Beef umami peptide, method for extracting the same, and related uses

Abstract

The application provides a beef salty peptide, an extraction method thereof and related uses. The amino acid sequence of the beef salty peptide is VKSD, and the beef salty peptide has high saltiness and salt-enhancing effect. The extraction method comprises the following steps: adding a beef sample into an ammonium bicarbonate aqueous solution containing a neutral protease, and hydrolyzing; performing first centrifugal treatment on a product obtained through the hydrolyzing, to obtain a first supernatant, and adding formic acid to perform protein precipitation; performing second centrifugal treatment on a product obtained through the protein precipitation, to obtain a second supernatant, and adding dichloromethane to perform extraction; performing liquid chromatography-mass spectrometry detection on a water phase obtained through the extraction, to obtain a plurality of polypeptides; screening a plurality of candidate polypeptides without toxicity, having water solubility, umami and saltiness from the plurality of polypeptides; performing stability analysis and saltiness analysis on the plurality of candidate polypeptides, to obtain the beef salty peptide. The beef salty peptide has good stability and use in preparation of a salty additive.

Description

Technical Field

[0001] This application relates to the field of polypeptide technology, and in particular to a beef savory peptide, its extraction method and related uses. Background Technology

[0002] Saltiness is an important taste characteristic, but high salt intake has seriously threatened human health, potentially leading to hypertension, cardiovascular disease, and other chronic illnesses. Exploring flavor compounds that can reduce salt consumption while maintaining the perception of saltiness has become a key research focus in the food industry. Summary of the Invention

[0003] In view of this, the purpose of this application is to provide a beef salty peptide, its extraction method and related uses.

[0004] For the purposes described above, this application provides a beef savory peptide, wherein the amino acid sequence of the beef savory peptide is VKSD.

[0005] In some embodiments, the amino acids of the beef savory peptide are all L-type.

[0006] In some embodiments, the beef saponin is obtained by extraction from beef.

[0007] This application also provides a method for extracting beef savory peptides, the extraction method comprising:

[0008] The beef sample was added to an aqueous solution of ammonium bicarbonate containing neutral protease for hydrolysis. The hydrolysis product was subjected to a first centrifugation process to obtain a first supernatant, and formic acid was added to precipitate the protein. The product obtained after protein precipitation was subjected to a second centrifugation to obtain a second supernatant, which was then extracted with dichloromethane. The aqueous phase obtained from the extraction was analyzed by liquid chromatography-mass spectrometry to obtain multiple peptides; The candidate peptides were screened from the multiple peptides to select those that were non-toxic, water-soluble, umami-tasting, and salty. Stability and saltiness analyses were performed on the multiple candidate peptides to obtain the beef salty peptide.

[0009] In some embodiments, the mass ratio of ammonium bicarbonate to the beef sample is 1:(31-32).

[0010] In some embodiments, the stability analysis includes molecularly docking the candidate peptide with the TMC4 receptor to determine binding affinity.

[0011] This application also provides the use of beef salty peptides as described in any of the preceding embodiments, or beef salty peptides extracted by any of the preceding embodiments, in the preparation of salty additives.

[0012] In some embodiments, the use in the preparation of savory additives includes use in the preparation of savory additives for food, or use in the preparation of savory additives for animal feed.

[0013] This application also provides a salty additive, including beef salty peptides as described in any of the previous embodiments or beef salty peptides extracted by the extraction method described in any of the previous embodiments.

[0014] This application also provides a feed additive, including beef savory peptides as described in any of the preceding embodiments or beef savory peptides extracted by the extraction method described in any of the preceding embodiments.

[0015] The beef-flavored peptides provided in this application have a high saltiness and salt-enhancing effect. They exhibit good stability, are non-toxic, and have good water solubility. They also possess advantages such as small molecular weight (composed of 4 amino acids), readily available raw materials, and simple extraction. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in this application or related technologies, the drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a technical roadmap for the discovery and preparation of the beef savory peptide of this application.

[0018] Figure 2 This is a flowchart of the method for extracting beef salty peptides according to this application.

[0019] Figure 3 This is a schematic diagram illustrating the frequency analysis of the C-terminal amino acid residues of the flavor peptide in this application.

[0020] Figure 4A The results show the identification of 2 to 4 peptides at different collision energies for the collision-induced dissociation mode of this application.

[0021] Figure 4B The results of identifying 2 to 4 peptides under different collision energies for the two fragmentation modes of high-energy collision dissociation in this application.

[0022] Figure 5A The results show the comparison of the number of 2 to 4 peptides identified in different extraction solution treatment groups of this application.

[0023] Figure 5B This is a comparison of the number of 2 to 4 peptides identified before and after ultrafiltration membrane treatment in this application.

[0024] Figure 5C The results show the comparative identification of 2 to 4 peptides of different types of acid-precipitated proteins in this application.

[0025] Figure 5D The results show the comparison of the identification numbers of 2 to 4 peptides treated with different solid phase extraction columns in this application.

[0026] Figure 5E The results show a comparison of the identification quantities of 2 to 4 peptides processed by different extraction methods in this application.

[0027] Figure 6A This is the compound matching score in the stability evaluation of the detection method in this application.

[0028] Figure 6B This represents the relative standard deviation of the retention times of 20 short peptides in the stability evaluation of the detection method of this application.

[0029] Figure 6C This represents the relative standard deviation of the signal responses of 20 short peptides in the stability evaluation of the detection method of this application.

[0030] Figure 7 This is a schematic diagram of the interaction between the TMC4 receptor and the beef savory peptide V2D molecule of this application.

[0031] Figure 8 The experimental results are for the electronic tongue analysis of the six peptides in this application.

[0032] Figure 9 The results of the saltiness test for the three peptides in this application are shown.

[0033] Figure 10 The results are from the stability test of the beef salty peptide V2D of this application. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings.

