CN109680027B - Grifola frondosa small peptide iron chelate as well as preparation method and application thereof - Google Patents
Grifola frondosa small peptide iron chelate as well as preparation method and application thereof Download PDFInfo
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- CN109680027B CN109680027B CN201811629128.9A CN201811629128A CN109680027B CN 109680027 B CN109680027 B CN 109680027B CN 201811629128 A CN201811629128 A CN 201811629128A CN 109680027 B CN109680027 B CN 109680027B
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- grifola frondosa
- iron chelate
- iron
- protein
- peptide
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Abstract
The invention discloses a Grifola frondosa small peptide iron chelate as well as a preparation method and application thereof, wherein the preparation method of the Grifola frondosa small peptide iron chelate comprises the following steps of (1) extracting Grifola frondosa fruiting body protein; (2) carrying out proteolysis on the grifola frondosa; (3) separating and purifying the grifola frondosa protein peptide; (4) preparing a grifola frondosa small peptide iron chelate. The invention extracts grifola frondosa protein from grifola frondosa sporocarp, obtains small peptide through enzymolysis, and chelates with iron salt after separation and purification to obtain the grifola frondosa peptide-iron chelate, wherein the iron bioavailability of the peptide-iron chelate is superior to that of inorganic iron salt, and the peptide-iron chelate has stronger immunological activity. The Grifola frondosa small peptide iron chelate prepared by the invention has stable property and is easy to dissolve in water, and compared with the traditional oral iron agent ferrous sulfate, the small peptide iron chelate has less damage to gastrointestinal tracts, can be better absorbed and can be used as a novel iron supplement agent. The preparation process is simple, convenient and quick, and the synthesized polypeptide iron chelate has high efficiency and high chelated iron content.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a grifola frondosa small peptide iron chelate as well as a preparation method and application thereof.
Background
Grifola frondosa (Grifola frondosa) is one of rare and edible fungi, belongs to the class of Basidiomycotina, class of Hymenomycetes, class of Ascomycotina, order Aphyllophorales, family of Polyporaceae, genus Grifola, also called Maitake (Maitake) in Japan, and is also called Bayesian, Qianfo, chestnut mushroom, Pleurotus ostreatus, Yunzhu, Lotus flower fungus, etc. Wild grifola frondosa is mostly grown in subtropical to temperate forests, and widely distributed in japan, russia, north america, and the Changbai mountain areas of china, Hebei, Sichuan, and the like. The Grifola frondosa fruiting body has delicious taste, the aroma of Tricholoma matsutake, and high nutrition and health promotion value. The grifola frondosa has the highest content of saccharides, the protein content of the grifola frondosa is second to that of polysaccharide, and the protein content of the grifola frondosa is higher than that of mushrooms, needle mushrooms, agarics and the like. The fruiting body contains 17 amino acids, wherein 7 essential amino acids account for 40.0% of total amino acids of Grifola frondosa. The content of isoleucine (Ile), aspartic acid (Asp) and glutamic acid (Glu) in the fruit body of the Grifola frondosa is high, and the Grifola frondosa has important effects of keeping full-bodied delicate flavor, promoting chelation with metal ions, relieving fatigue, protecting cranial nerves and the like.
The Grifola frondosa is rich in nutrition and has certain health care function. However, the research on the grifola frondosa mostly stays in the aspects of polysaccharide extraction and biological functions thereof, the grifola frondosa polypeptide has the functional activities of antioxidation, immunity enhancement, anti-tumor and the like, and the current researches on grifola frondosa protein and derivatives thereof are rarely reported.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a grifola frondosa small peptide iron chelate and a preparation method thereof.
The invention also provides application of the grifola frondosa small peptide iron chelate.
The technical scheme is as follows: in order to achieve the above object, the preparation method of the grifola frondosa small peptide iron chelate is characterized by comprising the following steps:
(1) extracting the protein of the fruit body of the grifola frondosa:
pulverizing dried Maitake Mushroom fruiting body into superfine powder, adding distilled water, stirring, extracting at constant temperature under stirring, cooling to room temperature, centrifuging, collecting supernatant, standing at low temperature overnight, centrifuging, removing supernatant, precipitating, washing, vacuum drying, pulverizing, and sieving to obtain Maitake Mushroom fruiting body protein powder;
(2) carrying out proteolysis on the grifola frondosa:
weighing Grifola frondosa sporophore protein powder, adding distilled water, adding protease, performing enzymolysis, centrifuging hydrolysate after reaction, dialyzing supernatant, lyophilizing to obtain crude Grifola frondosa protein peptide, and storing;
(3) and (3) separating and purifying the grifola frondosa protein peptide:
performing first-step purification on the obtained crude grifola frondosa protein peptide through ultrafiltration; separating and purifying by Sephadex column to obtain Polyporus frondosus polypeptide;
(4) preparing a grifola frondosa small peptide iron chelate:
weighing Polyporus frondosus polypeptide, dissolving in distilled water, adding ascorbic acid solution, and adding FeCl2And (3) carrying out oscillation reaction on the solution, centrifuging after the reaction is finished, taking the supernatant, adding absolute ethyl alcohol, standing overnight for precipitation, centrifuging to take the precipitate, washing and drying to obtain the grifola frondosa small peptide iron chelate.
Wherein, the supernatant fluid is firstly evaporated to 1/2 of the original volume after the supernatant fluid is taken in the step (1), and the pH value is adjusted to 3.5 to the isoelectric point of the grifolan.
