CN117486969A - Preparation method and application of bamboo shoot flavor peptide - Google Patents

Abstract

The application provides an application of the bamboo shoot flavor peptide in enhancing the fresh and sweet flavor of food or seasoning, or an application in preparing a flavor enhancer for enhancing the fresh and sweet flavor of food or seasoning, wherein the amino acid sequence of the bamboo shoot flavor peptide is as follows: gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe or gamma-L-Val-L-Gly. The five bamboo shoots taste peptide can effectively enhance the fresh and sweet taste of food or seasonings, increase preference and improve the flavor quality of products.

Description

Preparation method and application of bamboo shoot flavor peptide

Technical Field

The invention belongs to the technical field of biology, and relates to a preparation method of a bamboo shoot flavor peptide and application thereof in enhancing fresh and sweet flavor of food or condiment or in preparing flavor enhancer for enhancing fresh and sweet flavor of food or condiment.

Background

Natural flavoring for food is an important direction of the development of composite flavoring market in recent years, and the product not only has natural flavor, but also retains healthy ingredients. The research and development of natural and nutritional natural flavor substances has strong practical significance, can meet the demands of consumers and markets, and has broad prospects. The flavor peptide participates in and influences the formation of the color, the aroma and the taste of the seasoning, so that the flavor of the seasoning is stronger, the texture is more perfect, the overall taste is more coordinated, thick and fine, and the seasoning is an important raw material of compound high-grade seasonings, monosodium glutamate, chicken essence and various spices.

The bamboo shoots have rich nutritive value, are very beneficial to human bodies, have no pollution to the growth environment, are considered as cleanest green food, and are purely natural health food which is deeply touted by modern people. The bamboo shoots have flavor development functions such as delicate flavor, sweet taste and the like, can be used as novel natural seasonings for application, and can enhance the flavor of foods and improve the preference. At present, the research on the flavor peptide of the bamboo shoots is not reported.

Disclosure of Invention

The invention aims to provide the application of the bamboo shoot flavor peptide in enhancing the fresh and sweet flavor of food or seasoning or in preparing flavor base materials for enhancing the fresh and sweet flavor of food or seasoning.

Another object of the invention is to provide a method for preparing the bamboo shoot flavor peptide. The method comprises the steps of taking bamboo shoots as raw materials to release and extract bamboo shoot proteins, preparing bamboo shoot polypeptides by an enzymolysis technology, separating polypeptides with different molecular masses by ultrafiltration fractionation and gel filtration chromatography, determining polypeptide fragments with the most taste function by using technologies such as sensory evaluation, electronic tongue and the like, and purifying and identifying the bamboo shoot taste peptide by using a reversed phase chromatography and liquid chromatography-mass spectrometer.

The invention adopts the technical scheme that:

the invention provides an application of bamboo shoot flavoring peptide in enhancing the fresh and sweet flavor of food or condiment, or an application in preparing flavoring agent for enhancing the fresh and sweet flavor of food or condiment, wherein the amino acid sequence of the bamboo shoot flavoring peptide is as follows: gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe or gamma-L-Val-L-Gly.

Preferably, the amino acid sequence of the bamboo shoot taste peptide is: gamma-L-Asn-L-Pro or gamma-L-Glu-L-Phe.

In some embodiments of the application, the bamboo shoot flavor peptide is used to enhance the sweet flavor and richness of a food or seasoning.

In some embodiments of the application, the method for preparing the bamboo shoot flavor peptide comprises the following steps:

(1) Preparing bamboo shoot protein by adopting a two-step enzymolysis method of cellulase and pectase;

(2) Carrying out enzymolysis on the bamboo shoot protein by adopting a combined enzyme of 1:1 composite protease and papain to prepare a bamboo shoot polypeptide;

(3) Separating the bamboo shoot polypeptide by ceramic membrane equipment, grading and separating filtrate by ultrafiltration membrane equipment, and collecting components with molecular weight less than 1 kDa;

(4) Performing gel filtration chromatography on bamboo shoot polypeptide with molecular weight less than 1kDa, performing electronic tongue analysis, selecting the component with highest freshness, performing reverse phase chromatography separation and purification, and performing structural identification on the separated component to obtain 5 flavor peptides with primary structures of gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe and gamma-L-Val-L-Gly.

