Stropharia rugoso-annulata umami peptide as well as screening method and application thereof

Abstract

The invention belongs to the field of mushroom active peptides. The invention provides a Stropharia rugoso-annulata umami peptide as well as a screening method and application thereof. The amino acid sequence of the Stropharia rugoso-annulata umami peptide is KAELDLH and / or LGKSEDDVSK. The freshness threshold value of the stropharia rugoso-annulata freshness peptide ranges from 0.15 mM to 0.23 mM, and the stropharia rugoso-annulata freshness peptide has high freshness activity, is small in molecular weight, capable of being more easily perceived by human body sensory taste bud receptors, safe and non-toxic, can serve as a preparation raw material of seasonings and plant meat and has the freshness increasing effect when cooperating with MSG. The invention provides an effective and practical method for further deep excavation of the Stropharia rugoso-annulata umami peptide, provides a new umami peptide raw material for development of seasonings, and has important application value.

Description

Technical Field

[0001] The invention belongs to the field of mushroom active peptides, in particular to Stropharia rugosa umami peptides and a screening method and application thereof. Background Art

[0002] Umami is one of the five basic tastes and an important taste that produces a sense of pleasure. It has a very important impact on improving people's physical and mental health. In 1908, Japanese scholar Kikunae Ikeda first isolated glutamate from kelp and proposed the concept of umami. But it was not until nearly 100 years later that it was officially recognized as the fifth basic taste and described as the taste of monosodium glutamate. Studies have found that umami substances intensify the taste of saltiness and sweetness, balance bitterness and sourness, and help improve the overall palatability of certain foods. At the same time, umami substances can be used as a substitute for salt to reduce the risk of hypertension and cardiovascular disease in adults by reducing dietary sodium intake. Currently known umami substances can be divided into two categories: monosodium glutamate and umami peptides. Monosodium glutamate includes L-glutamic acid, organic acids and nucleotides. Despite commercial applications, their long-term use is controversial for health.

[0003] There are many methods for extracting umami peptides, among which chemical hydrolysis and bio-enzyme hydrolysis are the main extraction methods in recent years. Compared with chemical hydrolysis, bio-enzyme hydrolysis has mild conditions, high efficiency, good selectivity, and the amino acids produced will not be destroyed during the whole process. It is suitable for the food industry and has a wider application. Because different enzymes have different cleavage sites and selectivity specificities, the selection of enzymes is the key to producing umami substances. Small peptides containing Glu and Asp at the end are the main flavor substances. Therefore, selecting enzymes with good selectivity for Glu or Asp is likely to produce more umami substances. The enzymes currently used to obtain umami substances include acid proteases, neutral proteases, alkaline proteases, and flavor proteases. When it is difficult to achieve the desired effect with only one enzyme, two or more enzymes can be used in synergy. However, it is worth noting that if the degree of enzymatic hydrolysis of the protein is low, many peptides containing hydrophobic amino acids will be produced, resulting in an unpleasant bitter taste.

[0004] Stropharia rugosoannulata, also known as "red pine mushroom", is a precious edible mushroom recommended by the Food and Agriculture Organization of the United Nations (FAO) for cultivation in developing countries. Stropharia rugosoannulata is a good source of protein, fiber, amino acids, mineral elements, polysaccharides, phenols and a variety of bioactive substances. So far, there is little in-depth research on Stropharia rugosoannulata umami peptides, and there is also a lack of an efficient and directional enzymatic hydrolysis technology to prepare Stropharia rugosoannulata umami peptides.

[0005] Therefore, further research and development of Stropharia flavored products is still necessary. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a stropharia cap mushroom umami peptide and a screening method and application thereof. The stropharia cap mushroom umami peptide has strong umami activity, salty taste and rich taste characteristics. The umami threshold of the stropharia cap mushroom umami peptide provided by the present invention is between 0.15-0.23mM, the umami intensity of KAELDLH is 7.80, and the umami intensity of LGKSEDDVSK is 6.24, both of which are higher than the umami value of 4.93 of 0.05% MSG solution. At the same time, the stropharia cap mushroom umami peptide has a small molecular weight, is easier to be perceived by human sensory taste bud receptors, is safe and non-toxic, and can be used as a raw material for preparing condiments.

