Salt-reducing and flavor-enhancing peptides derived from deer antler mushroom, their preparation methods and applications

Abstract

This invention relates to a salt-reducing and flavor-enhancing peptide derived from *Amanita muscaria*, its preparation method, and its application, belonging to the field of food seasoning technology. This invention's salt-reducing and flavor-enhancing peptide is the first to be identified from *Amanita muscaria*, possessing a novel, natural, and safe sequence. It can be obtained through enzymatic hydrolysis or chemical synthesis, with a clear material basis, facilitating industrial-scale quality control and production. Molecular docking and sensory evaluation verification showed that this peptide can stably bind to the umami receptor T1R3 and the saltiness receptor TMC4, significantly enhancing saltiness and umami in low-salt systems (sodium chloride reduced by 30%–50%), achieving "reduced salt without reduced saltiness, and increased umami with reduced salt content." It not only has no unpleasant off-flavors and excellent palatability but also exhibits good stability, adapting to food processing conditions, providing a material basis for natural, efficient, and stable salt-reducing and flavor-enhancing ingredients.

Description

Technical Field

[0001] This invention belongs to the field of food seasoning technology, specifically relating to salt-reducing and flavor-enhancing peptides derived from deer antler mushrooms, their preparation methods, and applications. Background Technology

[0002] A long-term high-sodium diet is a significant risk factor for chronic diseases such as hypertension and cardiovascular disease, and "salt reduction" has become a global consensus in the field of public health. In the food industry, directly reducing the amount of NaCl added is the most efficient salt reduction strategy, but this directly leads to a decrease in the perceived saltiness of the product, bland flavor, and even off-flavors, severely limiting consumer acceptance of low-sodium foods. Currently, industrial techniques such as potassium salt substitution and the addition of flavor enhancers can partially compensate for the loss of saltiness, but they are often accompanied by bitterness, metallic taste, or limited enhancement effects, making them difficult to apply in high-quality seasonings.

[0003] In recent years, food-derived salty-enhancing peptides have been considered an ideal solution to the aforementioned problems due to their natural origin, high safety, and good synergy with taste receptors. These small molecule peptides themselves possess salty properties and can significantly enhance the perception of sodium ions by activating human salty taste receptors at low salt concentrations, thus achieving the effect of "reducing salt without reducing saltiness." However, research on food-derived salty-enhancing peptides is still in its infancy, with a limited number of reported peptide sequences, mostly derived from animal proteins or marine organisms, while the development of peptide resources from plants and edible fungi is severely insufficient. This situation leads to significant limitations in the current technology regarding raw material scalability and industrial-scale targeted preparation.

[0004] Deer antler mushroom ( Lyophyllum decastes As an edible fungus rich in protein and flavor-enhancing amino acids, *Agaricus esculentus* is an excellent raw material for preparing natural seasoning bases. However, to date, no specific small-molecule peptide sequences with clear salt-reducing and flavor-enhancing functions have been selectively isolated, purified, and screened from *Agaricus esculentus*. Furthermore, there is a lack of development schemes for *Agaricus esculentus* salt-reducing and flavor-enhancing peptides based on molecular docking with the novel salty taste receptor TMC4 and dual sensory verification. Therefore, developing a highly efficient salt-reducing and flavor-enhancing peptide derived from *Agaricus esculentus* that can be industrially prepared and has stable effects is of significant practical importance. Summary of the Invention

[0005] The purpose of this invention is to address the current lack of effective salt reduction methods in the food industry by providing a food-derived salt-enhancing peptide with clearly defined components that combines salt reduction and flavor enhancement functions. To this end, this invention provides a salt-reducing and flavor-enhancing peptide derived from *Deer Antler Mushroom*, its preparation method, and its applications.

[0006] This invention provides a salt-reducing and flavor-enhancing peptide derived from *Deer Antler Mushroom*, wherein the salt-reducing and flavor-enhancing peptide comprises one or more polypeptides with amino acid sequences as shown in SEQ ID NO. 1~5.

[0007] The present invention also provides a method for preparing the salt-reducing and flavor-enhancing peptides described in the above technical solution, including separation and extraction from deer antler mushroom and / or solid-phase synthesis using Fmoc.

[0008] Preferably, the separation and extraction of *Deer Antler Fungus* includes the following steps: Using deer antler mushroom as raw material, enzymatic hydrolysis with bromelain was performed to obtain deer antler mushroom enzymatic hydrolysate; The enzymatic hydrolysate of the deer antler mushroom was subjected to ultrafiltration treatment, and the permeate with a molecular weight of less than 1 kDa was collected. The permeate was separated by Sephadex G-15 gel filtration chromatography, with water as the mobile phase, and the third fraction was collected in order of peak elution time. The third fraction was purified by reversed-phase high-performance liquid chromatography using acetonitrile-water as the mobile phase and a C18 column, and a single high-purity main peak fraction was collected, which included the salt-reducing and flavor-enhancing peptide.

[0009] Preferably, the enzymatic hydrolysis treatment includes the following steps: Mix deer antler mushrooms with water at a ratio of 1 g: (4~10) mL, add 3~5% of the weight of deer antler mushroom raw material bromelain, and enzymatically hydrolyze for 120~150 min.

[0010] Preferably, the bromelain has an enzyme activity of 450,000 to 550,000 U / g.

[0011] The present invention also provides the application of the salt-reducing and flavor-enhancing peptides described in the above technical solutions or the salt-reducing and flavor-enhancing peptides prepared by the preparation methods described in the above technical solutions in the preparation of salt-reducing and flavor-enhancing seasonings.

[0012] The present invention also provides a salt-reducing and flavor-enhancing seasoning composition, comprising the salt-reducing and flavor-enhancing peptides described in the above technical solution, sodium chloride, and a food-grade acceptable carrier or excipient.

[0013] Preferably, the flavoring composition comprises, by weight percentage, 0.1% to 0.3% salt-reducing and flavor-enhancing peptides, 0.30% to 0.42% sodium chloride, and food-grade acceptable carriers or excipients to make up the balance.

[0014] Preferably, the flavoring composition further comprises monosodium glutamate, wherein the content of monosodium glutamate is less than 0.2%.

[0015] The present invention also provides the application of the salt-reducing and flavor-enhancing peptides described in the above technical solutions, or the salt-reducing and flavor-enhancing peptides prepared by the preparation methods described in the above technical solutions, or the flavoring compositions described in the above technical solutions, in food flavoring.

