CN117617478A - Natural flavor enhancer for pinus koraiensis and preparation method thereof - Google Patents
Natural flavor enhancer for pinus koraiensis and preparation method thereof Download PDFInfo
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- CN117617478A CN117617478A CN202311514690.8A CN202311514690A CN117617478A CN 117617478 A CN117617478 A CN 117617478A CN 202311514690 A CN202311514690 A CN 202311514690A CN 117617478 A CN117617478 A CN 117617478A
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a natural flavor enhancer for pinus koraiensis and a preparation method thereof, wherein the natural flavor enhancer for pinus koraiensis is prepared by taking pinus koraiensis as a raw material and adopting a method of biological enzymolysis for two times; firstly, performing enzymolysis for one time by adopting cellulase to break cell walls of the pinus koraiensis so as to fully dissolve soluble proteins in the cell walls; then, acid protease is adopted for secondary enzymolysis, so that cell walls are broken more completely, soluble proteins are hydrolyzed, and flavor-developing polypeptides and amino acids in the soluble proteins are fully dissolved; the two biological enzymolysis are combined, so that the raw materials are subjected to enzymolysis more thoroughly, the effective components and related flavor substances in the pinus koraiensis are fully extracted, the enzymolysis rate and the enzymolysis efficiency are improved, the technical problems of low utilization rate, low added value, resource waste and serious environmental pollution of the existing pinus koraiensis are solved, and the technology belongs to the technical field of manufacturing of natural marine flavoring agents.
Description
Technical Field
The invention relates to the technical field of manufacturing of natural marine flavor enhancers, in particular to a natural flavor enhancer for pinus koraiensis and a preparation method thereof.
Background
The flavoring agent is also called flavor enhancer, and is a compound capable of enhancing the flavor of food. Along with the high-speed development of social economy, the requirements of people on the quality of life at a high level are higher and higher, and the flavor enhancer is gradually developed from the original one to meet the taste requirement, so that the current pursuit of nutrition, safety, health care and other aspects is gradually developed. Research has shown that natural marine flavoring agents are favored by more consumers due to the fact that the natural marine flavoring agents contain rich amino acids, polypeptides, nucleotides, organic acids and other flavoring substances and polysaccharide, taurine and other active substances.
The pinus koraiensis is a large-scale seaweed widely distributed in the temperate sea area, and has medicinal value and edible value. The protein content in the pinus koraiensis is higher and accounts for 21.67%, the percentage of essential amino acids is 42.2% of the total amino acids, the amino acid score is 86, which is higher than that of kelp and laver, wherein the proportion of glutamic acid, aspartic acid, glycine, alanine and the like which are delicious amino acids is higher, so that the pinus koraiensis has better development and application potential of the flavoring agent. At present, research on the pinus koraiensis at home and abroad is mostly focused on extraction of macromolecular active substances such as polysaccharide and small molecular compounds such as low-polarity plant sterols, and release and extraction of flavor substances are not reported. Therefore, the deep processing of the pinus koraiensis is performed, the utilization rate of the pinus koraiensis is improved, the added value of the pinus koraiensis is increased, the resource waste and the environmental pollution are reduced, and the pinus koraiensis has wide application prospect.
Disclosure of Invention
The invention adopts the technical scheme of providing the natural flavor enhancer for the pinus koraiensis and the preparation method thereof, so as to solve the technical problems of low utilization rate, low added value, resource waste and serious environmental pollution of the existing pinus koraiensis.
In order to solve the technical problems, the embodiment of the application provides a preparation method of a natural flavor enhancer for pinus koraiensis, which comprises the following steps:
(1) Cleaning and drying: cleaning and drying the pinus koraiensis;
(2) Crushing: crushing the dried pinus koraiensis to obtain pinus koraiensis granules;
(3) Dissolving: dissolving the pinus koraiensis granules in a buffer solution to obtain a pinus koraiensis solution;
(4) Primary enzymolysis: performing primary enzymolysis on the pinus koraiensis solution to obtain primary enzymolysis liquid;
(5) Enzyme deactivation: sterilizing the primary enzymolysis liquid;
(6) And (3) secondary enzymolysis: performing secondary enzymolysis on the sterilized primary enzymolysis liquid to obtain secondary enzymolysis liquid;
(7) And (3) centrifuging: centrifuging the secondary enzymolysis liquid to obtain a supernatant;
(8) Concentrating: concentrating the supernatant to obtain a concentrated solution;
(9) Granulating: mixing the concentrated solution with carrier, drying, and granulating to obtain natural flavoring agent.
Preferably, in the step (1), the drying temperature is 40-55 ℃, and the drying is carried out until the moisture content is less than 15%.
Preferably, in the step (2), the dried pinus koraiensis is crushed to have a particle size of 40-60 meshes.
Preferably, in the step (3), the buffer solution is prepared from 0.1mol/L citric acid solution and 0.2mol/L disodium hydrogen phosphate solution according to a volume ratio of 12.63:7.37, and the dosage of the buffer solution is that each gram of the pinus koraiensis granules is dissolved in 20mL of the buffer solution.
Preferably, in the step (4), cellulase is adopted for primary enzymolysis, the specification is 400U/mg, and the addition amount of the cellulase is 9-13% of the mass of the echinocystis spinosa particles; the enzymolysis temperature of the primary enzymolysis is 40-60 ℃, the enzymolysis time is 90-210 min, and the initial pH value is 5.5-7.5.
