Separation and purification process of Landaise bone paste umami peptide and umami peptide screening process

Abstract

The invention discloses a separation and purification process of Landaise goose bone paste umami peptide and an umami peptide screening process, and the separation and purification process comprises the following steps: S11, preparing goose bone paste hydrolysate, and taking supernate for later use; s12, performing a separation experiment on supernate of the goose bone mud hydrolysate; s13, sensory evaluation is conducted on the separated goose bone paste through an electronic tongue, according to the separation and purification process of the Landaise goose bone paste flavor peptide and the screening process of the flavor peptide, separation, purification and peptiomics analysis are conducted on the bone mud hydrolysate as a research object through an NF, UF, GFC, RP-HPLC and a nano-liter liquid chromatography-ultrahigh resolution quadrupole electrostatic field orbitrap mass spectrometer. The method comprises the following steps: optimizing bone mud hydrolysate, evaluating the umami strength of the optimized bone mud hydrolysate in combination with electronic tongue detection, screening umami peptides by utilizing a computer software technology through peptidomics analysis, and laying a theoretical foundation for research and development of umami peptides of Landaise goose bone paste through the research of the invention.

Description

Technical Field

[0001] The present invention relates to the separation and purification of umami peptides from Langde goose bone paste and the technical field, specifically to a separation and purification process for umami peptides from Langde goose bone paste and an umami peptide screening process. Background Art

[0002] Landes geese were first produced in the Landes province in western France, near the Bay of Biscay. In China, they are mainly distributed in more than 10 provinces (municipalities and autonomous regions), including Shandong, Jilin, Zhejiang, Hebei, Shanxi, Guangdong, Guangxi and Anhui. Landes geese are a world-famous breed for foie gras, with delicious meat, low fat and low cholesterol. Goose liver has the characteristics of unique flavor, rich nutrition and delicate taste. In terms of breeding, the focus of Landes geese is on the breeding of goslings, including the insulation of goslings, the hatching method of goslings, and the feeding of goslings. Newly hatched goslings should be fed with high-protein, easy-to-eat, and complete feed due to their weak digestive ability. As herbivorous waterfowl, geese can be fed 20% to 40% green fodder in areas with rich pasture resources. When feeding green fodder, it is necessary to pay attention to chopping it and mixing it with concentrate. Research on Landes geese mainly focuses on feeding management, nutritional requirements and growth performance. In the future, research on the Landais goose can further focus on its breed improvement, disease prevention and control, and ecological breeding to promote the sustainable development of the Landais goose industry.

[0003] As a new flavoring substance, umami peptides have attracted wide attention in the field of food science. Mainly derived from animals and plants, umami peptides not only have a soft taste, but also have good functionality and processing characteristics. This coincides with modern people's pursuit of "natural, nutritious and safe" food, making umami peptides an important direction for food umami research. The soft taste of umami peptides comes from the interaction between its molecular structure and taste buds, which makes it not too strong or irritating while providing umami. Its functional properties include enhancing food flavor and improving the nutritional value of food. Umami agents are a new natural flavor enhancer discovered in recent years. They can not only make food taste delicious, increase its freshness and mellowness, but also play a synergistic role with other ingredients. Umami peptides have become a research hotspot in the current field of food preservation due to their high nutritional and flavor activity. Umami peptides have the characteristics of reducing or masking bitterness and improving food flavor. This is mainly because umami peptides can enhance the bitter taste of bitter ingredients, thereby reducing the bitter taste and making the overall flavor of food more mellow and soft; umami peptides have good thermal stability, which means that during food processing, they can withstand heat treatment without losing their flavor characteristics. This is an important advantage for the food industry because many foods need to be heated during processing, such as cooking, canning or heat sterilization.

[0004] The prior art lacks a process for separating, purifying and screening the umami peptides from Langde goose bone paste. To this end, we propose a process for separating, purifying and screening the umami peptides from Langde goose bone paste. Summary of the invention

[0005] The object of the present invention is to provide a separation and purification process for umami peptides from Langde goose bone paste and a umami peptide screening process to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a separation and purification process of Langde goose bone paste umami peptide, characterized in that it comprises the following steps:

[0007] S11, prepare goose bone mud hydrolyzate, and take the supernatant for later use;

[0008] S12, performing a separation experiment on the supernatant of the goose bone mud hydrolyzate;

[0009] S13. Perform sensory evaluation on the separated goose bone paste using an electronic tongue.

[0010] Preferably, the separation experiment in S12 includes a nanofiltration separation experiment, and the nanofiltration separation experiment includes the following steps:

[0011] S21, using a 200Da nanofiltration membrane to perform nanofiltration on the hydrolyzate to remove free amino acids and salts in the solution;

[0012] S22, adjusting the solution pH to 5-9, the total solution concentration to 0.1%-1%, and the solution temperature to 5-45°C;

[0013] S23, connect the instrument, filter three times, and perform formaldehyde potentiometric titration on the filtrate of the filtered polypeptide solution to determine the amino acid content therein, thereby determining the nanofiltration effect of the polypeptide solution;

[0014] S24, ultrafiltering the polypeptide solution filtrate and freeze-drying it.

[0015] Preferably, the separation experiment in S12 includes an ultrafiltration separation experiment, and the ultrafiltration separation experiment includes the following steps:

[0016] S31, pre-filtering it with a 0.45 μm organic filter membrane;

[0017] S32, filtering it using an ultrafiltration membrane with a molecular weight cut-off of less than 3000Da;

[0018] S33, use UF centrifuge tube, temperature is 4 ℃, rotation speed is 6000r / min, after centrifugation for 15min, freeze-dry the filtrate and store at -80℃.

