A salty peptide derived from dry-cured ham and preparation method thereof

Abstract

A salt-enhancing peptide derived from dry-cured ham, the amino acid sequence of which is: Asp-Leu, Phe-Met-Ser-Ala-Leu-Phe or His-Val-Arg-Arg-Lys. The salt-enhancing peptide of the invention has a small molecular weight, is easy to separate and purify, has a strong salt-enhancing effect, has a simple sequence, is easy to synthesize, and can be applied to the fields of food, health products, and pharmaceutical biology. It is expected to replace the salt substitute products currently on the market.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a saltiness-enhancing peptide derived from dry-cured ham and a preparation method thereof. Background Art

[0002] At present, the main source of saltiness in food is still table salt (mainly composed of NaCl). However, it is reported that the daily salt intake of domestic residents is usually higher than 10g, and excessive salt intake may damage the balance of the human body and the stability of blood pressure, and induce hypertension, stroke and other cardiovascular diseases. Research on increasing the taste of salt without increasing the sodium content in salt and achieving salt reduction without reducing saltiness has always been a research hotspot.

[0003] Salty peptides can increase the saltiness of food without increasing the salt content. Taste is an important factor when considering food quality. Peptides extracted from food proteins are also dietary components that may affect the taste of food, so they may be valuable ingredients when considering adjusting the taste of food and using them as salt enhancers. Such salty peptides can be recommended as health foods suitable for preventing civilization diseases such as obesity or cardiovascular disease.

[0004] Good ham contains many polypeptide substances, amino acid molecules, etc., and is closely related to these small molecules. Among them, small molecules: amino acids, polypeptides, etc., especially small molecule polypeptides, may have a strong saltiness enhancement effect, that is, salty peptides.

[0005] Therefore, using ham as the source, through separation, purification, identification technology and sensory characteristics analysis, a dry-cured ham-derived salty peptide with a clear structure and significant salty-enhancing effect and its preparation method are developed, which have great market value and application value. Summary of the invention

[0006] The purpose of the present invention is to provide a dry-cured ham-derived polypeptide capable of alleviating alcoholic liver damage and a preparation method thereof.

[0007] To achieve the above-mentioned object and other related objects, the technical solution provided by the present invention is: a salty peptide derived from dry-cured ham, characterized in that: the amino acid sequence is: Asp-Leu, Phe-Met-Ser-Ala-Leu-Phe or His-Val-Arg-Arg-Lys.

[0008] The preferred technical solution is: the amino acid sequence is: Asp-Leu.

[0009] To achieve the above-mentioned object and other related objects, the technical solution provided by the present invention is: a method for preparing salty peptides derived from dry-cured ham, comprising the following steps:

[0010] S1. Preparation of ham-derived extract

[0011] The ham is processed to obtain powder, the powder is dispersed in a hydrochloric acid buffer solution, homogenized and then centrifuged, and then the supernatant is filtered to obtain a ham source extract;

[0012] S2. Dialysis separation of ham source extract

[0013] The ham-derived extract is dialyzed to obtain a dialyzate, and the dialyzate is concentrated, and the concentrated solution is freeze-dried to obtain a crude polypeptide;

[0014] S3. Separation and purification of crude peptides

[0015] The crude polypeptide was dissolved in pure water, filtered and loaded onto a Sephadex G-15 gel filtration column for purification to obtain a separated and purified polypeptide, which was then further purified by ion exchange chromatography. The purified components were subjected to sensory evaluation according to the spectral peaks, and the components with the best salt-enhancing effect were screened out through sensory evaluation and electronic tongue-assisted sensory characteristic analysis.

[0016] S4. Amino acid sequence test of salt-enhancing peptide

[0017] The component with the best salt-enhancing effect obtained in S3 was identified by ultra-performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry, and the three amino acid sequences were determined to be Asp-Leu, Phe-Met-Ser-Ala-Leu-Phe and His-Val-Arg-Arg-Lys.