Preferably, in the step (5), the sterilization temperature is 90-95 ℃ and the sterilization time is 1-2 min.
Preferably, in the step (6), the secondary enzymolysis adopts acid protease with the specification of 50U/mg and the addition amount of the acid protease is 20-70% of the mass of the echinocystis particles; the enzymolysis temperature of the secondary enzymolysis is 35-55 ℃, the enzymolysis time is 60-210 min, and the initial pH value is 2.0-4.5.
Preferably, in the step (7), the rotational speed of the centrifugal treatment is 3500-4500 r/min, and the time is 20-30 min.
Preferably, in the step (9), the carrier is any one or a combination of at least two of maltodextrin, corn starch, tapioca starch, potato starch and sweet potato starch, and the addition amount of the carrier is 5-15% of the mass of the chaetonia ostii particles; the drying mode is low-temperature freeze drying, the temperature is-65 to-55 ℃, and the pressure is 8 to 12Pa.
The embodiment of the application also provides the natural flavor enhancer of the pinus koraiensis prepared by the preparation method of any one of the above.
The invention provides a natural flavor enhancer for pinus koraiensis and a preparation method thereof, and the beneficial effects are that: the invention takes the pinus koraiensis as a raw material and adopts a method of twice biological enzymolysis to prepare the flavoring agent with the aromatic smell of the pinus koraiensis. Firstly, performing enzymolysis for one time by adopting cellulase to break cell walls of the pinus koraiensis so as to fully dissolve soluble proteins in the cell walls; then, acid protease is adopted for secondary enzymolysis, so that cell walls are broken more completely, soluble proteins are hydrolyzed, and flavor-developing polypeptides and amino acids in the soluble proteins are fully dissolved; the combination of the two biological enzymolysis realizes more thorough enzymolysis of the raw materials, fully extracts the effective components and related flavor substances in the pinus koraiensis, and improves the enzymolysis rate and the enzymolysis efficiency; finally, a low-temperature freeze drying process is adopted, so that active ingredients and nutritional ingredients in the pinus koraiensis are reserved to the maximum extent, and the comprehensive utilization rate of the pinus koraiensis is greatly improved.
The preparation method and the using equipment are simple, and the prepared natural flavor enhancer for the pinus koraiensis is rich in a plurality of flavor-developing substances and nutritional ingredients such as amino acids, small peptides, trace elements, minerals, polysaccharides and the like, is delicious in taste, can be dissolved in water, and is more suitable for the current social health preserving and healthy diet concept; in addition, the invention provides technical parameters for extracting the flavor-developing substances of the pinus koraiensis, promotes the development of the flavor-developing agent, improves the utilization rate and the added value of the marine low-value protein, reduces the environmental pollution caused by resource waste, and promotes the healthy and sustainable development of marine economy.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following description will briefly introduce the drawings that are needed in the embodiments or the description of the prior art, it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a standard curve of an amino acid drawn by the standard curve method of the present invention;
FIG. 2 is a graph showing the effect of different cellulase addition amounts on the extraction effect of soluble proteins in Zostera Marina;
FIG. 3 is a graph showing the effect of different enzymolysis temperatures on the extraction of soluble proteins from the Zostera Marinae;
FIG. 4 is a graph showing the effect of different initial pH values on the extraction of soluble proteins from Zostera Marina;
FIG. 5 is a graph showing the effect of different enzymolysis times on the extraction effect of soluble proteins in the Zostera Marina;
FIG. 6 is a graph showing the effect of the addition of different acidic proteases on the extraction of amino acids from soluble proteins;
FIG. 7 is a graph showing the effect of different enzymolysis temperatures on the extraction of amino acids in soluble proteins;
FIG. 8 is a graph showing the effect of different enzymolysis times on the extraction of amino acids in soluble proteins;
FIG. 9 is a graph showing the effect of different initial pH values on the extraction of amino acids from soluble proteins.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved by the present application more clear, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. The methods used in the invention are all conventional methods unless specified otherwise; the raw materials and devices used, unless otherwise specified, are all conventional commercial products.