In some embodiments of the application, the method for preparing the bamboo shoot flavor peptide comprises the following steps:

(1) Preparation of bamboo shoot protein

Respectively carrying out enzymolysis on cellulase and pectase by adopting a two-step enzymolysis method, wherein the feed-liquid ratio is 1:5; adding 1% of cellulase, hydrolyzing at 55deg.C for 120min at natural pH; after enzyme deactivation, adding pectase for enzymolysis, wherein the enzyme addition amount of pectase is 1%, the hydrolysis temperature is 50 ℃, the natural pH is carried out for 60min, after enzyme deactivation, sieving with a 100-mesh sieve, centrifuging for 20min at 6000r/min, filtering, and freeze-drying to obtain the bamboo shoot protein. The protein extraction rate reaches 19.35%.

(2) Preparation of bamboo shoot Polypeptides

And (3) carrying out enzymolysis on the bamboo shoot protein prepared in the step (1), adopting a combined enzyme of 1:1 composite protease and papain, wherein the total enzyme dosage is 5000U/g substrate, pH7.0, the hydrolysis temperature is 50 ℃, the hydrolysis time is 180min, and inactivating enzyme at 90 ℃ for 20min after the reaction is finished to obtain the bamboo shoot polypeptide. The yield and hydrolysis degree of the short peptide were 59.83% and 23.47%, respectively.

(3) Ultrafiltration fractionation of bamboo shoot polypeptides

Separating bamboo shoot polypeptide by ceramic membrane equipment, and grading the filtrate by ultrafiltration membrane equipment, wherein the operation pressure of primary ultrafiltration is 20psi, the temperature is 30 ℃ and the material concentration is 4%; the operating pressure of the secondary ultrafiltration is 25psi, the temperature is 30 ℃, and the material concentration is 6%; the operation pressure of the three-stage ultrafiltration is 25psi, the temperature is 30 ℃, and the material concentration is 6%;

the bamboo shoot polypeptide is subjected to ultrafiltration fractionation to obtain four component fragments of more than 5kDa, 2.5-5kDa, 1-2.5kDa and <1kDa, and the bamboo shoot polypeptide with molecular weight of <1kDa is selected. In the experiment, the ratio of the molecular weight <1kDa is the highest and 71.24%, and the sensory indexes such as delicate flavor and preference are the best, so that the bamboo shoot polypeptide with the molecular weight <1kDa is selected for the next separation.

(4) Gel filtration chromatography separation of bamboo shoot polypeptides

Performing gel filtration chromatography on bamboo shoot polypeptide with molecular weight less than 1kDa, taking Sephadex G-15 Sephadex as a filler, taking deionized water as eluent, eluting at a flow rate of 2mL/min, collecting by an automatic fraction collector at a flow rate of 0.6mL/min of a constant flow pump, collecting from the outflow of chromatography effluent, collecting 67-104 tube number samples at intervals of 3min, separating and merging for multiple times, and concentrating to obtain the final product with highest freshness.

(5) Reversed phase purification of flavor peptide of bamboo shoots

Purifying by reverse phase chromatography under the conditions of XDB C18, 250X4.6mm, 5um; the mobile phase adopts A:0.05% tfa-water, B:0.05% tfa-acetonitrile elution; the flow rate is 1.0mL/min; the detection wavelength is 220nm; the column temperature is 30 ℃ and the sample injection amount is 20uL; collecting chromatographic peak component with peak time 14.253-15.397min by fraction collector, and lyophilizing.

(6) Identification of flavor peptide of bamboo shoots

Separating and identifying by using a liquid chromatography-mass spectrometry instrument to determine 5 flavor peptides, wherein the primary structures of the flavor peptides are gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe and gamma-L-Val-L-Gly respectively.

In some embodiments, the conditions for the ceramic membrane separation are a solution concentration of 10%, an operating pressure of 20psi, a temperature of 25 ℃, and a membrane flux of 22.56L/m ² ·h。

The invention has the following advantages and beneficial effects:

the invention combines the modes of bamboo shoot proteolysis, ultrafiltration separation, gel chromatography separation, reversed phase chromatography purification and the like to prepare 5 bamboo shoot flavor peptide, and the amino acid sequence of the flavor peptide is gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe and gamma-L-Val-L-Gly. The 5 bamboo shoot flavor peptide can effectively enhance the fresh and sweet flavor of foods or seasonings, increase the preference and improve the flavor quality of the product.

