CN111334554A - Salmon oligopeptide and preparation method thereof - Google Patents

Salmon oligopeptide and preparation method thereof Download PDF

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CN111334554A
CN111334554A CN202010422285.3A CN202010422285A CN111334554A CN 111334554 A CN111334554 A CN 111334554A CN 202010422285 A CN202010422285 A CN 202010422285A CN 111334554 A CN111334554 A CN 111334554A
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salmon
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王善恒
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Yantai Huaxin Biotechnology Co ltd
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Abstract

The invention discloses an oligopeptide from salmon and a preparation method thereof, belonging to the field of active substance extraction. The preparation method comprises the following steps: (1) pretreating microorganisms: mincing salmon leftovers by using a mincing machine, adding distilled water to adjust the concentration, then adding a buffering agent to adjust the pH value, and finally adding activated strains to perform fermentation treatment to obtain salmon leftovers microbial pretreatment base stock; (2) hydrolysis: adding distilled water into the salmon leftover microbial pretreatment base stock to adjust the concentration of the base stock; (3) carrying out first enzymolysis; (4) carrying out second enzymolysis; (5) centrifuging: centrifuging the enzymolysis product to obtain supernatant; (6) adsorption drying: and (5) performing activated carbon adsorption drying on the centrifugal supernatant to obtain a finished product. The invention has the beneficial effects that: the method solves the problem that the leftovers are difficult to treat, introduces a microbial fermentation method to treat the leftovers of the salmon, improves the hydrolysis effect of enzyme, saves the cost and improves the utilization rate.

Description

Salmon oligopeptide and preparation method thereof
Technical Field
The invention relates to the technical field of active substance extraction, in particular to an oligopeptide from salmon and a preparation method thereof.
Background
Salmon, also known as scamung fish or salmons, is one of the world's famous economic fishes. The salmon is not only rich in unsaturated fatty acid, but also contains 18 amino acids (including 8 essential amino acids), the protein content is obviously higher than that of other fishes, and the salmon also contains various mineral elements such as Ca, P, Mg and the like, and is known as 'rare in water'. The salmon in the market is usually sold in fresh or frozen slices, and a large amount of salmon powder is generated in the processing process
And (5) leftover material. The salmon has high grease content, heavy color and large fishy smell, and brings difficulty to the comprehensive utilization of the salmon leftovers.
In order to solve the problem, the invention fully utilizes the high-quality resource to fully extract the protein in the salmon leftovers, and the main method is to hydrolyze the protein. At present, methods for hydrolyzing proteins include acid method, alkaline method, enzymatic hydrolysis and the like. Compared with an acid method or an alkaline method, the method has the advantages of high enzyme hydrolysis efficiency, mild conditions and obvious advantage in the aspect of retaining nutrient components. The fish protein is degraded into small molecular oligopeptide and amino acid, which is beneficial to the absorption and utilization of human body. According to the introduction of experts, oligopeptides with small molecular weight can have higher skin permeability than polypeptides and are easier to be absorbed by human skin, and due to the small molecular weight to a certain degree, qualitative leap of biological activity occurs. The smaller the molecular weight of the peptide, the shorter the "amino acid chain" is, the easier it is to be absorbed and utilized by the human body.
According to the traditional enzymatic hydrolysis method, raw materials are subjected to simple treatment and then directly subjected to enzymolysis, and most of the selected raw materials are complete fish meat, and the raw materials are used for preparing oligopeptide products after finishing and cleaning. However, the leftover bits and pieces include fishbone, fish head, fish viscera and the like, and account for about 40 percent of the fish. The leftovers not only contain a large amount of protein, but also contain fat and a plurality of bioactive substances, and if the fish meat is separated independently, manpower and material resources are wasted, so the leftovers which cannot be separated at present are generally processed into feed or treated as wastes, thereby not only wasting resources, but also destroying the ecological environment. Therefore, the development and utilization of the fish leftovers are very important.
The prior domestic process for preparing polypeptide products by fish products is mainly as follows:
raw materials, pretreating, mashing, homogenizing, adding enzyme for hydrolysis, inactivating enzyme, centrifuging, performing suction filtration on supernate to adjust the value, adsorbing and drying, and obtaining a finished product. The processes have the problems that fish resources are not fully utilized, the adopted raw materials are fish meat, the leftovers are more, the waste is larger, the purity is not high, and the energy consumption is larger. The full utilization of the leftovers is particularly important. And how to improve the hydrolysis effect of the enzyme in the enzymolysis process is also the field of current research.
Disclosure of Invention
The invention provides salmon oligopeptide and a preparation method thereof, aiming at fully utilizing salmon leftovers and solving the problem that the leftovers are difficult to treat. The invention not only carries out enzyme hydrolysis on salmon, but also introduces a microbial fermentation method to treat salmon leftovers aiming at the characteristics of salmon, so that the salmon leftovers can be utilized without being excessively treated, the cost is saved, the utilization rate is improved, and the enzyme hydrolysis effect is improved in the enzymolysis process.
In order to achieve the above object, the present invention provides a method for preparing an oligopeptide from salmon, which is characterized in that the method for preparing the oligopeptide comprises the following steps:
(1) pretreating microorganisms: adding a small amount of water into appropriate amount of salmon leftovers, shearing, stirring with a stirrer to form an emulsion, adding distilled water into a small amount of emulsion sample to adjust the concentration, wherein the solid-liquid ratio is 1:1, and shaking the sample to fully mix the sample and the water;
(2) hydrolysis: adding distilled water into the salmon leftover microbial pretreatment base stock to adjust the concentration of the base stock; (3) carrying out first enzymolysis: adding pepsin into the base material obtained in the step (2), performing enzymolysis, inactivating enzymes, and cooling;
(4) and (3) carrying out second enzymolysis: adding trypsin into the enzymolysis product obtained in the step (3), carrying out enzymolysis, and inactivating enzyme;
(5) centrifuging: centrifuging the enzymolysis product obtained in the step (4) to obtain supernatant, and adjusting the pH value;
(6) adsorption drying: and (5) carrying out activated carbon adsorption drying on the centrifugal supernatant obtained in the step (5) to obtain a finished product.
Preferably, a buffer, KH, is added2PO4、K2HPO4And CaCO3The concentration of the buffer is 35g/L, and the ratio of the buffer is KH2PO4:K2HPO4:CaCO3: 1.3:1.5:1, then adjusting the initial pH value to 6.0-6.5;
preferably, the sample is heat sterilized and the fermentation strain is added, wherein the ratio of bacillus circulans: the preservation number is CGMCC No.1.2411, and the lactobacillus acidophilus: the preservation number is as follows: CGMCC N0.1.2919
Preferably, when the fermentation strain is a mixed strain, the concentration of the strain is 160g/L, and the ratio of the bacillus circulans to the lactobacillus acidophilus is 4: 2.
Preferably, the sample is placed in an incubator, the fermentation temperature is 35-38 ℃, and the fermentation time is 40-45 hours.
