Fish solubilized calcium chelate protein peptide and method for preparing the same

Abstract

The application provides a fish solubilized pulp calcium chelating protein peptide and a preparation method thereof. The fish solubilized pulp calcium chelating protein peptide is prepared by using fish solubilized pulp protein as raw material, adopting neutral protease and alkaline protease for enzyme hydrolysis, and then performing separation and purification and freeze-drying. The fish solubilized pulp calcium chelating protein peptide has a molecular weight of 792.413 Da and an amino acid sequence of Arg-Val-Phe-Asp-Lys-Glu. The fish solubilized pulp calcium chelating protein peptide prepared by the application can be used for producing a novel peptide calcium chelating agent, and has the characteristics of excellent performance, high bioavailability, safety and non-toxicity. The application provides a new idea for high-value utilization of low-value marine fish and processing waste.

Description

Technical Field

[0001] This invention relates to a calcium chelating protein peptide, and more specifically to a fish lysate calcium chelating protein peptide and its preparation method, belonging to the field of biotechnology. Background Technology

[0002] Currently, my country's annual fish production exceeds 30 million tons. Byproducts generated during processing, such as fish heads, bones, viscera, and cooking liquid, account for approximately 40%-55% of the total fish feed. These byproducts are rich in bioactive substances such as protein, chondroitin, and fat, but their utilization rate is extremely low. Cooking is a key step in fishmeal production, generating a large amount of fish lysate. This lysate is rich in protein, polypeptides, free amino acids, and trace elements, making it highly nutritious. However, due to limitations in processing technology, most of this fish lysate is currently directly discharged, resulting in a certain degree of resource waste. In recent years, numerous studies have reported that bioactive peptides possess physiological functions such as antioxidation, lowering blood lipids, lowering blood pressure, and anti-tumor effects. Fish lysate, a byproduct of fishmeal processing, is an excellent source of bioactive peptides. Therefore, how to rationally utilize fish lysate generated during fish processing can provide a theoretical basis for promoting the high-value utilization of animal protein resources, and also has positive significance for reducing environmental pollution and developing a green and healthy marine economy.

[0003] Calcium is one of the most abundant mineral elements in the human body, playing a vital role in essential life activities such as bone growth, muscle contraction, and phagocytosis. Currently, most people cannot obtain sufficient calcium from their daily diet, making the development of calcium supplements crucial. The most common calcium supplements for humans include first-generation and second-generation supplements, such as calcium carbonate, calcium lactate, and calcium citrate. However, these supplements have a drawback: they easily form precipitates in the gastrointestinal tract, reducing their bioavailability. Therefore, improving calcium bioavailability is key to solving the problem of calcium deficiency. With further scientific research, it has been discovered that calcium... 2+ Forming stable peptide-calcium chelates with polypeptides can overcome two major limitations of ionic calcium: (1) biotoxicity at high levels; and (2) low absorption and bioavailability at low concentrations. On the one hand, it can inhibit the hydrolytic activity of peptidases on the brush border, preventing peptide hydrolysis and avoiding the loss of calcium. 2+ In the intestines, precipitation occurs due to factors such as oxalic acid and phytic acid; on the other hand, Ca... 2+ In its chelated state, the peptide is absorbed by the human body through the absorption channels of small peptides, avoiding the competitive inhibition caused by other metal ions existing in the same channels. Therefore, obtaining protein peptides with high calcium chelating activity has become an urgent research direction for novel calcium supplements. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes a fish lysate calcium chelating protein peptide and its preparation method, enabling efficient realization of calcium chelation activity.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] A fish lysate calcium chelating protein peptide, wherein the amino acid sequence of the fish lysate calcium chelating protein peptide is Arg-Val-Phe-Asp-Lys-Glu, and the molecular weight is 792.413 Da.

[0007] The above-mentioned method for preparing fish lysate calcium chelate peptides specifically involves using fish lysate protein as raw material, enzymatically hydrolyzing it with a combination of neutral protease and alkaline protease, and then separating, purifying, and freeze-drying the hydrolysate to obtain the fish lysate calcium chelate peptides.

[0008] The enzymatic hydrolysis conditions are as follows: fish lysate protein concentration 100 g / L, enzyme addition amount 500 U / g, pH 8.2, enzymatic hydrolysis temperature 54.5℃, enzymatic hydrolysis time 45 min, and enzyme activity ratio of neutral protease to alkaline protease 1:1.

[0009] The separation and purification steps are as follows: the enzymatic hydrolysis product is first separated using Sephadex G-25 gel filtration chromatography with a sample loading volume of 1 mL, deionized water as the eluent, a flow rate of 0.36 BV / h, and a detection wavelength of 220 nm; the component with the highest calcium chelating activity is collected and further separated using RP-HPLC-C18 reversed-phase high-performance liquid chromatography with gradient elution using 0-90% (v / v) acetonitrile solution as the eluent at a flow rate of 2 mL / min; the component with the highest calcium chelating activity is collected and freeze-dried to obtain the fish lysate calcium chelating protein peptide.

[0010] The above-mentioned application of a fish lysate calcium chelate peptide in food preparation.