Drawings

FIG. 1 is a graph showing the yield and hydrolysis degree of short peptides of the bamboo shoot protein by single protease in example 2;

FIG. 2 is a graph showing the yield and hydrolysis degree of the short peptides of the bamboo shoot proteins by the enzyme hydrolysis of the different combination enzymes in example 2;

FIG. 3 is a graph of the effect of operating pressure on primary ultrafiltration in example 3;

FIG. 4 is a graph showing the effect of operating temperature on primary ultrafiltration in example 3;

FIG. 5 is a graph showing the effect of material concentration on primary ultrafiltration in example 3;

FIG. 6 is a graph of the effect of operating pressure on secondary ultrafiltration in example 3;

FIG. 7 is a graph of the effect of operating temperature on secondary ultrafiltration in example 3;

FIG. 8 is a graph showing the effect of material concentration on secondary ultrafiltration in example 3;

FIG. 9 is a graph of the effect of operating pressure on tertiary ultrafiltration in example 3;

FIG. 10 is a graph of the effect of operating temperature on tertiary ultrafiltration in example 3;

FIG. 11 is a graph showing the effect of material concentration on tertiary ultrafiltration in example 3;

FIG. 12 gel separation of the molecular weight <1kD ultrafiltration fraction;

FIG. 13 is a graph showing the results of analysis of principal components of four separated components of the electronic tongue, P1-P4;

FIG. 14 is a diagram of four separate component electronic tongue flavor radars P1-P4;

FIG. 15 is a RP-HPLC separation spectrum of P2;

FIG. 16 is a mass spectrum of gamma-L-Ala-L-Phe;

FIG. 17 is a mass spectrum of gamma-L-Glu-L-Phe;

FIG. 18 is a mass spectrum of gamma-L-Asn-L-Pro;

FIG. 19 is a mass spectrum of gamma-L-Ile-L-Phe;

FIG. 20 is a mass spectrum of gamma-L-Val-L-Gly;

Detailed Description

The following detailed description of embodiments of the invention is exemplary and intended to be illustrative of the invention and not to be construed as limiting the invention.

The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The terms "comprising," "including," and "comprising" are open-ended, meaning the terms including the elements of the invention, but not excluding other elements.

The terms "peptide", "polypeptide", "peptide fragment" refer to a molecular chain of amino acid residues, which may be modified at each of its amino acid residues, if desired, for example by mannosylation, glycosylation, amidation (e.g. C-terminal amide), carboxylation or phosphorylation.

The material source is as follows:

cellulase: purchased from novelin (china) biotechnology limited.

Pectase: purchased from novelin (china) biotechnology limited.

Papain: purchased from novelin (china) biotechnology limited.

Alkaline protease: purchased from novelin (china) biotechnology limited.

Complex protease: purchased from Guangxi nan Ning Pang Bo Bio-engineering Co., ltd.

Neutral protease: purchased from Guangxi nan Ning Pang Bo Bio-engineering Co., ltd.

Flavourzyme: purchased from novelin (china) biotechnology limited.

EXAMPLE 1 preparation of bamboo shoot protein

In this example, four preparation methods of cellulase, pectase, combined enzyme and two-step enzyme were studied, and the effects of the different preparation methods were examined using the protein extraction rate as an index, as shown in Table 1.

And (3) performing enzymolysis: bamboo shoots cleaning, shelling, dicing, debitterizing, draining, crushing (feed-liquid ratio 1:5,5 min), enzymolysis (cellulase 1%,55 ℃ and natural pH 2 h), enzyme deactivation (90 ℃ for 20 min), sieving (100 meshes), centrifugation (6000 r/min,20 min), filtration and freeze drying.

Pectase enzymolysis method: bamboo shoots cleaning, shelling, dicing, debitterizing, draining, crushing (feed-liquid ratio 1:5,5 min), enzymolysis (pectase 1%,50 ℃ and natural pH 1 h), enzyme deactivation (90 ℃ for 20 min), sieving (100 meshes), centrifugation (6000 r/min,20 min), filtration and freeze drying.

Combined enzyme enzymatic hydrolysis (combined enzyme: cellulase-pectinase (1:1)): bamboo shoots cleaning, shelling, dicing, debitterizing, draining, crushing (feed-liquid ratio 1:5,5 min), enzymolysis (combined enzyme 1%,50 ℃, natural pH 2 h), enzyme deactivation (90 ℃,20 min), sieving (100 meshes), centrifugation (6000 r/min,20 min), filtration and freeze drying.

