CN111153959A - Hypsizygus marmoreus flavor-developing peptide and preparation method and application thereof - Google Patents
Hypsizygus marmoreus flavor-developing peptide and preparation method and application thereof Download PDFInfo
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
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Abstract
The invention provides a white beech mushroom flavor-developing peptide and a preparation method and application thereof. The amino acid sequence is shown in SEQ ID NO. 1. The invention also provides a preparation method of the flavor peptide, which comprises the steps of firstly obtaining water-soluble components of white beech mushroom through compound enzyme enzymolysis; filtering, centrifuging, and ultrafiltering to cut off components with molecular weight of 200Da and 3000 Da; further separating and purifying the membrane separation component to obtain the amino acid sequence SEQ ID NO.1 of the Hypsizygus marmoreus taste peptide. The invention also provides application of the Hypsizygus marmoreus taste-presenting peptide in food. The invention also provides a food seasoning containing the flavor-developing peptide. The white beech mushroom flavor peptide provided by the invention is used as one of flavor development substances, and can be applied to the field of food. The white beech mushroom Maillard reaction peptide with flavor enhancement effect can be obtained after the white beech mushroom flavor development peptide prepared by the embodiment is subjected to Maillard reaction.
Description
Technical Field
The invention belongs to the field of dietetics, relates to a seasoning, and particularly relates to a white beech mushroom flavor-developing peptide.
Background
White beech mushroom (Hypsizygus marmoreus) belonging to Basidiomycotina, Agaricaceae, Agaricales, Lyophyllaceae, Hypsizygus, is an excellent edible fungus with white color, hemispherical shape, tender meat, and high yield. White beech mushroom is rich in polysaccharides, vitamins and amino acids. Research shows that the white beech mushroom contains a novel glycoprotein capable of inhibiting human leukemia, and the white beech mushroom can generate active substances for inhibiting hypertension and gout in a water extraction environment. Therefore, the development of the deep processing industry of the white beech mushroom has certain development prospect.
Fresh mushrooms are thick, hard to touch, light and high in moisture content. The research on white beech mushroom is mainly focused on the cultivation and the medicine aspect, and the research on flavor peptide is rarely reported. However, practical tests show that the dried white beech mushroom contains more protein than other varieties, so that the extraction of the flavor peptide from the white beech mushroom is feasible.
Food taste research scientists have found that the taste of foods is diverse in exploring the principles of food taste presentation. These flavors are first stimulated and sensed by taste sensitive receptors on the various taste buds and then transmitted to the human brain nerve center via specific conduction pathways. Umami taste is a taste that is one of the other five basic tastes and was first discovered and named by japanese scientists. Besides having a certain taste, the Umami peptide also can increase the overall thickness of food, and is called Umami peptide in English. The presence of umami peptide in combination with salt or sodium glutamate can significantly reduce the salt and sodium glutamate threshold, which can also be referred to as a taste enhancer. The delicious peptide is added into the chicken soup, so that the taste characteristics of the chicken soup can be enriched, and the chicken soup is more delicious and rich.
Disclosure of Invention
The invention aims to provide a white beech mushroom flavor peptide, a preparation method and an application thereof.
In order to achieve the purpose, the invention provides a white beech mushroom flavor peptide which is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
Preferably, the Hypsizygus marmoreus taste peptide is derived from Hypsizygus marmoreus extract.
Preferably, the Hypsizygus marmoreus taste peptide is artificially synthesized.
The invention also provides a preparation method of the pleurotus cornucopiae flavor-developing peptide, which is characterized by comprising the following steps of:
step 1: extracting water soluble components from white beech mushroom by using a method of compound enzyme enzymolysis;
step 2: intercepting components with molecular weight of 200Da to 3000Da by ultrafiltration membrane separation method;
and step 3: the obtained ultrafiltration component is subjected to chromatography and high performance liquid separation and purification to obtain the white beech mushroom flavor peptide with the amino acid sequence shown as SEQID NO. 1.
Preferably, the step 1 specifically includes: cleaning white beech mushroom, cutting, adding water, inactivating enzyme at water bath temperature above 80 deg.C, adjusting pH to 3.0-4.0, adding cellulase for enzymolysis for 1-3 hr, adjusting pH to 4.0-6.0, adding flavor protease for enzymolysis for 1-3 hr, filtering, centrifuging, and collecting supernatant.
