Preparation method of termitomyces albuminosus flavor peptide

Abstract

The invention discloses a preparation method of black-skin termitomyces albuminosus flavor peptide, and belongs to the technical field of polypeptide seasonings. The preparation method comprises the following steps: soaking black-skin termitomyces albuminosus mushroom powder in water, and boiling the black-skin termitomyces albuminosus mushroom powder; after cooling, adding a compound enzyme, and carrying out enzymolysis; after the enzymolysis is finished, heating for enzyme deactivation; performing subcritical dynamic extraction on the enzymatic hydrolysate; heating the extract liquor, cooling, and centrifuging to obtain supernate; and concentrating the supernate to obtain concentrated paste, namely the termitomyces albuminosus flavor peptide. The prepared termitomyces albuminosus flavor peptide is rich in umami amino acid content and excellent in flavor, so that the termitomyces albuminosus flavor peptide has better application value and prospect in the technical field of polypeptide seasonings.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide seasonings, and particularly relates to a method for preparing a flavor peptide from Termitomyces albuminosa. Background Art

[0002] Termitomyces albuminosa is rich in protein, polysaccharide, minerals, amino acids, various vitamins, etc., and has extremely high nutritional value and various physiological functions such as antibacterial, anti-cancer, hypoglycemic and hypolipidemic effects. The industrialized cultivation technology of Termitomyces albuminosa has been relatively mature (such as three-dimensional shelf cultivation), and the fruiting bodies are mostly used for fresh sales or extraction of primary products such as polysaccharides. Although its protein content is rich (such as the high proportion of flavor amino acids such as glutamic acid and aspartic acid), the existing processes mostly focus on the overall utilization of mycelia or fruiting bodies (such as meal replacement powder, polysaccharide extraction), and do not design an efficient enzymatic hydrolysis scheme for protein resources, only staying in the physical fragmentation or simple water extraction stage, and the yield of flavor peptides is less than 1%. For example, the degree of hydrolysis of proteins in the two-way fermented mycelium is usually less than 30%, and the molecular weight distribution of polypeptides after enzymatic hydrolysis is broad (1 - 10 kDa), and the yield of target flavor peptides is insufficient. In addition, multiple separation and purification steps (such as impurity removal, desalting) further increase the production cost and restrict industrial application.

[0003] Termitomyces albuminosa is often used to prepare mycelium with plant matrix (such as soybeans) through two-way fermentation technology and used in meal replacement foods. For example, most of the existing technologies extract proteins from fermented mycelium and hydrolyze them into polypeptides, but their core goal is to screen antioxidant active ingredients rather than optimize flavor characteristics. Although such processes can release some polypeptides, they lack directional regulation of enzymatic hydrolysis conditions (such as protease selection, degree of hydrolysis control), resulting in a relatively high proportion of bitter peptides in the products, and the sensory characteristics such as umami and sweetness are not fully explored.

[0004] In addition, traditional mycelium fermentation relies on the mixed enzyme system of natural strains, and the protein hydrolysis path is random, making it difficult to precisely control the peptide chain length and flavor characteristics, resulting in uncontrollable enzymatic hydrolysis process. Moreover, the liquid-fermented mycelium is separated from the subsequent enzymatic hydrolysis steps, and most of them need to add additional proteases for multi-step treatment, resulting in high energy consumption and long cycle, which causes fragmentation of the process. In addition, the current process lacks specific enzymatic hydrolysis technology and does not design a composite enzyme system according to the amino acid composition of Termitomyces albuminosa protein (such as a high proportion of acidic amino acids), resulting in low enzymatic hydrolysis efficiency. These problems make the process controllability and integration of Termitomyces albuminosa flavor peptides poor. Summary of the Invention

[0005] The present invention provides a method for preparing a flavor peptide from Termitomyces albuminosa, comprising the following steps:

[0006] Soak the black-skinned Termitomyces albuminosus mushroom powder in water and boil it; after cooling, add a composite enzyme and carry out enzymatic hydrolysis; after the enzymatic hydrolysis is completed, heat to inactivate the enzyme; subject the enzymatic hydrolysate to subcritical dynamic extraction; then heat-treat the extract, cool it, and centrifuge to obtain the supernatant; concentrate the supernatant to obtain a concentrated paste, namely the black-skinned Termitomyces albuminosus mushroom flavor peptide.

