CN115925800A - Fish soluble calcium chelate protein peptide and preparation method thereof - Google Patents
Fish soluble calcium chelate protein peptide and preparation method thereof Download PDFInfo
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Abstract
The invention provides a fish soluble calcium chelating protein peptide and a preparation method thereof. The fish soluble calcium chelate protein peptide is obtained by taking fish soluble protein as a raw material, carrying out enzymolysis on the fish soluble protein by adopting neutral protease and alkaline protease, then carrying out separation, purification and freeze drying on the fish soluble calcium chelate protein peptide, wherein the molecular weight of the fish soluble calcium chelate protein peptide is 792.413 Da, and the amino acid sequence of the fish soluble calcium chelate protein peptide is Arg-Val-Phe-Asp-Lys-Glu. The fish soluble calcium chelating protein peptide prepared by the invention can be used for producing novel peptide calcium chelating agents, and has the characteristics of excellent performance, high bioavailability, safety, no toxicity and the like. The invention provides a new idea for high-value utilization of low-value marine fishes and processing leftovers thereof.
Description
Technical Field
The invention relates to a calcium chelating protein peptide, in particular to a fish soluble plasma calcium chelating protein peptide and a preparation method thereof, belonging to the technical field of biology.
Background
At present, the annual output of fish in China exceeds more than 3000 million tons, and leftovers such as fish heads, fish bones, fish viscera, fish cooking liquor and the like generated in the processing process account for about 40-55 percent of the total amount of fish materials. The by-products are rich in bioactive substances such as protein, chondroitin, fat and the like, but the utilization rate is extremely low. Cooking is one of the key steps for producing the fish meal, a large amount of fish soluble paste is generated in the cooking process, and the fish soluble paste contains rich protein, polypeptide, free amino acid, trace elements and the like and has extremely high nutritional value. However, due to the limitation of processing technology, most of the fish dissolving pulp is directly discharged at present, which causes a certain waste of resources. In recent years, a large number of researches report that the bioactive peptide has physiological functions of resisting oxidation, reducing blood fat, reducing blood pressure, resisting tumors and the like, and fish soluble pulp which is leftover generated in the fish meal processing process is a high-quality source of the bioactive peptide. Therefore, how to reasonably utilize the fish soluble slurry generated in the fish processing process can provide a certain theoretical basis for promoting the high-value utilization of animal protein resources, and has positive significance for reducing environmental pollution and developing green and healthy ocean economy.
Calcium is one of the most abundant mineral elements in the human body, and plays a very important role in life activities such as bone growth, muscle contraction and cell phagocytosis of the human body. At present, most people cannot obtain sufficient calcium from the daily diet, so the development of calcium supplement preparations is very important. The most common calcium supplement preparations for human beings include first generation calcium supplement preparations and second generation calcium supplement preparations, such as calcium carbonate, calcium lactate, calcium citrate, and the like. However, these calcium supplement preparations have a disadvantage in that they are liable to form precipitates in the digestive environment of the gastrointestinal tract, reducing their bioavailability. Therefore, improving the bioavailability of calcium is the key to solving the calcium deficiency problem. With the progress of scientific research, ca is found 2+ Form stable peptide calcium chelate with polypeptideThe substance is able to overcome two main limitations of ionic calcium: (1) biological toxicity at high levels; (2) low absorption and bioavailability at low concentrations. On one hand, the hydrolysis activity of peptidase on brush edge can be inhibited, the hydrolysis of peptide is prevented, and Ca is avoided 2+ Precipitation phenomenon occurs in the intestinal tract due to the influence of factors such as oxalic acid, phytic acid and the like; on the other hand, ca 2+ The polypeptide is absorbed by human body through the absorption channel of the small peptide in a chelating state, so that the competitive inhibition effect existing in the same channel with other metal ions is avoided. Therefore, how to obtain protein peptides with high calcium chelating activity becomes an urgent research direction for novel calcium supplements.
Disclosure of Invention
In order to solve the problems, the invention provides a fish soluble calcium chelating protein peptide and a preparation method thereof, so that the calcium chelating activity can be efficiently realized.
In order to achieve the purpose, the invention adopts the following technical scheme:
the amino acid sequence of the fish soluble calcium chelating protein peptide is Arg-Val-Phe-Asp-Lys-Glu, and the molecular weight is 792.413 Da.
