CN115316672B - Preparation method of peanut peptide-calcium chelate - Google Patents
Preparation method of peanut peptide-calcium chelate Download PDFInfo
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- 235000017060 Arachis glabrata Nutrition 0.000 title claims abstract description 39
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- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
- A23L33/165—Complexes or chelates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Inorganic Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method of a peanut peptide-calcium chelate, and relates to the technical field of chemical synthesis of functional nutrients. The method comprises the following steps: dissolving 100mg of peanut peptide ultrafiltration component in 5mL of deionized water, placing in a constant-temperature heating magnetic stirrer at 50 ℃ and pH 8.0, adding 100mg of anhydrous calcium chloride, sealing with a preservative film, and stirring for 1h under constant-temperature shaking conditions; adding 9 times of absolute ethyl alcohol with volume to stand for 3 hours after the reaction is completed; and centrifuging the obtained precipitate at 9000r/min for 20min, collecting the precipitate, and freeze-drying to obtain the target product, namely the peanut peptide-calcium chelate. The preparation method is simple, and the prepared peanut peptide-calcium chelate has good calcium chelating activity effect.
Description
Technical Field
The invention relates to the technical field of chemical synthesis of functional nutrients, in particular to a preparation method of a peanut peptide-calcium chelate.
Background
The peanut peptide is a deep processing product of peanut protein, has rich nutrition, complete amino acid types and high content of necessary amino acid up to 38.29 percent, and has a completely independent absorption mechanism. The solubility and stability of the peanut peptide are better than those of peanut protein, the peanut peptide can be directly absorbed by small intestine to quickly supplement nutrition, directly participate in the synthesis of human amino acid and protein, promote the active group of the protein to play a role, enhance the immunity, and have biological activities such as antioxidation. In addition, the peanut peptide has good metal ion chelating ability, and the preparation of peptide-calcium and zinc chelate by using the peanut peptide has great advantages, and the presence of dietary fibers and phytate in food prevents the intestinal tract from absorbing calcium and zinc, because the dietary fibers and phytate can form insoluble complexes with metal ions, and the bioavailability of the calcium and zinc chelate is reduced. The chelate formed by chelating the peanut peptide with calcium and zinc can avoid the phenomena, remarkably improves the solubility and stability of metal ions and improves the absorption and utilization rate of the metal ions. Therefore, the calcium and zinc chelating peptide has high bioavailability and quick absorption, and is an ideal calcium and zinc co-supplement.
Calcium is a necessary nutrient element for the human body to maintain normal physiological metabolism, and plays an extremely important role in the growth and development process of the human body. Calcium deficiency can lead to a number of diseases such as osteoporosis, rickets, etc. Because of defects in the dietary structure of residents in China, high-quality sources of calcium are lacking in the diet of people, and the lack of calcium becomes a common nutritional disease in China. In fact, daily average calcium intake of residents in China is only half of recommended amount, and the daily average calcium intake is seriously insufficient. In view of the important role of calcium in the body, as well as the diseases caused by calcium deficiency, the preparation of calcium nutritional supplements has become a research hotspot.
The existing preparation method of the peanut peptide-calcium chelate is complex, and the prepared peanut peptide-calcium chelate has poor calcium chelating activity, so the invention designs a more effective preparation method of the peanut peptide-calcium chelate.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims at providing a preparation method of peanut peptide-calcium chelate, which is simple, has good calcium chelating activity effect,
in order to achieve the above object, the present invention is realized by the following technical scheme: the preparation method of the peanut peptide-calcium chelate comprises the following steps: 100mg of peanut peptide ultrafiltration component (< 3kDa,3-10kDa, >10 kDa) is dissolved in 5mL of deionized water, placed in a constant temperature heating magnetic stirrer at 50 ℃ and pH 8.0, then 100mg of anhydrous calcium chloride is added and sealed with a preservative film, and stirred for 1h under constant temperature shaking. Adding 9 times of absolute ethyl alcohol with volume to stand for 3 hours after the reaction is completed; and centrifuging the obtained precipitate at 9000r/min for 20min, collecting the precipitate, and freeze-drying to obtain the target product, namely the peanut peptide-calcium chelate.
