Kokumi polypeptide, preparation and application thereof

Abstract

The present application relates to the field of food science and technology, and more particularly to a kind of kokumi polypeptide and its preparation and application.The amino acid sequence of the kokumi polypeptide provided in the present application is shown in SEQ ID NO.3;The kokumi polypeptide can be prepared by solid-phase synthesis method or through double-spore mushroom extraction and purification.The present application first discovers and identifies the kokumi polypeptide RHE, which has a significant salty taste enhancing effect;The binding mechanism of polypeptide RHE and ENaC receptor is clarified by molecular docking and molecular dynamics simulation, and the neural mechanism of polypeptide RHE is improved by electroencephalography technology, which provides a theoretical basis for its application in food.

Description

Technical Field

[0001] This invention relates to the field of food science and technology, and in particular to a kokumi polypeptide and its preparation and application. Background Technology

[0002] Excessive sodium intake has been recognized by the World Health Organization as one of the major dietary risk factors leading to hypertension, cardiovascular disease, and increased kidney burden. With the popularization of healthy eating concepts and the promotion of "salt reduction initiatives" worldwide, how to reduce the sodium chloride content in food while maintaining its original saltiness and pleasant flavor has become a core technical challenge that urgently needs to be solved in the field of food science.

[0003] Currently, one of the most common salt reduction strategies employed in industry is the use of salt substitutes such as potassium chloride and magnesium chloride. However, these inorganic salt substitutes have significant sensory defects in practical applications: when the substitution ratio exceeds 30%, they often introduce noticeable bitterness, astringency, or metallic taste, severely impacting the overall flavor acceptability of food. Furthermore, simple salt substitution strategies cannot mimic the unique physiological role of sodium ions in taste transmission, making it difficult to balance "salt reduction" with "tasting good."

[0004] Button mushrooms (Agaricus bisporus), one of the most consumed and widely cultivated edible fungi globally, are rich in high-quality protein, various free amino acids, and flavor precursors, making them an ideal natural source for developing novel functional flavor peptides. Their unique metabolite composition, including various small molecule peptides and flavor nucleotides, provides a high-quality raw material basis for the targeted preparation of kokumi bioactive peptides through enzymatic hydrolysis or fermentation. However, despite limited research reports on salt-reducing peptides, commercially available products generally face two major technical bottlenecks: firstly, the saltiness enhancement is limited, making it difficult to meet the significant salt reduction requirements in actual production; secondly, some peptides are prone to producing bitter peptides during separation and purification, leading to undesirable flavors in the final product and limiting their application in clean-label foods.

[0005] Therefore, developing novel kokumi peptides and conducting in-depth research on their salt-increasing mechanisms has significant scientific and practical value. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a kokumi polypeptide and its preparation and application.

[0007] The objective of this invention can be achieved through the following technical solutions: The first object of the present invention is to provide a kokumi polypeptide, the amino acid sequence of which is shown in SEQ ID NO.3.

[0008] In one embodiment of the present invention, its chemical structural formula is shown below: .

[0009] A second objective of this invention is to provide a method for preparing a kokumi polypeptide, wherein the preparation of the kokumi polypeptide is selected from a solid-phase synthesis method or by extraction and purification via Agaricus bisporus.

[0010] In one embodiment of the present invention, the method for extraction and purification via Agaricus bisporus is as follows: (A1) After cutting the button mushrooms into sections, add water and steam. After steaming, filter and centrifuge in sequence. Dissolve the precipitate and centrifuge. Combine the supernatants and then process to obtain the water-soluble extract of button mushrooms. (A2) The water-soluble extract of Agaricus bisporus prepared in step (A1) is purified to obtain kokumi polypeptide.

[0011] In one embodiment of the present invention, in step (A1), the post-processing is to sequentially perform rotary evaporation concentration, ultrafiltration, and freeze-drying.

[0012] In one embodiment of the present invention, in step (A2), purification is performed using RP-HPLC.

[0013] A third objective of this invention is to provide an application of kokumi polypeptide in the preparation of food additives.