Extraction of soluble protein in the Zostera Marina by cellulase
(1) Influence of the addition amount of cellulase on the extraction effect of soluble protein in the Zostera Marinae
Example 1
A preparation method of a natural flavor enhancer for a pinus koraiensis comprises the following steps:
(1) Cleaning and drying: cleaning the pinus koraiensis, and then drying the pinus koraiensis at the temperature of 40-55 ℃ until the water content of the pinus koraiensis is less than 15%, thus obtaining dried pinus koraiensis;
(2) Crushing: crushing the dried pinus koraiensis by adopting a crusher until the granularity is 40-60 meshes to obtain pinus koraiensis granules;
(3) Dissolving: preparing a buffer solution from 0.1mol/L citric acid solution and 0.2mol/L disodium hydrogen phosphate solution according to a volume ratio of 12.63:7.37, and then dissolving the pinus koraiensis particles in the buffer solution according to a ratio that each gram of pinus koraiensis particles is dissolved in 20mL of the buffer solution to obtain a pinus koraiensis solution;
(4) Primary enzymolysis: adding 400U/mg cellulase into the Zostera Marina solution, and performing primary enzymolysis, wherein the addition amount of cellulase is 9% of the quality of Zostera Marina granule, the enzymolysis temperature is 45deg.C, the enzymolysis time is 120min, and the initial pH value is 6.0 to obtain primary enzymolysis solution;
(5) Enzyme deactivation: sterilizing the primary enzymolysis liquid at 90-95 ℃ for 1-2 min to obtain sterilized primary enzymolysis liquid;
(6) And (3) secondary enzymolysis: adding 50U/mg acid protease into the sterilized primary enzymolysis liquid, and performing secondary enzymolysis, wherein the addition amount of the acid protease is 20% of the mass of the particle of the pinus koraiensis, the enzymolysis temperature is 45 ℃, the enzymolysis time is 150min, and the initial pH value is 3.0, so as to obtain secondary enzymolysis liquid;
(7) And (3) centrifuging: centrifuging the secondary enzymolysis liquid at the rotating speed of 3500-4500 r/min for 20-30 min until no macroscopic particles of the pinus koraiensis are in the supernatant, and separating to obtain the supernatant;
(8) Concentrating: concentrating the supernatant to obtain a concentrated solution;
(9) Granulating: the dry maltodextrin is used as a carrier, the maltodextrin with the addition amount of 10 percent of the mass of the chaetoceros particles is added into the concentrated solution, the mixture is uniformly mixed, after the pre-freezing treatment, the low-temperature vacuum freeze drying is carried out under the environment of-60 ℃ and 10Pa, and the pale yellow particles are obtained, namely the natural flavor enhancer of the chaetoceros.
In the preparation process, in the step (4), the content of soluble protein in the primary enzymolysis liquid is detected by adopting a Coomassie brilliant blue method, and the specific process is as follows:
sample solution: and (3) centrifuging the primary enzymolysis liquid at the rotating speed of 2500r/min for 10-30 min, separating, taking supernatant, and diluting to obtain a sample solution.
Protein standard solution: protein standard solution (0.524 g/L) in a protein quantitative determination kit of Nanjing established biotechnology Co.
Coomassie brilliant blue color development liquid: the coomassie brilliant blue stock solution in the kit for quantitative determination of proteins by the company of biosciences, established in south kyo, was used according to the coomassie brilliant blue stock solution: distilled water=1:4 was formulated as coomassie brilliant blue color solution.
Blank, standard and assay tubes were configured in the amounts shown in table 1:
TABLE 1 configuration of blank tube, standard tube and measurement tube
| Blank pipe | Standard tube | Measuring tube | |
| Distilled water/mL | 0.05 | - | - |
| Protein standard solution/mL | - | 0.05 | - |
| Sample solution/mL | - | - | 0.05 |
| Coomassie brilliant blue color solution/mL | 3.0 | 3.0 | 3.0 |
Uniformly mixing the solutions in the blank tube, the standard tube and the measuring tube, standing for 10min, respectively detecting the absorbance of the blank tube, the standard tube and the measuring tube by using a spectrophotometer, setting the absorption wavelength to 595nm in the detection process, setting the optical path to 1cm, and zeroing by using distilled water; after detection, the content of soluble protein in the primary enzymolysis liquid is calculated according to the following formula:
wherein: c: the content of soluble protein in the primary enzymolysis liquid, g/L;
A measurement : measuring the absorbance of the tube;
A blank space : absorbance of the blank tube;
A standard of : absorbance of standard tube;
C standard of : concentration of protein standard solution, g/L;
n: dilution factor of the sample solution.
In the step (6), the content of amino acid in the secondary enzymolysis liquid is detected by adopting a standard curve method, and the specific process is as follows:
an amino acid standard solution (50 mu g/mL) in an Amino Acid (AA) detection kit (ninhydrin colorimetric method) of Shanghai Ye Biotech company is used as a standard substance, deionized water is used for dilution to obtain standard substance solutions with the concentration of 0.4 mu g/mL, 0.8 mu g/mL, 1.2 mu g/mL, 1.6 mu g/mL and 2.0 mu g/mL respectively, a spectrophotometer is used for detecting the absorbance of the standard substance solution at an absorption wavelength of 570nm respectively, the amino acid concentration of the standard substance solution is used as an abscissa, the absorbance is used as an ordinate, a scatter diagram is drawn, then a standard curve is drawn through linear regression, as shown in figure 1, the standard curve of the amino acid concentration and the absorbance is calculated to be Y=0.081X+0.0042, and the standard variance R thereof is calculated 2 =0.9930 > 0.99, indicating that the standard curve is well-linear and can be used to calculate the α -amino acid content.
Sample solution: and (3) centrifuging the secondary enzymolysis liquid at the rotating speed of 2500r/min for 10-30 min, separating, taking supernatant, and diluting to obtain a sample solution.
Detecting the absorbance of a sample solution at 570nm absorption wavelength by using a spectrophotometer, calculating the corresponding amino acid concentration by using a standard curve Y, substituting the amino acid concentration into the following formula, and calculating the content of the amino acid in the secondary enzymolysis liquid:
wherein: c (C) 2 : the content of amino acid in the secondary enzymolysis liquid is 1g/100g;
C 1 : amino acid concentration calculated by standard curve Y, μg/mL;
V T : total volume of sample solution dilution, mL;
w: the weight of the diluted sample solution, g;
n: dilution factor of the sample solution.