A two-step enzymolysis method: bamboo shoots cleaning, shelling, dicing, debitterizing, draining, crushing (feed-liquid ratio 1:5,5 min), enzymolysis (cellulase 1%,55 ℃, natural pH,2 h), enzyme deactivation (90 ℃,20 min), enzymolysis (pectase 1%,50 ℃, natural pH,1 h), enzyme deactivation (90 ℃,20 min), sieving (100 mesh), centrifugation (6000 r/min,20 min), filtration and freeze drying.

Table 1 protein extraction rates for four preparation methods

Through the table 1, the protein extraction rate of the two-step enzymolysis method is the highest, so the embodiment of the invention determines that the two-step enzymolysis method adopting cellulase and pectase is adopted for preparing the bamboo shoot protein, and the extraction process conditions are as follows: the feed liquid ratio is 1:5; adding enzyme amount of cellulase at 1%,55 ℃ and natural pH for 2 hours; the pectase has an enzyme adding amount of 1%, a natural pH value at 50 ℃ and an enzymolysis time of 1h, and the protein extraction rate can reach 19.35+/-1.85%.

Example 2: preparation of bamboo shoot polypeptide

The method takes bamboo shoot protein as a raw material, selects a protease enzyme method to prepare the bamboo shoot polypeptide, and takes the hydrolysis degree and the short peptide yield as indexes to mainly examine the influence of enzyme types on the preparation of the polypeptide.

Screening of Single proteases

The method comprises the steps of selecting 5 enzymes of papain, alkaline protease, compound protease, neutral protease and flavourzyme, performing single enzymolysis on bamboo shoot protein according to enzymolysis conditions in table 2, and screening out the most suitable protease as compound protein by using the short peptide yield and the hydrolysis degree as indexes, wherein the result is shown in figure 1.

TABLE 2 Single protease enzymatic hydrolysis conditions

Experimental results show that after the bamboo shoot proteins are respectively subjected to the action of five proteases, the effect of papain, alkaline protease and compound protease is close to that of the other two proteases by taking the yield of the short peptide as an index, and the effect is obviously superior to that of the other two proteases; the action effect of the flavourzyme is obviously higher than that of other four proteases by taking the degree of hydrolysis as an index, but the yield of enzymolysis products is lower; the hydrolysis degree of the hydrolysis products of the alkaline protease and the compound protease is relatively close, and is better than the hydrolysis effect of papain and neutral protease. In conclusion, the analysis shows that the enzymolysis of the bamboo shoot protein can achieve better effect by adopting the alkaline protease and the compound protease, but a large amount of alkali liquor is required to be added in the enzymolysis of the alkaline protease, so that the compound protease is finally selected as the optimal single enzyme, and the yield and the hydrolysis degree of the short peptide are 45.67 +/-1.23% and 11.59 +/-0.41% respectively.

(II) screening of Combined proteases

The papain, the neutral protease and the flavourzyme are respectively combined with the compound protease for enzymolysis under the condition that the dosage ratio of the compound protease is 1:1, the total dosage of the enzyme is 5000U/g substrate, pH7.0, the hydrolysis temperature is 50 ℃, the hydrolysis time is 180min, the enzyme is inactivated for 20min at 90 ℃ after the reaction is finished, and the enzymolysis effect is analyzed by taking the short peptide yield and the hydrolysis degree as indexes, and the result is shown in figure 2 in detail.

Experimental results show that after the bamboo shoot proteins are subjected to combined enzymolysis, the short peptide yield is used as an index, and the combined effect of the compound protease and the papain is best; the combination effect of the compound protease and the flavor protease is best by taking the hydrolysis degree as an index, and the combination effect of the compound protease and the papain is next. The papain is taken as the optimal combination enzyme, and the yield and the hydrolysis degree of the short peptide are 59.83 +/-1.17% and 23.47+/-0.78% respectively.

And finally determining the conditions of the proteolysis of the bamboo shoots as follows: the compound protease and papain compound are used as combined enzyme, the dosage ratio of the compound enzyme is 1:1, the total dosage of the enzyme is 5000U/g substrate, pH7.0, the hydrolysis temperature is 50 ℃, the hydrolysis time is 180min, the enzyme is inactivated at 90 ℃ for 20min after the reaction is finished, and the yield and the hydrolysis degree of the short peptide are 59.83 +/-1.17% and 23.47+/-0.78%, respectively.