[0007] The object of the present invention is to provide a stropharia cap mushroom umami peptide, the amino acid sequence of the stropharia cap mushroom umami peptide is KAELDLH and / or LGKSEDDVSK.

[0008] The present invention also aims to provide a method for screening umami peptides of Pleurotus eryngii, comprising the following steps:

[0009] (1) Directed enzymatic hydrolysis: dried Stropharia officinalis fruiting body powder is added with deionized water, and alkaline protease and DNPEP are added for step-by-step enzymatic hydrolysis to obtain Stropharia officinalis enzymatic hydrolyzate;

[0010] (2) The enzymatic hydrolysate of Psoralea corylifolia was ultrafiltrated to obtain components with a molecular weight less than 3KD, and the components were separated by gel chromatography. The fractions were collected and the fractions with the strongest umami taste were selected. The peptides were identified by LC-MS / MS. The BIOPEP-UWM sensory peptide and amino acid module tool was used to predict the taste of the peptides identified by LC-MS / MS to obtain Psoralea corylifolia umami peptides.

[0011] Furthermore, the conditions for the above-mentioned directional enzymatic hydrolysis are: firstly, alkaline protease is added for enzymatic hydrolysis for 50-65 minutes, then DNPEP is added for further enzymatic hydrolysis for 75-90 minutes, and then centrifuged to obtain the Stropharia rugosa enzymatic hydrolysate.

[0012] The dosage ratio of the dried Stropharia fruiting body powder to deionized water is 1g:10-30mL, the dosage of alkaline protease is 0.4-0.6% of the Stropharia fruiting body powder, and the dosage of DNPEP is 0.25-0.35% of the Stropharia fruiting body powder.

[0013] The present invention also provides application of the Stropharia rugosa umami peptide in improving the umami taste of food.

[0014] The present invention also provides the use of the Stropharia rugosa umami peptide in the preparation of condiments and / or vegetable meat.

[0015] The invention also provides a condiment, wherein the umami component of the condiment comprises the umami peptide of Pleurotus eryngii.

[0016] The invention also provides application of the seasoning in providing fresh taste of food.

[0017] The invention recombined and expressed a new aspartate aminopeptidase DNPEP, and used the enzyme in combination with other proteases to enzymolyze Pleurotus eryngii, and proved that the optimal conditions for directional enzymolysis were a temperature of 55°C, a solid-liquid ratio of 1:20 (g / mL), alkaline protease enzymolysis (60min, 0.5%, pH 9.0) and DNPEP enzymolysis (80min, 0.3%, pH 8.0). The umami peptides in the enzymolysis solution were separated and purified by ultrafiltration (UF) and gel filtration chromatography (GFC). The purified peptide structure was identified by nanoliter liquid chromatography-quadrupole orbital trap mass spectrometer (Easy nLC1200 / Q Exactive plus). The components were analyzed by sensory evaluation combined with electronic tongue determination to explain their umami characteristics and umami enhancement effects.

[0018] The present invention has the beneficial effects:

[0019] The present invention proposes a new directional enzymatic hydrolysis strategy, prepares the stropharia umami peptide by directional enzymatic hydrolysis, separates by ultrafiltration and gel chromatography, and uses LC-MS / MS combined with a database to identify and screen the umami peptide, and provides the role of the umami peptide in enhancing the freshness, and studies the structure and formation mechanism of its umami. The structures of the umami peptides of the present invention are KAELDLH and LGKSEDDVSK, and the molecular weights are 824.44 and 1076.53Da, respectively. Sensory evaluation shows that the two polypeptides have rich taste characteristics, the umami thresholds are 0.15 and 0.23mM, respectively, and have strong umami activity. The umami intensity of KAELDLH is 7.80, and the umami intensity of LGKSEDDVSK is 6.24, both of which are higher than the umami value of 4.93 of 0.05% MSG solution. It belongs to a small molecular weight and is more easily perceived by the human sensory taste bud receptors. The stropharia umami peptide of the present invention has a synergistic effect of enhancing the freshness with MSG, and can be used as a raw material for preparing condiments and plant meat.