Wherein, the protease in the step (2) is one of flavor enzyme, compound protease, pepsin, neutral protease, alkaline protease and trypsin.
Wherein, the adding amount of the protease in the step (2) is 1600-2100U/g grifola frondosa sporocarp protein powder.
Preferably, the enzymolysis time in the step (2) is 1-3.5 h.
Most preferably, the optimum process conditions for hydrolyzing the grifola frondosa protein by adopting the alkaline protease are that the pH is 9, the enzyme adding amount is 1800U/g grifola frondosa sporocarp protein powder, the enzymolysis temperature is 55 ℃, the concentration of the grifola frondosa protein is 0.5g/100mL, and the enzymolysis time is 2.5 h. Under the optimal enzymolysis condition, the ferrous chelation capacity is (1.854 +/-0.025) mg/g, and the hydrolysis degree is (6.14 +/-0.05)%.
And (3) performing ultrafiltration on the crude grifola frondosa protein peptide to purify the crude grifola frondosa protein peptide in the first step to prepare a solution, filtering to obtain a supernatant, performing ultrafiltration on the solution, dialyzing, and freeze-drying to obtain grifola frondosa polypeptide components with the KDa being more than 5KDa, 1KDa to 5KDa and the KDa being less than 1 KDa.
Separating and purifying the purified product by a glucan gel column after ultrafiltration purification in the step (3) by adding the glucan gel powder into distilled water for full swelling, removing suspended particles and degassing; and adding the degassed sephadex solution into a chromatographic column, taking a PBS (phosphate buffer solution) as a mobile phase, and loading and eluting the ultrafiltered grifola frondosa polypeptides with different molecular weights to obtain the purified component grifola frondosa polypeptides.
Wherein, in the step (4), 100-; adding 100-120 mu LFeCl2A solution; oscillating the reaction temperature to 20-25 ℃, and reacting for 4-5 min.
Most preferably, the optimal process conditions for the chelation reaction are: when the pH value of each 100mg of grifola frondosa polypeptide is 5 and the total volume of a reaction system is 10mL, adding 1mol/L of 100 mu L of ferrous chloride, reacting at the temperature of 20 ℃ for 5min, wherein the highest content of the obtained chelate iron is (31.51 +/-1.32) mg/g.
The invention relates to a Grifola frondosa small peptide iron chelate prepared by the preparation method of the Grifola frondosa small peptide iron chelate.
The application of the grifola frondosa small peptide iron chelate prepared by the preparation method of the grifola frondosa small peptide iron chelate in preparing a bioactive iron supplement agent.
The reagents of the present invention are commercially available, and alkaline protease (200U/mg), neutral protease (14000U/g), pepsin (15000U/g), complex protease (120U/mg), and flavourzyme (20U/mg) are purchased from Shanghai-derived Yellowski Co., Ltd; trypsin (2500U/g) Aladdin reagent Inc; sephadex G-25, Sephadex G-15, Beijing Solaibao Tech Co., Ltd.
Double resistance: (penicillin 100U/mL, streptomycin 0.1 mg/mL); non-essential amino acids (L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine and glycine); fetal bovine serum, an optional amino acid solution, and MEM culture medium were purchased from Gibco, USA; the mixed penicillin-streptomycin solution was purchased from Biyuntian biotechnology Co.
The material used in the invention is grifola frondosa, and the protein content of the grifola frondosa is second to that of polysaccharide. The protein of the fruit body of the grifola frondosa contains 17 amino acids, wherein 7 amino acids which are necessary for human bodies account for 40.0 percent of the total amount of the grifola frondosa amino acids. The content of isoleucine (Ile), aspartic acid (Asp) and glutamic acid (Glu) in the fruit body of the Grifola frondosa is high, and the Grifola frondosa has important effects of keeping full-bodied delicate flavor, promoting chelation with metal ions, relieving fatigue, protecting cranial nerves and the like. The grifola frondosa polypeptide obtained by enzymolysis of grifola frondosa protein generally has certain biological activity, is easy to digest and absorb by human bodies, and has low antigenicity and the like. The polypeptide iron chelate synthesized by utilizing the grifola frondosa polypeptide is brown powder, has no peculiar smell, has no free iron ions, is easy to dissolve in water, is insoluble in organic solvents such as ethanol and the like, and has stable property. The polypeptide iron chelate can be absorbed by an animal in a molecular form, and is targeted to a specific tissue to release iron elements, so that the condition that endogenous free radicals generated by free iron ions damage cell membranes is reduced while iron is supplemented. In addition, the human immune system is closely related to iron deficiency, and the grifola frondosa polypeptide iron can effectively promote the enhancement of the immune system of the body. The Grifola frondosa small peptide iron chelate prepared by the method has stable property, high safety, easy absorption and certain bioactivity.
The invention not only provides a new research direction for promoting the high-efficiency utilization of the industries and resources of medicinal and edible fungi, but also provides a raw material for preparing the peptide iron chelate. The invention researches the preparation process, the basic structure and the absorption mechanism of the grifola frondosa protein peptide iron chelate, researches the immunocompetence of the grifola frondosa polypeptide and the iron chelate thereof, provides a basis for the grifola frondosa protein peptide iron as a safe and effective functional food additive with certain bioactivity, and simultaneously improves the economic and social values of the grifola frondosa.
