A soybean paste polypeptide and a preparation method and application thereof

Abstract

The application discloses a kind of soybean paste polypeptide and its preparation method and application, it is related to biotechnology field, polypeptide is extracted from soybean paste, amino acid sequence is respectively:Asp-Asn-Val-Ile-Ser-Gln-Ile-His.The preparation method of polypeptide includes the following steps: first, extract water-soluble component from soybean paste, using ultrafiltration separation cut-off molecular weight less than 3kDa ultrafiltration component, further separate and purify after using RP-HPLC method to ultrafiltration component and obtain polypeptide, the polypeptide in the application has the effect of enhancing food fresh taste, can be applied to fish meat essence production, improve the fresh taste of fish meat essence;Meanwhile, the polypeptide has higher ACE inhibition rate, can be used for preparing antihypertensive health food, safe.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a soybean paste polypeptide, its preparation method, and its applications. Background Technology

[0002] Peptides are small molecule compounds formed by α-amino acids linked together by peptide bonds. They are also intermediate products of protein hydrolysis. A class of small molecule peptides from food sources that contribute to flavor are named flavor peptides. Peptides in food mainly originate from protein breakdown and microbial metabolism. The flavor effects of peptides in food vary depending on their amino acid composition, peptide chain length, and arrangement, resulting in different taste sensations such as sour, sweet, bitter, salty, and umami. After ingestion, most proteins are digested and absorbed by the digestive tract through enzymatic action, primarily in the form of low-peptides. The proportion absorbed as free amino acids is very small. Further experiments have revealed that peptides are digested and absorbed more readily than free amino acids. Therefore, the application of flavor peptides in food can not only improve human absorption but also address the issue of insufficient flavor richness from single-ingredient seasonings, thus improving food quality. Therefore, flavor peptides have significant market potential in the food industry.

[0003] Doubanjiang (fermented broad bean paste) is a type of condiment made primarily from broad beans, chili peppers, and salt through a long fermentation process. It is a semi-fluid, viscous, or semi-solid condiment. Rich in various amino acids, proteins, vitamins, lipids, and other flavor compounds, doubanjiang is bright red in color, spicy in taste, and has a rich aroma, making it an important condiment in Sichuan cuisine. Current research on doubanjiang mainly focuses on the extraction, separation, identification, and related trends of its flavor components, particularly volatile aroma components. However, research on non-volatile components is scarce, and studies on flavor peptides in doubanjiang have not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a soybean paste polypeptide, its preparation method, and its application. This polypeptide can be used in the preparation of fish flavoring, enriching the taste of fish flavoring and making it rich and delicious.

[0005] This invention is achieved through the following technical solution: A polypeptide with the following amino acid sequence: Asp-Asn-Val-Ile-Ser-Gln-Ile-His (abbreviated as: DNVISQIH).

[0006] Preferably, the polypeptide is extracted from fermented soybean paste.

[0007] The polypeptide described in this invention is obtained by the following preparation method, comprising the following steps: a. Grind the fermented soybean paste into a paste to prepare an extract of water-soluble substances from the fermented soybean paste; b. Use ultrafiltration to separate ultrafiltration components with a molecular weight cutoff of less than 3 kDa from the extract; c. The ultrafiltration fraction was subjected to gel permeation chromatography; d. Separate and purify the components obtained after chromatography.

[0008] More specifically, it includes the following steps: In step a, the fermented soybean paste is pulped, and ultrapure water is added at a mass ratio of 1:3~5. The mixture is shaken in a water bath at 37~40℃ for 1~2 hours. The filtrate is centrifuged at 8000r / min for 20~30 minutes at 4℃, filtered through a 0.22μm microporous membrane, and the filtrate is dispensed into a separatory funnel. Petroleum ether is added at a volume ratio of 1:1~2 for defatting. The lower layer of filtrate is then collected and freeze-dried to obtain freeze-dried powder. In step b, the lyophilized powder is dissolved in distilled water, filtered through a 0.22 μm microporous membrane, and the molecular weight of component F below 3 kDa is retained. After freeze-drying, it is stored at -18°C. In step c, the ultrafiltration component F is prepared into a solution of 20~30 mg / ml and filtered through a 0.22 μm aqueous microporous membrane. It is then separated by chromatography using Sephdex G-15 dextran gel, and the separated peak components are collected, freeze-dried, and stored for later use. In step d, the component with the strongest flavor and richness obtained after chromatography is separated and purified by RP-HPLC, and then the amino acid sequence is obtained by LC-MS / MS identification of the component.

