Polypeptides capable of enhancing saltiness and uses thereof

Abstract

The application discloses a kind of polypeptide capable of enhancing salty taste and application thereof, and belongs to the technical field of food processing.The polypeptide capable of enhancing salty taste is obtained by being separated and extracted from the fermentation liquor of flower clam meat, and the amino acid sequence of the polypeptide is Asn-Pro-Asp.The basic taste effect evaluation, salty taste enhancement effect evaluation and equivalent sodium chloride concentration determination are verified by experiments, and it is confirmed that the crude polypeptide prepared by the application has good salt-reducing effect, can reduce the amount of salt while ensuring that salty taste is not affected, realizes the purpose of salt reduction, and ensures that no significant changes occur in the perception of taste, which can provide a new way for "salt reduction without salty taste".It provides a new idea for the development of healthy and delicious food.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, in particular to a polypeptide capable of enhancing saltiness and application thereof. Background Art

[0002] Saltiness, as one of the most basic taste sensations, determines, to a certain extent, consumers' acceptance of the sensory qualities of food. In the food industry, salt (NaCl) is one of the most common condiments, used to enhance the saltiness of food, improve its flavor, and make it more appealing to consumers. However, according to statistics, the average daily salt intake per person in my country has exceeded 10g, far exceeding the World Health Organization (WHO) recommended daily salt intake of 5g for adults. Although salt is an important ingredient in food processing, enhancing saltiness, improving product taste perception, extending the shelf life and shelf life of food, and inhibiting the growth and reproduction of pathogenic and spoilage bacteria, excessive salt intake can have adverse effects on the human body, leading to a series of health problems, such as hypertension and cardiovascular disease. In light of the national initiative to reduce salt intake, how to reduce the amount of added salt has become a hot topic of research both domestically and internationally. Current approaches to reducing salt include changing the physical form of salt and using non-mineral salt substitutes. For example, KCl is widely used. However, studies have shown that excessive addition of non-sodium salts can create metallic or bitter tastes, affecting food flavor. Therefore, it is crucial to develop new methods or products that can reduce salt without compromising flavor.

[0003] In recent years, bioactive peptides have been widely studied and applied. People use different methods to extract flavoring substances from natural foods to study flavor changes and the main flavoring substances. Flavoring peptides have been widely reported as new taste enhancers. Among them, the currently isolated salty peptides have been shown to be able to replace part of the table salt, thereby reducing salt intake and increasing people's perception of saltiness. They also have certain functional properties that meet people's current pursuit of health, deliciousness, and safety. For example, one study used the obtained salty peptides to increase the saltiness perception intensity of a 50mmol / L sodium chloride solution by 26.20%. Another study applied the isolated salty peptides to salt-free chicken soup and found that they could enhance the saltiness and umami flavor of the chicken soup. Therefore, exploring the flavor effects and mechanisms of salty peptides in practical applications can provide a new reference for the food industry and a new strategy for salt reduction. Summary of the Invention

[0004] The present invention aims to provide a polypeptide capable of enhancing saltiness and its use to address existing problems in the prior art. The present invention isolates and extracts a polypeptide capable of enhancing saltiness from the fermentation broth of clam meat. Experimental verification demonstrates that the polypeptide prepared in the present invention has a significant salt-reducing effect, reducing salt intake without altering saltiness, providing a new approach to the development of healthy and delicious foods.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a polypeptide capable of enhancing salty taste. The amino acid sequence of the polypeptide is Asn-Pro-Asp.

[0007] The present invention also provides a convenient food ingredient comprising the polypeptide.

[0008] Furthermore, the invention also includes auxiliary materials, which include one or more of brewed soy sauce, brewed vinegar, salt, white sugar, sodium glutamate, concentrated lemon juice, potassium sorbate, edible oil or minced meat.

[0009] The present invention also provides a method for preparing the polypeptide, comprising the following steps:

[0010] The clam meat is shelled and cleaned, drained and added into ultrapure water for homogenization, and then preliminarily filtered after fermentation to obtain a crude extract; the crude extract is centrifuged, the supernatant is taken, and filtered through an ultrafiltration membrane to obtain a crude peptide extract; the crude peptide extract is purified by gel exclusion chromatography and reversed-phase high-performance liquid chromatography in sequence to obtain a crude peptide; the crude peptide is identified by mass spectrometry and then synthetically verified to screen and obtain the polypeptide.

[0011] Furthermore, the mass volume ratio of the clam meat to the ultrapure water is 1 kg:3-5 L.

[0012] Furthermore, the fermentation temperature is 30-35°C and the fermentation time is 24-48 hours.