[0020] The invention provides an effective and practical method for further exploring the umami peptides of Pleurotus eryngii, provides a new umami peptide raw material for the development of condiments, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of AKTA protein purification test for DNPEP enzyme;

[0022] Figure 2 Verify protein molecular weight and purification effect by SDS-PAGE;

[0023] Figure 3 The results of the DNPEP enzyme selectivity experiment are as follows;

[0024] Figure 4 The results of the umami value determination of Stropharia rugosa umami peptides under different enzymatic hydrolysis conditions;

[0025] Figure 5 It is the synergistic flavor-enhancing effect of umami peptides and MSG. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1 Expression, purification and activity determination of recombinant DNPEP enzyme

[0028] The aminopeptidase gene (NCBI: NM_001012919) was connected to the vector Pet15b and introduced into Escherichia coli BL21 (DE3) cells. The recombinant Escherichia coli BL21 (DE3) cells were cultured in LB medium containing 50 mg / mL ampicillin at 37°C, 220 rpm, and the cells were cultured to an OD value of 600 (0.8-1.0). IPTG was added to induce the culture for 12 hours at 25°C. The cells collected by centrifugation were frozen overnight, thawed, and then ultrasonically decomposed. The cell lysate was treated with a HisTrap HP affinity column, and DNPEP was eluted with an imidazole solution, pH 8.0. The results of AKTA purification of DNPEP enzyme are shown in Figure 2. Figure 1 As shown, two peaks can be seen in the figure, the left peak is 200mM imidazole eluent, and the right peak is 500mM imidazole eluent, and the proteins in the two peaks are collected.

[0029] The molecular weight of the enzyme was obtained by 8% SDS-PAGE analysis, and the enzyme was concentrated by centrifugation at 4800×g and 4°C for 20 min using Amicon Ullra (Millipore). The concentration of the target protein was determined using a BCA protein assay kit (Shanghai Biyuntian Biotechnology Co., Ltd.). SDS-PAGE was used to verify the molecular weight of the protein and the purification effect. Figure 2As shown. M is a marker, and the molecular weight of DNPEP is about 52kDa. Lanes 1-3 are 200mM imidazole eluents (AKTA left peak), and lanes 4-8 are 500mM imidazole eluents (AKTA right peak). Lanes 4-8 are DNPEP collected in the experiment, and the concentrations are different, but it can be seen that the band size is basically consistent with the target molecular weight and the target protein expression is high, with few foreign proteins. The protein eluents of the left peaks of lanes 1-3 have more protein bands, indicating that the target protein amount is very small, and other protein types are many. It is also shown that the imidazole elution concentration of the target protein is 500mM, and the DNPEP required for subsequent experiments is directly eluted using 500mM imidazole.

[0030] The aminopeptidase activity was determined by the hydrolysis of the substrate Glu-pNA into p-nitroaniline and glutamate by DNPEP. The reaction was completed within 10 min at 50 °C. The OD value was measured at 405 nm using a Multiskan Sky microplate spectrophotometer (Thermo Scientific) to determine the initial absorbance of p-nitroaniline. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of p-nitroaniline from glutamate per minute.

[0031] The enzyme molecule reacts with the substrate L-glutamic acid p-nitroaniline (L-Glu-pNA). The substrate is hydrolyzed to release p-nitroaniline, making the solution appear yellow. The depth of the color is proportional to the enzyme activity.

