CN114376197A

CN114376197A - Freshness enhancing agent and preparation method thereof

Info

Publication number: CN114376197A
Application number: CN202210083812.1A
Authority: CN
Inventors: 马磊; 杜雨桐; 梁必诚; 薛长湖; 孙建安; 毛相朝
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-04-22

Abstract

The invention discloses a preparation method of a freshener, which is prepared by the following steps: the euphausia superba head and oyster skirt meat are used as raw materials, and are obtained by enzymolysis through protease YDCP and fermentation; the amino acid sequence of the protease YDCP is shown in SEQ ID NO. 1. The freshener is prepared by a biological method by taking the shrimp heads and the oyster skirts as raw materials, fully utilizes the shrimp heads and the oyster skirts of the antarctic krill, and realizes high-value utilization of byproducts; inducing the euphausia superba heads to autolyze by adopting ultraviolet rays to generate delicious substances, and extracting the delicious substances, small molecular peptides and amino acids; high-heat processing is not used in the process of extracting the delicate flavor, a medium-low temperature processing mode is adopted, the flavor is retained to the maximum extent, side reaction harmful products generated by high-heat processing are avoided, and the nutrition is ensured. The freshener can be used as a seasoning bag in instant food, and can ensure the flavor and nutrition of the instant food.

Description

Freshness enhancing agent and preparation method thereof

Technical Field

The invention relates to a freshener and a preparation method thereof, belonging to the technical field of flavoring agents.

Background

The flavor enhancer, also called flavor enhancer, flavor enhancer and flavor enhancer, is a substance for supplementing or enhancing the original flavor of food. Some foods are added with flavoring agents, so that the foods have delicious taste, increase appetite and enrich nutrition. The food flavor enhancer does not affect the taste stimulation of 4 basic tastes such as sour, sweet, bitter, salty and the like and other flavor development substances, but enhances the respective flavor characteristics, thereby improving the palatability of the food. Most of the seasonings in the prior art mainly rely on salt and monosodium glutamate (sodium glutamate) to improve the delicate flavor.

Aquatic products are the main sources of the delicate flavour extracts, and shrimps, crabs, oysters and the like are all raw materials which can contribute to the delicate flavour to a great extent. Most of the salt-reducing and freshness-increasing methods in the prior art are realized through Maillard reaction, but the Maillard reaction is easy to generate some uncontrollable side reactions.

Antarctic krill is a large type of aquatic product produced in Antarctic regions with extremely high yield. Currently, Antarctic krill is mostly developed to extract functional substances such as shrimp oil and protein from Antarctic krill, but the utilization of byproducts such as shrimp heads is low.

Oyster is a shellfish food which is consumed in large quantity, but the eating rate of the oyster skirt is not high.

Disclosure of Invention

Aiming at the prior art, the invention provides a freshener and a preparation method thereof for improving the utilization rate of the shrimp heads and the oyster skirts of Antarctic krill.

The invention is realized by the following technical scheme:

a freshener is prepared from Euphausia superba head and oyster skirt meat as raw materials, by enzymolysis with protease YDCP, and fermenting; the protease YDCP is a protease from Geobacillus stearothermophilus, and the amino acid sequence of the protease is shown as SEQ ID NO. 1.

The amino acid sequence of the amino acid sequence (shown as SEQ ID NO. 1): MKKPELLVTP TSVSHIEELA EAGADAVMIG EQRYGLRLAG EFSRADVAAA IEAAHRHGMN VYVAMNAIFH NDKVDELGDY IAFVAAAGAD AIVFGDPAVL LTVREAAPHM KLHWSTETTA TNWYTCNYWG RKGAKRAVLA RELNMDAILE IKAHAEVEIE VQVHGATCMY QSKRSLIGNY FEYQGKVMEI ERKKYEKGMF LYDKERDNKY PIFEDENGTH IMSPNDVCII DELGDMIEAG IDSFKIDGIL HEPRYITEVT KLYRRAIDMC AKDREQYERE KEELLAAVEA LQPPHRRLDT GFFFKETIY are provided.