[0013] Furthermore, the centrifugation is carried out at 4°C, 8500-10000 rpm, and 15-20 min.

[0014] Furthermore, the molecular weight cut-off of the ultrafiltration membrane is less than 3000Da.

[0015] The present invention also provides applications of the polypeptide, including any of the following applications:

[0016] (1) Preparation of convenience food ingredients;

[0017] (2) preparing low-sodium foods;

[0018] (3) Enhance the saltiness of food.

[0019] The present invention discloses the following technical effects:

[0020] The present invention isolates and extracts a polypeptide capable of enhancing saltiness from the fermentation broth of clam meat. Experimental verification, including basic taste effect evaluation, saltiness enhancement evaluation, and equivalent sodium chloride concentration determination, confirms that the crude peptide prepared by the present invention has a good salt-reducing effect. It can reduce the amount of added salt while ensuring that the saltiness is not affected. This achieves the goal of salt reduction while ensuring that there is no significant change in taste perception. This provides a new approach to "reducing salt without reducing saltiness" and offers a new approach to the development of healthy and delicious foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 HPLC diagram (A) and mass spectrometry (B) of solid phase synthesized peptides;

[0023] Figure 2 for the taste modulation effects of peptides in different model matrices;

[0024] Figure 3 is the dose-response curve of the peptide in sodium chloride solution;

[0025] Figure 4 is the standard curve of the concentration and saltiness intensity of sodium chloride solution;

[0026] Figure 5 The taste effects of polypeptide solutions with different concentrations in low concentration sodium chloride solution. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Unless otherwise specified, the materials used in the embodiments of the present invention can be obtained from common commercial sources.

[0033] Example 1 Extraction and identification of polypeptides

[0034] 1. Preparation of Crude Peptide Extracts

[0035] The clam meat was washed and drained, and ultrapure water was added at a mass volume ratio of 1kg:5L. The slurry was homogenized at 20,000rpm for 3 times, each time for 30s, and the starter was inoculated (the starter was prepared by a mixture of Lactobacillus plantarum and Staphylococcus xylosus, with a strain ratio of 1:1 and an inoculation ratio of 3%). After fermentation at 30°C for 24h, the crude extract was obtained after preliminary filtration with gauze; the crude extract was centrifuged at 8500rpm and 4°C for 20min, the supernatant was taken, and the crude peptide extract was obtained by ultrafiltration membrane (<3000Da).

[0036] 2. Gel Exclusion Chromatography Purification

[0037] The crude peptide extract obtained by ultrafiltration was lyophilized at -40°C and dissolved in ultrapure water at a concentration of 0.1 g / mL. The extract was passed through a 0.22 μm filter membrane before injection. Ultrapure water was used as the eluent at a rate of 0.5 mL / min for gel exclusion chromatography. The fractions were collected using an automated collector and lyophilized at -40°C.

[0038] 3. Reversed-Phase High-Performance Liquid Chromatography Separation

[0039] Each component obtained after gel exclusion chromatography purification was prepared into a 10 mg / mL solution and separated by reverse-phase high-performance liquid chromatography (HPLC). The eluents A and B were ultrapure water and 100% acetonitrile, respectively, at a flow rate of 0.4 mL / min, and detected under a 220 nm UV detector. The fractions were collected and lyophilized at -20°C. The taste of each component was then tested (the taste test standard composition was 0.35% NaCl, 0.35% MSG, 1% sucrose, 0.08% citric acid, and 0.08% L-leucine). The component with the strongest saltiness was obtained.

[0040] 4. Mass Spectrometry Identification

[0041] The saltiest component obtained above was analyzed and identified to obtain a polypeptide capable of enhancing saltiness, whose amino acid sequence is: Asn-Pro-Asp. The sequence was subjected to solid phase synthesis, and the synthesis results are shown in FIG. Figure 1 shown.

[0042] Example 2 Verification of the taste effect of the polypeptide

[0043] The polypeptide capable of enhancing saltiness obtained in Example 1 was subjected to solid phase synthesis to verify its saltiness effect.

[0044] 1. Basic taste effects

[0045] The saltiness-enhancing polypeptide lyophilized powder obtained in Example 1 was prepared into a 5 mg / mL solution and compared with a standard solution, and the basic taste effects were recorded. The standard solution consisted of the following components, calculated by mass percentage: 0.35% NaCl, 0.35% MSG, 1% sucrose, 0.08% citric acid, and 0.08% L-leucine. The sensory properties of the polypeptides are evaluated as shown in Table 1.