EXAMPLE 3 Ultrafiltration of bamboo shoot Polypeptides

The experiment adopts ultrafiltration method to separate the bamboo shoot protein enzymolysis liquid, the enzymolysis liquid is firstly separated by ceramic membrane under the conditions that the solution concentration is 10%, the operating pressure is 20psi, the temperature is 25 ℃, and the membrane flux is 22.56L/m ² H. Fractionation was performed using ultrafiltration membrane apparatus. The ultrafiltration membrane apparatus was configured with a molecular weight cut-off of 5kDa (one3 ultrafiltration membranes of the ultrafiltration membranes of level), 2.5kDa (second level) and 1kDa (third level), classifying the flavor-imparting peptides of the bamboo shoots into more than 5kDa, 2.5-5kDa, 1-2.5kDa,<Four components of 1 kDa.

First-level ultrafiltration process parameter study

The bamboo shoot protein zymolyte ceramic membrane is used as the initial solution of the primary ultrafiltration after separation, and the influence of three single factors of operating pressure (figure 3), operating temperature (figure 4) and material concentration (figure 5) on the primary ultrafiltration effect is examined by taking membrane flux and membrane efficiency as indexes.

As can be seen from fig. 3, the membrane flux of the primary ultrafiltration gradually increases with increasing operating pressure, and when the operating pressure reaches 20psi, the membrane flux is significantly higher than that of the membrane under the condition that the operating pressure is 10-15psi under the same conditions, and the pressure is continuously increased to 25psi, so that the increase of the membrane flux is not obvious, and therefore 20psi is selected as the optimal operating pressure of the primary ultrafiltration.

As can be seen from fig. 4, the membrane flux of the primary ultrafiltration gradually increases with an increase in the operating temperature, and when the operating temperature reaches 30 ℃, the membrane flux is significantly higher than that of the membrane flux at the operating temperature of 20 ℃ and 25 ℃ under the same conditions, and the membrane flux is hardly increased by continuing to increase the temperature to 35 ℃, so that 30 ℃ is selected as the optimum operating temperature for the primary ultrafiltration.

As can be seen from fig. 5, the membrane flux decreases with increasing material concentration, and in the concentration range of 1% -2%, the membrane flux decreases rapidly, and in the range of 2% -4%, the membrane flux becomes gentle, and then decreases gradually; in the range of 1-6% of material concentration, the membrane efficiency increases along with the increase of the material concentration, the increase is rapid when the material concentration is 1-4%, the increase tends to be slow when the material concentration is 4-6%, the membrane efficiency reaches the maximum value when the material concentration is 6%, and then the material concentration is continuously increased, and the membrane efficiency starts to decrease. The membrane flux and the membrane efficiency are combined, and the material concentration of 4% is used as the optimal material concentration in the primary ultrafiltration process.

Referring to FIGS. 3-5, the operating conditions for the primary ultrafiltration were determined to be an operating pressure of 20psi, a temperature of 30℃and a material concentration of 4%.

(II) two-stage Ultrafiltration Process parameter study

The filtrate after primary ultrafiltration is used as the initial liquid of secondary ultrafiltration, and the influence of three single factors of operating pressure (figure 6), operating temperature (figure 7) and material concentration (figure 8) on the secondary ultrafiltration effect is examined by taking membrane flux and membrane efficiency as indexes, so that the optimal operating condition is determined to be the operating pressure of 25psi, the temperature of 30 ℃ and the material concentration of 6%.

Three-stage ultrafiltration process parameter study

The filtrate after the secondary ultrafiltration is used as the initial liquid of the tertiary ultrafiltration, and the influence of three single factors of operating pressure (figure 9), operating temperature (figure 10) and material concentration (figure 11) on the tertiary ultrafiltration effect is examined by taking membrane flux and membrane efficiency as indexes. Thus, the optimum operating conditions were determined to be an operating pressure of 25psi, a temperature of 30 ℃, and a material concentration of 6%.

EXAMPLE 4 gel separation of bamboo shoot Polypeptides

Preparing 5% water solution from the lyophilized powder of bamboo shoot polypeptide with molecular weight less than 1kDa, and passing through 0.22um microporous membrane. And (3) loading 3mL of the solution onto a column, eluting with deionized water as eluent at a flow rate of 2mL/min, and carrying out fractional collection by using an automatic fraction collector, wherein the flow rate of a constant flow pump is set to be 0.6mL/min, and the interval time of the fraction collector is set to be 3min. The absorbance of each tube of the eluate at 220nm wavelength was measured, and the eluate was separated a plurality of times and collected according to the peaks of different components to obtain a gel separation chart 12. The same component peak products were combined and freeze-dried. The peptide freeze-dried powder solution is separated by G15 sephadex and has four peaks P1-P4, wherein P1 is used for collecting 44-66 tube number samples, P2 is used for collecting 67-104 tube number samples, P3 is used for collecting 105-131 tube number samples, and P4 is used for collecting 132-161 tube number samples. And (3) concentrating after multiple separation and combination to obtain four separated components P1-P4.

