Edible mushroom peptide with synergistic salty-increasing and salt-reducing functions as well as screening method and application of edible mushroom peptide

Abstract

The invention relates to an edible mushroom peptide with a synergistic salt increasing and reducing function. The amino acid sequence of the edible mushroom peptide is LPAPGADDGSGTVTLL. The edible mushroom peptide with the synergistic salty-increasing and salt-reducing functions has a good salty-increasing effect after being chelated with calcium chloride, 0.01% (w / v) of edible mushroom peptide interacts with 1mmol / L of calcium chloride, the generated salty taste is equivalent to the salty taste generated by 5mmol / L of sodium chloride, salt can be partially replaced, and the purposes of reducing salt without reducing salty taste and reducing salt without reducing taste are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to an edible mushroom peptide with a synergistic salt-increasing and salt-reducing function, a screening method thereof, and an application thereof. Background Art

[0002] Sodium chloride is not only a fundamental factor for maintaining life activity regulation but also an essential saltiness carrier in the food industry. However, the disorder of sodium ion metabolism caused by excessive intake will significantly increase the burden on the cardiovascular system and become a major hidden danger to contemporary dietary health.

[0003] In the field of research and development of salt-reduced foods, a new flavoring system based on the principle of flavor compensation is gradually replacing the traditional way of using salt. Among them, salty peptides and their functional derivatives, as an important branch of bioactive peptides, have attracted much attention due to their unique flavor performance and intermolecular interaction mechanisms. These low-molecular-weight protein components derived from nature can synergistically enhance the flavor with the flavor components in the food matrix, maintain a similar salty perception while reducing the sodium chloride addition amount, and provide an innovative solution for constructing a healthy salt-reduced food system.

[0004] Coprinus comatus is widely distributed in the forests of the southeastern region of China. Its fruiting body is rich in protein, fungal polysaccharides, and various bioactive components, showing significant development potential. However, due to the traditional fresh sales and drying processing modes, the utilization rate of its nutritional components is relatively low. Therefore, developing and preparing salty peptides and their functional derivatives from Coprinus comatus not only makes full use of resources but also promotes the development of deep processing of Coprinus comatus. However, there are few relevant reports at present. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides an edible mushroom peptide with a synergistic salt-increasing and salt-reducing function, a screening 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 peptide with a synergistic salt-increasing and salt-reducing function, and the amino acid sequence of the edible mushroom peptide is LPAPGADDDGSGTVTLL.

[0008] An edible mushroom peptide with a synergistic salt-increasing and salt-reducing function proposed by the present invention has a good salt-increasing effect after chelating with calcium chloride. The interaction between 0.01% (w / v) edible mushroom peptide and 1 mmol / L calcium chloride produces a saltiness equivalent to that produced by 5 mmol / L sodium chloride, and can partially replace the use of table salt, achieving the purpose of reducing salt without reducing saltiness and flavor.

[0009] Preferably, the molecular weight of the edible mushroom peptide is 1598.97 Da.

[0010] On the other hand, the present invention provides a method for screening edible mushroom peptides with the functions of synergistically enhancing saltiness and reducing salt content as described above, comprising the following steps:

[0011] S1. Use flavor protease to enzymatically hydrolyze Coprinus comatus, subject the obtained Coprinus comatus enzymatic hydrolysate to ultrafiltration treatment, collect the permeate of the fraction with a molecular weight less than 3000 Da to obtain the crude extract of Coprinus comatus saltiness-enhancing peptide, and freeze-dry it;

[0012] S2. Use nano-liquid chromatography-quadrupole orbitrap mass spectrometer to determine the amino acid sequence of the crude extract of Coprinus comatus saltiness-enhancing peptide, and then screen out the polypeptide sequences that are edible, have a hydrophilicity > 0, and can bind to the saltiness receptor TMC4 through computer-aided screening, which are the edible mushroom peptides with the functions of synergistically enhancing saltiness and reducing salt content.

[0013] The present invention uses Coprinus comatus as a raw material and obtains edible mushroom peptides with the functions of synergistically enhancing saltiness and reducing salt content by using biotechnology. It not only breaks through the processing bottleneck and realizes the efficient conversion of resources, but also the characteristics of the natural source of Coprinus comatus endow the edible mushroom peptides with the advantages of balanced amino acid composition, excellent thermal stability, and safety and non-toxicity. The enzymatic hydrolysis technology adopted by the present invention is simple and efficient, can track the activity of the enzymatic hydrolysis products to achieve directional enzymatic cleavage, and avoids cost waste; at the same time, the present invention screens edible mushroom peptides through computer simulation, which has the advantages of simple operation, low production cost, and high accuracy, and provides a reference for the large-scale preparation of saltiness-enhancing peptide-related products using Coprinus comatus as a raw material.