[0009] The aforementioned polypeptides can also be synthesized using amino acids as raw materials through chemical methods, which is well known in the art.

[0010] Preferably, in step c, the sample loading volume of Sephdex G-15 dextran gel chromatography is 2-4 ml, the elution flow rate is 1-2 ml / min, and the detection wavelength is 220 nm.

[0011] In this invention, the polypeptide can be used in fish flavoring.

[0012] In this invention, the polypeptide has high ACE inhibitory activity.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention provides a polypeptide that can significantly enhance the saltiness, umami, and richness of food, and harmonize its taste. Its amino acid sequence is disclosed, along with a method for obtaining the polypeptide. The polypeptide can be obtained according to a specific preparation method, or it can be synthesized chemically based on the disclosed amino acid sequence. This polypeptide can be used to regulate the umami flavor of fish flavorings, significantly enhancing their flavor. Furthermore, the polypeptide exhibits high ACE inhibitory activity. Attached Figure Description

[0014] Figure 1 The chromatograms are those of ultrafiltration fractions with molecular weights below 3 kDa after separation by Sephadex G-15 gel chromatography.

[0015] Figure 2 The results of the taste dilution analysis of ultrafiltration component F and chromatography components F1, F2, and F3 are presented.

[0016] Figure 3 This is the ultra-high performance liquid chromatography (UHPLC) separation chromatogram of component F2.

[0017] Figure 4 This is the secondary mass spectrum of the DNVISQIH sequence.

[0018] Figure 5 It is a fish flavoring containing soybean paste peptides. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the present invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] This invention provides a polypeptide with the amino acid sequence: Asp-Asn-Val-Ile-Ser-Gln-Ile-His. The polypeptide is extracted from fermented soybean paste.

[0021] This invention provides a method for preparing soybean paste polypeptides, comprising the following steps: Step 1: Grind the fermented soybean paste into a paste, add ultrapure water at a mass ratio of 1:3~5, shake in a water bath at 37~40℃ for 1~2 hours, centrifuge the filtrate at 8000r / min for 20~30 minutes at 4℃, filter it through a 0.22μm microporous membrane, dispense the filtrate into a separatory funnel, add petroleum ether at a volume ratio of 1:1~2 for defatting, collect the lower layer of filtrate and freeze-dry it to obtain freeze-dried powder; Step 2: The above lyophilized powder is dissolved in distilled water and filtered through a 0.22μm microporous membrane. The filtrate is then ultrafiltered at 25℃ and 0.4MPa using an ultrafiltration cartridge to retain the component F with a molecular weight below 3kDa. The filtrate is collected, freeze-dried, and stored at -18℃. Step 3: Prepare an ultrafiltration fraction F solution of 20-30 mg / ml and filter it through a 0.22 μm aqueous microporous membrane. Separate the fractions by chromatography using Sephdex G-15 dextran gel. Collect the separated peaks and freeze-dry them for later use. Step 4: The component with the strongest flavor and richness obtained after chromatography (based on artificial sensory evaluation) was separated and purified by RP-HPLC, and the amino acid sequence was obtained by LC-MS / MS after identification. Asp-Asn-Val-Ile-Ser-Gln-Ile-His (DNVISQIH).

[0022] In step 3, the sample loading volume for Sephadex G-15 gel chromatography is 2-4 ml, and 4 ml is preferred to ensure chromatographic effect. The elution flow rate is 1-2 ml / min, and the detection wavelength is 220 nm.