[0032] In enzyme selectivity experiments, such as Figure 3 As shown, serial number 1 represents the control, serial numbers 2, 3, and 4 represent the enzyme activity of aspartate aminopeptidase at an enzyme concentration of 3 mg / ml. Serial numbers 2, 3, and 4 are three parallels. At the end of the reaction, it can be clearly seen that the color of the solution changes from colorless to yellow, as shown in FIG. Figure 3 Tubes 2, 3, and 4 are shown. This indicates that the expressed DNPEP has good selectivity for the glutamic acid site. The enzyme activity of DNPEP was determined using an enzyme reader using the change in the absorbance value of p-nitroaniline, the product of glutamic acid p-nitroaniline, at 405 nm. The results are shown in Table 1 below.

[0033] Table 1 Enzyme activity of DNPEP against L-glutamic acid p-nitroaniline

[0034]

[0035] Example 2 Optimization of the directional enzymatic hydrolysis process of Stropharia rugosotis

[0036] 1. Single enzyme hydrolysis

[0037] In order to screen proteases with high enzymatic hydrolysis efficiency, alkaline protease, DNPEP, neutral protease, flavor protease, papain and cellulase were selected to enzymatically hydrolyze the fruiting body powder of Pleurotus eryngii. Take the dried fruiting body powder of Pleurotus eryngii (the fruiting body of Pleurotus eryngii comes from the Edible Fungi Research Institute of Shanghai Academy of Agricultural Sciences) and add deionized water. Under the respective optimum temperature and pH conditions, the optimum temperature, pH and enzyme type of the enzyme are shown in Table 2. Alkaline protease, neutral protease, cellulase, flavor protease, papain and aspartic acid aminopeptidase were used for single enzyme hydrolysis. The umami value of the enzymatic hydrolyzate was used as the screening index to compare the enzymatic hydrolysis and freshness enhancement effects of these six enzymes.

[0038] Table 2 Enzymatic hydrolysis characteristics of six proteases

[0039]

[0040] Weigh an equal amount of each component sample and dissolve it, then measure the electronic tongue and conduct sensory evaluation.

[0041] 2. Electronic tongue measurement method

[0042] The taste of the enzymatic supernatant was determined using the INSENT SA-402B taste analysis system. The device contains six sensors for bitterness, astringency, sweetness, saltiness, umami and sourness. The sensors were activated and self-checked, and the sample was tested after the values ​​stabilized within the specified range. During the test, the sample was diluted to the same multiple and added to the sample test cup. Each cup contained about 35 mL of sample and tested at room temperature. Each sample was tested four times for 2 minutes, and the relevant data were analyzed using software. The obtained electronic tongue umami intensity is shown in Table 3. The enzymatic hydrolysis of the six proteases on Stropharia rugosa was statistically significant (P<0.05). From the results of Table 3, it can be seen that the order of umami intensity from high to low is: DNPEP>alkaline protease>neutral protease>flavor protease>papain>cellulase. Therefore, alkaline protease and DNPEP were selected for enzymatic hydrolysis.

[0043] 3. Selection of synergistic enzymatic hydrolysis method

[0044] Take the powder of Stropharia officinalis and add it to deionized water, select alkaline protease and DNPEP for synergistic enzymolysis, and determine the synergistic enzymolysis method. Use the following two enzymolysis methods to perform enzymolysis and compare the umami intensity of the enzymolysis products.

[0045] Step-by-step enzymatic hydrolysis: first add 0.5% alkaline protease to react for 60 minutes, inactivate the enzyme in boiling water for 10 minutes, then add 0.5% DNPEP to continue enzymatic hydrolysis for 60 minutes, inactivate the enzyme in boiling water for 10 minutes, cool to room temperature and centrifuge, and retain the supernatant.