Example 2
The difference between the implementation method and the embodiment 1 is that in the step (4), the addition amount of cellulase is 10% of the mass of the echinocystis spinosa particles, and other steps are the same, so that primary enzymolysis liquid is obtained, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 3
The difference between the implementation method and the embodiment 1 is that in the step (4), the addition amount of cellulase is set to 11% of the mass of the echinocystis spinosa particles, and other steps are the same, so that primary enzymolysis liquid is obtained, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 4
The difference between the implementation method and the embodiment 1 is that in the step (4), the addition amount of cellulase is set to be 12% of the mass of the echinocystis spinosa particles, and other steps are the same, so that primary enzymolysis liquid is obtained, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 5
The difference between the implementation method and the embodiment 1 is that in the step (4), the addition amount of the cellulase is set to be 13% of the mass of the echinocystis spinosa particles, and other steps are the same, so that primary enzymolysis liquid is obtained, and the content of soluble protein in the primary enzymolysis liquid is detected.
The effect of the different cellulase addition amounts of examples 1-5 on the extraction of soluble proteins from the Zostera Marinae was plotted and the results are shown in FIG. 2. As can be seen from fig. 2, as the cellulase addition amount increases, the content of soluble protein in the primary enzymatic hydrolysate increases significantly, and when the cellulase addition amount is 12%, the content of soluble protein is the highest, and the cellulase addition amount is further increased, so that the extraction effect tends to decrease; this is because the cellulase hydrolyzes cellulose in the cell wall of the pinus koraiensis to release the soluble protein in the cell, and the release amount of the soluble protein increases with the increase of the addition amount of the cellulase, however, when the addition amount of the cellulase is too large, the cellulases may adhere to each other, so that the mass transfer efficiency between the cellulase and the pinus koraiensis is reduced, thereby slowing down the reaction efficiency and reducing the release amount of the soluble protein; therefore, the addition amount of cellulase is preferably 12%.
(2) Influence of enzymolysis temperature on extraction effect of soluble protein in Zostera Marinae
Example 6
The difference between the method and the embodiment 4 is that in the step (4), the enzymolysis temperature is set to 40 ℃, other steps are the same, primary enzymolysis liquid is obtained, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 7
The difference between the method and the embodiment 4 is that in the step (4), the enzymolysis temperature is set to 50 ℃, other steps are the same, primary enzymolysis liquid is obtained, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 8
The difference between the method and the example 4 is that in the step (4), the enzymolysis temperature is set to 55 ℃, other steps are the same, primary enzymolysis liquid is obtained, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 9
The difference between the method and the embodiment 4 is that in the step (4), the enzymolysis temperature is set to 60 ℃, other steps are the same, primary enzymolysis liquid is obtained, and the content of soluble protein in the primary enzymolysis liquid is detected.
The effect of the different enzymolysis temperatures of examples 4, 6-9 on the extraction of soluble proteins in the pinus koraiensis is plotted, and the results are shown in fig. 3. As can be seen from fig. 3, as the enzymolysis temperature increases, the content of soluble protein in the primary enzymolysis liquid increases significantly, when the enzymolysis temperature is 45 ℃, the content of soluble protein is higher, and when the enzymolysis temperature increases to 50 ℃, the content of soluble protein is highest, but the increase is not obvious; when the enzymolysis temperature is further increased, the extraction effect is obviously reduced, because on one hand, the activity of the cellulase is reduced due to the excessively high temperature, and on the other hand, the content of the soluble protein in the primary enzymolysis liquid is reduced due to the bad changes such as denaturation and the like of the soluble protein at high temperature; therefore, from the economical point of view, the enzymolysis temperature is preferably 45 ℃.
(3) Effect of initial pH on the extraction Effect of soluble proteins in the Zostera Marinae
Example 10
The difference between the method and the example 7 is that in the step (4), the initial pH value is set to 5.5, and other steps are the same, so as to obtain primary enzymolysis liquid, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 11
The difference between the method and the example 7 is that in the step (4), the initial pH value is set to 6.5, and other steps are the same, so as to obtain primary enzymolysis liquid, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 12
The difference between the method and example 7 is that in the step (4), the initial pH value is set to 7.0, and other steps are the same, so as to obtain a primary enzymatic hydrolysate, and the content of soluble protein in the primary enzymatic hydrolysate is detected.
Example 13
The difference between the method and example 7 is that in the step (4), the initial pH value is set to 7.5, and other steps are the same, so as to obtain a primary enzymatic hydrolysate, and the content of soluble protein in the primary enzymatic hydrolysate is detected.
The effect of the different initial pH values of examples 7, 10-13 on the extraction of soluble proteins from Zostera Marinae was plotted and the results are shown in FIG. 4. As can be seen from fig. 4, with the increase of the initial pH, the content of soluble protein in the primary enzymatic hydrolysate increases significantly, and when the initial pH is 6.0, the content of soluble protein is higher, and when the initial pH is increased to 6.5, the content of soluble protein is highest, but the increase is not obvious; when the initial pH value is further increased, the extraction effect is obviously reduced, and the pH value of the reaction system is not matched with the optimal condition of the cellulase, so that the activity of the cellulase is reduced, even part of the cellulase is inactivated, the enzymolysis rate is reduced, and the content of soluble protein is reduced; therefore, from an economical point of view, the initial pH is preferably 6.0.
