ratio of 1:2, a temperature of 55°C, a pH of 8.0, an enzyme dosage of 7000 U / g, and a hydrolysis time of 6 hours. These process parameters were chosen because a comparison of trypsin, alkaline protease, papain, pepsin, and neutral protease was conducted, and the results showed that alkaline protease had the best hydrolysis effect. Therefore, this set of conditions was ultimately determined as the preferred conditions for large-scale preparation of SCPs.

[0026] After enzymatic hydrolysis, the hydrolysate is subjected to enzyme inactivation treatment, followed by centrifugation to remove incompletely hydrolyzed solid residues, yielding a clear hydrolysate. If necessary, fine suspended solids can be further removed by filtration after centrifugation to improve the stability of subsequent membrane fractionation. The instructions clearly state that the lyophilized powder will be collected for subsequent experiments, indicating that the obtained hydrolysate can be further purified and dried for storage.

[0027] The clarified enzymatic hydrolysate was subjected to ultrafiltration fractionation to collect low molecular weight marine shrimp oligopeptide components. As clearly stated in the disclosure, the final enzymatic hydrolysate was subjected to amino acid composition analysis and peptide structure identification after ultrafiltration, and the molecular weight of SCPs was mainly concentrated below 3kDa.

[0028] The oligopeptide fraction obtained by ultrafiltration was concentrated and then freeze-dried to obtain a freeze-dried powder of marine shrimp peptide intestinal flora regulator, which was stored at -20℃ for later use.

[0029] Example 2 The lyophilized marine shrimp peptide powder obtained in Example 1 was analyzed for its amino acid composition. See Table 1 for details. Table 1. Amino acid composition of SCPs

[0030] Note: * indicates essential amino acids, # indicates flavor amino acids. Seventeen amino acids were detected in the SCPs, with a total free amino acid content of 246.821 mg / g. Essential amino acids accounted for 97.138 mg / g, or 39.356% of the total amino acid mass. Amino acids with relatively high content included arginine (34.627 mg / g), phenylalanine (25.455 mg / g), glycine (24.451 mg / g), and tyrosine (23.467 mg / g). These results indicate that the obtained components have a rich short peptide and free amino acid base.

[0031] The peptides in the lyophilized marine shrimp peptide powder were further identified using ultra-high performance liquid chromatography tandem Q-TOF mass spectrometry, and the relevant results are shown in Table 2.

[0032] Table 2. Peptides identified in SCPs

[0033] A total of 17 peptides exhibited an ALC ≥ 95%, with each peptide consisting of 4-8 amino acid residues and a molecular weight less than 1 kDa. Among these, 15 contained proline, 9 contained alanine, and 9 contained leucine, indicating that the oligopeptide component exhibits the characteristics of proline-rich short peptides. Representative peptides include APPPPP, HPPPP, MPLPP, MLPLP, LPLP, and ELPP.

[0034] Based on the ultrafiltration fractionation results and peptide identification results, it can be determined that the marine shrimp peptide gut microbiota regulator obtained in this embodiment belongs to the oligopeptide component mainly composed of low molecular weight short peptides. Its molecular weight is mainly concentrated below 3kDa, and the core short peptide is a proline-rich short peptide composed of 4-8 amino acid residues.

[0035] The molecular weight distribution of the obtained oligopeptide components was detected by gel permeation chromatography or liquid chromatography-mass spectrometry. A calibration relationship was established using standard peptides or standards with known molecular weights. The molecular weight distribution range of the oligopeptide components was determined based on the peak area distribution. The detection results were used to confirm that the molecular weight of the obtained oligopeptide components was mainly concentrated below 3 kDa.

[0036] Example 3 The lyophilized marine shrimp peptide powder obtained in Example 1 was used to prepare sample solutions of different concentrations. LS174t cells were seeded in culture plates, and marine shrimp peptide samples were added after the cells had stabilized. The manual states that SCPs can promote LS174t cell proliferation in the range of 400-1000 μg / mL; in the range of 1000-1400 μg / mL, the relative cell proliferation rate decreased, but there was no significant difference compared with the control group. Therefore, 200, 600, and 1000 μg / mL were selected as the concentrations for subsequent experiments.

