Seafood mushroom sweet peptide and its extraction method and application
Sweetened peptides were isolated from seafood mushrooms by protein complex enzyme enzymatic method and molecular interception technology, and the peptide sequence was determined through virtual screening, which solved the problem of lack of sweet peptide research on seafood mushrooms in the prior art, and achieved the separation and application of sweet peptides with high sweetness intensity and easy absorption.
Patent Information
- Application Number
- CN202311436951.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-31
- Publication Date
- 2025-03-14
- Estimated Expiration
- 2043-10-31
AI Technical Summary
The lack of reports in the prior art on isolating and purifying sweetened peptides from seafood mushrooms, limiting the application of seafood mushrooms in the fields of food flavor and health care.
The protein complex enzyme enzymatic method was used to enzymatically dissolve seafood mushrooms, combined with the molecular interception technology of freeze-dried, nanofiltration membrane and ultrafiltration membrane, and the sweet-taste peptide of seafood mushrooms with sweet taste was isolated and purified, and T1R2 was used as a sweet receptor, and the peptide sequence was determined using molecular docking virtual screening technology.
Two sweet peptides were successfully isolated from seafood mushrooms. They have good sweetness and richness, are easy to be absorbed by the human body, have high sweetness intensity, and can be used instead of sucrose, which has great application significance in food.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of protein active peptides, and specifically relates to seafood mushroom sweet peptides and an extraction method and application thereof. Background Art
[0002] Seafood mushroom is an edible fungus with high nutritional value. It is rich in flavonoids, total phenols, saponins, choline, peroxidase, superoxide dismutase and other natural antioxidant active substances, and plays an important role in health care and diet therapy. At present, the research on edible fungus protein peptides is mostly limited to the research of simple volatile aroma, and there are few reports on the research of seafood mushroom sweet peptides.
[0003] Sweet peptides refer to oligopeptides with a molecular weight of less than 5000u 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, and are present in various foods such as fruits and vegetables. Sweet peptides, as a new type of active peptide sweetener, can not only improve the sweetness of food, but are also very important for the flavor of processed food.
[0004] For the study of sweet peptides, Bian et al. used multi-frequency countercurrent ultrasonic treatment to treat defatted mulberry seed protein, and then separated and purified the sweet peptides by ultrafiltration-gel filtration chromatography and reversed-phase high-performance liquid chromatography. The sweet peptides were identified by MALDI-TOF / TOF MS / MS technology, and DMSPH was obtained with a strong sweet taste. (Bian, YR, Li, WJ, Pan, LH, Peng, QM, You, S., Sheng, S., et al. (2022). Sweet-flavored peptides with biological activities from mulberry seed protein treated by multifrequency countercurrent ultrasonic technology. Food Chemistry, 367, 130647.). W. Tsang et al. prepared two sweet peptides, L-aspartyl-D-alaninebenzyl ester and L-aspartyl-D-cu-aminobutyric acid benzyl ester (Tsang, JW, Schmied, B., Nyfeler, R., & Goodman, M. (2002). Peptide sweeteners. 6. Structural studies on the C-terminal amino acid of L-aspartyl dipeptidesweeteners. Journal of Medicinal Chemistry, 27 (12), 1663-1668.). These sweet peptides contribute significantly to taste and can affect the taste of food.
[0005] So far, many researchers have isolated and purified sweet peptides from fruits such as mulberries, but there are no reports on the isolation and purification of sweet peptides from mushrooms. 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 sweet peptides and extraction methods and applications thereof.
[0007] In order to achieve the above object, the present invention adopts the following technical solutions:
[0008] The invention discloses a seafood mushroom sweet peptide, which has sweet taste and can be combined with a sweet taste receptor T1R2. The amino acid sequence of the seafood mushroom sweet peptide is shown in SEQ ID NO.1 and SEQ ID NO.2.
[0009] Preferably, the seafood mushroom sweet peptide is separated and extracted from seafood mushroom.