(4) Influence of enzymolysis time on extraction effect of soluble protein in pinus koraiensis
Example 14
The difference between the implementation method and the embodiment 7 is that in the step (4), the enzymolysis time is set to 90min, and other steps are the same, so as to obtain primary enzymolysis liquid, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 15
The difference between the implementation method and the embodiment 7 is that in the step (4), the enzymolysis time is set to 150min, other steps are the same, primary enzymolysis liquid is obtained, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 16
The difference between the implementation method and the embodiment 7 is that in the step (4), the enzymolysis time is set to 180min, other steps are the same, primary enzymolysis liquid is obtained, and the content of soluble protein in the primary enzymolysis liquid is detected.
Example 17
The difference between the implementation method and the embodiment 7 is that in the step (4), the enzymolysis time is set to 210min, and other steps are the same, so as to obtain primary enzymolysis liquid, and the content of soluble protein in the primary enzymolysis liquid is detected.
The effect of the different enzymolysis times of examples 7, 14-17 on the extraction of soluble proteins in the Zostera Marinae was plotted as a graph, and the results are shown in FIG. 5. As can be seen from fig. 5, as the enzymolysis time is prolonged, the content of soluble protein in the primary enzymolysis liquid is significantly increased, when the enzymolysis time is 150min, the content of soluble protein is highest, and when the enzymolysis time is further prolonged, the extraction effect is significantly reduced, which is probably because the enzymolysis time is too long, the soluble protein is aggregated, and the soluble protein is precipitated together with residues during centrifugation in the detection process, so that the numerical value is low; therefore, the enzymolysis time is preferably 150min.
(5) Orthogonal experiments of optimal enzymolysis conditions of cellulase
Four factors and three levels were set according to the above-mentioned enzymolysis conditions of examples 1 to 17 to obtain 9 experiments using A (enzymolysis temperature, °C), B (enzymolysis time, min), C (initial pH value), D (enzyme addition,%) as independent variables and soluble protein content (g/L) as response variables, and simulation was performed using Origin software, and experimental data are shown in tables 2 to 4.
TABLE 2 orthogonal experimental Table of enzymatic hydrolysis conditions of cellulases
TABLE 3 orthogonal experimental design and results of enzymatic hydrolysis conditions of cellulases
TABLE 4 orthogonal experimental analysis of variance table of enzymatic hydrolysis conditions of cellulases
As can be seen from the K value and the range R value in table 4, the effects of 4 factors on the enzymolysis effect are different in significance, and the effects are B > a > D > C according to the influence, namely the enzymolysis time > the enzymolysis temperature > the enzyme addition amount > the initial pH value; as can be seen from the optimal levels in Table 4, the optimal enzymolysis conditions of the cellulase on the Zostera Marinae are A1, B1, C3 and D3, namely the enzymolysis temperature is 40 ℃, the enzymolysis time is 90min, the initial pH value is 6.5, and the enzyme addition amount is 13%.
(II) extraction of amino acids from soluble proteins by acid proteases (i.e., hydrolysis of soluble proteins from Zostera Marinae)
(1) Influence of the amount of acid protease added on the extraction effect of amino acids in soluble proteins
Example 18
The difference between the implementation method and the embodiment 1 is that in the step (6), the addition amount of the acid protease is set to be 30% of the mass of the echinocystis spinosa particles, and other steps are the same, so that secondary enzymolysis liquid is obtained, and the content of amino acid in the secondary enzymolysis liquid is detected.
Example 19
The difference between the implementation method and the embodiment 1 is that in the step (6), the addition amount of the acid protease is set to be 40% of the mass of the echinocystis spinosa particles, and other steps are the same, so that secondary enzymolysis liquid is obtained, and the content of amino acid in the secondary enzymolysis liquid is detected.
Example 20
The difference between the implementation method and the embodiment 1 is that in the step (6), the addition amount of the acid protease is set to be 50% of the mass of the echinocystis spinosa particles, and other steps are the same, so that secondary enzymolysis liquid is obtained, and the content of amino acid in the secondary enzymolysis liquid is detected.
Example 21
The difference between the implementation method and the embodiment 1 is that in the step (6), the addition amount of the acid protease is set to be 60% of the mass of the echinocystis spinosa particles, and other steps are the same, so that secondary enzymolysis liquid is obtained, and the content of amino acid in the secondary enzymolysis liquid is detected.
Example 22
The difference between the implementation method and the embodiment 1 is that in the step (6), the addition amount of the acid protease is 70% of the mass of the echinocystis spinosa particles, and other steps are the same, so that secondary enzymolysis liquid is obtained, and the content of amino acid in the secondary enzymolysis liquid is detected.
The effect of the addition amounts of the different acidic proteases of examples 1 and 18 to 22 on the extraction of amino acids in the soluble protein was plotted, and the results are shown in FIG. 6. As shown in fig. 6, the content of amino acid in the secondary enzymolysis liquid is obviously increased along with the increase of the addition amount of the acid protease, when the addition amount of the acid protease is 60%, the reactant in the primary enzymolysis liquid is basically combined with the acid protease completely, the enzymolysis rate is directly proportional to increase, so that the cell wall is broken more completely, the content is fully discharged, the content of amino acid in the secondary enzymolysis liquid is very high, and when the addition amount of the acid protease exceeds 60%, the extraction effect is continuously increased, but the rising trend is greatly reduced, namely the increasing speed of the amino acid content is slow; therefore, the amount of the acid protease to be added is preferably 60% for the overall economic reasons.
