Edible mushroom polypeptide composition, and directional enrichment method and application thereof

Abstract

This invention discloses an edible fungus polypeptide composition, its targeted enrichment method, and its application, relating to the field of food biology. The targeted enrichment method includes the following steps: pretreatment of the edible fungus substrate; pulsed electric field pretreatment and inoculation; constant temperature and humidity solid-state fermentation; ultrasonic-microwave synergistic extraction and inactivation; and multi-stage targeted separation and enrichment. The edible fungus polypeptide composition includes the umami peptide AARTA. The invention also discloses the application of the edible fungus polypeptide composition in food flavoring. This invention is the first to discover that the extract of edible fungi fermented with *Aspergillus cristatus* contains the umami peptide AARTA. By combining pulsed electric field pretreatment with *Aspergillus cristatus* fermentation, and using two-stage ultrafiltration for fractional enrichment, the targeted enrichment of the specific hexapeptide AARTA in the fermentation product of edible fungi is achieved, which can be applied to food flavoring.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, specifically to an edible fungus polypeptide composition and its targeted enrichment method and application. Background Technology

[0002] Umami is the fifth basic taste recognized internationally, following sour, sweet, bitter, and salty, and plays an important role in food flavor design. Natural umami peptides, due to their safe origin, natural flavor, and diverse functions, have become a research hotspot in the food flavoring field and are considered an important direction for replacing chemically synthesized flavor enhancers.

[0003] King oyster mushroom (Pleurotus eryngii) is one of the most produced edible fungi in my country. It is rich in protein (18%–35% dry basis), polysaccharides, minerals, and various umami amino acids (glutamic acid, aspartic acid, etc.), making it a high-quality raw material for preparing natural umami peptides. However, the cell walls of king oyster mushrooms are mainly composed of cellulose, hemicellulose, and chitin, resulting in a dense structure that limits protein extraction rate and enzymatic hydrolysis efficiency.

[0004] *Eurotium cristatum* (commonly known as "Golden Flower Fungus") is a unique probiotic found in Fu brick tea. It is listed in the national food safety standards published by the National Health Commission of China and is classified as a food-grade safe microorganism. During its growth, *Eurotium cristatum* secretes abundant extracellular proteases (including acidic and neutral proteases), cellulases, and chitinases, effectively degrading polysaccharides in the cell walls of edible fungi and hydrolyzing plant proteins into small polypeptides and amino acids, demonstrating unique biotransformation capabilities.

[0005] However, the existing technology for preparing umami substances by fermenting king oyster mushrooms with *Aspergillus cristatus* presents the following key technical problems: the cell walls of king oyster mushrooms are dense, making it difficult for *Aspergillus cristatus* to fully penetrate the matrix, limiting protease secretion, resulting in low protein degradation rate and insufficient umami peptide yield; the fermentation products contain a wide variety of small molecule peptides with complex composition, and the content of the target umami peptide ATA is extremely low, lacking a targeted enrichment process for this specific peptide segment.

[0006] Traditional water bath extraction methods are time-consuming (1-2 hours) and inefficient, and prolonged high-temperature extraction easily leads to the loss of activity of heat-sensitive umami peptides; existing separation and purification processes lack specificity for ATATA (m / z=589.3184, [M+H)). + ) has the ability to target and collect information in a specific direction. Summary of the Invention

[0007] The purpose of this invention is to provide an edible fungus polypeptide composition and its targeted enrichment method and application. By combining pulsed electric field pretreatment with *Aspergillus cristatus* fermentation and two-stage ultrafiltration fractional enrichment, the targeted enrichment of the specific hexapeptide AARTA in edible fungus fermentation products is achieved, which can be applied to food flavoring.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A method for targeted enrichment of edible fungal polypeptide compositions includes the following steps:

[0010] S100. Mix the dried edible fungus powder with distilled water evenly, sterilize with high-temperature steam, and cool to obtain the sterilized edible fungus substrate.

[0011] S200. The sterilized edible fungus substrate is first pretreated with a pulsed electric field, and then inoculated with a suspension of *Aspergillus cristatus* bacteria, stirred evenly, to obtain the inoculated substrate.

[0012] S300: Place the inoculated substrate in an artificial climate chamber and ferment until the surface and interior of the substrate are covered with golden yellow closed capsules to obtain the fermentation product.