The preparation method of the fish soluble calcium chelating protein peptide specifically comprises the steps of taking fish soluble protein as a raw material, carrying out enzymolysis on the fish soluble protein by compounding neutral protease and alkaline protease, and then separating, purifying and freeze-drying an enzymolysis product to obtain the fish soluble calcium chelating protein peptide;
wherein, the enzymolysis conditions are as follows: the concentration of the fish soluble protein is 100 g/L, the enzyme adding amount is 500U/g, the pH value is 8.2, the enzymolysis temperature is 54.5 ℃, the enzymolysis time is 45 min, and the enzyme activity ratio of neutral protease and alkaline protease is 1:1;
wherein, the separation and purification steps are as follows: firstly, separating an enzymolysis product by Sephadex G-25 gel filtration chromatography, wherein the sample loading amount is 1 mL, deionized water is used as an eluent, the flow rate is 0.36 BV/h, and the detection wavelength is 220 nm; collecting the component with the highest calcium chelating activity, further separating by RP-HPLC-C18 reversed phase high performance liquid chromatography, performing gradient elution by taking 0-90% (v/v) acetonitrile solution as eluent at the flow rate of 2 mL/min, collecting the component with the highest calcium chelating activity, and freeze-drying to obtain the fish plasma calcium chelating protein peptide.
The application of the fish soluble calcium chelating protein peptide in preparing food.
The application of the fish soluble calcium chelating protein peptide in preparing health care products.
The application of the fish soluble calcium chelating protein peptide in preparing the peptide calcium chelate.
The invention has the remarkable advantages that:
the invention is based on the fact that metal calcium ions can form stable peptide calcium chelate with polypeptide, on one hand, the hydrolysis activity of peptidase on the brush edge can be inhibited, the hydrolysis of peptide is prevented, and the phenomenon that calcium ions are precipitated in intestinal tracts due to the influence of factors such as oxalic acid, phytic acid and the like is avoided; on the other hand, calcium ions are absorbed by the human body through the absorption channel of the small peptide in a chelating state, so that the competitive inhibition effect existing in the same channel with other metal ions is avoided. The method takes fish soluble protein from low-value marine fish as a raw material, and separates and prepares protein peptide with high calcium chelating activity by accurately controlling enzymolysis conditions, so that the calcium chelating activity is efficiently realized. The invention provides a new idea for high-value utilization of low-value marine fish and processing leftovers thereof.
Drawings
FIG. 1 shows the effect of single factor enzyme digestion temperature.
FIG. 2 shows the effect of single-factor enzymatic pH.
FIG. 3 is a graph showing the effect of single factor enzymatic time.
FIG. 4 is a graph showing the effect of the ratio of one-way alkaline protease to neutral protease.
FIG. 5 is a graph of the response of enzymatic pH and temperature.
FIG. 6 is a response surface diagram of complex enzyme ratio and temperature.
FIG. 7 is a graph of the response of complex enzyme ratio and pH.
FIG. 8 shows the result of Sephadex G-25 gel filtration chromatography.
FIG. 9 is a peptide fingerprint of fish plasma calcium chelating protein peptide.
Detailed Description
The technical solution of the present invention will be described below with reference to specific embodiments. The following are representative examples of the present invention, which can be used to explain and support the present invention without any limitation, and any simple modification, equivalent change and modification made to the following examples according to the technical essence of the present invention are within the scope of the technical solution of the present invention.
Example 1
A fish soluble calcium chelating protein peptide and a preparation method thereof, which comprises the following process operation steps:
the fish soluble protein adopted by the invention is from Fuzhou Haiki Biotechnology industry Co., ltd, the alkaline protease (200000U/g) is purchased from Beijing Soilebao technology Co., ltd, and the neutral protease (60000U/g) is purchased from Beijing Omboxing biotechnology Limited liability company. The trichloroacetic acid nitrogen solubility index (TCA-NSI) value can accurately reflect the enzymolysis condition of protein, is a main index for detecting the degree of protein hydrolysis, and the higher the TCA-NSI value is, the higher the polypeptide yield is. The specific determination steps are that the enzymolysis liquid is precipitated and stood for 10min by using a 15-percent TCA solution, then the enzymolysis liquid is centrifuged for 10min under the condition of 4000 r/min, the protein concentration of the supernatant liquid before and after precipitation is determined by a biuret method, and the protein concentration is substituted into the following formula for calculation:
TCA-NSI = (protein concentration of supernatant after TCA precipitation/protein concentration of supernatant before TCA precipitation) × 100%.
