A fish-derived active peptide with dual functions of salty taste enhancement and inhibition of adipocyte differentiation and application thereof

Abstract

This invention discloses a fish-derived bioactive peptide with dual functions of enhancing saltiness and inhibiting adipocyte differentiation, and its applications, belonging to the field of biotechnology. The bioactive peptide is a γ-glutamylarginine peptide with the general formula γ-[Glu]n-Arg, where n is an integer from 1 to 4. This invention efficiently prepared a series of γ-[Glu]n-Arg peptides through a targeted enzymatic synthesis method, further elucidating the dual effects of this bioactive peptide at the molecular mechanism level. The bioactive peptide can effectively enhance saltiness perception by binding to the saltiness receptor TMC4, and inhibit adipocyte differentiation and reduce lipid accumulation through multiple targets by downregulating PPAR-γ, C / EBPα, and FAS genes, and upregulating AMPK and HSL genes. This invention provides a reliable scientific basis and technical support for developing innovative food ingredients with both flavoring and health functions.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a fish-derived bioactive peptide with dual functions of enhancing saltiness and inhibiting adipocyte differentiation, and its applications. Background Technology

[0002] High-salt diets are a common risk factor for chronic diseases such as hypertension, cardiovascular disease, and obesity. Currently, salt reduction has become a major global health concern. To reduce sodium intake, the scientific community is actively seeking effective salt substitutes or saltiness enhancers. Against this backdrop, a series of natural or synthetic flavor peptides have been extensively studied, especially arginine-containing dipeptides or polypeptides, which have been proven to enhance saltiness perception in various food systems, thus helping to maintain product saltiness while reducing added salt. However, existing saltiness-modifying peptides are mainly concentrated in dipeptide or short peptide forms, with limited flavor intensity and stability, and relatively singular functions. They typically focus only on sensory characteristics, lacking simultaneous exploration of potential physiological activities.

[0003] The flavor characteristics and physiological activities of γ-glutamyl peptides are closely related to their molecular structure, amino acid sequence, and peptide chain length. Therefore, the development of novel γ-glutamyl peptides holds great promise. Studies have reported that arginine peptides can enhance the saltiness of NaCl aqueous solutions, simulated broth solutions, and cheese models as saltiness modifiers, potentially replacing table salt and reducing sodium intake. This could benefit the prevention or alleviation of a range of cardiovascular diseases caused by high-salt diets. Furthermore, arginine-rich peptides may also have anti-obesity effects. Theoretically, combining the saltiness-modifying potential of arginine with the characteristics of the γ-glutamyl group, oligo-γ-glutamylarginine peptides (OERs) are expected to be ideal candidates for both saltiness enhancement and physiological activity. However, existing synthetic techniques mostly focus on known γ-glutamyl peptides such as glutathione, and targeted, efficient synthesis of OERs is still rarely reported. Existing synthetic methods face challenges such as low product specificity, numerous side reactions, and low conversion efficiency. Especially under high solids concentrations, the efficiency of enzymatic reactions decreases significantly, severely restricting their large-scale preparation and application. Furthermore, although arginine and its derivatives have shown potential in regulating lipid metabolism, to date, no studies have systematically integrated saltiness enhancement and anti-obesity activity on the same peptide molecule and conducted empirical studies. This has resulted in existing salty peptide products having limited functions and failing to achieve the synergistic effect of "flavoring" and "health".

[0004] Therefore, existing technologies mainly suffer from the following problems: First, there is a lack of efficient and targeted methods for synthesizing γ-glutamylarginine oligopeptides with specific structures, especially in high-concentration reaction systems where enzymatic synthesis efficiency is low; second, existing research on salty peptides is mostly limited to sensory evaluation, lacking analysis of the molecular mechanisms of interaction with taste receptors; and third, the synergistic design and functional verification of the same peptide molecule in terms of salt-enhancing and anti-obesity activities have not yet been achieved. These deficiencies limit the further development and application of such peptides in low-sodium health foods. This invention aims to overcome the above difficulties by providing a new technical solution for developing fish-derived bioactive peptides with both salt-enhancing and anti-obesity activities through targeted enzymatic synthesis, combined sensory and molecular mechanism analysis, and cellular function verification. Summary of the Invention

[0005] The purpose of this invention is to provide a fish-derived bioactive peptide with dual functions of enhancing saltiness and inhibiting adipocyte differentiation, and its applications, to address the problems existing in the prior art. The fish-derived γ-[Glu]n-Arg peptide developed in this invention can effectively enhance saltiness perception by binding to the saltiness receptor TMC4, and inhibit adipocyte differentiation and reduce lipid accumulation through multiple targets by downregulating PPAR-γ, C / EBPα, and FAS genes, and upregulating AMPK and HSL genes. This peptide provides a new approach for developing food ingredients with both salt-reducing and anti-obesity functions.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a fish-derived active peptide with dual functions of enhancing saltiness and inhibiting adipocyte differentiation. The active peptide is a γ-glutamylarginine peptide with the general formula γ-[Glu]n-Arg, where n is an integer from 1 to 4.

[0007] Optional, n is 1 or 2.