[0013] S400. After inactivating the fermentation product, add ultrapure water and extract using ultrasound-microwave combined extraction. After extraction, centrifuge to collect the supernatant to obtain crude polypeptide solution.

[0014] S500 and crude polypeptide solution are subjected to two-stage ultrafiltration. The ultrafiltration components are collected and freeze-dried to obtain an edible fungus polypeptide composition enriched with these components.

[0015] The inventors, during their systematic study of *Aspergillus cristatus* fermentation of *Pleurotus eryngii*, discovered that by employing pulsed electric field pretreatment combined with optimized fermentation parameters, the hexapeptide with the amino acid sequence Ala-Thr-Ala-Arg-Thr-Ala (ATARTA) could be significantly enriched in the fermentation product. AATARTA is not present in unfermented *Pleurotus eryngii*, but is only produced by the hydrolysis of *Pleurotus eryngii* proteins by *Aspergillus cristatus* proteases under specific fermentation conditions. Currently, research on targeted enrichment processes for AATARTA is lacking.

[0016] This invention constructs a complete process chain of sterilization of dried Pleurotus eryngii powder → pulsed electric field pretreatment → Aurorum cristatum inoculation and fermentation → ultrasonic-microwave synergistic extraction → two-stage ultrafiltration → freeze drying, achieving targeted enrichment of the specific hexapeptide ATA.

[0017] This invention places pulsed electric field pretreatment before the fermentation process. By altering the microstructure of the Pleurotus eryngii substrate through the physical field effect of the pulsed electric field, it creates more favorable conditions for the subsequent invasion and enzyme hydrolysis by Aspergillus cristatus. This synergistic model of physical field pretreatment and microbial directional fermentation is unprecedented in the preparation of edible fungi peptides.

[0018] This invention achieves the targeted enrichment of the ATATA hexapeptide with a specific amino acid sequence. Existing reports on umami peptides from *Pleurotus eryngii* mainly include sequences such as VIIIIH, FVPISGW, and VIILIH, and immunomodulatory active peptides such as DFPALR and LLGVD have also been disclosed. However, the sequence Ala-Thr-Ala-Arg-Thr-Ala has not been recorded in any currently published literature or patents.

[0019] The unique feature of this peptide is that it is not an endogenous substance naturally present in *Pleurotus eryngii*, and is completely absent in unfermented samples. It only appears after pretreatment with a pulsed electric field and a specific fermentation process using *Aspergillus cristatus*. This means that the production of this peptide depends on the specific recognition between the strain and the substrate, the targeted cleavage of *Pleurotus eryngii* proteins by the protease, and the precise control of fermentation environmental parameters on the enzymatic hydrolysis process. Furthermore, there are many uncertainties regarding the strain-substrate matching relationship, the selectivity of the cleavage site, and the accumulation conditions of the target peptide. The causal relationship cannot be predicted through conventional reasoning or limited exploratory experiments.

[0020] The extraction process employs a synergistic extraction mode combining ultrasonic power (200-300W) and microwave power (300-400W), primarily to ensure smooth process flow and improve extraction efficiency. For product enrichment, this invention utilizes a two-stage ultrafiltration scheme, sequentially passing the crude peptide solution through ultrafiltration membranes with molecular weight cutoffs of 3kDa and 1kDa, selectively collecting ultrafiltration fractions smaller than 1kDa. As a hexapeptide, ATATA has a molecular weight of approximately 600Da, falling precisely within this range. While existing technologies employ multi-stage ultrafiltration for peptide fractionation, the molecular weight cutoffs are mostly concentrated at conventional levels such as 100kDa, 10kDa, and 3kDa. This invention sets the cutoff precision at the 1kDa level and employs a two-stage tandem cutoff strategy of 3kDa and 1kDa, resulting in more targeted fractionation accuracy and focus. Essentially, it is a refined enrichment method designed specifically for the molecular weight characteristics of the target peptide.

[0021] Further, in step S100, fresh edible fungi slices are taken, dried with hot air at 60℃~70℃ to constant weight, and pulverized through a 60~80 mesh sieve to obtain dried edible fungi powder; the dried edible fungi powder and distilled water are mixed at a material-to-liquid ratio of 1:1~2.5 to make the moisture content of the edible fungi substrate reach 45%~55%; the edible fungi include king oyster mushroom.

[0022] Furthermore, in step S200, the electric field strength of the pulsed electric field is 10~15kV / cm, the number of pulses is 30~50, and the pulse width is 2~5μs.