[0007] In the above preparation method, the mass ratio of the black-skinned Termitomyces albuminosus mushroom powder to water is (0.5 - 1.5):(1 - 3), preferably 1:2.

[0008] In the above preparation method, the soaking time is selected from 7 - 16 min, preferably 10 min; the boiling time is selected from 8 - 12 min, preferably 10 min.

[0009] In the above preparation method, the addition amount of the composite enzyme is 0.5 - 5% by mass percentage; preferably 1%.

[0010] In the above preparation method, the composite enzyme is composed of alkaline protease, cellulase, and flavor protease, and the mass ratio of the three is selected from (2 - 4):(1 - 2):(0.3 - 0.5), preferably 3:1.5:0.4.

[0011] In the above preparation method, the conditions for enzymatic hydrolysis are selected as follows: pH is 6.0 - 7.5, temperature is 45 - 55 °C, and enzymatic hydrolysis time is 3 - 6 h; preferably: pH is 7.0, temperature is 50 °C, and enzymatic hydrolysis time is 4.5 h.

[0012] In the above preparation method, the conditions for heat inactivation of the enzyme are selected as follows: heat at 100 °C for 10 min; subject to sufficient enzyme inactivation.

[0013] In the above preparation method, the extractant for subcritical dynamic extraction is an ethanol aqueous solution, and the mass concentration of ethanol is 50%.

[0014] In the above preparation method, the extraction conditions for subcritical dynamic extraction are selected as follows: temperature 90 - 110 °C, pressure 2 - 4 MPa, solid-liquid ratio 1 - 15:1 - 25, extraction time 25 - 35 min; preferably: temperature 100 °C, pressure 3 MPa, solid-liquid ratio 1:20, extraction time 30 min.

[0015] In the above preparation method, the conditions for heat treatment are selected as follows: heat at 100 °C for 10 min.

[0016] In the above preparation method, the concentration is microwave concentration, and the microwave condition is power 20.

[0017] The present invention provides the black-skinned Termitomyces albuminosus mushroom flavor peptide prepared by the above method.

[0018] The present invention provides the application of the above-mentioned Oudemansiella raphanipes flavor peptide in the preparation of food seasonings.

[0019] The beneficial effects of the present invention are as follows:

[0020] According to the protein characteristics of Oudemansiella raphanipes, alkaline protease, cellulase and flavor protease are used as composite enzymes, and an ethanol-water mixed system is used as the subcritical dynamic extraction agent to enzymatically hydrolyze and subcritically dynamically extract Oudemansiella raphanipes protein. There is a synergistic effect among the three enzymes, which can significantly increase the content of umami amino acids in the enzymatic hydrolysate and improve the product flavor. At the same time, the subcritical dynamic extraction technology can also effectively extract various volatile substances in the enzymatic hydrolysate, retain and enhance various volatile substances, and perform excellently in retaining flavor.

[0021] The Oudemansiella raphanipes flavor peptide prepared by the present invention is rich in umami amino acids and has excellent flavor, thus having good application value and prospects in the field of polypeptide seasoning technology. Description of the Drawings

[0022] Figure 1 It is the result of DPPH free radical scavenging assay;

[0023] Figure 2 It is the result of flavor sensory evaluation;

[0024] Figure 3 It is the result of electronic nose analysis of the enzymatic hydrolysate;

[0025] Figure 4 It is the result of PCA analysis and electronic nose analysis of the subcritical dynamic extraction solution;

[0026] Figure 5 It is the result of PCA analysis and electronic nose analysis of different concentration methods. Detailed Embodiments

[0027] In the present invention, Oudemansiella raphanipes (mushroom) was purchased from the wholesale market in Chengyang District, Qingdao. Alkaline protease (100000U / g), cellulase (20000U / g), and flavor protease (20000U / g) were all purchased from Nanning Pangbo Bioengineering Co., Ltd.

[0028] Other materials used in the present invention, unless otherwise specified, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise stated, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are only for illustrative purposes of the present invention and do not limit the scope of the present invention in any way.

[0029] Example 1

[0030] Preparation of black-skinned Termitomyces albuminosus flavor peptide is as follows:

[0031] 1. Pretreatment

[0032] After washing the black-skinned Termitomyces albuminosus raw materials with water, freeze them at -18°C for 24 h, and then grind them at low temperature for 20 min. Then, use ultrasonic-assisted water extraction (frequency 40 kHz, power 300 W, time 20 min) to remove soluble polysaccharides and impurities, retain the protein matrix, and obtain black-skinned Termitomyces albuminosus mushroom powder after drying.