[0008] This invention also provides a method for preparing the aforementioned fish-derived bioactive peptide, comprising the following steps: Fish meat is used as raw material, and enzymatic hydrolysis is performed using protease to obtain protein hydrolysate; the protein hydrolysate is then subjected to a transpeptidation reaction under the action of glutaminase to generate the γ-glutamylarginine peptide.

[0009] Optionally, the protease is a complex enzyme prepared by mixing chymotrypsin, trypsin and papain in an enzyme activity ratio of 1:1:1.

[0010] Optionally, the enzyme activity of the chymotrypsin, the trypsin, and the papain is 5000 U / g.

[0011] Optionally, the enzymatic hydrolysis step includes: mixing fish meat and water at a ratio of 1g:2mL until homogenized, adding enzyme, and then hydrolyzing at pH 7.5 and a temperature of 55℃ for 3 hours.

[0012] Optionally, the conditions for the transpeptide reaction are: pH 10.0 and temperature 37°C.

[0013] The present invention also provides the application of the aforementioned fish-derived active peptide in the preparation of a product that has both salty flavor enhancement function and helps control body fat.

[0014] The present invention also provides a product that has both salty flavor enhancement function and helps control body fat, the active ingredient of which includes the aforementioned fish-derived active peptide.

[0015] Optionally, the products include food and health supplements.

[0016] The present invention discloses the following technical effects: This invention successfully developed a fish-derived bioactive peptide with dual functions of enhancing saltiness and inhibiting adipocyte differentiation. A series of γ-[Glu]n-Arg peptides were efficiently prepared using a targeted enzymatic synthesis method. This invention further elucidates the dual efficacy of this bioactive peptide at the molecular mechanism level. This peptide enhances saltiness perception by stably binding to the saltiness receptor TMC4, while effectively inhibiting 3T3-L1 preadipocyte differentiation, reducing lipid accumulation, and promoting lipolysis. Its anti-obesity effect involves downregulation of multiple adipogenic genes such as PPAR-γ, C / EBPα, and FAS, and upregulation of catabolic genes such as AMPK and HSL, achieving multi-target synergistic regulation of lipid metabolism. This invention provides a reliable scientific basis and technical support for developing innovative food ingredients with both flavoring and health benefits. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The docking results of γ-Glu-Arg (A), γ-Glu-Glu-Arg (B), γ-Glu-Glu-Glu-Arg (C) and γ-Glu-Glu-Glu-Glu-Arg (D) with TMC4 are shown in the figure. Figure 2 Ion interaction diagrams of γ-Glu-Arg (A), γ-Glu-Glu-Arg (B), γ-Glu-Glu-Glu-Arg (C) and γ-Glu-Glu-Glu-Glu-Arg (D) with TMC4; Figure 3The diagram shows the hydrophobic interactions between γ-Glu-Arg (A), γ-Glu-Glu-Arg (B), γ-Glu-Glu-Glu-Arg (C), and γ-Glu-Glu-Glu-Glu-Arg (D) and TMC4. Figure 4 The charge interaction diagrams of γ-Glu-Arg (A), γ-Glu-Glu-Arg (B), γ-Glu-Glu-Glu-Arg (C) and γ-Glu-Glu-Glu-Glu-Arg (D) with TMC4 are shown. Figure 5 The diagram shows the hydrogen bond interactions between γ-Glu-Arg (A), γ-Glu-Glu-Arg (B), γ-Glu-Glu-Glu-Arg (C), and γ-Glu-Glu-Glu-Glu-Arg (D) and TMC4. Figure 6 Aromatic interactions of γ-Glu-Arg (A), γ-Glu-Glu-Arg (B), γ-Glu-Glu-Glu-Arg (C) and γ-Glu-Glu-Glu-Glu-Arg (D) with TMC4; Figure 7 SAS interaction diagram of γ-Glu-Arg (A), γ-Glu-Glu-Arg (B), γ-Glu-Glu-Glu-Arg (C) and γ-Glu-Glu-Glu-Glu-Arg (D) with TMC4; Figure 8 Oil Red O staining results of different samples 8 days after induced differentiation; Figure 9 A graph showing the changes in OD values ​​of cells from different samples after 8 days of induced differentiation. Figure 10 A graph showing the changes in triglyceride content in cells of different samples after 8 days of induced differentiation; Figure 11 A graph showing the changes in glycerol content in cells of different samples after 8 days of induced differentiation; Figure 12 The expression levels of genes related to cell proliferation and differentiation in different sample groups. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1: Theoretical peptide profile prediction and enzymatic hydrolysis scheme design for fish protein source OER This embodiment provides a theoretical design method for preparing oligo-γ-glutamylarginine peptides (OER), aiming to directionally screen target peptides from oval pomfret meat protein. The specific steps are as follows: 1. Substrate selection and sequence acquisition: The amino acid sequences of actin and myosin from the oval pomfret were used as the research object.

[0025] 2. Virtual Enzymatic Digestion: The above protein sequences were virtually digested using the "ENZYME(S) ACTION" module in the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) and the ExPASy PeptideCutter tool (http: / / web.expasy.org / peptide_cutter / ). The selected proteases and their combinations were: papain, trypsin, chymotrypsin, and a complex enzyme composed of the three in a 1:1:1 enzyme activity ratio.