The invention firstly adopts a single factor experiment to investigate the influence of enzymolysis temperature (37.5, 47.5, 57.5, 67.5 and 77.5 ℃), enzymolysis pH (4.5, 5.5, 6.5, 7.5, 8.5, 9.5 and 10.5), enzymolysis time (5, 15, 30, 45, 60, 90 and 120 min), alkaline protease and neutral protease on TCA-NSI (3:1, 2:1, 1:1, 1:2 and 1:3) in terms of enzyme activity ratio under the conditions that the total enzyme addition amount is 500U/g (E/S) and the concentration of fish-soluble protein is 100 g/L (figure 1-4). Then, 3 influencing factors of enzymolysis temperature, enzymolysis pH and complex enzyme ratio (enzyme activity ratio) are used as test factors, TCA-NSI is used as a response value, and a three-factor three-level orthogonal analysis test is designed, wherein the test factors and the level design are shown in table 1. Weighing a certain mass of fish soluble protein, dissolving the fish soluble protein in deionized water, and then adjusting the pH value by using 2mol/L sodium hydroxide. Firstly, the solution is heated to a corresponding temperature in a water bath, then a certain amount of enzyme is added according to different compounding proportions, and the reaction is started according to the preset reaction time. Inactivating in boiling water bath for 10min after enzymolysis, cooling, centrifuging for 10min at 10000 r/min, collecting supernatant, and performing TCA-NSI determination.
TABLE 1 independent variable factors and levels of response surface testing
The response surface experiment results are shown in table 2 and fig. 5 to 7, and after corresponding TCA-NSI and various factors are subjected to regression fitting by using Design-Expert 8.0.6, the optimal regression equation of the TCA-NSI to the enzymolysis temperature (a), the enzymolysis pH (B) and the complex enzyme ratio (C) is obtained as follows:
Y=77.63-3.64A-2.54B-3.70C-0.52AB+0.26AC+1.43BC-17.01A 2 -10.12B 2 -5.95C 2 ，R 2 =0.9993。
as can be seen from Table 3, the model was constructedFValues less than 0.0001 indicate that the model is extremely significant. Goodness of fitR 2 =0.9993, adjust fitting degreeR 2 Adj =0.9983, indicating that the quadratic regression equation accommodates 99.83% change in response value. Where the items are mishapedPAnd =0.3423 > 0.05, which indicates that the model mismatching item is not significant, and indicates that the model obtained by the test result can well predict TCA-NSI. In this prediction model, A, B, C, BC, A 2 、B 2 、C 2 The influence on TCA-NSI is obvious, while the influence on AB and AC is not obvious, which shows that the influence of the 3 factors on TCA-NSI is not simple linear relation.
Table 2 response surface experimental protocol and results
TABLE 3 significance test and variance analysis of the coefficients of the regression equations
Note: * Obviously, P is less than 0.05; * Remarkable, P < 0.01
The optimal enzymolysis conditions obtained by Design-Expert software are as follows: the temperature is 54.49 ℃, the pH is 8.18, and the enzyme activity ratio of alkaline protease to neutral protease is 1.32. In order to verify the reliability of the model and consider the convenience of actual operation, the obtained optimal process is adjusted, and the verification enzymolysis process is designed as follows: the temperature is 54.50 ℃, the pH is 8.20, the enzyme activity of the complex enzyme is 1:1, and the verification is repeated for 3 times. The results are shown in table 4, the response surface predicts that the obtained TCA-NSI is 78.32%, the verification result is 76.51%, and the verification result is similar to the predicted value, which indicates that the regression model can better predict the enzymolysis condition of the fish soluble protein.
TABLE 4 Process determination and design of confirmatory experiments for preparing protein peptides by enzymatic hydrolysis
Separation and purification means such as Sephadex G-25 gel filtration chromatography, RP-HPLC-C18 reversed-phase high performance liquid chromatography and the like are adopted to realize separation and purification of the high-calcium chelating activity protein peptide.
10.0 g fish plasm protein is weighed and dissolved in 100 mL deionized water, and then the pH value is adjusted to 8.2 by 2mol/L sodium hydroxide. The solution is heated to 54.5 ℃ in a water bath, and then 500U/g (E/S) of enzyme is added for reaction for 45 min according to the enzyme activity ratio of the neutral protease to the alkaline protease 1:1. Inactivating in boiling water bath for 10min after enzymolysis, cooling, centrifuging for 10min at 10000 r/min, and taking supernatant to obtain fish soluble protein hydrolysate for later use.