[0018] The preferred technical solution is: in S1, the concentration of the hydrochloric acid buffer is 0.01 mol / L, and the ratio of powder to hydrochloric acid buffer is 50 g:100-200 mL; during homogenization: homogenize at 15000-18000 r / min for 3-5 times, each time for 8-12 seconds; during centrifugation, centrifuge at a speed of 10000-14000 r / min for 15-25 minutes at -3~-5ºC; the supernatant is filtered with a 0.45μm filter membrane.

[0019] The preferred technical solution is: in S2, the ham source extract is placed in a dialysis bag less than 3000 Da, dialyzed at -3~-5ºC for 20-28h to obtain a dialysate, and then rotary evaporated at 60-70 ºC to obtain a concentrated solution.

[0020] The preferred technical solution is: in S3, the chromatographic parameters are: sample mass concentration 40 mg / mL; injection volume 200µL; eluent is 0.01M hydrochloric acid buffer, flow rate 0.5mL / min, primary UV detection wavelength is 214 nm, and secondary UV detection wavelength is 250 nm.

[0021] The preferred technical scheme is: in S3, ion exchange chromatography is used for further purification, and the specific detection conditions are: sample mass concentration 10 mg / mL; injection volume 2 mL; chromatographic column: DEAE anion column, mobile phase A is 20 mM Tris-HCl buffer, mobile phase B is 20 mM Tris-HCl buffer + 1M NaCl; gradient elution: first gradient: mobile phase A 100%, mobile phase B 0%, elution volume 60 mL; second gradient: mobile phase A 90%, mobile phase B 10%, elution volume 60 mL; third gradient: mobile phase A 100%, mobile phase B 0%, elution volume 40 mL; elution rate is 5 mL / min, the main ultraviolet detection wavelength is 214 nm, and the secondary ultraviolet detection wavelength is 250 nm; the pH values ​​of mobile phase A and mobile phase B are both 9.0.

[0022] The preferred technical scheme is: the chromatographic conditions of ultra-high performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry are: the chromatographic column is Agilent Eclipse Plus-C18; mobile phase A: ultrapure water containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid; linear gradient elution: The chromatographic conditions of ultra-high performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry are: the chromatographic column is Agilent Eclipse Plus-C18; mobile phase A: ultrapure water containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid; linear gradient elution:

[0023] 100% mobile phase A, 0% mobile phase B 0-1 min;

[0024] 100%-70% mobile phase A, 0%-30% mobile phase B for 1-10 min;

[0025] 70%-0% mobile phase A, 30%-100% mobile phase B for 10-15 min;

[0026] 0% mobile phase A, 100% mobile phase B for 15-18 min;

[0027] 0%-100% mobile phase A, 100%-0% mobile phase B 18-22 min;

[0028] 100% mobile phase A, 0% mobile phase B for 22-25 min.

[0029] The preferred technical solution is: the inner diameter of the Sephadex G-15 gel filtration column is 3.0 cm and the column length is 200 cm; the inner diameter of the DEAE anion column is 16 mm and the column height is 25 mm; the inner diameter of the C18 chromatographic column is 4.6 mm, the column length is 150 mm, and the particle size is 3.5 µm.

[0030] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0031] 1. The salt-increasing peptide of the present invention has a small molecular weight, is easy to separate and purify, has a strong salt-increasing effect, has a simple sequence, is easy to synthesize, and can be applied to the fields of food, health products, medicine and biology. It is expected to replace the salt substitute products in the existing market.

[0032] 2. The present invention selects the famous dry-cured Jinhua ham, uses dialysis to enrich crude peptides, and then uses a dextran gel Sephadex G-15 gel filtration column and a DEAE anion column for purification to screen out the component with the best saltiness enhancement effect. The amino acid sequence is identified as Asp-Leu (DL) by liquid chromatography-mass spectrometry.

[0033] 3. The best salt-enhancing components can be accurately screened out through sensory evaluation and electronic tongue-assisted evaluation, and electronic tongue-assisted evaluation can show that these salt-enhancing peptide sequences also have a synergistic effect of umami. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a Sephadex G-15 gel chromatogram.

[0035] Figure 2 It is a sensory evaluation table of gel-purified components.