Preferably, finally adding activated strains for fermentation treatment to obtain salmon leftover microorganism pretreatment base stock;
preferably, the microbial pretreatment base material of the salmon leftovers is added with distilled water to adjust the concentration of the base material, and the concentration of the base material is adjusted to 4.0-4.5%.
Preferably, the pH value of the base material is adjusted to 6.9-7.2, pepsin is added, the concentration of the pepsin is 3-5%, the enzymolysis temperature is 45-48 ℃, the enzymolysis time is 1-1.5 hours, then enzyme deactivation treatment is carried out, and cooling is carried out after sterilization.
Preferably, the pH value of the base material is adjusted to 1.5-3, pepsin is added, the concentration of the pepsin is 3-5%, the temperature for enzymolysis is 45-48 ℃, the enzymolysis time is 1-1.5 hours, then enzyme deactivation treatment is carried out, and cooling is carried out after sterilization.
Preferably, the pH value of the enzymolysis product is adjusted to 6.5-7.0, trypsin is added, the concentration of the trypsin is 4-6%, the enzymolysis temperature is 48-52 ℃, the enzymolysis time is 2-3 hours, and the enzyme is inactivated after enzymolysis.
Preferably, the pH value of the enzymolysis product is adjusted to 7.5-8.0, trypsin is added, the concentration of the trypsin is 4-6%, the enzymolysis temperature is 35-40 ℃, the enzymolysis time is 2-3 hours, and the enzyme is inactivated after enzymolysis.
Preferably, centrifuging the enzymolysis product for 10-20 minutes under the condition of 4000-5000r/min to obtain a supernatant, and adjusting the pH value of the supernatant to 3-4;
preferably, the centrifugal supernatant is subjected to activated carbon adsorption drying, the volume ratio of the mass of the supernatant to the activated carbon particles is 10-20:1, the adsorption time is 10-20 minutes, and the salmon oligopeptide product is obtained after adsorption and filtration.
The invention has the beneficial effects that: provides an oligopeptide from salmon and a preparation method thereof, which makes full use of salmon leftovers and solves the problem that the leftovers are difficult to treat. Aiming at the characteristics of salmon, a microbial fermentation method is introduced to treat the salmon leftovers, so that the salmon leftovers can be utilized without over-treatment, the cost is saved, and the utilization rate is improved.
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FIG. 1 is a graph showing the effect of base concentration on the degree of hydrolysis in test example 1 of the present invention;
FIG. 2 is a graph showing the effect of enzyme dosage on the degree of hydrolysis in test example 2 of the present invention;
FIG. 3 is a graph showing the effect of pH on the degree of hydrolysis in test example 3 of the present invention;
FIG. 4 is a graph showing the effect of temperature on the degree of hydrolysis in test example 4 of the present invention;
FIG. 5 is a graph showing the effect of enzyme dosage on the degree of hydrolysis in test example 6 of the present invention;
FIG. 6 is a graph showing the effect of pH on the degree of hydrolysis in test example 7 of the present invention;
FIG. 7 is a graph showing the effect of temperature on the degree of hydrolysis in test example 8 of the present invention.
FIG. 8 is a graph showing the effect of base concentration on the degree of hydrolysis in test example 10 of the present invention;
FIG. 9 is a graph showing the effect of enzyme dosage on degree of hydrolysis in example 11 of the present invention;
FIG. 10 is a graph showing the effect of pH on the degree of hydrolysis in test example 12 of the present invention;
FIG. 11 is a graph showing the effect of temperature on the degree of hydrolysis in test example 13 of the present invention;
FIG. 12 is a graph showing the effect of enzyme dosage on the degree of hydrolysis in test example 15 of the present invention;
FIG. 13 is a graph showing the effect of pH on the degree of hydrolysis in test example 16 of the present invention;
FIG. 14 is a graph showing the effect of temperature on the degree of hydrolysis in test example 17 of the present invention.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
The protease can hydrolyze food protein under mild conditions, no great environmental pollution is caused, the protease can hydrolyze the protein without causing the change of amino acid configuration, the original efficacy of the amino acid is well preserved, the protease hydrolysis can realize the positioning hydrolysis, the control of the hydrolysis process is easy to realize, thus the applicability and the functionality of the peptide are widened, and the market demand is met. However, protein enzymolysis is not a simple process involving many steps. The first step is to select proper enzyme, because the enzyme not only affects the reaction rate and the product yield, but also directly affects the flavor, physicochemical properties and physiological activity of the product, and the selection basis is not only related to the specificity of the action site of the enzyme, but also has great relation with the substrate and the process conditions.
The protein controlled enzymolysis has great relation with the operation condition, the substrate concentration, the enzyme adding amount, the hydrolysis time and the temperature influence the hydrolysis process, and the hydrolysis degree is the most intuitive index of the protein controlled enzymolysis. The degree of hydrolysis, which controls the size of the peptide fragments and the relative content of free amino acids, is an important factor affecting the quality and yield of the peptides. If the degree of hydrolysis is small, the hydrolysis is insufficient, the active amino acid residue cannot be broken, the corresponding activity cannot be shown, if the hydrolysis is too complete, the short peptide chain is further hydrolyzed into free amino acids, some active short peptides are also hydrolyzed, and the physiological activity of the free amino acids is not as good as that of the short peptides, so that the activity is obviously reduced, and therefore, it is very necessary to terminate the enzymolysis reaction at a proper time to control the degree of hydrolysis.
1. Nitrogen utilization calculation
Nitrogen utilization ratio (%) = protein amount in enzymolysis liquid/total protein amount in raw material X100%
2. Method for calculating hydrolysis degree
DH（%）=Free amino nitrogen content in hydrolysis-free amino nitrogen before hydrolysisX100%
Total nitrogen content in the feedstock-non-protein nitrogen in the feedstock
Comparative example 1
The embodiment of the invention provides a preparation method of salmon oligopeptide, which comprises the following specific implementation steps:
(1) pretreatment: adding a small amount of water into appropriate amount of salmon leftovers, shearing, stirring with a stirring machine to obtain an emulsion, adding distilled water into a small amount of emulsion sample to adjust the concentration, wherein the solid-liquid ratio is 1:1, and shaking the sample to fully mix the sample and the water.
(2) Hydrolysis: adding distilled water into the salmon leftover microbial pretreatment base stock to adjust the concentration of the base stock, wherein the concentration of the base stock is adjusted to 4.0%.
(3) Carrying out first enzymolysis: adjusting the pH value of the base material to 6.9, adding pepsin, wherein the concentration of the pepsin is 3%, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 1, then carrying out enzyme deactivation treatment, sterilizing and cooling.
(4) And (3) carrying out second enzymolysis: adjusting the pH value of the enzymolysis product to 6.5, adding trypsin, wherein the concentration of the trypsin is 4%, the enzymolysis temperature is 48 ℃, the enzymolysis time is 2 hours, and inactivating enzyme after enzymolysis.
(5) Centrifuging: centrifuging the enzymolysis product for 10 minutes under the condition of 4000r/min to obtain supernatant, and adjusting the pH value of the supernatant to 3;
(6) adsorption drying: and (5) performing activated carbon adsorption drying on the centrifugal supernatant obtained in the step (5), wherein the volume ratio of the mass of the supernatant to the volume of the activated carbon particles is 10:1, the adsorption time is 10 minutes, and filtering after adsorption to obtain the salmon oligopeptide product.