Separating the supernatant by Sephadex G-25 gel filtration chromatography with the sample amount of 1 mL, using deionized water as eluent at the flow rate of 0.36 BV/h and the detection wavelength of 220 nm, and collecting the component P1 with the highest calcium chelating activity as shown in FIG. 8. And then further separating the component P1 by RP-HPLC-C18 reversed-phase high performance liquid chromatography, performing gradient elution by taking 0-90% (v/v) acetonitrile solution as eluent, wherein the flow rate is 2 mL/min, starting the eluent until the volume of the eluent is 100 percent, ending the gradient elution until the volume of the eluent is mixed solution of 90 percent acetonitrile and 10 percent water, collecting a component S7 with the highest calcium chelating activity (the retention time is 22.586 min), and performing freeze drying to obtain the fish plasma calcium chelating protein peptide.
And (3) determining the chelation of the fish soluble pulp calcium chelating protein peptide on calcium ions by adopting an o-cresolphthalein colorimetric method. 2 mL phosphate buffer solution (0.2 moL/L, pH 8.0.0) and 1 mL calcium chloride solution (5 mmol/L) were added to stoppered test tube, followed by 1 mL fish soluble calcium chelating protein peptide solution (1.25 g/L), placed in 37 ℃ constant temperature water bath shaker for 2 h reaction, and then the reaction solution was centrifuged at 10000 r/min for 10 min. Taking 1 mL supernatant, adding o-cresolphthalein color development liquid 5 mL, and shaking up. After standing for 10min, measuring the light absorption value at 570 nm by using an ultraviolet spectrophotometer, and substituting the value into a standard curve to calculate the calcium chelating amount.
The purified fish soluble calcium chelating protein peptide has high calcium chelating activity, and as shown in table 5, compared with the fish soluble protein enzymolysis liquid and the P1 component, the calcium chelating amount of S7 is obviously improved.
TABLE 5 calcium chelation amounts of different component protein peptides
The amino acid sequence of the purified S7 fraction was determined by a mass spectrometer (Q-active Plus mass spectrometer, thermo Scientific, san Jose, USA) and the amino acid sequence was Arg-Val-Phe-Asp-Lys-Glu, as shown in FIG. 9.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A fish soluble calcium chelating protein peptide is characterized in that: the amino acid sequence of the fish plasma calcium chelating protein peptide is Arg-Val-Phe-Asp-Lys-Glu.
2. A method for preparing the fish soluble calcium chelating protein peptide of claim 1, which comprises: taking fish soluble protein as a raw material, carrying out enzymolysis on the fish soluble protein by adopting neutral protease and alkaline protease, and then separating, purifying, freezing and drying an enzymolysis product to obtain the fish soluble calcium chelate protein peptide.
3. The method for preparing a fish soluble calcium chelating protein peptide according to claim 2, wherein: the enzymolysis conditions are that the concentration of the fish soluble protein is 100 g/L, the enzyme adding amount is 500U/g, the enzymolysis pH is 8.2, the enzymolysis temperature is 54.5 ℃, the enzymolysis time is 45 min, and the enzyme activity ratio of neutral protease and alkaline protease is 1:1.
4. The method for preparing a fish soluble calcium chelating protein peptide according to claim 2, wherein the method comprises the following steps: the separation and purification steps are as follows: firstly, separating an enzymolysis product by Sephadex G-25 gel filtration chromatography, wherein the sample loading amount is 1 mL, deionized water is used as an eluent, the flow rate is 0.36 BV/h, and the detection wavelength is 220 nm; collecting the component with the highest calcium chelating activity, further separating by RP-HPLC-C18 reversed phase high performance liquid chromatography, performing gradient elution by using 0-90% v/v acetonitrile solution as eluent at the flow rate of 2 mL/min, collecting the component with the highest calcium chelating activity, and freeze-drying to obtain the fish soluble plasma calcium chelating protein peptide.
5. Use of the fish soluble calcium chelating protein peptide of claim 1 in the preparation of a food product.
6. The use of the fish soluble calcium chelating protein peptide of claim 1 in the preparation of a health product.
7. The use of the fish plasma calcium chelating protein peptide of claim 1 in the preparation of peptide calcium chelates.
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