[0014] A fourth objective of this invention is to provide a food additive comprising the aforementioned kokumi polypeptide.

[0015] The fifth object of the present invention is to provide an application of the kokumi polypeptide in the preparation of low-salt foods or compositions.

[0016] A sixth object of the present invention is to provide a low-salt food or composition comprising the aforementioned kokumi polypeptide.

[0017] In one embodiment of the present invention, the food includes low-salt or low-sodium foods; The amount of kokumi peptide added was 0.1%. Compared with the prior art, the present invention has the following beneficial effects: This invention provides a novel, safe, naturally sourced kokumi peptide that enhances saltiness. This kokumi peptide has advantages such as high activity and no off-flavor. This invention is the first to combine molecular simulation and EEG technology to systematically reveal its salt-enhancing mechanism, providing theoretical and technical support for the development of low-salt foods. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the docking pattern between the kokumi peptide and the CaSR receptor molecule. Figure 2 (A) is a schematic diagram of the docking pattern of the kokumi peptide with the ENaC receptor molecule; (B) is a descriptive evaluation analysis diagram showing the results of four synthetic peptides (YWS, WME, RHE, WDND) and the salty standard arginine dipeptide RS in 3 mg / mL and 6 mg / mL NaCl solutions, respectively; (C) is a QDA analysis diagram showing the results of four synthetic peptides (YWS, WME, RHE, WDND) and the salty standard arginine dipeptide RS in 3 mg / mL and 6 mg / mL NaCl solutions, respectively; (E) is a TI analysis diagram showing the results of four synthetic peptides (YWS, WME, RHE, WDND) and the salty standard arginine dipeptide RS in 3 mg / mL and 6 mg / mL NaCl solutions, respectively; (GH) is a QDA analysis diagram showing the results of four synthetic peptides (YWS, WME, RHE, WDND) and the salty standard arginine dipeptide RS in 3 mg / mL and 6 mg / mL NaCl solutions, respectively; The results of electronic tongue analysis of four synthetic peptides (YWS, WME, RHE, WDND) and the salty standard arginine dipeptide RS in NaCl solution are presented in the figure.

[0019] Figure 3 The following figures illustrate the effects of the kokumi peptide in different salt solutions: (A) Root mean square deviation (RMSD) of the synthetic peptide RHE and the salty receptor ENaC. (B) Radius of gyration (Rg) of the synthetic peptide RHE and the salty receptor ENaC. (C) Soluble surface area (SASA) of the synthetic peptide RHE and the salty receptor ENaC. (D) Hydrogen bond results between the synthetic peptide RHE and the salty receptor ENaC. (E) Root mean square fluctuation (RMSF) of the synthetic peptide RHE and the salty receptor ENaC. (F) Free energy landscape (FEL) results of the synthetic peptide RHE and the salty receptor ENaC.

[0020] Figure 4 Results of molecular dynamics simulations of the kokumi peptide-ENaC complex; (RMSD value; Rg value; SASA value; HBonds value; RMSF value; free energy landscape plot). Figure 5 Power spectrum analysis of brain responses to different salty tastes; (A) Power spectrum analysis of brain under water stimulation (PSD); (B) Power spectrum analysis of brain under 3 mg / mL NaCl stimulation (PSD); (C) Power spectrum analysis of EEG under 3 mg / mL NaCl + 0.1% RHE stimulation (PSD); (D) Power spectrum analysis of EEG under 3 mg / mL NaCl + 0.1% RS stimulation (PSD).

[0021] Figure 6 The following are electroencephalogram amplitude analysis diagrams of the brain's response to different salinity levels: (A) Time characteristics of brain neural response under water stimulation (852ms); (B) Time characteristics of brain neural response under 3mg / mL NaCl stimulation (852ms); (C) Time characteristics of brain neural response under 3mg / mL NaCl + 0.1%RHE stimulation (852ms); (D) Time characteristics of brain neural response under 3mg / mL NaCl + 0.1%RS stimulation (852ms).