[0035] High-salt diets are a significant risk factor for diseases such as hypertension, stroke, and stomach cancer. Currently, efforts are being made to actively promote salt reduction, advocate for healthy eating, and lower residents' salt intake.

[0036] With rising living standards and health awareness, consumers are increasingly pursuing healthy foods and paying attention to their red meat intake. Beef, due to its high protein and low fat content, is gradually becoming a preferred choice for meat consumption. Beef contains abundant flavor compounds, including water-soluble nitrogenous compounds such as amino acids, nucleotides, and peptides, which are important sources of beef's umami flavor; fat-soluble substances such as saturated fatty acids, unsaturated fatty acids, and cholesterol, which decompose into aldehydes and ketones when heated, producing a distinctive meaty aroma and enriching the taste; and volatile aroma components, mainly including aldehydes (such as nonanal), sulfur-containing compounds (such as methanethiol), and heterocyclic compounds (such as pyrazines). Although these substances are present in low amounts, they are diverse and mainly generated through Maillard reactions and fat oxidation, which are key to the rich aroma produced when beef is heated.

[0037] Therefore, preparing savory peptides from beef protein is of great significance for reducing salt intake without reducing saltiness, for promoting healthy eating, and for solving the problem of excessive salt intake.

[0038] Firstly, this application provides a beef savory peptide. The beef savory peptide can be beef savory peptide V2D. The amino acid sequence of the beef savory peptide is shown in SEQ ID NO:1, which is VKSD, i.e., Val-Lys-Ser-Asp. All amino acids in the beef savory peptide are L-type. The molecular weight of the beef savory peptide is 447.48 Da, and the isoelectric point is 6.6. It should be understood that the beef savory peptide of this application can be extracted from beef or obtained through artificial synthesis. This application does not limit the specific source of the beef savory peptide.

[0039] The beef salty peptide provided in this application has a saltiness score of 6.14 at a concentration of 0.5 mg / mL, as measured by an electronic tongue, indicating a high saltiness level. Furthermore, this beef salty peptide exhibits a binding affinity of -5.425 kcal / mol to the TMC4 salty receptor, with both the lower and upper limits of the root mean square deviation being 0, demonstrating good stability. It is non-toxic and possesses good water solubility. It also boasts advantages such as small molecular weight (composed of 4 amino acids), readily available raw materials, and simple extraction.

[0040] The beef savory peptide provided in this application exhibits excellent resistance to enzymatic hydrolysis and good stability against pepsin. Therefore, it can avoid degradation by enzymes in savory additives or food products during preparation. Consequently, the beef savory peptide provided in this application can stably exist in environments with abundant enzymes, maintaining its savory properties.

[0041] The beef-based salty peptide provided in this application can be used to prepare salty additives, including saltiness agents or saltiness enhancers, and has good application prospects. It is of great significance for reducing salt content without reducing saltiness.

[0042] Secondly, embodiments of this application also provide a method for extracting beef savory peptides. For example... Figure 1 and Figure 2 As shown, the extraction method may include: In step S100, the beef sample is added to an ammonium bicarbonate aqueous solution containing a neutral protease for hydrolysis. The mass ratio of the neutral protease to the beef sample can be (1:20)-(1:50), for example, 1:(29-31). The mass ratio of the ammonium bicarbonate to the beef sample is 1:(31-32). The concentration of the ammonium bicarbonate aqueous solution can be 49-51 mmol / L. Using an ammonium bicarbonate aqueous solution, compared to water and acetonitrile aqueous solutions, allows for better dissolution of short peptides from the muscle matrix.

[0043] In some embodiments, the tissue can be homogenized before hydrolysis. Hydrolysis conditions can be 20-40°C for 2-8 hours.

[0044] In step S200, the hydrolysis product is subjected to a first centrifugation treatment to obtain a first supernatant, and formic acid is added to precipitate the protein. The final concentration of formic acid can be 1.8-2.2% (υ:υ). Formic acid is chosen as the protein precipitant because, compared with trichloroacetic acid (TCA) and trifluoroacetic acid (TFA), it effectively precipitates proteins while minimizing the loss of short peptides.

[0045] In step S300, the product obtained after protein precipitation is subjected to a second centrifugation to obtain a second supernatant, which is then extracted with dichloromethane. The concentration of dichloromethane during extraction can be water:dichloromethane = 1:1 (v:v). Using an aqueous dichloromethane solution for extraction, compared to a methanol:water:dichloromethane system, can effectively remove non-polar interfering substances such as fats without losing short peptides, significantly reducing the background interference of the high-fat beef matrix on the mass spectrometry signal.

[0046] In step S400, the aqueous phase obtained from the extraction is subjected to liquid chromatography-mass spectrometry to obtain multiple peptides.

[0047] Step S500: Screening multiple candidate peptides from the plurality of peptides for non-toxicity, water solubility, umami flavor, and saltiness. Toxicity, water solubility, umami flavor, and saltiness can be predicted using online tools. Toxicity can be predicted using online tools, such as Virtual Scanning of Toxic Peptides using ToxinPred, to screen for non-toxic peptides. Water solubility can be predicted using online tools, such as Proteins tools, to screen for moderately or highly water-soluble peptides. Umami flavor can be predicted using online tools, such as UMPred-FRL and Umami YYDS respectively, to screen for peptides that show umami flavor in both tests (e.g., scores not lower than 0.95). Saltiness can be predicted using online tools, such as Umami YYDS, to screen for peptides with a saltiness score not lower than 0.5.

[0048] Step S600 involves performing stability and saltiness analysis on the multiple candidate peptides to obtain the beef salty peptide. The stability analysis is performed by molecularly docking the candidate peptides with the TMC4 receptor and determining the binding affinity and the lower and upper limits of the root mean square deviation. After stability analysis, candidate peptides with a binding affinity less than -5 kcal / mol are selected for saltiness analysis, which can be performed using an electronic tongue. The peptide with the highest saltiness value is selected, thus obtaining the beef salty peptide V2D.