[0037] A H2O2-induced oxidative damage model of LS174t cells was established. Before oxidative damage treatment, marine shrimp peptide samples were pretreated. Results showed that, compared with the oxidative damage group, the 200, 600, and 1000 μg / mL dose groups significantly improved the survival rate of LS174t cells. There was no significant difference between the 600 μg / mL and 1000 μg / mL doses, indicating that the marine shrimp peptide components have a protective effect against oxidative stress.

[0038] The above results demonstrate that the marine shrimp oligopeptide components prepared in Example 1 not only structurally conform to the characteristics of low molecular weight, proline-rich short peptides, but also possess the ability to alleviate oxidative damage at the cellular level. These results provide a foundation for subsequent evaluation of its function from the perspective of intestinal barrier protection.

[0039] Example 4 To further illustrate the potential activities of the representative short peptides in Example 2, bioinformatics prediction and molecular docking analysis were performed on HPPPP, APPPPP, and MPLPP. The disclosure states that among the 17 peptides with ALC ≥ 95%, 7 peptides had an activity score ≥ 0.5, including 3 peptides with a free radical scavenging ability score ≥ 0.5: HPPPP, APPPPP, and MPLPP. Simultaneously, LPLP was found to have antihypertensive activity and could be used as an ACE inhibitor.

[0040] The disclosure also states that Keap1 forms four hydrogen bonds with APPPPP, two hydrogen bonds with HPPPP, and three hydrogen bonds with MPLPP. These peptides can interact with residues such as VAL-467, VAL-420, CYS-368, THR-560, VAL-465, ASN-381, THR-388, and SER-391, indicating that the representative short peptide has strong antioxidant-related binding potential. It should be noted that the focus of this application is not on using the aforementioned single short peptide as the sole active ingredient, but rather on using the entire marine shrimp oligopeptide component containing the aforementioned representative short peptide as the active basis for intestinal flora regulation.

[0041] Example 5 C57BL / 6J mice were selected and, after acclimatization, were induced to develop a model by feeding them a high-fat diet for 7 consecutive weeks. The data showed that after 7 weeks of high-fat diet induction, the average weight of the high-fat group mice reached approximately 32g, which was 1.23 times the average weight of the normal diet group, indicating successful model establishment.

[0042] After the model was established, a normal control group, a model group, a positive control group, a low-dose marine shrimp peptide group, and a high-dose marine shrimp peptide group were set up, and the intervention continued for 4 weeks.

[0043] After the intervention, the body weight, food intake, liver, kidney, spleen, epididymal fat, and perirenal fat weight of mice in each group were measured. See Table 3: Table 3. Changes in mouse body weight after 4 weeks of drug administration

[0044] See Table 4 for details: Table 4 Weight of mouse organs and adipose tissue

[0045] Marine shrimp peptide intervention reduced the final weight of model mice and decreased the weight of epididymal and perirenal fat. Taking the high-dose group as an example, its final weight was 27.204±1.82g, which was lower than that of the model group (29.892±1.11g); the weight of epididymal and perirenal fat was also lower than that of the model group.

[0046] Further analysis of serum lipid metabolism and liver function indicators was conducted. The data disclosed that, compared to the model group, SCPs-H significantly improved indicators such as TC, TG, LDL-C, ALT, AST, and HDL-C, indicating that marine shrimp peptide oligopeptide components can improve metabolic abnormalities and liver function damage induced by high lipid levels.

[0047] The liver was stained with Oil Red O and H&E, and the levels of MDA, GSH-Px, CAT, and SOD were measured. The results showed that intervention with marine shrimp peptides reduced lipid droplet accumulation and improved liver tissue structure. Simultaneously, MDA levels decreased, while the activities of GSH-Px, CAT, and SOD increased, indicating that this component can alleviate lipid-induced hepatic steatosis and oxidative stress.

[0048] Example 6 After the animal experiments in Example 5, ileal and colonic tissues were collected for H&E staining and AB-PAS staining. The results showed that, compared with the model group, the marine shrimp peptide intervention improved the ileal crypt structure, increased villus length, and increased goblet cell number; the colonic U-shaped crypt integrity was restored, and the number of goblet cells increased significantly, thereby promoting mucin secretion and mucosal barrier formation.

