Hypsizygus marmoreus umami peptide and extraction method and application thereof

Abstract

The invention discloses a hypsizygus marmoreus umami peptide as well as an extraction method and application thereof, and belongs to the technical field of protein active peptides. The amino acid sequences of the ten flavor peptides are as shown in SEQ ID NO. 1 to SEQ ID NO. 10. The ten umami peptides are extracted from hypsizygus marmoreus, sensory detection results show that the ten umami peptides have good umami and richness, can replace monosodium glutamate and have great application significance in food, and the umami peptides are small-molecular-weight peptides and are easy to absorb by human bodies.

Description

[0001] This invention patent application is a divisional application of the invention patent application with application date of October 31, 2023, application number CN202311436934.5, and invention name as “Seafood Mushroom Umami Peptide, Extraction Method and Application thereof”. Technical Field

[0002] The invention belongs to the technical field of protein active peptides, and in particular relates to seafood mushroom umami peptides and an extraction method and application thereof. Background Art

[0003] Umami peptides refer to oligopeptides with a molecular weight of less than 5000 u that are extracted from food or synthesized from amino acids and contribute to the flavor of food to a certain extent. They include characteristic flavor peptides and flavor precursor peptides. They exist in various foods such as meat, fruits and vegetables. Umami peptides, as a new type of active peptide flavor enhancer, can not only improve the umami taste of food, but are also very important for the flavor of processed food.

[0004] In 1972, Arai et al. first separated and purified three dipeptides, namely Glu-Asp, Glu-Ser and Glu-Glu, and one tripeptide, namely Glu-Gly-Ser, from soy protein hydrolysates. These peptides have a taste similar to that of broth. It was not until 1978 that Yamasaki et al. processed beef with papain and obtained an octapeptide, Beefy Meaty Peptide (BMP), which was named Umami Peptide. Since then, the study of umami peptides has received widespread attention. Zhang et al. used Nano-LC-Q-TOF-MS / MS technology to identify umami peptides in chicken soup. After synthesis, they identified 9 flavor peptides with umami taste through sensory analysis: PPQEAAQF, AEEHVEAVN, NEFGYSNR, LPLQD, GPSGKLW, DGGRYY, GPVGYGYE, GPVGYGYEY and AGPSIVH, among which PPQEAAQF had the highest umami intensity (Zhang, JC, Zhang, JC, Liang, L., Sun, BG, & Zhang, YY (2023). Identification and virtual screening of novel umami peptides from chicken soup by molecular docking. Food Chemistry, 404, 134414.). Wang et al. discovered new umami peptides from marine protein resources. Based on the prediction of umami activity, water solubility and toxicity, they screened out 23 possible umami peptides from the virtual enzymatic hydrolysate of Atlantic cod myosin, among which GGR, AGCD and SGDAW had high umami intensity (Wang, YY, Luan, JJ, Tang, XH, Zhu, WH, Xu, YX, Bu, Y., et al. (2023). Identification of umami peptides based on virtual screening and molecular docking from Atlantic cod (Gadus morhua). Food&Function, 14(3), 1510-1519.). These umami peptides contribute significantly to taste and affect the taste of food.

[0005] So far, many researchers have isolated and purified umami peptides from chicken, shiitake mushrooms, soybean paste and ham, but there are few reports on the isolation and purification of umami peptides from edible fungi. Summary of the invention

[0006] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide seafood mushroom umami peptides and an extraction method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The invention discloses a seafood mushroom umami peptide, which has umami taste and enhances umami taste and can bind to umami receptor T1R1. The amino acid sequence of the seafood mushroom umami peptide is shown in SEQ ID NO.1 to SEQ ID NO.3, SEQ ID NO.7 and SEQ ID NO.8.

[0008] Preferably, the seafood mushroom umami peptide is separated and extracted from seafood mushroom.

[0009] Preferably, the seafood mushroom umami peptide is artificially synthesized.

[0010] Preferably, the umami threshold of the seafood mushroom umami peptide is 0.18-0.71 mmol / mL.

[0011] Preferably, the umami flavor enhancement threshold of the seafood mushroom umami peptide is 0.09-0.35 mM.

[0012] The present invention also discloses a method for extracting the seafood mushroom umami peptides, which comprises enzymatically hydrolyzing the seafood mushrooms with a protein complex enzyme method, freeze-drying, and using a nanofiltration membrane and an ultrafiltration membrane to retain components with a molecular weight of 200 to 1000 Da, separating and purifying to obtain the seafood mushroom flavor peptides; using T1R1 as an umami receptor, and obtaining the seafood mushroom umami peptides shown in SEQ ID NO.1 to SEQ ID NO.3, SEQ ID NO.7 and SEQ ID NO.8 through molecular docking virtual screening technology.

[0013] Preferably, the protease is neutral protease and flavor protease.