Specifically, the preparation method comprises the following steps:

the method comprises the following steps of (1) preparing an enzymolysis delicate flavor extracting solution by using Antarctic krill head and oyster skirt meat as raw materials:

(1) soaking: cleaning the head of Antarctic krill, and soaking in a preservative; cleaning oyster skirts and soaking the oyster skirts in a preservative; taking out after soaking for later use.

(2) Pulping: making the soaked shrimp heads and oyster skirt meat into homogenate respectively.

(3) Treating homogenate: adjusting the pH value of the shrimp head homogenate to 7.0-8.0 (preferably 7.5), carrying out ultraviolet irradiation for 15-25 minutes (preferably 20 minutes) under the water bath condition of 50 +/-2 ℃, and inducing endogenous enzyme to accelerate the autolysis process; and (3) after ultraviolet irradiation, standing and autolyzing for 1.5-2.5 hours (preferably 2 hours) under the water bath condition of 50 +/-2 ℃.

(4) Enzymolysis: and mixing the shrimp head homogenate and the oyster skirt meat homogenate uniformly, adding a protease YDCP, and carrying out enzymolysis for 2-3 hours (preferably 2.5 hours) under the conditions of the pH value of 7.5 and the temperature of 50 +/-2 ℃.

(5) Enzyme deactivation: after the enzymolysis, the enzyme is inactivated for 15 minutes under the condition of water bath at 90 +/-2 ℃.

(6) Debitterizing: after the enzyme deactivation, active carbon is added (bitter peptides are generated in the enzymolysis process, so that the bitter taste is removed by adopting an active carbon adsorption method), and debittering is carried out for 30 minutes under the water bath condition of 50 +/-2 ℃.

(7) Centrifuging: and after debitterizing, centrifuging, taking supernate, and concentrating by rotary evaporation to 1/4-1/2, preferably 1/3 of the original volume to obtain the enzymolysis delicate flavor extracting solution.

(II) fermenting the extracting solution:

inoculating a fermentation strain to the prepared enzymolysis delicate flavor extracting solution, adding 4.5-5.5% of salt (mass volume ratio, unit g/ml, calculated by the volume of the enzymolysis delicate flavor extracting solution), and fermenting for more than 60 hours at the temperature of 33-37 ℃; after fermentation, sterilizing (121 deg.C for 20min) to obtain the freshener;

the fermentation strain is composed of bacillus subtilis, saccharomycetes and lactic acid bacteria, and the weight ratio of the bacillus subtilis to the saccharomycetes to the lactic acid bacteria is 4.5-5.5: 1.5-2.5: 1, and preferably 5:2: 1.

Further, in the step (one) (1), the preservative is selected from a potassium sorbate solution, and the concentration of the potassium sorbate solution is 0.5-1.5% (mass-to-volume ratio, unit g/ml), preferably 1%.

Further, in the step (a) (1), the soaking time is 30 seconds.

Further, in the step (a) and (2), the ratio of the homogenate to the liquid is 1: 2-5 (mass to volume, unit g/ml), preferably 1: 3.

Further, in the step (a) (3), the pH of the shrimp head homogenate is adjusted using an arginine solution.

Further, in the step (a) and (3), the ultraviolet irradiation is performed by using an ultraviolet lamp with the wavelength of 250nm and the power of 30W.

Further, in the step (I) (4), the addition amount of the protease YDCP is 500U/g (based on the total weight of the shrimp heads and the oyster skirt meat).

Further, in the step (one) and (6), the adding amount of the activated carbon is 0.5-1.5% (mass-volume ratio, unit g/ml), preferably 1%.

Further, in the step (a) (7), the centrifugation conditions are as follows: centrifuge at 8000r/min for 15 min. The conditions of rotary evaporation were: the vacuum degree is 0.1MPa, and the temperature is 60 ℃.

Further, in the step (II), the inoculation amount of the zymophyte is 0.1 percent (mass-volume ratio, unit g/ml).

Further, in the step (two), the fermentation conditions are as follows: fermenting at 35 deg.C for 72 hr.