Example 1
The embodiment of the invention provides a preparation method of salmon oligopeptide, which comprises the following specific implementation steps:
(1) pretreatment: adding a small amount of water into appropriate amount of salmon leftovers, shearing, stirring with a stirring machine to obtain an emulsion, adding distilled water into a small amount of emulsion sample to adjust the concentration, wherein the solid-liquid ratio is 1:1, and shaking the sample to fully mix the sample and the water.
Then adding a buffer, KH2PO4、K2HPO4And CaCO3The concentration of the buffer is 35g/L, and the ratio of the buffer is KH2PO4:K2HPO4:CaCO3: 1.3:1.5:1, and then adjusting the initial pH to 6.0.
Heating and sterilizing the sample, and adding fermentation strains with the concentration of 160 g/L; the fermentation strain is bacillus circulans, and the preservation number is as follows: CGMCC No. 1.2411;
and (3) putting the sample into an incubator, wherein the fermentation temperature is 35 ℃, and the fermentation time is 40 hours.
Selecting and finally adding activated strains for fermentation treatment to obtain salmon leftover microbial pretreatment base stock;
(2) hydrolysis: adding distilled water into the salmon leftover microbial pretreatment base stock to adjust the concentration of the base stock, wherein the concentration of the base stock is adjusted to 4.0%.
(3) Carrying out first enzymolysis: adjusting the pH value of the base material to 6.9, adding pepsin, wherein the concentration of the pepsin is 3%, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 1, then carrying out enzyme deactivation treatment, sterilizing and cooling.
(4) And (3) carrying out second enzymolysis: adjusting the pH value of the enzymolysis product to 6.5, adding trypsin, wherein the concentration of the trypsin is 4%, the enzymolysis temperature is 48 ℃, the enzymolysis time is 2 hours, and inactivating enzyme after enzymolysis.
(5) Centrifuging: centrifuging the enzymolysis product for 10 minutes under the condition of 4000r/min to obtain supernatant, and adjusting the pH value of the supernatant to 3;
(6) adsorption drying: and (5) performing activated carbon adsorption drying on the centrifugal supernatant obtained in the step (5), wherein the volume ratio of the mass of the supernatant to the volume of the activated carbon particles is 10:1, the adsorption time is 10 minutes, and filtering after adsorption to obtain the salmon oligopeptide product.
Example 2
The embodiment of the invention provides a preparation method of salmon oligopeptide, which refers to the specific implementation steps of embodiment 1, but is different from embodiment 1 in that the fermentation strain is lactobacillus acidophilus with the collection number of CGMCC N0.1.2919; the concentration of the bacterial strain is 160 g/L.
Example 3
The embodiment of the invention provides a preparation method of salmon oligopeptide, and the specific implementation steps refer to embodiment 1, but the difference from embodiment 1 is that the fermentation strain is a mixed strain of bacillus circulans and lactobacillus acidophilus in a ratio of 4:2, and the concentration of the strain is 160 g/L; wherein, bacillus circulans: the preservation number is CGMCC No.1.2411, and the lactobacillus acidophilus: the preservation number is as follows: n0.1.2919, respectively;
example 4
The embodiment of the invention provides a preparation method of salmon oligopeptide, and the specific implementation steps refer to embodiment 3, but the difference from embodiment 3 is that the sample is placed in an incubator, the fermentation temperature is 37 ℃, and the fermentation time is 43 hours.
Example 5
The embodiment of the invention provides a preparation method of salmon oligopeptide, and the specific implementation steps refer to embodiment 3, but the difference from embodiment 3 is that the sample is placed into an incubator, the fermentation temperature is 38 ℃, and the fermentation time is 45 hours.
The following results were obtained by comparing comparative example 1 and examples 1 to 5 above:
TABLE 1 Effect of different pretreatment methods on the enzymatic hydrolysis
Numbering Comparative example 1 Example 1 Example 2 Example 3 Example 4 Example 5
Bacterial strain Untreated Bacillus circulans Lactobacillus acidophilus Mixed strain Mixed strain Mixed strain
pH value - 6.0 6.0 6.0 6.2 6.5
Fermentation temperature (degree) - 35 35 35 37 38
Fermentation time (hours) - 40 40 40 43 45
Nitrogen utilization rate% 82 85 86 90 92 89
DH（%） 11 13 12 15 16 15
As can be seen from the influence of different pretreatment modes on the enzymolysis effect in Table 1, the nitrogen utilization rate and the hydrolysis degree of salmon leftovers after microbial fermentation treatment in the enzymolysis process are better than those of untreated raw materials. Wherein the mixed strain is better than a single strain. The adjustment of the pH value, the fermentation temperature and the fermentation time also has obvious influence on the enzymolysis effect.
Example 6
The embodiment of the invention provides a preparation method of salmon oligopeptide, which comprises the following specific implementation steps:
(1) pretreatment: adding a small amount of water into appropriate amount of salmon leftovers, shearing, stirring with a stirring machine to obtain an emulsion, adding distilled water into a small amount of emulsion sample to adjust the concentration, wherein the solid-liquid ratio is 1:1, and shaking the sample to fully mix the sample and the water.
Then adding a buffer, KH2PO4、K2HPO4And CaCO3The concentration of the buffer is 35g/L, and the ratio of the buffer is KH2PO4:K2HPO4:CaCO3: 1.3:1.5:1, and then adjusting the initial pH to 6.0.
Heating and sterilizing the sample, adding fermentation strain which is Lactobacillus acidophilus with a preservation number of CGMCC N0.1.2919; the concentration of the strain is 160 g/L;
the samples were placed in an incubator at 37 ℃ for 43 hours.
Selecting and finally adding activated strains for fermentation treatment to obtain salmon leftover microbial pretreatment base stock;
(2) hydrolysis: adding distilled water into the salmon leftover microbial pretreatment base stock to adjust the concentration of the base stock, wherein the concentration of the base stock is adjusted to 4.0%.
(3) Carrying out first enzymolysis: adjusting the pH value of the base material to 2.5, adding pepsin, wherein the concentration of the pepsin is 3%, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 1, then carrying out enzyme deactivation treatment, sterilizing and cooling.
(4) And (3) carrying out second enzymolysis: adjusting the pH value of the enzymolysis product to 7.9, adding trypsin, wherein the concentration of the trypsin is 4%, the enzymolysis temperature is 37 ℃, the enzymolysis time is 2 hours, and inactivating enzyme after enzymolysis.
(5) Centrifuging: centrifuging the enzymolysis product for 10 minutes under the condition of 4000r/min to obtain supernatant, and adjusting the pH value of the supernatant to 3;
(6) adsorption drying: and (5) performing activated carbon adsorption drying on the centrifugal supernatant obtained in the step (5), wherein the volume ratio of the mass of the supernatant to the volume of the activated carbon particles is 10:1, the adsorption time is 10 minutes, and filtering after adsorption to obtain the salmon oligopeptide product.