[0046] Table 1 Sensory properties evaluation of peptides

[0047]

[0048] As shown in Table 1, compared with the standard product, the polypeptide solution exhibits a stronger salty and umami taste, a slightly sweet aftertaste, and no bitterness.

[0049] 2. Salty taste enhancement effect of peptides

[0050] 2.1 Verification of the effect of peptides on salty taste enhancement

[0051] A sodium chloride solution with a mass concentration of 0.35% (referred to as 0.35% NaCl solution) and a mixed solution of sodium chloride and monosodium glutamate (sodium chloride: monosodium glutamate = 1:1, m / m) with a mass concentration of 0.35% (referred to as MB solution) were prepared respectively.

[0052] The peptide freeze-dried powder was added to the 0.35% NaCl solution and MB solution respectively to make the concentration 5 mg / mL. The 0.35% NaCl solution without peptide freeze-dried powder was used as the control. The saltiness intensity of each group of solutions was measured. The results are as follows: Figure 2 shown.

[0053] Depend on Figure 2 It can be seen that the saltiness of the 0.35% NaCl solution with the addition of P solution is significantly enhanced compared to the 0.35% NaCl solution without the addition of P solution; the saltiness of the MB solution with the addition of P solution is significantly enhanced. These results all indicate that the polypeptide prepared in Example 1 of the present invention has a good saltiness enhancement effect.

[0054] 2.2 Effect of peptide addition on salty taste enhancement

[0055] Using the 0.35% sodium chloride solution prepared in 2.1 as the matrix, mixed solutions with salty peptide concentrations of 0, 1, 2, 3, 4, 8, and 10 mg / mL were prepared and their saltiness intensity was measured. Figure 3 .

[0056] Depend on Figure 3 It can be seen that in a sodium chloride solution with a mass concentration of 0.35%, as the polypeptide concentration increases, the saltiness intensity increases significantly, and generally shows a trend of first increasing and then decreasing. When the polypeptide concentration is 8 mg / mL, the saltiness intensity reaches the highest point, after which the saltiness intensity decreases and the solution is accompanied by a sour taste, indicating that the amount of polypeptide added is subject to certain restrictions.

[0057] 3. The salt reduction effect of peptides

[0058] A 50 mmol / L NaCl solution is closest to the concentration of broth, so a 50 mmol / L NaCl solution (≈0.29% NaCl solution) was selected as a benchmark. The salt reduction effect was explored by combining NaCl solutions below 50 mmol / L with peptide solutions of different concentrations.

[0059] 3.1 Preparation of standard curve

[0060] Prepare NaCl solutions of different concentrations (0, 15, 30, 45, 60, 75, 90 mmol / L), plot the standard curve with the concentration of the salt solution as the horizontal axis and the saltiness intensity as the vertical axis ( Figure 4 ).

[0061] 3.2 Verification of the effectiveness of peptide salt reduction

[0062] Prepare a 0.2% NaCl solution, add 1, 2, 3, 4, and 5 mg / mL peptide solutions to prepare sodium chloride solutions AE containing different peptide concentrations. Use the NaCl solution without peptide solution as the control (Cont) to measure the saltiness intensity of each solution ( Figure 5 ).

[0063] by Figure 4 The obtained standard curve was used to calculate the saltiness intensity at a NaCl concentration of 0.29%. Comparison with the saltiness intensity of the above-mentioned solutions showed that the saltiness intensity of the mixed solution gradually increased with increasing peptide concentration. When the peptide concentration reached 4 mg / mL, the saltiness intensity of the mixed solution exceeded that of the NaCl solution with a mass concentration of 0.29%. These results show that it is possible to reduce salt without reducing saltiness by replacing part of the sodium chloride with peptides, providing a new salt reduction strategy that meets the public's pursuit of health and deliciousness.

[0064] Example 3 Preparation of Low-Sodium Sauce

[0065] (1) Preparation of polypeptides: Shell the clam meat, wash it, and drain the water. Add ultrapure water at a mass volume ratio of 1kg:3L, homogenize at 22000rpm for 4 times, each time for 30s, filter it with gauze, and then add the fermentation agent described in part 1 of Example 1. Heat it at 35℃ for 24h, filter it with gauze to obtain a crude extract; centrifuge the crude extract (10000rpm, 4℃, 15min), take the supernatant, and filter it through an ultrafiltration membrane (<3000Da) to obtain a crude peptide extract; purify the crude peptide extract by gel exclusion chromatography and reversed-phase high-performance liquid chromatography to obtain a crude peptide; identify the crude peptide by mass spectrometry and then perform synthesis verification to screen and obtain the polypeptide capable of enhancing saltiness in Example 1 of the present invention.