[0049] Thirdly, the embodiments of this application also provide the use of the beef salty peptide described in any of the above technical solutions or the beef salty peptide prepared by the preparation method described in the above technical solutions in the preparation of salty additives.

[0050] In some embodiments, the use in the preparation of savory additives may include the use in the preparation of savory additives for food. The savory additive may function as a saltiness agent or a saltiness enhancer.

[0051] In some embodiments, the use in the preparation of salty additives may include the use in the preparation of salty additives for animal feed. The salty additive may function as a saltiness agent or a saltiness enhancer.

[0052] Fourthly, this application also provides a salty flavoring additive, including beef salty peptides as described in any of the preceding embodiments or beef salty peptides extracted by the extraction method described in any of the preceding embodiments. The salty flavoring additive can be used as a saltiness agent or a saltiness enhancer.

[0053] In some embodiments, the feed additive may also include food-grade excipients, etc.

[0054] In some embodiments, the savory additive may be a powder, granules, or liquid formulation.

[0055] It should be understood that the salty additives in the embodiments of this application include the beef salty peptides as described in any of the previous embodiments, and have the effects of the beef salty peptide embodiments as described above, which will not be repeated here.

[0056] Fifthly, embodiments of this application also provide a feed additive, including beef salicylic peptides as described in any of the preceding embodiments or beef salicylic peptides extracted by the extraction method described in any of the preceding embodiments.

[0057] In some embodiments, the feed additive may also include feed-grade acceptable adjuvants, etc.

[0058] In some embodiments, the feed additive may be a powder, granule, or liquid formulation, etc.

[0059] It should be understood that the feed additives in the embodiments of this application include the beef salty peptides as described in any of the previous embodiments, and have the effects of the beef salty peptides as described in the previous embodiments, which will not be repeated here.

[0060] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0061] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0062] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0063] Example 1: Optimization of Short Peptide Identification Method Objective: To improve the efficiency of identifying endogenous peptides in beef.

[0064] Experimental methods: 1.1 Main Reagents and Instruments Main reagents: Acetonitrile and methanol (chromatographic grade) were purchased from Thermo Fisher Scientific, Inc., USA; formic acid (purity >98%) was purchased from Aladdin Biochemical Technology Co., Ltd., China; ultrapure water was purchased from Watsons; all reagents were stored under specified conditions, and samples were stored at -20℃.

[0065] Main instruments: Analytical balance (JA3003, Shanghai Lichen Instrument Technology Co., Ltd.); Vortex mixer (VORTEX-5, Qilinbell Instrument Manufacturing Co., Ltd.); Small high-speed refrigerated centrifuge (5810R, Eppendorf GmbH, Germany); Ultrasonic cleaner (KQ-500DE, Beijing Victory Technology Co., Ltd.); Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, San Jose, CA, USA); Discovery HS F5-3 column (15 cm × 2.1 mm, 3 μm, Sigma-Aldrich).

[0066] 1.2 One-pot synthesis of short peptide standards A mixture of short peptide standards was prepared using a Fmoc solid-phase synthesis combined with a one-pot method. The synthesis processes for dipeptides, tripeptides, and tetrapeptides were as follows: 1.2.1 Dipeptide Standards Ten C-terminal amino acids (A, D, E, G, K, L, P, R, S, V) were pre-coupled to Wang resins, with each type of resin synthesized independently in a one-pot process. After removing the Fmoc protecting groups from the C-terminal amino acids, a mixture of 19 amino acids (excluding Cys, in equimolar proportions) was added to the reaction system for coupling, yielding a C-terminal fixed dipeptide mixture (sequence XC, C-terminus fixed, where X represents one of the 19 amino acids).

[0067] 1.2.2 Tripeptide Standards Nineteen C-terminal amino acids (excluding Cys) were loaded onto Wang resin as C-terminal immobilized amino acids. After two consecutive one-pot coupling processes, a tripeptide mixture (sequence XXC, C-terminus immobilized, where X represents one of the 19 amino acids) was obtained.

[0068] 1.2.3 Tetrapeptide Standard Ten C-terminal amino acids identical to those in the dipeptide were loaded onto Wang resin as C-terminal immobilized amino acids. After three consecutive one-pot couplings, each using a mixture of 19 amino acids, a tetrapeptide mixture was obtained (sequence XXXC, C-terminus immobilized, where X represents one of the 19 amino acids).

[0069] After the above peptides were coupled, the peptide chains were cleaved from the resin using trifluoroacetic acid (TFA), precipitated with diethyl ether, and freeze-dried to obtain mixed short peptide powder. After the above synthesis was completed, a total of 39 groups of mixed peptides were obtained, theoretically including 190 dipeptides, 6,859 tripeptides, and 68,590 tetrapeptides.

[0070] 1.3 Optimization of Detection Conditions for Liquid Chromatography-Tandem Mass Spectrometry Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis was performed using an Orbitrap Fusion Tribrid mass spectrometer (ThermoScientific, San Jose, CA) equipped with a nanospray ionization source. Addressing the challenges of short peptides' high polarity, short retention time on traditional C18 columns, and severe co-elution, the separation performance of three columns—Agilent Eclipse Plus C8 (2.1 × 100 mm, 1.8 μm), Waters ACQUITY Premier CSH C18 (2.1 × 100 mm, 1.7 μm), and Discovery HS F5-3 (15 cm × 2.1 mm, 3 μm)—for short peptide standard mixtures was systematically compared. The optimal column type was determined using the uniformity of compound distribution, ion abundance, and signal response intensity in the total ion chromatogram (TIC).