The invention carries out ultraviolet and infrared spectrum scanning on the grifola frondosa protein peptide and the iron chelate thereof, and the atlas shows that the grifola frondosa protein peptide and the iron chelate thereof have the characteristic absorption of amino acid. But the maximum absorption peak of the chelate ultraviolet generates blue shift, and the number of peaks, the peak intensity and the positions of the peaks in the infrared spectrum are all changed and an N-Fe absorption peak appears, which indicates that the grifola frondosa peptide iron chelate is generated, and simultaneously illustrates that the chelating mechanism is as follows: iron binds to both amino and carboxyl groups.
In vitro digestion experiments show that GFP-Fe can keep higher solubility and stability in gastrointestinal tract. And the solubility ratio of GFP-Fe to FeSO in the intestinal environment4Higher GFP-Fe absorption of small intestine can be promoted. The results of cytotoxicity experiments prove that GFP-Fe and FeSO4Compared with low toxicity. Research on absorption effect and absorption mechanism of grifolan iron chelate by Caco-2 cell model method shows that GFP-Fe absorption is better than FeSO absorption4Oligopeptide transporter (PepT1) mediates the active transport of GFP-Fe, while multidrug resistance protein-mediated efflux exists.
In an immune activity experiment, CGFP, GFP-2, CGFP-Fe and GFP-Fe have obvious enhancement effect on the proliferation of splenocytes and macrophages of mice, which shows that grifolan peptide and iron chelate thereof have promotion effect on both specific immunity and non-specific immunity process, wherein the promotion effect of the grifolan iron chelate is stronger. Meanwhile, the grifolan peptide and the iron chelate thereof can promote macrophage to secrete NO and can also enhance the release of IL-6 and TNF-alpha cytokines to a certain extent.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the Grifola frondosa small peptide iron chelate prepared by the invention has stable property and is easy to dissolve in water, and compared with the traditional oral iron agent ferrous sulfate, the small peptide iron chelate has less damage to gastrointestinal tracts, can be better absorbed and can be used as a novel iron supplement agent.
2. The grifola frondosa polypeptide iron chelate prepared by the invention has certain immunocompetence, strengthens the immune system of an organism, and has certain recovery effect on the damage of the immune system of the organism caused by iron deficiency.
3. Compared with the traditional chemical method, the method for preparing the grifola frondosa polypeptide by the enzymolysis method has the advantages of better retention of the product structure and activity, mild reaction conditions and high selectivity.
4. The method purifies the crude grifola frondosa protein peptide by ultrafiltration and sephadex methods, and the ultrafiltration is widely applied to separation and purification of polypeptide liquid, and compared with the traditional separation methods such as ion exchange chromatography and high-efficiency chromatographic analysis, the method has the advantages of low energy consumption, small equipment, good selectivity, low temperature, high separation speed and high separation efficiency.
5. The invention prepares the grifola frondosa small peptide iron chelate by a chemical method, the method is simple, convenient and quick, and the synthesized polypeptide iron chelate has high efficiency and high chelated iron content.
Drawings
FIG. 1 is a schematic drawing showing the elution curve of GFPU-2 by Sephadex G-25;
FIG. 2 is a schematic drawing showing the elution curve of GFPU-3 by Sephadex G-15;
FIG. 3 is a schematic diagram of Sephadex G-15 for determining the molecular weight of a standard substance;
FIG. 4 is an ultraviolet spectrum of Grifola frondosa protein peptide and iron chelate thereof;
FIG. 5 is an infrared spectrum of Grifola frondosa protein peptide;
FIG. 6 is an infrared spectrum of Grifola frondosa small peptide iron chelate;
FIG. 7 is a schematic diagram of digestion of Grifola frondosa small peptide iron chelate by in vitro simulated gastric environment;
FIG. 8 is a schematic diagram showing in vitro simulated intestinal digestion of Grifola frondosa small peptide iron chelate;
FIG. 9 is a schematic diagram showing the MTT method for determining the influence of Grifola frondosa small peptide iron chelate and ferrous sulfate on Caco-2 cells; wherein A, B, C are intervention 12h, 24h and 48h MTT results respectively. And indicate P <0.05 and P <0.01, respectively, compared to the blank group;
FIG. 10 is a schematic representation of the effect of Grifola frondosa protein peptides and iron chelates thereof on mouse splenocyte proliferation;
FIG. 11 is a schematic representation of the effect of Grifolan peptides and their iron chelates on mouse macrophage proliferation;
FIG. 12 is a graph showing the effect of Grifolan peptides and iron chelates on NO production by macrophages;
FIG. 13 is a graph showing the effect of Grifolan peptides and iron chelates on IL-6 production by macrophages;
FIG. 14 is a graph showing the effect of grifolan peptides and iron chelates thereof on TNF- α production by macrophages.
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1
(1) Pulverizing dried Grifola frondosa fruiting body with an ultrafine pulverizer, sieving with 300 mesh sieve to obtain Grifola frondosa fruiting body ultrafine powder, weighing 90g Grifola frondosa fruiting body powder, adding distilled water according to the material-liquid ratio of 1:50(g/mL), stirring uniformly, adjusting pH to 9 with 1mol/L NaOH solution, placing into a 70 ℃ constant temperature water bath kettle, stirring and extracting for 3h, and keeping the pH of the system constant with 1mol/L NaOH solution. After the reaction is finished, the mixture is cooled to room temperature and centrifuged for 20min at 4000 r/min. The supernatant was rotary evaporated to 1/2 of the original volume, adjusted to pH 3.5 (isoelectric point of grifolan) with 2mol/L HCl solution, left overnight at low temperature, centrifuged at 4000r/min for 25min, decanted, the precipitate washed once with 95% ethanol solution and dried in vacuo. Pulverizing, and sieving with 60 mesh sieve to obtain Maitake Mushroom fruiting body protein powder.