Example 7
The embodiment of the invention provides a preparation method of salmon oligopeptide, which refers to the specific implementation steps of embodiment 6, but is different from embodiment 6 in that the fermentation strain is lactobacillus acidophilus with the collection number of CGMCC N0.1.2919; the concentration of the bacterial strain is 160 g/L.
Example 8
The embodiment of the invention provides a preparation method of salmon oligopeptide, and the specific implementation steps refer to embodiment 6, but the difference from embodiment 6 is that the fermentation strain is a mixed strain of bacillus circulans and lactobacillus acidophilus in a ratio of 4:2, and the concentration of the strain is 160 g/L; wherein, bacillus circulans: the preservation number is CGMCC No.1.2411, and the lactobacillus acidophilus: the preservation number is as follows: n0.1.2919, respectively;
example 9
The embodiment of the invention provides a preparation method of salmon oligopeptide, and the specific implementation steps refer to embodiment 8, but the difference from embodiment 8 is that the sample is placed in an incubator, the fermentation temperature is 35 ℃, and the fermentation time is 40 hours.
Example 10
The embodiment of the invention provides a preparation method of salmon oligopeptide, and the specific implementation steps refer to embodiment 8, but the difference from embodiment 8 is that the sample is placed into an incubator, the fermentation temperature is 38 ℃, and the fermentation time is 45 hours.
The following results were obtained by comparing comparative example 1 and examples 6 to 10 above:
TABLE 2 Effect of different pretreatment methods on the enzymolysis
Numbering Comparative example 1 Example 6 Example 7 Example 8 Example 9 Example 10
Bacterial strain Untreated Bacillus circulans Lactobacillus acidophilus Mixed strain Mixed strain Mixed strain
pH value - 6.0 6.0 6.0 6.2 6.5
Fermentation temperature (degree) - 37 37 37 37 38
Fermentation time (hours) - 43 43 43 43 45
Nitrogen utilization rate% 82 86 88 95 90 91
DH（%） 11 14 14 17 15 15
As can be seen from the influence of different pretreatment modes on the enzymolysis effect in Table 2, the nitrogen utilization rate and the hydrolysis degree of the salmon leftovers after the microbial fermentation treatment in the enzymolysis process are better than those of the untreated raw materials. Wherein the mixed strain is better than a single strain. The adjustment of the pH value, the fermentation temperature and the fermentation time also has obvious influence on the enzymolysis effect.
Comparative example 2
The embodiment of the invention provides a preparation method of salmon oligopeptide, which comprises the following specific implementation steps:
1) pretreatment: adding a small amount of water into appropriate amount of salmon leftovers, shearing, stirring with a stirring machine to obtain an emulsion, adding distilled water into a small amount of emulsion sample to adjust the concentration, wherein the solid-liquid ratio is 1:1, and shaking the sample to fully mix the sample and the water.
Then adding a buffer, KH2PO4、K2HPO4And CaCO3The concentration of the buffer is 35g/L, and the ratio of the buffer is KH2PO4:K2HPO4:CaCO3: 1.3:1.5:1, then adjust to initial 6.2.
Heating and sterilizing the sample, and adding fermentation strains with the concentration of 160 g/L; wherein the fermentation strain is a mixed strain of bacillus circulans and lactobacillus acidophilus in a ratio of 4: 2; bacillus circulans: the preservation number is CGMCC No.1.2411, and the lactobacillus acidophilus: the preservation number is as follows: n0.1.2919 are provided.
The samples were placed in an incubator at 37 ℃ for 43 hours.
Selecting and finally adding activated strains for fermentation treatment to obtain salmon leftover microbial pretreatment base stock;
(2) hydrolysis: adding distilled water into the salmon leftover microbial pretreatment base stock to adjust the concentration of the base stock, wherein the concentration of the base stock is adjusted to 4.0%.
(3) Carrying out first enzymolysis: adjusting the pH value of the base material to 6.9, adding papain, controlling the concentration of the comparative protease to be 3%, controlling the enzymolysis temperature to be 45 ℃ and controlling the enzymolysis time to be 1, then carrying out enzyme deactivation treatment, sterilizing and cooling.
(4) And (3) carrying out second enzymolysis: adjusting the pH value of the enzymolysis product to 6.5, adding trypsin, wherein the concentration of the trypsin is 4%, the enzymolysis temperature is 48 ℃, the enzymolysis time is 2 hours, and inactivating enzyme after enzymolysis.
(5) Centrifuging: centrifuging the enzymolysis product for 10 minutes under the condition of 4000r/min to obtain supernatant, and adjusting the pH value of the supernatant to 3;
(6) adsorption drying: and (5) performing activated carbon adsorption drying on the centrifugal supernatant obtained in the step (5), wherein the volume ratio of the mass of the supernatant to the volume of the activated carbon particles is 10:1, the adsorption time is 10 minutes, and filtering after adsorption to obtain the salmon oligopeptide product.
Comparative example 3
The protease added in the first enzymolysis is flavourzyme.
The other steps of the preparation method are the same as those of comparative example 2.
Comparative example 4
The protease added in the first enzymolysis is neutral protease.
The other steps of the preparation method are the same as those of comparative example 2.
Example 11
The embodiment of the invention provides a preparation method of salmon oligopeptide, which comprises the following specific implementation steps:
1) pretreatment: adding a small amount of water into appropriate amount of salmon leftovers, shearing, stirring with a stirrer to form an emulsion, adding distilled water into a small amount of emulsion sample to adjust the concentration, wherein the solid-liquid ratio is 1:1, and shaking the sample to fully mix the sample and the water;
then adding a buffer, KH2PO4、K2HPO4And CaCO3The concentration of the buffer is 35g/L, and the ratio of the buffer is KH2PO4:K2HPO4:CaCO3: 1.3:1.5:1, then adjust initial 6.2;
heating and sterilizing the sample, and adding fermentation strains with the concentration of 160 g/L; wherein the fermentation strain is a mixed strain of bacillus circulans and lactobacillus acidophilus in a ratio of 4: 2; bacillus circulans: the preservation number is CGMCC No.1.2411, and the lactobacillus acidophilus: the preservation number is as follows: n0.1.2919, respectively; (ii) a
Putting the sample into an incubator, wherein the fermentation temperature is 37 ℃, and the fermentation time is 43 hours;
and finally adding activated strains for fermentation treatment to obtain the salmon leftover microbial pretreatment base material.
(2) Hydrolysis: adding distilled water into the salmon leftover microbial pretreatment base stock to adjust the concentration of the base stock, wherein the concentration of the base stock is adjusted to 4.0%.
(3) Carrying out first enzymolysis: adjusting the pH value of the base material to 6.9, adding pepsin, wherein the concentration of the pepsin is 3%, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 1, then carrying out enzyme deactivation treatment, sterilizing and cooling.
(4) And (3) carrying out second enzymolysis: adjusting the pH value of the enzymolysis product to 6.5, adding trypsin, wherein the concentration of the trypsin is 4%, the enzymolysis temperature is 48 ℃, the enzymolysis time is 2 hours, and inactivating enzyme after enzymolysis.