[0066] (2) Preparation of auxiliary ingredients: 2 parts by weight of brewed soy sauce; 0.4 parts by weight of brewed vinegar; 0.8 parts by weight of salt; 0.3 parts by weight of white sugar; 0.5 parts by weight of monosodium glutamate; 0.8 parts by weight of polypeptide; 0.1 parts by weight of concentrated lemon juice; 0.2 parts by weight of potassium sorbate; and 0.5 parts by weight of water. Mix and stir the above auxiliary ingredients evenly, let them stand for 15 minutes, then sterilize, inspect, bag, and seal to prepare the compound sauce.

[0067] The prepared sauce utilizes the characteristics of flavor peptides, reduces the use of sodium chloride during the preparation process, and partially replaces it with peptides, so that the product has the advantages of low sodium, deliciousness and health.

[0068] Example 4 Preparation of Low-Sodium Filling

[0069] (1) Preparation of polypeptides: Shell the clam meat, clean it, and drain the water. Add ultrapure water at a mass volume ratio of 1kg:4L, homogenize the mixture at 21000rpm / min for 30 seconds each time, filter it with gauze, and then add the fermentation agent described in Section 1 of Example 1. Ferment it at 30°C for 48 hours to obtain a crude extract; centrifuge the crude extract (9500rpm / min, 4°C, 15 minutes), take the supernatant, and filter it through an ultrafiltration membrane (<3000Da) to obtain a crude peptide extract; purify the crude peptide extract by gel exclusion chromatography and reversed-phase high-performance liquid chromatography to obtain a crude peptide; identify the crude peptide by mass spectrometry and then perform synthesis verification to screen and obtain the polypeptide capable of enhancing saltiness in Example 1 of the present invention.

[0070] (2) Preparation of auxiliary materials: 40 parts by weight of ground pork; 2 parts by weight of cooking oil; 1 part by weight of brewed soy sauce; 1 part by weight of salt; 0.5 parts by weight of white sugar; 0.5 parts by weight of monosodium glutamate; 0.9 parts by weight of polypeptide; 0.3 parts by weight of pepper; 0.2 parts by weight of concentrated lemon juice; 1 part by weight of scallion and ginger juice. Mix and stir the above auxiliary materials until evenly combined.

[0071] (3) Place the auxiliary materials in the dough and crust, press them into shape through a mold, and then freeze and package them.

[0072] The low-sodium filling prepared utilizes the characteristics of flavor peptides, reduces the use of sodium chloride during the preparation process, and partially replaces it with peptides, so that the product has the advantages of low sodium, deliciousness and health.

[0073] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a polypeptide, characterized in that: The following steps are involved: (1) Preparation of crude peptide extract The clam meat is cleaned, drained and then added into ultrapure water for homogenization; after homogenization, a fermentation agent is inoculated for fermentation, and after fermentation, preliminary filtration is performed to obtain a crude extract; The crude extract was centrifuged, and the supernatant was taken, and the supernatant was filtered through an ultrafiltration membrane to obtain a crude peptide extract; (2) Gel exclusion chromatography purification The crude peptide extract was freeze-dried and dissolved in ultrapure water at a concentration of 0.1 g / mL. The sample was filtered through a 0.22 µm filter membrane, and the eluent was ultrapure water at an elution rate of 0.5 mL / min. Gel exclusion chromatography was performed to purify the components, and the components were collected and freeze-dried. (3) Reversed-phase high performance liquid chromatography separation Each component obtained after gel exclusion chromatography purification was prepared into a 10 mg / mL solution and separated by reverse-phase high-performance liquid chromatography (RP-HPLC). The eluent A was ultrapure water and the eluent B was 100% acetonitrile. The flow rate was 0.4 mL / min and the detection was carried out under 220 nm ultraviolet light. The components were collected and freeze-dried, and then the taste effect was tested to obtain the component with the strongest saltiness. (4) Mass spectrometry identification The component with the strongest saltiness obtained in step (3) is subjected to mass spectrometry identification to obtain the polypeptide; the amino acid sequence of the polypeptide is Asn-Pro-Asp; Wherein, in step (1), the mass volume ratio of the clam meat to the ultrapure water is 1 kg: 3-5 L; The fermentation temperature is 30-35°C and the fermentation time is 24-48h; The centrifugation conditions are 4°C, 8500-10000 rpm, and 15-20 min; The molecular weight cut-off of the ultrafiltration membrane is less than 3000Da; The fermentation agent is prepared by mixing Lactobacillus plantarum and Staphylococcus xylosus, with a strain ratio of 1:1 and an inoculation ratio of 3%.

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