(2) Accurately weighing a certain amount of grifola frondosa sporophore protein powder, adding distilled water according to a proportion, dissolving grifola frondosa sporophore protein to ensure that the mass concentration of the solution is 0.5g/100mL, adjusting the optimum pH value of alkaline protease to 9.0, then preserving heat at the optimum temperature of 55 ℃ for 10min, adding the alkaline protease according to the enzyme addition amount of 1800U/g of grifola frondosa sporophore protein powder, carrying out enzymolysis for 2.5h at the temperature of 55 ℃, keeping the pH value of the system constant during the enzymolysis, carrying out enzyme inactivation at the temperature of 95 ℃ for 10min, centrifuging hydrolysate for 25min at 4000r/min, and fixing the volume of the supernatant to 250mL to obtain crude grifola protein peptide (CGFP) stored at the temperature of 4 ℃.
(3) The obtained crude grifolan protein peptide (CGFP) is purified by an ultrafiltration method in the first step, the CGFP is prepared into a solution of 10mg/mL, and the solution passes through a 0.45-micron water system filter membrane to obtain a supernatant. And then, sequentially passing the solution through 5kDa and 1kDa ultrafiltration membranes at normal temperature and under the pressure of 0.3MPa to obtain three components of 5kDa,5 kDa-1 kDa and <1kDa, which are respectively named as GFPU-1, GFPU-2 and GFPU-3, reserving GFPU-2, and carrying out separation and purification on the GFPU-3 by using a sephadex column. Firstly, 20g of glucan gel powder is added into 500mL of distilled water for full swelling, and suspended particles are removed for degassing treatment; secondly, adding the degassed sephadex solution into a chromatographic column (1.6X 60cm) to avoid faults and bubbles; then 5 column volumes were equilibrated with 0.05mol/L PBS (pH 6.8) buffer as mobile phase; and finally, carrying out sample loading and elution to obtain a purified component.
(4) Further purifying by Sephadex G-25 and Sephadex G-15 chromatographic columns respectively according to different molecular weights of GFPU-2 and GFPU-3, wherein chromatographic conditions of the Sephadex G-25 are as follows: the sample loading amount is 15 mg/mL; PBS (pH 6.8) buffer with a mobile phase of 0.05 mol/L; the flow rate is 0.2 mL/min; 5mL of each tube was collected. The chromatographic conditions for Sephadex G-15 were: the sample loading amount is 25 mg/mL; PBS (pH 6.8) buffer with a mobile phase of 0.05 mol/L; the flow rate is 0.4 mL/min; 6mL of each tube was collected to obtain GFP-1 and GFP-2 Grifola frondosa polypeptides, respectively. The effluents were measured at 220nm and the major components (GFP-1 and GFP-2) were collected for protein content and ferrous chelation capacity.
(5) Accurately weighing 0.1g Grifola frondosa polypeptide (GFP-2) and crude Grifola frondosa protein peptide (CGFP), dissolving in 10mL distilled water, adding 100 μ L0.2 g/L ascorbic acid solution, adjusting pH to 5.0, adding 100 μ L1 mol/L FeCl2The solution was reacted for 5min with shaking at 20 ℃. After the reaction is finished, centrifuging for 10min at 4000r/min, taking the supernatant, adding absolute ethyl alcohol according to the proportion of 1:5(v: v), and precipitating at 4 ℃ overnight. Centrifuging at 4000r for 15min, collecting precipitate, washing with 25mL of 95% (v/v) ethanol, and drying in a vacuum drying oven at 50 deg.C for 8h to obtain purple solid including Grifola frondosa small peptide iron chelate (GFP-Fe) and crude Grifola frondosa protein peptide iron chelate (CGFP-Fe).
Example 2
Example 2 is the same as the preparation method of example 1, except that in the step (2), alkaline protease is added into the grifola frondosa sporocarp protein powder with the enzyme addition amount of 2100U/g for enzymolysis for 1 hour; adding 0.1g of grifola frondosa protein peptide into 120 mu L of 0.2g/L ascorbic acid solution in the step (5); adding 120 mu L of 1mol/L FeCl2A solution; the reaction temperature was 25 ℃ with shaking and the reaction time was 4 min.
Example 3
Example 3 is the same as the preparation method of example 1, except that in step (2), alkaline protease is added according to the enzyme adding amount of 1600U/(g grifola frondosa sporocarp protein powder), and enzymolysis is carried out for 3.5 h.
Example 4
Example 4 the same preparation method as example 1 except that in step (2), the alkaline protease is replaced by flavor enzyme, complex protease, pepsin, neutral protease or trypsin, the optimum pH of each enzyme is adjusted to 7.0, 2.0, 7.0 and 8.0, and the optimum enzymolysis temperature is 50 ℃, 45 ℃, 37 ℃, 50 ℃ and 37 ℃.