[0023] Further, in step S200, the concentration of the *Eurotium cristatum* bacterial suspension is 10. 6 ~10 7 CFU / mL, inoculation amount is 5%~10%.

[0024] Furthermore, in step S300, the temperature of the artificial climate chamber is 28℃~30℃, the relative humidity is 85%~90%, and the fermentation time is 10~15 days.

[0025] Furthermore, in step S400, the inactivation temperature of the fermentation product is 90℃~100℃, and the inactivation time is 15~20 minutes; the ultrasonic power is 200~300W, the microwave power is 300~400W, and the extraction time is 30~60 minutes; the ratio of fermentation product to ultrapure water is 1:10~20.

[0026] Further, in step S500, the two-stage ultrafiltration includes the following: passing the crude polypeptide solution sequentially through ultrafiltration membranes with molecular weight cutoffs of 3kDa and 1kDa, and collecting ultrafiltration components with molecular weight cutoffs less than 1kDa.

[0027] An edible fungus polypeptide composition obtained by the directional enrichment method described above, wherein the edible fungus polypeptide composition includes the umami peptide ATATA.

[0028] Application of the aforementioned edible fungus polypeptide composition in food seasoning.

[0029] Furthermore, the food products include compound seasonings, meat products, plant protein foods, and pre-prepared foods.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] 1. This invention is the first to discover that the extract of *Pleurotus eryngii* fermented with *Aurorus cristatus* contains the umami peptide ATA. Pretreatment with a pulsed electric field of 10-15 kV / cm effectively disrupts the dense structure of the *Pleurotus eryngii* cell wall, allowing *Aurorus cristatus* to more fully penetrate the matrix and secrete proteases, significantly promoting ATA production. Through optimized fermentation process parameters and multi-stage separation and purification conditions, the ATA content in the final composition is stable. Furthermore, the extract of *Pleurotus eryngii* not fermented with *Aurorus cristatus* does not contain ATA, indicating that ATA is entirely derived from the *Aurorus cristatus* fermentation process.

[0032] 2. This invention utilizes the synergistic effect of ultrasonic cavitation and microwave thermal effects to shorten the extraction time from 1-2 hours in the traditional water bath method to 30-60 minutes. Furthermore, the process parameters of this invention are clearly defined, exhibiting good repeatability, good batch-to-batch stability, and are easily scaled up for production. Attached Figure Description

[0033] Figure 1 The UHPLC-QTOF-MS / MS mass spectrum of ATATA obtained in Example 1 of this invention;

[0034] Figure 2 This is a bar chart comparing the ATA content in Example 1 and Comparative Examples 1-3 of the present invention. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to embodiments and comparative examples, but the scope of protection of the present invention is not limited thereto. The king oyster mushrooms used in the embodiments were purchased from a local farmers' market, and *Aspergillus cristatus* CICC 2422 was purchased from the China Industrial Microbial Culture Collection Center. UHPLC-QTOF-MS / MS analysis was performed using a Waters ACQUITY UPLC system equipped with a Xevo G2-XS QTOF mass spectrometer, with data acquisition in positive ion mode (ESI+). ATATA content was quantified using external standard method high-performance liquid chromatography.

[0036] Example 1

[0037] 1. Pretreatment of King Oyster Mushroom Substrate

[0038] Take fresh king oyster mushrooms (moisture content approximately 92%), wash them, and slice them into thin slices 4±1 mm thick. Dry them in hot air at 65℃ until constant weight (approximately 8 hours), then pulverize and pass them through a 70-mesh sieve to obtain dried king oyster mushroom powder (moisture content ≤8%). Mix the dried king oyster mushroom powder with distilled water at a material-to-liquid ratio of 1:1.8 (m / v) to achieve a moisture content of 50%. Pour the mixture into 500 mL Erlenmeyer flasks, filling each flask with 100 g (based on dry powder). Seal the flasks with cotton plugs and autoclave them at 121℃ for 20 minutes. Cool to room temperature before use.

[0039] 2. Pulsed electric field pretreatment and inoculation

[0040] The sterilized king oyster mushroom substrate was transferred to a pulsed electric field treatment chamber, where the electric field strength was set to 12 kV / cm, the number of pulses to 40, and the pulse width to 3 μs for pretreatment. The activated slant of *Aspergillus cristatus* CICC2422 was rinsed with sterile physiological saline (0.9% NaCl), counted using a hemocytometer, and the spore concentration was adjusted to 5 × 10⁻⁶. 6 CFU / mL. Inoculate the bacterial suspension into the pretreated king oyster mushroom substrate at an inoculation rate of 8% (v / w), mix thoroughly, and then reseal with cotton plugs.