[0019] Preferably, the separation experiment in S12 includes a gel chromatography separation experiment, and the gel chromatography separation experiment includes the following steps:

[0020] S41, dissolving goose bone mud hydrolyzate with a molecular weight lower than 3000Da in pure water, and loading into a Sephadex G-15 chromatographic column at 25°C for separation;

[0021] S42, eluting with ultrapure water under the chromatographic conditions of 5 mL, 100 mg / mL, at a flow rate of 1.0 mL / min, and detecting under 220 nm ultraviolet light;

[0022] S43. Collect, concentrate and freeze-dry the components in order of their peaks.

[0023] Preferably, the components separated in the gel chromatography separation experiment are dissolved in pure water, filtered with a 0.22 μm organic filter membrane, and the sample is purified by reverse phase high performance liquid chromatography, and the steps of the purification process are as follows:

[0024] S51, sample volume is 100 μL, injection concentration is 10 mg / mL, C18 column is used, column temperature is 30 °C;

[0025] S52, in the sample, the mobile phase is 0.05% trifluoroacetic acid-acetonitrile, the mobile phase B is 0.05% trifluoroacetic acid-ultrapure water, VA:VB=2:8, and the flow rate is 1 mL / min, and the isocratic elution is performed for 40 min;

[0026] S53. Determine with an ultraviolet spectrophotometer at a wavelength of 220 nm, collect each component in the order of spectral peaks, and freeze-dry.

[0027] Preferably, the electronic tongue is a taste detection system composed of seven taste sensors, and the seven taste sensors are respectively sour, salty, NMS, sweet, bitter, PKS and CPS sensory flavor sensors.

[0028] Preferably, the electronic tongue measurement method is: dissolving 10 mg of freeze-dried powder of bone mud hydrolysate in 70 mL of pure water for electronic tongue measurement, and the dissolved concentration is 0.14 mg / mL.

[0029] A screening process for Langde goose bone paste umami peptide, the process comprising the following steps:

[0030] S61, peptidomic analysis of goose bone mud hydrolysate by ultra-performance liquid chromatography-tandem mass spectrometry;

[0031] S62, using the online database PeptideRanker to predict the biological activity of all peptide sequences obtained by analyzing the amino acid sequences after LC-MS analysis using PEAKSOnline software;

[0032] S63. The screening method is as follows: first, the amino acid sequence must contain umami amino acids, then it must not contain amino acids that may produce toxic peptides, and the umami peptides found again are mostly peptides with less than 10 amino acids, and finally screening is carried out based on the peptide confidence ALC being greater than 90%.

[0033] Preferably, the steps of performing peptidomics analysis on goose bone mud hydrolysate by ultra-performance liquid chromatography-tandem mass spectrometry are:

[0034] S71, pre-treatment sample:

[0035] The freeze-dried sample of goose bone mud hydrolyzate was dissolved in double distilled water, then DTT was added for 1 hour at 56°C, placed in a light-proof room for 40 minutes to remove excess DTT, and eluted to the separated and purified peptides based on a desalting column and a 1.5 mL centrifuge tube as a template, and excess reactants were removed in a vacuum centrifuge concentrator at 45°C;

[0036] S72, Capillary liquid chromatography conditions:

[0037] a. Column information: Pre-column: 300μm×5mm, AcclaimPepMapRPLCC18, 5μm, Chromatographic column: 150μm×150mm, AcclaimPepMapRPLCC18, 1.9μm,

[0038] b. Mobile phase information:

[0039] 0.1% formic acid water was used as mobile phase A, and 0.1% formic acid and 80% acetonitrile were used as mobile phase B; gradient elution was performed for 90 min, and the conditions were 0-10 min 6%-9% B, 10-30 min 9%-14% B, 30-70 min 14%-30% B, 70-87 min 30%-40% B, 87-90 min 40%-95% B, and the flow rate was 600 nL / min;

[0040] S73, mass spectrometry detection:

[0041] The polypeptide fragments were dissolved in the sample solution, centrifuged at 13200r / min at 4°C for 20min, and the supernatant was directly input into the mass spectrometer for online analysis. The specific parameters were as follows: the first-stage mass spectrometer parameters were as follows: resolution: 70000; AGCtarget: 3e6; MaximumITzz: 40ms; the second-stage mass spectrometer parameters were: resolution: 17500; AGCtarget: 1e5; MaximumITzz: 60ms; TopN: 20; the secondary mass spectrometer data were subjected to secondary Denovo sequencing using the manual Denovo method.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention adopts NF, UF, GFC, RP-HPLC and nano-liquid chromatography-ultra-high resolution quadrupole electrostatic field orbital trap mass spectrometry to separate and purify bone mud hydrolysate as the research object and conducts peptide group analysis. The umami intensity of the optimized bone mud hydrolysate is evaluated by combining electronic tongue detection, and then the umami peptides are screened by peptide group analysis and computer software technology. Through the research of this application, a theoretical foundation is laid for the research and development of umami peptides of Langde goose bone mud. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of components obtained by GFC separation (Note: F1 is peak 1, F2 is peak 2, and F3 is peak 3);

