Fungus protein hydrolysate as well as preparation method and application thereof in meat flavor

Abstract

The invention belongs to the field of microorganisms and food processing, and particularly discloses a fungal protein hydrolysate, a preparation method thereof and application of the fungal protein hydrolysate in meat flavor. According to the method disclosed by the invention, firstly, fungi are treated by a combined means of alkali liquor suspension, high-pressure homogenization and heating, so that the extractability of fungal protein is improved; then, protease is adopted for carrying out directional cutting on hydrophobic amino acid residue sites, and the protein hydrolysate rich in branched chain amino acid is obtained. The hydrolysate has the following advantages: (1) the content of branched chain amino acids (leucine, isoleucine and valine) is high; the total content of branched chain amino acids accounts for more than 20% of the total content of free amino acids; (2) the proportion of low-molecular-weight peptide is high; the mass percentage content of the protein peptide fragment with the molecular weight of less than 3 kD is not less than 80%; and (3) good Maillard reaction activity is achieved, and rich meat flavor substances can be generated under the heating condition.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and food processing, specifically relating to a fungal protein hydrolysate, its preparation method, and its application in meat flavorings. Background Technology

[0002] In the field of flavor formulations, protein hydrolysates, as a key nitrogen source in the Maillard reaction, can also be used in the preparation of meat flavorings. This process typically involves enzymatic or acid hydrolysis of animal, plant, or edible fungi proteins to obtain protein hydrolysates containing polypeptides and free amino acids. These hydrolysates are then reacted with reducing sugars and sulfur-containing amino acids (such as cysteine) under heat to generate flavor compounds characterized by meat, nutty, and roasted aromas. Several patents have already addressed the application of protein hydrolysates in flavoring preparation. For example, patent CN202210532129.1, "A Method for Preparing Meat Flavorings Using High-Temperature Sesame Cake," describes a liquid flavoring with a rich meat aroma obtained through defatting, high-temperature enzymatic hydrolysis, hydrolysis, Maillard reaction, and membrane separation. Patent CN202210223021.4, "A Vegan Meat Flavoring and Its Preparation Method," describes a pure vegan thermally reacted meat flavoring obtained by compounding edible fungi enzymatic hydrolysate with thermally oxidized vegetable oil.

[0003] Branched-chain amino acids (leucine, isoleucine, valine) possess physiological functions such as promoting muscle synthesis, preventing muscle breakdown, improving exercise endurance, relieving fatigue, regulating immune function, and anti-inflammation, and are widely used in the field of functional nutritional foods. Currently, protein or oligopeptide products rich in branched-chain amino acids have received widespread attention in the food industry, mainly prepared using plant-derived raw materials. For example, patent CN202110402951.1, "A Processing Method for Producing High-Branched-Chain Amino Acid Plant Protein Meat Using Pea Protein," uses pea protein as raw material, combining deep enzymatic hydrolysis and extrusion puffing processes to destroy the molecular chain structure of pea protein, thereby increasing its branched-chain amino acid content and developing a plant protein meat product with a textured structure. Patent CN202210639536.2, "A Preparation Method and Application of Flaxseed Hydrolysate Rich in Branched-Chain Amino Acids," obtains flaxseed hydrolysate rich in branched-chain amino acids through pH-shift modification followed by enzymatic hydrolysis. Patent CN202211332268.6, "A protein peptide rich in branched-chain amino acids and its preparation method and application", uses papain to enzymatically hydrolyze zein to obtain a protein peptide rich in branched-chain amino acids that can promote the proliferation of C2C12 cells and prevent and alleviate apoptosis.