[0041] 3. Constant temperature and humidity solid-state fermentation

[0042] The inoculated substrate was placed in an artificial climate chamber (model: RXZ-380B) for solid-state fermentation, with the temperature controlled at 29℃, relative humidity at 88%, and natural light. The fermentation cycle was 12 days, during which the substrate was turned over every 3 days to ensure uniform aeration. The fermentation endpoint was determined by the uniform distribution of golden-yellow cleistothecia (characteristic morphology of *Eurotium cristatum*) on the surface and inside of the substrate.

[0043] 4. Ultrasonic-microwave synergistic extraction and inactivation

[0044] After fermentation, the fermentation product was heated in a 95℃ water bath for 18 minutes to kill *Aspergillus cristatus* and inactivate the protease (to ensure that ATATA is not further hydrolyzed during subsequent separation). Ultrapure water was added at a material-to-liquid ratio of 1:15 (m / v), and the mixture was transferred to an ultrasonic-microwave co-extraction instrument (model: SCIENTZ-VIIP). The ultrasonic power was set to 250W, the microwave power to 350W, the extraction time to 45 minutes, and the extraction temperature to be controlled below 50℃. After extraction, the mixture was centrifuged at 9000 r / min for 15 minutes (4℃), and the supernatant was collected and filtered through a 0.45 μm microporous membrane to obtain the crude peptide broth from *Pleurotus eryngii* fermentation.

[0045] 5. Multi-level targeted separation and enrichment

[0046] The crude polypeptide solution was sequentially passed through polyethersulfone ultrafiltration membranes with molecular weight cutoff (MWCO) of 3 kDa and 1 kDa (operating pressure 0.20 MPa, operating temperature 25 °C). The ultrafiltration fraction with MWCO <1 kDa was collected, pre-frozen at -80 °C, and then freeze-dried for 72 hours to obtain ultrafiltration lyophilized powder.

[0047] The ultrafiltration lyophilized powder was reconstituted to a concentration of 10 mg / mL and loaded onto a Sephadex G-15 gel chromatography column (2.6 cm × 60 cm, GE Healthcare). Ultrapure water was used as the eluent, the flow rate was 1.0 mL / min, the column temperature was 25 °C, and the column was monitored at 214 nm using a UV detector. The elution peak fraction with the strongest umami activity (based on electronic tongue umami score) was collected, combined, and lyophilized.

[0048] After reconstitution of the above-mentioned gel chromatography active components, the sample was loaded onto a C18 semi-preparative high-performance liquid chromatography column (10 mm × 250 mm, 5 μm, Waters). Gradient elution was performed using acetonitrile (phase A) – ultrapure water containing 0.1% trifluoroacetic acid (phase B): 0–30 min, phase A 5% → 30%; flow rate 3.0 mL / min; detection wavelength 214 nm. Based on the pre-determined AARTA retention time using UHPLC-QTOF-MS / MS, the corresponding characteristic target peaks were collected directionally, combined, and freeze-dried to obtain the composition enriched with the umami peptide AARTA.

[0049] 6. Confirmation of ATATA Structure

[0050] The purified fraction was dissolved in 0.1% formic acid aqueous solution and analyzed by UHPLC-QTOF-MS / MS.

[0051] Chromatographic conditions: ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm), flow rate 0.3 mL / min, gradient elution (water / acetonitrile, containing 0.1% formic acid). Mass spectrometry conditions: ESI positive ion mode, capillary voltage 3.5 kV, cone voltage 40 V, collision energy 20–40 eV, scan range m / z 50–2000.

[0052] Measurement results: such as Figure 1 As shown, the measured mass-to-charge ratio of the precursor ion [M+H]⁺ = 589.3184, while the theoretical value is 589.3199 (Ala-Thr-Ala-Arg-Thr-Ala), with a mass error of 2.5 ppm (<5 ppm, meeting the accuracy requirements of the QTOF instrument). Each amino acid residue was individually confirmed using secondary fragment ion sequences (b / y ion series), confirming the amino acid sequence as Ala-Thr-Ala-Arg-Thr-Ala (ATARTA).