[0014] Preferably, in step S1, the specific method of the enzymatic hydrolysis is: take the pulverized Coprinus comatus, add deionized water and stir to form a Coprinus comatus slurry solution, adjust the pH value to neutral and add flavor protease for enzymatic hydrolysis, perform enzyme inactivation treatment after the enzymatic hydrolysis ends, and centrifuge to collect the supernatant, that is, obtain the Coprinus comatus enzymatic hydrolysate.

[0015] More preferably, the conditions of the enzymatic hydrolysis are: the enzyme-substrate ratio is 8.00% w / w, the enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis time is 3.70 h.

[0016] Preferably, in step S1, the specific method of the ultrafiltration treatment is: add the Coprinus comatus enzymatic 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, repeat the operation, and combine all the permeates.

[0017] 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 edible polypeptide sequences with a hydrophilicity > 0, and then the Chem3D Pro software is used to generate polypeptide models for the edible polypeptide sequences with a hydrophilicity > 0. Next, the polypeptide models are imported into DiscoveryStudio 2019 and molecular docking is performed 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 peptides with the function of synergistically enhancing saltiness and reducing salt content.

[0018] Another aspect of the present invention provides an application of an edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt content as described in any one of the above technical solutions or an edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt content obtained by the screening method as described in any one of the above technical solutions in the preparation of a salt substitute seasoning.

[0019] The edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt content of the present invention exhibits good saltiness enhancement ability, has the potential to replace table salt, and can be applied to fields such as food. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the secondary mass spectrometry diagram of the edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt content in Example 1;

[0021] Figure 2 It is the structural model diagram of the edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt content in Example 1 and the schematic diagram of its molecular docking structure with the TMC4 receptor;

[0022] Figure 3 It is the determination of the saltiness enhancement activity of the edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt content and calcium chloride in Example 2. SeqLPA represents 0.01% (w / v) of the edible mushroom peptide LPAPGADDDGSGTVTLL with the function of synergistically enhancing saltiness and reducing salt content; SeqLPA + CaCl2 represents a mixture of 0.01% (w / v) SeqLPA and 1 mmol / L calcium chloride. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The following further describes the present invention with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0024] The edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt content of the present invention has an amino acid sequence of LPAPGADDDGSGTVTLL, which can be extracted from the enzymolysis solution 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 method.

[0025] Example 1

[0026] A screening method for edible mushroom peptides with a synergistic salt-increasing and salt-reducing function, comprising the following steps:

[0027] S1. Use flavor protease to enzymatically hydrolyze Coprinus comatus, ultrafilter the obtained Coprinus comatus enzymatic hydrolysate, collect the permeate of the fraction with a molecular weight less than 3000 Da, obtain the crude extract of Coprinus comatus saltiness-enhancing peptide, and freeze-dry it;

[0028] S2. Use nano-liquid chromatography-quadrupole orbitrap mass spectrometer to determine the amino acid sequence of the crude extract of Coprinus comatus saltiness-enhancing peptide, and then screen out the polypeptide sequences that are edible, hydrophilicity > 0, and can bind to the saltiness receptor TMC4 through computer-aided screening, which are the edible mushroom peptides with a synergistic salt-increasing and salt-reducing function.

[0029] The specific steps are as follows:

[0030] (1) Crush Coprinus comatus, weigh 50 g of Coprinus comatus powder, add 0.5 L of deionized water and stir to form a Coprinus comatus slurry, adjust the pH = 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 enzymatic hydrolysate.

[0031] (2) Add the above Coprinus comatus enzymatic hydrolysate to an ultrafiltration centrifuge tube with a molecular weight cut-off of 3000 Da, ultrafilter at a speed of 12000 rpm for 1 h, collect the permeate of the fraction with a molecular weight less than 3000 Da, repeat this operation, and combine all the permeates to obtain the crude extract of Coprinus comatus saltiness-enhancing peptide, and freeze-dry it.