[0014] The invention also discloses application of the seafood mushroom umami peptide in preparing food seasoning.

[0015] Preferably, the food seasoning is a flavor-enhancing food additive.

[0016] The invention also discloses a product containing the seafood mushroom umami peptide.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The seafood mushroom umami peptides provided by the present invention are: 1. all obtained by separation and purification from seafood mushrooms, and have good affinity with the umami receptor T1R1; using sensory detection, the results show that the five umami peptides have good umami and richness. 2. The extracted umami peptides are all small molecular weight peptides, which are easily absorbed by the human body. 3. The extracted umami peptides have high umami intensity and can replace the use of monosodium glutamate, and have great application significance in food. 4. The extraction method of umami peptides is simple to operate, without any pollution, and the edible fungus umami peptides obtained have outstanding umami taste, and are high-quality flavor base materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a 2D diagram of the docking of taste receptor protein T1R1 and seafood mushroom flavor peptide; A is VLPVPQK, and B is GPFPILV; Figure 2 This is a purity verification result diagram of the flavor peptide KENINELSK extracted from seafood mushrooms; Figure 3 This is the mass spectrometry result of the artificially synthesized flavor peptide KENINELSK in seafood mushroom; Figure 4 This is a purity verification result diagram of the flavor peptide VLPVPQK extracted from seafood mushrooms; Figure 5 This is the mass spectrometry result of the artificially synthesized flavor peptide VLPVPQK in seafood mushroom; Figure 6 This is a purity verification result diagram of the flavor peptide VLVLDTDYKK extracted from seafood mushroom; Figure 7 This is the mass spectrometry result of the artificially synthesized flavor peptide VLVLDTDYKK in seafood mushroom; Figure 8 This is a purity verification result diagram of the flavor peptide EGNPAHQK extracted from seafood mushrooms; Fig. 9 This is the mass spectrometry result of the artificially synthesized flavor peptide EGNPAHQK in seafood mushroom; Fig.10 This is a purity verification result diagram of the flavor peptide LDSPATPEK extracted from seafood mushrooms; Fig.11 This is the mass spectrometry result of the artificially synthesized flavor peptide LDSPATPEK in seafood mushroom; Fig.12 This is a purity verification result diagram of the flavor peptide VVEGEPSLK extracted from seafood mushrooms; Fig.13 This is the mass spectrometry result of the artificially synthesized flavor peptide VVEGEPSLK in seafood mushroom; Fig.14This is a purity verification result diagram of the flavor peptide QKLPEKPER extracted from seafood mushroom; Fig.15 This is the mass spectrometry result of the artificially synthesized flavor peptide QKLPEKPER in seafood mushroom; Fig.16 This is a purity verification result diagram of the flavor peptide GPFPILV extracted from seafood mushroom; Fig.17 This is the mass spectrometry result of the artificially synthesized flavor peptide GPFPILV in seafood mushroom; Fig.18 This is a purity verification result diagram of the flavor peptide AGLELLK extracted from seafood mushrooms; Fig.19 This is the mass spectrometry result of the artificially synthesized flavor peptide AGLELLK in seafood mushroom; Fig. 20 This is a purity verification result diagram of the flavor peptide GPFPILN extracted from seafood mushroom; Fig.21 This is the mass spectrometry result of the artificially synthesized flavor peptide GPFPILN in seafood mushroom; Fig. 22 0.3 g / mL of MSG was added to ten umami peptide solutions (1 mg / mL) to evaluate the umami enhancement effect diagram; among them, P1~P10 represent KENINELSK, VLPVPQK, VLVLDTDYKK, EGNPAHQK, LDSPATPEK, VVEGEPSLK, QKLPEKPER, GPFPILV, AGLELLK and GPFPILN, respectively, and a~g represent significant difference analysis; Fig.23 0.3 g / ml NaCl was added to ten umami peptide solutions (1 mg / ml) to evaluate the salty taste enhancement effect; among them, P1~P10 represent KENINELSK, VLPVPQK, VLVLDTDYKK, EGNPAHQK, LDSPATPEK, VVEGEPSLK, QKLPEKPER, GPFPILV, AGLELLK, GPFPILN, respectively, and a~g represent significant difference analysis. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0020] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0021] In the specification of the present invention and the following examples, unless otherwise specified, seafood mushroom umami peptide refers to seafood mushroom umami peptide obtained by, for example, but not limited to, the extraction method described herein. Those skilled in the art will appreciate that the seafood mushroom umami peptide of the present invention can be obtained by conventional methods or the extraction method described in the specification, or can be obtained by artificial synthesis according to the amino acid sequence provided in the specification.

[0022] The invention provides seafood mushroom umami peptide and an extraction method and application thereof.

[0023] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0024] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and ratio means weight ratio. "MSG" means "monosodium glutamate" or "monosodium glutamate".