[0045] Figure 2 A schematic diagram of the relative ranking of sour, salty and fresh directly given by the taste ranking function (Note: F1 is peak one, F2 is peak two, and F3 is peak three);

[0046] Figure 3 This is a radar chart of relative taste ranking {Note: AHS (sour), CTS (salty), NMS (umami), ANS (sweet), SCS (bitter), PKS and CPS (universal sensor), A1 is peak one, A2 is peak two, and A3 is peak three.};

[0047] Figure 4 This is a schematic diagram of the 220nm wavelength of reversed-phase high performance liquid chromatography;

[0048] Figure 5 This is a schematic diagram of the optimal flow rate for reversed-phase high performance liquid chromatography;

[0049] Figure 6 Schematic diagram of reversed-phase high performance liquid phase VA:VB=2:8;

[0050] Figure 7 This is a schematic diagram of the reverse phase ultra-high chromatography of the GFCF1 peak;

[0051] Figure 8 This is a schematic diagram of the reverse phase ultra-high chromatography of the GFCF2 peak;

[0052] Fig. 9 This is a schematic diagram of the reverse phase ultra-high chromatography of the GFCF3 peak;

[0053] Fig.10 It is a schematic diagram of the signal intensity bar graph of 6 samples on 7 sensors;

[0054] Fig.11Schematic diagram of the flavor distribution of 06 samples (with the response signal intensity of 7 sensors as the variable);

[0055] Fig.12 This is a diagram of the relative ranking of sour, salty and fresh directly given by the taste ranking function;

[0056] Fig.13 This is a schematic diagram of a relative taste ranking radar chart;

[0057] Fig.14 Schematic diagram of peptide length and peptide number distribution;

[0058] Fig.15 Schematic diagram of the ratio of different peptide segments;

[0059] Fig.16 Venn diagram for umami peptide screening (Note: A. Contains umami amino acids; B. Contains hydrophilic amino acids at both ends of the sequence; C. Does not contain amino acids that may produce toxic peptides; D. Peptides with less than 10 amino acids; E. Peptide confidence ALC is greater than 90%.). DETAILED DESCRIPTION

[0060] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0061] The present invention provides a technical solution: a separation and purification process of Langde goose bone paste umami peptide, characterized in that it comprises the following steps:

[0062] S11, prepare goose bone mud hydrolyzate, and take the supernatant for later use;

[0063] S12, performing a separation experiment on the supernatant of the goose bone mud hydrolyzate;

[0064] S13. Perform sensory evaluation on the separated goose bone paste using an electronic tongue.

[0065] The separation experiment in S12 includes a nanofiltration (NF) separation experiment. A 200Da filter membrane is used to perform nanofiltration on the hydrolyzate to remove free amino acids and salts in the solution. The pH value of the solution is adjusted to 5-9. The total concentration of the solution is preferably within 0.1%, and the maximum cannot exceed 1%. The solution temperature is 5-45°C. The instrument is connected and filtered three times. The clean water (free amino acid solution) and waste water (polypeptide solution) are extracted three times. The filtrate of the waste water (polypeptide solution) is subjected to formaldehyde potentiometric titration to determine the amino acid content therein, thereby determining the nanofiltration effect of the polypeptide solution. The waste liquid is ultrafiltered and freeze-dried.

[0066] The separation experiment in S12 includes an ultrafiltration (UF) separation experiment. Currently, many studies have adopted flavor evaluation technology, but most of their molecular weight does not exceed 3000Da. Therefore, this study intends to select the hydrolyzate of Langde goose bone mud with a molecular weight below 3000Da as the research object, and filter it with an ultrafiltration membrane with a cutoff molecular weight below 3000Da, and pre-filter it with a 0.45μm organic filter membrane to prevent the membrane assembly from being scratched. On this basis, a UF centrifuge tube was used, and the filtrate was centrifuged for 15 minutes at a temperature of 4℃ and a speed of 6000r / min. The filtrate was freeze-dried and stored at -80℃.

[0067] The separation experiment in S12 includes a gel chromatography (GFC) separation experiment. Samples with a molecular weight lower than 3000 Da are dissolved in pure water and loaded into a Sephadex G-15 column (2.6 cm × 60 cm) for separation at 25°C to determine the optimal chromatographic conditions: 5 mL, 100 mg / mL; eluted with ultrapure water at a flow rate of 1.0 mL / min, detected under 220 nm ultraviolet light, and the components were collected in the order of spectral peaks, concentrated, freeze-dried, and subjected to electronic tongue measurement.

[0068] Gel selection: Colloidal particles are usually spherical, expressed by their diameter or mesh number. The size of gel particles determines the resolution of the chromatographic column and the sample flow rate. Larger particle sizes and pores generally have higher flow rates, but poor separation efficiency; small pores and slow sample flow rates result in poor separation efficiency. Dextran gel specifications are marked with G, and the Arabic number after G is 10 times the water content of the gel. For example, G-25 absorbs 2.5 grams of water per gram of gel when it swells. Dextran has G-10, 15, 25, 50, 75, 100, 150, and 200. "G" indicates the degree of cross-linking, swelling, and distribution of the gel. See Table 1 for dextran specification parameter indicators.

[0069] Table 1 Specifications and parameters of dextran

[0070]

[0071] Pretreatment of gel medium: Before use, swell it with water (gel: water = 1:10). Natural swelling takes a long time, but it can be accelerated by heating, that is, heating the gel to boiling in a boiling water bath, and it will swell in 1-2 hours. Add water or buffer to the beaker, stir, let it stand, pour out the supernatant, remove the colloidal fragments in the supernatant, and repeat several times until the supernatant becomes clear.