[0026] 3. Peptide Screening: All peptides generated from the virtual enzymatic hydrolysis were collected and statistically analyzed. Considering absorption efficiency, only peptides with fewer than 10 amino acid residues were screened. The statistical results (as shown in Table 1) indicate that, theoretically, hydrolysis of actin by the three enzymes, individually and in combination, could produce 243 acceptable peptides; for myosin, it could produce 170 acceptable peptides. Theoretically, these three proteases can hydrolyze to obtain hydrolysates with a relatively large number of polypeptides. Based on this, these three enzymes and their mixtures were used in subsequent actual enzymatic hydrolysis.

[0027] Table 1. Statistical analysis of the number of active peptides obtained from virtual enzymatic hydrolysis with different hydrolases. Example 2: Directed enzymatic synthesis, isolation, and structural identification of OER This embodiment describes the directed enzymatic synthesis and identification of oligomeric γ-glutamyl arginine peptide (OER) from oval pomfret meat. The specific steps are as follows: 1. Raw material pretreatment: Grind the oval pomfret meat thoroughly using a meat grinder.

[0028] 2. Enzymatic hydrolysis: The minced fish meat was mixed with distilled water at a ratio of 1:2 (g / mL). The initial pH of the mixture was adjusted to 6.0 and thoroughly homogenized. The system was placed in a constant temperature water bath, heated to 55℃ and continuously stirred, while the pH was precisely adjusted to 7.5. Subsequently, a complex of papain, trypsin, and chymotrypsin (1:1:1) with an enzyme activity of 5000 U / g (based on raw protein) was added, and enzymatic hydrolysis was carried out at 55℃ with continuous stirring for 3 hours.

[0029] 3. Termination of enzyme activity: After the enzymatic hydrolysis is completed, the reaction system is heated in a 100°C water bath for 10 minutes to inactivate the protease, and then cooled rapidly.

[0030] 4. Glutaminant transpeptidation: Adjust the pH of the enzyme hydrolysate to 10.0 after enzyme inactivation. Add glutaminase (0.2 g / 100 mL) and react in a 37°C water bath for 24 hours. After the reaction is complete, inactivate the enzyme again in a 100°C water bath for 15 minutes.

[0031] The reaction solution was centrifuged at 4°C and 10,000 g for 10 minutes, and the supernatant was collected. The supernatant was then separated and purified using a semi-preparative liquid chromatography system equipped with an XSelect HSST3 column (5 μm × 4.6 mm × 250 mm), and the fraction containing the target peptide was collected.

[0032] 5. Drying and Structural Identification: The collected fractions were subjected to alkaline drying using a vacuum freeze dryer. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was used to analyze and identify the freeze-dried samples.

[0033] Primary mass spectrometry analysis revealed five precursor ions, with the main precursor ions having m / z values ​​of 175.1188, 304.1619, 433.2041, 562.2456, and 691.2871. Secondary mass spectrometry further showed fragment ion information for these substances; the fragment ion of γ-Glu-Arg included [Gln-COOH]. + (m / z=84.0418, b1 type ion), [M-γ-glutamyl residue-guanidinyl] + (m / z=116.0684, y1 type ion), [M-Arg+H] + (m / z=130.0955, b1 type ion), [Arg+H] + (m / z = 175.1172, y1 type ion). γ-Glu-Glu-Arg also contains ions with m / z values ​​of 84.0412, 116.0694, and 175.1172, in addition to [M-γ-glutamyl residue + H]. + (m / z=304.1625, y2 type ion); γ-Glu-Glu-Glu-Arg and γ-Glu-Glu-Glu-Glu-Arg also contain fragment ions similar to the two polypeptides mentioned above.

[0034] By comparing with the theoretical molecular weight and analyzing the fragment ions by secondary mass spectrometry (fragment ion information is shown in Table 2), it was confirmed that these ions correspond to arginine (Arg), γ-glutamylarginine (γ-Glu-Arg), γ-glutamyl-glutamylarginine (γ-Glu-Glu-Arg), γ-triglutamylarginine (γ-Glu-Glu-Glu-Arg), and γ-tetraglutamylarginine (γ-Glu-Glu-Glu-Glu-Arg), thus confirming the successful synthesis of the OER series peptides.

[0035] Table 2 Ion fragments in OER identified by UPLC-MS / MS Example 3: Sensory evaluation of OER and determination of saltiness enhancement effect This embodiment evaluates the saltiness-enhancing effect of OER and its different components. The specific steps are as follows: 1. Establish a saltiness standard curve: Prepare sodium chloride (NaCl) aqueous solutions with concentration gradients of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg / mL. Trained sensory evaluators (no fewer than 8 people) evaluate the saltiness intensity of the above solutions and determine the perceived NaCl concentration with reference to the standard. Establish a standard curve with the actual NaCl concentration as the x-axis (x, mg / mL) and the average perceived NaCl concentration as the y-axis (y, mg / mL). The standard curve equation is obtained as y = 1.0657x – 0.0952 (R²). 2 = 0.94).