[0071] After determining the column, analysis was performed using a Discovery HS F5-3 column (15 cm × 2.1 mm, 3 μm) with an injection volume of 2 μL and a flow rate of 300 μL / min. The mobile phase A was an aqueous solution containing 0.1% formic acid, and phase B was an acetonitrile solution containing 0.1% formic acid. The gradient elution program was as follows: 0–2 min, 2% phase B; 2–15 min, 2%–40% phase B; 15–20 min, 40%–100% phase B; 20–22 min, 100% phase B; 22–22.1 min, 100%–2% phase B; 22.1–25 min, 2% phase B. Mass spectrometry was performed in electrospray ionization (ESI) positive ion mode with an Orbitrap resolution of 120,000, and the mass axis measurement error was controlled within 5 ppm.

[0072] Optimization of fragmentation methods: The system compared two fragmentation modes: collision-induced dissociation (CID) and high-energy collisional dissociation (HCD). Using the same batch of short peptide standards, the number of peptides identified under both modes was statistically analyzed, and the optimal fragmentation mode was determined by combining the types and abundance of fragment ions. To balance fragment ion coverage and abundance, the mixed collision energy acquisition function of the Orbitrap Fusion mass spectrometer was used to overlay secondary fragment ion spectra at collision energies of 15, 30, and 45 eV, obtaining more comprehensive fragment information while significantly improving spectral acquisition efficiency.

[0073] 1.4 Data Processing and Statistical Analysis Excel 2019 was used for statistical analysis; GraphPad Prism 9.5 was used for graphing; PEAKS Studio was used for mass spectrometry data processing and de novo sequencing analysis; mzConvert was used for raw data format conversion; mzVault was used for database format conversion; and Compound Discoverer 3.3 was used for searching a new short peptide database and identifying short peptides. Identification results were screened with a matching score ≥60 as the confidence threshold, retention time matching tolerance was set to ±0.2 min, and mass error tolerance was set to ±5 ppm.

[0074] Experimental results: such as Figures 3 to 4B As shown. Among them, Figure 3 Frequency analysis of C-terminal amino acid residues of flavor peptides. Figures 4A to 4B The results of identifying 2-4 peptides under different collision energies for two fragmentation modes: collision-induced dissociation and high-energy collision dissociation. Figure 4A The results of identifying 2 to 4 peptides under three collision energy gradients (CID-15, CID-30, and CID-45) are shown for collision-induced dissociation (CID). Figure 4B High-energy collision dissociation (HCD) results were obtained for the identification of 2 to 4 peptides at three collision energy gradients: HCD-15, HCD-30, and HCD-45.

[0075] Results analysis: 1.5 Determination of flavor amino acids In metabolomics analysis, establishing a metabolite mass spectrometry database using standards can improve the accuracy and efficiency of metabolite identification. Therefore, drawing on the workflow for establishing metabolite mass spectrometry databases, a short peptide mass spectrometry database was established using peptide standards. Theoretically, establishing a database of short peptides composed of 2-4 amino acids requires obtaining 168,400 peptides, which is currently unavailable for all peptide standards. To reduce the scale of short peptide synthesis, the focus was on establishing a flavor peptide database, considering that the C-terminal amino acid is a key amino acid for activating taste receptors and influencing the formation and intensity of peptide flavor. 978 flavor peptide sequences were obtained from the TastePeptidesDB flavor database (as of April 2024), and the frequency of C-terminal amino acids in these flavor peptides was analyzed. The results showed that the 10 most frequently occurring amino acids at the C-terminus of flavor peptides were, in descending order, A, D, E, G, K, L, P, R, S, and V. This result provides data support for the subsequent one-pot synthesis design of short peptides. Using the high-frequency amino acids at the C-terminus of flavor peptides as the C-terminal amino acids of mixed peptides, a total of 39 sets of mixed dipeptides, tripeptides and tetrapeptides with clearly defined C-terminal amino acids were obtained using a one-pot method.

[0076] 1.6 Determination of Detection Conditions for Liquid Chromatography-Mass Spectrometry To achieve effective separation and detection of short peptides, three chromatographic columns were first systematically compared: Agilent EclipsePlus C8 (2.1 × 100 mm, 1.8 μm), Waters ACQUITY Premier CSH C18 (2.1 × 100 mm, 1.7 μm), and Discovery HS F5-3 (15 cm × 2.1 mm, 3 μm), to examine their separation performance for mixtures of short peptide standards. Results from the C18 and C8 columns showed that a large number of short peptides co-eluted within the first 0–5 minutes of elution, with significant peak overlap and uneven ion abundance distribution, resulting in many peptides failing to obtain effective secondary mass spectrometry information. The F5 column, however, performed significantly differently: the compounds were evenly distributed throughout the 22-minute detection window, with significantly higher signal response intensity and ion abundance than the other two, and the peak shape was also more symmetrical. This is thanks to the pentafluorophenylpropyl (PFP) functional group on the F5 stationary phase, which can generate dipole-dipole interactions and π-π stacking interactions with polar short peptides, thereby prolonging the retention time of polar peptides and achieving better separation. Therefore, the Discovery HS F5-3 column was ultimately selected for subsequent experiments.

[0077] To compare the identification effects of collision-induced dissociation (CID) and high-energy collisional dissociation (HCD) on mixtures of short peptide standards, the same batch of samples was analyzed using both CID and HCD methods. The results are as follows: Figure 4A and Figure 4B As shown, the HCD mode significantly outperforms CID in terms of the number of identified peptides. This is mainly because HCD utilizes higher collision energies to fragment peptide ions more thoroughly, generating not only more b / y ions but also imine ions, side-chain feature fragments, and other small molecule ions, providing more evidence for short peptide sequence deduction. In contrast, CID, due to its relatively mild energy transfer and low-mass truncation, fails to effectively detect some feature fragments. Therefore, HCD was chosen as the fragmentation mode for subsequent experiments.