[0036] Figure 3 This is a radar chart of the electronic tongue taste analysis of gel-purified components.

[0037] Figure 4 This is the radar chart of the electronic tongue taste analysis of gel-purified component 2.

[0038] Figure 5 This is a radar chart of the electronic tongue taste analysis of gel-purified fraction three.

[0039] Figure 6 This is a radar chart of electronic tongue taste analysis of four gel-purified components.

[0040] Figure 7 It is the PCA plot of gel-purified fractions.

[0041] Figure 8 It is an ion exchange chromatogram.

[0042] Fig. 9 It is a sensory evaluation table of ion exchange purified components.

[0043] Fig.10 It is the secondary mass spectrum of the best salt-enhancing component. DETAILED DESCRIPTION

[0044] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0045] See also Figure 1-10 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions under which the present invention can be implemented. Therefore, they have no substantive technical significance, and any structural modification, change in proportional relationship or adjustment of size. The following examples are provided to better understand the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0046] Example 1: A salty peptide derived from dry-cured ham and its preparation method

[0047] The saltiness-increasing peptide of the present invention specifically corresponds to Figure 1 Component 1, Figure 5 Peak 1 in.

[0048] A saltiness-enhancing peptide derived from dry-cured ham, wherein the amino acid sequence of the saltiness-enhancing peptide is: Asp-Leu (DL).

[0049] The salty-enhancing peptide has a strong salty-enhancing effect and can be used in the preparation of salt substitutes or salty taste enhancers and health foods.

[0050] S1. Preparation of ham-derived extract

[0051] Take the hind leg meat of Jinhua ham, a dry-cured ham, and process it into powder. Weigh 50 g of the powder sample and dissolve it in 150 mL of 0.01 mol / L hydrochloric acid buffer solution, homogenize it at 16000 r / min for 4 times, 10 s each time, and then centrifuge it at 12000 r / min at -4 ºC for 20 min. Take the supernatant and filter it with a 0.45 μm filter membrane to obtain the ham source extract.

[0052] S2. Dialysis separation of ham source extract

[0053] The ham-derived extract obtained above was placed in a dialysis bag of less than 3000 Da and dialyzed at -4 ºC for 24 h. The dialysate obtained was concentrated by rotary evaporation at 65 ºC. The concentrated solution was freeze-dried using a freeze dryer to obtain a crude polypeptide.

[0054] S3. Separation and purification of crude peptides

[0055] The crude polypeptide in S2 was dissolved in pure water, filtered with a 0.22 μm aqueous phase filter membrane, and then loaded onto a Sephadex G-15 gel filtration column (3.0×200 cm) for purification at 25 °C. Specific chromatographic parameters: sample mass concentration 40 mg / mL; injection volume 200 µL; eluent 0.01 M hydrochloric acid buffer, flow rate 0.5 mL / min, primary UV detection wavelength 214 nm, secondary UV detection wavelength 250 nm. Sensory evaluation was performed on each purified component according to the spectral peaks. The component with the best salt-enhancing effect was screened out through sensory evaluation and electronic tongue-assisted sensory characteristic analysis.

[0056] The best salt-enhancing component separated from the gel was further purified by ion exchange chromatography. Specific detection conditions: sample mass concentration 10 mg / mL; injection volume 2 mL; chromatographic column: DEAE anion column (16×25 mm), mobile phase A is 20 mM Tris-HCl buffer (pH=9.0), mobile phase B is 20 mM Tris-HCl buffer + 1M NaCl (pH=9.0); gradient elution: first gradient: mobile phase A 100%, mobile phase B 0%, elution volume 60 mL; second gradient: mobile phase A 90%, mobile phase B 10%, elution volume 60 mL; third gradient: mobile phase A 100%, mobile phase B 0%, elution volume 40 mL; elution rate 5 mL / min, main UV detection wavelength 214 nm, secondary UV detection wavelength 250 nm. Sensory evaluation of each purified component was performed according to the spectral peak. The component with the best salt-enhancing effect was screened out through sensory evaluation and electronic tongue-assisted sensory characteristic analysis.