[0049] Simultaneously, serum DAO and liver tissue LPS were measured. The disclosure stated that DAO was significantly elevated in the model group, and LPS content in liver tissue was significantly increased; after intervention with marine shrimp peptides, DAO decreased and LPS decreased, indicating that the oligopeptide components of the marine shrimp peptides can reduce intestinal permeability and reduce the transport of enterogenic endotoxins to the liver. This section directly supports the description of its use in reducing intestinal permeability.

[0050] Therefore, based on three aspects—tissue morphology, goblet cell number, mucin secretion trend, and DAO and LPS indicators—it can be confirmed that the marine shrimp peptide gut microbiota regulator described in this application not only affects the microbiota structure but also improves the intestinal mucosal barrier function.

[0051] Example 7 Fecal samples from mice in each group of Example 5 were used for gut microbiota sequencing analysis. The disclosure states that intervention with marine shrimp peptides can partially reverse species structure changes induced by a high-fat diet, and has a reversing effect on the relative abundance changes of Firmicutes, Verrucomicrobia, and Actinobacteria at the phylum level.

[0052] At the scientific level, the disclosure clearly states that marine shrimp peptides can improve the relative abundance of Lachnospiraceae and Erysipelotrichaceae, and increase the relative abundance of Eubacterium coprostanoligenes group, Akkermansiaceae, and Lactobacillaceae. These results indicate that the oligopeptide components of the marine shrimp peptides have a tendency to regulate the intestinal flora structure and increase the abundance of beneficial bacteria.

[0053] At the genus level, the data disclosed that peptide intervention in marine shrimp increased the relative abundance of *norank_f_Eubacterium_coprostanoligenes_group* and *Turicibacter*, while decreasing the levels of *Coriobacteriaceae_UCG-002* and *Negativibacillus*. Combined with the correlation analysis results in the data disclosure, a correlation was observed between changes in gut microbiota and lipid metabolism, liver function, and intestinal permeability indicators.

[0054] The above results indicate that the marine shrimp peptide gut microbiota regulator described in this application does not rely solely on a single antioxidant mechanism, but rather achieves a comprehensive regulatory effect through a continuous chain of action: "low molecular weight proline-rich short peptide components—improvement of gut microbiota structure—improvement of goblet cell and mucin secretion—intestinal barrier repair—reduction of DAO / LPS".

[0055] Example 8 The marine shrimp peptide oligopeptide fraction obtained in Example 1 was added to the fecal microbiota of the model group for anaerobic fermentation to obtain the supernatant of intestinal microbiota metabolites. This supernatant was then applied to LS174t cells. The manual clearly states that, using an in vitro gastrointestinal digestion and anaerobic fermentation workstation, the Chinese whip shrimp peptide was added to the fecal microbiota of obese mice in the model group for anaerobic fermentation. Combined with the LS174t cell model, the protective effects of the metabolites on goblet cells were investigated from two aspects: antioxidant and regulation of mucin secretion.

[0056] The disclosure also clearly states that SCPs can protect the intestinal barrier by regulating gut microbiota homeostasis and increasing the MUC2 secretion level of LS174t cells. Figure 4 This study investigated the effect of gut microbiota metabolites on MUC2 secretion in LS174t cells. It demonstrates that the marine shrimp peptide oligopeptide components described in this application can directly act on cells or indirectly enhance MUC2 secretion through gut microbiota metabolism, further illustrating that its technical effect of improving intestinal mucosal barrier function has a clear implementation pathway.

[0057] Example 9 A method for preparing an intestinal flora regulator based on marine shrimp peptides includes the following steps: S1. Pre-treat the marine shrimp raw material to obtain marine shrimp protein raw material liquid; S2. Add protease to the marine shrimp protein raw material solution for enzymatic hydrolysis, so that the protein in the marine shrimp protein raw material solution is broken down into oligopeptide components, and an enzymatic hydrolysate containing oligopeptide components is obtained. S3. The enzyme hydrolysate is subjected to enzyme inactivation treatment and solid-liquid separation to obtain a clear enzymatic hydrolysate. S4. Perform membrane fractionation on the clarified enzymatic hydrolysis product to separate and collect oligopeptide components with a molecular weight not greater than 3kDa. S5. The oligopeptide component is concentrated and dried to obtain the intestinal flora regulator.