[0078] In mass spectrometry fragmentation, the collision energy directly affects the type and abundance of fragment ions. At too low an energy, the parent ion hardly fragments, making sequence information unavailable; at too high an energy, fragmentation may occur excessively, resulting in the loss of crucial b / y ions. Therefore, optimizing the appropriate collision energy is a key step in improving peptide identification efficiency. To better compare fragment information under different fragmentation modes and energy conditions, using the tetrapeptide SLYE (Ser-Leu-Tyr-Glu) as a model peptide, HCD-15, HCD-30, HCD-45, and a mixed spectrum combining the three HCD energies (15, 30, and 45) were collected. The results show that HCD-15 yields a more complete b / y ion series, aiding in the confirmation of the peptide's sequence backbone; HCD-30 produces more low-mass small molecule feature fragments (such as imine ions and side chain fragments), providing additional evidence for amino acid composition. The fragment information from both methods is highly complementary. However, with HCD-45, the fragments are too small, and the signal of intact b / y ions is significantly weakened, making it less than ideal for use alone.

[0079] To fully utilize the complementary information from fragments at different collision energies, subsequent experiments will involve real-time overlay of secondary fragment spectra at energies of 15, 30, and 45 energies. This not only provides more comprehensive fragment information (covering both the sequence backbone and characteristic small fragments) but also improves spectral acquisition efficiency by approximately 50%, as a single injection can effectively complete multiple data acquisitions at different energies. Especially for isomers with very similar retention times, the rich diversity of fragment ions in the mixed collision energy spectrum provides ample evidence for distinguishing and resolving sequences, significantly improving the accuracy and reliability of short peptide sequence identification.

[0080] Example 2: Optimization of the pretreatment method for extracting short peptides from beef Experimental objective: To systematically optimize the pretreatment method for short peptide extraction.

[0081] Experimental methods: Basic extraction procedure: Weigh an appropriate amount of sample, add neutral protease and extraction solution, homogenize at 60 Hz for 2 min, centrifuge at 14,000 rpm for 3 min, and collect the supernatant. Add acid (2% v / v) to precipitate the protein, and centrifuge at 14,000 rpm for 5 min. Adjust the pH of the supernatant to 3-4 with ammonia. Determine whether to use a 10 kDa ultrafiltration membrane based on the optimization results. Select the purification method based on the optimization results: purification methods include solid-phase extraction purification optimization and liquid-liquid extraction optimization. After purification, freeze-dry the sample under vacuum. Reconstitute with 0.5 mL of an aqueous solution containing 0.1% formic acid / 2% acetonitrile. Centrifuge at 14,000 rpm for 10 min and transfer to a vial for analysis.

[0082] The optimized experimental design is as follows, with the total number of identified 2-4 peptides used as the evaluation index for each optimization step: (1) Optimization of extraction solution: Three extraction solutions were compared: water, 50% acetonitrile aqueous solution, and 50 mM ammonium bicarbonate aqueous solution.

[0083] (2) Optimization of protein precipitation acid: Compare the precipitation effects of three acids: formic acid, trichloroacetic acid (TCA), and trifluoroacetic acid (TFA).

[0084] (3) Ultrafiltration membrane treatment optimization: Compare the changes in the number of short peptides identified before and after passing through a 10 kDa ultrafiltration membrane.

[0085] (4) Solid phase extraction purification optimization: compare the purification effects of three solid phase extraction columns: Waters MCX column, TUP HLB column and custom column.

[0086] (5) Liquid-liquid extraction optimization: The number of short peptides identified by comparing two extraction systems, water:dichloromethane (1:1, υ:υ) and methanol:water:dichloromethane (4:4:3, υ:υ:υ), with the unextracted control.

[0087] Experimental results: such as Figures 5A to 5E As shown. Among them, Figure 5A Results for different extraction solution treatment groups. Figure 5B This is a comparison of the results before and after ultrafiltration membrane treatment. Figure 5C Comparison results of different types of acid-precipitated proteins. Figure 5D Comparison results of different solid phase extraction column treatments. Figure 5E Comparison results for different extraction methods.

[0088] Results Analysis: Regarding the optimization of the extraction solution, the extraction effects of water, 50% acetonitrile aqueous solution, and 50 mM ammonium bicarbonate aqueous solution were compared. Figure 5A It was found that the number of dipeptides and tripeptides extracted by 50 mM ammonium bicarbonate aqueous solution was significantly higher than that extracted by the other two solvents. This indicates that the weakly alkaline buffer facilitates the dissolution of short peptides from the muscle matrix, which may be related to the ionic strength effect of ammonium bicarbonate and its disruption of protein-peptide interactions. Therefore, 50 mM ammonium bicarbonate aqueous solution was selected as the extraction solution.

[0089] Ultrafiltration is often used as an important method for preparing peptides while eliminating protein interference. Therefore, in optimizing the selection of ultrafiltration membranes, the effects of treatment with a 10 kDa ultrafiltration membrane and direct treatment without a membrane were compared. Figure 5B It was observed that the number of dipeptides increased after passing through a 10 kDa ultrafiltration membrane, but the number of tripeptides and tetrapeptides decreased significantly. This indicates that 10 kDa ultrafiltration membrane treatment causes non-specific adsorption loss of some short peptides, with the net loss exceeding the net gain. Therefore, ultrafiltration membranes were not used in subsequent sample processing; instead, protein precipitation was employed to remove large protein molecules.

[0090] In the selection of protein precipitants, the precipitation effects of formic acid, trichloroacetic acid (TCA), and trifluoroacetic acid (TFA) were compared. Figure 5C As shown, the results indicated that the number of tripeptides and tetrapeptides identified after formic acid precipitation was significantly higher than that in the TCA and TFA treatment groups. This suggests that formic acid effectively precipitates proteins while minimizing the loss of short peptides, possibly due to its weak acidity (pKa 3.75) having a relatively small impact on the structure of short peptides. Therefore, formic acid (final concentration 2%, υ:υ) was selected as the protein precipitant.