[0022] Figure 7 This is a graph showing the overall average brain response of the subjects to the taste stimuli of four samples. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0024] In the following embodiments, the chemical structural formula of the kokumi polypeptide is shown below: ; Unless otherwise specified, all reagents used are commercially available, and all detection methods and techniques used are conventional in this field.

[0025] Example 1 This embodiment provides a method for screening kokumi peptides, including the following steps: Fresh button mushrooms were derooted and cut into 0.5-1 cm segments. Deionized water was added at a material-to-liquid ratio of 1:1.5 (W / V), and the mixture was placed in a high-pressure cooking device and cooked at 85-90 kPa and 121 °C for 35 min. The cooking liquid was cooled to room temperature and filtered through a 200-mesh nylon filter. The filtrate was centrifuged at 10000 r / min and 4 °C for 15 min. The supernatant was collected, and the precipitate was dissolved in an appropriate amount of deionized water at room temperature for 2 h. The supernatant was then centrifuged again. The two supernatants were combined and concentrated by rotary evaporation (45 °C, volume reduced to 1 / 10 of the original volume). The mixture was then treated with a 1000 Da ultrafiltration membrane and freeze-dried (-80 °C, 24 h) to obtain a water-soluble extract of button mushrooms, which was stored at -20 °C for later use.

[0026] Peptide identification of Agaricus bisporus aqueous extract was performed using LC-MS / MS, identifying a total of 295 pure peptides. To narrow down the peptide screening range, preliminary screening was conducted, and the screening steps are as follows: Peptides are required to be non-toxic (predicted using the ToxinPred tool), non-allergenic (predicted using the AllerTOP v. 2.0 tool), have good water solubility (predicted using the Innovagen tool), be stable (predicted using the Expasy-ProtParam tool), have potential biological activity, and have a PeptideRanker value greater than 0.5 (predicted using the PeptideRanker tool).

[0027] After the above series of screenings, 18 qualified peptides were finally obtained. These 18 peptides were then molecularly docked with the calcium-sensitive receptor (CaSR). Four peptides (SEQ ID NO.1: YWS; SEQ ID NO.2: WDND; SEQ ID NO.3: RHE; SEQ ID NO.4: WME) showed higher docking scores compared to GSH (binding energy -7.8), which has been confirmed as a kokumi peptide. Their binding energies were -9.1, -8.98, -8.81, and -8.77, respectively. Figure 1 Next, these four kokumi peptides were molecularly docked with the salty taste receptor ENaC to virtually verify whether the four screened kokumi peptides possess salt-enhancing properties. The results showed that the binding energies of these four kokumi peptides to epithelial sodium channels (ENaC) were -9.2, -8.6, -7.9, and -8.4 kcal / mol, respectively. Figure 2 The above results indicate that the four kokumi peptides have a strong affinity for ENaC.

[0028] Example 2 This embodiment provides a method for preparing kokumi peptide, including the following steps: Fresh button mushrooms were derooted and cut into 0.5-1 cm segments. Deionized water was added at a material-to-liquid ratio of 1:1.5 (W / V), and the mixture was placed in a high-pressure cooking device and cooked at 85-90 kPa and 121 °C for 35 min. The cooking liquid was cooled to room temperature and filtered through a 200-mesh nylon filter. The filtrate was centrifuged at 10000 r / min and 4 °C for 15 min. The supernatant was collected, and the precipitate was dissolved in an appropriate amount of deionized water at room temperature for 2 h. The supernatant was then centrifuged again. The two supernatants were combined (at 45 °C, the volume was reduced to 1 / 10 of the original volume), and then treated with a 1000 Da ultrafiltration membrane. After that, the mixture was freeze-dried (-80 °C, 24 h) to obtain a water-soluble extract of button mushrooms, which was stored at -20 °C for later use.

[0029] The water-soluble extract of Agaricus bisporus was purified by RP-HPLC to prepare kokumi peptides: YWS, WDND, RHE, and WME.