(5) Centrifuging: centrifuging the enzymolysis product for 10 minutes under the condition of 4000r/min to obtain supernatant, and adjusting the pH value of the supernatant to 3;
(6) adsorption drying: and (5) performing activated carbon adsorption drying on the centrifugal supernatant obtained in the step (5), wherein the volume ratio of the mass of the supernatant to the volume of the activated carbon particles is 10:1, the adsorption time is 10 minutes, and filtering after adsorption to obtain the salmon oligopeptide product.
The following results were obtained by comparing the above comparative examples 2 to 4 with example 6:
TABLE 3 Primary protease hydrolysis conditions and Effect
Name (R) Concentration of base material% The amount of enzyme used% pH value Temperature (degree) Time (hours) Nitrogen utilization (%) DH（%）
Comparative example 2 4 3 6.9 45 1 76 12
Example 11 4 3 6.9 45 1 92 16
Comparative example 3 4 3 6.9 45 1 63 10
Comparative example 4 4 3 6.9 45 1 72 11
As can be seen from the conditions and effects of the first protease hydrolysis in Table 3, the hydrolysis effect of pepsin is more prominent and the best.
Comparative example 5
The protease added in the first enzymolysis is papain.
The preparation method was otherwise the same as in comparative example 2, except that the pH was adjusted to 6 and the temperature of the enzymatic hydrolysis was adjusted to 55 ℃.
Comparative example 6
The protease added in the first enzymolysis is flavourzyme.
The preparation method was otherwise the same as in comparative example 2, except that the pH was adjusted to 6.5 and the enzymolysis temperature was 53 ℃.
Comparative example 7
The protease added in the first enzymolysis is neutral protease.
The preparation method was otherwise the same as in comparative example 2, except that the pH was adjusted to 7 and the enzymolysis temperature was 50 ℃.
The following results were obtained by comparing the above comparative examples 5 to 7 with example 8:
TABLE 4 Primary protease hydrolysis conditions and Effect
Name (R) Concentration of base material% The amount of enzyme used% pH value Temperature (degree) Time (hours) Nitrogen utilization (%) DH（%）
Comparative example 5 4 3 6 55 1 80 12
Example 8 4 3 2.5 45 1 95 17
Comparative example 6 4 3 6.5 53 1 68 11
Comparative example 7 4 3 7 50 1 75 12
As can be seen from the conditions and effects of the first protease hydrolysis in Table 4, the hydrolysis effect of pepsin is more prominent and the best.
Comparative example 8 the embodiment of the present invention provides a method for preparing an oligopeptide from salmon, comprising the following specific steps:
1) pretreatment: adding a small amount of water into appropriate amount of salmon leftovers, shearing, stirring with a stirring machine to obtain an emulsion, adding distilled water into a small amount of emulsion sample to adjust the concentration, wherein the solid-liquid ratio is 1:1, and shaking the sample to fully mix the sample and the water.
Then adding a buffer, KH2PO4、K2HPO4And CaCO3The concentration of the buffer is 35g/L, and the ratio of the buffer is KH2PO4:K2HPO4:CaCO3: 1.3:1.5:1, then adjust to initial 6.2.
Heating and sterilizing the sample, and adding fermentation strains with the concentration of 160 g/L; wherein the fermentation strain is a mixed strain of bacillus circulans and lactobacillus acidophilus in a ratio of 4: 2; bacillus circulans: the preservation number is CGMCC No.8864, and the lactobacillus acidophilus: the preservation number is as follows: CGMCC N0.2122;
the samples were placed in an incubator at 37 ℃ for 43 hours.
Selecting and finally adding activated strains for fermentation treatment to obtain salmon leftover microbial pretreatment base stock;
(2) hydrolysis: adding distilled water into the salmon leftover microbial pretreatment base stock to adjust the concentration of the base stock, wherein the concentration of the base stock is adjusted to 4.2%.
(3) Carrying out first enzymolysis: adjusting the pH value of the base material to 7, adding pepsin, wherein the concentration of the pepsin is 4.5%, the enzymolysis temperature is 47 ℃, the enzymolysis time is 1, then carrying out enzyme deactivation treatment, sterilizing and cooling.
(4) And (3) carrying out second enzymolysis: adjusting the pH value of the enzymolysis product to 6.5, adding papain, controlling the concentration of trypsin to be 4%, controlling the enzymolysis temperature to be 48 ℃, controlling the enzymolysis time to be 2 hours, and inactivating enzyme after enzymolysis.
(5) Centrifuging: centrifuging the enzymolysis product for 10 minutes under the condition of 4000r/min to obtain supernatant, and adjusting the pH value of the supernatant to 3;
(6) adsorption drying: and (5) performing activated carbon adsorption drying on the centrifugal supernatant obtained in the step (5), wherein the volume ratio of the mass of the supernatant to the volume of the activated carbon particles is 10:1, the adsorption time is 10 minutes, and filtering after adsorption to obtain the salmon oligopeptide product.
Comparative example 9
The preparation method is the same as that of comparative example 6, and only differs from that of comparative example 6 in that: adding protease for the second enzymolysis to obtain flavourzyme.
Comparative example 10
The preparation method is the same as that of comparative example 6, and only differs from that of comparative example 6 in that: the protease added in the first enzymolysis is neutral protease.
Example 12
The embodiment of the invention provides a preparation method of salmon oligopeptide, which comprises the following specific implementation steps:
1) pretreatment: adding a small amount of water into appropriate amount of salmon leftovers, shearing, stirring with a stirring machine to obtain an emulsion, adding distilled water into a small amount of emulsion sample to adjust the concentration, wherein the solid-liquid ratio is 1:1, and shaking the sample to fully mix the sample and the water.
Then adding a buffer, KH2PO4、K2HPO4And CaCO3The concentration of the buffer is 35g/L, and the ratio of the buffer is KH2PO4:K2HPO4:CaCO3: 1.3:1.5:1, then adjust to initial 6.2.
Heating and sterilizing the sample, and adding fermentation strains with the concentration of 160 g/L; wherein the fermentation strain is a mixed strain of bacillus circulans and lactobacillus acidophilus in a ratio of 4: 2; bacillus circulans: the preservation number is CGMCC No.8864, and the lactobacillus acidophilus: the preservation number is as follows: CGMCC N0.2122;
the samples were placed in an incubator at 37 ℃ for 43 hours.
Selecting and finally adding activated strains for fermentation treatment to obtain salmon leftover microbial pretreatment base stock;
(2) hydrolysis: adding distilled water into the salmon leftover microbial pretreatment base stock to adjust the concentration of the base stock, wherein the concentration of the base stock is adjusted to 4.0%.
(3) Carrying out first enzymolysis: adjusting the pH value of the base material to 7, adding pepsin, wherein the concentration of the pepsin is 4.5%, the enzymolysis temperature is 47 ℃, the enzymolysis time is 1, then carrying out enzyme deactivation treatment, sterilizing and cooling.