[0057] S4. Amino acid sequence test of salt-enhancing peptide

[0058] The component with the best salt-enhancing effect in S3 was finally identified using ultra-performance liquid chromatography tandem triple quadrupole time-of-flight mass spectrometry, referred to as UPLC-Triple-TOF / MS, to identify the sequence of the salt-enhancing peptide. The chromatographic conditions were as follows: the chromatographic column was Agilent Eclipse Plus-C18; mobile phase A: ultrapure water containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid; linear gradient elution: 100% mobile phase A, 0% mobile phase B (0-1 min); 100%-70% mobile phase A, 0%-30% mobile phase B (1-10 min); 70%-0% mobile phase A, 30%-100% mobile phase B (10-15 min); 0% mobile phase A, 100% mobile phase B (15-18 min); 0%-100% mobile phase A, 100%-0% mobile phase B (18-22 min); 100% mobile phase A, 0% mobile phase B (22-25 min). The LC-MS was used to determine the amino acid sequence, and the three amino acid sequences were determined to be Asp-Leu (DL), Phe-Met-Ser-Ala-Leu-Phe (FMSALF), and His-Val-Arg-Arg-Lys (HVRRK). Among them, Asp-Leu (DL) can be matched in insilico.

[0059] The steps of the method for separating and purifying the salty peptides from dry-cured ham described in this example are the same as those in Example 2, except that:

[0060] (1) The mobile phase for gel filtration in step S3 is 0.01 M Tris-HCl buffer at a flow rate of 0.4 mL / min;

[0061] (2) Chromatographic conditions for ion exchange in step S3: Gradient elution: first gradient: mobile phase A 100%, mobile phase B 0%, elution volume 120 mL; second gradient: mobile phase A 95%, mobile phase B 5%, elution volume 40 mL; third gradient: mobile phase A 90%, mobile phase B 10%, elution volume 40 mL.

[0062] Example 2: A method for preparing a salty peptide derived from dry-cured ham

[0063] Each step of the method for separating and purifying the salty peptides from dry-cured ham described in this embodiment is the same as that in Example 2, except that:

[0064] (1) The mobile phase for gel filtration in step S3 is ultrapure water at a flow rate of 0.4 mL / min;

[0065] (2) the detection wavelength in the gel filtration in step S3 is 220 nm;

[0066] (3) Chromatographic conditions for ion exchange in step S3: mobile phase A is ultrapure water, and mobile phase B is 2 M NaCl.

[0067] Example 3: A salty peptide derived from dry-cured ham and its preparation method

[0068] A method for preparing a salty peptide derived from dry-cured ham comprises the following steps:

[0069] S1. Preparation of ham-derived extract

[0070] The ham is processed to obtain powder, the powder is dispersed in a hydrochloric acid buffer solution, homogenized and then centrifuged, and then the supernatant is filtered to obtain a ham source extract;

[0071] S2. Dialysis separation of ham source extract

[0072] The ham-derived extract is dialyzed to obtain a dialyzate, and the dialyzate is concentrated, and the concentrated solution is freeze-dried to obtain a crude polypeptide;

[0073] S3. Separation and purification of crude peptides

[0074] The crude polypeptide was dissolved in pure water, filtered and loaded onto a Sephadex G-15 gel filtration column for purification to obtain a separated and purified polypeptide, which was then further purified by ion exchange chromatography. The purified components were subjected to sensory evaluation according to the spectral peaks, and the components with the best salt-enhancing effect were screened out through sensory evaluation and electronic tongue-assisted sensory characteristic analysis.

[0075] S4. Amino acid sequence test of salt-enhancing peptide

[0076] The component with the best salt-enhancing effect obtained in S3 was identified by ultra-performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry, and the three amino acid sequences were determined to be Asp-Leu, Phe-Met-Ser-Ala-Leu-Phe and His-Val-Arg-Arg-Lys.