Example 5
Accurately weighing a certain amount of grifola frondosa sporophore protein powder by adopting the step (2) in the example 1, adding distilled water according to a proportion, dissolving the grifola frondosa sporophore protein to ensure that the mass concentration of the solution is 0.5g/100mL, and keeping the temperature at the optimum temperature for 10min after adjusting the optimum pH value of each enzyme. Adding 6 proteases (flavor enzyme, compound protease, pepsin, neutral protease, alkaline protease and trypsin) according to the enzyme dosage of 1800U/g, respectively, performing enzymolysis for 0.5, 1, 2, 3, 4 and 5h, keeping the pH value (7.0, 2.0, 7.0, 9.0 and 8.0) of the system constant, performing hydrolysis at the optimum temperature (50 ℃, 45 ℃, 37 ℃, 50 ℃, 55 ℃ and 37 ℃) of each enzyme, and inactivating the enzyme at 95 ℃ for 10min to finish the reaction. And (4) centrifuging the hydrolysate for 25min at 4000r/min, metering the volume of the supernatant to 250mL, and storing at 4 ℃. Each group of enzymolysis reaction is subjected to 3 parallel tests, and the ferrous chelation capacity and hydrolysis degree determination tests are performed for 3 times in parallel.
The result shows that the alkaline protease is adopted to hydrolyze the grifola frondosa sporocarp protein optimally, and the optimal process conditions are that the pH is 9, the enzyme adding amount is 1800U/g, the enzymolysis temperature is 55 ℃, the substrate mass concentration is 0.5%, and the enzymolysis time is 2.5 h. Under the optimal enzymolysis condition, the ferrous chelating capacity is (1.854 +/-0.025) mg/g, and the hydrolysis degree is (6.14 +/-0.05)%.
The crude grifolan peptide (CGFP) obtained in step (3) of example 1 was purified in the first step by ultrafiltration, and the CGFP was prepared as a 10mg/mL solution and passed through a 0.45 μm aqueous membrane to obtain a supernatant. Then, at normal temperature and under the pressure of 0.3MPa, the solution is sequentially passed through 5kDa and 1kDa ultrafiltration membranes to obtain three components of 5kDa,5 kDa-1 kDa and <1kDa, which are respectively named as GFPU-1, GFPU-2 and GFPU-3, the protein content of each component is determined by a biuret method, and the ferrous chelation capacity of the components is determined at the same time, and the results are shown in Table 1.
TABLE 1 determination of protein content and ferrous chelation Capacity of Ultrafiltration fraction
As can be seen from Table 1, the protein contents of the three fractions obtained by ultrafiltration, the protein content of the fraction >5kDa were the lowest and the protein peptides having generally better chelating activity were all small molecule peptides, so this fraction was discarded and further studies were carried out on the other two fractions GFPU-2 and GFPU-3 in example 1.
GFPU-2 and GFPU-3 were separated and purified on a column of Sephadex by the procedure (3) of example 1. Firstly, 20g of glucan gel powder is added into 500mL of distilled water to be fully swelled, and suspended particles are removed; secondly, adding the degassed sephadex solution into a chromatographic column (1.6X 60cm) to avoid faults and bubbles; then 5 column volumes were equilibrated with 0.05mol/L PBS (pH 6.8) buffer as mobile phase; and finally, carrying out sample loading and elution to obtain a purified component.
Further purifying by Sephadex G-25 and Sephadex G-15 chromatographic columns respectively according to different molecular weights of GFPU-2 and GFPU-3, wherein chromatographic conditions of the Sephadex G-25 are as follows: the sample loading amount is 15 mg/mL; PBS (pH 6.8) buffer with a mobile phase of 0.05 mol/L; the flow rate is 0.2 mL/min; 5mL of each tube was collected. The chromatographic conditions for Sephadex G-15 were: the sample loading amount is 25 mg/mL; PBS (pH 6.8) buffer with a mobile phase of 0.05 mol/L; the flow rate is 0.4 mL/min; 6mL of each tube was collected to obtain GFP-1 and GFP-2 Grifola frondosa polypeptides, respectively. The effluents were measured at 220nm and the major components (GFP-1 and GFP-2) were collected for protein content and ferrous chelation capacity.
The elution profile of GFPU-2 through Sephadex G-25 column is shown in FIG. 1. As can be seen from FIG. 1, after GFPU-2 passed through Sephadex G-25, three chromatographic peaks flowed out, but since the second and third peaks were much smaller than the first peak, i.e., the protein content was lower over 20 tubes, only the sample solution of the first peak was collected. The collected liquid is distilled under reduced pressure at 50 ℃, and is freeze-dried after dialysis to obtain light yellow GFP-1 solid powder.
The elution profile of GFPU-3 through Sephadex G-15 column is shown in FIG. 2. The chromatographic peak obtained as shown in fig. 2 is a single symmetrical peak, which indicates that the obtained grifolan peptide is a relatively uniform small peptide. The fractions were collected, concentrated, dialyzed and lyophilized to obtain white GFP-2 solid powder.
The molecular weight of the grifolan peptide was measured with a Sephadex G-15 column chromatography on GFP-2. Vitamin B12(1350Da), oxidized glutathione (613Da), reduced glutathione (307Da) and hippuric acid (178Da) are used as standard substances, a standard curve is formed according to the linear relation between the logarithm of molecular weight and the elution volume, and the measurement is carried out according to the elution volume of a sample, and the result is shown in figure 3.
The linear relationship between the elution volume and the molecular weight of the standard substance was-0.0144 x +3.7612 for y and 0.9965 for R2. The molecular weight of GFP-2 was calculated to be 963Da from the elution volume of GFP-2, and it was estimated that GFP-2 was composed of 7 to 8 amino acids.