[0036] 2. Evaluation of the saltiness enhancement of OER in NaCl solution: A NaCl solution with a concentration of 3 mg / mL was prepared as the base solution. OER mixtures prepared in Example 2 were added to the solution at final concentrations of 0, 1, 5, 10, 20, and 40 mg / mL, respectively. Sensory evaluators assessed the saltiness intensity, and based on the standard curve from step 1, the perceived intensity was converted into "perceived NaCl concentration" and "saltiness equivalent concentration" (i.e., the NaCl concentration required to produce the same level of saltiness). The results are shown in Table 3: In the NaCl solution, as the amount of OER added gradually increased from 1 mg / mL to 40 mg / mL, the perceived NaCl concentration first increased from 3.15 mg / mL to 4.71 mg / mL, and finally decreased to 3.18 mg / mL, equivalent to the saltiness produced by NaCl concentrations of 3.05 mg / mL, 3.41 mg / mL, 3.96 mg / mL, 4.51 mg / mL, and 3.07 mg / mL, respectively. Therefore, within a certain range, the concentration of OER added is positively correlated with the saltiness enhancement effect, and can enhance the saltiness by about 1.5 times (at a concentration of 20 mg / mL); however, when the concentration is increased to 40 mg / mL, there is basically no effect on enhancing the saltiness, possibly because the amount of peptide added is too large, which enhances the bitterness.

[0037] The above results indicate that OER has a significant saltiness-enhancing effect in the concentration range of 1-20 mg / mL, with the optimal addition concentration being 20 mg / mL, which can enhance the perceived saltiness by about 1.5 times.

[0038] 3. Evaluation of OER's Saltiness Enhancement in Simulated Broth: A NaCl-free simulated broth solution was prepared. The NaCl equivalent concentration (e.g., 2.83 mg / mL) corresponding to its initial saltiness intensity was determined through sensory evaluation. OER with the same concentration gradient as in step 2 was added to this broth model, and sensory evaluation was performed to calculate the saltiness equivalent concentration. The results are shown in Table 3: First, the initial saltiness level of the simulated broth model was determined to be equivalent to the saltiness intensity produced by a 2.83 mg / mL NaCl aqueous solution. The saltiness enhancement effect of OER on the model broth was similar to that in the NaCl aqueous solution, both ranging from 1 mg / mL to 20 mg / mL. The larger the amount of OER added, the better the saltiness enhancement effect, with a maximum increase of 1.7 times in the saltiness of the simulated broth model.

[0039] Table 3. OER's activity in enhancing the saltiness of sodium chloride and simulated broth solutions. 4. Flavor Characteristics Analysis of OER Components: The OER obtained in Example 2 was separated into four main components by semi-preparative liquid chromatography: γ-Glu-Arg, γ-Glu-Glu-Arg, γ-Glu-Glu-Glu-Arg, and γ-Glu-Glu-Glu-Glu-Arg. The flavor thresholds (using a three-point selection method) and basic taste attributes (sweet, salty, umami) in aqueous solution were determined for each component. For evaluation, the flavor intensity of standard sweet (sucrose), salty (NaCl), and umami (monosodium glutamate) solutions was set as a baseline score of 2.5, and the intensity of the test peptide solution compared to these baselines was evaluated.

[0040] The results are shown in Table 4. γ-Glu-Arg, γ-Glu-Glu-Arg, γ-Glu-Glu-Glu-Arg, and γ-Glu-Glu-Glu-Glu-Arg exhibit only slight astringent and bitter tastes in aqueous solution. The threshold concentrations of γ-Glu-Arg, γ-Glu-Glu-Arg, γ-Glu-Glu-Glu-Arg, and γ-Glu-Glu-Glu-Glu-Arg in aqueous solution were 1.25, 2.32, 2.49, and 2.61 mg / mL, respectively. With a taste score of 2.5 for the basic sweet, salty, and umami solutions, the taste scores of γ-Glu-Arg, γ-Glu-Glu-Arg, γ-Glu-Glu-Glu-Arg, and γ-Glu-Glu-Glu-Glu-Arg were all greater than 2.5 after the addition of these four peptides, indicating that all four peptides could enhance the original sweet, salty, and umami tastes. At the same added concentration (1 mg / mL), the enhancement effect of the four peptides in OER on sweetness, saltiness and umami was as follows: the longer the peptide chain, the better the enhancement effect on sweetness, saltiness or umami.

[0041] Table 4 Flavor characteristics and thresholds of different peptides in OER in aqueous solution. Example 4: Analysis of the OER Saltiness Enhancement Mechanism Based on Molecular Docking This embodiment utilizes molecular docking technology to investigate the interaction mechanism between the OER and the salty taste receptor TMC4. The specific steps are as follows: Molecular structure preparation: The three-dimensional protein structure model of the salty taste receptor TMC4 was obtained from the Protein Database (PDB) or through homology modeling, and preprocessed by hydrogenation and charge distribution. Simultaneously, the 3D molecular structures of four peptides—γ-Glu-Arg, γ-Glu-Glu-Arg, γ-Glu-Glu-Glu-Arg, and γ-Glu-Glu-Glu-Glu-Arg—were constructed, and energy was optimized.