[0011] The above-mentioned application of a fish lysate calcium chelate peptide in the preparation of health products.

[0012] The above-mentioned fish lysate calcium chelating protein peptide is used in the preparation of peptide-calcium chelates.

[0013] The significant advantages of this invention are:

[0014] This invention is based on the principle that metallic calcium ions can form stable peptide-calcium chelates with polypeptides. On one hand, this inhibits the hydrolytic activity of peptidases on the brush border, preventing peptide hydrolysis and avoiding the precipitation of calcium ions in the intestine due to factors such as oxalic acid and phytic acid. On the other hand, calcium ions in the chelated state are absorbed by the human body through the absorption channels of small peptides, avoiding the competitive inhibition caused by other metal ions in the same channels. Using fish lysate protein from low-value marine fish as raw material, and through precise control of enzymatic hydrolysis conditions, protein peptides with high calcium chelating activity are isolated and prepared, thus achieving efficient calcium chelation. This invention provides a new approach for the high-value utilization of low-value marine fish and their processing byproducts. Attached Figure Description

[0015] Figure 1 The effect of single-factor enzymatic hydrolysis temperature.

[0016] Figure 2 The effect of pH on single-factor enzymatic hydrolysis.

[0017] Figure 3 The effect of single-factor enzymatic hydrolysis time.

[0018] Figure 4 The effect of the ratio of alkaline protease and neutral protease on single factors.

[0019] Figure 5 This is a response surface plot of pH and temperature for enzymatic hydrolysis.

[0020] Figure 6 This is a response surface plot of the ratio of the complex enzyme and temperature.

[0021] Figure 7 This is a response surface plot of the complex enzyme ratio and pH.

[0022] Figure 8 The results are from Sephadex G-25 gel filtration chromatography.

[0023] Figure 9 The peptide fingerprint of fish lysate calcium chelate peptide. Detailed Implementation

[0024] The technical solution of the present invention will be described below with reference to specific embodiments. The following are representative embodiments of the present invention, which can be used to explain and support the present invention, but do not limit the present invention in any way. Any simple modifications, equivalent changes and modifications made to the following embodiments without departing from the content of the technical solution of the present invention and based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

[0025] Example 1

[0026] A fish lysate calcium chelate peptide and its preparation method, comprising the following process steps:

[0027] The fish lysate protein used in this invention was obtained from Fuzhou Haihui Biotechnology Co., Ltd., the alkaline protease (200,000 U / g) was purchased from Beijing Solarbio Science & Technology Co., Ltd., and the neutral protease (60,000 U / g) was purchased from Beijing Aoboxing Biotechnology Co., Ltd. The trichloroacetic acid nitrogen solubility index (TCA-NSI) value can accurately reflect the enzymatic hydrolysis of proteins and is a major indicator for detecting the degree of protein hydrolysis. The higher the TCA-NSI value, the higher the peptide yield. The specific determination procedure is as follows: the enzymatic hydrolysate is precipitated with 15% TCA solution and allowed to stand for 10 min, then centrifuged at 4000 r / min for 10 min. The protein concentration of the supernatant before and after precipitation is determined by the biuret method, and then calculated using the following formula:

[0028] TCA-NSI = (protein concentration in supernatant after TCA precipitation / protein concentration in supernatant before TCA precipitation) × 100%.

[0029] This invention first investigated the effects of enzymatic hydrolysis temperature (37.5, 47.5, 57.5, 67.5, 77.5℃), enzymatic hydrolysis pH (4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5), enzymatic hydrolysis time (5, 15, 30, 45, 60, 90, 120 min), and the ratio of alkaline protease to neutral protease (based on enzyme activity ratio, 3:1, 2:1, 1:1, 1:2, 1:3) on TCA-NSI using single-factor experiments under the conditions of a total enzyme addition of 500 U / g (E / S) and a fish lysate protein concentration of 100 g / L. Figures 1 to 4 Then, the three influencing factors—enzymatic hydrolysis temperature, enzymatic hydrolysis pH, and the ratio of compound enzymes (enzyme activity ratio)—were used as experimental factors. A three-factor, three-level orthogonal analytical experiment was designed with TCA-NSI as the response value. The experimental factors and levels are shown in Table 1. A certain mass of fish lysate protein was weighed and dissolved in deionized water, and then the pH was adjusted with 2 mol / L sodium hydroxide. The solution was first heated to the appropriate temperature in a water bath, and then a certain amount of enzyme was added according to different compound ratios, and the reaction was started at the predetermined reaction time. After enzymatic hydrolysis, the enzyme was inactivated in a boiling water bath for 10 min, cooled, and then centrifuged at 10000 r / min for 10 minutes. The supernatant was collected for TCA-NSI determination.

[0030] Table 1. Independent variables and levels in response surface methodology experiments

[0031]

[0032] The results of the response surface methodology experiments are shown in Table 2 and Figures 5 to 7As shown, after using Design-Expert 8.0.6 to perform regression fitting between the corresponding TCA-NSI and each factor, the optimal regression equation for TCA-NSI on enzymatic hydrolysis temperature (A), enzymatic hydrolysis pH (B), and compound enzyme ratio (C) is obtained as follows:

[0033] 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.