[0030] Example 3 This embodiment provides a sensory evaluation of the kokumi peptide and a molecular dynamics simulation of the salty taste receptor ENaC, as detailed below: In this embodiment, the kokumi peptides used—YWS, WDND, RHE, and WME—were prepared by Nanjing Peptide Valley Biotechnology Co., Ltd. using a solid-phase synthesis method.

[0031] Sensory analysis and electronic tongue detection were performed on YWS, WDND, RHE, WME and arginine dipeptide (SEQ ID NO.5: RS, as a control).

[0032] Sensory Evaluation: In accordance with ISO 8586 guidelines, a sensory evaluation team of 10 members (5 men and 5 women, aged 25-28) was recruited from Shanghai University of Applied Technology (Shanghai, China), and they gave informed consent to participate in the sensory testing. The sensory evaluation was approved by the Ethics Committee of Shanghai University of Applied Technology. All members had prior experience in similar sensory experiments and had received at least one year of training. The training used an aqueous solution of 0.08% citric acid, 1% sucrose, 0.01% quinine sulfate, 0.4% sodium chloride, and 0.35% monosodium glutamate as the standard for the five basic tastes: sour, sweet, bitter, salty, and umami. A model chicken broth solution prepared with 1% glutathione was used to train the evaluators' ability to identify "richness." After training, all members of the team were able to accurately distinguish the above six basic taste characteristics.

[0033] The descriptive sensory evaluation of kokumi peptides was conducted using the QDA method. YWS, WDND, RHE, WME, and RS (as controls) were added to blank chicken broth and saline solutions of different concentrations (3 mg / mL NaCl and 6 mg / mL NaCl) to achieve a final peptide concentration of 1 mg / mL. During the evaluation, each evaluator took 10 mL of the solution to be evaluated, tasted it in their mouth for 5 seconds, spat it out, and then rinsed their mouth with water to remove any residual taste. The intensity of five basic tastes (sour, sweet, bitter, salty, and umami) and three taste-related attributes (richness, fullness, and continuity) were recorded on a 15-point scale (0 points represent no corresponding taste, 15 points represent an extremely strong taste; 3 mg / mL NaCl and 6 mg / mL NaCl represent 5 and 10 points respectively for saltiness). Each solution was evaluated three times, with a one-hour interval between each evaluation.

[0034] The saltiness-enhancing effect of kokumi peptide was assessed using a time-intensity (TI) method. First, standard control solutions of 3 mg / mL NaCl and 6 mg / mL NaCl were prepared. Then, YWS, WDND, RHE, WME, and RS (as controls) were added to these two standard control solutions and dissolved to ensure a final peptide concentration of 1 mg / mL. Before the experiment, evaluators rinsed their mouths with water, then held 10 mL of the prepared solution in their mouths for 5 seconds before spitting it out. From the spitting-out moment, the intensity of the taste properties produced by the solution in the oral cavity was continuously evaluated at preset time intervals (5, 10, 20, 30, 45, 60, 75, 90, 120, 150, 180, 210, and 240 s) until the taste completely disappeared. According to the 15-point scale (0 points represent no corresponding taste, 15 points represent an extremely strong taste, where 3 mg / mL NaCl and 6 mg / mL NaCl represent 5 points and 10 points of salinity standards, respectively), each sample needs to be evaluated three times, and the sample solution is provided to the evaluator in a random order each time.

[0035] Electronic Tongue: Taste measurement experiments were conducted using a TS-5000Z electronic tongue. Six types of sensors were used: an AAE sensor for umami, a CTO sensor for saltiness, a CA0 sensor for sourness, a CO0 sensor for bitterness, an AE1 sensor for astringency, and a GL1 sensor for sweetness. For sample preparation, YWS, WDND, RHE, WME, and RS were dissolved at 1 mg / mL in 3 mg / mL and 6 mg / mL NaCl solutions, respectively. These solutions served as blank control groups. Each sample was measured four times using the electronic tongue. Due to the variability of the initial measurement data, the first reading was discarded, and the remaining three measurements were considered valid. During the measurement, the intensity of five basic tastes (sour, sweet, bitter, salty, and umami), as well as the intensity values ​​of three taste-related attributes—richness, fullness, and continuity—were recorded simultaneously.