(4) And (3) carrying out second enzymolysis: adjusting the pH value of the enzymolysis product to 6.5, adding trypsin, wherein the concentration of the trypsin is 4%, the enzymolysis temperature is 48 ℃, the enzymolysis time is 2 hours, and inactivating enzyme after enzymolysis.
(5) Centrifuging: centrifuging the enzymolysis product for 10 minutes under the condition of 4000r/min to obtain supernatant, and adjusting the pH value of the supernatant to 3;
(6) adsorption drying: and (5) performing activated carbon adsorption drying on the centrifugal supernatant obtained in the step (5), wherein the volume ratio of the mass of the supernatant to the volume of the activated carbon particles is 10:1, the adsorption time is 10 minutes, and filtering after adsorption to obtain the salmon oligopeptide product.
The following results were obtained by comparing the above comparative examples and examples:
TABLE 5 second protease hydrolysis conditions and Effect
Name (R) The amount of enzyme used% pH value Temperature (degree) Time (hours) Nitrogen utilization (%) DH（%）
Comparative example 8 4 6.5 48 2 72 10
Example 12 4 6.5 48 2 89 15
Comparative example 9 4 6.5 48 2 75 13
Comparative example 10 4 6.5 48 2 80 8
As can be seen from the conditions and effects of the second protease hydrolysis in Table 5, the hydrolysis effect of trypsin is more prominent and the best.
Comparative example 11
The protease added in the second enzymolysis is papain.
The preparation method was otherwise the same as that of example 8, except that the pH was adjusted to 6 and the enzymolysis temperature was 55 ℃.
Comparative example 12
Adding protease for the second enzymolysis to obtain flavourzyme.
The preparation method was otherwise the same as that of example 8, except that the pH was adjusted to 6.5 and the enzymolysis temperature was 53 ℃.
Comparative example 13
Adding protease for the second enzymolysis to obtain neutral protease.
The preparation method was otherwise the same as that of example 8, except that the pH was adjusted to 7 and the enzymolysis temperature was 50 ℃.
The following results were obtained by comparing the above comparative examples and examples:
TABLE 6 second protease hydrolysis conditions and Effect
Name (R) The amount of enzyme used% pH value Temperature (degree) Time (hours) Nitrogen utilization (%) DH（%）
Comparative example 11 4 6 55 2 78 12
Example 8 4 7.9 37 2 95 17
Comparative example 12 4 6.5 53 2 79 14
Comparative example 13 4 7 50 2 82 10
As can be seen from the conditions and effects of the second protease hydrolysis in Table 6, the hydrolysis effect of trypsin is more prominent and the best.
Test example 1
This test example 1 actually contained 6 examples of the present invention; 6 embodiments respectively provide 6 preparation methods of oligopeptides, and the specific steps are basically consistent with those of embodiment 4; the differences are only in the part of the parameters, in particular,
the conditions for the first enzymatic hydrolysis in the 6 examples were all: the dosage of the enzyme: 3%, pH: 6.9, temperature: 45 degrees, time: 1 hour;
the base concentrations for the 6 examples were: 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%.
The influence of the concentration of the substrate on the degree of hydrolysis in the first enzymatic hydrolysis was examined by 6 examples contained in test example 1;
the test results are shown in FIG. 1. As can be seen from fig. 1, the degree of hydrolysis gradually increases with increasing concentration of the base material, and starts to decrease when a certain degree is reached. The degree of hydrolysis was highest when the binder concentration was 4.2%. At base concentrations of 4.0% and 4.5%, the degree of hydrolysis was relatively low, but still allowed the degree of pepsin hydrolysis to be satisfactory.
Test example 2
This test example 2 actually contained 5 examples of the present invention; 5 embodiments provide 5 methods for preparing oligopeptides, respectively; the specific steps are basically the same as those in example 4; the differences are only in the part of the parameters, in particular,
the conditions for the first enzymatic hydrolysis of the 5 examples were all: concentration of the base material: 4.2%, pH: 6.9, the temperature is 45 ℃, and the time is 1 hour;
the enzyme dosage in the first enzymolysis of 5 embodiments is respectively as follows: 3%, 3.5%, 4%, 4.5%, 5%.
The influence of the amount of enzyme for the first enzymatic hydrolysis on the degree of hydrolysis was examined by 5 examples contained in test example 2; the test results are shown in FIG. 2. As shown in FIG. 2, the degree of hydrolysis was gradually increased with the amount of the enzyme, and when the amount of the enzyme was increased beyond 4%, the degree of hydrolysis was not significantly changed. At enzyme levels of 3.0% and 4%, the degree of hydrolysis is relatively low, but still allows the degree of pepsin hydrolysis to be satisfactory.
Test example 3
This test example 3 actually included 4 examples of the present invention; 4 examples provide 4 methods for preparing oligopeptides, respectively; the specific steps are basically the same as those in example 4; the differences are only in the part of the parameters, in particular,
the conditions for the first enzymatic hydrolysis in all 4 examples are: concentration of the base material: 4.2%, enzyme dosage: 4.5%, temperature 45 ℃ and time 1 hour;
in 4 examples, the pH values of the base materials are respectively adjusted as follows during the first enzymolysis: 6.9,7.0,7.1,7.2.
The influence of pH adjustment on the base material on the degree of hydrolysis during the first enzymatic hydrolysis was examined by 4 examples in test example 3; the test results are shown in fig. 3; as can be seen from FIG. 3, the pH value has a significant effect on the degree of hydrolysis, with the highest degree of hydrolysis being observed at a pH of 7.0. At pH 6.9 and 7.2, the degree of hydrolysis is relatively low, but still allows the degree of pepsin hydrolysis to be satisfactory.
Test example 4
The present test example 4 actually comprises 4 examples of the present invention, and 4 examples respectively provide 4 methods for preparing oligopeptides; the specific steps are basically the same as those in example 4; the differences are only in the part of the parameters, in particular,
the conditions for the first enzymatic hydrolysis in all 4 examples are: concentration of the base material: 4.2%, enzyme dosage: 4.5%, pH: 7.0, time 1 hour;
in the 4 embodiments, the enzymolysis temperature for the first enzymolysis is respectively as follows: 45 degrees, 46 degrees, 47 degrees and 48 degrees.
The influence of the enzymolysis temperature of the first enzymolysis on the hydrolysis degree is examined through 4 examples of the experimental example 4; the test results are shown in FIG. 4. As can be seen from fig. 4, the hydrolysis temperature also has an important effect on the degree of hydrolysis, and the hydrolysis effect is best at a hydrolysis temperature of 46 degrees. When the enzymolysis temperature is 45 ℃ and 48 ℃, the hydrolysis degree is relatively slightly low, but the hydrolysis degree of the pepsin can still meet the requirement.
Test example 5
The preparation method is the same as that of example 4; the difference lies in that:
the enzymolysis conditions are as follows: concentration of the base material: 4.2%, enzyme dosage: 4.5%, pH: 7.0;
the first time of enzymolysis is considered as follows: 1 hour, 1.5 hours.