The flavor enhancer prepared by the method can be added into sauce to prepare a flavoring agent, and due to the improvement of the delicate flavor, the use of soy sauce and salt is required to be reduced to ensure the reasonable sense of the flavoring agent, so that the sodium content is also reduced, and the aim of reducing salt and enhancing the flavor is fulfilled. Specifically, the preparation method of the flavoring agent can be as follows: curing the prepared base material, adding 10% (volume percentage) of concentrated solution and prepared ingredients, immediately adding 10% (mass volume percentage, unit g/ml) of starch for gelatinization, and stopping heating immediately after the viscosity of the sauce becomes viscous, thus finishing sauce preparation. Starch gelatinization is a process that changes the ordered structure of starch molecules into a disordered state. The gelatinized starch has increased viscosity, and can form gel with low cost. The gelatinization degree is a key control point of the process, the gelatinization is insufficient, the sauce is not good in texture, the soup is turbid when the sauce is eaten, uneven suspended matters exist, and the mixing effect with the oil is not ideal; but the gelatinization is excessive, and the sauce is not well formed and has poor texture quality. In the production control, the gelatinization should be stopped by immediately cooling as soon as a viscosity increase occurs.

The freshener is prepared from shrimp heads and oyster skirts as raw materials. According to the preparation method, endogenous enzymes in the shrimp heads are adopted to generate autolysis under controlled conditions, so that the flavor substances are generated. The shrimp heads are likely to decay in the autolysis process, so that the shrimp heads are soaked in the potassium sorbate solution for antiseptic pretreatment, the environment is strictly controlled in the operation process, the clean production environment is ensured as much as possible, and the microbial pollution is avoided. The flavor substance of the head of Antarctic krill is utilized, and meanwhile, the oyster skirt zymolyte is also utilized as the flavor substance. In the experimental process, the bitter substances are generated by referring to other literature methods, and after the methods such as an embedding method, a microbiological method, an active carbon removing method and the like are adopted, the bitter taste cannot be effectively removed by various methods, but the original flavor can be greatly reserved by an active carbon treating method, and the removing effect is best, so the bitter taste is removed by the active carbon.

The freshener can be used as a seasoning bag in instant food (such as instant noodles, instant soup and other foods), and can ensure the flavor and nutrition of the instant food. The compound seasoning can be used as a compound seasoning for daily cooking, the compound seasoning is used for replacing sugar, soy sauce, monosodium glutamate and the like, the compound seasoning becomes an important choice for more families and catering enterprises, the compound seasoning is rich in taste, convenient to operate, strong in delicate flavor and high in nutritional value, and the compound seasoning can be reasonably used for improving the enjoyment of food and improving the nutritional value when daily cooking such as soup cooking, dish frying and dish stewing.

The freshener and the preparation method have the following advantages:

(1) the contents of the flavor-producing amino acid and flavor peptide are higher, and the sodium content is lower, so that the purposes of reducing salt and not reducing freshness and reasonably using the hypertension and other people are realized.

(2) The method is prepared by adopting a biological method, and the shrimp heads and the oyster skirts of the Antarctic krill are fully utilized, so that high-value utilization of byproducts is realized, compared with flavor substances obtained by processing common shrimp meat, shrimp meal or oyster peptides and the like, the method has higher delicate flavor degree, is a common synergistic effect of raw materials and methods, the shrimp heads and the oyster skirts belong to the byproducts, and the utilization value is lower, but the method can effectively utilize the byproducts, and the effect is not inferior to that of the traditional shrimp meat and the like. In the method, different enzymes are utilized for hydrolysis, and the flavor fingerprint shows that the delicate flavor substances prepared by utilizing YDCP enzyme hydrolysis are more obvious or unique, and the raw materials are cheap.

(3) The Antarctic krill heads and the oyster skirts contain various nutritional ingredients, are rich in substances such as protein and amino acid, and contain taurine, mineral elements and the like.

(4) High-heat processing is not used in the process of extracting the delicate flavor, a medium-low temperature processing mode is adopted, the flavor is retained to the maximum extent, side reaction harmful products generated by high-heat processing are avoided, and the nutrition is ensured.

