Edible mushroom source salty peptide as well as preparation method and application thereof

Abstract

The invention relates to an edible mushroom source salty peptide. The edible mushroom source salty peptide comprises any one or more of amino acid sequences as shown in SEQ ID NO.1 to SEQ ID NO.3. The edible mushroom source salty peptide has the advantages that the edible mushroom source salty peptide can be used for preparing edible mushroom source salty peptide; the edible mushroom source salty peptide shows good salty taste characteristics, and experiments show that the salty taste of 0.1% (w / v) salty peptide is equivalent to the flavor generated by 2-3mmol / L NaCl. The edible mushroom source salty peptide disclosed by the invention can be used for replacing table salt, high-sodium intake is avoided while a salty effect is ensured, the effect of reducing salt but not reducing salty is achieved, and the edible mushroom source salty peptide has a good application prospect in the fields of seasonings and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and particularly to an edible mushroom-derived savory peptide, a preparation method thereof, and an application thereof. Background Art

[0002] As one of the five basic tastes in addition to sour, sweet, bitter, and umami, salt is known as the "king of all flavors". It can not only endow food with a rich flavor but also improve the quality of food. Therefore, it occupies a crucial position in the field of food processing. The main component of salt is sodium chloride, in which Na + and Cl - are essential minerals for the human body, which not only maintain the normal osmotic pressure but also regulate the water-salt balance in the body, and are important substances for maintaining physiological functions. However, long-term intake of high-concentration sodium salts will disrupt the stability of the internal environment and cause varying degrees of damage to organs such as the heart, liver, and kidneys. If only the amount of salt added is reduced without taking other remedial measures, it will inevitably lead to problems such as reduced perception of food saltiness, insufficient flavor, deteriorated texture, and shortened shelf life. With the popularization of the concept of healthy diet, people are increasingly concerned about the health risks brought by excessive salt intake. Therefore, the solution of "reducing salt without reducing saltiness" has attracted wide attention globally, and savory peptides are gradually becoming a research hotspot. Compared with other flavor regulators, naturally extracted savory peptides, due to their green, healthy, safe, and easily acceptable characteristics, can not only provide saltiness but also supplement essential amino acids for the human body, thus becoming a green and healthy salt substitute, which has great utilization value and broad development prospects for people who need a low-sodium diet.

[0003] As a kind of food material rich in various nutrients, Coprinus comatus can provide the human body with a variety of important nutrients, which helps to maintain health and promote the normal operation of various physiological functions. So far, there is no report on the process of preparing savory peptides from Coprinus comatus.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides an edible mushroom-derived savory peptide, a preparation method thereof, and an application thereof.

[0006] The technical solution adopted by the present invention is as follows:

[0007] On the one hand, the present invention provides an edible mushroom-derived savory peptide, and the savory peptide includes any one or more of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3.

[0008] An edible mushroom-derived salty peptide proposed by the present invention exhibits good salty characteristics. Experiments have shown that the saltiness of 0.1% (w / v) of the salty peptide is equivalent to the flavor produced by 2 - 3 mmol / L of NaCl. The edible mushroom-derived salty peptide of the present invention can replace the use of table salt, ensuring a salty effect while avoiding high sodium intake, achieving the effect of reducing salt without reducing saltiness, and having good application prospects in the fields of seasonings and the like.

[0009] On the other hand, the present invention provides a method for preparing the above-mentioned edible mushroom-derived salty peptide, which includes the following steps:

[0010] S1. Using Coprinus comatus as the raw material, performing protease hydrolysis on it, subjecting the obtained Coprinus comatus enzymatic hydrolysate to ultrafiltration treatment, collecting the permeate, and freeze-drying to obtain the crude extract of Coprinus comatus salty peptide;

[0011] S2. Using nano liquid chromatography-quadrupole orbitrap mass spectrometer to determine the amino acid sequence of the crude extract of Coprinus comatus salty peptide, and then screening out the polypeptide sequences that are non-toxic, have a hydrophilicity > 0, and can bind to the salty receptor TMC4 through computer-aided screening, which are the edible mushroom-derived salty peptides.

[0012] Starting from Coprinus comatus, the present invention focuses on the process control of protease hydrolysis, prepares active polypeptides with specific amino acid sequences, and enables the efficient realization of salty activity. The enzymatic hydrolysis method adopted by the present invention is simple and efficient, can perform activity tracking on the enzymatic hydrolysis products to achieve directional enzymatic cleavage, and avoids cost waste; at the same time, with the aid of computer-aided screening, it has the advantages of simple operation, low production cost, and high accuracy. The combination of the two helps to realize the large-scale production and wide application of salty peptides.

[0013] Preferably, in step S1, the specific method of the protease hydrolysis is: using flavor protease to hydrolyze the Coprinus comatus, inactivating the enzyme after the hydrolysis ends, centrifuging to collect the supernatant to obtain the Coprinus comatus enzymatic hydrolysate.

[0014] More preferably, the conditions for the enzymatic hydrolysis are: the enzyme-substrate ratio is 8.00% w / w, the pH is 7.0, the enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis time is 3.70 h.