[0072] Selection of chromatographic columns: The selection of parameters such as column volume, column length, and column diameter is an important factor affecting the efficiency of column chromatography. When performing group analysis, most of the columns used are 2-30 cm in length. The column volume is 5 times the volume of the sample solution, and the column ratio is usually 5-10; but multi-stage chromatography usually requires a column volume of 100 cm, a column volume of not less than 25 times, and a column ratio of 20-100.

[0073] Filling of gel column: The difference between gel chromatography and other chromatography techniques is that in the chromatography process, there is no interaction force between the solute molecules and the stationary phase, and the separation of the components depends only on their own flow rates. When filling the column, close the bottom of the column, add about 1 / 3 of water or buffer to the column, stir the buffer evenly along one side of the column, and slowly inject it into the column again and again. When the colloid thickness reaches 5cm, open the opening at the bottom of the column and control the flow rate to 1mL / min.

[0074] Sample processing and loading: According to the type of sample to be tested and the purification of the sample to be tested, select an appropriate buffer solution to ensure the quality of the sample, usually 1-5% of the column volume. It needs to be concentrated before loading so that its content does not exceed 4%. However, for materials with larger molecular weight, when the concentration increases, the viscosity of the solution will increase, thereby limiting the movement of molecules and affecting the speed. Before injection, use a membrane filter or centrifugation method to remove impurities that may block the column layer.

[0075] Elution and collection: One or a salt-containing buffer can be used as a buffer for gel filtration chromatography. There are two reasons for this. One is solubility and the other is stability. The buffer used should ensure that the protein sample will not denature and precipitate. The pH value should be selected so that the sample is relatively stable and has good solubility. During this process, a certain amount of salt (NaCl) must be added to the buffer to achieve stability and protection of the protein. During elution, a constant working pressure must be maintained and the flow rate should not be too large, maintained at 0.5-3.0mL / min.

[0076] Post-use maintenance and protection of chromatography columns:

[0077] (1) Gel sterilization method: After washing the column with ultrapure water, flush the column forward with 0.5 mol / L sodium hydroxide at a rate of 3 mL / min, and flush 3 column volumes;

[0078] (2) Equilibration: After the sodium hydroxide treatment, wash 2 column volumes with ultrapure water, and then wash 5-10 times the column volume with pH = 7.0 buffer (200 mmol / L NaCl and 20 mml / L phosphate buffer solution);

[0079] (3) Sampling: After equilibration, select a sampling pump with a sample flow rate of 3 mL / min and a sample volume of 1 mL;

[0080] (4) Elution: After loading, use a balanced buffer for elution;

[0081] (5) Cleaning and storage: After purification, backwash 2 column volumes with 0.5 mol / L sodium hydroxide for 30-60 min. After cleaning, forward wash 5 columns with ultrapure water and wash 3 columns with 20% ethanol. Remove the columns, seal both ends, and store in a freezer.

[0082] Reverse phase high performance liquid chromatography (RP-HPLC) purification process:

[0083] Detection wavelength selection, reverse ultra-high liquid phase wavelength selection 201-224nm, select the best wavelength;

[0084] Flow rate selection: The reverse phase ultra-high liquid phase flow rate is set to 0.3-1.0mL / min, and the optimal flow rate is selected;

[0085] Selection of mobile phase ratio: As shown in Table 2, the reversed phase ultra-high liquid phase mobile phase ratio is set;

[0086] Table 2 Grouping of mobile phase conditions

[0087] Serial number ATrifluoroacetic acid & acetonitrile B Trifluoroacetic acid & pure water A:B Remark 1 0.05％ 0.05％ 1:9 5-15min 2 0.05％ 0.05％ 1.5:8.5 5-15min 3 0.05％ 0.05％ 2:8 5-15min 4 0.05％ 0.05％ 2.5:7.5 5-15min 5 0.05％ 0.05％ 3:7 5-15min 6 0.05％ 0.05％ 3.5:6.5 5-15min

[0088] Pretreatment of mobile phase:

[0089] (1) Filtration: First, remove insoluble impurities and bubbles by filtering. Then use ultrasound to remove bubbles. Ultrasound also helps dissolve, mix more evenly, and reduce baseline noise. When preparing the mobile phase, if the mobile phase is a mixed mobile phase, it is necessary to filter it with a 0.22 μm filter membrane; if the mobile phase is a pure mobile phase, no filtration is required.

[0090] (2) Ultrasonic degassing is more effective. For many organic solvents or organic solvent / water mixtures, ultrasonic degassing can be performed for 10-20 minutes (usually 500 mL of solution requires ultrasonic treatment for 20-30 minutes). For the ultrasonic treatment time, each laboratory has a time of 5-30 minutes. Generally, when the mobile phase composition is inorganic (buffered salt solution), 5-15 minutes is sufficient. When the mobile phase is a mixture of water and organic volume, the ultrasonic treatment time is relatively long. This method can only remove 30% of the dissolved gas. In this process, the presence of bubbles will have a certain impact on the baseline stability and signal-to-noise ratio of the chromatographic peak. However, in general, 15 minutes of ultrasonic treatment is sufficient. This has no effect on the solvent composition. During the ultrasonic treatment process, care should be taken not to allow the bottom surface of the container to directly contact the inner wall of the ultrasonic treatment, otherwise the container will break.