Evaluation of flavor of components separated by bamboo shoot polypeptide gel

The four separated components P1-P4 are subjected to flavor evaluation by using an electronic tongue, and a principal component analysis chart 13 and a flavor radar chart 14 are obtained.

As can be seen from fig. 13, the sum of the contribution rates of the first principal component (PC 1) and the second principal component (PC 2) reaches 90.630%, which can well reflect the actual situation of the sample. And the identification index of the principal component analysis of all samples was 98, which indicates that different gel separation fragments can be well distinguished.

As can be seen from fig. 14, 4 components P1-P4 are generally higher in umami taste and sweetness and generally lower in salty taste intensity; wherein the freshness of P2 is highest, and the freshness intensity of 4 samples is ranked as P2> P3> P1> P4; the sweetness intensity of P3 is highest, and the sweetness intensity of 4 samples is ranked as P3> P2> P4> P1; the acidity of P3 is highest, and the acidity intensity of 4 samples is ordered as P3> P4> P2> P1; p1 is the most bitter, and the other 3 samples are obviously lower than P1. P2 has better freshness and sweetness, proper acidity, smaller bitter degree and optimal preference, and P2 is selected for purification.

EXAMPLE 5 reverse purification and identification of bamboo shoot taste peptide

P2 with optimal preference is further purified by reverse-phase high performance liquid chromatography, and the separation and purification conditions are as follows: column XDB C18, 250×4.6mm,5um; the mobile phase adopts A:0.05% tfa-water, B:0.05% tfa-acetonitrile elution, VA: vb=2:8; the flow rate is 1.0mL/min; the detection wavelength is 220nm; the column temperature is 30 ℃ and the sample injection amount is 100uL. RP-HPLC separation spectrum of P2 is shown in FIG. 15.

As can be seen from fig. 15, 16 absorption peaks can be obtained by separation; collecting 2.073-2.300min, 2.300-2.540min, 2.540-2.708min, 2.708-2.964min, 5.553-6.020min, 10.119-10.830min,

14.253-15.397min、21.146-21.563min、22.606-23.804min、25.293-25.879min、36.981-39.576min、39.939-40.723min、45.126-45.966min、46.413-47.488min、

47.746-49.626min and 53.599-55.273min to obtain the separated components, repeatedly combining, lyophilizing, and performing sensory evaluation.

The sensory panel consisted of eight panelists trained in the sensory technology profession and possessing a rich sensory experience. And (3) evaluating the 16 freeze-dried components with 5% clear water, and removing adverse characteristics such as bitter, astringent and acid, wherein the remaining 4 freeze-dried components with absorption peaks are 5.553-6.020min, 10.119-10.830min, 14.253-15.397min and 21.146-21.563 min. Wherein 14.253-15.397min is the component with the highest freshness, and the subsequent identification experiment is determined.

Further separating and identifying components of the purified freeze-dried product by utilizing a liquid chromatography-mass spectrometer, and detecting conditions: mass mode esi+/ESI-, ionization voltage 3.2kV, ion source temperature 120 ℃, atomization temperature 350 ℃, atomization gas flow rate 600L/h, cone hole gas flow rate 50L/h, cone hole voltage 30V. 5 polypeptides were identified. The mass spectrum identification results of the gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe and gamma-L-Val-L-Gly are shown in FIG. 16-FIG. 20.

EXAMPLE 5 application of bamboo shoot flavor peptide

(1) Clear water evaluation

5 tasting peptides were evaluated in 0.1% clear water, and each index was scored in terms of intensity in the range of 0-9 points. The sensory panel consisted of eight panelists trained in the sensory technology profession and having a rich sensory experience, with the final score being the average of 8 scores. The results are shown in Table 3, the flavor development effect is mainly fresh, sweet, sour and bitter, wherein the freshness of the gamma-L-Asn-L-Pro is highest, and the flavor development effect has the characteristics of obvious fresh flavor, slight sweet and convergence; the two peptides gamma-L-Glu-L-Phe and gamma-L-Ala-L-Phe have obvious delicate flavor, the gamma-L-Val-L-Gly has obvious bitter taste and slightly delicate flavor, and the gamma-L-Ile-L-Phe has stronger bitter taste and lower freshness.