Preferably, the step 3 specifically includes: and (3) after the ultrafiltration component is subjected to chromatography, selecting the component with the strongest thickening effect through artificial sensory evaluation, separating and purifying the component with the strongest thickening effect, and finally performing structural identification on the separated component.
More preferably, the chromatography adopts a Sephadex G-15 gel chromatography method, the separation and purification adopts an RP-HPLC method, and the structural identification is carried out by UPLC-Q-TOF-MS.
The invention also provides application of the Hypsizygus marmoreus taste-presenting peptide in food.
Preferably, the food product is a seasoning.
The invention also provides white beech mushroom Maillard peptide which is characterized in that the white beech mushroom Maillard peptide is obtained by carrying out Maillard reaction on the white beech mushroom flavor-presenting peptide and sugar.
The method comprises the steps of firstly obtaining water-soluble components from the white beech mushroom through compound enzyme enzymolysis, then carrying out membrane separation on the water-soluble components, and further separating and purifying. The white beech mushroom flavor peptide is used as one of flavor development substances in the field of food, such as a base material or an auxiliary material for food seasoning, which are conventional applications of flavor development substances.
Compared with the prior art, the invention has the advantages that the technical progress is remarkable:
the white beech mushroom is taste peptide, tastes delicious, and brings a thick taste effect when added into food. The research of the invention finds that the Maillard reaction is carried out by utilizing the white beech mushroom flavor development peptide prepared by the embodiment, and the obtained Maillard peptide has good flavor enhancement effect.
Drawings
FIG. 1 is a chromatographic chart of a membrane separation component with the molecular weight of 200Da-3000Da after Sephadex G-15 gel chromatographic separation;
FIG. 2 shows taste dilution analysis results of membrane separation fraction U2 and chromatographic fractions F1-F4;
FIG. 3 shows the results of taste intensity of chromatographic fractions F1-F4 in chicken broth;
FIG. 4 is an RP-HPLC separation spectrum of chromatographic fraction F1;
FIG. 5 shows the results of the taste intensity of three components F1a, F1b and F1c in chicken broth;
FIG. 6 is a time-of-flight mass spectrometry (TOF MS) primary mass spectrum of component F1c, and graphs A and B are the results of two tests on TOF MS of the same component, respectively;
FIG. 7 is a time of flight mass spectrometry (TOF MS) second order mass spectrum of component F1 c.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
In the specific implementation mode of the invention, after the membrane separation components are subjected to chromatography, the components with the most delicious and full-bodied taste are selected, the Sephadex G-15 gel chromatography method is adopted for chromatography, and then the components with the most delicious taste are selected for RP-HPLC separation, and the separated components are identified.
Example 1
The embodiment provides a preparation method of white beech mushroom flavor peptide, which comprises the following specific steps:
step (1): preparing white beech mushroom enzymolysis liquid:
cleaning white beech mushroom (purchased from Shanghai Fengyu Co., Ltd.) and removing impurities, homogenizing with a homogenizer, adding deionized water at a ratio of 1:4, inactivating enzyme at 90 deg.C for 5min, cooling to 40 deg.C, adjusting pH to 3.5, adding cellulase (Celluclast,1.5L, Denmark Novit China investment Co., Ltd.) in an amount of 0.5% of fresh mushroom weight, and performing enzymolysis for 2 h. Then heating to 50 ℃, adjusting the pH value to 6.0, adding Flavourzyme (1000L, China investment Co., Ltd., Denmark Novo Nutgen) accounting for 0.4 percent of the weight of the fresh mushrooms, and continuing enzymolysis for 2 hours; finally heating to 90 deg.C, inactivating enzyme for 5min, and cooling to room temperature; then filtering with double-layer gauze, centrifuging the filtrate at 4 ℃ for 10 minutes at the rotating speed of 10000r, and collecting the supernatant;
step (2): separating and purifying the white beech mushroom thick peptide extracting solution:
carrying out ultrafiltration separation on the supernatant obtained in the step (1) by using an ultrafiltration membrane with molecular weight cut-off ranges of 3000Da and 200Da, collecting ultrafiltration components (marked as U3) with molecular weights of less than 200Da (marked as U1), 200-3000Da (marked as U2) and more than 3000Da, freezing and drying the ultrafiltration components, and storing the ultrafiltration components in a refrigerator at the temperature of-20 ℃;
according to sensory tests, only U2 has a remarkable effect of increasing the freshness of chicken soup, so that the membrane separation component U2 (freeze-dried sample powder) is prepared into a solution with the concentration of 25mg/mL, and is further separated by Sephadex G-15 gel chromatography under the conditions that ultrapure water is used as an eluent and the elution flow rate is 1.2 mL/min; the chromatogram results are shown in FIG. 1, the abscissa represents elution time (min), and the ordinate represents abundance at a detection wavelength of 220 nm; from FIG. 1, 4 absorption peaks can be seen, i.e. 4 separated components are obtained by chromatography, and 4 chromatographic components obtained in sequence in the elution process are collected (sequentially represented by F1, F2, F3 and F4); freeze-drying the sample powder, and storing the freeze-dried sample powder at-20 ℃ for later use;
and (3): the taste characteristics of the ultrafiltration fraction U2 and the chromatography fractions F1-F4 were analyzed using artificial sensory evaluation analysis.