The peanut peptide ultrafiltration component adopts <3kDa.
Determination of the chelation yield of the peanut peptide-calcium chelate:
and (3) measuring the total mass of reactants in the reaction solution and the total mass of chelates after chelation:
chelate yield (%) = (m 1/m 2) ×100% (1)
Wherein: m1 is the total mass (g) of chelate after chelation; m2 is the total mass (g) of reactants in the reaction solution.
And (3) separating and purifying the ultrafiltration component with high calcium chelating activity of <3kDa by using a Sephadex G-25 Sephadex separation chromatographic column to obtain an FPPDVA amino acid sequence with the best calcium chelating activity effect, and preparing the peptide-calcium chelate from the FPPDVA.
The invention has the beneficial effects that: the preparation method is simple, and the calcium chelating activity effect of the ultrafiltration component <3kDa is best in the measurement of the calcium chelating force and chelate yield of the ultrafiltration component (< 3kDa,3-10kDa, >10 kDa).
Drawings
The invention is described in detail below with reference to the drawings and the detailed description;
FIG. 1 is a graph of calcium ion concentration of the present invention.
Detailed Description
The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand.
Referring to fig. 1, the present embodiment adopts the following technical scheme: the preparation method of the peanut peptide-calcium chelate comprises the following steps: 100mg of peanut peptide ultrafiltration component (< 3kDa,3-10kDa, >10 kDa) is dissolved in 5mL of deionized water, placed in a constant temperature heating magnetic stirrer at 50 ℃ and pH 8.0, then 100mg of anhydrous calcium chloride is added and sealed with a preservative film, and stirred for 1h under constant temperature shaking. Adding 9 times of absolute ethyl alcohol with volume to stand for 3 hours after the reaction is completed; and centrifuging the obtained precipitate at 9000r/min for 20min, collecting the precipitate, and freeze-drying to obtain the target product, namely the peanut peptide-calcium chelate.
Notably, the peanut peptide ultrafiltration fraction was <3kDa.
Notably, the determination of the chelation yield of the peanut peptide-calcium chelate:
and (3) measuring the total mass of reactants in the reaction solution and the total mass of chelates after chelation:
chelate yield (%) = (m 1/m 2) ×100% (1)
Wherein: m1 is the total mass (g) of chelate after chelation; m2 is the total mass (g) of reactants in the reaction solution.
In addition, an ultrafiltration component <3kDa with high calcium chelating activity is separated and purified by using a Sephadex G-25 Sephadex separation chromatographic column to obtain an FPPDVA amino acid sequence with the best calcium chelating activity, and the FPPDVA is subjected to peptide-calcium chelate preparation.
The peanut peptide of the specific embodiment is subjected to enzymolysis and ultrafiltration treatment to obtain three components with different molecular weights (< 3kDa,3-10kDa and >10 kDa). And screening out the optimal component of the calcium chelating activity by using the calcium chelating force as an index. In order to obtain peanut calcium chelating peptide with higher calcium chelating activity, and the amino acid sequence is clear, sephadex G-15 Sephadex gel chromatography is adopted for separation and purification, then NANO-HPLC-MS/MS structure identification is carried out on the obtained optimal component of the calcium chelating activity, finally synthetic peptide is obtained through a solid phase synthesis technology, and the active peptide with optimal calcium chelating activity effect is screened out from the synthetic peptide for preparing peanut peptide-calcium chelate.
Example 1:
1. 100mg of laboratory-supplied peanut peptide ultrafiltration component (< 3kDa,3-10kDa, >10 kDa) was dissolved in 5mL of deionized water, placed in a constant temperature heating magnetic stirrer at 50℃and pH 8.0, then 100mg of anhydrous calcium chloride was added and sealed with a preservative film, and stirred under constant temperature shaking for 1 hour. After the reaction was completed, 9 volumes of absolute ethanol were added and the mixture was allowed to stand for 3 hours. And centrifuging the obtained precipitate at 9000r/min for 20min, collecting the precipitate, and freeze-drying to obtain the target product, namely the peanut peptide-calcium chelate.