[0091] Optimal conditions: After the best flavor component separated from GFC is dissolved in pure water, it is filtered with a 0.22 μm organic filter membrane, and the sample is separated and purified by RP-HPLC. The specific detection conditions are: the sample volume is 100 μL, the injection concentration is 10 mg / mL; a C18 column (4.6 mm×250 mm, 5 μm) is used, and the column temperature is 30° C. In the sample, the mobile phase is 0.05% trifluoroacetic acid-acetonitrile, and the mobile phase B is 0.05% trifluoroacetic acid-ultrapure water, VA:VB=2:8, and at a flow rate of 1 mL / min, an isocratic elution is performed for 40 minutes, and the sample is measured by an ultraviolet spectrophotometer with a wavelength of 220 nm. The components are collected in the order of the spectral peaks, and the sensory quality is evaluated after freeze-drying.

[0092] Electronic tongue measurement method is:

[0093] Sample preparation method of hydrolyzate: The hydrolyzate of goose bone mud was filtered with filter paper to remove solid residues and adjust the pH to neutral, then centrifuged at 6000r / min for 10min, and the supernatant after centrifugation was freeze-dried for 48h at a freeze-drying temperature of -60℃, then the powder was obtained and refrigerated at -80℃ for electronic tongue analysis;

[0094] Sample preparation method after ultrafiltration: The sample with a molecular weight of less than 3000Da of the hydrolyzed liquid of Langde goose bone mud was used as the object, and an ultrafiltration membrane with a molecular weight cutoff of 3000Da was used for filtering the goose bone mud extract. In order to prevent the membrane assembly from being scratched, a 0.45μm organic filter membrane was first used for pre-filtration. On this basis, a UF centrifuge tube was used, and centrifuged at 4℃, 6000r / min for 15min. The filtrate was freeze-dried and stored at -80℃ to obtain powder for electronic tongue analysis;

[0095] Sensory evaluation after gel filtration chromatography: ultrapure water was used as the mobile phase, and UV detection was performed at 220 nm. In order to bind the gel more strongly, the mobile phase was washed on the gel filtration column at a flow rate of 1 mL / min for 5 hours. After that, the umami components after ultrafiltration were prepared into a 5 mg / mL solution with ultrapure water, passed through a 0.22 μm organic filter membrane, and then added to the gel filtration column at a flow rate of 1 mL / min. The components of the peak were collected, and all the components corresponding to one peak were mixed, freeze-dried, and stored at -80°C.

[0096] Electronic tongue sensory evaluation: 10 mg of bone mud hydrolyzate freeze-dried powder was dissolved in 70 mL of pure water for electronic tongue measurement (dissolution concentration was about 0.14 mg / mL). The electronic tongue analysis used a taste detection system containing 7 taste sensors (Astree Electrical Take), which consisted of five sensory flavors: sour (AHS), salty (CTS), NMS (umami), sweet (ANS), bitter (SCS), PKS and CPS. Astree electronic tongue is specially developed for taste analysis. The analysis principle is very similar to human taste. The sensor array analyzes the synthesized global information and uses sensor chips, pattern recognition and other methods to achieve high-sensitivity recognition of odor fingerprints. The instrument mainly includes three aspects: automatic sampling, sensor array and data processing. It uses six groups of sensors, consisting of seven highly sensitive sensors. It only takes 2 minutes to measure a sample. Test sample preparation: take a certain amount of sample, filter it with filter paper, take 1 mL of filtrate, dilute it 30 times with distilled water, put 25 mL of the dilution in a special electronic tongue flask, and place it in an automatic sampling device for analysis.

[0097] Peptide-omics analysis of goose bone mud hydrolysate by ultra-performance liquid chromatography-tandem mass spectrometry:

[0098] Pre-treatment of samples: The freeze-dried samples of goose bone mud hydrolyzate were dissolved in double distilled water, and then DTT was added at 56°C for 1 hour to prevent the egg white from being oxidized; it was placed in a light-proof room for 40 minutes to remove excess DTT; based on the desalting column, a 1.5 mL centrifuge tube was used as a template to elute it to the separated and purified peptides, and the excess reactants were removed in a vacuum centrifuge concentrator at 45°C;

[0099] Capillary liquid chromatography conditions:

[0100] a. Column information: Pre-column: 300μm×5mm, AcclaimPepMapRPLCC18, 5μm, Chromatographic column: 150μm×150mm, AcclaimPepMapRPLCC18, 1.9μm,

[0101] b. Mobile phase information:

[0102] 0.1% formic acid water was used as mobile phase A, and 0.1% formic acid and 80% acetonitrile were used as mobile phase B; gradient elution was performed for 90 min, and the conditions were 0-10 min 6%-9% B, 10-30 min 9%-14% B, 30-70 min 14%-30% B, 70-87 min 30%-40% B, 87-90 min 40%-95% B; flow rate was 600 nL / min;

[0103] Mass spectrometry detection: the polypeptide fragments were dissolved in the sample solution (0.1% methyl acid water), centrifuged at 13200 r / min at 4°C for 20 min, and the supernatant was directly input into the mass spectrometer (ThermoScientific Q Exactive) for online analysis. The specific parameters are as follows: the first-stage mass spectrometry parameters are as follows: resolution: 70000; AGC target: 3e6; Maximum ITzz: 40 ms; second-stage mass spectrometry parameters: resolution: 17500; AGC target: 1e5; Maximum ITzz: 60 ms; TopN: 20; the secondary mass spectrometry data were subjected to secondary Denovo sequencing using the manual Denovo method. The sequencing results showed that the relative molecular weight deviation of the measured protein was ±0.02 Da.