[0046] Simultaneous enzymatic hydrolysis: 0.5% alkaline protease and 0.5% DNPEP were added to the substrate at the same time, enzymatic hydrolysis was carried out for 60 minutes, the enzyme was inactivated by boiling water for 10 minutes, and the supernatant was retained after cooling to room temperature. The enzymatic hydrolysis temperature and pH were both the optimal temperature and pH of the enzyme, and the umami intensity of the supernatant was measured using an electronic tongue.

[0047] Compared with simultaneous enzymatic hydrolysis, the umami intensity of step-by-step enzymatic hydrolysis was 17.47, which was 15.93% higher than the umami intensity of 15.07 of simultaneous enzymatic hydrolysis ((17.47-15.07) / 15.07=15.93), showing a softer taste attribute, as shown in Table 3.

[0048] Table 3 Electronic tongue test results of different Stropharia officinalis fruiting body powder enzymatic hydrolysates

[0049]

[0050] 4. Optimization of step-by-step enzymatic hydrolysis conditions

[0051] According to the enzymatic hydrolysis process, other factors were fixed respectively, and the umami intensity of the enzymatic hydrolysate under the following conditions was measured using an electronic tongue: material-liquid ratio (1:15, 1:20, 1:25, 1:30, 1:35, 1:40, g / mL), enzymatic hydrolysis temperature (35°C, 40°C, 45°C, 50°C, 55°C, 60°C), enzymatic hydrolysis time (20min, 40min, 60min, 80min, 100min, 120min), alkaline protease addition amount (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% relative to the amount of Stropharia fruiting body powder), DNPEP addition amount (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% relative to the amount of Stropharia fruiting body powder). Figure 4 The results of umami value determination under different enzymatic hydrolysis conditions show that, Figure 4 As shown in Figure b, when the solid-liquid ratio is 1:20, the umami intensity reaches a maximum value of 15.78. As the solid-liquid ratio continues to decrease, the degree of enzymatic hydrolysis also decreases. This may be due to the decrease in substrate concentration, which leads to a decrease in the frequency of contact between the enzyme and the substrate. Figure 4 As shown in a, the umami intensity increases with the increase of enzyme dosage. When the dosage of alkaline protease is 0.5%, the umami intensity reaches the maximum value of 14.71, and the binding of enzyme and substrate becomes saturated. Therefore, further increasing the enzyme dosage does not promote enzymolysis. Figure 4 As shown in Figure c, the umami intensity reaches its maximum value at 50 min, and gradually decreases with time. This may be due to excessive enzymatic hydrolysis of alkaline protease, which exposes more bitter amino acids and reduces the umami intensity of the solution. Figure 4As shown in Figure d, within the optimal temperature range of enzymatic hydrolysis, the umami intensity gradually increased with the increase of temperature and reached the maximum value at 55°C.

[0052] Therefore, based on the above optimal alkaline protease conditions, the effects of DNPEP concentration and enzymatic hydrolysis time on the umami intensity of Pleurotus eryngii were further explored. Figure 4 As shown in Figure f, when 0.5% alkaline protease was added, the amount of DNPEP added gradually increased from 0.1% to 0.6%, and the umami intensity first increased and then decreased. When the amount of DNPEP added was 0.3%, the umami intensity reached a maximum value of 15.54, and the flavor characteristics were good. Figure 4 As shown in Figure e, after alkaline protease hydrolysis for 60 minutes, DNPEP was added to continue hydrolysis for 20-120 minutes. The umami intensity reached a maximum value of 15.22 when DNPEP hydrolysis was performed for 80 minutes.

[0053] 5. Response Surface Optimization

[0054] Based on the results of the single factor experiment, three factors, namely, the material-liquid ratio (A), the amount of alkaline protease added (B), and the amount of DNPEP added (C), were selected as independent variables, and the umami intensity was taken as the response value (Y). According to the Box-Behnken principle, a response surface optimization experiment was designed, as shown in Table 4.