(2) Effect of enzymolysis temperature on extraction effect of amino acids in soluble proteins
Example 23
The difference between the method and the example 19 is that in the step (4), the enzymolysis temperature is set to 35 ℃, the other steps are the same, the secondary enzymolysis liquid is obtained, and the content of the amino acid in the secondary enzymolysis liquid is detected.
Example 24
The difference between the method and the example 19 is that in the step (4), the enzymolysis temperature is set to 40 ℃, the other steps are the same, the secondary enzymolysis liquid is obtained, and the content of the amino acid in the secondary enzymolysis liquid is detected.
Example 25
The difference between the method and the example 19 is that in the step (4), the enzymolysis temperature is set to 50 ℃, the other steps are the same, the secondary enzymolysis liquid is obtained, and the content of the amino acid in the secondary enzymolysis liquid is detected.
Example 26
The difference between the method and the example 19 is that in the step (4), the enzymolysis temperature is set to 55 ℃, the other steps are the same, the secondary enzymolysis liquid is obtained, and the content of the amino acid in the secondary enzymolysis liquid is detected.
The effect of the different enzymatic hydrolysis temperatures of examples 19, 23-26 on the extraction of amino acids from soluble proteins was plotted and the results are shown in FIG. 7. As shown in fig. 7, the content of amino acid in the secondary enzymolysis liquid tends to increase and decrease with increasing enzymolysis temperature, and the content of amino acid is highest when the enzymolysis temperature is 45 ℃, because the activity of acid protease is proportional to the enzymolysis temperature in the range of 35-45 ℃, the activity of acid protease increases with increasing enzymolysis temperature, macromolecular glycoprotein on the cell wall is hydrolyzed, so that the content in the cell flows out more, but when the enzymolysis temperature exceeds the optimal temperature of acid protease, the activity of acid protease decreases, the hydrolysis effect of acid protease on soluble protein decreases, the content in the secondary enzymolysis liquid decreases, and the content of amino acid also decreases correspondingly; therefore, the enzymolysis temperature is preferably 45 ℃.
(3) Effect of enzymolysis time on extraction effect of amino acids in soluble proteins
Example 27
The difference between the method and the example 19 is that in the step (4), the enzymolysis time is set to 60min, and other steps are the same, so as to obtain a secondary enzymolysis liquid, and the content of amino acid in the secondary enzymolysis liquid is detected.
Example 28
The difference between the method and the example 19 is that in the step (4), the enzymolysis time is set to 90min, and other steps are the same, so as to obtain a secondary enzymolysis liquid, and the content of amino acid in the secondary enzymolysis liquid is detected.
Example 29
The difference between the method and the example 19 is that in the step (4), the enzymolysis time is set to 120min, the other steps are the same, the secondary enzymolysis liquid is obtained, and the content of the amino acid in the secondary enzymolysis liquid is detected.
Example 30
The difference between the method and the example 19 is that in the step (4), the enzymolysis time is set to 180min, the other steps are the same, the secondary enzymolysis liquid is obtained, and the content of the amino acid in the secondary enzymolysis liquid is detected.
Example 31
The difference between the method and the example 19 is that in the step (4), the enzymolysis time is set to 210min, and other steps are the same, so as to obtain a secondary enzymolysis liquid, and the content of amino acid in the secondary enzymolysis liquid is detected.
The effect of the different enzymolysis times of examples 19, 27-31 on the extraction of amino acids in the soluble protein was plotted as a graph, and the results are shown in FIG. 8. As can be seen from FIG. 8, the amino acid content in the secondary enzymolysis liquid basically tends to increase rapidly and then slowly to a stable state within the enzymolysis time of 0-210min, and the amino acid content is basically unchanged after the enzymolysis time of 150min, and is 2.38 times of that of the enzymolysis time of 60 min; the method is characterized in that along with the prolongation of the enzymolysis time, the cell wall of the pinus koraiensis is broken more completely, and meanwhile, the hydrolysis rate of the soluble protein is increased, so that the soluble protein is quickly converted into amino acid and polypeptide, and after the time of enzymolysis reaches 150min, the substrate is reacted completely, and even if the enzymolysis time is prolonged, more amino acid can not be hydrolyzed; therefore, from the viewpoint of economic efficiency, the enzymolysis time is preferably 150 minutes.
(4) Effect of initial pH on the extraction Effect of amino acids in soluble proteins
Example 32
The difference between the method and example 19 is that in the step (4), the initial pH value is set to 2.0, and the other steps are the same, so as to obtain a secondary enzymolysis liquid, and the content of amino acid in the secondary enzymolysis liquid is detected.
Example 33
The difference between the method and example 19 is that in the step (4), the initial pH is set to 2.5, and the other steps are the same, so as to obtain a secondary enzymatic hydrolysate, and the content of amino acid in the secondary enzymatic hydrolysate is detected.
Example 34
The difference between the method and example 19 is that in the step (4), the initial pH is set to 3.5, and the other steps are the same, so as to obtain a secondary enzymatic hydrolysate, and the content of amino acid in the secondary enzymatic hydrolysate is detected.
Example 35
The difference between the method and example 19 is that in the step (4), the initial pH value is set to 4.0, and the other steps are the same, so as to obtain a secondary enzymolysis liquid, and the content of amino acid in the secondary enzymolysis liquid is detected.