[0104] Screening conditions for umami peptides: The amino acid sequences after liquid-mass analysis were analyzed by PEAKSOnline software, and the biological activity of the peptides was predicted using the online database PeptideRanker. First, the amino acid sequence must contain umami amino acids such as glutamic acid (E), aspartic acid (D), asparagine (N), glutamine (Q), etc., which are important factors for the peptide to produce umami. Secondly, both ends of the sequence must contain hydrophilic amino acids, such as glutamine (Q), asparagine (N), glycine (G), tyrosine (W), serine (S), threonine (T), etc.; then, it must not contain amino acids that may produce toxic peptides, such as cysteine ​​residues (C) and methionine (M); thirdly, most of the umami peptides discovered so far are peptides with less than 10 amino acids; finally, screening is carried out based on the peptide confidence ALC being greater than 90%.

[0105] Design-Expert7.0 and Origin2021 were used to process and plot the experimental data, and Duncan's new multiple range test was adopted.

[0106] Analysis of gel filtration chromatography separation results: GFC is a new separation method for active polypeptide compounds, which has been widely used. GFC is based on the basic principle of molecular sieves. Through the network structure of the gel network, it can effectively separate different molecular weights. The GFC method is used to prepare goose bone slurry hydrolyzed flavor components with a molecular weight below 3000Da. Figure 1 As shown, the retention time of each component is 80-260min. The F1 group is separated first because of its large molecular weight. It cannot pass through the pores and can only pass through the gel particles. Analysis shows that F1 (peak one) is the component with the largest molecular weight; F2 (peak two) has a relatively small molecular weight and can flow out quickly through the internal pores of the gel; F3 (peak three) has a molecular weight between F1 and F2, so it is finally screened out, and the components are collected and freeze-dried, and electronic tongue detection is performed on them.

[0107] Electronic tongue analysis of gel filtration fractions: Figure 2-3 As shown, the umami taste of F2 in the hydrolyzate after gel filtration obtained by electronic tongue experiment is greater than that of F1 and F3, indicating that F2 is more likely to contain umami and the probability of umami peptides appearing is also higher. The order of sour taste is: A2>A3>A1; the order of salty taste is: A1>A3>A2; the order of umami taste is: A2>A3>A1; that is, the three-peak umami taste is: F2>F3>F1.

[0108] Choice of wavelength for reversed-phase HPLC: Figure 4 As shown in the liquid chromatogram, when the wavelength is 220nm, the peaks are separated relatively clearly, and the slope and sharpness of the peaks are more obvious and the effect is better. Therefore, the reversed-phase high performance liquid chromatography has the best effect when the wavelength is 220nm.

[0109] Selection of reversed-phase HPLC flow rate: Figure 5 As shown, it can be observed from the liquid chromatogram that when the flow rate is 1.0 mL / min, the peaks are separated relatively clearly, and the slope and sharpness of the peaks are more obvious and the effect is better. Therefore, the best effect is achieved when the flow rate of reversed-phase HPLC is 1.0 mL / min.

[0110] Choice of mobile phase for reversed-phase HPLC: Figure 6 As shown, it can be observed from the liquid chromatogram that when the mobile phase volume ratio is 2:8, the peaks are separated relatively clearly, and the slope and sharpness of the peaks are more obvious and the effect is better. Therefore, the best effect is achieved when the mobile phase volume ratio of reversed-phase HPLC is 2:8.

[0111] Reverse phase RP-HPLC separation results analysis: Figure 7-9As shown, reversed-phase RP-HPLC is a chromatography method based on hydrophobicity. This technology is the final step in the purification and separation of peptides, proteins, etc. It is safe and efficient. The results show that after GFC separation, three groups of peaks were obtained, indicating that the material was impure. After reversed-phase RP-HPLC purification, several groups of peaks were obtained, which made the bone mud hydrolyzate more delicately purified and provided raw materials for subsequent identification.

[0112] The electronic tongue test results are:

[0113] Please refer to Figure 11-13 , PCA can well reflect the actual situation of the samples, and the sample discrimination index reaches 98, indicating that the electronic tongue can effectively distinguish the six samples based on the difference in taste; the six samples are compared with each other, and the overall taste of samples 5 and 6 is the closest, and the overall taste of samples 3 and 4 is the largest. The electronic tongue TasteRanking function obtains the sour, salty and fresh taste ranking results of the six samples: sour taste ranking: 4>5>6>1>2>3; salty taste ranking: 3>2>1>6>5>4; fresh taste ranking: 4>5>6>1>2>3. The umami intensity after ultrafiltration is mainly due to the fact that all the components are concentrated together after ultrafiltration, resulting in the umami components being all inside, while the umami intensity after gel filtration is slightly weaker than that of the sample after ultrafiltration because the gel filtration divides the sample into three peaks, resulting in a slight weakening of the umami components. The reason why the sample after hydrolysis has less umami is that the salt content is relatively high. The sample information is shown in Table 3.