[0058] The pretreatment includes washing, impurity removal, crushing, and homogenization.

[0059] The protease is an alkaline protease, trypsin, papain, pepsin, or neutral protease.

[0060] In step S2, alkaline protease is used for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: material-to-liquid ratio of 1:2, hydrolysis temperature of 55℃, pH value of 8.0, enzyme addition amount of 7000U / g, and hydrolysis time of 6h.

[0061] The solid-liquid separation is performed by centrifugal separation or filtration separation; The membrane grading is ultrafiltration grading; The drying process employs either freeze drying or spray drying.

[0062] During membrane fractionation, it is preferred to use an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to clarify the enzymatic hydrolysis product, and collect the permeate through the ultrafiltration membrane as the oligopeptide component. When it is necessary to further enrich lower molecular weight peptides, an ultrafiltration membrane with a molecular weight cutoff of 1 kDa can be used for fractionation, and the corresponding low molecular weight components can be collected for subsequent concentration and drying.

[0063] Example 10 In this embodiment, the gut microbiota regulator based on marine shrimp peptides is not evaluated using a single indicator, but rather a comprehensive evaluation system is established around gut microbiota homeostasis, intestinal mucosal barrier function, and immune-related responses. Gut microbiota regulation is the core evaluation component, while intestinal barrier function and immune function are extended evaluation components related to changes in the microbiota. This evaluation system can determine whether the oligopeptide component can form a continuous action process in the intestinal environment, i.e., first affecting the gut microbiota structure, then improving the barrier state through microbiota metabolism and intestinal mucosal responses, and further alleviating abnormal immune responses related to gut-derived stimuli.

[0064] The immune function evaluation in this embodiment is used to help explain the changes in inflammation-related responses after intestinal flora imbalance and intestinal mucosal barrier damage, and does not limit the marine shrimp peptide oligopeptide components to a single immunotherapeutic drug or immunotherapeutic drug component.

[0065] In the evaluation of gut microbiota regulation, fecal samples were collected from subjects, and the composition of the microbiota was sequenced and analyzed. Changes in α-diversity, β-diversity, and abundance of microbiota at the phylum, family, and genus levels were compared among different treatment groups. The evaluation focused on whether the microbiota structure shift caused by a high-fat diet or other modeling factors was partially reversed by the marine shrimp peptide oligopeptide component. Further analysis was conducted on the relative abundance changes of beneficial bacteria, short-chain fatty acid-producing bacteria, and bacteria associated with inflammation or metabolic disorders. This approach allows for confirmation at the microbiota structure level whether the regulator has a role in improving gut microbiota imbalance.

[0066] In evaluating intestinal barrier function, assessments are made by combining histological observations, changes in goblet cell count, mucin secretion levels, and intestinal permeability indicators. Specifically, ileal and colonic tissues are subjected to H&E staining and AB-PAS staining to observe villus integrity, crypt structure, goblet cell count, and mucosal layer condition. Simultaneously, mucin-related indicators such as MUC2 are detected to reflect goblet cell secretory function. Serum diamine oxidase levels and lipopolysaccharide levels in liver tissue or serum are also measured to reflect intestinal permeability and enterogenic endotoxin transport. If treatment with the marine shrimp peptide oligopeptide component results in improved villus structure, restored goblet cell count, enhanced mucin secretion, and decreased diamine oxidase and lipopolysaccharide levels, it indicates that the component can improve intestinal mucosal barrier function.

[0067] In evaluating immune function, a comprehensive assessment can be made by combining intestinal barrier status and inflammatory response-related indicators. Intestinal flora imbalance and barrier disruption make it easier for enterogenic irritants to enter the circulation or liver tissue, thereby inducing local or systemic immune responses. Therefore, this embodiment can detect inflammatory-related factors in intestinal tissue, serum, or liver tissue, and analyze them in conjunction with immune cell infiltration, histopathological changes, and lipopolysaccharide levels. If, after treatment with the marine shrimp peptide oligopeptide component, abnormal inflammatory infiltration is reduced, inflammatory-related indicators decrease, and intestinal barrier-related indicators improve simultaneously, it indicates that it can alleviate abnormal immune responses caused by intestinal microecological imbalance by regulating the intestinal flora and maintaining barrier integrity.

