Flavor peptides and their applications

Abstract

The present invention discloses three flavor peptides, whose amino acid sequences are shown in SEQ ID NOs. 1-3. The present invention also discloses compositions containing the same. The present invention also discloses the use of the aforementioned products for enhancing flavor. The present invention synthesizes the three selected flavor peptides and verifies their umami and umami-enhancing effects through sensory evaluation and electronic tongue analysis. Ultimately, it is verified that all three peptides have umami and umami-enhancing effects.

Description

Technical Field

[0001] The present invention relates to three flavor peptides and their preparation. Background Art

[0002] Watermelon bean paste is a well-known condiment in my country with a unique flavor and excellent nutritional value. The traditional production process of watermelon bean paste is similar to that of fermented bean paste, but differs in that a large amount of watermelon juice is added during the fermentation process, providing more carbohydrates and free sugars for microbial fermentation. Therefore, compared with traditional fermented bean paste, the relative abundance of bacterial communities and the content of their metabolites in watermelon bean paste samples are higher. Peptides are one of the most important metabolites in fermented soy products, produced by the action of microbial proteolytic enzymes on proteins during the fermentation process. These peptides are typically composed of 2-20 amino acids and have functions including antioxidant, antihypertensive, antitumor, and antidiabetic properties. In recent years, flavor peptides such as umami in fermented soy products have gradually attracted the attention of researchers.

[0003] Umami, one of the five basic flavors, brings a pleasant feeling and enhances the taste of food. Previous studies have shown that umami can be attributed to free amino acids, organic acids, nucleotides, organic bases, and low-molecular-weight peptides. Summary of the Invention

[0004] The first object of the present invention is to provide three new flavor peptides for watermelon bean paste, whose amino acid sequences are Ala-Lys-Glu-Lys-Phe-Asp, Leu-Ala-Glu-Leu-Lys and Leu-Thr-Phe-Val-Glu-Arg respectively.

[0005] The second object of the present invention is to provide a composition containing the above peptide, such as a seasoning or food.

[0006] The present invention also discloses a method for preparing flavor peptides in watermelon bean paste water extract, comprising the following steps:

[0007] (1) preparing watermelon bean paste into a watermelon bean paste aqueous extract and collecting the supernatant;

[0008] (2) ultrafiltration supernatant to obtain ultrafiltration fraction;

[0009] (3) performing gel chromatography on the ultrafiltration fraction to obtain a gel chromatography fraction;

[0010] (4) Perform LC-MS / MS on the gel chromatography fractions to identify the peptides they may contain and further screen for possible flavor peptides;

[0011] (5) Synthesize and verify the flavor peptides.

[0012] Furthermore, the specific operation of step (1) includes: taking a certain amount of watermelon bean paste and grinding it, adding a certain amount of ultrapure water to make up the volume, shaking and extracting it at a certain temperature for a certain time, centrifuging it at 4000 rpm for 15 minutes, and taking the supernatant.

[0013] Furthermore, the material-liquid ratio of the watermelon bean paste to ultrapure water is 1:10, the extraction temperature is 55° C., and the extraction time is 4 hours.

[0014] The invention also discloses the use of the technical solution for enhancing the freshness of food. Specific implementation methods

[0015] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any way. Example

[0016] The preparation method of three flavor peptides from watermelon bean paste water extracts comprises the following specific steps:

[0017] (1) Preparation of watermelon bean paste aqueous extract: Grind a certain amount of watermelon bean paste, add ultrapure water to the volume at a solid-liquid ratio of 1:10, and extract at 55°C for 4 h. Centrifuge at 4000 rpm for 15 min and collect the supernatant.

[0018] (2) Ultrafiltration separation of supernatant: Use ultrafiltration tubes with a relative molecular weight cutoff of 3kD to centrifuge at 4°C, 4000r / min for 15min, and collect the filtrate. The retained fraction is recorded as Group A (>3kDa), and the filtered fraction is recorded as Group B (<3kDa). Component B is selected for subsequent experiments;

[0019] (3) Gel chromatography separation of ultrafiltration components: Ultrapure water was used to balance the gel chromatography column, and the filler was Sephadex G-15 dextran gel resin. 1.5 mL of the ultrafiltration separation component was loaded onto the column and eluted with deionized water at a flow rate of 0.75 mL / min. The eluate was detected and collected using an ultraviolet spectrophotometer at a wavelength of 220 nm. The eluate was collected every four minutes. The fractions were combined according to the elution curve, and three gel chromatography components F1, F2, and F3 were separated.