[0042] Molecular docking: Using molecular docking software such as Autodock Vina, molecular docking simulations were performed on the four peptides and their respective active pocket regions of TMC4. A docking search space was set to cover the hypothesized binding region of TMC4. After docking, the conformation with the lowest binding free energy (binding energy) was selected as the most probable binding mode.

[0043] Interaction analysis: Analyze the interaction forces between the peptide and TMC4 in the optimal docking conformation, including: hydrogen bonds (e.g., ... Figure 5 ), ion interactions (such as Figure 2 ), hydrophobic interactions (such as Figure 3 ), charge transfer ( Figure 4 ), aromatic interactions (such as Figure 6 ) and solvent-accessible surface area (SAS, e.g. Figure 7 )change.

[0044] Results Explanation: The docking results are displayed (e.g., ...). Figure 1 The binding energies of the four peptides to TMC4 were -4.9, -5.0, -5.0, and -6.3 kcal / mol, respectively, indicating that they could all bind spontaneously, and the binding strength was positively correlated with the peptide chain length. Among them, the γ-Glu-Glu-Glu-Glu-Arg binding was the most stable. Detailed interaction diagram (…) Figure 2-7 This indicates that the hydrogen bond network is a key force stabilizing the complex. This result reveals at the molecular level the potential mechanism by which OERs, particularly long-chain OERs, enhance saltiness perception by stabilizing the binding of the saltiness receptor TMC4.

[0045] Example 5: Study on the activity and mechanism of OER in inhibiting the differentiation of 3T3-L1 preadipocytes This embodiment describes a method for evaluating the anti-obesity activity of OER at the cellular level and preliminarily exploring its mechanism of action. The specific steps are as follows: 1. Prediction and screening of target peptide properties Based on online bioinformatics tools, preliminary physicochemical properties of target peptides are predicted, providing a theoretical basis for cell experiment screening.

[0046] I. Experimental Methods 1. γ-[Glu] (n=1, 2, 3, 4) -Arg property prediction The cell membrane penetration ability, hydrophilicity, and isoelectric point of Arg and γ-[Glu](n=1, 2, 3, 4)-Arg were predicted using CPPpred (http: / / distilldeep.ucd.ie / CPPpred / ), a protein hydrophilicity analysis tool (http: / / www.detaibio.com / sms2 / protein_gravy.html), and PepDraw (http: / / pepdraw.com / ), respectively, to preliminarily screen peptides.

[0047] 2. Culture of 3T3-L1 preadipocytes Cell resuscitation: The frozen 3T3-L1 preadipocytes were removed from liquid nitrogen and quickly thawed (37°C). They were first disinfected with alcohol and then added to the culture medium (5 mL). After low-speed centrifugation, the supernatant was discarded and the culture medium was added again. The cells were gently pipetted until they were suspended, transferred to culture flasks, placed in an incubator, and the medium was changed and observed regularly.

[0048] Cell passage: When the cells adhere to the culture flask and cover 80% of the flask, wash several times with PBS buffer, then digest with trypsin for 1.5 min. When the cells become round, stop the digestion with DMEM complete medium, pipette until the cells are suspended, centrifuge (760g, 4℃, 4 min), discard the supernatant, add fresh medium, and continue culturing.

[0049] Cell cryopreservation: When cells reach the logarithmic growth phase, digest and centrifuge them according to the passage procedure, add cryopreservation solution to suspend the cells, and count them using a hemocytometer to achieve a cell density of 1-10 × 10⁻⁶ cells / mL. 6 The sample was dispensed at 1.5 mL per tube into sterile cryovials, pre-cooled at 4°C for 20 min, then placed at -20°C for 4 h, and finally stored in liquid nitrogen.

[0050] 3. Standard curve for determining the number of 3T3-L1 preadipocytes using the MTT assay. When the cells have grown to 80% of the volume of a hemocytometer flask, they are digested with trypsin, thoroughly mixed by pipetting, counted using a hemocytometer, and adjusted to a cell concentration of 36 × 10⁻⁶. 4 cell / mL, 18×10 4cell / mL, 9×10 4 cell / mL, 4.5 × 10 4 cell / mL, 1.5 × 10 4 cell / mL, 0.5×10 4 cell / mL, 0.25×10 4 Cells / mL were seeded in 6 parallel copies at each density into a 96-well plate. 50 μL of MTT was added to each well, and the plate was incubated at 37°C for 4 h. The liquid was gently aspirated with a pipette, and then 150 μL of DMSO was added. The plate was shaken at low speed for 10 min to fully dissolve the crystals. The absorbance was measured at 490 nm.

[0051] 4. Effects of different concentrations of samples on the proliferation of 3T3-L1 preadipocytes Cells in the logarithmic growth phase were selected. First, the cells were washed twice with PBS, then trypsinized and the supernatant was discarded. 1 mL of culture medium was added, and the cell concentration was adjusted to 1.5 × 10⁻⁶ cells. 4 Cells / mL; 100 μL / well was seeded into a 96-well plate and cultured at 37°C as usual. After the cells were fully attached (24 h), the culture medium was removed and replaced with culture medium containing different concentrations of the sample. After culturing for 24 h, the proliferation was detected by the MTT assay, and the cell number was calculated according to the standard curve.