[0032] (3) Use nano-liquid chromatography-quadrupole orbitrap mass spectrometer to determine the amino acid sequence of the above crude extract of Coprinus comatus saltiness-enhancing peptide (take 100 μg of freeze-dried powder).

[0033] (4) Screen out the edible and hydrophilicity > 0 polypeptide sequences from the polypeptide sequences obtained by mass spectrometry sequencing through the ToxinPred database, then use Chem3D Pro software to generate a polypeptide model for the edible and hydrophilicity > 0 polypeptide sequences, and then import the polypeptide model into Discovery Studio 2019 for ligand minimization to obtain the best polypeptide model, perform molecular docking with the saltiness receptor TMC4 model, and further screen out the polypeptide sequences that can bind to the saltiness receptor TMC4, which are the edible mushroom peptides with a synergistic salt-increasing and salt-reducing function, and its amino acid sequence is LPAPGADDDGSGTVTLL, the hydrophilicity is 0.04, and the molecular weight is 1598.97 Da;

[0034] 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.

[0035] The secondary mass spectrometry diagram of the edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt content 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.

[0036] Example 2

[0037] The edible mushroom peptide sequence LPAPGADDDGSGTVTLL with the function of synergistically enhancing saltiness and reducing salt content obtained in Example 1 was synthesized by solid-phase synthesis method. Its saltiness enhancement ability with calcium chloride was measured by an electronic tongue, and its interaction with calcium chloride was characterized by a calcium chelating activity experiment.

[0038] (1) Determination of the synergistic saltiness enhancement activity of the edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt content and calcium chloride

[0039] Determination method: An aqueous solution of calcium chloride with the same concentration of 1 mmol / L was mixed with 0.01% (w / v) LPAPGADDDGSGTVTLL respectively as test samples, and sodium chloride solutions with different concentrations were used as saltiness reference samples (1, 2, 3, 4, 5 mmol / L). The data was measured by an electronic tongue, the signal amplification factor of the electronic tongue was adjusted to 100, and each sample was measured in parallel 6 times. The obtained sample signal data reflected the difference in saltiness strength among the samples through principal component analysis.

[0040] From Figure 3 it can be seen 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 data points in the PC1 direction represents its saltiness strength. It can be seen from this figure that the saltiness intensity of 0.01% (w / v) edible mushroom peptide is equivalent to that produced by 2 - 3 mmol / L sodium chloride, and when it is mixed with a 1 mmol / L calcium chloride standard solution, the saltiness of the mixed solution is equivalent to that produced by 5 mmol / L sodium chloride. The above results show that the edible mushroom peptide of the present invention has good saltiness activity, and its saltiness effect can be enhanced after chelating with calcium chloride, and it can be applied to the preparation of salt substitute seasonings.

[0041] (2) Determination of the chelating activity of the edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt content and calcium chloride

[0042] Determination method: Prepare 0.5 mol / L HCl solution; 10 mmol / L calcium chloride solution; 0.2 mol / L phosphate buffer (PBS, pH = 8.00); ethanolamine-borate buffer (dissolve 1.8 g boric acid in 5 mL deionized water, transfer to a 50 mL light-proof volumetric flask, and make up the volume with ethanolamine); o-cresolphthalein solution (dissolve 40.0 mg o-cresolphthalein complexone in a 50 mL brown volumetric flask, add 12.5 mL deionized water, 0.2 mL 1 mol / L KOH, shake until completely dissolved, make up the volume with deionized water, and finally add 0.25 mL glacial acetic acid and mix well); 8-hydroxyquinoline solution (dissolve 2.5 g 8-hydroxyquinoline in a 50 mL brown volumetric flask and make up the volume with 95% ethanol); 1 mg / mL stock calcium standard solution. Mix 1 mL of edible mushroom peptide sample (1 mg / mL), 2 mL of phosphate buffer and 1 mL of calcium chloride (10 mmol / L) in a 10 mL centrifuge tube, place it in a water bath at 37 °C and react for 120 min with continuous oscillation. After balancing, centrifuge at 4000 r / min for 20 min in a centrifuge, and the supernatant is LPAPGADDDGSGTVTLL-Ca. Dilute 2.5 mL of the calcium stock solution to a 50 mL volumetric flask to obtain the standard calcium working solution. Take 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL of the working solution in different 4 mL centrifuge tubes respectively, and then add 1.0, 0.8, 0.6, 0.4, 0.2, 0 mL of deionized water respectively to make up to 1 mL, shake well, and prepare the calcium standard solution for making the calcium standard curve. Take 6 mL of ethanolamine-borate buffer, 1.8 mL of 8-hydroxyquinoline stock solution, and 6 mL of o-cresolphthalein solution and make up the volume to 100 mL in a volumetric flask to obtain the working color-developing solution. Pipette 30 μL of the standard calcium working solution or sample dilution into a 96-well plate, and then add 150 μL of the working color-developing solution to the corresponding wells respectively. Use an enzyme-labeled instrument to measure the absorbance at a wavelength of 570 nm as the standard curve or sample measurement data.