[0091] To remove fat from the samples, Waters MCX columns, TUP HLB columns, and custom-made lipid adsorption columns were compared. Figure 5D As shown in the results, all three solid-phase extraction columns resulted in a significant reduction in the number of tripeptides and tetrapeptides identified. Therefore, subsequent sample processing did not employ solid-phase adsorption to remove lipids, but instead used liquid-liquid extraction.

[0092] In optimizing the liquid-liquid extraction reagents, two extraction systems were compared: water:dichloromethane (1:1, υ:υ) and methanol:water:dichloromethane (4:4:3, υ:υ:υ), with the unextracted sample serving as a control. As shown in Figure 5E, the results indicate that water:dichloromethane (1:1) extraction can effectively remove nonpolar interfering substances such as fats without losing short peptides, significantly reducing the background interference of the high-fat matrix of beef on the mass spectrometry signal. Therefore, water:dichloromethane (1:1, υ:υ) liquid-liquid extraction was selected as the purification step.

[0093] Therefore, the optimal pretreatment method for extracting short peptides from beef was determined to be: extraction with 50 mM ammonium bicarbonate aqueous solution, protein precipitation with formic acid (final concentration 2%, υ:υ), liquid-liquid extraction with water:dichloromethane (1:1, υ:υ), vacuum freeze-drying, reconstitution with 0.1% formic acid / 2% acetonitrile aqueous solution, centrifugation, and then injection for detection. This method effectively reduces background interference from the complex high-fat matrix of beef on the mass spectrometry signal while ensuring the extraction efficiency of short peptides, providing a reliable sample preparation method for the high-throughput identification of endogenous peptides from three beef varieties.

[0094] Example 3: Stability Evaluation of the Detection Method Experimental Objective: To test the stability of the method obtained through screening in Examples 1 and 2 of this application. To verify the reliability of the established beef polypeptide identification process, to investigate the impact of large-scale sample detection on mass spectrometry stability and database identification stability, and to evaluate the stability of the detection method.

[0095] Experimental Methods: The stability of the detection method was evaluated by continuously injecting the same beef sample (with 20 short peptide standards synthesized separately with known sequences) 50 times (the instrument ran continuously for about 24 hours). The results of the 1st, 10th, 20th, 30th, 40th and 50th injections were selected for analysis. The evaluation indicators included: (1) the distribution of compound matching score (with a score ≥60 as the confidence threshold); (2) the relative standard deviation (RSD) of the retention time of the 20 standards; and (3) the RSD of the signal response value of the above 20 standards. The retention times of the 20 standards were evenly distributed within the detection window of 0~15 min, covering short peptides with different polarities and molecular weight ranges, ensuring the representativeness of the stability evaluation.

[0096] Experimental results: such as Figures 6A to 6C As shown. Among them, Figure 6A The compound matching score is given. Figure 6B The relative standard deviation of retention times for 20 short peptides. Figure 6C The values ​​represent the relative standard deviations of the signal responses of 20 short peptides. RSD stands for Relative Standard Deviation.

[0097] Results analysis: During continuous detection, most compounds scored above 60. Figure 6A Regarding chromatographic retention time stability ( Figure 6B The RSD of the polar retention time of 20 known short peptide sequences was analyzed. The results showed that the RSD of 16 peptides was <2%, and the RSD of 4 peptides was between 2% and 5%, all meeting the routine quality control requirements for the stability of liquid chromatography methods (RSD < 5%), indicating that the chromatographic system maintained good stability during continuous detection. Regarding signal response stability ( Figure 6C The RSD results of the signal response values ​​of the above 20 standards showed that the RSD of 2 peptides was <10%, the RSD of 12 peptides was between 10% and 20%, and the RSD of 6 peptides was between 20% and 30%. The coefficient of variation of the signal response was slightly higher than that of the retention time. This is related to the fact that the mass spectrometry signal is easily affected by matrix effects and fluctuations in ionization efficiency, which is within the normal range of the LC-MS / MS method and does not affect the reliability of qualitative identification. In summary, the detection method established in this application has excellent retention time stability and acceptable signal response repeatability under continuous 24-hour operation conditions, and can meet the practical needs of short peptide identification in large batches of beef samples.

[0098] Example 4: Extraction of salty peptides from beef Experimental methods: such as Figure 1 As shown.

[0099] Extraction of endogenous peptides from beef: (1) Weigh 0.5 g of beef, add 15 g of neutral protease and 4 mL of 50 mM ammonium bicarbonate aqueous solution; (2) Add 1 3 mm steel ball and homogenize at 60 Hz for 2 min; (3) Centrifuge at 14,000 rpm for 3 min; (4) Add 2% formic acid (υ:υ) for protein precipitation; (5) Centrifuge at 14,000 rpm for 5 min; (6) Adjust the pH of the supernatant to 3-4 using ammonia water; (7) Centrifuge at 14,000 rpm for 5 min; (8) Perform liquid-liquid extraction of the supernatant with dichloromethane at a ratio of 1:1 (υ:υ) and collect the aqueous phase; (9) Perform vacuum freeze-drying of the aqueous phase; (10) Redissolve in 30 μL of 0.1% formic acid / 2% acetonitrile aqueous solution; (11) Centrifuge at 14,000 rpm for 10 min and transfer to a vial for testing.