[0004] Studies have shown that branched-chain amino acids can serve as precursor nitrogen sources for the Maillard reaction, participating in the deamination and decarboxylation processes. Their side-chain structures can guide the generation of specific aroma pathways, which is beneficial for the formation of key flavor compounds such as branched-chain aldehydes and pyrazines, thereby significantly enhancing the meaty, nutty, and roasted aromas of the Maillard reaction system (Cerny C. Thermal generation of aroma-active volatiles in food. The chemistry and biology of volatiles. 2010,20:231-52.). Furthermore, the molecular weight distribution of protein hydrolysates also significantly influences the flavor profile of Maillard reaction products: compared to large peptides, low molecular weight peptides and free amino acids (less than 3000 Da) react more readily with reducing sugars to generate higher levels of volatile aldehydes, thiols, and pyrazines, resulting in a more pronounced flavor profile (Aslam N, Liu S, He S, Cao X, Ma Y, Al-Dalali S, Aslam I, Sun H. Effect of molecular weight distribution of sesame meal hydrolysates on the flavor profile of cysteine ​​Maillard reaction products. Journal of Food Science and Technology. 2024, doi:10.1007 / s13197-024-06191-x).

[0005] Fusarium venenatum, an edible filamentous fungus, has a fermented mycelium dry matter with a protein content of up to 50%, rich in all essential amino acids required by the human body. Its nutritional composition is close to that of animal protein, making it considered one of the most promising meat protein substitutes. Currently, several companies have developed commercial fungal meat products based on this strain, and these products are sold in multiple countries. For example, CN202510207212.5, "A Separation and Preparation Method for Improving the Purity and Emulsifying Properties of Fusarium venenatum Protein Based on Cell Wall Simplification Engineering Strategy," describes a method for obtaining high-purity Fusarium venenatum protein through alkaline treatment, high-pressure homogenization, acid precipitation, and chitinase hydrolysis, but does not further address its application in flavor development. CN202311496768.8, "A Concentrated Fungal Protein, Its Preparation Method and Its Application in Cell Cultured Meat," describes a concentrated fungal protein with a protein content higher than 75% and a branched-chain amino acid content higher than 40% obtained through a combined separation technique of alkalization, high-pressure homogenization and isoelectric point precipitation. It is mainly used in cell cultured meat, and no further research or development in the direction of flavoring and fragrance has been found. Summary of the Invention

[0006] This invention, through research, firstly improves the extractability of Fusarium venetum protein by combining alkaline treatment, high-pressure homogenization, and heating; then, it establishes a method for hydrolyzing Fusarium venetum cells by directional cleavage of hydrophobic amino acid residues using proteases, which can yield protein hydrolysates rich in branched-chain amino acids. These hydrolysates have the following advantages: (1) High content of branched-chain amino acids (leucine, isoleucine, valine); the total content of branched-chain amino acids accounts for more than 20% of the total free amino acids; (2) High proportion of low molecular weight peptides; the mass percentage of protein peptides with a molecular weight less than 3kD is not less than 80%; (3) Possesses good Maillard reaction activity, and can generate rich meat flavor substances under heating conditions.

[0007] This invention provides a method for preparing fungal protein hydrolysates, comprising the following steps:

[0008] S1. Raw material selection: The raw material is Fusarium venenatum wet substrate or dry substrate powder; for example, Fusarium venenatum TB01 is used.

[0009] S2. Cell disruption and solubilization treatment:

[0010] S2-1. Add alkaline solution to the bacterial raw material;

[0011] S2-2 undergoes pressurized homogenization;

[0012] S2-3. Heat treatment of the filtrate after filtration of the obtained turbid liquid;

[0013] S3. Add protease to hydrolyze and obtain the product;

[0014] Optionally, the following steps may also be included:

[0015] S4. After enzyme inactivation, filter, concentrate, and spray dry to obtain bacterial protein hydrolysate powder.

[0016] Specifically, in S2-1, an aqueous solution of sodium hydroxide or potassium hydroxide is used to adjust the pH of the system to 10.0–12.0;

[0017] Preferably, the homogenization pressure in S2-2 is not less than 800 bar, and the number of cycles is not less than 1.

[0018] More preferably, the homogenization pressure in S2-2 is 800-1200 bar, and the number of cycles is 2-4.