The sensory evaluation method adopts a Taste Dilution Analysis (TDA) method to determine the dilution factors of each component obtained by gel separation and the membrane separation component U2, and comprises the following specific operations: preparing freeze-dried component samples of U2 and F1-F4 into 25mg/mL solutions respectively, gradually diluting the solutions by deionized water in a ratio of 1:1 to prepare a series of solutions with concentration gradients, and evaluating each dilution level by a three-point test method until the taste cannot be tasted by a certain dilution factor; recording the dilution factor, namely the dilution factor (TD); the sensory evaluator is 8 persons (4 men and 4 women, age 25 to 30 years), before sensory analysis, 1% sucrose solution is used as a sweet taste standard, 0.35% sodium chloride solution is used as an umami taste standard, 0.08% quinine solution is used as a bitter taste standard, 0.35% sodium glutamate solution is used as an umami taste standard, 0.08% citric acid solution is used as an sour taste standard, training is carried out on sensory analysis panelists, sipping is carried out at 9:00am-12:00am every day, and the sensory analysis is carried out for five days to train the taste sense of the panelists; the dilution factor when the taste difference between a certain dilution level solution and two deionized water is just identified is the taste intensity dilution factor, namely the TD value; the TD value is the average value of the evaluation results of each sensory evaluator, and each sample is evaluated at normal temperature after being repeated three times in different time. Each sensory evaluator needs to perform descriptive sensory evaluation on the taste characteristics of each sample in water;
the results of the flavor intensity dilution factors of the membrane separation fraction U2 and the chromatographic fractions F1-F4 are shown in FIG. 2, in which the abscissa represents the fraction name and the ordinate represents the flavor intensity dilution factor of each fraction.
As can be seen from FIG. 2, the TD values of the membrane separation fraction U2 and the chromatography fraction F1 were the highest, and the taste was the strongest in the same range.
The results of evaluation of artificial organoleptic properties of the ultrafiltration component U2 and the chromatography components F1-F4 are shown in the following table.
TABLE 1 sensory evaluation results
As can be seen from the results in Table 1, the flavor profiles of the chromatographic fraction F1 and the membrane separation fraction U2 are most similar, and the umami taste is more distinct and the taste sensation is strong.
In order to further determine the taste of each component, sensory analysis was performed by using the interaction of chicken soup and flavor peptide using a 5-point scale method. Sensory evaluation score 0 indicated no taste, and 5 indicated that the richness was the most intense. When the taste of the flavor development peptide is subjected to strong sensory evaluation, the temperature of the sample is maintained at 40 +/-2 ℃.
Preparing 0.2g/L solution from each chromatographic component F1-F4, adding 10mL of each chromatographic component into 50mL of chicken soup reference solution to prepare sample solution, and adding 10mL of deionized water into the pure chicken soup reference solution as blank control. The chicken soup reference solution is prepared by using stewed chicken soup thick soup treasure purchased from union lihua company, diluting with 1000ml deionized water per 128g thick soup treasure, and sealing for later use. The scoring results are shown in fig. 3.