[0077] The preferred implementation manner is: in S1, the concentration of the hydrochloric acid buffer is 0.01 mol / L, and the ratio of the powder to the hydrochloric acid buffer is 50 g:100 mL; during homogenization: homogenize 3 times at 15000 r / min, each time for 8 s; during centrifugation, centrifuge at 10000 r / min for 15 min at -3°C; and the supernatant is filtered using a 0.45 μm filter membrane.

[0078] A preferred implementation manner is: in S2, the ham source extract is placed in a dialysis bag of less than 3000 Da, dialyzed at -3°C for 20 hours to obtain a dialysate, and then rotary evaporated at 60°C to obtain a concentrated solution.

[0079] The preferred implementation manner is: in S3, the chromatographic parameters are: sample mass concentration 40 mg / mL; injection volume 200 µL; eluent is 0.01 M hydrochloric acid buffer, flow rate 0.5 mL / min, primary UV detection wavelength is 214 nm, and secondary UV detection wavelength is 250 nm.

[0080] The preferred implementation manner is: in S3, ion exchange chromatography is used for further purification, and the specific detection conditions are: sample mass concentration 10 mg / mL; injection volume 2 mL; chromatographic column: DEAE anion column, mobile phase A is 20 mM Tris-HCl buffer, and mobile phase B is 20 mM Tris-HCl buffer + 1M NaCl; gradient elution: first gradient: mobile phase A 100%, mobile phase B 0%, elution volume 60 mL; second gradient: mobile phase A 90%, mobile phase B 10%, elution volume 60 mL; third gradient: mobile phase A 100%, mobile phase B 0%, elution volume 40 mL; elution rate is 5 mL / min, the main ultraviolet detection wavelength is 214 nm, and the secondary ultraviolet detection wavelength is 250 nm; the pH values ​​of mobile phase A and mobile phase B are both 9.0.

[0081] A preferred embodiment is as follows: the chromatographic conditions of ultra-high performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry are as follows: the chromatographic column is Agilent Eclipse Plus-C18; mobile phase A: ultra-pure water containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid; linear gradient elution: The chromatographic conditions of ultra-high performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry are as follows: the chromatographic column is Agilent Eclipse Plus-C18; mobile phase A: ultra-pure water containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid; linear gradient elution:

[0082] 100% mobile phase A, 0% mobile phase B 0-1 min;

[0083] 100%-70% mobile phase A, 0%-30% mobile phase B for 1-10 min;

[0084] 70%-0% mobile phase A, 30%-100% mobile phase B for 10-15 min;

[0085] 0% mobile phase A, 100% mobile phase B for 15-18 min;

[0086] 0%-100% mobile phase A, 100%-0% mobile phase B 18-22 min;

[0087] 100% mobile phase A, 0% mobile phase B for 22-25 min.

[0088] The preferred embodiment is: the inner diameter of the Sephadex G-15 gel filtration column is 3.0 cm and the column length is 200 cm; the inner diameter of the DEAE anion column is 16 mm and the column height is 25 mm; the inner diameter of the C18 chromatographic column is 4.6 mm and the column length is 150 mm, and the particle size is 3.5 µm.

[0089] Example 4: A salty peptide derived from dry-cured ham and its preparation method

[0090] A method for preparing a salty peptide derived from dry-cured ham comprises the following steps:

[0091] S1. Preparation of ham-derived extract

[0092] The ham is processed to obtain powder, the powder is dispersed in a hydrochloric acid buffer solution, homogenized and then centrifuged, and then the supernatant is filtered to obtain a ham source extract;

[0093] S2. Dialysis separation of ham source extract

[0094] The ham-derived extract is dialyzed to obtain a dialyzate, and the dialyzate is concentrated, and the concentrated solution is freeze-dried to obtain a crude polypeptide;

[0095] S3. Separation and purification of crude peptides

[0096] The crude polypeptide was dissolved in pure water, filtered and loaded onto a Sephadex G-15 gel filtration column for purification to obtain a separated and purified polypeptide, which was then further purified by ion exchange chromatography. The purified components were subjected to sensory evaluation according to the spectral peaks, and the components with the best salt-enhancing effect were screened out through sensory evaluation and electronic tongue-assisted sensory characteristic analysis.