[0033] 2. Enzymatic hydrolysis

[0034] Add water to the black-skinned Termitomyces albuminosus mushroom powder according to a mass ratio of 1:2, soak for 10 min, then boil at 100°C for 10 min, cool down to 50°C, maintain the pH at 7.0, then add 1% enzyme by mass percentage, stir and hydrolyze for 4.5 h, and heat at 100°C for 10 min to inactivate the enzyme after the enzymatic hydrolysis is completed.

[0035] Control of the enzymatic hydrolysis end point: Terminate the reaction by monitoring the degree of hydrolysis (DH≥15%) and the peptide molecular weight distribution (the proportion of 500 - 2000 Da ≥ 70%) in real time.

[0036] In this step, the enzyme is selected from the following groups: (1) alkaline protease; (2) cellulase; (3) flavor protease; (4) alkaline protease + cellulase 2:1; (5) alkaline protease + flavor protease 2:1; (6) alkaline protease + cellulase + flavor protease 3:1.5:0.4.

[0037] Perform amino acid determination on the enzymatic hydrolysates of black-skinned Termitomyces albuminosus with different enzymatic hydrolysis methods. The determination method uses the acid hydrolysis method in GB5009.124 - 2016, and the test indexes include umami amino acids, sweet amino acids, bitter amino acids, and tasteless amino acids to compare the flavor of each enzymatic hydrolysate.

[0038] The test results are shown in Table 1:

[0039] Table 1 Determination results of amino acid contents of different enzymatic hydrolysis methods

[0040]

[0041]

[0042] As can be seen from Table 1, when the three enzymes are used in combination, the total amount of amino acids after enzymatic hydrolysis is the highest. Among them, the content of umami amino acids increased by 35.55% compared with that of enzymatic hydrolysis using alkaline protease alone, and the total amount also increased by 21.78%. Thus, it can be seen that on the premise of the same dosage of enzymatic hydrolysis, the flavor intensity of the combined enzymatic hydrolysis of the three enzymes is higher than that of single enzymatic hydrolysis and pairwise compounding, indicating that there is a synergistic effect among the three. Therefore, the combined enzymatic hydrolysis of the three enzymes was selected for the next experiment.

[0043] 3. Subcritical dynamic extraction

[0044] The enzymatic hydrolysate was subjected to subcritical dynamic extraction using an ethanol-water mixed system (ethanol mass concentration 50%). The extraction conditions were as follows: temperature 100 °C, pressure 3 MPa, solid-liquid ratio 1:20, and extraction time 30 min.

[0045] 4. Concentrated paste

[0046] The extract was heated at 100 °C for 10 min, cooled, and then centrifuged at 4000 rmp for 20 min at 4 °C to obtain the supernatant. The supernatant was concentrated to obtain a concentrated paste.

[0047] In this step, the concentration is selected from the following groups: (1) Rotary evaporation concentration (50 °C); (2) Microwave concentration (Galanz T70TPDFZB1, power 20).

[0048] I. Effect of enzymatic hydrolysis time on the performance of enzymatic hydrolysate

[0049] The black chicken mushroom powder was enzymatically hydrolyzed using a composite enzyme (alkaline protease + cellulase + flavor protease). The enzymatic hydrolysis process refers to step (2) of Example 1 above. The enzymatic hydrolysis times were 3 h, 4.5 h, and 6 h respectively. The supernatant of the enzymatic hydrolysate was taken for DPPH free radical scavenging determination, flavor sensory evaluation, and electronic nose analysis. Non-enzymatic hydrolysis was used as a control.

[0050] The flavor sensory evaluation criteria are shown in Table 2:

[0051] Table 2 Flavor sensory evaluation criteria

[0052]

[0053] The results of DPPH free radical scavenging determination are as Figure 1 shown: The antioxidant ability of the enzymatic hydrolysate with an enzymatic hydrolysis time of 4.5 h was significantly higher than that of other treatment groups.

[0054] The results of flavor sensory evaluation are as Figure 2 shown: With the increase of enzymatic hydrolysis time, the better the enzymatic hydrolysis effect, the stronger the flavor, and the higher the score. The sensory scores of enzymatic hydrolysis for 4.5 h and 6 h were similar. Considering the comprehensive antioxidant ability and production cost, 4.5 h was selected as the optimal enzymatic hydrolysis time.