[0100] Mass spectrometry identification of endogenous peptides in beef: Nanoscale LC-MS / MS analysis was performed using an Orbitrap Fusion Tribrid mass spectrometer equipped with a nanospray flexible ion source. 2 μL of sample was injected into a Discovery HS F5-3 column (15 cm × 2.1 mm, 3 μm) for separation. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Gradient elution was used: 0–2 min, 2% B phase; 2–15 min, 2%–40% B phase; 15–20 min, 40%–100% B phase; 20–22 min, 100% B phase; 22–22.1 min, 100%–2% B phase; 22.1–25 min, 2% B phase. Mass spectrometry acquisition was performed in DDA mode. The first-stage mass spectrometry had a resolution of 120,000 and a scan range of 100–1,500 m / z. The second-stage mass spectrometry used HCD fragmentation with mixed collision energies (15, 30, and 45 eV superimposed) and a resolution of 30,000. Data analysis employed a dual-track strategy: short peptides (2–4 amino acids) were matched with the short peptide mass spectrometry database (27,235 short peptides) constructed in Chapter 2 using CompoundDiscoverer 3.3 software, with identification thresholds set at retention time deviation <0.2 min, mass deviation <5 ppm, and a matching score of at least 60. Long peptides (5 amino acids or more) were matched with UniProt using PEAKS Studio software. Bos taurus Database comparison, setting FDR < 1%, using -|log10( p )|≥20 is the significance threshold.

[0101] Virtual screening of candidate flavor peptides: Using 1800 identified peptide sequences as the research object, a multi-model cross-prediction strategy was adopted to conduct virtual screening of flavor activities: (1) Bitterness prediction: The BERT4Bitter online tool was used, with a bitterness score of not less than 0.5 as the positive threshold; (2) Saltiness prediction: The SaltyPep prediction model was used, with a saltiness score of not less than 0.5 as the positive threshold; (3) Umami prediction: Two independent models, Umami_YYDS and UMPred-FRL, were used, with a double positive threshold of umami scores of not less than 0.95 for both models, to screen candidate umami peptides; (4) Toxicity screening: The ToxinPred 3.0 tool was used to exclude toxic positive peptides and retain non-toxic peptides; (5) Water solubility screening: The PeptideAnalyzer tool was used to retain moderately water-soluble or highly water-soluble peptides; (6) Novelty verification: The peptides were compared with the TastePeptidesDB database to exclude verified peptides and obtain 213 candidate peptides. The results of the physicochemical properties and toxicity analysis of beef savory peptide V2D are shown in Table 1. The results of umami prediction of beef savory peptide V2D using the online tools Umami_YYDS and UM Pred-FRL are shown in Table 2.

[0102]

[0103]

[0104] Molecular docking of candidate peptides with umami and saltiness receptors and synthesis of target peptides: 213 candidate peptides were ranked according to their umami prediction scores. The top 50 were molecularly docked with the 1EWK receptor using AutoDock Vina software. Binding affinity (kcal / mol) and RMSD were used as evaluation indicators. The 20 peptides with the lowest binding affinity were selected for further analysis. Saltiness receptor molecular docking: 133 peptides with 9 or fewer amino acid residues from the 213 candidate peptides were molecularly docked with the TMC4 receptor using AutoDock Vina software. The docking box was centered near the Arg437 key residue. Binding affinity (kcal / mol) and RMSD were used as evaluation indicators. The 20 peptides with the lowest binding affinity were selected for further analysis.

[0105] The TMC4 receptor model from the Uniport database was used, and the V2D structure was drawn using ChemDraw software. Before molecular docking, water molecules were removed from the TMC4 receptor and hydrogenation was performed. The V2D small molecule underwent hydrogenation and torsion bond setting. After the operations, both the receptor and the small molecule were saved in .pdbqt format. AutoDockTools software was used to perform molecular docking between the TMC4 receptor and the beef savory peptide V2D. The results of the TMC4 receptor-beef savory peptide V2D molecular docking are shown in Table 3. The interaction generated by the TMC4 receptor-beef savory peptide V2D molecular docking is as follows: Figure 7 As shown.

[0106] Table 3. Results of molecular docking between TMC4 receptor and beef savory peptide V2D

[0107] Table 3 shows the molecular docking results, indicating that the optimal conformational binding affinity between the TMC4 receptor and beef savory peptide V2D is -5.425 kcal / mol, with both the lower and upper limits of the root mean square deviation being 0. This suggests a tight binding and high spatial overlap between the TMC4 receptor and beef savory peptide V2D. The interaction forces generated during molecular docking are as follows: Figure 7 As can be seen in the figure, 8 hydrogen bonds and 2 hydrophobic interactions are generated during molecular docking, making the conformation more stable during binding.

[0108] Based on the combined docking results of the T1R1 / T1R3 umami receptor and the TMC4 salty receptor, and taking binding energy as the core criterion, while also considering peptide length (preferably short peptides of 2-4 amino acids), amino acid composition and physicochemical properties, a multi-dimensional screening was conducted. Finally, six target peptides, namely SLYE, EATA, VKSD, VAD, EYE, and QAD, were selected for solid-phase synthesis and in vitro functional verification.

[0109] Taste characteristics analysis of synthetic peptides using an electronic tongue: The electronic tongue evaluation used an SA402B type electronic tongue taste sensor (Insent Corporation, Japan). The sensors used in the experiment were AAE (umami sensor), CTO (salty sensor), CA0 (sour sensor), CO0 (bitter sensor), and AE1 (astringent sensor); Reference solution: 30 mM potassium chloride, 0.3 mM tartaric acid; Negative electrode cleaning solution: 100 mM hydrochloric acid, 30% ethanol (volume); Positive electrode cleaning solution: 10 mM potassium hydroxide, 100 mM potassium chloride, 30% ethanol (volume).

[0110] The specific steps for electronic tongue measurement are as follows: Cleaning: Immerse the positive and negative electrodes in the negative electrode cleaning solution and the positive electrode cleaning solution respectively for 90 seconds, and then clean them twice with the reference solution for 120 seconds each time. Test: After cleaning, the sensor is balanced at the equilibrium position for 30 seconds to obtain the potential Vr of the reference solution. After the sensor reaches equilibrium, it tests the sample solution to obtain the potential Vs of the sample (the sampling time in the sample is 30 seconds). The difference between the two is Vs – Vr = initial potential. Then, after cleaning in two separate reference solutions for 3 seconds each, the sensor is inserted into the new reference solution to measure the membrane potential Vr' (sampling time 30 seconds). Vr' – Vr = afterpotential potential (defined as the membrane potential difference CPA caused by adsorption).