[0020] (4) Sensory evaluation and electronic tongue analysis were performed on the gel chromatography fractions, and the fractions with the strongest umami taste were selected for subsequent experiments: Sensory evaluation: The concentration of each gel chromatography fraction in the sensory evaluation was 5% (mass concentration). The samples were evaluated using a scoring method ranging from 0 to 10, with 0 indicating that the sample had no taste and 10 indicating that the sample had a significant taste. The standard substances for sweetness, bitterness, sourness, saltiness, and umami were sucrose (1%), caffeine (0.08%), citric acid (0.08%), sodium chloride (0.35%), and monosodium glutamate (0.35%), respectively. These standards were rated 5 points by the sensory evaluation at this concentration. The taste of the samples that were lower than the standard substances was scored on a scale of 0-5, and the taste of the samples that were higher than the standard substances was scored on a scale of 5-10. Finally, a chart was drawn. The evaluators tasted each sample (approximately 10 ml) and performed a sensory evaluation of each sample's sourness, sweetness, bitterness, saltiness, and umami. After evaluating each sample, they rinsed their mouths and rested for 1 minute before tasting the next sample. The analysis results of the five sensory indicators of umami, saltiness, sweetness, bitterness, and sourness for each component were plotted using a radar chart.

[0021] Electronic Tongue Analysis: The taste profile of gel chromatography samples was determined using an electronic tongue. The nine sensors (richness, aftertaste-A, aftertaste-B, sourness, saltiness, bitterness, astringency, sweetness, and umami) and the electrodes were immersed in a buffer solution for at least 24 hours before use. The electrodes were immersed in a 3.3 mM KCl solution, and the taste sensors were immersed in a reference solution (30 mM KCl and 0.3 mM tartaric acid). The sensors were calibrated for approximately 30 minutes before measurement. The default measurement method was selected. Experiments were performed at room temperature. Each sample was measured four times, and the data from the three replicates were used, with the data from the first cycle discarded.

[0022] Based on the sensory evaluation and electronic tongue analysis results, the F2 component with the strongest umami taste was selected for subsequent identification;

[0023] (5) LC-MS / MS analysis was performed on the gel chromatography fractions to determine the peptides that may be contained therein: The reductive alkylation and desalting of fraction F2 were performed as follows: (1) an appropriate amount of DTT solution was added to the sample to a final concentration of 10 mmol / L, and the sample was reduced in water at 56°C for 1 hour; (the final concentration of the IAM solution was 50 mmol / L, and the reaction was protected from light for 40 minutes; (3) a self-packed desalting column was used for desalting, and the solvent was evaporated in a vacuum centrifugal concentrator at 45°C. The liquid chromatography column was 150 μm id × 150 mm, packed with Acclaim PepMap RPLC C18, 3 μm, 100Å. The mobile phase A was 0.1% formic acid, and the mobile phase B was 0.1% formic acid and 80% acetonitrile. The flow rate was 600 nL / min, and each fraction was analyzed for 66 minutes. The chromatographic gradient was as follows: 0-2 minutes, 4-8% B; 2-45 minutes, 8-28% B; 45-55 min, 28-40% B; 55-56 min, 40-95% B; 56-66 min, 95% B. The mass spectrometer was operated in data-dependent acquisition mode and automatically switched between MS and MS / MS modes. The parameters were set as follows: (1) MS: resolution: 70,000; automatic gain control (AGC) target: 3e6; maximum injection time: 100 ms; scan range (m / z): 300-1800; (2) MS / MS: resolution: 17,500; AGC target = 1e5; maximum injection time = 50 ms; NCE / stepped NCE: 28.

[0024] Parse peptide sequences from raw mass spectrometry files.

[0025] (6) The possible peptides identified by LC-MS / MS were further screened to determine the final flavor peptides. The peptides in component F2 with the strongest umami taste were identified by LC-MS / MS, and 2447 possible peptides were identified. Peptides with ALC greater than 95% were selected and further screened to screen out peptides with 4 to 6 amino acids, leaving 39 peptides that met all the above requirements. The 39 peptides were then docked with the homology model of T1R1 / T1R3 and ranked by docking energy. Solubility and flavor predictions were performed on 36 of these peptides, and three possible flavor peptides were selected (AKEKFD, LAELK, and LTFVER, numbered as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 in the sequence list);

[0026] (7) Three possible flavor peptides were synthesized and their umami taste was verified by sensory evaluation: the synthetic peptide solution (1 mg / ml, 10 ml) was gradually diluted with ultrapure water in a ratio of 1:1, and the samples at each dilution level were measured using the 3-point method. When the panelists could only distinguish between the previous sample and the blank control (ultrapure water), but could not distinguish between the next level of sample and the blank control, the average of the two levels was the umami threshold. The umami thresholds of the three synthetic peptides are shown in the table below:

[0027] Table 1

[0028] Flavor peptides Basic taste Umami threshold (mg / mL) AKEKFD Sour, sweet, fresh 0.106 LAELK Sour, fresh 0.106 LTFVER fresh 0.375

[0029] It can be seen from the table that all three peptides have umami taste and are new flavor peptides.

Claims

1. The polypeptide represented by SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO.

3.

2. Use of the polypeptide according to claim 1 for increasing the umami taste of food.

3. A composition comprising the polypeptide according to claim 1, characterized in that: The composition is a seasoning.