[0055] The analysis results of the electronic nose are as Figure 3 shown below:

[0056] There are obvious differences in the response values of several supernatant liquids on sensors W5S and W1W. Analyzing individual samples shows that the overall sensor response value after enzymatic hydrolysis for 4.5 h is better than that of other treatment groups. Generally speaking, the overall flavor of the enzymatic hydrolysate obtained by adding alkaline protease, cellulase, and flavor protease for enzymatic hydrolysis for 4.5 h is better than that of other treatments.

[0057] II. Analysis of the influence of subcritical dynamic extraction

[0058] The extraction liquid after subcritical dynamic extraction is analyzed by an electronic nose. Using non-enzymatic hydrolysis and enzymatic hydrolysis as controls, neither of them undergoes subcritical dynamic extraction. The process of enzymatic hydrolysis refers to step (2) of Example 1 above, and the process of subcritical dynamic extraction refers to step (3) of Example 1.

[0059] The analysis results of the electronic nose are as Figure 4 shown below:

[0060] The sample without enzymatic hydrolysis treatment has fewer volatile substances and its odor characteristics are not obvious. Enzymatic hydrolysis treatment can significantly increase the release of these volatile substances and produce specific odor characteristics. The subcritical extraction has the highest response values on W1S and W1W, indicating that subcritical extraction can more effectively extract various volatile substances, performs better in retaining flavor, and can retain and enhance various volatile substances.

[0061] Other methods (ultrasonic extraction and hot water extraction) are used to extract the enzymatically hydrolyzed solution, and its amino acid content is measured and compared with the subcritical dynamic extraction technology.

[0062] The conditions for ultrasonic extraction: The enzymatically hydrolyzed solution is subjected to ultrasonic extraction with a power of 300 w and a time of 20 min. Hot water extraction: The enzymatically hydrolyzed solution is kept at 85 °C for extraction for 1 h.

[0063] The test results are shown in Table 3:

[0064] Table 3 Determination results of amino acid content in different extraction methods

[0065]

[0066] The results show that ultrasonic extraction and hot water extraction technologies can increase the amino acid content in the enzymatically hydrolyzed solution, but the improvement effect of subcritical extraction is more significant. The content of umami amino acids has increased by 54.43%, and the total amino acid content has increased by 66.47%.

[0067] III. Analysis of the influence of different concentration methods

[0068] PCA analysis and electronic nose analysis were performed on the extracts obtained by two concentration methods, rotary evaporation and microwave, with the extract before concentration as the control, as Figure 5 shown.

[0069] According to the calculation method, the closer the obtained value is to 1, the more significant the sample discrimination is. The total contribution rate of PC1 and PC2 reaches 99.3%, indicating that the three samples can be clearly distinguished in PCA.

[0070] The curve graph of the electronic nose response value shows that there are obvious differences in the response values of the three groups (the supernatant and the concentrated paste are calculated by weight) on the W1S, W1W, and W2S sensors. The aroma components change significantly after concentration. However, in the two concentration methods, the overall sensor response value of microwave is better than that of rotary evaporation, indicating that the microwave reaction can increase the overall flavor of the enzymolysis solution of Termitomyces heimii.

[0071] The amino acid contents of the extracts obtained by the above two concentration methods were measured, and the measurement results are shown in Table 4:

[0072] Table 4 Measurement results of amino acid contents by different concentration methods

[0073]

[0074] The results show that the total amount of amino acids in the microwave-treated sample is higher than that in the rotary evaporation-treated sample, with an increase of 21.85%. After microwave treatment, the content of umami amino acids increased by 21.54% compared with rotary evaporation treatment, and the content of sweet amino acids increased by 15.85%.

[0075] Microwave concentration is a non-contact heating method. The temperature distribution inside the solution is more uniform, avoiding local overheating. The heating time is short, reducing the exposure time of amino acids at high temperatures, thereby reducing the risk of degradation due to high temperatures. Rotary evaporation relies on water bath to conduct heat from the outside. It is easy to form a temperature gradient between the surface and the inside of the solution. Local high-temperature areas and long heating times lead to thermal decomposition or Maillard reactions of some amino acids (such as tryptophan and cystine), resulting in relatively large losses. Therefore, compared with rotary evaporation, microwave is beneficial to reducing the loss of amino acids during evaporation due to uniform and rapid heating, so that the amino acid content in the extract obtained by microwave concentration is higher than that by rotary evaporation.