Test example 6
This test example 6 actually contains 5 examples of the present invention, and 5 examples respectively provide 5 methods for preparing oligopeptides; the specific steps are substantially the same as those in example 12; the differences are only in the part of the parameters, in particular,
the conditions for the second enzymatic hydrolysis in the 5 examples were all: pH value: 6.5, the temperature is 48 ℃, and the time is 2 hours;
in the 5 embodiments, the enzyme dosage in the second enzymolysis is respectively as follows: 4%, 4.5%, 5%, 5.5%, 6%.
The influence of the enzyme dosage on the hydrolysis degree in the second enzymolysis is examined through 5 examples of the test example 6; the test results are shown in FIG. 5. As can be seen from fig. 5, the degree of hydrolysis gradually increases with increasing concentration of the base material, and the degree of hydrolysis does not change significantly when a certain degree is reached. When the base concentration is 4.5%, the degree of hydrolysis tends to be smooth. At enzyme levels of 4.0% and 6%, the degree of hydrolysis was relatively low, but still allowed the degree of trypsin hydrolysis to be satisfactory.
Test example 7
This test example 7 actually contains 6 examples of the present invention, and 6 examples respectively provide 6 methods for preparing oligopeptides; the specific steps are substantially the same as those in example 12; the differences are only in the part of the parameters, in particular,
the second enzymatic hydrolysis conditions of the 6 examples were all: enzyme dosage: 4.5%, temperature 45 ℃ and time 2 hours;
in 6 embodiments, when performing the second enzymatic hydrolysis, the PH of the first enzymatic hydrolysis product is adjusted as follows: 6.5,6.6,6.7,6.8,6.9,7.0.
The 6 examples of test example 7 were used to examine the effect of different pH values of the first enzymatic hydrolysate on the degree of hydrolysis before the second enzymatic hydrolysis; the test results are shown in fig. 6. As can be seen from FIG. 6, the pH value has a significant effect on the degree of hydrolysis, with the highest degree of hydrolysis being observed at a pH of 6.7. At pH 6.5 and 7.0, the degree of hydrolysis was relatively low, but still allowed the degree of trypsin hydrolysis to be satisfactory.
Test example 8
This test example 8 actually contains 5 examples of the present invention, and 5 examples respectively provide 5 methods for preparing oligopeptides; the specific steps are substantially the same as those in example 12; the differences are only in the part of the parameters, in particular,
the second enzymatic conditions of the 5 examples were all enzyme dosages: 4.5%, pH: 6.7, the time is 2 hours;
in 5 embodiments, the enzymolysis temperature during the second enzymolysis is respectively: 48 degrees, 49 degrees, 50 degrees, 51 degrees and 52 degrees.
The influence of the enzymolysis temperature on the degree of hydrolysis at the second enzymolysis was examined by 5 examples of test example 7, and the test results are shown in FIG. 7. As can be seen from fig. 7, the hydrolysis temperature also has an important effect on the degree of hydrolysis, and the hydrolysis effect is best at a hydrolysis temperature of 50 ℃. When the enzymolysis temperature is 48 ℃ and 52 ℃, the degree of hydrolysis is relatively slightly low, but the degree of hydrolysis of the trypsin can still meet the requirement.
Test example 9
The enzymolysis conditions are as follows: enzyme dosage: 4.5%, pH: 7.0; temperature 50 DEG C
The second enzymolysis time is considered as follows: 1 hour, 2.5 hours, 3 hours
The other steps of the preparation method are the same as those of example 12.
Test example 10
This test example 10 actually included 6 examples of the present invention; 6 embodiments respectively provide 6 preparation methods of oligopeptides, and the specific steps are basically consistent with those of embodiment 8; the differences are only in the part of the parameters, in particular,
the conditions for the first enzymatic hydrolysis in the 6 examples were all: the dosage of the enzyme: 3%, pH: 2.5, temperature: 45 degrees, time: 1 hour;
the base concentrations for the 6 examples were: 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%.
The influence of the concentration of the substrate on the degree of hydrolysis in the first enzymatic hydrolysis was examined by 6 examples contained in test example 10;
the test results are shown in fig. 8. As can be seen from fig. 8, the degree of hydrolysis gradually increased with increasing concentration of the base material, and began to decrease when reaching a certain level. The degree of hydrolysis was highest when the binder concentration was 4.2%. At base concentrations of 4.0% and 4.5%, the degree of hydrolysis was relatively low, but still allowed the degree of pepsin hydrolysis to be satisfactory.
Test example 11
This test example 11 actually included 5 examples of the present invention; 5 embodiments provide 5 methods for preparing oligopeptides, respectively; the specific steps are basically the same as those in example 8; the differences are only in the part of the parameters, in particular,
the conditions for the first enzymatic hydrolysis of the 5 examples were all: concentration of the base material: 4.2%, pH: 2.5, the temperature is 45 ℃, and the time is 1 hour;
the enzyme dosage in the first enzymolysis of 5 embodiments is respectively as follows: 3%, 3.5%, 4%, 4.5%, 5%.
The influence of the amount of enzyme used for the first enzymatic hydrolysis on the degree of hydrolysis was examined by 5 examples contained in test example 11; the test results are shown in fig. 9. FIG. 9 shows that the degree of hydrolysis gradually increased with the amount of enzyme, and that the degree of hydrolysis did not change significantly with increasing amounts of enzyme above 4%.
Test example 12
This test example 12 actually included 4 examples of the present invention; 4 examples provide 4 methods for preparing oligopeptides, respectively; the specific steps are basically the same as those in example 8; the differences are only in the part of the parameters, in particular,
the conditions for the first enzymatic hydrolysis in all 4 examples are: concentration of the base material: 4.2%, enzyme dosage: 4.0 percent, the temperature is 45 ℃ and the time is 1 hour;
in 4 examples, the pH values of the base materials are respectively adjusted as follows during the first enzymolysis: 1.5,2,2.5,3.
The influence of pH adjustment on the base material on the degree of hydrolysis in the first enzymatic hydrolysis was examined by 4 examples in test example 12; the test results are shown in fig. 10; as can be seen from FIG. 10, the pH value has an important influence on the degree of hydrolysis, with the highest degree of hydrolysis being observed at a pH of 2.5. At pH 1.5 and 3, the degree of hydrolysis is relatively low, but still allows the degree of pepsin hydrolysis to be satisfactory.
Test example 13
This test example 13 actually contains 4 examples of the present invention, and 4 examples respectively provide 4 methods for preparing oligopeptides; the specific steps are basically the same as those in example 8; the differences are only in the part of the parameters, in particular,
the conditions for the first enzymatic hydrolysis in all 4 examples are: concentration of the base material: 4.2%, enzyme dosage: 4.0%, pH: 7.0, time 1 hour;
in the 4 embodiments, the enzymolysis temperature for the first enzymolysis is respectively as follows: 45 degrees, 46 degrees, 47 degrees and 48 degrees.
The influence of the enzymolysis temperature of the first enzymolysis on the hydrolysis degree is examined through 4 examples of the experimental example 4; the test results are shown in fig. 11. As can be seen from fig. 11, the hydrolysis temperature also has an important effect on the degree of hydrolysis, and the hydrolysis effect is best at a hydrolysis temperature of 45 ℃.