[0016] Beneficial effects: This invention provides salt-reducing and flavor-enhancing peptides derived from *Amanita muscaria*, which contain one or more polypeptides with amino acid sequences as shown in SEQ ID NO. 1-5. The five peptide sequences EAEFS, ENLSG, ESKDR, ETEAQ, and GQKES described in this invention are all newly identified from *Amanita muscaria*, possessing novel sequences, natural origin, and high safety. They can be reliably obtained through either enzymatic separation and purification or chemical synthesis, with a clear material basis, facilitating industrial quality control and standardized production. Verified by both molecular docking and sensory evaluation, the salt-reducing and flavor-enhancing peptides can stably bind to the umami receptor T1R3 and the novel saltiness receptor TMC4, significantly restoring and enhancing the perceived saltiness intensity in low-salt systems (sodium chloride content reduced by 30%-50%), while exhibiting a prominent flavor-enhancing effect, achieving a synergistic flavor enhancement of "reduced salt without reducing saltiness, and reduced salt with enhanced flavor". Furthermore, the peptides described in this invention have no bitter or metallic taste or other unpleasant odors, exhibit excellent overall palatability, and demonstrate good thermal stability, storage stability, and pH stability. They can be widely adapted to processing conditions such as cooking, sterilization, and storage in the food industry, providing a clear material basis for natural, efficient, and stable salt-reducing and flavor-enhancing functional ingredients. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The figure shows the results of peptide content detection in the enzymatic hydrolysis substrate of *Mucuna arvense* using different proteases; among them, represent P <0.05, represent P <0.01, represent P <0.001, represent P <0.0001; Figure 2 The image shows the results of detecting the amino acid nitrogen content in the enzymatic hydrolysis substrate of *Mucuna arvensis* using different proteases; among them, represent P <0.05, represent P <0.0001; Figure 3 The image shows the results of umami value detection in different proteases from the enzymatic hydrolysis substrate of *Mucuna arvense*. represent P <0.0001; Figure 4 Figure 1 shows the results of peptide content detection in enzymatic hydrolysis substrates of *Mushroom reticulata* prepared with different enzyme dosages and hydrolysis times; among which, represent P <0.05, represent P <0.01, represent P <0.001, represent P <0.0001; Figure 5 Response surface methodology for enzymatic hydrolysis of Peptide-based materials from *Deer Antler Mushroom*; Figure 6 This is a graph showing the relationship between observed and predicted peptide content values. Figure 7 3D diagram of molecular docking between EAEFS, MSG and T1R3; Figure 8 3D diagram of molecular docking between ELNSG, MSG and T1R3; Figure 9 3D diagram of molecular docking between ESKDR, MSG and T1R3; Figure 10 3D diagram of molecular docking between ETEAQ, MSG and T1R3; Figure 11 3D diagram of molecular docking between GQKES, MSG and T1R3; Figure 12 3D diagram of molecular docking between pentapeptide-MSG and T1R3; Figure 13 3D diagram of molecular docking between EAEFS, NaCl and TMC4; Figure 14 3D diagram of molecular docking of ELNSG, NaCl and TMC4; Figure 15 3D diagram of molecular docking between ESKDR, NaCl and TMC4; Figure 16 3D diagram of molecular docking between ETEAQ, NaCl and TMC4; Figure 17 3D diagram of molecular docking of GQKES, NaCl and TMC4; Figure 18 Figure showing the thermal stability test results of enzymatically hydrolyzed peptide substrate from *Mushroom arganum*. Figure 19 The results of the storage stability test of the enzymatic hydrolysed peptide substrate of *Flammulina velutipes* are shown in the figure. Figure 20 The pH stability test results for the enzymatic hydrolysed peptide substrate of *Pleurotus ostreatus* are shown in the figure. Figure 21 The LC-MS / MS mass spectrometry identification chromatogram of EAEFS; Figure 22 The LC-MS / MS mass spectrometry identification chromatogram of ENLSG; Figure 23 The LC-MS / MS mass spectrometry identification chromatogram of ESKDR; Figure 24 The LC-MS / MS mass spectrometry identification chromatogram of ETEAQ; Figure 25 This is the LC-MS / MS mass spectrometry identification chromatogram of GQKES. Detailed Implementation

[0018] This invention provides salt-reducing and flavor-enhancing peptides derived from *Agrocybe aegerita*, wherein the salt-reducing and flavor-enhancing peptides contain one or more polypeptides with amino acid sequences as shown in SEQ ID NO. 1-5. The amino acid sequences of the salt-reducing and flavor-enhancing peptides of this invention are: EAEFS (SEQ ID NO. 1), ELNSG (SEQ ID NO. 2), ESKDR (SEQ ID NO. 3), ETEAQ (SEQ ID NO. 4), and GQKES (SEQ ID NO. 5). The salt-reducing and flavor-enhancing peptides of this invention are all newly identified from *Agrocybe aegerita*, with novel sequences, natural sources, and high safety. They can be reliably obtained through either enzymatic separation and purification or chemical synthesis, with a clear material basis, which is beneficial for industrial quality control and standardized production.

[0019] The present invention also provides a method for preparing the salt-reducing and flavor-enhancing peptides described in the above technical solution, including separation and extraction from deer antler mushroom and / or solid-phase synthesis using Fmoc.

[0020] As one embodiment, the separation and extraction of *Deer Antler Fungus* according to the present invention includes the following steps: Using deer antler mushroom as raw material, enzymatic hydrolysis with bromelain was performed to obtain deer antler mushroom enzymatic hydrolysate; The enzymatic hydrolysate of the deer antler mushroom was subjected to ultrafiltration treatment, and the permeate with a molecular weight of less than 1 kDa was collected. The permeate was separated by Sephadex G-15 gel filtration chromatography, with water as the mobile phase, and the third fraction was collected in order of peak elution time. The third fraction was purified by reversed-phase high-performance liquid chromatography using acetonitrile-water as the mobile phase and a C18 column, and a single high-purity main peak fraction was collected, which included the salt-reducing and flavor-enhancing peptide.

[0021] This invention uses *Volvariella antler* as raw material and employs bromelain for enzymatic hydrolysis to obtain *Volvariella antler* enzymatic hydrolysate. As one embodiment, the *Volvariella antler* raw material of this invention is *Volvariella antler* powder. As another embodiment, the *Volvariella antler* powder of this invention has a moisture content of less than 5% and can pass through a 100-mesh sieve.