[0025] 1. Isolation and extraction of seafood mushroom flavor peptides The fresh and intact seafood mushrooms available on the market were first washed with running water, blanched and sterilized for 10 minutes, and then beaten into a fine seafood mushroom homogenate with a beater, and water was added to make the material-liquid ratio 1:10. First, 0.4% cellulase was added, the pH value was adjusted to 5, and the enzyme was hydrolyzed in a constant temperature magnetic mixer at a temperature of 50°C for 5 hours, and then the enzyme activity was quickly inactivated for 10 minutes to fully lose its enzyme activity. Protease (0.32% neutral protease + 0.64% flavor protease) was added again, the pH value was adjusted to 6, and the enzyme was hydrolyzed in a constant temperature magnetic mixer at a temperature of 50°C for 5.5 hours, and the enzyme activity was quickly inactivated for 10 minutes. The supernatant was separated by centrifugation, freeze-dried, and the freeze-dried sample was re-dissolved in ultrapure water. The nanofiltration membrane with a molecular weight cutoff of 200 Da and the ultrafiltration membrane with a molecular weight cutoff of 1000 Da were used for ultrafiltration separation at 25°C, and the components with a molecular weight of 200-1000 Da were collected. The crude seafood mushroom flavor peptide was obtained by vacuum freeze-drying technology. The crude seafood mushroom flavor peptide was reconstituted with ultrapure water, passed through a gel chromatography column, and eluted with ultrapure water. The elution conditions were: 1 mL sample volume, 0.4 mL / min flow rate, 1.0 UV detector sensitivity, 220 nm detection wavelength, updated the collection tube every 6 min, collected multiple times, and then concentrated and freeze-dried again to obtain the seafood mushroom flavor peptide.

[0026] 2. Identification of umami peptides from seafood mushrooms The seafood mushroom flavor peptide obtained in step 1 was sequenced by mass spectrometry, and then seafood mushroom flavor peptides (umami peptides) with umami taste were obtained by virtual screening.

[0027] Specifically, T1R1 was used as the umami receptor, and the model of umami receptor T1R1 and seafood mushroom flavor peptide was established using AlphaFold2 software. The pdb files of T1R1 and seafood mushroom flavor peptide were selected, and molecular docking was performed using Discovery Studio 2019. The results are shown in Figure 1It can be seen that Asn, Asp, Arg, Ser, Glu and Lys in the peptide sequence play an important role in binding to the taste receptor T1R1. Combined with sensory evaluation, the seafood mushroom flavor peptide with the best affinity to the umami receptor T1R1 was screened as the umami peptide. The sequences are shown in SEQ ID NO.1 to SEQ ID NO.10 in Table 1, which are Lys-Glu-Asn-Ile-Asn-Glu-Leu-Ser-Lys, Val-Leu-Pro-Val-Pro-Gln-Lys, Val-Leu-Val-Leu-Asp-Thr-Asp-Tyr-Lys-Lys, Glu-Gly-Asn-Pro-Ala-His-Gln-Lys, Leu-Asp-Ser-Pro-Ala-Th r-Pro-Glu-Lys, Val-Val-Glu-Gly-Glu-Pro-Ser-Leu-Lys, Gln-Lys-Leu-Pro-Glu-Lys-Pro-Glu-Arg, Gly-Pro-Phe-Pro-Ile-Leu-Val, Ala-Gly-Leu-Glu-Leu-Leu-Lys or Gly-Pro-Phe-Pro-Ile-Leu-Asn. Among them, the umami taste is more obvious: Val-Leu-Pro-Val-Pro-Gln-Lys, Val-Val-Glu-Gly-Glu-Pro-Ser-Leu-Lys, Gly-Pro-Phe-Pro-Ile-Leu-Val, Ala-Gly-Leu-Glu-Leu-Leu-Lys, Gly-Pro-Phe-Pro-Ile-Leu-Asn. The purity verification results of the ten umami peptides are shown in the figure. Figure 2 , Figure 4 , Figure 6 , Figure 8 , Fig.10 , Fig.12 , Fig.14 , Fig.16 , Fig.18 and Fig. 20 As shown, the purity of all seafood mushroom umami peptides is above 97%.

[0028] According to the sequences SEQ ID NO.1 to SEQ ID NO.10 in Table 1, the umami peptides were artificially synthesized. The mass spectrometry results of the artificially synthesized umami peptides are as follows: Figure 3 , Figure 5 , Figure 7 , Fig. 9 , Fig.11 , Fig.13 , Fig.15 , Fig.17 , Fig.19 and Fig.21 As shown in the figure, it can be seen that the molecular weight and molecular mass-to-charge ratio of the seafood mushroom umami peptide are consistent with the theoretical molecular weight and molecular mass-to-charge ratio of the corresponding sequence umami peptide, confirming that the sample is the target peptide.