[0010] Preferably, the seafood mushroom sweet peptide is artificially synthesized.
[0011] The present invention also discloses a method for extracting the above-mentioned seafood mushroom sweet peptide, which comprises enzymatically hydrolyzing the seafood mushroom 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 peptide; using T1R2 as a sweet taste receptor, and obtaining the seafood mushroom sweet peptides shown in SEQ ID NO.1 and SEQ ID NO.2 through molecular docking virtual screening technology.
[0012] Preferably, before the seafood mushroom is enzymatically hydrolyzed by the protein complex enzyme method, cellulose enzyme is used for enzymatic hydrolysis.
[0013] Preferably, the protease is neutral protease and flavor protease.
[0014] The invention also discloses application of the seafood mushroom sweet peptide in preparing food.
[0015] Preferably, the food is a food seasoning.
[0016] Preferably, the food seasoning is a sweetening food additive.
[0017] The invention also discloses a product containing the seafood mushroom sweet peptide.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The seafood mushroom sweet peptides provided by the present invention have the following characteristics: 1. Both sweet peptides are obtained by separation and purification from seafood mushrooms, and have good affinity with the sweet receptor T1R2; using sensory detection, the results show that both sweet peptides have good sweetness and richness. 2. The extracted sweet peptides are small molecular weight peptides, which are easily absorbed by the human body. 3. The extracted sweet peptides have high sweetness intensity and can replace the use of sucrose, and have great application significance in food. 4. The extraction method of the two sweet peptides is simple and pollution-free, and the obtained seafood mushroom sweet peptides have outstanding sweetness, and are high-quality flavor base materials. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 2D diagram of the docking of taste receptor protein T1R2 and sweet peptide; A to D represent EGNPAHQK, QEQEAKKR, GPFPILV and AGLELLK, respectively;
[0021] Figure 2 This is a purity verification result diagram of the flavor peptide EGNPAHQK extracted from seafood mushrooms;
[0022] Figure 3 This is the mass spectrometry result of the artificially synthesized flavor peptide EGNPAHQK in seafood mushroom;
[0023] Figure 4 This is a purity verification result diagram of the flavor peptide QEQEAKKR extracted from seafood mushroom;
[0024] Figure 5 This is the mass spectrometry result of the artificially synthesized flavor peptide QEQEAKKR in seafood mushroom;
[0025] Figure 6 This is a purity verification result diagram of the flavor peptide AGLELLK extracted from seafood mushrooms;
[0026] Figure 7 This is the mass spectrometry result of the artificially synthesized flavor peptide AGLELLK in seafood mushroom. DETAILED DESCRIPTION
[0027] 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.
[0028] 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.
[0029] Herein, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values within the range (including integers and fractions).
[0030] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”
[0031] In the specification of the present invention and the following examples, unless otherwise specified, seafood mushroom sweet peptide refers to seafood mushroom sweet 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 sweet 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.
[0032] 1. Isolation and extraction of seafood mushroom flavor peptides
[0033] The fresh and intact seafood mushrooms available on the market were first cleaned with running water, blanched and sterilized for 10 minutes, and then beaten into a fine seafood mushroom slurry 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, and after freeze-drying, the freeze-dried sample was redissolved in ultrapure water, and ultrafiltration separation was performed at 25°C using a nanofiltration membrane with a molecular weight cutoff of 200Da and an ultrafiltration membrane with a molecular weight cutoff of 1000Da, and the components with a molecular weight of 200-1000Da were collected, and 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 sensitivity of the UV detector, 220 nm detection wavelength, updating the collection tube every 6 minutes, multiple collections, and then concentrated and freeze-dried again to obtain the seafood mushroom flavor peptide.
[0034] 2. Identification of sweet peptides from seafood mushrooms
[0035] The seafood mushroom flavor peptide obtained in step 1 is sequenced by mass spectrometry, and then a seafood mushroom flavor peptide (sweet peptide) with sweet taste is obtained by virtual screening.