[0023] In the following specific embodiments of the present invention, the ultrafiltration components are subjected to Sephadex G-15 gel chromatography to select the components with the strongest flavor and richness (artificial sensory evaluation), which are then further separated by RP-HPLC and identified by LC-MS / MS.

[0024] The human sensory evaluation uses taste dilution analysis (TDA) to determine the dilution factor, or TD value, of each sample. The higher the TD value, the stronger the taste characteristics of the sample.

[0025] The TD value is determined as follows: The component to be tested is dissolved in an equal proportion of pure water and diluted 1:1 (volume ratio). A three-point test method is used to evaluate each dilution level until the taste is no longer perceptible at a certain dilution factor. This dilution factor is the taste dilution factor (TD) of the solution; the higher the TD value, the stronger the taste of the component. Sensory evaluation is conducted by eight sensory evaluators (4 men and 4 women, aged 25 to 30), who are trained before the experiment.

[0026] This invention proposes the application of a polypeptide in fish flavoring, comprising the following steps: Step 1: Using white mullet as raw material, the white mullet is pre-treated, cleaned, cut, and minced into a paste. Step 2: Take the above fish paste and mix it with pure water at a ratio of 1:7. Add 2.4% of compound enzyme (compound protease and flavor protease in a ratio of 1:3) according to the weight of the fish paste. Adjust the pH to 7.0 and enzymatically hydrolyze at 50℃ for 6 hours. Then, inactivate the enzyme at 90℃ for 10 minutes. After completion, quickly cool to room temperature, filter, collect the filtrate, seal and store it to obtain the enzymatic hydrolysate.

[0027] Step 3: Add reducing sugar, thiamine and other ingredients and the polypeptide obtained by the present invention to the enzymatic hydrolysate. After mixing thoroughly, place it in a CJF-0.1L small high-pressure reactor for reaction. After cooling to room temperature, the fragrance is obtained.

[0028] Example 1 A method for preparing a polypeptide, the specific steps of which are as follows: Step 1: Grind the fermented soybean paste into a paste, add ultrapure water at a ratio of 1:4, shake in a 40℃ water bath for 1 hour, centrifuge the filtrate at 8000r / min for 20 minutes at 4℃, filter it through a 0.22μm microporous membrane, dispense the filtrate into a separatory funnel, add petroleum ether at a volume ratio of 1:1 for defatting, and then collect the lower layer of filtrate for freeze-drying to obtain freeze-dried powder.

[0029] Step 2: Dissolve the above lyophilized powder in distilled water, filter it with a 0.22μm microporous membrane, and ultrafilter the filtrate at 25℃ and 0.4MPa using an ultrafiltration cartridge to retain the component F with a molecular weight below 3kDa. Collect the filtrate, freeze-dry it, and store it at -18℃.

[0030] Step 3: Prepare a 20 mg / ml solution of ultrafiltration fraction F and filter it through a 0.22 μm aqueous microporous membrane. Separate the solution using Sephdex G-15 dextran gel chromatography. The sample loading volume for gel chromatography is 4 ml, the elution flow rate is 1 ml / min, and the detection wavelength is 220 nm. Collect the separated peak components, freeze-dry them, and use them for later use. The chromatographic results are shown below. Figure 1As shown, three separate components were obtained successively and identified as F1, F2, and F3.

[0031] Step 4: The F1, F2, F3 and F fractions obtained after chromatography were subjected to sensory evaluation using taste dilution analysis (TDA). The results are shown in [Figure number missing]. Figure 2 The horizontal axis represents each component, and the vertical axis represents the flavor intensity dilution factor of each component.

[0032] like Figure 2 Components F and F2 have the highest TD values ​​and the strongest taste within the same range.

[0033] The results of the artificial sensory evaluation of the taste characteristics of ultrafiltration component F and chromatography components F1, F2, and F3 are shown in Table 1. As can be seen from Table 1, the taste descriptions of chromatography component F2 and component F are the most similar.