[0053] 7. Determination of ATA content

[0054] Quantitative analysis was performed using external standard HPLC: a standard curve was established using synthetic ATATA standard (purity ≥98%, HPLC method), with a linear range of 0.01–1.0 mg / mL. 2 =0.9997. After three parallel experiments (n=3), the ATA content in the obtained compositions was 0.62%, 0.88%, and 0.91%, respectively, with a mean of 0.80% and an RSD of 19.7% (inter-batch variation is acceptable).

[0055] Example 2

[0056] In step (2), the pulse electric field strength was adjusted to 10 kV / cm, the number of pulses was 30, and the pulse width was 2 μs. The remaining steps were the same as in Example 1. The ATATA content in the resulting composition was detected to be 0.45% (single experiment), slightly lower than in Example 1, but still detectable, indicating that the 10 kV / cm electric field strength is at the lower limit of the scope of the claims of this invention and has a certain effect.

[0057] Example 3

[0058] In step (2), the pulse electric field intensity was adjusted to 15 kV / cm, the number of pulses was 50, and the pulse width was 5 μs. The remaining steps were the same as in Example 1. The ATATA content in the obtained composition was detected to be 0.83% (single experiment), which is similar to the result of Example 1, indicating that the process is stable and effective within the upper limit of the scope of the claims of this invention.

[0059] Example 4

[0060] 1. Pretreatment of King Oyster Mushroom Substrate

[0061] Take fresh king oyster mushrooms (moisture content approximately 92%), wash them, and slice them into thin slices 4±1 mm thick. Dry them in hot air at 60℃ until constant weight (approximately 8 hours), then pulverize and pass them through a 60-mesh sieve to obtain king oyster mushroom powder (moisture content ≤8%). Mix the king oyster mushroom powder with distilled water at a material-to-liquid ratio of 1:1 (m / v) to achieve a moisture content of 45%. Pour the mixture into 500 mL Erlenmeyer flasks, filling each flask with 100 g (based on dry powder). Seal the flasks with cotton plugs and autoclave them at 121℃ for 20 minutes. Cool to room temperature before use.

[0062] 2. Pulsed electric field pretreatment and inoculation

[0063] The sterilized *Pleurotus eryngii* substrate was transferred to a pulsed electric field treatment chamber. The electric field strength was set to 10 kV / cm, the number of pulses to 30, and the pulse width to 2 μs for pretreatment. *Aspergillus cristatus* CICC2422 activation slant was rinsed with sterile physiological saline (0.9% NaCl), counted using a hemocytometer, and the spore concentration was adjusted to 1 × 10⁻⁶. 6 CFU / mL. Inoculate the bacterial suspension into the pretreated king oyster mushroom substrate at an inoculation rate of 5% (v / w), mix thoroughly, and then reseal with cotton plugs.

[0064] 3. Constant temperature and humidity solid-state fermentation

[0065] The inoculated substrate was placed in an artificial climate chamber (model: RXZ-380B) for solid-state fermentation, with the temperature controlled at 28℃, relative humidity at 85%, and natural light. The fermentation cycle was 10 days, during which the substrate was turned over every 3 days to ensure uniform aeration. The fermentation endpoint was determined by the uniform distribution of golden-yellow cleistothecia (characteristic morphology of *Eurotium cristatum*) on the surface and inside of the substrate.

[0066] 4. Ultrasonic-microwave synergistic extraction and inactivation

[0067] After fermentation, the fermentation product was heated in a 90℃ water bath for 15 minutes to kill *Aspergillus cristatus* and inactivate the protease (to ensure that ATATA is not further hydrolyzed during subsequent separation). Ultrapure water was added at a material-to-liquid ratio of 1:10 (m / v), and the mixture was transferred to an ultrasonic-microwave co-extraction instrument (model: SCIENTZ-VIIP). The ultrasonic power was set to 200W, the microwave power to 300W, the extraction time to 30 minutes, and the extraction temperature to be controlled below 50℃. After extraction, the mixture was centrifuged at 8000 r / min for 15 minutes (4℃), and the supernatant was collected and filtered through a 0.45 μm microporous membrane to obtain the crude peptide liquid from *Pleurotus eryngii* fermentation.

[0068] The rest is the same as in Example 1.

[0069] The ATA content in the obtained composition was measured to be 0.41% (single experiment).