[0019] The specific operation of S2-3 is as follows: stir the bacterial suspension obtained in the previous step at 200–300 rpm for 0.5–2 hours, filter to remove insoluble residue, and obtain supernatant; treat at a temperature of 80–98℃ for 0.5–2 hours.

[0020] The specific operation of S3 is as follows: Adjust the pH of the supernatant from the previous step to 7.0–8.0, and add protease to hydrolyze it at 50–60℃ for 2–6 hours. The protease used is one or a combination of the following: alkaline protease, neutral protease, papain, and flavor protease; wherein, the amount of protease used is 0.2–0.8% of the dry weight of the bacterial powder, preferably 0.4–0.6%.

[0021] Preferably, after adding alkaline protease for hydrolysis, flavor protease is added for further hydrolysis.

[0022] This invention also provides a fungal protein hydrolysate rich in branched-chain amino acids prepared by the aforementioned method. Its characteristics are: branched-chain amino acid content: the total content of leucine, isoleucine, and valine accounts for no less than 20% of the total free amino acids; protein peptide distribution: the percentage of peptides with a relative molecular mass less than 3 kDa is no less than 80%.

[0023] The present invention further provides the application of the protein hydrolysate rich in branched-chain amino acids in the preparation of meat flavorings.

[0024] This invention provides a method for preparing a meat flavoring paste base, comprising the following steps:

[0025] Take the protein hydrolysate rich in branched-chain amino acids, add reducing sugar (such as glucose, xylose, ribose or a combination thereof), sulfur-containing compound (a combination of cysteine ​​and thiamine) and vegetable oil (such as sunflower seed oil, rapeseed oil, palm oil, coconut oil or a combination thereof); after mixing evenly, adjust the pH of the system to 6.5-9.0, and react at 100-120℃ for 0.5-3 hours to obtain the final product.

[0026] The present invention further provides a meat flavoring paste base material prepared by the method.

[0027] The protein hydrolysate rich in branched-chain amino acids can participate in the Maillard reaction with the following components to prepare meat flavor base: reducing sugar: selected from glucose, xylose, ribose or a combination thereof; sulfur-containing compound: selected from cysteine ​​and thiamine; vegetable oil: selected from sunflower oil, rapeseed oil, palm oil, coconut oil or a combination thereof.

[0028] During the heating reaction, the amino acids and low-molecular-weight peptides in the protein hydrolysate undergo Maillard reactions with the above components to generate typical meat flavor compounds such as pyrazines, thiophenes, and furans, thereby forming flavor products with rich meat aroma, caramel aroma, and good aftertaste. Attached Figure Description

[0029] Figure 1 Flowchart for the preparation of Fusarium vesicator protein hydrolysate from Venice.

[0030] Figure 2 Molecular weight mass spectrometry detection of Fusarium vesicola protein hydrolysate.

[0031] Figure 3 Distribution of peptide fragments in Fusarium vesicola protein hydrolysates.

[0032] Figure 4 Sensory evaluation of Maillard reaction products of Fusarium vesicola protein hydrolysate. Detailed Implementation

[0033] The present invention will be described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0034] Example 1:

[0035] according to Figure 1 The flowchart shown illustrates the preparation of Fusarium vesicatoria protein hydrolysate. 6 kg of Fusarium vesicatoria TB01 (published in CN202210314499.8) powder was suspended in 100 L of deionized water. The pH was adjusted to 11 using sodium hydroxide. The solution was homogenized under high pressure at 850, 1020, and 1200 bar, respectively. After stirring for 2 hours, the resulting turbid liquid was centrifuged, and the supernatant and residue were collected separately. The effect of combined alkaline treatment, high-pressure homogenization, and heating on the cell disruption and solubilization of Fusarium vesicatoria was calculated based on the weight difference between the powder and the dried residue. It was found that with increasing cell disruption pressure and the number of cycles, the compact cell structure was disrupted, and solubility increased. High-pressure homogenization at alkaline pH was more effective in disrupting the cell structure, further enhancing the solubilization effect. The collected supernatant was heated, and the nitrogen solubility index was tested. It was found that with prolonged heating time, the nitrogen solubility index of the protein extract gradually increased, indicating that heating helps to disrupt the protein structure and enhance solubility, promoting the release of nitrogen sources. Enzymatic hydrolysis of the heated supernatant was performed using proteases. The results showed an increased degree of protein hydrolysis in the supernatant, with an increased proportion of short peptides with molecular weights less than 3 kDa. Papain, neutral protease, alkaline protease, and flavor protease showed better hydrolytic effects than pepsin and trypsin.