The hydrolysate was purified by ultrafiltration, Sephadex G-25 and Sephadex G-15 to obtain the fraction with the strongest ferrous chelating ability, GFP-2, with chelating ability (269.7 + -3.9) μ G/G. Molecular weight determination by gel chromatography gave a GFP-2 relative molecular weight of 963 Da. The composition analysis of the grifolan amino acid shows that the grifolan has higher contents of aspartic acid, glutamic acid and histidine and plays a main role in chelating ferrous ions.
Example 6
Ultraviolet spectrum and infrared spectrum analysis of Grifola frondosa small peptide iron chelate (GFP-Fe)
The purified grifola frondosa protein peptide component (GFP-2) and the iron chelate (GFP-Fe) thereof in example 1 were scanned by ultraviolet spectroscopy to qualitatively observe the change of the ultraviolet absorption spectrum of the sample. Weighing a certain amount of GFP-2 and GFP-Fe respectively, dissolving the GFP-2 and GFP-Fe with distilled water to prepare a sample solution with the concentration of 0.2mg/mL, and scanning an ultraviolet spectrum within the wavelength range of 190-400 nm.
Infrared spectroscopic analysis of GFP-2 and GFP-Fe was performed by KBr pellet method. KBr was dried in an infrared drying oven to constant weight, and 1.5mg of GFP-2 and GFP-Fe samples were accurately weighed and ground with 150mg of KBr powder, respectively. After being uniformly ground, the mixture is pressed into thin slices by a tablet machine. 150mg of KBr was then weighed out accurately as a background control. Scanning and measuring a sample in a Fourier transform infrared spectrometer, wherein the scanning range is 4000-400 cm-1Scanning times of 32 times and resolution of 2cm-1。
The ultraviolet absorption spectrogram of the grifola frondosa protein peptide component (GFP-2) and the grifola frondosa small peptide iron chelate (GFP-Fe) thereof is shown in FIG. 4, the ultraviolet absorption of characteristic amino acid residues is mainly at 280nm, the ultraviolet absorption of peptide bonds (amido bonds at the protein fracture part) is mainly at 190-220 nm, and the ultraviolet absorption of the peptide bonds after the protein fracture is mainly shown in a sample, so that the sample contains more peptide fragment proteins.
Comparing the ultraviolet absorption spectra of the grifola frondosa protein peptide component (GFP-2) and the grifola frondosa small peptide iron chelate (GFP-Fe), the highest absorption peak is changed from 194nm to 192nm after chelation, and the absorption peak is blue-shifted because the oxygen atom on the carbonyl group participates in Fe2+Complexation of (1), Fe2+The addition of (2) causes charge transfer transition and coordination field transition, resulting in the change of the charge of the carbonyl group and blue shift of the absorption band. Thus, the chelate is two different species than before.
FIGS. 5 and 6 are infrared spectra of peptide fraction of Grifola frondosa protein (GFP-2) and its Grifola frondosa small peptide iron chelate (GFP-Fe), respectively.
3377.97cm in FIG. 5-1The absorption peak is the stretching vibration peak of N-H, belonging to the characteristic absorption peak of amino acid. 1654.15cm-1The strong absorption peak is an amide I band and is caused by C ═ O asymmetric stretching vibration of a protein polypeptide skeleton; 1547.95cm-1And 1259.66cm-1The absorption peaks are respectively assigned to an amide II band and an amide III band, and are respectively the absorption peaks caused by bending vibration of N-H and stretching vibration of C-H, thereby deducing that the peptide structure of the grifolan is probably beta-sheet. 1400.58cm-1Has an absorption peak ofSymmetric stretching vibration peak of carboxylate radical. At the same time, 1068.53cm-1Is shown (Pt-NH)2) Strong absorption peak at 517.34cm-1(Pr-NH2) The characteristic absorption peak of (A) is obvious.
As can be seen by comparing FIG. 5 with FIG. 6, the characteristic absorption peaks of the protein peptide substances are still retained in the infrared spectrum of the chelate, but the infrared spectra of the Grifola frondosa protein peptide and the Grifola frondosa small peptide iron chelate are obviously different in the number, intensity and position of the absorption peaks, which shows that the two are different in structure. The stretching vibration peak of N-H moves from low wave number to high wave number and the intensity is weakened, the peak intensity of amide I band is weakened, Pt-NH2The absorption peak appears at 1053.41cm-1The strong absorption peak indicates that Fe is present2+and-NH2Has stronger combination. The intensities of the C ═ O symmetric stretching vibration peak and the asymmetric stretching vibration peak are weakened to a large extent, which shows that Fe2+It also has strong binding with-COOH. After chelation, 800cm-1～500cm-1The peak of the fingerprint region of (2) disappeared, a new peak of 526.85cm-1It appears because of the stretching vibration of N-Fe.