[0036] The results are as follows Figure 3 As shown, through Figure 3 It can be observed that the kokumi peptide RHE has the best saltiness-enhancing effect, significantly increasing the saltiness of the salt solution. Electronic tongue detection also shows that it can significantly increase the salinity of the salt solution itself.

[0037] Molecular dynamics simulations of the salty taste receptor ENaC, such as Figure 4 As shown, through Figure 4It was observed that during molecular dynamics simulations, RHE exhibited high stability when binding to the ENaC protein receptor, with minimal structural changes in the complex and good hydrogen bonding. Therefore, the small molecule demonstrates good binding to the target protein.

[0038] Example 4 This embodiment provides a study on the neural mechanism of the kokumi polypeptide RHE, as detailed below: Participants in the EEG experiment: This study included 10 participants aged 20-30 years, all of whom were right-handed, with no olfactory or gustatory dysfunction, and no smoking or drinking habits. Participants with a history of neurological diseases, head trauma, long-term medication use, or gustatory / olfactory disorders currently in an acute phase were excluded. All participants were required to fast, abstain from alcohol, and refrain from smoking for one hour prior to the test. Basic information such as gender, age, height, and weight was collected during the study. The entire experiment was conducted in a dedicated EEG testing room, which was required to be safe, quiet, and odorless, with the room temperature controlled at (21±1)℃. Before the experiment officially began, researchers explained the experimental procedure to the participants in detail through a questionnaire, covering information such as whether they had previous EEG research experience, the expected duration and cycle of the experiment, etc. After fully understanding the information, all participants signed informed consent forms confirming their voluntary participation in this study.

[0039] Stimulus selection: Pure water was used as a negative control, and 3 mg / mL NaCl solution was used as a positive control. Experimental group 1 used kokumi peptide RHE (3 mg / mL NaCl + 1 mg / mL RHE, both are final concentrations), and experimental group 2 used the salty standard arginine dipeptide RS (3 mg / mL NaCl + 1 mg / mL RS, both are final concentrations).

[0040] EEG device: The device used is the EEG-1200C electroencephalogram manufactured by Nippon Photonics Co., Ltd. The sampling frequency of the EEG signal is 1000 Hz, and it is used with the YM-23 gel-free, no-wash EEG cap (also known as saline cap).

[0041] EEG pre-test: EEG signals were collected from subjects in different scenarios, including at rest, with eyes open / closed, swallowing, eye movements, smiling, and changes in inner emotions. The core purpose of this step is to complete preliminary monitoring and status assessment by observing the subjects' behavior and emotional fluctuations at rest.

[0042] Experimental Procedure: After donning a saline cap, subjects maintained a normal respiratory rate, were awake and not asleep, and the EEG device began recording signals once the baseline stabilized. Simultaneously with biomarker recording, the sample was rapidly injected into the oral cavity (1-2 seconds). The sample remained stationary in the oral cavity for 30 seconds, during which time the tongue remained relatively still to record the taste changes induced by the taste stimulus. The experimental design included a negative control group, a positive control group, and an experimental group, with a total of four stimulation groups, each repeated ten times. To prevent interactions between different stimuli, previous stimuli were rinsed three times and subjects were allowed to rest for 5 minutes before testing the next stimulus.

[0043] Data Analysis: The waveforms of the raw EEG signals were preprocessed using EEGLAB software, including filtering, removal of electrooculogram (EOG) artifacts, independent component analysis (ICA), and rereference. The preprocessed EEG data were segmented into time segments of 0–1000 ms post-stimulation. First, time-frequency domain feature analysis was performed, and repeated measures ANOVA was conducted on the EEG amplitude. Different salty stimulus types were used as between-group factors, and time windows (0–100 ms to 900–1000 ms, a total of 10 windows) were used as within-group factors to examine the interaction effect of these two factors on amplitude, clarifying the dynamic differences in neural responses to different salty stimuli over time. Simultaneously, two-way ANOVA was performed on the power spectral density at 2 Hz, 6 Hz, 10 Hz, 22 Hz, and 40 Hz to examine the main effects and interaction between stimulus type and frequency. For brain topography data, repeated measures ANOVA with electrode points as within-group factors was used to compare amplitude differences in brain regions such as the frontal, central, and parietal regions under different stimulation conditions; the statistical significance level was set at p<0.05.