[0022] As one embodiment, the enzymatic hydrolysis treatment of the present invention includes the following steps: Mix *Deer Antler Mushroom* with water at a ratio of 1 g:(4~10) mL, add 3~5% (by weight of *Deer Antler Mushroom* raw material) of bromelain, and hydrolyze for 120~150 min. In one embodiment, *Deer Antler Mushroom* is mixed with water at a ratio of 1 g:6 mL. In another embodiment, the bromelain activity of the bromelain is 450,000~550,000 U / g. In yet another embodiment, the bromelain activity is 500,000 U / g. In one embodiment, 3.9% (by weight of *Deer Antler Mushroom* raw material) of bromelain is added during the hydrolysis, and hydrolysis is carried out for 128 min. In another embodiment, the hydrolysis temperature is 50℃.

[0023] After obtaining the enzymatic hydrolysate of *Mucuna arvensis*, this invention subjectes the enzymatic hydrolysate to ultrafiltration, collecting the permeate with a molecular weight less than 1 kDa. As one embodiment, the ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, collecting the permeate with a molecular weight less than 1 kDa. As another embodiment, the permeate is a small-molecule flavor peptide enrichment solution. As yet another embodiment, freeze-drying the permeate yields a pale yellow powder.

[0024] After collecting the permeate with a molecular weight less than 1 kDa, the present invention performs Sephadex G-15 gel filtration chromatography separation using water as the mobile phase, collecting the third fraction in elution time order. As one embodiment, the chromatographic column for the Sephadex G-15 gel filtration chromatography separation is 1.6 cm × 60 cm. As one embodiment, the gel filtration chromatography separation uses ultrapure water as the mobile phase, a flow rate of 1.0 mL / min, a detection wavelength of 220 nm, and a sample loading volume of 5 mL. As one embodiment, the present invention collects four main fractions (F1, F2, F3, and F4) sequentially in elution order, and the third fraction is the F3 fraction. As one embodiment, the present invention evaluates the umami intensity and saltiness enhancement effect of each fraction, and the F3 fraction has the highest umami value and the most significant salt reduction and saltiness enhancement effect, thus being identified as the target active fraction.

[0025] After collecting the third fraction, this invention uses acetonitrile-water as the mobile phase and a C18 column for reversed-phase high-performance liquid chromatography (RP-HPLC) purification, collecting a single high-purity main peak fraction, which includes the reduced-salt flavor-enhancing peptides. As one embodiment, the RPI purification of this invention uses a C18 reversed-phase column with acetonitrile-water as the mobile phase for gradient elution, and online detection at 220 nm. As one embodiment, this invention collects the single high-purity main peak fraction presented in the chromatogram. As one embodiment, the single high-purity main peak fraction is analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and compared with database sequences to obtain the aforementioned five reduced-salt flavor-enhancing peptides (SEQ ID NO. 1~5).

[0026] This invention also provides the application of the salt-reducing and flavor-enhancing peptides described in the above-described technical solutions, or the salt-reducing and flavor-enhancing peptides prepared by the preparation methods described in the above-described technical solutions, in the preparation of salt-reducing and flavor-enhancing seasonings. As one embodiment, when preparing salt-reducing and flavor-enhancing seasonings, the salt-reducing and flavor-enhancing peptides can maintain or enhance the perceived saltiness intensity while reducing the amount of added salt by 30% to 50%. As one embodiment, under 30% salt reduction conditions (0.42% NaCl), adding 0.20% of the salt-reducing and flavor-enhancing peptides can restore the saltiness intensity to 7.5 points, exceeding the 7.2 points of the normal salt control. As one embodiment, under 50% salt reduction conditions (0.30% NaCl), adding 0.20% of the salt-reducing and flavor-enhancing peptides can increase the saltiness to 6.5 points, far exceeding the 3.1 points of the blank salt-reducing group. In one embodiment, the salt-reducing and flavor-enhancing peptides, while improving the perception of saltiness, also exhibit a flavor-enhancing effect. A 0.20% salt-reducing and flavor-enhancing peptide combined with 0.42% NaCl achieves a flavor intensity of 8.0 points, exceeding the 7.5 points of the 0.05% MSG positive control, demonstrating its potential to replace or partially replace MSG. In another embodiment, the salt-reducing and flavor-enhancing seasoning is in the form of a solid powder or a liquid seasoning. In yet another embodiment, the salt-reducing and flavor-enhancing seasoning is applied to low-salt soy sauce, low-salt mushroom broth, or low-salt minced meat products. In yet another embodiment, this invention, through molecular docking, confirms that all five peptides in the salt-reducing and flavor-enhancing peptides can stably bind to the umami receptor T1R3 and the saltiness receptor TMC4. Among them, GQKES exhibits a unique binding mode with TMC4, and the NaCl binding site is closer to the active cavity, indicating optimal salt-reducing effect.

[0027] This invention also provides a salt-reducing and flavor-enhancing seasoning composition, comprising the salt-reducing and flavor-enhancing peptides described in the above-mentioned technical solution, sodium chloride, and a food-grade acceptable carrier or excipient. As one embodiment, the seasoning composition is in solid powder or liquid form. As one embodiment, the food-grade acceptable carrier or excipient is selected from one or more of maltodextrin, edible starch, white sugar, yeast extract, thickener, and drinking water. As one embodiment, the seasoning composition is prepared using conventional physical mixing. As one embodiment, the GQKES, ESKDR, and ETEAQ compound system in the salt-reducing and flavor-enhancing peptides of this invention has no bitterness or astringency and excellent palatability; EAEFS and ENLSG in the salt-reducing and flavor-enhancing peptides have a slightly astringent taste and are suitable for compound use.

[0028] In one embodiment, the flavoring composition of the present invention, by weight percentage, comprises 0.1% to 0.3% of a salt-reducing and flavor-enhancing peptide, 0.30% to 0.42% of sodium chloride, and a food-grade acceptable carrier or excipient to make up the balance. In one embodiment, the amount of the salt-reducing and flavor-enhancing peptide added is 0.2%, and the amount of sodium chloride added is 0.42%. In another embodiment, the amount of the salt-reducing and flavor-enhancing peptide added is 0.2%, and the amount of sodium chloride added is 0.30%.

[0029] In one embodiment, the flavoring composition of the present invention further comprises monosodium glutamate (MSG), wherein the MSG content is 0.2% or less. In another embodiment, the amount of MSG added is 0.05%.