2. Determination of chelation yield of peanut peptide-calcium chelate
And (3) measuring the total mass of reactants in the reaction solution and the total mass of chelates after chelation:
chelate yield (%) = (m 1/m 2) ×100% (formula 2-1)
Wherein: m1 is the total mass (g) of chelate after chelation; m2 is the total mass (g) of reactants in the reaction solution.
3. Determination of the calcium chelating force of the peanut peptide-calcium chelate:
3.1 the experiment adopts an o-cresolphthalein complexation colorimetric method to measure the content of calcium ions in a sample, and the principle is that calcium and a complexing agent react in an alkaline buffer solution with the pH value of 11.0+/-0.1 to form a mauve complex, the maximum absorption wavelength is 570nm, wherein the interference of divalent ions can be eliminated by 8-hydroxyquinoline, and the concentration of the calcium ions and the absorption value are in linear correlation.
3.2 Standard Curve
And respectively taking 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of standard calcium working solution (10 mug/mL) into test tubes, respectively adding deionized water to prepare solutions with the volume of 1mL, respectively adding 5mL of working color development solution, shaking uniformly, measuring the light absorption value at 570nm, and drawing a standard curve.
According to the test method, the absorbance is taken as an X (abscissa) axis, the calcium ion concentration is taken as a Y (ordinate) axis, a standard curve is obtained as shown in fig. 1, and a regression equation of the standard curve is as follows: y=0.0584x+0.0281, relative coefficient r2= 0.9911.
3.3 determination of calcium chelating force
Adding 1mL of CaCl2 solution with the concentration of 5mmol/L and 2mL of phosphate buffer with the pH of 0.2mol/L into a test tube, adding 1mL of sample with the concentration of 1mg/mL, placing into a shaking table water bath kettle for reaction at 37 ℃ for 2h, taking out, centrifuging at normal temperature for 10min at 10000r/min, and measuring the soluble calcium binding capacity in the supernatant by adopting an o-cresolphthalein complexometric colorimetric method.
Sephadex G-15 sephadex gel chromatography separation and purification
The component with optimal calcium chelating force and chelate yield in the peanut peptide Sephadex G-25 component (F1, F2, F3, F4) is separated and purified by Sephadex G-15 Sephadex gel chromatography (gel filtration column 60cm multiplied by 1 cm). The detection wavelength was 291nm. The sample was dissolved (concentration: 80mg/mL; loading: 1.5 mL), passed through a 0.22 μm pore-size filter, and then injected into a column, the column was eluted with a gradient of 0.8mL/min flow rate, an elution profile was drawn, and the different sample fractions were collected and lyophilized immediately for storage at-20 ℃.
5. NANO-HPLC-MS/MS structure identification
The peptides collected were analyzed via LC-MS/MS equipped with an online nano-spray ion source. The application system is Q exact of series EASY-nanoLC 1200 TM Plus mass spectrometer (Thermo Fisher Scientific, MA, USA). Peptides were isolated using an Acclaim PepMap C18 analytical column (75. Mu. m x 25 cm). A total of 3. Mu.L of sample was applied, the column flow was controlled at 300nL/min, the column temperature was 40℃and the electrospray voltage was 2kV, the chromatographic gradient (mobile phase A: 0.1% formic acid in water; mobile phase B: ACN solution containing 0.1% formic acid). The mass spectrometer operates in a data dependent acquisition mode, automatically switching between MS and MS/MS acquisition. The mass spectral parameters were set as follows: (1) MS: scan range (m/z) =200-1800; resolution=70000; AGC target=3e6; maximum injection time = 50ms; scan charge = 1-6; (2) HCD-MS/MS (top 20): resolution=17500; isolation window = 2m/z; AGC target=1e5; maximum injection time = 45ms; collision energy = 28; dynamic exclusion time = 30s. The amino acid sequence was determined using the analytical software PEAKS.
Synthesis of 6 calcium chelating active peptides
The LC-MS/MS identified peptide was selected, and the peptide was purified by using high performance liquid chromatography using a solid phase synthesis method for synthesizing peanut active peptide from Jiangsu Jitai peptide technology Co. With solvent a, each containing 0.01% tfa: water (H2O) and solvent B: the peptides were eluted with a linear gradient of Acetonitrile (ACN) at a flow rate of 1mL/min. The separation was monitored at 220nm using UV detection. The peptides were then subjected to Agilent-6125B Mass Spectrometry (MS) analysis. And finally, measuring the molecular weight of the synthesized peptide by adopting a liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) technology. The purity of the synthetic peptide was checked by RP-HPLC and was >95%.