[0070] Example 5

[0071] 1. Pretreatment of King Oyster Mushroom Substrate

[0072] Take fresh king oyster mushrooms (moisture content approximately 92%), wash them, and slice them into thin slices 4±1 mm thick. Dry them in hot air at 70℃ until constant weight (approximately 8 hours), then pulverize and pass them through an 80-mesh sieve to obtain dried king oyster mushroom powder (moisture content ≤8%). Mix the dried king oyster mushroom powder with distilled water at a material-to-liquid ratio of 1:2.5 (m / v) to achieve a moisture content of 55%. Pour the mixture into 500 mL Erlenmeyer flasks, filling each flask with 100 g (based on dry powder). Seal the flasks with cotton plugs and autoclave them at 121℃ for 20 minutes. Cool to room temperature before use.

[0073] 2. Pulsed electric field pretreatment and inoculation

[0074] The sterilized king oyster mushroom substrate was transferred to a pulsed electric field treatment chamber, where the electric field strength was set to 15 kV / cm, the number of pulses to 50, and the pulse width to 5 μs for pretreatment. The activated slant of *Aspergillus cristatus* CICC2422 was rinsed with sterile physiological saline (0.9% NaCl), counted using a hemocytometer, and the spore concentration was adjusted to 1 × 10⁻⁶. 7 CFU / mL. Inoculate the bacterial suspension into the pretreated king oyster mushroom substrate at an inoculation rate of 10% (v / w), mix thoroughly, and then reseal with cotton plugs.

[0075] 3. Constant temperature and humidity solid-state fermentation

[0076] The inoculated substrate was placed in an artificial climate chamber (model: RXZ-380B) for solid-state fermentation, with the temperature controlled at 30℃, relative humidity at 90%, and natural light. The fermentation period was 15 days, during which the substrate was turned over every 3 days to ensure uniform aeration. The fermentation endpoint was determined by the uniform distribution of golden-yellow cleistothecia (characteristic morphology of *Eurotium cristatum*) on the surface and inside of the substrate.

[0077] 4. Ultrasonic-microwave synergistic extraction and inactivation

[0078] After fermentation, the fermentation product was heated in a 100℃ water bath for 20 minutes to kill *Aspergillus cristatus* and inactivate the protease (to ensure that ATATA is not further hydrolyzed during subsequent separation). Ultrapure water was added at a material-to-liquid ratio of 1:20 (m / v), and the mixture was transferred to an ultrasonic-microwave co-extraction instrument (model: SCIENTZ-VIIP). The ultrasonic power was set to 300W, the microwave power to 400W, the extraction time to 60 minutes, and the extraction temperature to be controlled below 50℃. After extraction, the mixture was centrifuged at 10000 r / min for 15 minutes (4℃), and the supernatant was collected and filtered through a 0.45 μm microporous membrane to obtain the crude peptide broth from *Pleurotus eryngii* fermentation.

[0079] The rest is the same as in Example 1.

[0080] The ATA content in the obtained composition was measured to be 0.85% (single experiment).

[0081] Comparative Example 1

[0082] Without performing the pulsed electric field pretreatment in step 2, the remaining steps were identical to those in Example 1. The ATATA content of the obtained composition was determined using the same HPLC external standard method as in Example 1. The results showed that ATATA was completely undetectable (below the detection limit of 0.01 mg / mL), i.e., the content was 0. Because the pulsed electric field pretreatment disrupts the dense structure of the Pleurotus eryngii cell wall through electroporation, it is a prerequisite for the full contact between the Aspergillus cristatus protease and the substrate protein, leading to hydrolysis and the production of ATATA. Without this step, the ATATA generation process is completely inhibited.

[0083] Comparative Example 2

[0084] The ultrasonic-microwave synergistic extraction in step 4 was replaced with a 60°C water bath oscillation extraction (100 rpm, 2 hours), with the remaining steps identical to those in Example 1. ATATA was also undetectable in the resulting composition (below the detection limit of 0.01 mg / mL). Because traditional water bath extraction involves a high temperature (60°C, 2 hours) and lacks the assistance of ultrasonic cavitation, ATATA undergoes thermal denaturation or binds to matrix components during extraction, ultimately failing to be effectively recovered. Ultrasonic-microwave synergistic extraction, at a lower temperature (≤50°C), efficiently releases ATA through physical cavitation, which is a key factor in ensuring its recovery rate.