Table 30.1% sensory evaluation of taste peptide clear water

(2) The synergistic effect of different flavoring substances has a great influence on the flavoring effect, and the synergistic effect of substances cannot be fully reflected by simple clear water evaluation, so that the flavoring solution is prepared for sensory evaluation. The 5 flavor peptides were formulated into a solution containing 0.05% polypeptide, 0.5% pork bone soup powder, and 0.2% NaCl, and compared with the blank, conventional flavor agent glutathione to evaluate the flavor effect in terms of freshness, richness, preference, etc., and the results are shown in Table 4.

The freshness of the 5 polypeptides is higher than that of the blank in the perfuming effect, and meanwhile, the 5 polypeptides show sweet, sour and bitter tastes, and the preference and the mellow feeling of the 5 polypeptides under the synergistic effect are better than those of the blank; wherein the freshness, sweetness, richness and preference of the 3 flavor peptides of gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe and gamma-L-Asn-L-Pro are superior or equivalent to those of glutathione. The gamma-L-Asn-L-Pro and gamma-L-Glu-L-Phe have stronger freshness and mellowness, can effectively enhance the fresh and sweet characteristics and the mellowness of foods, improve the preference and can be used as key raw materials for product upgrading and quality improvement.

Table 4 organoleptic evaluation of flavoring of flavor peptide chicken powder solutions

Name of the name	Fresh flavor	Sweet taste	Sour taste	Bitter taste	Mellow feeling	Preference of a person
							Blank space	5.5	4.5	2.5	3.5	6.0	6.1
γ-L-Ala-L-Phe	6.2	6.1	3.2	4.2	6.5	6.8
							γ-L-Glu-L-Phe	6.7	6.8	4.0	4.0	7.1	7.2
γ-L-Asn-L-Pro	7.1	6.5	3.3	3.8	7.0	7.5
							γ-L-Ile-L-Phe	5.6	5.1	5.2	5.5	6.1	6.5
γ-L-Val-L-Gly	6.0	6.2	5.8	4.8	6.2	6.3
							GlutathioneGlycine peptides	6.0	4.8	4.5	4.6	6.8	6.8

As can be seen from Table 4, the taste peptides gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe and gamma-L-Val-L-Gly of 5 bamboo shoots are superior to the blank in taste effect, mainly fresh and sweet taste, and preference and mellow feeling of 5 polypeptides under the synergistic effect.

In the 5 peptide fragments, the gamma-L-Asn-L-Pro and the gamma-L-Glu-L-Phe have stronger delicate flavor, sweet taste and mellow feel, are applied as novel flavoring base materials, and have great application value.

EXAMPLE 6 use of bamboo shoot flavor peptide

The bamboo shoot flavor peptide (gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe and gamma-L-Val-L-Gly) provided by the invention can be used as a flavor base material for preparing seasonings and other foods. Non-limiting examples are condiments such as sauce, soy sauce, soup stock, instant noodle sauce or monosodium glutamate, and the like; other foods such as conventional flour products, baked goods, snacks, and the like.

In 5 peptide segments, the gamma-L-Asn-L-Pro and gamma-L-Glu-L-Phe show stronger functions of increasing the delicate flavor, sweetness, richness and preference of foods or seasonings, and have larger application value.

For purposes of this disclosure, the terms "one embodiment," "some embodiments," "example," "a particular example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (6)

1. The application of the bamboo shoot flavoring peptide in enhancing the fresh and sweet flavor of food or seasoning or in preparing flavoring for enhancing the fresh and sweet flavor of food or seasoning is characterized in that the amino acid sequence of the bamboo shoot flavoring peptide is as follows:

gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe or gamma-L-Val-L-Gly.

2. The use according to claim 1, characterized in that the amino acid sequence of the bamboo shoot taste peptide is: gamma-L-Asn-L-Pro or gamma-L-Glu-L-Phe.

3. The use according to claim 2, wherein the bamboo shoot flavor peptide is used to enhance the sweet flavor and richness of a food or seasoning.