[0044] The neurological mechanism of action of the peptide RHE was discovered through electroencephalography (EEG) experiments. Figure 5 The power spectral density (PSD) of the brain in response to four stimuli was measured. The results showed that RHE, upon the addition of 3 mg / mL NaCl solution, significantly enhanced neural activity in the parietal lobe region of the brain at the 10 Hz frequency band, manifested as a higher power density signal. This indicates that the kokumi peptide RHE can strengthen the neural response in the core brain region responsible for salty taste perception, thereby enhancing the subjective experience of saltiness. Figure 6 The temporal maps of the brain in response to the four stimuli further confirmed that the ERP waveforms of the RHE group showed a significant positive amplitude increase after stimulation, and the high response area was still concentrated in the central-parietal brain region. This indicates that the kokumi peptide RHE mainly achieves its saltiness-enhancing effect by increasing the neural activity intensity of the core salty brain region, rather than expanding the response range. Figure 7A topographic map showing the evolution of the average EEG signal intensity of the brain in response to four stimuli over time revealed that the kokumi peptide RHE did not alter the latency of salty taste perception, but significantly prolonged the activation duration in the core brain regions (central-apical), increasing it from approximately 700 ms in the positive control to over 900 ms. This mechanism differs from that of the control peptide RS, which primarily enhances salty taste perception by delaying latency and expanding the range of responding brain regions. These EEG experimental data objectively confirm the true effectiveness of this peptide at the neurosensory level, overcoming the inherent subjectivity of sensory evaluation and providing a new dimension for quality control in the standardized food industry.

[0045] In summary, this invention is the first to discover and identify the kokumi polypeptide RHE, which has a significant saltiness-enhancing effect; the binding mechanism of polypeptide RHE to the ENaC receptor was elucidated through molecular docking and molecular dynamics simulation; and the neural mechanism of polypeptide RHE was further refined through electroencephalography (EEG) technology, providing a theoretical basis for its application in food.

[0046] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A kokumi polypeptide, characterized in that, The amino acid sequence of the kokumi polypeptide is shown in SEQ ID NO.

3.

2. The kokumi polypeptide according to claim 1, characterized in that, Its chemical structural formula is shown below: 。 3. A method for preparing the kokumi polypeptide as described in claim 1, characterized in that, The kokumi polypeptide was prepared by solid-phase synthesis or by extraction and purification from Agaricus bisporus.

4. The method for preparing a kokumi polypeptide according to claim 3, characterized in that, The specific method for extraction and purification from Agaricus bisporus is as follows: (A1) After cutting the button mushrooms into sections, add water and steam. After steaming, filter and centrifuge in sequence. Dissolve the precipitate and centrifuge. Combine the supernatants and then process to obtain the water-soluble extract of button mushrooms. (A2) The water-soluble extract of Agaricus bisporus prepared in step (A1) is purified to obtain kokumi polypeptide.

5. The method for preparing a kokumi polypeptide according to claim 4, characterized in that, In step (A1), the post-processing involves sequentially performing rotary evaporation concentration, ultrafiltration, and freeze-drying.

6. The method for preparing a kokumi polypeptide according to claim 4, characterized in that, In step (A2), purification is performed using RP-HPLC.

7. The use of the kokumi polypeptide as described in claim 1 in the preparation of food additives.

8. A food additive, characterized in that, Includes the kokumi polypeptide as described in claim 1.

9. The use of the kokumi polypeptide as described in claim 1 in the preparation of low-salt foods or compositions.

10. A low-salt food or composition, characterized in that, It contains the kokumi polypeptide as described in claim 1.

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