[0034] As can be seen from Table 3, the proposed model... F A value less than 0.0001 indicates that the model is extremely significant. Goodness of fit. R 2 =0.9993, adjust the goodness of fit R 2 Adj =0.9983, indicating that the quadratic regression equation adapts to 99.83% of the response value changes. The lack-of-fit term... P =0.3423>0.05, indicating that the model lack of fit term is not significant, meaning that the model obtained from this experiment can predict TCA-NSI well. In this prediction model, A, B, C, BC, A 2 B 2 C 2 The effect of TCA-NSI is significant, while the effects of AB and AC are not significant, indicating that the influence of these three factors on TCA-NSI is not a simple linear relationship.

[0035] Table 2 Response Surface Experiment Scheme and Results

[0036]

[0037] Table 3. Significance tests and variance analysis of the coefficients of the regression equation.

[0038]

[0039] Note: *Significant, P < 0.05; **Highly significant, P < 0.01

[0040] The optimal enzymatic hydrolysis conditions obtained from Design-Expert software were: temperature 54.49℃, pH 8.18, and an enzyme activity ratio of alkaline protease to neutral protease of 1.32:1. To verify the reliability of the model and considering the convenience of actual operation, the optimal process was adjusted, and the validation enzymatic hydrolysis process was designed as follows: temperature 54.50℃, pH 8.20, and a compound enzyme activity ratio of 1:1, with three repeated validations. The results are shown in Table 4. The TCA-NSI predicted by the response surface methodology was 78.32%, while the validation result was 76.51%. The validation result is close to the predicted value, indicating that the regression model can predict the enzymatic hydrolysis of fish lysate proteins well.

[0041] Table 4. Process determination and verification experimental design for the preparation of protein peptides by enzymatic hydrolysis.

[0042]

[0043] The separation and purification of high-calcium chelating active protein peptides were achieved using Sephadex G-25 gel filtration chromatography and RP-HPLC-C18 reversed-phase high-performance liquid chromatography.

[0044] Weigh 10.0 g of fish lysate protein and dissolve it in 100 mL of deionized water. Then adjust the pH to 8.2 with 2 mol / L sodium hydroxide. First, heat the solution to 54.5℃ in a water bath. Then, add 500 U / g (E / S) of enzyme at a neutral protease to alkaline protease activity ratio of 1:1 and react for 45 min. After enzymatic hydrolysis, inactivate the enzyme in a boiling water bath for 10 min. After cooling, centrifuge at 10000 r / min for 10 min and collect the supernatant, which is the fish lysate protein hydrolysate, for later use.

[0045] The supernatant was separated by Sephadex G-25 gel filtration chromatography with a sample volume of 1 mL, using deionized water as the eluent, a flow rate of 0.36 BV / h, and a detection wavelength of 220 nm. The elution chromatogram is shown below. Figure 8 As shown, fraction P1, exhibiting the highest calcium chelating activity, was collected. Fraction P1 was then further separated using RP-HPLC-C18 reversed-phase high-performance liquid chromatography, employing a gradient elution with 0-90% (v / v) acetonitrile solution as the eluent at a flow rate of 2 mL / min. The eluent was initially set to contain 100% water by volume and ended with a mixture of 90% acetonitrile and 10% water by volume. Fraction S7, exhibiting the highest calcium chelating activity (retention time 22.586 min), was collected and freeze-dried to obtain the aforementioned fish lysate calcium chelating protein peptide.

[0046] The chelating effect of the fish lysate calcium chelating peptide on calcium ions was determined using the o-cresolphthalein colorimetric method. 2 mL of phosphate buffer (0.2 mol / L, pH 8.0) and 1 mL of calcium chloride solution (5 mmol / L) were added to a stoppered test tube, followed by 1 mL of fish lysate calcium chelating peptide solution (1.25 g / L). The mixture was placed in a 37°C water bath with a shaker for 2 h, and then centrifuged at 10000 r / min for 10 min. 1 mL of the supernatant was collected, and 5 mL of o-cresolphthalein colorimetric solution was added and mixed well. After standing for 10 min, the absorbance was measured at 570 nm using a UV spectrophotometer. The calcium chelation amount was then calculated by substituting the absorbance into a standard curve.

[0047] The purified fish lysate calcium chelating protein peptides have high calcium chelating activity, as shown in Table 5. Compared with the fish lysate protein hydrolysate and P1 component, the calcium chelation amount of S7 is significantly improved.

[0048] Table 5. Calcium chelation amount of different protein peptide components

[0049]

[0050] The purified S7 fraction was sequenced using a Q-Exactive Plus mass spectrometer (ThermoScientific, San Jose, USA). The obtained amino acid sequence was Arg-Val-Phe-Asp-Lys-Glu. Figure 9 As shown.

[0051] The above description is merely a representative embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fish lysate calcium chelating protein peptide, characterized in that: The amino acid sequence of the fish lysate calcium chelate peptide is Arg-Val-Phe-Asp-Lys-Glu.

2. The application of the fish lysate calcium chelating protein peptide as described in claim 1 in the preparation of peptide-calcium chelates.

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