Test example 1
Grifola frondosa small peptide iron chelate for in vitro simulation of gastric environment digestion
Simulated gastric fluid: accurately weighing 0.2g of sodium chloride and 3g of pepsin (enzyme activity 15000U/g), weighing 80mL of water, putting the mixture into a 100mL beaker, stirring the mixture to fully dissolve the mixture, adjusting the pH of the mixture to 2.0 by using 1mol/L HCl, and fixing the volume in a 100mL volumetric flask. 5mg/mL CGFP-Fe and GFP-Fe aqueous solutions were prepared for the crude Grifola frondosa protein iron chelate (CGFP-Fe), Grifola frondosa small peptide iron chelate (GFP-Fe) and 0.5mg/mL FeSO obtained in example 14An aqueous solution. Respectively sucking 10mL of the solution into 50mL conical flasks, adjusting the pH value to 2.0 by using 0.5mol/L hydrochloric acid, putting the conical flasks into a constant-temperature shaking incubator at 37 ℃ to preheat for 5min, respectively adding 10mL of simulated gastric juice, uniformly mixing, reacting in the constant-temperature shaking incubator at 37 ℃ at 150rpm for 0, 10, 30, 60, 120 and 180min, taking out the conical flasks from the constant-temperature shaking incubator at 95 ℃ to inactivate enzyme for 10min, standing and cooling, centrifuging at 4000r/min for 10min, collecting supernatant, and measuring the concentration of iron in the sample solution by using a phenanthroline colorimetric method.
As shown in FIG. 7, in overview, FeSO4CGFP-Fe and GFP-Fe can be kept in a stable dissolved state in the stomach environment. Thus, the grifolan iron peptide chelate can stably exist in an acid environment and resist digestion by pepsin.
Test example 2
Grifola frondosa small peptide iron chelate for in vitro intestinal environment digestion simulation
Simulating intestinal juice: accurately weighing 0.68g of potassium dihydrogen phosphate, dissolving in 25mL of water, adding 7.7mL of 0.2mol/L sodium hydroxide solution and 50mL of water, uniformly mixing, adding 6.0g of bovine bile salt and 1.0g of trypsin (2500U/mg), stirring to completely dissolve, finally adjusting the pH value of the mixed solution to 7.5 by using 0.2mol/L sodium hydroxide solution, and fixing the volume to 100 mL. Respectively preparing 5mg/mL crude Grifola frondosa protein peptide iron chelate (CGFP-Fe), Grifola frondosa small peptide iron chelate (GFP-Fe) aqueous solution and 0.5mg/mL FeSO4An aqueous solution. Respectively taking 10mL of the three solutions into a 50mL conical flask, adjusting the pH value to 2.0 by using 0.5mol/L hydrochloric acid, putting the conical flask into a constant-temperature shaking incubator at 37 ℃ for preheating for 5min, respectively adding 10mL of simulated gastric juice prepared in experimental example 1, uniformly mixing, reacting in the constant-temperature shaking incubator at 37 ℃ at 150rpm for 1h, taking out, and using 0.9mol/L NaHCO3The pH value of the sample is adjusted to 5.3, the pH value is adjusted to 7.5 by 2mol/L NaOH, 1mL of simulated intestinal fluid is added, and the beaker is placed in a constant temperature oscillation box at 37 ℃ and is reacted for 0.5, 1, 2, 4 and 6 hours at 150 rpm. And (3) after the reaction is finished, inactivating the enzyme at 95 ℃ for 10min, centrifuging at 4000r/min for 10min, collecting supernatant, and measuring the iron content in the solution by using a phenanthroline colorimetric method to obtain the solubility.
As shown in FIG. 8, after digestion of gastric juice-digested grifolan iron peptide for a period of time via simulated intestinal juice, GFP-Fe was compared with FeSO4Compared with the traditional method, the solubility of the protein can be kept higher, thereby better promoting the absorption of GFP-Fe by the small intestine. Because the grifola frondosa protein peptide iron has no free Fe2+And the existence of the grifolan peptide plays a certain role in protecting and buffering iron elements, so that the reduction of iron solubility caused by iron insoluble substances formed in the alkaline environment of the intestinal tract can be reduced, and the soluble iron can be better absorbed by the intestinal tract. CGFP-Fe in the gut over timeThe iron concentration gradually decreases and finally reaches a stable state, because the existence of other impurities in the crude grifola frondosa protein peptide influences the stability of the chelate, thereby influencing the solubility of iron in the chelate.
In vitro digestion experiments show that GFP-Fe can keep higher solubility and stability in gastrointestinal tract. And the solubility ratio of GFP-Fe to FeSO in the intestinal environment4Higher GFP-Fe absorption of small intestine can be promoted.
Test example 3
Caco-2 cytotoxicity test of grifola frondosa small peptide iron chelate
Caco-2 cells were cultured in MEM complete medium containing 20% (v/v) FBS (fetal bovine serum), 1% (v/v) diabody (penicillin-streptomycin) and 1% (v/v) nonessential amino acids at 37 ℃ with 5% CO2Culturing in a cell culture box with relative humidity of 90%, replacing culture medium at intervals of two days, and performing experiments after cells grow to about 80% and are digested by pancreatin. Caco-2 cell suspension was seeded in 96-well plates at 5X 10 per well3One cell, put 5% CO2And culturing for 72 hours in an incubator at 37 ℃. When the cells grow to about 70-80%, 150 μ L of the Grifola frondosa small peptide iron chelate (GFP-Fe) and FeSO prepared in the step (5) of example 1 and having the concentrations of 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0mg/L are added respectively4The MEM medium (10% FBS) was intervened for 12h, 24h, 48h, respectively, with 6 duplicate wells per concentration, followed by a blank. Thereafter, 5mg/mL MTT solution was added and the incubation was continued in the incubator for 4 h. The cell supernatant was discarded, dimethyl sulfoxide DMSO (150. mu.L/well) was added, shaken, and the absorbance of each well was measured at 492nm on a microplate reader. The cell viability was calculated as follows:
wherein A isExperimental groupIs the absorbance of the sample; a. theControl groupAbsorbance of blank.