[0030] This invention also provides the application of the salt-reducing and flavor-enhancing peptides described in the above-described technical solutions, or the salt-reducing and flavor-enhancing peptides prepared by the preparation methods described in the above-described technical solutions, or the flavoring compositions described in the above-described technical solutions, in food seasoning. As one embodiment, the salt-reducing and flavor-enhancing peptides of this invention, or the flavoring compositions containing the salt-reducing and flavor-enhancing peptides, can be applied to low-salt seasonings, meat products, convenience foods, soup bases, sauces, or pickled foods. As one embodiment, the application includes seasoning applications in low-salt soy sauce, low-salt mushroom soup bases, or low-salt minced meat products.

[0031] To further illustrate the present invention, the salt-reducing and flavor-enhancing peptides derived from *Agaricus esculentus*, their preparation methods, and applications are described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 Preparation and enzymatic hydrolysis process screening of deer antler mushroom powder 1. Preparation of deer antler mushroom powder Fresh deer antler mushroom fruiting bodies (purchased from Shanghai Guosen Biotechnology Co., Ltd.) were cleaned, sliced, dried, and pulverized. The powder was then passed through a 100-mesh sieve to obtain deer antler mushroom powder, with the moisture content controlled to be below 5%. The powder was then set aside for later use.

[0033] 2. Protease screening Take *Deer Antler Mushroom* powder and add distilled water at a material-to-liquid ratio of 1:6 (g / mL). Add 4% (based on mushroom powder mass) of neutral protease, hemicellulase, alkaline protease, trypsin, pepsin, and bromelain, respectively. Enzymatically hydrolyze for 120 min at the optimal temperature (see Table 1) with the pH at its natural setting. Use water-extracted mushroom powder without added protease as a control. After enzymatic hydrolysis, inactivate the enzymes in a 100℃ water bath for 10 min, dry in hot air at 50℃ for 48 h, and pulverize through a 100-mesh sieve to obtain *Deer Antler Mushroom* enzymatically hydrolyzed powder.

[0034] Table 1. Selection and Conditions of Protease

[0035] The optimal protease was screened using peptide content, amino acid nitrogen content, and umami value as evaluation indicators.

[0036] Peptide content was determined using a kit method (Peptide Content Assay Kit, Suzhou Mengxi Biomedical Technology Co., Ltd.): 0.1 g of enzymatic hydrolysate was weighed and added to 1 mL of the kit's extraction buffer (10% trichloroacetic acid). The mixture was homogenized on ice and allowed to stand for 30 min. After centrifugation at 12000 rpm and 4℃ for 10 min, the supernatant was collected. 10 μL of the supernatant sample was taken and 190 μL of the kit's working solution was added. The mixture was incubated at 60℃ for 30 min, and the absorbance was measured at 562 nm. The peptide content was calculated using the following formula: Peptide content (mg / g dry weight) = Standard concentration × (Sample absorbance - Blank absorbance) ÷ (Standard absorbance - Blank absorbance) × V 提取液体积 ÷ W 基料质量 , Formula I.

[0037] The standard was glycine-glycine-tyrosine-arginine with a mass concentration of 0.5 mg / mL, the blank was water, the volume of extract V was 1 mL, and the mass of substrate W was 0.1 g.

[0038] The amino acid nitrogen content was determined using the method specified in GB5009.235—2016.

[0039] Umami value was determined using the TS-5000 Z Insent electronic tongue taste analysis system: 0.1 g of enzymatic hydrolysate was weighed and dissolved in 100 mL of pure water. 25 mL of the solution was added to the electronic tongue sample cup. Each sample was tested for 30 s. After washing with reference solution for 3 s (washing twice), the aftertaste test time was 30 s. Each sample was tested 4 times, and the last 3 results were taken as the test results.

[0040] The results are shown in Table 2 and Figures 1-3 As shown, the peptide content of all six protease-hydrolyzed substrates was significantly higher than that of unhydrolyzed mushroom powder (63.15 mg / g). Among them, the substrate hydrolyzed with bromelain had the highest peptide content (224.13 mg / g), the highest amino acid nitrogen content (1.71 g / 100g), and the highest umami value (4.78). Considering all factors, bromelain was selected as the best enzyme preparation.

[0041] Table 2. Peptide content, amino acid nitrogen content, and umami value of different proteases in *Mucuna stolonifera* hydrolysate.

[0042] 3. Single-factor experiment Enzyme dosage screening: Deer antler mushroom powder and water were mixed at a material-to-liquid ratio of 1:6 (g / mL). Bromelain was added at concentrations of 1%, 2%, 3%, 4%, and 5% (based on mushroom powder mass), respectively. Enzymatic hydrolysis was performed at 50℃ for 120 min, with the pH set to natural. After hydrolysis, the enzyme was inactivated in a 100℃ water bath for 10 min, followed by hot air drying at 50℃ for 48 h. The resulting powder was then pulverized and passed through a 100-mesh sieve to obtain deer antler mushroom enzymatic hydrolysate. Peptide content was determined.

[0043] The results are shown in Table 3 and Figure 2 As shown, the peptide content first increases and then tends to level off as the amount of enzyme added increases, with higher peptide content when the amount of enzyme added is 4% to 5%.

[0044] Enzymatic hydrolysis time screening: With a fixed enzyme dosage of 4%, enzymatic hydrolysis was performed for 60, 90, 120, 150, and 180 min, respectively, with other conditions remaining the same. Peptide content was then determined.

[0045] The results are shown in Table 3 and Figure 4 As shown, the peptide content first increases and then decreases with increasing enzymatic hydrolysis time, with higher peptide content observed at 120-150 min hydrolysis time.

[0046] Based on the combined results of enzyme dosage and hydrolysis time screening, an enzyme dosage of 3%–5% and a hydrolysis time of 90–150 min were selected as the factor level range for subsequent response surface optimization.

[0047] Table 3. Peptide content in *Mucuna spp.* enzymatic hydrolysate prepared with different enzyme dosages and hydrolysis times.

[0048] Example 2 Response surface optimization of enzymatic hydrolysis process Based on the single-factor experimental results of Example 1, enzyme dosage (A) and enzymatic hydrolysis time (B) were selected as independent variables, and peptide content was used as the response value. A response surface methodology was designed using Design-Expert 8.0.6 software. The factor levels are shown in Table 4.

[0049] Table 4 Enzymatic response surface methodology settings

[0050] Each experimental group underwent enzymatic hydrolysis under the set conditions. The enzyme was inactivated in a water bath at 100℃ for 10 min, centrifuged at 4000 rpm for 10 min, and the supernatant was collected and freeze-dried at -70℃ for 48 h. The peptide content was then determined. The experimental design and results are shown in Table 5.