Combining the results of FIGS. 1-3 and Table 1, it can be considered that: the component F1 has the most delicious taste and the most intense taste, so that the component F1 is selected for the next RP-HPLC separation and purification.
And (4): further separating and purifying F1 component:
further separation and purification of the F4 fraction were carried out by RP-HPLC. The column used was Spursil C18(5 μm, 250 x 4.6 mm; Dikma Technologies Inc.). The RP-HPLC separation conditions were: isocratic elution, 30% methanol and 70% ultrapure water, flow rate: 0.8mL/min, column temperature: the sample loading was 10. mu.L at 25 ℃ and the detection wavelength was 220 nm.
The RP-HPLC separation spectrum of chromatography component F1 is shown in FIG. 4. The abscissa of the graph represents elution time (min), and the ordinate represents abundance at a detection wavelength of 220 nm.
From the figure, three absorption peaks can be seen, and the absorption peaks in the elution process are collected to obtain 3 separated components (sequentially marked as F1a, F1b and F1 c). The components are freeze-dried and stored at-20 ℃.
And (5): the taste characteristics of the RP-HPLC separated fractions were analyzed using artificial sensory evaluation analysis.
Sensory evaluation was performed by 8 sensory evaluators (4 men and 4 women, age 25 to 30 years). And (4) training the evaluator according to the training method in the step (3). Sensory samples were prepared on a 5-point scale. RP-HPLC components F1a, F1b and F1c are prepared into 0.2g/L solution, 10ml of each component is added into 50ml of chicken soup reference solution respectively to prepare sample solution. A blank was prepared by adding 50ml of chicken broth to 10ml of deionized water. Evaluation was performed by a sensory evaluator. Sensory evaluation score 0 indicated no taste, 5 indicated the most intense umami taste, and the sample temperature was evaluated at 40 ± 2 ℃. The sensory evaluation results are shown in fig. 5.
As can be seen in FIG. 5, component F1c was the most umami taste and therefore F1c was selected for polypeptide sequence identification.
Step (6) identification of polypeptide sequence Structure of component F1c
Preparing a sample: dissolving the F1c component sample in chromatographic pure water, mixing on a vortex mixer to fully dissolve the component, centrifuging and taking the supernatant for later use.
The UPLC-Q-TOF-MS measurement conditions were as follows:
liquid phase conditions: a BEH C18 column (5 cm. times.2.1 mm, 1.7 μm) was used: the sample volume is 10 mu L; flow rate: 0.3 ml/min; gradient elution is carried out by adopting two kinds of eluent as mobile phases, wherein the eluent A is 0.1% acetonitrile water solution, and the eluent B is 0.1% formic acid water solution; the column temperature is 45 ℃; the gradient elution conditions were as follows: 0-2 min: 100% of B; 2-3 min: 90% of B; 3-10 min: 0% of B.
The mass spectrometry conditions were as follows: ionization mode: ESI +, capillary voltage of 3.2Kvolts, cone hole voltage of 20Kvolts, ion source temperature of 100 ℃, desolvation vaporization temperature of 400 ℃, cone hole flow rate of 50L/h, ion energy of 1Volt, collision energy of 6Volts and 20Volts, scanning time of 1s, detection voltage of 1700Volts, and mass range of 20-1000 m/z.
After separation and identification of component F4b by UPLC-Q-TOF-MS, the relative molecular weight of the separated component F1c was identified by Biolynx in Masslynx. The abscissa of FIG. 6 is the molecular weight (m/z) and the ordinate represents the abundance. The results from FIG. 6 show the major ion fragment ([ M + H ] of F1c]+) 505.8, and finally analyzed for a relative molecular mass of 503.99 Da. The amino acid sequence of the F1c fraction was identified by secondary mass spectrometry and analyzed to give the final peptide sequence Leu-Ala-Gly-His, the results are shown in FIG. 7. The abscissa of fig. 7 is the molecular weight (m/z) of the ion fragment and the ordinate is the abundance.
In conclusion, the polypeptide sequence structure of the flavor peptide of the RP-HPLC separation component F1c can be obtained, and the polypeptide sequence structure is Leu-Ala-Gly-His (sequence of SEQ ID NO. 1).
And (7) preparing the white beech mushroom Maillard peptide.