[0034] Table 1 Sensory evaluation results

[0035] comprehensive Figure 2 Based on Table 1, it can be concluded that the F2 component has the most intense peptide flavor and is most similar to the ultrafiltration component F. Therefore, the F2 component was selected for the next step of separation and purification.

[0036] Step 6: The F2 ultrafiltration fraction was separated and purified using liquid chromatography-mass spectrometry (LC-MS). LC conditions: Analytical column: 150 μm id × 150 mm, packed with Acclaim PepMap RPLC C18, 3 μm, 100A; Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid, 80% ACN; Flow rate: 600 nL / min; Gradient elution: 0 min (4% B), 0-2 min (4%-8% B), 2-45 min (8%-28% B), 45-55 min (28%-40% B), 55-56 min (40%-95% B), 56-66 min (95% B); Sample loading volume: 10 μL. Results are as follows: Figure 3 .

[0037] Mass spectrometry conditions: Primary mass spectrometry parameters: Resolution: 70,000; AGC target: 3e6; Maximum IT: 100 ms; Scan range: 100 to 1500 m / z. Secondary mass spectrometry parameters: Resolution: 17,500; AGC target: 1e5; Maximum IT: 50 ms; Top N: 20; NCE / stepped NCE: 28. Results reference. Figure 4 .

[0038] The amino acid sequence obtained after identification is as follows: Asp-Asn-Val-Ile-Ser-Gln-Ile-His (DNVISQIH).

[0039] Table 2 Peptide Information

[0040] The properties of the obtained polypeptide (DNVISQIH) were further investigated.

[0041] (1) Flavor threshold and characteristics of polypeptides Prior to the experiment, the evaluation team members were trained. The reference standards for umami, sweetness, saltiness, bitterness, and sourness were monosodium glutamate solution (0.35%, w / v), sucrose solution (1%, w / v), sodium chloride (0.35%, w / v), L-isoleucine solution (0.25%, w / v), and citric acid solution (0.08%, w / v), respectively, in order to achieve more accurate sensory evaluation results.

[0042] The peptide obtained in Example 1 was prepared as a 1% synthetic peptide solution. Following the triangulation method, it was progressively diluted at a 1:1 ratio. Each dilution level was evaluated using three measurements until it was just distinguishable from two blank controls (pure water). The sample concentration at this point was taken as the taste threshold. The stimulus threshold for each evaluation panel was the average of the results from all evaluators. The results are shown in Table 3. Panel members were also asked to verbally describe their taste experiences, including umami, sweetness, sourness, saltiness, and bitterness.

[0043] Table 3. Taste and threshold properties of synthetic peptide aqueous solutions

[0044] The results in the table show that the DNVISQIH sequence has a low taste threshold of 0.38 mg / mL, which is lower than the umami standard of 3.5 g / mL sodium glutamate solution, indicating a good taste effect. It also presents a pleasant taste sensation and a harmonious mouthfeel, suggesting it may be a salty peptide. (2) Enhancement properties of peptide-salt solution The saltiness of a 0.35% salt solution was used as a sensory standard, with a score of 5. The polypeptide obtained in Example 1 was added to the salt solution, resulting in a polypeptide concentration of 2 mg / mL in the 0.35% salt solution, and its saltiness was scored. The standard salt solution was scored out of 5. The synergistic effect of the synthetic peptide and salt was investigated, and the final results were averaged. The results are shown in Table 4.

[0045] Table 4 Enhancement properties of peptide-saline solution

[0046] As shown in Table 4, the polypeptide DNVISQIH has a slightly salty taste.

[0047] (3) Umami-enhancing effect of peptides Using the umami flavor of a 0.35% monosodium glutamate (MSG) solution as a sensory standard with a score of 5, the polypeptide obtained in Example 1 was added to the MSG solution, resulting in a polypeptide concentration of 2 mg / mL in the 0.35% MSG solution. The umami flavor enhancement effect of the sample was then evaluated, and the final results were averaged. The results are shown in Table 5.