(5) Ultraviolet induction is adopted to carry out autolysis on the euphausia superba heads to generate umami substances, and the umami substances, small molecular peptides and amino acids are extracted.

The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.

Drawings

FIG. 1: SDS-PAGE electrophoresis detection result, wherein M is a protein molecular weight Marker; 1 is protein electrophoresis of cell disruption solution; 2, performing electrophoresis on the protease YDCP protein obtained after purification.

FIG. 2: and carrying out GC-IMS qualitative analysis on the obtained fingerprint.

Detailed Description

The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.

The fermentation strains used by the invention are as follows: yeast (aroma-producing active dry yeast, 1X 10)¹⁰cfu/g, Angel Yeast Co., Ltd.), Bacillus subtilis (Bacillus subtilis powder, 1X 10)¹⁰cfu/g, Saian, Kangze Biotech Co., Ltd.), Lactobacillus bulgaricus (Lactobacillus bulgaricus, 1X 10)¹⁰cfu/g, norson organism, Qingdao).

Experiment 1 obtaining protease YDCP

The special protease YDCP used in the invention is protease from Geobacillus stearothermophilus (Geobacillus stearothermophilus) which is widely existedThe gene sequence of the protease is expressed and is applied to the preparation of seasonings, so that a product with unique flavor is obtained, the amino acid sequence of the product is shown as SEQ ID NO.1, and the expression gene of the protease (after codon optimization, the gene sequence is shown as SEQ ID NO. 2) is obtained by whole-gene synthesis; after gene synthesis, connecting the gene to a plasmid pET-28a by using a seamless splicing technology to construct a recombinant expression vector, transforming the expression vector into escherichia coli BL21(DE3) according to a standard calcium chloride heat shock method, and screening the transformed escherichia coli in an LB culture medium plate containing ampicillin; selecting the successfully transformed positive monoclonal, transforming the positive monoclonal into a liquid LB culture medium, and culturing the positive monoclonal at 37 ℃ until the thallus OD₆₀₀0.6, adding 1mM inducer IPTG, culturing for 10h at 20 ℃, centrifugally collecting thalli after culturing, re-suspending the thalli by using a phosphate buffer solution with pH 7.0, ultrasonically crushing bacterial suspension for 15min, centrifugally collecting supernatant of the crushed solution, passing the supernatant through a nickel column, eluting by using 80mM imidazole solution, detecting eluent by using SDS-PAGE electrophoresis, and displaying a band with the molecular weight of about 35kDa by an electrophoresis result (shown in figure 1), wherein the band is consistent with the predicted molecular weight, which shows that the protease is successfully separated, and drying the eluent to obtain protease powder.

Experiment 2 comparison of the freshener obtained by enzymatic hydrolysis of different proteases

The preparation steps of the freshener are as follows:

(1) soaking: cleaning 50 g of Antarctic krill heads, and soaking in a 1% potassium sorbate solution for 30 s; cleaning 50 g of oyster skirt, and soaking in 1% potassium sorbate solution for 30 s; taking out after soaking.

(2) Pulping: adding water three times the weight of the soaked shrimp heads and oyster skirt meat into the soaked shrimp heads and oyster skirt meat respectively to prepare homogenate, wherein the material-liquid ratio of the homogenate is 1: 3.

(3) Treating homogenate: adjusting pH of the shrimp head homogenate to 7.5 (adjusted by 1% arginine solution), and irradiating with ultraviolet lamp with wavelength of 250nm and power of 30W for 20min in 50 deg.C water bath to induce endogenous enzyme to accelerate autolysis process; after the UV irradiation, the mixture is allowed to stand and autolyzed for 2 hours in a water bath at 50 ℃.