[0051] Table 5 Results of Response Surface Experiment

[0052] Software analysis yielded the following regression model equation for peptide content (Y) on enzyme dosage (A) and hydrolysis time (B): Y = 239.42 - 0.38A + 2.88B - 1.45AB - 5.31A 2 - 5.37B 2 Formula II.

[0053] The regression model and significance test results are shown in Table 6. The model p-value is less than 0.0001, indicating the regression model is significant; the p-value for the lack-of-fit term is 0.0732, indicating no significant lack of fit, meaning the model fits well. The model's coefficient of determination R0... 2 The value is 0.9943, so R is adjusted. 2 The predicted R is 0.9902. 2 The value was 0.9558, and all three values ​​were greater than 0.9, indicating that the model has good fit and predictive ability. The interaction between enzyme dosage and enzymatic hydrolysis time was significant. The response surface plot is shown below. Figure 5 As shown.

[0054] Table 6. Regression Model and Significance Test Results of Response Surface Experiment

[0055] Based on the response surface methodology, the software predicted the optimal enzymatic hydrolysis conditions as follows: enzyme dosage 3.91% and hydrolysis time 127.95 min. To verify the reliability of the model, a verification experiment was conducted with an enzyme dosage of 3.9% and a hydrolysis time of 128 min, with three parallel measurements. The verification showed that the average actual peptide content in the *Mushroom antler velvet* enzymatic hydrolysate prepared under these conditions was 238.54 mg / g dry weight, with a relative error of only 0.54% compared to the theoretical prediction of 239.83 mg / g dry weight. The relationship between the observed and predicted peptide content values ​​is shown below. Figure 6 As shown, the two have good consistency, indicating that the optimal enzymatic hydrolysis preparation process parameters predicted by the model are reliable.

[0056] The enzymatic hydrolysate of *Agaricus esculentus* prepared using the above-mentioned optimal process has an umami value of 6.3, which is 1.32 times higher than the original value (4.78); the amino acid nitrogen content is 2.023 g / 100g, which is 1.18 times higher than the original value (1.71 g / 100g). This enzymatic hydrolysate will be used as a raw material for subsequent separation and purification of salt-reduced and flavor-enhancing peptides.

[0057] In summary, this embodiment systematically optimized the process conditions for enzymatic hydrolysis of *Agaricus esculentus* powder using bromelain via response surface methodology. The optimal process parameters obtained were: a material-to-liquid ratio of 1:6 (g / mL), a bromelain addition of 3.9% (based on the mass of the mushroom powder), a hydrolysis temperature of 50℃, a hydrolysis time of 128 min, and a natural pH. Under this process, the peptide content can reach 238.54 mg / g dry weight, providing a high-quality hydrolysate for the subsequent separation and purification of active peptides.

[0058] Example 3 The optimal enzymatic hydrolysis process determined in Example 2 was used to prepare the enzymatic hydrolysis substrate for *Agaricus esculentus*. The specific steps are as follows: Take deer antler mushroom powder, add distilled water at a material-to-liquid ratio of 1:6 (g / mL) and mix well. Add 3.9% bromelain (enzyme activity 500,000 U / g) based on the mushroom powder weight, and enzymatically hydrolyze at 50℃ for 128 min, with the pH at natural. After enzymatic hydrolysis, inactivate the enzyme in a 100℃ water bath for 10 min, dry with hot air at 50℃ for 48 h, and pulverize through a 100-mesh sieve to obtain the deer antler mushroom enzymatic hydrolysate. The nutrient content of this nutrient is 238.54 mg / g dry weight, the umami value is 6.3, and the amino acid nitrogen content is 2.023 g / 100g.

[0059] Take the above-mentioned enzymatically hydrolyzed material, prepare a 10% (w / v) aqueous solution with ultrapure water, centrifuge at 10000 r / min for 15 min to remove insoluble impurities, and collect the supernatant for later use. This supernatant will serve as the starting material for subsequent separation, purification, and salt-reduced flavor-enhancing peptides.

[0060] Example 4 Isolation, purification and active fraction screening of salt-reduced and flavor-enhancing peptides 1. Ultrafiltration fractionation and enrichment of small molecule peptides The supernatant of the enzymatic hydrolysis substrate prepared in Example 3 was separated using an ultrafiltration membrane (GE, specification UFP-1-C-6, catalog number 56-4102-51) with a molecular weight cutoff of 1 kDa. The permeate with a molecular weight less than 1 kDa was collected, which is the enriched solution of small molecule flavor peptides. The permeate was freeze-dried to obtain a pale yellow powder for later use. Before the next step of gel filtration chromatography separation, it was reconstituted with ultrapure water to a concentration of 100 mg / mL as the loading solution.

[0061] 2. Gel filtration chromatography separation Separation was performed using a Sephadex G-15 gel chromatography column under the following chromatographic conditions: column size: 1.6 cm × 60 cm; mobile phase: ultrapure water; flow rate: 1.0 mL / min; detection wavelength: 220 nm; sample loading volume: 5 mL. The four main fractions, F1, F2, F3, and F4, were collected sequentially according to their elution time and then freeze-dried.

[0062] 3. Screening of active fractions Sensory evaluations were conducted on the umami intensity and saltiness enhancement effects of four fractions (F1, F2, F3, and F4). The results showed that the sensory evaluation values ​​for the four fractions were 2.92±0.89, 4.21±0.81, 6.28±0.63, and 2.72±0.65, respectively. Among them, fraction F3 had the highest sensory evaluation value, and was therefore identified as the target active fraction for further purification.

[0063] Example 5 Reversed-phase HPLC purification and LC-MS / MS mass spectrometry identification 1. Purification by reversed-phase high-performance liquid chromatography The F3 fraction obtained in Example 4 was further purified using a C18 reversed-phase column. The chromatographic conditions were: gradient elution with acetonitrile-water as the mobile phase and online detection at 220 nm. The single high-purity main peak fraction presented in the chromatogram was collected.

[0064] 2. Identification by LC-MS / MS mass spectrometry The collected single high-purity main peak fractions were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Combined with sequence comparison in the database, five salt-reducing and flavor-enhancing peptides were identified, with the following amino acid sequences: EAEFS (SEQ ID NO.1), ENLSG (SEQ ID NO.2), ESKDR (SEQ ID NO.3), ETEAQ (SEQ ID NO.4), and GQKES (SEQ ID NO.5). These are the salt-reducing and flavor-enhancing peptides derived from *Ilex chinensis* that are claimed in this invention.