[0055] Table 4 Response surface optimization experiment

[0056]

[0057] The optimal enzymatic hydrolysis conditions were determined by Box Behnken response surface design optimization: temperature 55°C, solid-liquid ratio 1:20 (g / mL), alkaline protease (0.5%) hydrolysis for 60 min, hydrolysis pH 9.0, DNPEP (0.3%) hydrolysis for 80 min, hydrolysis pH 8.0. The results were consistent with the results of the single factor test.

[0058] Example 3 Screening of Stropharia rugosa umami peptides

[0059] 1. Preparation of Stropharia rugosa enzymatic hydrolysate

[0060] Take the giant puffball powder and add deionized water, select alkaline protease and DNPEP for step-by-step enzymolysis, the enzymolysis temperature is 55°C, the solid-liquid ratio is 1:20 (g / mL), first add 0.5% alkaline protease for enzymolysis for 60 minutes (pH 9.0), boil water to inactivate the enzyme for 10 minutes, then add 0.3% DNPEP to continue enzymolysis for 80 minutes (pH 8.0), after the reaction is completed, boil in 100°C water bath for 10 minutes, cool to room temperature, centrifuge at 8000rpm for 10 minutes, collect the supernatant for use.

[0061] 2. Ultrafiltration and gel chromatography separation of Stropharia rugosa hydrolysate

[0062] Small peptides with molecular weights below 3KD have a strong umami taste. The enzymatic supernatant collected was filtered with a 0.45μm water filter membrane to remove impurities, and a tangential flow membrane filtration system equipped with a 3KD ultrafiltration tube was used for interception (25°C, △P=0.50bar, 300rpm). Two components, U1 (>3KD) and U2 (<3KD), were separated and freeze-dried for later use. The freeze-dried components were dissolved in deionized water to form a solution with a concentration of 1mg / mL, and the taste of U-1 and U-2 was measured using an electronic tongue.

[0063] Take the lyophilized component of U-2, add deionized water to make a 50 mg / mL solution, and then take 2 mL of the prepared solution and load it onto a Sephadex G-15 gel filtration column at room temperature. Before loading, the sample was filtered through a 0.22 μm microporous filter membrane, the flow rate was set to 0.75 mL / min, and deionized water was used as the eluent. The detection of ultraviolet absorbance was set to 220 nm, and four fractions F1, F2, F3 and F4 were collected and freeze-dried. The dried fraction was dissolved in deionized water to make a solution with a concentration of 1 mg / mL, and the electronic tongue was used to measure it in the same way as above, and freeze-dried for sensory taste evaluation. After ultrafiltration and gel chromatography separation of the giant puffball hydrolyzate, the component with the strongest umami taste was selected for peptide identification by nanoliter liquid chromatography-quadrupole orbital trap mass spectrometry.

[0064] 3. Nano-liquid chromatography-quadrupole orbital trap mass spectrometry to identify peptide sequences

[0065] The peptide sequence analysis of the component with the highest umami intensity separated in the previous step was performed using a nano-liquid chromatography-quadrupole orbital trap mass spectrometer (Easy nLC1200 / Q Exactive plus). Sample processing: Desalt the component to be measured using a ZipTip C18 column, dissolve the sample with 0.1% trifluoroacetic acid, and collect the supernatant after high-speed centrifugation for 20 minutes. Take 10 μL of eluent to elute the sample into a centrifuge tube, vacuum dry it and set aside. Chromatographic analysis: The freeze-dried sample is dissolved in deionized water. Then put it into a liquid phase bottle for analysis.

[0066] The sample was passed through a nanoViper C18 analytical column (75 μm × 150 mm, Acclaim PepMap RSLC C18, 2 μm, )C18 column (5cm×2.1mm, 1.7μm) was injected through an automatic sampler. Gradient elution was performed for 60min using mobile phase A (0.1% formic acid in water) and mobile phase B (80% acetonitrile, 0.1% formic acid). The gradient elution conditions were as follows: 0min, 2% B; 0-3min, 6% B; 3-42min, 20% B; 42-47min, 35% B; 47-48min, 100% B; 48-60min, 100% B. The column temperature was 45°C and the flow rate was 300nL / min. The injection volume was 8μL. The ionization mode was ESI+, the mass scan range was 200-2000m / z, and the scan time was 1s. Peaks 10.5 software was used for database retrieval.