[0052] The treatment was divided into a blank control group and an experimental group (Arg, γ-Glu-Arg and γ-Glu-Glu-Arg, with concentrations of 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL and 1.2 mg / mL, respectively).

[0053] 5. Effects of different concentrations of samples on the differentiation of 3T3-L1 preadipocytes Cells were loaded at 3.6 × 10 4 Cells were seeded at a density of 6-well plates and observed regularly, with the medium changed every other day. When the cells reached the contact inhibition level two days later (Day 0), the old medium was aspirated and replaced with induction medium I (1 μg / mL Insulin, 0.5 mmol / L IBMX and 1 μmol / L Dex) and the sample. After 48 h, the medium was replaced with induction medium II (medium containing 1 μg / mL Insulin).

[0054] Sample processing was divided into a model group (Control) without any added samples and an experimental group (Arg, γ-Glu-Arg and γ-Glu-Glu-Arg at 0.05 mg / mL or 0.2 mg / mL, denoted as ArgL, ArgH, γ-Glu-ArgL, γ-Glu-ArgH, γ-Glu-Glu-ArgL and γ-Glu-Glu-ArgH, respectively). Eight days after induction of differentiation, Oil Red O staining was used and absorbance was measured at 490 nm.

[0055] 6. Effects of different concentrations of induced differentiation on triglyceride and glycerol content The experimental procedures and treatment groups were the same as above. After culturing all cells for 8 days, cells and cell culture medium were collected separately, cells were lysed, and the triglyceride content was determined using a TG assay kit according to the GPO-PAP method. The glycerol content was determined using a glycerol assay kit.

[0056] 7. Extraction of total RNA Collect cells 8 days after the above-mentioned induced differentiation, and extract total RNA according to the following steps: (1) Aspirate the cell culture medium, wash with PBS, add 1 mL of Trizol to each well, place horizontally for a while, and pipette the cells to detach them; transfer to a 1.5 mL centrifuge tube, pipette repeatedly until there is no obvious precipitation in the lysis buffer, and let stand at room temperature for 5 min.

[0057] (2) Add 200 μL of chloroform, shake to mix, let stand at room temperature for 5 min, centrifuge at 12000 g and 4℃ for 15 min, discard the precipitate, and transfer the supernatant to another centrifuge tube.

[0058] (3) Add an equal volume of isopropanol to the supernatant, mix well, let stand at room temperature for 10 min, centrifuge at 12000 g and 4℃ for 15 min, and discard the supernatant; slowly add 1 mL of ethanol (75% concentration, pre-cooled) to the precipitate, mix well, centrifuge at 12000 g and 4℃ for 5 min, and discard the supernatant.

[0059] (4) Dry the precipitate at room temperature for 2-5 min, add an appropriate amount of DEPC water to dissolve the precipitate, gently blow and wait for the RNA precipitate to completely dissolve, measure the concentration, dispense and store in a -80℃ refrigerator for later use.

[0060] 8. Quantitative and purity detection of RNA Take 1 μL of total RNA and measure the OD using a nucleic acid protein analyzer. 260 and OD 280 These two values ​​represent nucleic acids and proteins, respectively. Using OD... 260 / OD 280The R value reflects the degree of contamination from proteins and other organic matter in RNA. An R value between 1.8 and 2.2 indicates good RNA quality. The total RNA concentration is calculated using the following formula: RNA concentration (μg / mL) = OD0 260 ×40×Total volume / Original sample volume.

[0061] 9. RNA agarose gel electrophoresis Take 5 μL of RNA, add 6×DNA Loading Buffer, and mix well; use 5 µL of standard molecular weight marker as a control, electrophoresis at 180 V in a 1% agarose gel (containing chromogenic agent) for 30 min. Stop electrophoresis when the band moves to 1-2 cm from the front of the gel, remove the gel, and observe the electrophoresis results on a UV gel spectrometer.

[0062] If two clearly visible rRNA bands are observed with a concentration ratio of approximately 2:1, the RNA is not degraded; if severe tailing is observed, the RNA has been degraded. If bands longer than 28S are observed, the sample may be contaminated with genomic DNA.

[0063] 10. Real-time quantitative PCR (1) Primer design and synthesis: Primers were designed using Primer Express 5.0 (ABI) software and synthesized by Thermo Fisher Scientific. The amplification product length of each primer pair was 90-150 bp, as shown in Table 5.

[0064] Table 5 Amplification Primer Information (2) After reverse transcription of RNA into cDNA, real-time quantitative PCR was performed: each sample and marker was set up in three separate wells on a 96-well plate. The genes in the test samples were amplified by qPCR in a Bio-Rad CFX 96 Real-time PCR instrument using the SYBR Green dye method. The reaction system is shown in Table 6.