[0114] Table 3 Sample information table

[0115] Sample name serial number Sample after hydrolysis 1 Sample after hydrolysis 2 Sample after hydrolysis 3 Ultrafiltration sample 1 4 Ultrafiltration sample 2 5 Gel filtration samples 6

[0116] Evaluation of peptide omics analysis results: Fig.14 The analysis showed that the length of the peptide is 2-39 sequences, but most of the polypeptides are short peptides (2-5 peptides), only a small part is long peptides, and more than 20 peptides are almost negligible. This may be because acid is used to hydrolyze bone mud. Acid has the characteristics of fast hydrolysis speed, high hydrolysis efficiency, thorough hydrolysis and more action points. Therefore, the product obtained after the reaction with protein is often a large number of short peptides. This is also in line with the research rules of umami peptides. Generally, umami peptide sequences are short peptides, and the probability of long peptides containing umami is extremely low, which also provides a better basis for the study of umami peptides.

[0117] The peptides in the bone mud hydrolysate were classified using an ensemble classifier: the profile quantification (PSMs) refers to the secondary profiles of peptides extracted from the sample, which contain secondary profiles of the same fragments, and the peptide identification quantity reflects the content of peptides produced in the sample after analysis and identification; from Table 4 and Fig.15It can be seen that the proportion of short peptides in the total number is much greater than that of long peptides. Since most umami peptides are composed of short peptides, this also confirms that there is no problem with the result of hydrolysis, and the separation goal is also met when separated again, which provides a basis for the subsequent umami peptide screening.

[0118] Table 4 Number of peptides in different peptide sequences

[0119]

[0120] Screening of potential umami peptides in goose bone paste: Fig.16 As shown in the figure, after acid hydrolysis of goose bone mud, more than 16,600 peptides were obtained, containing more than 120,000 amino acids. Peptides have attracted much attention for their high biological activity, low cost, high specificity and penetration. However, toxicity is still the main problem in peptide development. In addition, good water solubility is the key to the normal metabolism of peptides in the body. Therefore, the toxicity and solubility of peptides should be predicted by silicon. The results showed that 126 peptides were obtained after preliminary screening by machine, including 8 heptapeptides, 36 octapeptides, and 82 nonapeptides with molecular weights of 732Da-1180Da. As the length of the peptide chain increases, its water solubility decreases, which is related to the flavor characteristics and hydrophilicity of umami amino acids. The peptide has good water solubility and non-toxicity, and the peptide sequence confidence area is greater than 90%, and the peptide chain length is less than 10, which narrows the range for the subsequent molecular docking. The above virtual screening strategy adopted in this study has obvious advantages such as saving resources, saving time, and quick and convenient operation, but subsequent studies will further consider improving and optimizing the accuracy and reliability of distinguishing umami peptides from non-umami peptides.

[0121] in conclusion:

[0122] (1) The umami taste of F2 was greater than that of F1 and F3 in the hydrolyzate after gel filtration obtained by electronic tongue experiment, indicating that F2 was more likely to contain umami and the probability of umami peptides appearing was also higher. The order of sour taste was: A2>A3>A1; the order of salty taste was: A1>A3>A2; the order of umami taste was: A2>A3>A1; that is, the three-peak umami taste was: F2>F3>F1.

[0123] (2) HPLC conditions: The best effect was achieved when the wavelength was 220 nm, the flow rate was 1.0 mL / min, and the mobile phase volume ratio was 2:8.

[0124] (3) The umami intensity after ultrafiltration is mainly due to the fact that all the components are concentrated together after ultrafiltration, resulting in the umami components being all inside. The umami intensity after gel filtration is slightly weaker than that of the sample after ultrafiltration because gel filtration separates the sample into three peaks, resulting in a slight weakening of the umami components. The reason why the sample after hydrolysis has less umami is that the salt content is higher.

[0125] (4) The peptide sequences obtained from the hydrolyzate of Langde goose bone mud are all short peptides, and the proportion is as high as 75.45%. This may be because acid is used to hydrolyze bone mud. Acid has the characteristics of fast hydrolysis speed, high hydrolysis efficiency, thorough hydrolysis and more action points. Therefore, the products obtained after the reaction with protein are often a large number of short peptides.

[0126] (5) After preliminary screening by machine, 126 peptides were obtained, including 8 heptapeptides, 36 octapeptides, and 82 nonapeptides with molecular weights of 732Da-1180Da. As the length of the peptide chain increases, its water solubility decreases, which is related to the flavor characteristics and hydrophilicity of umami amino acids. The polypeptide has good water solubility and is non-toxic. The confidence region of the peptide sequence is greater than 90%, and the peptide chain length is less than 10, which narrows the range for subsequent molecular docking.

[0127] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0128] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A separation and purification process for Langde goose bone paste umami peptide, characterized in that: The following steps are involved: S11, prepare goose bone mud hydrolyzate, and take the supernatant for later use; S12, performing a separation experiment on the supernatant of the goose bone mud hydrolyzate; S13. Perform sensory evaluation on the separated goose bone paste using an electronic tongue.

2. The separation and purification process of the Langde goose bone paste umami peptide according to claim 1, characterized in that: The separation experiment in S12 includes a nanofiltration separation experiment, and the nanofiltration separation experiment includes the following steps: S21, using a 200Da nanofiltration membrane to perform nanofiltration on the hydrolyzate to remove free amino acids and salts in the solution; S22, adjusting the solution pH to 5-9, the total solution concentration to 0.1%-1%, and the solution temperature to 5-45°C; S23, connect the instrument, filter three times, and perform formaldehyde potentiometric titration on the filtrate of the filtered polypeptide solution to determine the amino acid content therein, thereby determining the nanofiltration effect of the polypeptide solution; S24, ultrafiltering the polypeptide solution filtrate and freeze-drying it.