[0067] Mass spectrometry conditions: The sample was dissolved in a mixture of 0.1% formic acid and 2% acetonitrile, centrifuged at high speed for 25 min at low temperature, and the supernatant was collected and detected by mass spectrometer. The specific parameters are as follows: (1) Primary mass spectrometry parameters: Resolution: 70,000 AGC target: 3e6 Maximum IT: 50 ms Scanrange: 350 to 1800 m / z; (2) Secondary mass spectrometry parameters: Resolution: 17,500 AGC target: 1e5 Maximum ITzz: 45 ms Scanrange: 200 to 2000 m / z TopN: 20 NCE / stepped NCE: 28

[0068] The peptide amino acid sequence was obtained by manual De novo analysis of the secondary mass spectrometry data, and the relative molecular mass error of the obtained peptide sequence was ±0.02Da.

[0069] 4. Virtual prediction of peptide taste characteristics

[0070] Using the BIOPEP-UWM sensory peptide and amino acid module tool (https: / / biochemia.UWM.edu.pl / bioprep-UWM), among the identified peptides, two peptide fragments with high iUmami-UWM umami prediction scores and BIOPEP-UWM umami prediction scores, KAELDLH and LGKSEDDVSK, were selected.

[0071] Example 4 Solid phase synthesis, taste description and threshold analysis of two peptide fragments

[0072] 1. Solid phase synthesis of peptides

[0073] The target peptides screened by BIOPEP-UWM were synthesized by Hefei Guopeptide Biotechnology Co., Ltd. using solid phase synthesis and desalting to obtain peptides with a purity of more than 98%. After synthesis, sensory evaluation and electronic tongue analysis were performed on their taste characteristics.

[0074] 2. Taste evaluation and threshold analysis of synthetic peptides

[0075] The electronic tongue results of the synthetic peptides are shown in Table 5. The taste characteristics of the two synthetic peptides are similar, among which the umami characteristics are prominent, followed by salty, sweet and bitter. The taste of sour is the weakest of the five basic tastes. Among the two peptides, KAELDLH has the highest umami intensity of 7.80, followed by LGKSEDDVSK, with an umami intensity of 6.24, both of which are higher than the umami value of 4.93 of 0.05% MSG solution. Similarly, KAELDLH has the highest saltiness score of 0.53, and LGKSEDDVSK has the highest sweetness score of 1.57, followed by KAELDLH, with a sweetness score of 0.04. In addition, both synthetic peptides show a certain sour taste, which may be because the dissociation strength of the carboxyl group in the peptide chain is greater than that of the amino group, and the solution is acidic.

[0076] Table 5 Electronic tongue analysis results of synthetic peptides

[0077]

[0078] The synthesized peptides were dissolved in ultrapure water to prepare a 1 mg / mL solution and the pH was adjusted to 6.5 for sensory evaluation of the synthesized peptides. The threshold of umami peptides was determined by the taste dilution method. The synthetic peptide solution with an initial mass concentration of 1 mg / mL was gradually diluted 1:1 (v:v), and the maximum dilution concentration that the evaluators could accurately evaluate was used as the threshold for peptide recognition.

[0079] According to the threshold analysis, the results are shown in Table 6. The umami thresholds of KAELDLH and LGKSEDDVSK were 0.15 and 0.23 mM, respectively, showing strong umami activity.