Example 36
The difference between the method and example 19 is that in the step (4), the initial pH is set to 4.5, and the other steps are the same, so as to obtain a secondary enzymatic hydrolysate, and the content of amino acid in the secondary enzymatic hydrolysate is detected.
The effect of the different initial pH values of examples 19, 32-36 on the extraction of amino acids from soluble proteins was plotted and the results are shown in FIG. 9. As can be seen from fig. 9, the content of amino acid in the secondary enzymolysis liquid shows a trend of increasing and then decreasing with the increase of the initial pH value, and at the initial pH of 3.5, the content of amino acid reaches the maximum value, and then the content of amino acid gradually decreases, because when the pH is more than 3.5, the pH value is too high, that is, the pH value of the reaction system is not matched with the optimal condition of the acid protease, so that the activity of the acid protease decreases, even part of the acid protease is deactivated, the enzymolysis rate decreases, and the content of amino acid also decreases; therefore, the initial pH is preferably 3.5.
(5) Orthogonal experiments on optimal enzymatic hydrolysis conditions of acid protease
Four factors and three levels were set according to the above-described enzymolysis conditions of examples 1 and 18 to 36 to obtain 9 experiments using A (enzymolysis temperature, °C), B (enzymolysis time, min), C (initial pH value), D (enzyme addition amount,%) as independent variables and amino acid content (g/100 g) as response variables, and simulation was performed using Origin software, and experimental data are shown in tables 5 to 7.
TABLE 5 orthogonal experimental Table of enzymatic hydrolysis conditions of acid proteases
TABLE 6 orthogonal experimental design and results of enzymatic hydrolysis conditions of acid proteases
TABLE 7 orthogonal experimental analysis of variance table of enzymatic hydrolysis conditions of acid protease
As can be seen from the K value and the extremely poor R value in Table 7, the effects of 4 factors on the enzymolysis effect are different in significance, and the effects are A > C > D > B according to the influence sizes, namely the enzymolysis temperature > initial pH value > enzyme addition amount > enzymolysis time; as can be seen from the optimum levels in Table 7, the optimum conditions for the enzymatic hydrolysis of soluble proteins by the acid protease were A3, B3, C2, D2, i.e., the enzymatic hydrolysis temperature was 50℃and the enzymatic hydrolysis time was 210min, the initial pH was 3.5, and the enzyme addition was 60%.
In summary, the optimal enzymolysis conditions for the primary enzymolysis are as follows: the enzymolysis temperature is 40 ℃, the enzymolysis time is 90min, the initial pH value is 6.5, and the enzyme addition amount is 13%; the optimal enzymolysis conditions for the secondary enzymolysis are as follows: the enzymolysis temperature is 50 ℃, the enzymolysis time is 210min, the initial pH value is 3.5, and the enzyme addition amount is 60 percent.
The invention also provides the natural flavor enhancer of the pinus koraiensis prepared in the examples 1-36.
In summary, the invention provides a natural flavor enhancer for the pinus koraiensis and a preparation method thereof, wherein the flavor enhancer with the aromatic flavor of the pinus koraiensis is prepared by taking the pinus koraiensis as a raw material and adopting a method of two times of biological enzymolysis. Firstly, performing enzymolysis for one time by adopting cellulase to break cell walls of the pinus koraiensis so as to fully dissolve soluble proteins in the cell walls; then, acid protease is adopted for secondary enzymolysis, so that cell walls are broken more completely, soluble proteins are hydrolyzed, and flavor-developing polypeptides and amino acids in the soluble proteins are fully dissolved; the combination of the two biological enzymolysis realizes more thorough enzymolysis of the raw materials, fully extracts the effective components and related flavor substances in the pinus koraiensis, and improves the enzymolysis rate and the enzymolysis efficiency; finally, a low-temperature freeze drying process is adopted, so that active ingredients and nutritional ingredients in the pinus koraiensis are reserved to the maximum extent, and the comprehensive utilization rate of the pinus koraiensis is greatly improved.
The preparation method and the using equipment are simple, and the prepared natural flavor enhancer for the pinus koraiensis is rich in a plurality of flavor-developing substances and nutritional ingredients such as amino acids, small peptides, trace elements, minerals, polysaccharides and the like, is delicious in taste, can be dissolved in water, and is more suitable for the current social health preserving and healthy diet concept; in addition, the invention provides technical parameters for extracting the flavor-developing substances of the pinus koraiensis, promotes the development of the flavor-developing agent, improves the utilization rate and the added value of the marine low-value protein, reduces the environmental pollution caused by resource waste, and promotes the healthy and sustainable development of marine economy.
It should be noted that:
(1) In the above embodiments 1 to 36, in the step (1), the echinacea is selected from the echinacea in sea area of the wilfordii city of shandong.
(2) In the above embodiments 1 to 36, in the step (9), maltodextrin is selected as the carrier of the flavoring agent, the addition amount thereof is 10% of the mass of the granule of the chaetonia Sparassis, and only as a preferred embodiment, in actual production, the carrier may be any one or a combination of at least two of maltodextrin, corn starch, tapioca starch, potato starch and sweet potato starch, and the addition amount thereof may be 5 to 15% of the mass of the granule of the chaetonia Sparassis, and in actual operation, the carrier is adaptively adjusted according to specific conditions.