[0065] Example 6 Chemical Synthesis and Purification of Salt-Reducing and Flavor-Enhancing Peptides The chemical synthesis and purification of five salt-reducing and flavor-enhancing peptides (EAEFS, ENLSG, ESKDR, ETEAQ, and GQKES) were commissioned to Jier Biochemical (Shanghai) Co., Ltd. Solid-phase synthesis was employed, followed by HPLC purification, resulting in peptides with a purity of ≥98%. The high-purity peptides were used for subsequent sensory evaluation and application research.

[0066] Example 7 Molecular docking analysis Using MOE (Molecular Operating Environment) software, five salt-reducing and flavor-enhancing peptides were molecularly docked with the umami receptor T1R3 and the saltiness receptor TMC4 to evaluate their potential molecular mechanisms of flavor enhancement and salt reduction.

[0067] 1. Docking with the umami receptor T1R3 Five peptides—EAEFS, ENLSG, ESKDR, ETEAQ, and GQKES—and monosodium glutamate (MSG) were molecularly docked with the umami receptor T1R3 (PDB ID: Q7RTX0). The MOE molecular docking software was used to optimize the receptor protein crystal structure, removing water molecules and adding hydrogen atoms. The MOE software was used to construct the 3D structure of the peptide molecules and to minimize their energy. The active site and cavity pocket of the receptor protein were determined using the MOE Site Finder module. Molecular docking binding analysis between the peptide molecules and the receptor protein was performed using the active pocket docking mode. Using the docking fraction, number of bonds, and bond energy as screening indicators, tight-binding complexes of peptide molecules and receptor proteins were selected. The MOE software was then used to analyze the binding sites and binding modes between the peptide molecules and the receptor protein.

[0068] The results are shown in Tables 7-11 and Figures 7-12 The results shown in Tables 7-11 correspond to the docking results of the five peptides with MSG-T1R3. Figures 7-12 (See corresponding 3D docking diagrams and overlay diagrams). The results show that the docking score of EAEFS and MSG with T1R3 is -13.10, the bond energy of EAEFS with T1R3 is -32.3 kcal / mol, and the bond energy of MSG with T1R3 is -8.8 kcal / mol, for a total total bond energy of -41.1 kcal / mol; the docking score of ENSG and MSG with T1R3 is -13.28, the bond energy of ENSG with T1R3 is -27.4 kcal / mol, and the bond energy of MSG with T1R3 is -4.9 kcal / mol, for a total total bond energy of -32.3 kcal / mol; the docking score of ESKDR and MSG with T1R3 is -13.97, the bond energy of ESKDR with T1R3 is -55.1 kcal / mol, and the bond energy of MSG with T1R3 is -9.2 kcal / mol, for a total total bond energy of -64.3 kcal / mol. The binding energy of ETEAQ and MSG to T1R3 was -14.36 kcal / mol, with ETEAQ binding to T1R3 at -28 kcal / mol and MSG binding to T1R3 at -7.6 kcal / mol, for a total binding energy of -35.6 kcal / mol. The binding energy of GQKES and MSG to T1R3 was -14.43 kcal / mol, with GQKES binding to T1R3 at -56 kcal / mol and MSG binding to T1R3 at -7.8 kcal / mol, for a total binding energy of -63.8 kcal / mol. Except for EAEFS, the other four peptides and MSG penetrated deep into the active cavity of T1R3. GQKES and ESKDR had the highest total binding energy, indicating that they bound most tightly to the umami receptor and had the most significant synergistic umami enhancement effect.

[0069] Table 7 Molecular docking results of EAEFS, MSG and T1R3

[0070] Table 8. Molecular docking results of ELNSG, MSG and T1R3

[0071] Table 9. Molecular docking results of ESKDR, MSG, and T1R3

[0072] Table 10 Molecular docking results of ETEAQ, MSG and T1R3

[0073] Table 11 Molecular docking results of GQKES, MSG and T1R3

[0074] 2. Docking with the salty taste receptor TMC4 Five peptides and NaCl were molecularly docked with the salty taste receptor TMC4 (Uniprot: A0A0G2JP95). The results are shown in Tables 12-16. Figures 13-17As shown in the diagram, the bonding scores for EAEFS, NaCl, and TMC4 are -10.60, with a bond energy of -61.9 kcal / mol between EAEFS and TMC4, and -2.9 kcal / mol between NaCl and TMC4, for a total bond energy of -64.8 kcal / mol. For ENLSG, NaCl, and TMC4, the bonding score is -10.65, with a bond energy of -72 kcal / mol between EAEFS and TMC4, and -2.9 kcal / mol between NaCl and TMC4, for a total bond energy of -74.9 kcal / mol. For ESKDR, NaCl, and TMC4, the bonding score is -11, with a bond energy of -70.2 kcal / mol between ESKDR and TMC4, and -2.9 kcal / mol between NaCl and TMC4, for a total bond energy of -73.1 kcal / mol. The binding energy of ETEAQ and NaCl to TMC4 was -10.24, with a bond energy of -72.3 kcal / mol for ETEAQ and -2.7 kcal / mol for NaCl, resulting in a total bond energy of -75 kcal / mol. The binding energy of GQKES and NaCl to TMC4 was -11.11, with a bond energy of -53.1 kcal / mol for GQKES and -2.5 kcal / mol for NaCl, resulting in a total bond energy of -55.6 kcal / mol. Notably, in the GQKES-NaCl-TMC4 complex, the binding sites of GQKES peptide molecules and NaCl were closer than in the other four complexes, and NaCl was located closer to the key region of the TMC4 active cavity. This suggests that GQKES and NaCl exhibited the best synergistic salt-increasing effect and the most significant salt-reducing effect.

[0075] Table 12 Molecular docking results of EAEFS, NaCl, and TMC4

[0076] Table 13 Molecular docking results of ELNSG, NaCl and TMC4

[0077] Table 14 Molecular docking results of ESKDR, NaCl, and TMC4

[0078] Table 15 Molecular docking results of ETEAQ, NaCl, and TMC4

[0079] Table 16 Molecular docking results of GQKES, NaCl, and TMC4

[0080] Example 8 Sensory evaluation verification Sensory evaluation methods were conducted in accordance with national standards: the 1-9 point quantitative scoring method followed GB / T 39501-2020 "Guidelines for the Use of Quantitative Response Scales in Sensory Analysis"; evaluator selection and training followed GB / T 16291.1-2012 "General Guidelines for the Selection, Training and Management of Sensory Evaluators - Part 1: Selection of Evaluators"; and the evaluation environment and operation followed GB / T 13868-2009 "General Guidelines for Establishing Sensory Analysis Laboratories". Evaluations were conducted by 20 trained sensory evaluators, with three parallel measurements taken and the average value recorded. Scoring scale: 1 point = no perception, 2 points = very weak, 3 points = weak, 4 points = weak, 5 points = moderate, 6 points = strong, 7 points = strong, 8 points = very strong, 9 points = extremely strong. Lower odor scores were better; ≤2 points indicated no noticeable odor.