[0097] S4. Amino acid sequence test of salt-enhancing peptide

[0098] The component with the best salt-enhancing effect obtained in S3 was identified by ultra-performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry, and the three amino acid sequences were determined to be Asp-Leu, Phe-Met-Ser-Ala-Leu-Phe and His-Val-Arg-Arg-Lys.

[0099] The preferred embodiment is: in S1, the concentration of the hydrochloric acid buffer is 0.01 mol / L, and the ratio of the powder to the hydrochloric acid buffer is 50 g:200 mL; during homogenization: homogenize 5 times at 18000 r / min, each time for 12 s; during centrifugation, centrifuge at 14000 r / min for 25 min at -5ºC; the supernatant is filtered with a 0.45 μm filter membrane.

[0100] A preferred embodiment is as follows: in S2, the ham source extract is placed in a dialysis bag with a diameter of less than 3000 Da, dialyzed at -5°C for 28 hours to obtain a dialysate, and then rotary evaporated at 70°C to obtain a concentrated solution.

[0101] The preferred implementation manner is: in S3, the chromatographic parameters are: sample mass concentration 40 mg / mL; injection volume 200 µL; eluent is 0.01 M hydrochloric acid buffer, flow rate 0.5 mL / min, primary UV detection wavelength is 214 nm, and secondary UV detection wavelength is 250 nm.

[0102] The preferred implementation manner is: in S3, ion exchange chromatography is used for further purification, and the specific detection conditions are: sample mass concentration 10 mg / mL; injection volume 2 mL; chromatographic column: DEAE anion column, mobile phase A is 20 mM Tris-HCl buffer, and mobile phase B is 20 mM Tris-HCl buffer + 1M NaCl; gradient elution: first gradient: mobile phase A 100%, mobile phase B 0%, elution volume 60 mL; second gradient: mobile phase A 90%, mobile phase B 10%, elution volume 60 mL; third gradient: mobile phase A 100%, mobile phase B 0%, elution volume 40 mL; elution rate is 5 mL / min, the main ultraviolet detection wavelength is 214 nm, and the secondary ultraviolet detection wavelength is 250 nm; the pH values ​​of mobile phase A and mobile phase B are both 9.0.

[0103] A preferred embodiment is as follows: the chromatographic conditions of ultra-high performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry are as follows: the chromatographic column is Agilent Eclipse Plus-C18; mobile phase A: ultra-pure water containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid; linear gradient elution: The chromatographic conditions of ultra-high performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry are as follows: the chromatographic column is Agilent Eclipse Plus-C18; mobile phase A: ultra-pure water containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid; linear gradient elution:

[0104] 100% mobile phase A, 0% mobile phase B 0-1 min;

[0105] 100%-70% mobile phase A, 0%-30% mobile phase B for 1-10 min;

[0106] 70%-0% mobile phase A, 30%-100% mobile phase B for 10-15 min;

[0107] 0% mobile phase A, 100% mobile phase B for 15-18 min;

[0108] 0%-100% mobile phase A, 100%-0% mobile phase B 18-22 min;

[0109] 100% mobile phase A, 0% mobile phase B for 22-25 min.

[0110] The preferred embodiment is: the inner diameter of the Sephadex G-15 gel filtration column is 3.0 cm and the column length is 200 cm; the inner diameter of the DEAE anion column is 16 mm and the column height is 25 mm; the inner diameter of the C18 chromatographic column is 4.6 mm and the column length is 150 mm, and the particle size is 3.5 µm.