[0080] Table 6 Sensory evaluation and threshold analysis of two synthetic peptides

[0081]

[0082]

[0083] 3. Synergistic effect of synthetic peptides and MSG

[0084] In order to understand the synergistic effect of the two synthetic peptides and monosodium glutamate solution, 0.05 mg / mL synthetic peptide solution was prepared and added to 0.05 mg / mL monosodium glutamate (MSG) solution, mixed evenly, and the umami intensity was measured using an electronic tongue, and compared with the umami values ​​of 0.05 mg / mL, 0.1 mg / mL MSG solution and 0.05 mg / mL synthetic peptide solution. Figure 5 It can be seen that after adding 0.05mg / mL synthetic peptide solution to 0.05mg / mL MSG solution, the umami intensity of the solution increased to varying degrees. Compared with 0.05mg / mL synthetic peptide, KAELDLH has the strongest umami enhancement ability, and the umami value increased by 98.3% after adding MSG. The second is LGKSEDDVSK, with an umami enhancement degree of 39.76%. The above experimental results show that umami peptides and MSG have a certain degree of synergistic umami enhancement ability, and the mixture of the two can increase the umami intensity.

[0085] In summary, the umami intensity of the umami peptide KAELDLH of the present invention is 7.80, and the saltiness score is 0.53, the umami intensity of LGKSEDDVSK is 6.24, and the umami thresholds of KAELDLH and LGKSEDDVSK are 0.15 and 0.23 mM, respectively. The umami peptide of the present invention has strong umami activity and rich taste characteristics. At the same time, the molecular weights of KAELDLH and LGKSEDDVSK are 824.44 and 1076.53, respectively, which are small molecular weights and are more easily perceived by human sensory taste bud receptors. Umami peptides and monosodium glutamate have a certain degree of synergistic freshness-enhancing ability. The mixture of the two can increase the umami intensity and can be used in the preparation of condiments, etc.

[0086] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Those skilled in the relevant technical field may make various changes or modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also belong to the scope of the present invention and should be defined by the claims.

Claims

1. A stropharia flavor peptide, characterized in that: Its amino acid sequence is KAELDLH and / or LGKSEDDVSK.

2. Use of the Stropharia rugosa umami peptide according to claim 1 in improving the umami taste of food.

3. Use of the Stropharia rugosa umami peptide according to claim 1 in the preparation of condiments and / or vegetable meat.

4. A seasoning, characterized in that: The umami component of the seasoning includes the Stropharia officinalis umami peptide according to claim 1.

5. Use of the seasoning according to claim 4 in providing umami taste to food.

6. A method for screening umami peptides from Pleurotus eryngii as claimed in claim 1, characterized in that: The following steps are included: (1) Directed enzymatic hydrolysis: dried Stropharia officinalis fruiting body powder is added with deionized water, and alkaline protease and DNPEP are added for step-by-step enzymatic hydrolysis to obtain Stropharia officinalis enzymatic hydrolyzate; (2) The enzymatic hydrolysate of Psoralea corylifolia was ultrafiltrated to obtain components with a molecular weight less than 3KD, and the components were separated by gel chromatography. The fractions were collected and the fractions with the strongest umami taste were selected. The peptides were identified by LC-MS / MS. The BIOPEP-UWM sensory peptide and amino acid module tool was used to predict the taste of the peptides identified by LC-MS / MS to obtain Psoralea corylifolia umami peptides.

7. The method according to claim 6, characterized in that The conditions of the directional enzymatic hydrolysis are: firstly, alkaline protease is added to hydrolyze for 50-65 minutes, then DNPEP is added to continue the enzymatic hydrolysis for 75-90 minutes, and centrifugation is performed to obtain the Stropharia rugosa hydrolyzate.

8. The method according to claim 6, characterized in that The dosage ratio of the dried Stropharia capillaris fruiting body powder to deionized water is 1g:10-30mL, the dosage of alkaline protease is 0.4%-0.6% relative to the dosage of the Stropharia capillaris fruiting body powder, and the dosage of DNPEP is 0.25%-0.35% relative to the dosage of the Stropharia capillaris fruiting body powder.

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