The data obtained in the experiment of this example are all expressed as mean ± Standard Deviation (SD) of triplicate experiments, and each index data was analyzed by one-way analysis of variance (ANOVE, LSD) using IBM SPSS Statistics 20.0.0 software, with P <0.05 indicating significant differences between the data (marked with different letters). The original 8.0 software was used for mapping.
7. Results:
(1) In the determination of the calcium chelating force and chelate yield of the ultrafiltration component (< 3kDa,3-10kDa, >10 kDa), the calcium chelating force (9.13+ -0.11 μg/mL) of the component <3kDa was significantly higher than the calcium chelating force (P < 0.05) of the components of 3-10kDa (7.82+ -0.02 μg/mL) and >10kDa (7.54+ -0.31 μg/mL). The chelate yield of the ultrafiltration fraction <3kDa (32.38%) is higher than the chelate yield of the 3-10kDa fraction (18.92%) (P > 0.05), but lower than the chelate yield of the >10kDa fraction (45.76%) (P < 0.05). Indicating that the calcium chelating activity of the ultrafiltration fraction <3kDa is best.
(2) The ultrafiltration component with high calcium chelating activity of <3kDa is separated and purified by a Sephadex G-25 Sephadex separation chromatographic column to obtain 4 components named as F1, F2, F3 and F4 respectively. The measurement of the calcium chelating force and the chelate yield was carried out on 4 components. The results showed that the calcium chelating force (9.31.+ -. 0.02. Mu.g/mL) of the F1 component was significantly higher than the calcium chelating force (P < 0.05) of the F2 (4.68.+ -. 0.28. Mu.g/mL), F3 (4.85.+ -. 0.09. Mu.g/mL), F4 (4.77.+ -. 0.06. Mu.g/mL) components. The chelate yield of the F1 component (43.42%) is significantly higher than that of the F2 (19.62%), F3 (9.63%), F4 (4.18%) components (P < 0.05). Indicating that the calcium chelating activity of the F1 component works best.
(3) Separating and purifying the F1 component with high calcium chelating activity by using a Sephadex G-15 Sephadex separating chromatographic column to obtain 2 components named as C1 and C2 respectively. The measurement of the calcium chelating force was performed on 2 components. The results showed that the calcium chelating force of the C1 component (7.95.+ -. 0.06. Mu.g/mL) was significantly higher than the calcium chelating force of the C2 component (3.63.+ -. 0.29. Mu.g/mL) (P < 0.05), indicating that the calcium chelating activity of the C1 component was best.
(4) And adopting NANO-HPLC-MS/MS to identify the molecular weight and the amino acid sequence of the C1 component. According to the amino acid sequence characteristics of the calcium chelating active peptide, screening is carried out in the identification result with the peptide fragment credibility more than or equal to 95%. 7 amino acid sequences satisfying amino acid having calcium chelating activity were selected, and the amino acid sequences were FPEFQ, FPPDVA, LPDPVP, LPEGQF, FPDL, FPDVP, LPEPV, respectively. The determination of the calcium sequestration force is then carried out. The results showed that the calcium chelating activity of FPPDVA was significantly better than that of the other 6 synthetic peptides (P < 0.05), the calcium chelating force reached 15.67+ -0.39 μg/mL, followed by FPEFQ (6.96+ -0.42 μg/mL), LPDPVP (7.06+ -0.36 μg/mL), LPEGQF (6.61+ -0.16 μg/mL), FPDL (6.37+ -0.16 μg/mL), FPDVP (9.70+ -0.25 μg/mL), LPEPV (5.77+ -0.48 μg/mL), indicating that the calcium chelating activity of FPPDVA was the best, and the FPPDVA was subjected to peptide-calcium chelate preparation.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (1)
1. A peanut peptide-calcium chelate, characterized in that the peptide has the amino acid sequence FPEFQ, FPPDVA, LPDPVP, LPEGQF, FPDL, FPDVP or LPEPV.
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