[0111] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A method for preparing a salty peptide derived from dry-cured ham, Features: The following steps are involved: S1. Preparation of ham-derived extract The Jinhua ham is processed to obtain powder, and the powder is dispersed in a hydrochloric acid buffer solution with a concentration of 0.01 mol / L, homogenized and then centrifuged, and then the supernatant is filtered to obtain a ham source extract; during homogenization: the homogenization is performed at 15000-18000 r / min for 3-5 times, each time for 8-12 seconds; S2. Dialysis separation of ham source extract The ham-derived extract is dialyzed to obtain a dialyzate, and the dialyzate is then concentrated. The concentrated solution is freeze-dried to obtain a crude polypeptide product; during the dialysis: the ham-derived extract is placed in a dialysis bag with a diameter of less than 3000 Da; S3. Separation and purification of crude peptides The crude polypeptide was dissolved in pure water, filtered and loaded onto a Sephadex G-15 gel filtration column for purification to obtain the separated and purified polypeptide. The specific chromatographic parameters were as follows: sample mass concentration 40 mg / mL; injection volume 200µL; eluent 0.01M hydrochloric acid buffer, flow rate 0.5mL / min, primary UV detection wavelength 214 nm, secondary UV detection wavelength 250 nm; sensory evaluation was performed on each purified component according to the spectral peaks, and the component with the best salt-enhancing effect was screened out through sensory evaluation and electronic tongue-assisted sensory characteristic analysis; The component with the best salt-enhancing effect obtained from the gel filtration column was further purified by ion exchange chromatography, and the specific purification conditions were as follows: sample mass concentration 10 mg / mL; injection volume 2 mL; chromatographic column: DEAE anion column, mobile phase A was 20 mM Tris-HCl buffer, and mobile phase B was 20 mM Tris-HCl buffer + 1M NaCl; gradient elution: first gradient: mobile phase A 100%, mobile phase B 0%, elution volume 60 mL; second gradient: mobile phase A 90%, mobile phase B 10%, elution volume 60 mL; third gradient: mobile phase A 100%, mobile phase B 0%, elution volume 40 mL; elution rate was 5 mL / min, the main UV detection wavelength was 214 nm, and the secondary UV detection wavelength was 250 nm; the pH values ​​of mobile phase A and mobile phase B were both 9.0; sensory evaluation was performed on each purified component according to the spectral peaks, and the component with the best salt-enhancing effect was screened out through sensory evaluation and electronic tongue-assisted sensory characteristic analysis; S4. Amino acid sequence test of salt-enhancing peptide The component with the best salt-enhancing effect obtained in S3 was identified by ultra-performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry, and three peptides with amino acid sequences of Asp-Leu, Phe-Met-Ser-Ala-Leu-Phe and His-Val-Arg-Arg-Lys were obtained.

2. The method for preparing the salty peptide derived from dry-cured ham according to claim 1, Features: In S1, the ratio of powder to hydrochloric acid buffer is 50g:100-200mL; during centrifugation, centrifuge at 10000-14000r / min for 15-25min at -3~-5ºC; filter the supernatant with a 0.45μm filter membrane.

3. The method for preparing the salty peptide derived from dry-cured ham according to claim 1, Features: In S2, dialyze at -3~-5ºC for 20-28h to obtain dialysate, and then rotary evaporate at 60-70 ºC to obtain a concentrated solution.

4. The method for preparing the salty peptide derived from dry-cured ham according to claim 1, Features: The chromatographic conditions of ultra-high performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry were as follows: chromatographic column: Agilent Eclipse Plus-C18; mobile phase A: ultrapure water containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid; linear gradient elution: 100% mobile phase A, 0% mobile phase B 0-1 min; 100%-70% mobile phase A, 0%-30% mobile phase B for 1-10 min; 70%-0% mobile phase A, 30%-100% mobile phase B for 10-15 min; 0% mobile phase A, 100% mobile phase B for 15-18 min; 0%-100% mobile phase A, 100%-0% mobile phase B for 18-22 min; 100% mobile phase A, 0% mobile phase B for 22-25 min.

5. The method for preparing the salty peptide derived from dry-cured ham according to claim 1, Features: The inner diameter of the Sephadex G-15 gel filtration column was 3.0 cm and the column length was 200 cm; the inner diameter of the DEAE anion column was 16 mm and the column height was 25 mm.

6. The method for preparing the salty peptide derived from dry-cured ham according to claim 4, Features: The inner diameter of the C18 column is 4.6 mm, the column length is 150 mm, and the particle size is 3.5 µm.

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