Test example 14
The preparation method is the same as that of example 8; the difference lies in that:
the enzymolysis conditions are as follows: concentration of the base material: 4.2%, enzyme dosage: 4.5%, pH: 2.5;
the first time of enzymolysis is considered as follows: 1 hour, 1.5 hours.
Test example 15
This test example 15 actually contains 5 examples of the present invention, and 5 examples respectively provide 5 methods for preparing oligopeptides; the specific steps are basically the same as those in example 8; the differences are only in the part of the parameters, in particular,
the conditions for the second enzymatic hydrolysis in the 5 examples were all: pH value: 7.9, the temperature is 37 ℃, and the time is 2 hours;
in the 5 embodiments, the enzyme dosage in the second enzymolysis is respectively as follows: 4%, 4.5%, 5%, 5.5%, 6%.
The influence of the enzyme dosage on the degree of hydrolysis in the second enzymatic hydrolysis was examined by 5 examples of test example 15; the test results are shown in fig. 12. As can be seen from fig. 12, the degree of hydrolysis gradually increased with the increase in the concentration of the base material, and the degree of hydrolysis did not change significantly when reaching a certain degree. When the base concentration is 4.5%, the degree of hydrolysis tends to be smooth. At enzyme levels of 4.0% and 6%, the degree of hydrolysis was relatively low, but still allowed the degree of trypsin hydrolysis to be satisfactory.
Test example 16
This test example 16 actually contains 6 examples of the present invention, and 6 examples respectively provide 6 methods for producing oligopeptides; the specific steps are basically the same as those in example 8; the differences are only in the part of the parameters, in particular,
the second enzymatic hydrolysis conditions of the 6 examples were all: enzyme dosage: 4.0%, temperature 37 ℃ and time 2 hours;
in 6 embodiments, when performing the second enzymatic hydrolysis, the PH of the first enzymatic hydrolysis product is adjusted as follows: 7.5,7.6,7.7,7.8,7.9,8.0.
The 6 examples of the test example 16 are used to examine the influence of different pH values of the first enzymatic hydrolysate on the degree of hydrolysis before the second enzymatic hydrolysis; the test results are shown in fig. 13. As can be seen from FIG. 13, the pH value has a significant effect on the degree of hydrolysis, with the highest degree of hydrolysis being observed at a pH of 7.9. At pH 7.5 and 8.0, the degree of hydrolysis was relatively low, but still allowed the degree of trypsin hydrolysis to be satisfactory.
Test example 17
This test example 17 actually contains 6 examples of the present invention, and 6 examples respectively provide 5 methods for preparing oligopeptides; the specific steps are basically the same as those in example 8; the differences are only in the part of the parameters, in particular,
the second enzymatic hydrolysis conditions of the 6 examples were enzyme dosage: 4.0%, pH: 7.9, time 2 hours;
in 6 embodiments, the enzymolysis temperature during the second enzymolysis is respectively: 35 degrees, 36 degrees, 37 degrees, 38 degrees, 39 degrees, 40 degrees.
The influence of the enzymolysis temperature on the degree of hydrolysis at the second enzymolysis was examined by 5 examples of test example 7, and the test results are shown in FIG. 14. As can be seen from fig. 14, the hydrolysis temperature also has an important effect on the degree of hydrolysis, and the hydrolysis effect is best at a hydrolysis temperature of 37 ℃. When the enzymolysis temperature is 35 ℃ and 40 ℃, the degree of hydrolysis is relatively slightly low, but the hydrolysis degree of trypsin can still meet the requirement
Test example 18
The enzymolysis conditions are as follows: enzyme dosage: 4.0%, pH: 7.9; temperature 37 deg.C
The second enzymolysis time is considered as follows: 1 hour, 2.5 hours, 3 hours
The other steps of the preparation method are the same as those of the example 8.
The experiment is characterized in that fixed parameters are set in the previous experiment to test the hydrolysis effect and corresponding effects under the same conditions, the reason is to reduce influence factors, particularly, the pH value is properly increased according to the factors of the salmon, the enzymolysis requirement, the environment and the like during the first enzymolysis, experiments can be carried out in the later period, and the adjustment on parameters is carried out, including the conditions of the pH value, the temperature and the like of the enzymolysis.
The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.

Claims (8)

1. A preparation method of salmon oligopeptide, which is characterized by comprising the following steps:
(1) pretreating microorganisms: mincing salmon leftovers by using a mincing machine, adding distilled water to adjust the concentration, then adding a buffering agent to adjust the pH value, adding activated strains to perform fermentation treatment, and obtaining salmon leftovers microbial pretreatment base stock;
(2) hydrolysis: adding distilled water into the salmon leftover microbial pretreatment base stock to adjust the concentration of the base stock;
(3) carrying out first enzymolysis: adding pepsin into the base material obtained in the step (2), performing enzymolysis, inactivating enzymes, and cooling;
(4) and (3) carrying out second enzymolysis: adding trypsin into the enzymolysis product obtained in the step (3), carrying out enzymolysis, and inactivating enzyme;
(5) centrifuging: centrifuging the enzymolysis product obtained in the step (4) to obtain supernatant, and adjusting the pH value;
(6) adsorption drying: and (5) carrying out activated carbon adsorption drying on the centrifugal supernatant obtained in the step (5) to obtain a finished product.
2. The method of claim 1, wherein the buffer is KH2PO4、K2HPO4And CaCO3The strain is bacillus circulans and lactobacillus acidophilus; wherein the bacillus circulans: the preservation number is CGMCC No.1.2411, and the lactobacillus acidophilus: the preservation number is as follows: CGMCC N0.1.2919.
3. The method for preparing salmon oligopeptide according to claim 2, wherein in the step (1), salmon leftovers are crushed by a crusher, distilled water is added, and the solid-to-liquid ratio is 1: 1; the buffer is added in an amount of 35g/L in a ratio of KH2PO4:K2HPO4:CaCO3: 1.3:1.5:1, and adjusting the initial pH value to 6.0-6.5; the addition amount of the activated strains is as follows: 160 g/L.
4. The method for preparing the salmon-derived oligopeptide according to any one of claims 1 to 3, wherein the microbial fermentation temperature in the step (1) is 35-38 ℃ and the fermentation time is 40-45 hours.
5. The method for preparing salmon oligopeptide according to any one of claims 1 to 3, wherein in the step (2), the salmon waste microbial pretreatment base material is added to distilled water, and the concentration of the base material is adjusted to 4.0 to 4.5%.
6. The method for preparing the oligopeptide from salmon according to any one of claims 1 to 3, wherein the fifth step comprises centrifuging the enzymatic hydrolysate for 10 to 20 minutes at 4000-.
7. The method for preparing salmon oligopeptide according to any one of claims 1-3, wherein activated carbon particles are used for adsorption, the volume ratio of the supernatant to the activated carbon particles is 10-20:1, the adsorption time is 10-20 minutes, and the salmon oligopeptide is obtained by filtering after adsorption.
8. An oligopeptide product prepared by the method for preparing the salmon oligopeptide according to the claim 1.
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