Dissolving the obtained white beech mushroom flavor peptide to prepare 20% peptide solution, adding xylose according to the ratio of 3:1 of peptide sugar, uniformly mixing, adjusting the pH value to 8.0, reacting for 3h at 120 ℃, and cooling by using ice water to terminate the reaction.
Sensory evaluation results show that the white beech mushroom flavor peptide has stronger delicate flavor and thick taste after undergoing the Maillard reaction. The white beech mushroom flavor peptide in the embodiment has obvious flavor enhancement effect, can be used in the field of food, can be used as a base material and an auxiliary material to be added with seasonings, and enriches the taste of the food.
SEQUENCE LISTING
<110> Suzhou cymbidium Chai Biotech Co., Ltd
<120> white beech mushroom flavor-developing peptide and preparation method and application thereof
<130>BCN1200137
<160>1
<170>PatentIn version 3.5
<210>1
<211>4
<212>PRT
<213>Hypsizygus marmoreus
<400>1
Leu Ala Gly His
1
Claims (9)
1. The white beech mushroom flavor-presenting peptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. The Hypsizygus marmoreus taste peptide of claim 1, wherein the Hypsizygus marmoreus taste peptide is derived from a Hypsizygus marmoreus extract.
3. The Hypsizygus marmoreus taste peptide of claim 1, wherein the Hypsizygus marmoreus taste peptide is artificially synthesized.
4. The method of preparing white beech mushroom flavor peptide of claim 1, comprising the steps of:
step 1: extracting water soluble components from white beech mushroom by using a method of compound enzyme enzymolysis;
step 2: intercepting components with molecular weight of 200Da to 3000Da by ultrafiltration membrane separation method;
and step 3: performing chromatography and high performance liquid separation and purification on the obtained ultrafiltration component to obtain the white beech mushroom flavor peptide with the amino acid sequence shown as SEQ ID NO. 1.
5. The method for preparing the white beech mushroom flavor-developing peptide according to claim 4, wherein the step 1 specifically comprises: cleaning white beech mushroom, cutting, adding water, inactivating enzyme at water bath temperature above 80 deg.C, adjusting pH to 3.0-4.0, adding cellulase for enzymolysis for 1-3 hr, adjusting pH to 4.0-6.0, adding flavor protease for enzymolysis for 1-3 hr, filtering, centrifuging, and collecting supernatant.
6. The method for preparing the white beech mushroom flavor-developing peptide according to claim 4, wherein the step 3 specifically comprises: and (3) after the ultrafiltration component is subjected to chromatography, selecting the component with the strongest thickening effect through artificial sensory evaluation, separating and purifying the component with the strongest thickening effect, and finally performing structural identification on the separated component.
7. The method for preparing the white beech mushroom flavor-developing peptide according to claim 6, wherein the chromatography is performed by SephadexG-15 gel chromatography, the separation and purification are performed by RP-HPLC, and the structural identification is performed by UPLC-Q-TOF-MS.
8. Use of the white beech mushroom flavor peptide of any one of claims 1 to 3 in a food product.
9. A Hypsizygus marmoreus Maillard peptide obtained by Maillard reaction of the Hypsizygus marmoreus taste peptide of any one of claims 1-3 and sugar.
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| CN113651869A (en) * | 2021-07-14 | 2021-11-16 | 南京财经大学 | Umami peptide and preparation method and application thereof |
| CN115947780A (en) * | 2022-08-29 | 2023-04-11 | 成都大学 | Polypeptide and preparation method and application thereof |
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| CN113390994A (en) * | 2021-06-16 | 2021-09-14 | 上海应用技术大学 | Method for extracting, separating, identifying and verifying bitter peptides in yellow wine |
| CN113651869A (en) * | 2021-07-14 | 2021-11-16 | 南京财经大学 | Umami peptide and preparation method and application thereof |
| CN113651869B (en) * | 2021-07-14 | 2023-02-24 | 南京财经大学 | Umami peptide and preparation method and application thereof |
| CN115947780A (en) * | 2022-08-29 | 2023-04-11 | 成都大学 | Polypeptide and preparation method and application thereof |
| CN115947780B (en) * | 2022-08-29 | 2023-09-19 | 成都大学 | Polypeptide and preparation method and application thereof |
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