[0015] Preferably, in step S1, the specific method of the ultrafiltration treatment is: adding the Coprinus comatus enzymatic hydrolysate to an ultrafiltration centrifuge tube with a molecular weight cut-off of 3000 Da, ultrafiltering at a speed of 12000 rpm for 1 h, collecting the permeate, repeating the operation, and combining all the permeates.

[0016] Preferably, in step S2, the specific method of computer-aided screening is as follows: the polypeptide sequences obtained by mass spectrometry sequencing are screened through the ToxinPred database to obtain non-toxic polypeptide sequences with a hydrophilicity > 0. Then, the Chem3D Pro software is used to generate polypeptide models of the non-toxic polypeptide sequences with a hydrophilicity > 0. Next, the polypeptide models are imported into DiscoveryStudio 2019 and molecularly docked with the salty taste receptor TMC4 model to further screen out the polypeptide sequences that can bind to the salty taste receptor TMC4, which are the edible mushroom-derived salty peptides.

[0017] Another aspect of the present invention provides an application of the above-mentioned edible mushroom-derived salty peptide or the edible mushroom-derived salty peptide obtained by the preparation method as described in any one of the above technical solutions in the preparation of a food flavor regulator.

[0018] The edible mushroom-derived salty peptide of the present invention has good salty taste activity and can be applied as products such as food flavor regulators. Description of the Drawings

[0019] Figure 1 It is the secondary mass spectrometry diagram of the edible mushroom-derived salty peptide in Example 1;

[0020] Figure 2 It is the structural model diagram of the edible mushroom-derived salty peptide in Example 1 and the schematic diagram of its molecular docking structure with the TMC4 receptor;

[0021] Figure 3 It is the salty taste activity characterization of the edible mushroom-derived salty peptide in Example 2. In the figure, SeqSQS represents SQSNARTF, SeqGFP represents GFPGAGEDGPS, and SeqLEV represents LEVESSDTIDNVK. Detailed Embodiments

[0022] To better understand the above technical solutions, the exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings. Although the exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more clear and thorough understanding of the present invention and to be able to fully convey the scope of the present invention to those skilled in the art.

[0023] The edible mushroom-derived salty peptide of the present invention includes any one or more of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3. It can be extracted from the enzymatic hydrolysate of Coprinus comatus and special purification steps can be designed according to special properties such as molecular weight, or it can be synthesized by solid-phase synthesis methods.

[0024] Example 1

[0025] A method for preparing an edible mushroom-derived salty peptide, characterized by comprising the following steps:

[0026] S1. Using Coprinus comatus as a raw material, performing protease hydrolysis on it, subjecting the obtained Coprinus comatus enzyme hydrolysate to ultrafiltration treatment, collecting the permeate, and freeze-drying to obtain a crude extract of Coprinus comatus salty peptide.

[0027] S2. Using a nano liquid chromatography-quadrupole orbitrap mass spectrometer to determine the amino acid sequence of the crude extract of Coprinus comatus salty peptide, and then screening out, through computer-aided means, polypeptide sequences that are non-toxic, have a hydrophilicity > 0, and can bind to the salty receptor TMC4, which are the edible mushroom-derived salty peptides.

[0028] The specific steps are as follows:

[0029] (1) Grind Coprinus comatus into powder, weigh 50 g of Coprinus comatus powder, add 0.5 L of deionized water, adjust the pH to 7.00, add 4 g of flavor protease, place it in a water bath at 50 °C for enzymatic hydrolysis for 3.70 h, after the enzymatic hydrolysis is completed, boil it in a boiling water bath for 10 min to inactivate the enzyme, centrifuge at 10000 rpm for 10 min, and collect the supernatant to obtain the Coprinus comatus enzyme hydrolysate.

[0030] (2) Add the above Coprinus comatus enzyme hydrolysate to an ultrafiltration centrifugal tube with a molecular weight cut-off of 3000 Da, ultrafilter at a speed of 12000 rpm for 1 h, collect the permeate (the fraction with a molecular weight less than 3000 Da), repeat this operation, combine all the permeates, and freeze-dry to obtain a crude extract of Coprinus comatus salty peptide.

[0031] (3) Use a nano liquid chromatography-quadrupole orbitrap mass spectrometer to determine the amino acid sequence of the above crude extract of Coprinus comatus salty peptide (take 100 μg of the freeze-dried powder).