[0076] In the present invention, subcritical extraction uses a subcritical solvent as the extractant. In a closed, oxygen-free, and low-pressure container, based on the principle of "like dissolves like" for organic substances, through the molecular diffusion process between the extraction material and the extractant during the soaking process, the target product components in the solid material are transferred into the liquid extractant. Then, through the process of vacuum evaporation, the extractant is separated from the target product, and finally the target product is obtained. The enzymatic hydrolysis process has decomposed proteins into smaller peptide segments and amino acids, but these amino acids may still be bound to or encapsulated by some macromolecular substances. Ethanol can precipitate polysaccharides or lipids, reducing their adsorption or encapsulation of amino acids. The lipid-soluble property of ethanol can disrupt the lipid bilayer of cell membranes, promoting the release of intracellular amino acids, thereby increasing the content of detectable free amino acids in the solution. Amino acids in edible fungi are polar molecules. Under subcritical conditions, the polarity of the ethanol-water mixed system matches the polarity of amino acids, making it easier for amino acids to dissolve in the solvent. High temperature can also accelerate the limited hydrolysis of proteins in the fungus, releasing free amino acids, thus increasing the concentration and purity of amino acids in the solution.

[0077] Microwave concentration utilizes the penetrability and selective heating characteristics of microwaves, enabling water molecules inside the material to rapidly absorb microwave energy and convert it into heat energy, thereby achieving rapid heating and evaporation. During the microwave concentration process, due to the intense movement of water molecules under the action of microwaves, a large amount of heat energy is generated, resulting in rapid evaporation of water. As the water evaporates, the concentration of solutes (such as amino acids) in the material gradually increases. Samples of the same weight are taken from the supernatant and the concentrated paste for measurement. The proportion of dry matter (including amino acids) in the concentrated paste is significantly higher than that in the supernatant. Therefore, the measured amino acid content (mg / g) per unit weight will also be higher.

[0078] The above are only the preferred embodiments of the present invention and do not limit the present invention in other forms. Any person skilled in the relevant art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A preparation method of an Oudemansiella raphanipies flavor peptide, characterized in that, It includes the following steps: Soak the powder of Termitomyces albuminosus with black skin in water and boil it; after cooling, add a complex enzyme and carry out enzymatic hydrolysis; after the enzymatic hydrolysis ends, heat to inactivate the enzyme; carry out subcritical dynamic extraction on the enzymatic hydrolysate; then heat-treat the extract, cool it, and centrifuge to obtain the supernatant; concentrate the supernatant to obtain a concentrated paste, namely the Termitomyces albuminosus peptide with black skin flavor.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the powder of Termitomyces albuminosus with black skin to water is (0.5 - 1.5):(1 - 3).

3. The preparation method according to claim 1, characterized in that, The addition amount of the complex enzyme is 0.5 - 5% by mass percentage.

4. The preparation method according to claim 1, wherein The complex enzyme is composed of alkaline protease, cellulase and flavor protease, and the mass ratio of the three is selected from (2 - 4):(1 - 2):(0.3 - 0.5).

5. The preparation method according to claim 1, characterized in that, The conditions of the enzymatic hydrolysis are selected from: pH is 6.0 - 7.5, temperature is 45 - 55 °C, and the enzymatic hydrolysis time is 3 - 6 h.

6. The preparation method according to claim 1, characterized in that, The extractant for the subcritical dynamic extraction is an aqueous ethanol solution, and the mass concentration of ethanol is 50%.

7. The preparation method according to claim 1, characterized in that, The extraction conditions for the subcritical dynamic extraction are selected from: temperature 90 - 110 °C, pressure 2 - 4 MPa, solid-liquid ratio 1 - 15:1 - 25, and extraction time 25 - 35 min.

8. The preparation method according to claim 1, wherein, The conditions for the heat treatment are selected from: heating at 100 °C for 10 min; the concentration is microwave concentration, and the microwave condition is power 20.

9. The Termitomyces albuminosus peptide with black skin flavor prepared by the method according to any one of claims 1 - 8.

10. Use of the Termitomyces albuminosus peptide with black skin flavor according to claim 9 in the preparation of food seasonings.

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