[0111] One data point is collected every second, and the data is recorded and analyzed by the electronic tongue software. The test value at 30 seconds is taken as the sensor's signal output value. Each sample is tested four times according to the above test procedure, and the measurement data from the last three tests are analyzed. The CPA value (Vr'–Vr) represents the aftertaste of the flavor. Only bitter, astringent, and umami substances have an aftertaste, which are respectively referred to as bitter aftertaste (Aftertaste-B), astringent aftertaste (Aftertaste-A), and richness.

[0112] Among them, the experimental results of electronic tongue analysis are as follows: Figure 8 As shown in Table 4, the umami intensity of the six peptides at a concentration of 0.5 mg / mL is similar to that of a 0.01% monosodium glutamate solution. VKSD, EYE, and QAD exhibit significant salty characteristics. Therefore, saltiness enhancement experiments were conducted on VKSD and EYE, as well as SLYE, which has the lowest binding energy when docking with salty taste receptor molecules.

[0113] Table 4. Taste intensity data (mean ± SD, n=3) of the electronic tongue for the six synthetic peptides in this application.

[0114] Saltiness enhancement experiment: The saltiness values ​​of 0.25 mg / mL NaCl solution, 0.5 mg / mL VKSD, EYE, and SLYE solution, and 0.5 mg / mL VKSD, EYE, and SLYE solution containing 0.25 mg / mL NaCl were measured separately using an electronic tongue to assess the changes. Each sample was tested four times, and the data from the last three tests were analyzed.

[0115] The results of the saltiness increase experiment are as follows Figure 9 As shown in Table 5, the specific values ​​indicate that the 0.25 mg / mL NaCl solution had an inconspicuous salty taste, while the 0.5 mg / mL VKSD solution had the highest salty value. Compared with the NaCl solution alone (0.25 mg / mL), the experimental group with added VKSD showed the greatest increase in salty taste.

[0116] Table 5. Electronic tongue saltiness value data (mean ± SD, n=3) of the three peptide saltiness experiments in this application.

[0117] Therefore, beef salty peptide V2D has a significant salty taste and a significant salt-enhancing effect.

[0118] Verification example: Resistance to enzymatic hydrolysis of beef salty peptide V2D Experimental conditions: A pH 2.0 simulated gastric juice was prepared, and pepsin was added to prepare a digestive solution. 10 μg / ml of VKSD peptide was incubated at 37 °C with shaking. Samples were taken at 0, 0.5 h, 1 h, and 2 h. Digestion was terminated by adjusting the pH to neutral. After centrifugation and filtration, the peptide content was analyzed using LC-MS to calculate the VKSD retention rate at different times and assess its enzymatic stability in a gastric environment. The stability time was set at 2 h.

[0119] Test results: such as Figure 10 As shown.

[0120] Results Analysis: Figure 10 It was found that VKSD remained relatively stable within 2 hours of enzymatic hydrolysis, with a decrease of no more than 10%. This confirms that the peptide has good resistance to gastric protein, which is beneficial for the subsequent development of functional peptide products.

[0121] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of this disclosure (including the claims) is limited to these examples; within the framework of this disclosure, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of the embodiments of this disclosure as described above, which are not provided in detail for the sake of brevity.

[0122] Although this disclosure has been described in conjunction with specific embodiments thereof, many substitutions, modifications and variations of these embodiments will be apparent to those skilled in the art from the foregoing description.

[0123] This disclosure is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure.

Claims

1. A beef-flavored savory peptide, characterized in that, The amino acid sequence of the beef savory peptide is VKSD.

2. The beef savory peptide according to claim 1, characterized in that, The amino acids in the beef savory peptides are all L-type.

3. The beef-flavored peptide according to claim 1, characterized in that, The beef saponin peptide is obtained by extraction from beef.

4. The method for extracting beef savory peptides as described in claim 1, characterized in that, The extraction method includes: The beef sample was added to an aqueous solution of ammonium bicarbonate containing neutral protease for hydrolysis. The hydrolysis product was subjected to a first centrifugation process to obtain a first supernatant, and formic acid was added to precipitate the protein. The product obtained after protein precipitation was subjected to a second centrifugation to obtain a second supernatant, which was then extracted with dichloromethane. The aqueous phase obtained from the extraction was analyzed by liquid chromatography-mass spectrometry to obtain multiple peptides; The candidate peptides were screened from the multiple peptides to select those that were non-toxic, water-soluble, umami-tasting, and salty. Stability and saltiness analyses were performed on the multiple candidate peptides to obtain the beef salty peptide.

5. The extraction method according to claim 4, characterized in that, The mass ratio of ammonium bicarbonate to the beef sample is 1:(31-32).

6. The extraction method according to claim 4, characterized in that, The stability analysis includes molecular docking of the candidate peptide with the TMC4 receptor to determine the binding affinity.

7. The use of the beef savory peptide as described in any one of claims 1 to 3 or the beef savory peptide extracted by the extraction method as described in any one of claims 4 to 6 in the preparation of savory additives.

8. The use according to claim 7, characterized in that, The use in the preparation of savory additives includes use in the preparation of savory additives for food, or use in the preparation of savory additives for feed.

9. A salty additive, characterized in that, This includes beef savory peptides as described in any one of claims 1 to 3, or beef savory peptides extracted by the extraction method described in any one of claims 4 to 6.

10. A feed additive, characterized in that, This includes beef savory peptides as described in any one of claims 1 to 3, or beef savory peptides extracted by the extraction method described in any one of claims 4 to 6.

Images