[0068] In the aforementioned evaluation system, there is a corresponding relationship between the preparation process and the functional evaluation. The marine shrimp protein raw material undergoes pretreatment, enzymatic hydrolysis, enzyme inactivation, solid-liquid separation, membrane fractionation, concentration, and drying to obtain an oligopeptide component mainly composed of low-molecular-weight short peptides. After entering the intestinal environment, this oligopeptide component serves as the basis for regulating the intestinal microecology, further influencing the composition of the gut microbiota, microbial metabolites, goblet cell secretion status, and the integrity of the intestinal mucosal barrier. Therefore, this application establishes a continuous technical pathway of "controlled preparation of oligopeptide components, regulation of intestinal microbiota structure, improvement of intestinal barrier function, and reduction of abnormal immune responses," which differs from schemes that only use ordinary shrimp protein hydrolysates for single nutritional supplementation or single antioxidant evaluation.

[0069] 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 gut microbiota regulator based on marine shrimp peptides, characterized in that, include: The oligopeptide component is obtained from marine shrimp protein raw material through enzymatic hydrolysis, enzyme inactivation, solid-liquid separation and membrane fractionation. In the oligopeptide component, peptides with a molecular weight not greater than 3kDa account for more than 60% of the total mass of the oligopeptide component, peptides with a molecular weight not greater than 1kDa account for more than 40% of the total mass of the oligopeptide component, and the oligopeptide component contains short peptides composed of 4 to 8 amino acid residues.

2. The intestinal flora regulator based on marine shrimp peptides according to claim 1, characterized in that, The marine shrimp protein raw material is derived from the shrimp meat, shrimp kernels, shrimp heads, or by-product protein extracts of marine shrimp.

3. The intestinal flora regulator based on marine shrimp peptides according to claim 2, characterized in that, The oligopeptide component contains proline-rich short peptides.

4. The intestinal flora regulator based on marine shrimp peptides according to claim 3, characterized in that, The gut microbiota regulator is used to regulate the gut microbiota structure, improve the intestinal mucosal barrier function, or reduce intestinal permeability.

5. The intestinal flora regulator based on marine shrimp peptides according to claim 4, characterized in that, The intestinal flora regulator is formulated as a powder, granule, tablet, capsule, oral liquid, or gavage preparation.

6. A method for preparing an intestinal flora regulator based on marine shrimp peptides, applicable to the intestinal flora regulator based on marine shrimp peptides as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Pre-treat the marine shrimp raw material to obtain marine shrimp protein raw material liquid; S2. Add protease to the marine shrimp protein raw material solution for enzymatic hydrolysis, so that the protein in the marine shrimp protein raw material solution is broken down into oligopeptide components, and an enzymatic hydrolysate containing oligopeptide components is obtained. S3. The enzyme hydrolysate is subjected to enzyme inactivation treatment and solid-liquid separation to obtain a clear enzymatic hydrolysate. S4. Perform membrane fractionation on the clarified enzymatic hydrolysis product to separate and collect oligopeptide components with a molecular weight not greater than 3kDa. S5. The oligopeptide component is concentrated and dried to obtain the intestinal flora regulator.

7. The method for preparing an intestinal flora regulator based on marine shrimp peptides according to claim 6, characterized in that, The pretreatment includes washing, impurity removal, crushing, and homogenization.

8. The method for preparing an intestinal flora regulator based on marine shrimp peptides according to claim 7, characterized in that, The protease is an alkaline protease, trypsin, papain, pepsin, or neutral protease.

9. The method for preparing an intestinal flora regulator based on marine shrimp peptides according to claim 8, characterized in that, In step S2, alkaline protease is used for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: material-to-liquid ratio of 1:2, hydrolysis temperature of 55℃, pH value of 8.0, enzyme addition amount of 7000U / g, and hydrolysis time of 6h.

10. A method for preparing an intestinal flora regulator based on marine shrimp peptides according to claim 9, characterized in that, The solid-liquid separation is performed by centrifugal separation or filtration separation; The membrane grading is ultrafiltration grading; The drying process employs either freeze drying or spray drying.

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