[0032] (4) Screen out polypeptide sequences that are non-toxic and have a hydrophilicity > 0 from the polypeptide sequences obtained by the above mass spectrometry sequencing through the ToxinPred database, then use Chem3D Pro software to generate a polypeptide model of the non-toxic and hydrophilicity > 0 polypeptide sequences, and then import the polypeptide model into Discovery Studio 2019 for molecular docking with the salty receptor TMC4 model, and further screen out polypeptide sequences that can bind to the salty receptor TMC4, which are the edible mushroom-derived salty peptides, and their amino acid sequences are shown as SEQ ID NO.1 to SEQ ID NO.3, specifically LEVESSDTIDNVK (hydrophilicity is 0.68, molecular weight is 1448.73 Da), GFPGAGEDGPS (hydrophilicity is 0.30, molecular weight is 990.14 Da), SQSNARTF (hydrophilicity is 0.08, molecular weight is 910.05 Da);

[0033] Among them, the salt taste receptor TMC4 model is obtained by the following method: obtaining the amino acid sequence of the salt taste receptor TMC4 from the NCBI website, and performing homology modeling on the SWISS-MODEL website according to the sequence, and screening to obtain the receptor model with the highest score as the molecular pair acceptor model.

[0034] The secondary mass spectrometry diagram of the edible mushroom-derived salt taste peptide in this example is as Figure 1 shown, and its structural model diagram and the schematic diagram of its docking structure with the TMC4 receptor are as Figure 2 shown.

[0035] Example 2

[0036] The edible mushroom-derived salt taste peptide sequences LEVESSDTIDNVK, GFPGAGEDGPS, and SQSNARTF obtained in Example 1 were synthesized by solid-phase synthesis method, and their salt taste activities were measured by an electronic tongue. The results are as Figure 3 shown.

[0037] Test method: During the test, the three synthesized polypeptides were respectively prepared into aqueous solutions with the same concentration and used as test samples, and sodium chloride solutions with different concentrations (1, 2, 3, 4, 5 mmol / L) were used as standard salt taste intensity reference products, and data were measured by an electronic tongue. Each sample was measured in parallel 6 times, and the obtained sample signal data reflected the difference in salt taste strength among samples through principal component analysis.

[0038] It can be seen from Figure 3 that the projections of the data points of sodium chloride in the PC1 direction are arranged in an orderly manner in the positive direction of the PC1 axis as the concentration of sodium chloride increases. Therefore, it is considered that the projection of the sample in the PC1 direction represents the salt taste intensity. It can be seen from this figure that the salt taste of 0.1% (w / v) LEVESSDTIDNVK, SQSNARTF, and GFPGAGEDGPS is equivalent to the flavor produced by 2 - 3 mmol / L of sodium chloride, indicating that the edible mushroom-derived salt taste peptide of the present invention has salt taste characteristics and can be applied to the preparation of food flavor regulators.

[0039] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An edible mushroom-derived salty peptide, characterized in that, The salty peptides include any one or more of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.

3.

2. A method for preparing an edible mushroom-derived salty peptide as described in claim 1, characterized in that, It includes the following steps: S1. Using Coprinus comatus as a raw material, performing proteolysis on it, subjecting the obtained Coprinus comatus enzymolysis solution to ultrafiltration treatment, collecting the permeate, and freeze-drying to obtain a crude extract of Coprinus comatus salty peptides; S2. Using a nano-liquid chromatography-quadrupole orbitrap mass spectrometer to determine the amino acid sequence of the crude extract of Coprinus comatus salty peptides, and then screening out non-toxic polypeptide sequences with a hydrophilicity > 0 and capable of binding to the salty receptor TMC4 through computer-aided means, which are the edible mushroom-derived salty peptides.

3. The preparation method of the edible mushroom-derived salty peptide according to claim 2, wherein In step S1, the specific method of the proteolysis is: using flavor protease to perform enzymolysis on the Coprinus comatus, inactivating the enzyme after the enzymolysis ends, centrifuging to collect the supernatant to obtain the Coprinus comatus enzymolysis solution.

4. The preparation method of the edible mushroom-derived salty peptide according to claim 3, wherein, The conditions of the enzymolysis are: the enzyme-substrate ratio is 8.00% w / w, the pH is 7.0, the enzymolysis temperature is 50 °C, and the enzymolysis time is 3.70 h.

5. The preparation method of the edible mushroom-derived salty peptide according to claim 2, wherein, In step S1, the specific method of the ultrafiltration treatment is: adding the Coprinus comatus enzymolysis solution into an ultrafiltration centrifugal tube with a molecular weight cut-off of 3000 Da, ultrafiltering at a speed of 12000 rpm for 1 h, collecting the permeate, repeating the operation, and combining all the permeates.

6. The preparation method of the edible mushroom-derived salty peptide according to claim 2, wherein, In step S2, the specific method of the computer-aided screening is: screening out non-toxic polypeptide sequences with a hydrophilicity > 0 from the polypeptide sequences obtained by mass spectrometry sequencing through the ToxinPred database, then using Chem3D Pro software to generate a polypeptide model of the non-toxic polypeptide sequences with a hydrophilicity > 0, and then importing the polypeptide model into Discovery Studio 2019 for molecular docking with the salty receptor TMC4 model, and further screening out polypeptide sequences capable of binding to the salty receptor TMC4, which are the edible mushroom-derived salty peptides.

7. Use of an edible mushroom-derived salty peptide as described in claim 1 or an edible mushroom-derived salty peptide obtained by the preparation method as described in any one of claims 2 - 6 in the preparation of a food flavor regulator.

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