[0065] Table 6 Reaction System The reaction program was set as follows: 95℃ pre-denaturation for 1 min, 95℃ denaturation for 15 s, 60℃ annealing for 1 min, 72℃ extension for 30 s, and repeated 40 times.

[0066] II. Experimental Results 1. γ-[Glu] (n=1, 2, 3, 4) -Arg property prediction Preliminary predictions of cell penetration ability were made using online tools, with samples showing a predicted value above 0.5 indicating cell penetration capability. As shown in Table 7, the cell penetration ability of the samples, from highest to lowest, was: Arg > γ-Glu-Arg > γ-Glu-Glu-Arg > γ-Glu-Glu-Glu-Arg > γ-Glu-Glu-Glu-Glu-Arg > Gln, demonstrating a trend where shorter peptide chains correlated with stronger cell penetration ability. The hydrophilicity ranking of these samples was identical to the cell penetration ability ranking. Therefore, given the relatively strong cell penetration and solubility of γ-Glu-Arg and γ-Glu-Glu-Arg, these two short peptides will be selected for further in-depth research on their effects on the proliferation and differentiation of 3T3-L1 preadipocytes.

[0067] Table 7. Cell penetration ability and properties of different samples 2. Oil Red O staining results of cells from different samples after 8 days of induced differentiation like Figure 8 As shown, a large number of lipid droplets were formed in the model group after 8 days of induction. However, the number of lipid droplets was significantly reduced after adding Arg, γ-Glu-Arg and γ-Glu-Glu-Arg samples to induce differentiation. This indicates that all three samples can inhibit the formation of lipid droplets, with Arg and γ-Glu-Glu-Arg having more significant inhibitory effects.

[0068] Oil Red O, which specifically binds to lipid droplets, can be separated by isopropanol extraction, and thus quantified by measuring OD values. Therefore, changes in cell OD values ​​can reflect the effect of sample-induced differentiation on 3T3-L1 preadipocytes, as shown in the results. Figure 9As shown, the model group had the highest OD value of 2.19, while the OD values ​​of all other samples decreased significantly, indicating that they all had a certain inhibitory effect on the differentiation of 3T3-L1 preadipocytes. After 8 days of induction with low concentration (0.05 mg / mL), the OD values ​​of the Arg, γ-Glu-Arg, and γ-Glu-Glu-Arg treatment groups were equivalent to 48%, 44%, and 44% of the model group, respectively, indicating that all three samples significantly inhibited cell differentiation. However, there was no significant difference in the inhibitory effect among the three groups at this time (p>0.05). The high concentration (0.2 mg / mL) was generally more effective than the low concentration group. The OD values ​​of the Arg, γ-Glu-Arg, and γ-Glu-Glu-Arg treatment groups were equivalent to 39%, 42%, and 35% of the blank control, respectively. Therefore, the inhibitory effect from strongest to weakest was γ-Glu-Glu-Arg>Arg>γ-Glu-Arg. This shows that at the high concentration (0.2 mg / mL), γ-Glu-Glu-Arg had a more significant inhibitory effect on the differentiation of 3T3-L1 preadipocytes than γ-Glu-Arg.

[0069] 3. Changes in triglyceride content in cells of different samples after 8 days of induced differentiation Triglycerides are the main lipids in adipocytes. Their continuous accumulation leads to a sustained increase in adipocyte volume; reducing their accumulation can inhibit adipocyte differentiation. For example... Figure 10Compared to the model group, all treatment groups showed a significant decrease in triglyceride content (p<0.05), indicating that all treatments had a certain inhibitory effect on adipocyte differentiation. At low concentrations, the Arg treatment group had the highest triglyceride content, indicating the weakest inhibitory effect, while there was no significant difference in triglyceride content between γ-Glu-Arg and γ-Glu-Glu-Arg (p>0.05), suggesting that γ-Glu-Arg and γ-Glu-Glu-Arg exerted similar inhibitory effects at this concentration. The high-concentration treatment groups had even lower triglyceride content than the corresponding low-concentration treatment groups, indicating that the inhibitory effect of all samples on cell differentiation was concentration-dependent to some extent. At this point, the triglyceride content from highest to lowest was Arg group > γ-Glu-Arg group > γ-Glu-Glu-Arg group, further demonstrating that γ-Glu-Glu-Arg had a stronger inhibitory effect on 3T3-L1 preadipocyte differentiation than γ-Glu-Arg, consistent with the change pattern of OD values. It has been reported that the synthesis and accumulation of triglycerides are directly affected by peroxisome proliferator-activated receptor γ (PPAR-γ). At the same time, sterol regulatory element binding protein family genes (SREBPs) can also promote triglyceride synthesis through the regulation of acetyl-CoA decarboxylase, stearoyl-CoA desaturase, and fatty acid synthase (FAS). Therefore, it is speculated that the decrease in triglyceride content may be related to the decrease in gene expression of these regulatory factors.