[0036] Specifically, T1R2 was used as the sweet taste receptor, and the model of sweet taste receptor T1R2 and seafood mushroom flavor peptide was established using AlphaFold2 software. The pdb files of T1R2 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 Pro, Ser, Trp, Tyr and Val in the peptide sequence play an important role in binding to the taste receptor T1R2. Combined with sensory evaluation, the seafood mushroom flavor peptide with the best affinity to the sweet taste receptor T1R2 was screened out as the sweet peptide. The sequence is shown in SEQ ID NO.1 to SEQ ID NO.3 in Table 1, specifically Gln-Glu-Gln-Glu-Ala-Lys-Lys-Arg, Ala-Gly-Leu-Glu-Leu-Leu-Lys, and Glu-Gly-Asn-Pro-Ala-His-Gln-Lys, and the ones with more obvious sweetness are: Gln-Glu-Gln-Glu-Ala-Lys-Lys-Arg, Ala-Gly-Leu-Glu-Leu-Leu-Lys. The purity of the three sweet peptides was verified, and the results are as follows Figure 2 , Figure 4 and Figure 6 As shown, it was found that the purity of all sweet peptides was above 97%.
[0037] According to the sequences SEQ ID NO.1 to SEQ ID NO.3 in Table 1, the artificially synthesized sweet peptides, the mass spectrometry results of the artificially synthesized sweet peptides are as follows Figure 3 , Figure 5 and Figure 7 As shown, the molecular weight and molecular mass-to-charge ratio test results of the three sweet peptides are consistent with the theoretical molecular weight and molecular mass-to-charge ratio of the corresponding sweet peptides, confirming that the samples are target peptides.
[0038] Table 1 Detection results of sweet peptides extracted from seafood mushrooms
[0039] Peptide sequence Serial number length Molecular weight (Da) Molecular mass-to-charge ratio Sweetness rating results QEQEAKKR SEQ ID NO.1 8 1016.13 1014.92 3.1±0.17 AGLELLK SEQ ID NO.2 7 742.92 745.95 3.1±0.18 EGNPAHQ SEQ ID NO.3 8 879.93 880.80 3.1±0.09
[0040] 3. Sensory evaluation and verification of seafood mushroom sweet peptide
[0041] 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. The volunteers were trained in advance to prepare for the basic taste of sweetness, namely sucrose. It is particularly important to rinse the mouth with 50 mL of pure water before tasting the next sweet peptide to avoid taste errors for the next sweet peptide solution.
[0042] Three sweet peptide solutions of 2 mg / mL were prepared respectively, and their sweetness intensity was tasted after being fully stirred. The sweetness of the seafood mushroom sweet peptide was scored, and the scores with large differences were removed. The remaining scores were averaged, and the scoring results are shown in Table 1. The sensory evaluation results showed that the screened sweet peptides all had sweetness, indicating that the seafood mushroom sweet peptide can enhance the sweetness characteristics of food, can be used in the food field, can be used as a base material and an auxiliary material as a condiment, and can meet the needs of the senses while being nutritionally safe.
[0043] 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. Seafood mushroom sweet peptide, characterized in that: It has a sweet taste and can bind to the sweet taste receptor T1R2. The amino acid sequence of the seafood mushroom sweet taste peptide is shown in SEQ ID NO.1 and SEQ ID NO.
2.
2. The seafood mushroom sweet peptide according to claim 1, characterized in that: The seafood mushroom sweet peptide is separated and extracted from seafood mushroom.
3. The seafood mushroom sweet peptide according to claim 1, characterized in that: The seafood mushroom sweet peptide is artificially synthesized.
4. Use of the seafood mushroom sweet peptide according to any one of claims 1 to 3 in preparing food.
5. The use according to claim 4, characterized in that: The food is a food seasoning.
6. The use according to claim 5, characterized in that: The food seasoning is a sweetening food additive.
7. A food seasoning containing the seafood mushroom sweet peptide according to any one of claims 1 to 3.