[0029] Table 1 Detection results of the extracted seafood mushroom umami peptides

[0030] 3. Sensory evaluation of seafood mushroom umami peptides Ten volunteers (5 males and 5 females, aged between 23 and 26 years old) with no taste disorder and no history of taste disorder were recruited and trained in advance to prepare for the basic taste of umami, i.e. MSG. It is particularly important to rinse the mouth with 50 mL of pure water before tasting the next umami peptide to avoid taste errors for the next umami peptide solution.

[0031] Eight umami peptide solutions with a concentration of 2 mg / mL were prepared respectively, and their umami intensity was tasted after being fully stirred to score the umami taste of the seafood mushroom umami peptides. The scores with large differences were removed, and the remaining scores were averaged. The scoring results are shown in Table 1. By calculating the umami threshold values, it can be obtained that the umami threshold values ​​of SEQ ID NO.1 to SEQ ID NO.10 are 0.18, 0.35, 0.35, 0.35, 0.71, 0.71, 0.71, 0.18, 0.18 and 1.40, respectively.

[0032] 3 mg MSG or 3 mg NaCl were added to each 20 mL ultrapure water, and then 10 mg of eight umami peptides were added as the experimental group. At the same time, 15 mg / mL MSG / NaCl was prepared as the control group to evaluate its umami and salty taste enhancement effects. The evaluation results are as follows: Fig. 22 and Fig.23 As shown, Fig. 22 As shown in the figure, it can be seen that P4 and P9 have the most significant umami enhancement effect, followed by P1 and P3; Fig.23 As shown, it can be seen that the salty taste enhancement effect of P8 is the most significant, followed by P4, the salty taste enhancement effect of P2 and P9 is the least significant, and P7 and P10 have no obvious salty taste enhancement effect. By calculating the freshness enhancement threshold and saltiness enhancement threshold of each salty peptide, it can be obtained that the freshness enhancement thresholds of SEQ ID NO.1 to SEQ ID NO.8 are 0.09, 0.18, 0.09, 0.02, 0.18, 0.18, 0.35, 0.18, 0.04, -, respectively, and the saltiness enhancement thresholds are -, -, -, 0.02, 0.09, 0.09, -, 0.02, 0.08, 0.08 (where "-" means no enhancement of the freshness / saltiness threshold).

[0033] The results of sensory evaluation found that edible fungus umami peptides can enhance the umami characteristics of food and, to a certain extent, can enhance the umami / salty intensity of MSG / NaCl. They can be applied in the food field as base materials and auxiliary materials as seasonings, which can meet sensory needs while being nutritionally safe.

[0034] The above embodiments are limitations on the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any changes, modifications, substitutions, combinations, and simplifications made within the spirit and principles of the present invention should be equivalent replacement methods and should be included in the protection scope of the claims of the present invention.

Claims

1. The use of seafood mushroom umami peptide in preparing food seasoning, characterized in that: The amino acid sequence of the seafood mushroom umami peptide is shown in SEQ ID NO.8, and can bind to the umami receptor T1R1.

2. The use according to claim 1, characterized in that: The seafood mushroom umami peptide has umami taste and the effect of enhancing umami taste.

3. The use according to claim 1, characterized in that: The seafood mushroom umami peptide is separated and extracted from seafood mushroom or artificially synthesized.

4. The use according to claim 1, characterized in that: The food seasoning is a flavor-enhancing food additive.

5. The use according to claim 1, characterized in that: The food seasoning is a flavor-enhancing seasoning for pet food.

6. A seasoning mixture, characterized in that It includes seafood mushroom flavor peptides with an amino acid sequence as shown in SEQ ID NO.8 and other flavor-enhancing food additives.

7. A seasoning mixture according to claim 6, characterized in that The other umami peptides are selected from at least one of the seafood mushroom umami peptides whose amino acid sequences are shown in SEQ ID NO.1 to SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.

10.

8. A food, characterized in that: The food contains food seasoning, and the food seasoning is seafood mushroom umami peptide with an amino acid sequence as shown in SEQ ID NO.

8.

9. A food, characterized in that: The food contains a seasoning mixture, which includes a seafood mushroom umami peptide with an amino acid sequence as shown in SEQ ID NO.8 and other food additives for enhancing flavor, and the other food additives for enhancing flavor are selected from at least one of the seafood mushroom umami peptides with amino acid sequences as shown in SEQ ID NO.1 to SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.

10.

10. A method for increasing the umami taste of food, characterized in that: A seafood mushroom umami peptide with an amino acid sequence as shown in SEQ ID NO.8 is added to the food, or at least one of a seafood mushroom umami peptide with an amino acid sequence as shown in SEQ ID NO.8 and an amino acid sequence as shown in SEQ ID NO.1 to SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.10 is added to the food.

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