[0036] Example 2:

[0037] Six kg of *Fusarium vesicatoria* TB01 mycelial powder was suspended in 100 L of deionized water, and the pH was adjusted to 12. The mixture was homogenized three times at 1200 bar, and the resulting turbid liquid was stirred at 200-300 rpm for 1 hour before filtration. The filtrate was heated at 90°C for 1 hour, cooled to 60°C, and the pH was adjusted to 8.0 using hydrochloric acid. 0.5% alkaline protease was added according to the dry weight of the mycelial powder for hydrolysis for 2 hours, and the pH was adjusted to 7.0. 0.2% flavor protease was added for hydrolysis for another 2 hours, and the enzyme activity was inactivated at 90°C. The mixture was then filtered, concentrated, and spray-dried to obtain 1.81 kg of mycelial protein hydrolysate powder.

[0038] Take 3 mL of bacterial protein hydrolysate solution with a concentration of 40 mg / mL, mix it thoroughly with 1 mL of 50% sulfosalicylic acid, let it stand for 15 minutes, centrifuge at 4℃ and 10000 r / min for 20 minutes, take the supernatant, dilute it 1-fold with 2 mM hydrochloric acid, and then inject it into an automated amino acid analyzer for testing. The results are shown in Table 1. It shows that the total amount of branched-chain amino acids (leucine, isoleucine, and valine) accounts for 20.65% of the total free amino acids. Mass spectrometry analysis shows that the mass percentage of protein peptides with a molecular weight less than 3 kD is 82.95%. Figure 2 ).

[0039] Table 1. Free amino acid composition of Fusarium vesicatoria protein hydrolysate

[0040]

[0041] "-" was not detected.

[0042] Example 3

[0043] 20 kg of wet Fusarium tumefaciens TB01 cells were added to 80 L of deionized water, and the pH was adjusted to 11. The mixture was then ground in a colloid mill and homogenized five times at 850 bar. The resulting suspension was stirred at 200-300 rpm for 1 hour, then filtered, and the filtrate was collected. The filtrate was heated at 80°C for 2 hours, then cooled to 60°C, and the pH was adjusted to 7.0 with hydrochloric acid. 0.5% neutral protease and 0.5% flavor protease were added according to the dry weight of the bacterial powder for hydrolysis for 3 hours. The enzyme activity was inactivated at 90°C, and the mixture was filtered, concentrated, and spray-dried to obtain 1.61 kg of bacterial protein hydrolysate powder.

[0044] Amino acid analyzer analysis showed that branched-chain amino acids accounted for 30.32% of the total free amino acids (Table 2).

[0045] Table 2. Free amino acid composition of Fusarium vesicatoria protein hydrolysate

[0046]

[0047] "-" was not detected.

[0048] Gel chromatography analysis showed that the percentage of protein peptides with a molecular weight less than 3 kDa was 83.70%. Figure 3 ).

[0049] Example 4:

[0050] Mix 3g of Fusarium vesicator protein hydrolysate from Example 1, 0.7g of glucose, 0.3g of ribose, 0.1g of cysteine, 0.02g of thiamine, 0.5mL of sunflower seed oil, and 20mL of water; adjust the pH to 7.0, react at 120℃ for 2 hours, and then concentrate under vacuum to obtain meat flavor base.