[0070] 4. Changes in glycerol content in cells of different samples after 8 days of induced differentiation like Figure 11 Eight days after differentiation, the glycerol content in adipocytes of the model group was 52.89 μmol / L. However, after eight days of differentiation, cells treated with 0.05 mg / mL Arg, γ-Glu-Arg, and γ-Glu-Glu-Arg showed significantly increased glycerol content to 81.58 μmol / L, 66.06 μmol / L, and 76.96 μmol / L, respectively. When the treatment concentration increased to 0.2 mg / mL, the glycerol content changed to 73.73 μmol / L, 81.95 μmol / L, and 83.49 μmol / L, respectively, still significantly higher than the model group, but not significantly different from the corresponding low-concentration treatment groups (p>0.05). Glycerol is a hydrolysis product of lipid triglycerides; therefore, the increased glycerol content in the above results may indicate that the tested samples can accelerate the fat breakdown process. However, the degree to which the three samples promote fat breakdown may not be significantly different, and this process is not concentration-dependent. The hydrolysis of triglycerides is regulated by hormone-sensitive lipases (HSL) and adipocyte triglyceride hydrolase (ATGL), as well as multiple other pathways. Therefore, the promoting effect of Arg and its corresponding γ-glutamyl peptide on glycerol hydrolysis may be related to the above factors.

[0071] 5. Expression levels of differentiation-related genes PPAR-γ is essential for the induction of adipocyte differentiation in vitro, and studies have shown that its absence prevents adipocytes from further differentiating. C / EBPα is another key factor in the induction of adipocyte differentiation, accelerating the differentiation of terminal adipocytes and further promoting adipocyte generation through its synergistic effect with PPAR-γ. Except for the γ-Glu-Arg treatment group, the other two sample treatments had some impact on the gene expression of PPAR-γ and C / EBPα. γ-Glu-Glu-Arg treatment downregulated the mRNA expression of both PPAR-γ and C / EBPα genes simultaneously, while Arg treatment only downregulated the mRNA expression of PPAR-γ, with no significant effect on C / EBPα.

[0072] AMPK, or AMP-dependent protein kinase, primarily regulates cellular metabolism. When the intracellular ATP:AMP ratio decreases (i.e., energy is insufficient), AMPK is activated, leading to increased catabolism and decreased anabolism. FAS (fatty acid synthase) and HSL (hormone-sensitive lipase) are more directly related to adipocyte metabolism. FAS catalyzes the synthesis of long-chain fatty acids from malonyl-CoA and acetyl-CoA, making it a key enzyme in fat synthesis. Conversely, activation of HSL triggers the hydrolysis of triglycerides, promoting adipocyte breakdown. Figure 12 Only γ-Glu-Glu-Arg upregulated AMPK mRNA expression, and in this group, a decrease in FAS mRNA expression and an increase in HSL gene expression were simultaneously observed, indicating that γ-Glu-Glu-Arg can affect these three genes simultaneously, leading to the inhibition of triglyceride synthesis and enhanced degradation. Arg only downregulated FAS mRNA expression, without significantly affecting AMPK and HSL expression; γ-Glu-Arg had no significant effect on the gene expression of AMPK, FAS, and HSL. These results suggest that γ-Glu-Glu-Arg may exert a stronger inhibitory effect on the proliferation and differentiation of 3T3-L1 preadipocytes than γ-Glu-Arg and Arg by co-regulating multiple genes, including PPAR-γ, C / EBPα, AMPK, FAS, and HSL. This result corresponds to the fact that the OD value and triglyceride content of Oil Red O staining in the γ-Glu-Glu-Arg treatment group were the lowest, as mentioned earlier. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fish-derived bioactive peptide with dual functions of enhancing saltiness and inhibiting adipocyte differentiation, characterized in that, The active peptide is γ-glutamylarginine peptide, with the general formula γ-[Glu]n-Arg, where n is an integer from 1 to 4.

2. The fish-derived active peptide according to claim 1, characterized in that, n is 1 or 2.

3. A method for preparing fish-derived bioactive peptides as described in claim 1 or 2, characterized in that, Includes the following steps: Fish meat is used as raw material, and enzymatic hydrolysis is performed using protease to obtain protein hydrolysate; the protein hydrolysate is then subjected to a transpeptidation reaction under the action of glutaminase to generate the γ-glutamylarginine peptide.

4. The preparation method according to claim 3, characterized in that, The protease is a complex enzyme prepared by mixing chymotrypsin, trypsin and papain in an enzyme activity ratio of 1:1:

1.

5. The preparation method according to claim 4, characterized in that, The enzyme activity of the chymotrypsin, the trypsin, and the papain is 5000 U / g.

6. The preparation method according to claim 3, characterized in that, The enzymatic hydrolysis steps include: mixing fish meat and water at a ratio of 1g:2mL until homogenized, adding enzyme, and then hydrolyzing for 3 hours at pH 7.5 and a temperature of 55℃.

7. The preparation method according to claim 3, characterized in that, The conditions for the transpeptide reaction were: pH 10.0 and temperature 37°C.

8. The use of a fish-derived bioactive peptide as described in claim 1 or 2 in the preparation of a product that has both salty flavor enhancement function and helps control body fat.

9. A product that combines the functions of enhancing saltiness and helping to control body fat, characterized in that, The active ingredients include the fish-derived active peptides as described in claim 1 or 2.

10. The product according to claim 9, characterized in that, The products include food and health supplements.

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