[0043] The determination results show that the calcium chelating activity of the edible mushroom peptide LPAPGADDDGSGTVTLL of the present invention is 22.337 ± 0.669 μg / mg, that is, 1 mg of LPAPGADDDGSGTVTLL can chelate with about 22.337 μg of calcium chloride and inhibit the bad flavor brought by free calcium chloride, further enhancing the salty taste.

[0044] 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 described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An edible mushroom peptide with a synergistic function of increasing saltiness and reducing salt content, characterized in that, The amino acid sequence of the edible mushroom peptide is LPAPGADDDGSGTVTLL.

2. The edible mushroom peptide with the function of synergistically increasing and decreasing salt as described in claim 1, wherein The molecular weight of the edible mushroom peptide is 1598.97 Da.

3. A screening method for edible mushroom peptides with a synergistic function of increasing and decreasing salt as described in claim 1, characterized in that, It includes the following steps: S1. Use flavor protease to enzymatically hydrolyze Coprinus comatus, ultrafilter the obtained Coprinus comatus enzymatic hydrolysate, collect the permeate of the fraction with a molecular weight less than 3000 Da to obtain the crude extract of Coprinus comatus salty flavor enhancing peptide, and freeze-dry it. S2. Use nano-liquid chromatography-quadrupole orbitrap mass spectrometer to determine the amino acid sequence of the crude extract of Coprinus comatus salty flavor enhancing peptide, and then screen out the polypeptide sequences that are edible, hydrophilicity > 0, and can bind to the salty taste receptor TMC4 through computer-aided screening, which are the edible mushroom peptides with the function of synergistically enhancing saltiness and reducing salt.

4. The screening method of the edible mushroom peptide with the function of synergistically increasing and decreasing salt as claimed in claim 3, wherein, In step S1, the specific method of enzymatic hydrolysis is as follows: Take the crushed Coprinus comatus, add deionized water and stir to form a Coprinus comatus slurry solution, adjust the pH value to neutral, add flavor protease for enzymatic hydrolysis, perform enzyme inactivation treatment after enzymatic hydrolysis, and centrifuge to collect the supernatant to obtain the Coprinus comatus enzymatic hydrolysate.

5. The screening method of edible mushroom peptides with a synergistic salt-increasing and salt-reducing function according to claim 4, wherein The conditions for enzymatic hydrolysis are: the enzyme-substrate ratio is 8.00% w / w, the enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis time is 3.70 h.

6. The screening method of the edible mushroom peptide with the function of synergistically increasing and decreasing salt according to claim 3, characterized in that, In step S1, the specific method of ultrafiltration treatment is as follows: Add the Coprinus comatus enzymatic 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, repeat the operation, and combine all the permeates.

7. The screening method of the edible mushroom peptide with the function of synergistically increasing and decreasing salt as claimed in claim 3, wherein, In step S2, the specific method of computer-aided screening is as follows: Screen out the edible and hydrophilicity > 0 polypeptide sequences from the polypeptide sequences obtained by mass spectrometry sequencing through the ToxinPred database, then use Chem3D Pro software to generate a polypeptide model of the edible and hydrophilicity > 0 polypeptide sequences, and then import the polypeptide model into Discovery Studio 2019 for molecular docking with the salty taste receptor TMC4 model, and further screen out the polypeptide sequences that can bind to the salty taste receptor TMC4, which are the edible mushroom peptides with the function of synergistically enhancing saltiness and reducing salt.

8. Application of an edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt as described in any one of claims 1-2 or an edible mushroom peptide with the function of synergistically enhancing saltiness and reducing salt obtained by the screening method as described in any one of claims 3-7 in the preparation of a salt substitute seasoning.

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