[0051] Sensory evaluation was conducted by a sensory evaluation team of 10 individuals with over one year of sensory evaluation experience, using descriptive analysis. Aroma attributes were identified as meaty, fatty, salty, roasted, and sweet. Aroma intensity scores ranged from 1 (weak) to 9 (strong). Sample evaluations were repeated three times. Results showed that the prepared meat flavor base possessed strong meaty, fatty, and roasted aroma attributes. Figure 4 ).

[0052] Example 5:

[0053] Mix 3g of Venetian Fusarium somatic protein hydrolysate, 0.5g of xylose, 0.5g of ribose, 0.08g of cysteine, 0.01g of thiamine, 0.5g of coconut oil, and 20mL of water from Example 2; adjust the pH to 7.0, and react at 110℃ for 3 hours; after vacuum concentration, obtain meat flavor paste base.

[0054] The volatile components of the Maillard reaction products were analyzed by gas chromatography-mass spectrometry (GC-MS), and 29 volatile meat flavoring substances were detected, mainly including aldehydes, ketones, furans, sulfur-containing compounds, and nitrogen-containing hybrid compounds (Table 3).

[0055] Table 3. Volatile meat flavoring substances in Maillard reaction products of Fusarium venetum protein hydrolysate as detected by GC-MS

[0056] .

Claims

1. A method for preparing fungal protein hydrolysate, characterized in that, The steps include: S1. Raw material selection: The raw material is either wet substrate Fusarium vesicant or dry substrate powder; S2. Cell disruption and solubilization treatment: S2-1. Add alkaline solution to the bacterial raw material; S2-2. Perform pressure homogenization treatment; S2-3. Heat treatment of the filtrate after filtering the obtained turbid liquid; S3. Add protease for hydrolysis; Optionally, the following steps may also be included: S4. After enzyme inactivation, filter, concentrate, and spray dry to obtain bacterial protein hydrolysate powder.

2. The preparation method according to claim 1, characterized in that, In S2-1, an aqueous solution of sodium hydroxide or potassium hydroxide is used to adjust the pH of the system to 10.0–12.

0.

3. The preparation method according to claim 1, characterized in that, In S2-2, the homogenization pressure shall not be less than 800 bar, and the number of cycles shall not be less than 1.

4. The preparation method according to claim 3, characterized in that, In S2-2, the homogenization pressure is 800-1200 bar, and the number of cycles is 2-4.

5. The preparation method according to claim 1, characterized in that, The specific operation of S2-3 is as follows: stir the bacterial suspension obtained in the previous step at 200–300 rpm for 0.5–2 hours, filter to remove insoluble residue, and obtain supernatant; treat at a temperature of 80–98℃ for 0.5–2 hours.

6. The preparation method according to claim 1, characterized in that, The specific operation of S3 is as follows: Adjust the pH of the supernatant from the previous step to 7.0–8.0, and add protease to hydrolyze it at 50–60℃ for 2–6 hours. The protease used is one or a combination of the following: alkaline protease, neutral protease, papain, and flavor protease; wherein, the amount of protease used is 0.2–0.8% of the dry weight of the bacterial powder, preferably 0.4–0.6%. Preferably, after adding alkaline protease for hydrolysis, flavor protease is added for further hydrolysis.

7. Fungal protein hydrolysate rich in branched-chain amino acids prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the protein hydrolysate rich in branched-chain amino acids as described in claim 7 in the preparation of meat flavoring.

9. A method for preparing a meat flavoring paste base, comprising the following steps: S1. The protein hydrolysate rich in branched-chain amino acids as described in claim 7, with the addition of reducing sugars (such as glucose, xylose, ribose or a combination thereof), sulfur-containing compounds (cysteine ​​and thiamine combination) and / or vegetable oils (such as sunflower oil, rapeseed oil, palm oil, coconut oil or a combination thereof). After mixing thoroughly, adjust the pH of the system to 6.5-9.0, and react at 100-120℃ for 0.5-3 hours to obtain the final product.

10. The meat flavoring paste base prepared by the method described in claim 9.

Images