(4) Enzymolysis: mixing the shrimp head homogenate and the oyster skirt meat homogenate uniformly, dividing into 4 parts (each part is 100g) and performing enzymolysis experiment in 4 groups, wherein,

adding papain (500U/g sample) into group B, and performing enzymolysis at 60 deg.C at pH 6 for 2.5 h;

adding flavourzyme (500U/g sample) into group C, and performing enzymolysis for 2.5h at the pH value of 6.5 and the temperature of 53 ℃;

adding protease YDCP 500U/g into group D, and performing enzymolysis at 55 deg.C and pH 7.5 for 2.5 h;

group A was used as a control, and was left standing at 50 ℃ for 2.5 hours at pH 7.5 without adding protease.

(5) Enzyme deactivation: after the above enzymatic hydrolysis, the enzyme was inactivated in a water bath at 90. + -. 2 ℃ for 15 minutes (the same treatment was also carried out for group A).

(6) Debitterizing: after the enzyme deactivation, 1% of active carbon (bitter peptides are generated in the enzymolysis process, so that the bitter taste is removed by adopting an active carbon adsorption method) is added, and debittering is carried out for 30 minutes under the condition of 50 ℃ water bath.

(7) Centrifuging: after the debitterizing, centrifuging at 8000r/min for 15min, and taking supernatant fluid, namely the enzymolysis delicate flavor extracting solution.

(II) fermenting the extracting solution:

respectively inoculating fermentation strains to the prepared 4 parts of enzymolysis delicate flavor extracting solution according to the inoculation amount of 0.1%, adding 5% of salt, and fermenting at 35 ℃ for 72 hours; after fermentation, sterilizing (121 ℃ for 20min) to obtain 4 parts of the freshener;

the fermentation strain is composed of bacillus subtilis, saccharomycetes and lactic acid bacteria, and the weight ratio of the bacillus subtilis to the saccharomycetes to the lactic acid bacteria is 5:2: 1.

Flavor analysis of the freshener prepared by enzymolysis of different proteases:

2g of each of the 4 parts of the prepared freshener is put into a 20ml headspace sample injection bottle and sealedPlacing in an automatic sample injector, incubating at 50 deg.C for 10min with a vibration heater, and sucking the upper layer with a sample needle at a sample injection temperature of 55 deg.C to obtain 500 μ L. The column temperature was set at 40 ℃, the drift gas flow rate was set at 150ml/min, the IMS drift tube temperature was set at 45 ℃, the ionization source in the drift tube was deuterium emission (beta ray), and the ionization mode was positive ions. The carrier gas is N in the determination process₂Setting the flow rate of the carrier gas: the initial flow rate is 2ml/min and is kept for 2min, then the flow rate is increased to 15ml/min and is kept for 8min, then the flow rate is increased to 50ml/min and is kept for 5min, then the flow rate is increased to 100ml/min and is kept for 5min, then the flow rate is increased to 150ml/min and is kept for 10min, and the total analysis time is 30 min. Each sample is parallelly measured for 2 times, the volatile flavor components of the freshener are collected by using self-provided LAV analysis software of GC-IMS, a fingerprint spectrum is established, and qualitative analysis is carried out according to a NIST gas phase retention index database and IMS migration time in the software of GC x IMS Library Search.

Sensory evaluation of the freshener prepared by enzymolysis of different proteases:

the 4 prepared fresheners are separately packaged in different sample bottles, 5 men and women engaged in food research are invited to perform sensory evaluation, each time the people taste the fresheners by observing for more than 5s under the light of a white fluorescent lamp, and the people taste fine in the mouth for more than 5s and stir the fresheners by using the tongue. The texture, flavor, taste and color were scored separately, and each time a mouth was rinsed thoroughly before tasting, with the scoring criteria shown in table 2.

The above analysis and evaluation led to the conclusion that:

after GC-IMS qualitative analysis, obtaining a fingerprint (shown in figure 2) and qualitative substance description (shown in Table 1), wherein the flavor substances special in the freshener without protease treatment (group A) comprise ethyl pelargonate, 2-octanone, ethyl propionate and ethyl isobutyrate; the special flavor substances in the papain treatment (group B) comprise isobutyl acetate, propyl acetate and 2, 3-pentanedione; the flavor substances of the freshener treated by the flavor protease (group C) and the YdcP protease (group D) are the same, and the flavor substances comprise n-valeric acid, methyl isobutyl ketone, dipropyl disulfide, phenethyl alcohol, propyl butyrate, cyclohexanone, hydroxyacetone, alpha-pinene and 2, 6-dimethyl pyrazine. It can be seen that there were more flavor substances after treatment with YDCP protease (group C had a higher concentration of flavor substances in the umami extract after treatment with group D compared to group D).