[0048] Table 5 shows the umami-enhancing effects of peptides.

[0049] As shown in Table 5, the DNVISQIH sequence has a strong umami-enhancing effect.

[0050] Example 2 Step 1: After pre-treating the white cuttlefish, clean, cut, and mince the meat into a paste. Step 2: Take the above fish paste and mix it with pure water at a ratio of 1:7. Add 2.4% of compound enzyme (compound protease and flavor protease in a ratio of 1:3) according to the weight of the fish paste. Adjust the pH to 7.0 and enzymatically hydrolyze at 50℃ for 6 hours. Then, inactivate the enzyme at 90℃ for 10 minutes. After completion, quickly cool to room temperature, filter, collect the filtrate, seal and store it to obtain the enzymatic hydrolysate.

[0051] Step 3: Add 4% reducing sugar (glucose:xylose ratio 4:1), 1% thiamine, and 1% DNVISQIH to the enzymatic hydrolysate. Mix thoroughly and place in a CJF-0.1L small high-pressure reactor. React at 110℃ for 20 minutes. After cooling to room temperature, the fragrance is obtained. Figure 5 .

[0052] Step 4: Sensory evaluation of fish flavoring adopts a scoring test method. Eight sensory evaluators were invited and, after unified training, conducted sensory evaluation based on the umami, sweetness, fishy taste, off-flavor, and richness of the fish flavoring (Table 6).

[0053] Table 6 Sensory Evaluation Criteria for Fish Flavoring

[0054] Comparative Example 1 In Example 2, step 3 does not include the polypeptide, while the remaining steps are the same as in Example 2.

[0055] Comparative Example 2 In Example 2, the polypeptide added in step 3 was replaced with three umami amino acids: L-cysteine, L-alanine, and L-serine. The remaining steps were the same as in Example 2.

[0056] The sensory evaluation results are shown in Table 7.

[0057] Table 7 Sensory evaluation results of Example 2 and Comparative Example 1

[0058] Compared with Comparative Example 1, Example 2 and Comparative Example 2 have a richer and more prominent umami flavor, and the unpleasant off-flavors are not obvious. It can be seen that the polypeptide described in this invention can significantly improve the umami flavor of fish meat.

[0059] Example 3 The activity assay of peptides with ACE inhibitory activity includes the following steps: Step 1: Dissolve the soybean paste peptides in a 0.1 mol / L borate buffer (containing 0.3 mol / L NaCl) at pH 8.3; Step 2: Add 50 μL of the above peptide solution and 50 μL of 5 mmol / L hippuryl-histyl-leucine (HHL) to a 1.5 mL centrifuge tube, and incubate in a 37 °C water bath for 5 min. Step 3: Add 50 μL of 0.1 U / mL ACE solution, incubate in a 37℃ water bath for 30 min, and then add 100 μL of 1 mol / L HCl to terminate the reaction.

[0060] Step 4: The blank control group was replaced with borate buffer. After centrifugation, the amount of hippuric acid produced was determined by HPLC. HPLC conditions: mobile phase: acetonitrile (containing 0.1% trifluoroacetic acid), water (containing 0.1% trifluoroacetic acid), isogradient elution: 0% to 28% and 28% to 47%; flow rate: 1 mL / min; detection wavelength: 228 nm; injection volume: 10 μL.

[0061] ACE inhibitory activity is calculated using the following formula: ACE inhibitory activity = (Control A - Sample A) / Control A × 100% The determination showed that the soybean paste peptide in Example 1 had a high ACE inhibition rate, which was 57.82%.

[0062] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is: Asp-Asn-Val-Ile-Ser-Gln-Ile-His.

2. The polypeptide according to claim 1, characterized in that, The polypeptide was extracted from fermented soybean paste.

3. The use of the polypeptide as described in any one of claims 1-2 in the preparation of fish flavoring.

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