3. The separation and purification process of the umami peptide from Langde goose bone paste according to claim 1, characterized in that: The separation experiment in S12 includes an ultrafiltration separation experiment, and the ultrafiltration separation experiment includes the following steps: S31, pre-filtering it with a 0.45 μm organic filter membrane; S32, filtering it using an ultrafiltration membrane with a molecular weight cut-off of less than 3000Da; S33, use UF centrifuge tube, temperature is 4 ℃, rotation speed is 6000r / min, after centrifugation for 15min, freeze-dry the filtrate and store at -80℃.

4. The separation and purification process of the Langde goose bone paste umami peptide according to claim 1, characterized in that: The separation experiment in S12 includes a gel chromatography separation experiment, and the gel chromatography separation experiment includes the following steps: S41, dissolving goose bone mud hydrolyzate with a molecular weight lower than 3000Da in pure water, and loading into a Sephadex G-15 chromatographic column at 25°C for separation; S42, eluting with ultrapure water under the chromatographic conditions of 5 mL, 100 mg / mL, at a flow rate of 1.0 mL / min, and detecting under 220 nm ultraviolet light; S43. Collect, concentrate and freeze-dry the components in order of their peaks.

5. The separation and purification process of the Langde goose bone paste umami peptide according to claim 4, characterized in that: The components separated in the gel chromatography separation experiment were dissolved in pure water, filtered with a 0.22 μm organic filter membrane, and purified by reverse phase high performance liquid chromatography. The steps of the purification process are as follows: S51, sample volume is 100 μL, injection concentration is 10 mg / mL, C18 column is used, column temperature is 30 °C; S52, in the sample, the mobile phase is 0.05% trifluoroacetic acid-acetonitrile, the mobile phase B is 0.05% trifluoroacetic acid-ultrapure water, VA:VB=2:8, and the flow rate is 1 mL / min, and the isocratic elution is performed for 40 min; S53. Determine with an ultraviolet spectrophotometer at a wavelength of 220 nm, collect each component in the order of spectral peaks, and freeze-dry.

6. The separation and purification process of the Langde goose bone paste umami peptide according to claim 1, characterized in that: The electronic tongue is a taste detection system composed of seven taste sensors, and the seven taste sensors are respectively sour, salty, NMS, sweet, bitter, PKS and CPS sensory flavor sensors.

7. The separation and purification process of the Langde goose bone paste umami peptide according to claim 6, characterized in that: The electronic tongue measurement method is: dissolve 10 mg of bone mud hydrolyzate lyophilized powder in 70 mL of pure water for electronic tongue measurement, and the dissolved concentration is 0.14 mg / mL.

8. A screening process for umami peptides from Langde goose bone paste, characterized in that: The process includes the following steps: S61, peptidomic analysis of goose bone mud hydrolysate by ultra-performance liquid chromatography-tandem mass spectrometry; S62, using the online database PeptideRanker to predict the biological activity of all peptide sequences obtained by analyzing the amino acid sequences after LC-MS analysis using PEAKSOnline software; S63. The screening method is as follows: first, the amino acid sequence must contain umami amino acids, then it must not contain amino acids that may produce toxic peptides, and the umami peptides found again are mostly peptides with less than 10 amino acids, and finally screening is carried out based on the peptide confidence ALC being greater than 90%.

9. The screening process of the umami peptide of Langde goose bone paste according to claim 8, characterized in that: The steps for peptidomic analysis of goose bone mud hydrolysate by ultra-performance liquid chromatography-tandem mass spectrometry are as follows: S71, pre-treatment sample: The freeze-dried sample of goose bone mud hydrolyzate was dissolved in double distilled water, then DTT was added for 1 hour at 56°C, placed in a light-proof room for 40 minutes to remove excess DTT, and eluted to the separated and purified peptides based on a desalting column and a 1.5 mL centrifuge tube as a template, and excess reactants were removed in a vacuum centrifuge concentrator at 45°C; S72, Capillary liquid chromatography conditions: a. Column information: Pre-column: 300μm×5mm, AcclaimPepMapRPLCC18, 5μm, Column: 150μm×150mm, Acclaim PepMapRPLCC18, 1.9μm, b. Mobile phase information: 0.1% formic acid water was used as mobile phase A, and 0.1% formic acid and 80% acetonitrile were used as mobile phase B; gradient elution was performed for 90 min, and the conditions were 0-10 min 6%-9% B, 10-30 min 9%-14% B, 30-70 min 14%-30% B, 70-87 min 30%-40% B, 87-90 min 40%-95% B, and the flow rate was 600 nL / min; S73, mass spectrometry detection: The polypeptide fragments were dissolved in the sample solution, centrifuged at 13200r / min at 4°C for 20min, and the supernatant was directly input into the mass spectrometer for online analysis. The specific parameters were as follows: the first-stage mass spectrometer parameters were as follows: resolution: 70000; AGCtarget: 3e6; MaximumITzz: 40ms; the second-stage mass spectrometer parameters were: resolution: 17500; AGCtarget: 1e5; MaximumITzz: 60ms; TopN: 20; the secondary mass spectrometer data were subjected to secondary Denovo sequencing using the manual Denovo method.

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