Sensory evaluation shows that the freshener subjected to enzymolysis by YdcP protease has the highest score, is particularly prominent in the aspects of fragrance and taste, and the score table obtained after only integral parts are reserved after the average scores of the scores of 10 participants are calculated is shown in Table 3. In terms of color and texture, the differences of the sauce material characteristics are not outstanding, so that the difference of 1 point above 10 points can indicate that the differences are obvious.

TABLE 1

TABLE 2

TABLE 3

The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.

Sequence listing

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Claims

1. A preparation method of a freshener is characterized in that: the euphausia superba head and oyster skirt meat are used as raw materials, and are obtained by enzymolysis through protease YDCP and fermentation; the amino acid sequence of the protease YDCP is shown in SEQ ID NO. 1;

the method comprises the following steps:

(1) soaking: cleaning the head of Antarctic krill, and soaking in a preservative; cleaning oyster skirts and soaking the oyster skirts in a preservative; taking out after soaking for later use;

(2) pulping: respectively making the soaked shrimp heads and oyster skirt meat into homogenate;

(3) treating homogenate: adjusting the pH value of the shrimp head homogenate to 7.0-8.0, and carrying out ultraviolet irradiation for 15-25 minutes under the water bath condition of 50 +/-2 ℃ to induce endogenous enzyme so as to accelerate the autolysis process; after ultraviolet irradiation, standing and autolyzing for 1.5-2.5 hours under the water bath condition of 50 +/-2 ℃;

(4) enzymolysis: mixing the shrimp head homogenate and the oyster skirt meat homogenate, adding protease YDCP, and carrying out enzymolysis for 2-3 hours at the temperature of 50 +/-2 ℃;

(5) enzyme deactivation;

(6) debitterizing;

(7) centrifuging: after debitterizing, centrifuging, taking supernatant, and concentrating to obtain enzymolysis delicate flavor extract;

(II) fermenting the extracting solution:

inoculating a fermentation strain to the prepared enzymolysis delicate flavor extracting solution, adding 4.5-5.5% of salt, and fermenting for more than 60 hours at the temperature of 33-37 ℃; after the fermentation is finished, sterilizing to obtain the freshener;

the fermentation strain is composed of bacillus subtilis, saccharomycetes and lactic acid bacteria, and the weight ratio of the bacillus subtilis to the saccharomycetes to the lactic acid bacteria is 4.5-5.5: 1.5-2.5: 1.

2. The method for preparing a freshener according to claim 1, wherein: in the step (I) (1), the preservative is selected from a potassium sorbate solution, the concentration of the potassium sorbate solution is 0.5-1.5%, and the soaking time is 30 seconds.

3. The method for preparing a freshener according to claim 1, wherein: in the step (I) and the step (2), the material-liquid ratio of the homogenate is 1: 2-5.

4. The method for preparing a freshener according to claim 1, wherein: in the steps (a) and (4), the addition amount of the protease YDCP is 500U/g.

5. The method for preparing a freshener according to claim 1, wherein: in the step (I) and the step (6), active carbon is added for debittering, and the adding amount of the active carbon is 0.5-1.5%.

6. The method for preparing a freshener according to claim 1, wherein: in the step (II), the weight ratio of the bacillus subtilis, the saccharomycetes and the lactic acid bacteria is 5:2: 1.

7. The method for preparing a freshener according to claim 1, wherein: in the step (II), the fermentation conditions are as follows: fermenting at 35 deg.C for 72 hr.

8. The freshener prepared by the preparation method of any one of claims 1 to 7.

9. Use of a flavour enhancer according to claim 8 in the preparation of a flavouring agent.

10. The application of the protease YDCP with an amino acid sequence shown as SEQ ID NO.1 in the preparation of a freshener.