[0081] 1. Basic Comparison 0.6% NaCl was set as the positive control for saltiness, 0.05% MSG as the positive control for umami, and 0.42% NaCl (30% salt reduction) and 0.30% NaCl (50% salt reduction) were set as blank salt reduction groups.

[0082] The sensory evaluation results of the baseline control samples are shown in Table 17: the saltiness intensity of the positive control for saltiness was 7.2 points, and the umami intensity of the positive control for umami was 7.5 points; the saltiness intensity of the 30% salt reduction blank decreased to 4.3 points, and the saltiness intensity of the 50% salt reduction blank decreased to 3.1 points, indicating that direct salt reduction led to a significant decrease in the perception of saltiness.

[0083] Table 17 Sensory evaluation results of the baseline control samples

[0084] Note: An odor score of ≤2 indicates no obvious odor.

[0085] 2. Effect of the 30% salt reduction system compound formulation Five peptides (EAEFS, ENLSG, ESKDR, ETEAQ, and GQKES) prepared as described in Example 6 were added to a 0.42% NaCl solution (30% salt reduction). The results are shown in Table 18: The sample with added GQKES achieved a saltiness intensity of 7.5 points, exceeding the positive control (7.2 points); an umami intensity of 8.0 points, exceeding the positive control (7.5 points); and no off-flavors, with an overall palatability score of 7.9 points, showing the best effect. ESKDR and ETEAQ also showed good saltiness restoration and umami enhancement effects, with no off-flavors. EAEFS and ENLSG were effective but had a slightly astringent taste.

[0086] Table 18. Validation of the effects of five peptide combinations in a 30% salt reduction system (0.42% NaCl).

[0087] 3. Effect of compounding the 50% salt reduction system Five peptides were added at 0.20% each to a 0.30% NaCl (50% reduced salt) solution. The results are shown in Table 19: The saltiness intensity of the sample with added GQKES increased to 6.5 points, far higher than the 50% reduced salt control group (3.1 points); the umami intensity reached 7.4 points; the overall palatability was 7.2 points, with no off-flavors. ESKDR and ETEAQ also significantly improved the perception of salty and umami flavor.

[0088] Table 19. Validation of the effect of five peptide combinations in a 50% salt reduction system (0.3% NaCl).

[0089] 4. Concentration gradient experiment Five peptides were added to 0.42% NaCl at concentrations of 0.10%, 0.20%, and 0.30%, respectively. The results are shown in Table 20: the enhancement of saltiness and umami flavor by the five peptides was dose-dependent. GQKES showed the best effect at all concentrations, reaching its optimal effect at 0.20%, with a saltiness score of 7.5 and an umami score of 8.0. EAEFS and ENLSG had a slightly astringent taste at the high concentration of 0.30%.

[0090] Table 20 Verification of Salt Reduction and Flavor Enhancement Effects of Five Peptide Concentration Gradients (0.1% / 0.2% / 0.3%)

[0091] 5. Comprehensive Ranking Based on four indicators—enhanced saltiness, enhanced umami, overall palatability, and control of off-flavors—the five peptides were ranked in terms of their salt-reduction and umami-enhancing effects as follows: GQKES > ESKDR > ETEAQ > ELNSG > EAEFS (see Table 21). Among them, GQKES performed best in all indicators.

[0092] Table 21 Ranking of the Salt Reduction and Flavor Enhancement Effects of Five Peptides

[0093] Example 9 Stability verification 1. Thermal stability The enzymatic hydrolysate of *Agaricus esculentus* prepared in Example 3 was diluted to a 1% aqueous solution and heated at 80℃, 90℃, and 100℃ for 30 min, respectively. After cooling, the peptide content and umami value were measured. The results are as follows: Figure 18As shown, the peptide content retention rate is ≥96.7%, the umami value retention rate is ≥95.2%, the flavor shows no significant change, no off-flavors are produced, and the thermal stability is excellent.

[0094] 2. Storage stability The above-mentioned enzymatically hydrolyzed substrate was sealed and stored at 4°C for 7 days, 15 days, and 30 days at 25°C. The results are as follows: Figure 19 As shown, after 30 days of storage at 4℃, the peptide content retention rate was ≥97.3%; after 30 days of storage at 25℃, the peptide content retention rate was ≥92.6%, with no clumping, no mold, no off-odor, and good storage stability.

[0095] 3. pH stability The above enzymatically hydrolyzed substrate was adjusted to pH 3.0 (acidic), pH 7.0 (neutral), and pH 9.0 (alkaline), respectively, and measured after standing for 2 hours. The results are as follows: Figure 20 As shown, within the pH range of 3.0 to 9.0, the peptide content and umami value did not decrease significantly, and the umami flavor was more prominent under acidic conditions, making it suitable for a wide range of food pH systems.

[0096] 4. Stability of the five bioactive peptides Purified peptides (≥98%) containing EAEFS, ENLSG, ESKDR, ETEAQ, and GQKES were subjected to heating at 100℃ for 30 min and storage at 4℃ for 7 days, respectively. Detection was performed by LC-MS / MS. Figures 21-25 The peptide structures remained intact, and no new degradation fragments were observed. Sensory evaluation confirmed that the flavor-enhancing and salt-reducing effects were not diminished. This indicates that all five bioactive peptides possess good thermal and storage stability.

[0097] In summary, the deer antler mushroom salt-reduced and flavor-enhancing peptide base and five active peptides have excellent thermal stability, storage stability and pH stability, and can adapt to industrial food processing conditions such as steaming, sterilization and storage.

[0098] Example 10 Typical application examples 1. Low-sodium soy sauce Based on regular soy sauce containing 8% NaCl, the NaCl content was reduced to 6.4% (20% salt reduction), and 0.2% GQKES peptide was added to prepare low-salt soy sauce.

[0099] Formula: 85% unsalted brewed soy sauce base, 6.4% NaCl, 0.2% GQKES (purity ≥98%), 0.1% lactic acid, 0.05% citric acid, 1.0% edible alcohol, water balance. Process: Dissolve at 50℃ for 15 min, filter through 100 mesh, sterilize at 85℃ for 15 min, cool and fill.