[0085] Comparative Example 3

[0086] The inoculation process in step 2 and the fermentation process in step 3 were omitted. Instead, the sterilized and pulsed electric field-treated *Pleurotus eryngii* substrate was directly subjected to ultrasonic-microwave extraction. The remaining steps were identical to those in Example 1. ATATA was not detected in the resulting composition (below the detection limit of 0.01 mg / mL). This is because ATATA is absent in unfermented *Pleurotus eryngii* substrate and is a specific product of the hydrolysis of *Pleurotus eryngii* proteins by a specific protease secreted by *Aspergillus cristatus* CICC2422. Its formation is entirely dependent on the fermentation process of this invention.

[0087] Application Example 1

[0088] The ATA-enriched umami peptide composition obtained in Example 1 was added to the compound seasoning base at an addition amount of 0.5%. After evaluation by a professional sensory evaluation group (n=10), the umami intensity was significantly improved compared with the group without addition (p<0.05), and was comparable to the umami intensity of the 0.3% monosodium glutamate group. The aftertaste was more mellow and lasting, without bitterness. It is suitable for enhancing the umami flavor of compound seasonings, meat products, plant protein foods and ready-made dishes.

Claims

1. A method for targeted enrichment of edible fungal polypeptide compositions, characterized in that, Includes the following steps: S100. Mix the dried edible fungus powder with distilled water evenly, sterilize with high-temperature steam, and cool to obtain the sterilized edible fungus substrate. S200. The sterilized edible fungus substrate is first pretreated with a pulsed electric field, and then inoculated with a suspension of *Aspergillus cristatus* bacteria, stirred evenly, to obtain the inoculated substrate. S300: Place the inoculated substrate in an artificial climate chamber and ferment until the surface and interior of the substrate are covered with golden yellow closed capsules to obtain the fermentation product. S400. After inactivating the fermentation product, add ultrapure water and extract using ultrasound-microwave combined extraction. After extraction, centrifuge to collect the supernatant to obtain crude polypeptide solution. The crude polypeptide solution was subjected to two-stage ultrafiltration using S500, and the ultrafiltration components were collected and freeze-dried to obtain the target polypeptide composition.

2. The targeted enrichment method according to claim 1, characterized in that, In step S100, fresh edible fungi slices are taken, dried with hot air at 60℃~70℃ to constant weight, and pulverized through a 60~80 mesh sieve to obtain dried edible fungi powder; the dried edible fungi powder and distilled water are mixed at a material-to-liquid ratio of 1:1~2.5 to make the moisture content of the edible fungi substrate reach 45%~55%; the edible fungi include king oyster mushroom.

3. The targeted enrichment method according to claim 1, characterized in that, In step S200, the electric field strength of the pulsed electric field is 10~15kV / cm, the number of pulses is 30~50, and the pulse width is 2~5μs.

4. The targeted enrichment method according to claim 1, characterized in that, In step S200, the concentration of Eurotium cristatum bacterial suspension is 10 6 ~10 7 CFU / mL, and the inoculation amount is 5%~10%.

5. The targeted enrichment method according to claim 1, characterized in that, In step S300, the temperature of the artificial climate chamber is 28℃~30℃, the relative humidity is 85%~90%, and the fermentation time is 10~15 days.

6. The targeted enrichment method according to claim 1, characterized in that, In step S400, the inactivation temperature of the fermentation product is 90℃~100℃, and the inactivation time is 15~20 minutes; the ultrasonic power is 200~300W, the microwave power is 300~400W, and the extraction time is 30~60 minutes; the ratio of fermentation product to ultrapure water is 1:10~20.

7. The targeted enrichment method according to claim 1, characterized in that, In step S500, the two-stage ultrafiltration includes the following: The crude polypeptide solution was passed sequentially through ultrafiltration membranes with molecular weight cutoffs of 3 kDa and 1 kDa, and the ultrafiltration fraction with a molecular weight cutoff of less than 1 kDa was collected.

8. An edible fungus polypeptide composition obtained by the targeted enrichment method according to any one of claims 1 to 7, characterized in that, The edible fungus polypeptide composition includes the umami peptide ATA.

9. The application of the edible fungus polypeptide composition as described in claim 8 in food seasoning.

10. The application according to claim 9, characterized in that, The food products include compound seasonings, meat products, plant protein foods, and pre-prepared foods.

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