(3) In the above embodiments 1 to 36, in the step (9), the low-temperature freeze-drying temperature is-60 ℃ and the pressure is 10Pa, and only as a preferred embodiment, in actual production, the low-temperature freeze-drying temperature may be-65 to-55 ℃ and the pressure may be 8 to 12Pa, and the method is specifically adapted according to actual conditions.
The above embodiments are only for illustrating the technical solution of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application, and are intended to be included in the scope of the present application.
Claims (10)
1. The preparation method of the natural flavor enhancer for the pinus koraiensis is characterized by comprising the following steps of:
(1) Cleaning and drying: cleaning and drying the pinus koraiensis;
(2) Crushing: crushing the dried pinus koraiensis to obtain pinus koraiensis granules;
(3) Dissolving: dissolving the pinus koraiensis granules in a buffer solution to obtain a pinus koraiensis solution;
(4) Primary enzymolysis: performing primary enzymolysis on the pinus koraiensis solution to obtain primary enzymolysis liquid;
(5) Enzyme deactivation: sterilizing the primary enzymolysis liquid;
(6) And (3) secondary enzymolysis: performing secondary enzymolysis on the sterilized primary enzymolysis liquid to obtain secondary enzymolysis liquid;
(7) And (3) centrifuging: centrifuging the secondary enzymolysis liquid to obtain a supernatant;
(8) Concentrating: concentrating the supernatant to obtain a concentrated solution;
(9) Granulating: mixing the concentrated solution with carrier, drying, and granulating to obtain natural flavoring agent.
2. The method for preparing natural flavor enhancer of echinocystis according to claim 1, wherein in the step (1), the drying temperature is 40-55 ℃, and the drying is carried out until the moisture content is less than 15%.
3. The method for producing a natural flavor enhancer for pinus koraiensis according to claim 1, wherein in the step (2), the dried pinus koraiensis is pulverized to a particle size of 40 to 60 mesh.
4. The preparation method of the natural flavor enhancer for the pinus koraiensis according to claim 1, wherein in the step (3), the buffer solution is prepared from 0.1mol/L citric acid solution and 0.2mol/L disodium hydrogen phosphate solution according to a volume ratio of 12.63:7.37, and the buffer solution is used in an amount of 20mL buffer solution per gram of pinus koraiensis particles.
5. The preparation method of the natural flavor enhancer for the pinus koraiensis according to claim 1, wherein in the step (4), cellulase is adopted for primary enzymolysis, the specification is 400U/mg, and the addition amount of the cellulase is 9-13% of the mass of the pinus koraiensis particles; the enzymolysis temperature of the primary enzymolysis is 40-60 ℃, the enzymolysis time is 90-210 min, and the initial pH value is 5.5-7.5.
6. The method for preparing natural flavor enhancer for chaetoceros according to claim 1, wherein in the step (5), the sterilization temperature is 90-95 ℃ and the sterilization time is 1-2 min.
7. The preparation method of the natural flavor enhancer for the pinus koraiensis according to claim 1, wherein in the step (6), acid protease with the specification of 50U/mg is adopted for secondary enzymolysis, and the addition amount of the acid protease is 20-70% of the mass of the pinus koraiensis particles; the enzymolysis temperature of the secondary enzymolysis is 35-55 ℃, the enzymolysis time is 60-210 min, and the initial pH value is 2.0-4.5.
8. The method for preparing natural flavor enhancer for pinus koraiensis according to claim 1, wherein in the step (7), the rotational speed of the centrifugal treatment is 3500-4500 r/min, and the time is 20-30 min.
9. The preparation method of the natural flavor enhancer for the pinus koraiensis according to claim 1, wherein in the step (9), the carrier is any one or a combination of at least two of maltodextrin, corn starch, tapioca starch, potato starch and sweet potato starch, and the addition amount of the carrier is 5-15% of the mass of the pinus koraiensis particles; the drying mode is low-temperature freeze drying, the temperature is-65 to-55 ℃, and the pressure is 8 to 12Pa.
10. A natural flavor enhancer of pinus koraiensis produced by the production method according to any one of claims 1 to 9.
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| CN110140802A (en) * | 2019-06-18 | 2019-08-20 | 天津市食品研究所有限公司 | A kind of preparation method of the modified processing tasty agents of mushroom stems |
| CN111374290A (en) * | 2018-12-28 | 2020-07-07 | 天津市绿禾食品添加剂有限公司 | Seaweed flavor enhancer |
| CN114403386A (en) * | 2022-02-10 | 2022-04-29 | 阳西美味鲜食品有限公司 | Method for preparing oyster juice by enzymolysis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2011089764A1 (en) * | 2010-01-20 | 2011-07-28 | 焼津水産化学工業株式会社 | Taste-improving agent for potassium-chloride-containing foods and beverages and process for producing potassium-chloride-containing foods and beverages |
| CN106262656A (en) * | 2016-08-02 | 2017-01-04 | 大连工业大学 | One main laminaria flavoring agent and production method thereof |
| CN111374290A (en) * | 2018-12-28 | 2020-07-07 | 天津市绿禾食品添加剂有限公司 | Seaweed flavor enhancer |
| CN110140802A (en) * | 2019-06-18 | 2019-08-20 | 天津市食品研究所有限公司 | A kind of preparation method of the modified processing tasty agents of mushroom stems |
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