[0100] The control soy sauce contained 8.0% NaCl, and the rest were the same.

[0101] The sensory evaluation results are shown in Table 22. It can be seen that the experimental group scored 7.3 points for saltiness, 7.8 points for umami, 7.5 points for richness, and 7.9 points for overall palatability, all of which are better than the control group (7.2 points for saltiness, 6.5 points for umami, 6.8 points for richness, and 7.0 points for palatability). A better salty and umami flavor was achieved under the condition of reducing sodium content by 20%.

[0102] Table 22 Application Trial of Low-Salt Soy Sauce

[0103] 2. Low-salt mushroom broth mix Based on a 0.60% NaCl bacterial broth, the NaCl content was reduced to 0.40% (a 33% reduction in salt), and 0.15% ESKDR peptide was added to prepare a low-salt bacterial broth.

[0104] Formula: Salt 0.40%, ESKDR 0.15%, deer antler mushroom powder 5%, yeast extract 2%, maltodextrin 89.45%, spices 3%. Process: Grind all components and mix for 15 minutes, then package into 5 g bags; when preparing, add 5 g to 250 mL of hot water. The control mushroom broth contains 0.60% salt, with the other components identical.

[0105] The sensory evaluation results are shown in Table 23. It can be seen that the experimental group scored 7.1 points for saltiness, 7.7 points for umami, 7.6 points for freshness, and 8.0 points for overall palatability. All of these scores were at or above those of the control group (7.0 points for saltiness, 6.2 points for umami, 6.5 points for freshness, and 7.1 points for palatability). The umami flavor was increased by about 25% while the salt content was reduced by 33%.

[0106] Table 23 Application Trial of Low-Salt Bacterial Broth

[0107] (3) Low-salt minced meat products Based on 0.60% NaCl minced meat, the NaCl content was reduced to 0.42% (salt reduction of 30%), and 0.20% GQKES peptide was added to prepare low-salt minced meat products.

[0108] Formula (100 g): 70 g lean pork, 10 g fatty pork, 6 g starch, 2 g soy protein isolate, 0.42 g salt, 0.2 g GQKES, 11.38 g ice water. Process: Mince → chop and mix → fill → heat to 75℃ and hold for 10 min → cool. Control minced meat contains 0.60 g salt and 11.40 g ice water, all other components are the same.

[0109] The sensory evaluation results are shown in Table 24. It can be seen that the experimental group scored 7.4 points for saltiness, 7.9 points for umami, 7.2 points for juiciness, and 8.1 points for overall acceptability, all of which are better than the control group (7.0 points for saltiness, 6.3 points for umami, 6.5 points for juiciness, and 7.0 points for acceptability). The flavor and taste were significantly improved while reducing salt by 30%.

[0110] Table 24 Application Trials of Low-Salt Minced Meat Products

[0111] The above application examples show that the deer antler mushroom-derived salt-reducing and flavor-enhancing peptides provided by the present invention can achieve a 30% to 50% reduction in salt in different food systems such as soy sauce, soup base, and minced meat products, while maintaining or even enhancing the perception of saltiness and the intensity of umami, making them suitable for a variety of low-salt food systems.

[0112] The salt-reducing and flavor-enhancing peptides of this invention are derived from the edible fungus *Deer Antler Mushroom*, a readily available and highly safe raw material. The preparation process is simple and controllable. The resulting peptides can be widely used in condiments, meat products, convenience foods, soup bases, sauces, and pickled foods, contributing to the development of high-quality foods that meet the health needs of salt reduction, and yielding significant economic and social benefits.

[0113] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A salt-reducing and flavor-enhancing peptide derived from deer antler mushroom, characterized in that, The salt-reducing and flavor-enhancing peptides contain one or more polypeptides with amino acid sequences as shown in SEQ ID NO. 1~5.

2. The method for preparing the salt-reducing and flavor-enhancing peptide according to claim 1, characterized in that, This includes separation and extraction from *Deer Antler Fungus* and / or solid-phase synthesis using Fmoc.

3. The method according to claim 2, characterized in that, The separation and extraction of *Deer Antler Mushroom* includes the following steps: Using deer antler mushroom as raw material, enzymatic hydrolysis with bromelain was performed to obtain deer antler mushroom enzymatic hydrolysate; The enzymatic hydrolysate of the deer antler mushroom was subjected to ultrafiltration treatment, and the permeate with a molecular weight of less than 1 kDa was collected. The permeate was separated by Sephadex G-15 gel filtration chromatography, with water as the mobile phase, and the third fraction was collected in order of peak elution time. The third fraction was purified by reversed-phase high-performance liquid chromatography using acetonitrile-water as the mobile phase and a C18 column, and a single high-purity main peak fraction was collected, which included the salt-reducing and flavor-enhancing peptide.

4. The method according to claim 3, characterized in that, The enzymatic hydrolysis process includes the following steps: Mix deer antler mushrooms with water at a ratio of 1 g: (4~10) mL, add 3~5% of the weight of deer antler mushroom raw material bromelain, and enzymatically hydrolyze for 120~150 min.

5. The method according to claim 3 or 4, characterized in that, The enzyme activity of the bromelain is 450,000 to 550,000 U / g.

6. The application of the salt-reducing and flavor-enhancing peptide according to claim 1 or the salt-reducing and flavor-enhancing peptide prepared by any one of claims 2 to 5 in the preparation of salt-reducing and flavor-enhancing seasonings.

7. A seasoning composition for reducing salt and enhancing flavor, characterized in that, It includes the salt-reducing and flavor-enhancing peptides as described in claim 1, sodium chloride, and a food-grade acceptable carrier or excipient.

8. The flavoring composition according to claim 7, characterized in that, The flavoring composition comprises, by weight percentage, 0.1% to 0.3% of a salt-reducing and flavor-enhancing peptide, 0.30% to 0.42% of sodium chloride, and the remainder being a food-grade acceptable carrier or adjuvant.

9. The flavoring composition according to claim 7 or 8, characterized in that, The flavoring composition also contains monosodium glutamate, wherein the content of monosodium glutamate is less than 0.2%.

10. The use of the salt-reducing and flavor-enhancing peptide of claim 1, or the salt-reducing and flavor-enhancing peptide prepared by the preparation method of any one of claims 2 to 5, or the flavoring composition of any one of claims 7 to 9, in food seasoning.

Images