4. The use according to claim 1, characterized in that the method for preparing the bamboo shoot taste peptide comprises the following steps:

(1) Preparing bamboo shoot protein by adopting a two-step enzymolysis method of cellulase and pectase;

(2) Carrying out enzymolysis on the bamboo shoot protein by adopting a combined enzyme of 1:1 composite protease and papain to prepare a bamboo shoot polypeptide;

(3) Separating the bamboo shoot polypeptide by ceramic membrane equipment, grading and separating filtrate by ultrafiltration membrane equipment, and collecting components with molecular weight less than 1 kDa;

(4) Performing gel filtration chromatography on bamboo shoot polypeptide with molecular weight less than 1kDa, performing electronic tongue analysis, selecting the component with highest freshness, performing reverse phase chromatography separation and purification, and performing structural identification on the separated component to obtain 5 flavor peptides with primary structures of gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe and gamma-L-Val-L-Gly.

5. The use according to claim 4, characterized in that the method for preparing the bamboo shoot taste peptide comprises the following steps:

(1) Preparation of bamboo shoot protein

Respectively carrying out enzymolysis on cellulase and pectase by adopting a two-step enzymolysis method, wherein the feed-liquid ratio is 1:5; adding 1% of cellulase, hydrolyzing at 55deg.C for 120min at natural pH; after enzyme deactivation, adding pectase for enzymolysis, wherein the enzyme addition amount of the pectase is 1%, the hydrolysis temperature is 50 ℃, the natural pH is carried out for 60min, after enzyme deactivation, sieving with a 100-mesh sieve, centrifuging for 20min at 6000r/min, filtering, and freeze-drying to obtain the bamboo shoot protein;

(2) Preparation of bamboo shoot Polypeptides

Carrying out enzymolysis on the bamboo shoot protein prepared in the step (1), adopting a combined enzyme of 1:1 composite protease and papain, wherein the total enzyme dosage is 5000U/g substrate, pH7.0, the hydrolysis temperature is 50 ℃, the hydrolysis time is 180min, and inactivating enzyme at 90 ℃ for 20min after the reaction is finished to obtain the bamboo shoot polypeptide;

(3) Ultrafiltration fractionation of bamboo shoot polypeptides

Separating bamboo shoot polypeptide by ceramic membrane equipment, and grading the filtrate by ultrafiltration membrane equipment, wherein the operation pressure of primary ultrafiltration is 20psi, the temperature is 30 ℃ and the material concentration is 4%; the operating pressure of the secondary ultrafiltration is 25psi, the temperature is 30 ℃, and the material concentration is 6%; the operation pressure of the three-stage ultrafiltration is 25psi, the temperature is 30 ℃, and the material concentration is 6%;

the bamboo shoot polypeptide is subjected to ultrafiltration fractionation to obtain four component fragments of more than 5kDa, 2.5-5kDa, 1-2.5kDa and <1kDa, and the bamboo shoot polypeptide with molecular weight of <1kDa is selected;

(4) Gel filtration chromatography separation of bamboo shoot polypeptides

Performing gel filtration chromatography on bamboo shoot polypeptide with molecular weight less than 1kDa, taking Sephadex G-15 Sephadex as a filler, taking deionized water as eluent, eluting at a flow rate of 2mL/min, collecting by an automatic fraction collector at a flow rate of 0.6mL/min of a constant flow pump, collecting from the outflow of chromatography effluent, collecting 67-104 tube number samples at intervals of 3min, separating and combining for multiple times, and concentrating;

(5) Reversed phase purification of flavor peptide of bamboo shoots

Purifying by reverse phase chromatography under the conditions of XDB C18, 250X4.6mm, 5um; the mobile phase adopts A:0.05% tfa-water, B:0.05% tfa-acetonitrile elution; the flow rate is 1.0mL/min; the detection wavelength is 220nm; the column temperature is 30 ℃ and the sample injection amount is 20uL; collecting chromatographic peak component with peak time 14.253-15.397min by fraction collector, and lyophilizing;

(6) Identification of flavor peptide of bamboo shoots

Separating and identifying by using a liquid chromatography-mass spectrometry instrument to determine 5 flavor peptides, wherein the primary structures of the flavor peptides are gamma-L-Ala-L-Phe, gamma-L-Glu-L-Phe, gamma-L-Asn-L-Pro, gamma-L-Ile-L-Phe and gamma-L-Val-L-Gly respectively.

6. The method according to claim 5, wherein the conditions for the separation of the ceramic membrane are a solution concentration of 10%, an operating pressure of 20psi, a temperature of 25 ℃ and a membrane flux of 22.56L/m ² ·h。