MTT assay results are shown in FIG. 9, GFP-Fe and FeSO4Time-dependent toxicity to cells; at the same time, experiments were carried outThe results show that: the toxicity of GFP-Fe is lower than that of FeSO in 24h and 48h4This suggests that GFP-Fe can be a safe novel iron supplement.
Test example 4
Determination of transepithelial cell resistance
The cells were subcultured at 105The cells were inoculated in each well in the upper chamber of the Transwell chamber, 0.5mL of the cell suspension was added to each well in the upper chamber, 1.5mL of fresh medium was added to each well in the lower chamber, and the medium was changed every other day for the first 7 days, and then changed every day for 21 days. Measuring transmembrane epithelial cell resistance (TEER) with cell resistance meter, which is greater than 300 Ω · cm2The cell monolayer membrane of (a) can be used in transport experiments. Firstly, putting an electrode into HBSS preheated to 37 ℃, and balancing for 20 min; removing the culture medium in the culture plate, adding preheated HBSS 0.5mL into the upper chamber side, adding preheated HBSS 1.5mL into the lower chamber side, balancing at 37 ℃ for 20min, and washing off impurities on the cell surface; removing HBSS, adding preheated HBSS again, and measuring transmembrane resistance; repeating the above steps with 1 blank vector to obtain blank values; the transmembrane resistance of the cells was calculated according to the following formula
TEER (measured resistance value-blank cell resistance value) X culture cell area (1.12 cm)2)
Selecting Caco-2 monolayer cells with the transmembrane resistance value of more than 300 omega cm2The cells of (3) were subjected to subsequent experiments.
Test example 5
Grifola frondosa small peptide iron chelate transport experiment
Cell culture As shown in test example 4, when the cell resistance value (TEER) measured was more than 300. omega. cm2In this case, the upper chamber cells were carefully washed 3 times with HBSS (pH 7.4) buffer, and HBSS was gently aspirated from the wells after incubation 3 times in a 37 ℃ incubator for 30 min.
Grifola frondosa small peptide iron chelate (GFP-Fe) and FeSO4Transport of drug from the upper chamber to the lower chamber side: 0.5mL of GFP-Fe and FeSO were added to the upper chamber side at different concentrations (0.5, 1.0, 2.0mg/L)4The solution was used as a supply reservoir, and 1.5mL of HBSS was added to the chamber side as a receiving reservoir. Transfer from the lower chamber side to the upper chamber side: 1.5mL of the same concentration of the drug solution was added to the lower chamber side as a supply reservoir, and was placed on the upper sideThe chamber side was charged with 0.5mL HBSS as a receiving pool. The Transwell plates with the added drug solution and the blank were placed in a 37 ℃ incubator, and 500. mu.L of the receiving well solution was aspirated at 30, 60, 90, and 120min, respectively, while the same volume of HBSS solution preheated to 37 ℃ was replenished. And measuring the content of iron in the sample by adopting an AAS method. The drug transport rate is expressed in terms of apparent permeability coefficient (Papp).
Different concentrations of GFP-Fe and FeSO4The apparent permeability coefficients in transcellular transport in Caco-2 cell monolayers are shown in Table 2. As shown in Table 2, the apparent permeability coefficients of GFP-Fe at different concentrations for the upper chamber → the lower chamber are all compared to FeSO4Has a certain degree of improvement, and the apparent permeability coefficient of the upper chamber → the lower chamber is obviously higher than that of FeSO under the concentration of 2mg/L4The apparent permeability coefficient of lower chamber → upper chamber is significantly lower than that of FeSO4. Therefore, the GFP-Fe intestinal tract absorption effect is better than that of FeSO4. At the same time, P of GFP-Fe at various concentrationsappAre all larger than 10 of the internationally specified well-absorbed medicines-6cm/s, indicating that GFP-Fe has good absorption effect.
On the other hand, the apparent permeability coefficient from the superior to inferior compartment surface was significantly greater than from the inferior to superior compartment surface, indicating that GFP-Fe is likely to be taken up by the transport vector on the apical side of the small intestine. Meanwhile, the ratio of the lower chamber → the upper chamber to the upper chamber → the lower chamber shows that the absorption of GFP-Fe is less affected by the efflux, further illustrating the good absorption effect of GFP-Fe.
TABLE 2 different concentrations of GFP-Fe and FeSO4Apparent permeability coefficient P in Caco-2 cell transport processapp(×10- 6cm/s)
Note: indicates GFP-Fe and FeSO at the same concentration under the same experimental conditions4Comparison with significant differences (P)<0.05)
The digestion and absorption of grifolan peptide iron chelate by gastrointestinal tract digestion and Caco-2 cell model transport in vitro simulation through the testThe effect was evaluated. Meanwhile, the absorption mechanism of the grifolan peptide iron chelate is researched through a Caco-2 cell model. The experimental result shows that GFP-Fe and FeSO are in the gastric environment4All have better stability, and GFP-Fe is compared with FeSO in the intestinal environment4The solubility is higher. The cytotoxicity experiments show that GFP-Fe and FeSO4Compared with the prior art, the toxicity is lower, and the apparent permeability coefficient of the upper chamber → the lower chamber of GFP-Fe is found to be larger than that of FeSO through the successfully constructed Caco-2 cell model transfer experiment4It shows better absorption than FeSO4Therefore, GFP-Fe can be developed into a novel iron supplement agent with safety and high bioavailability. Research on absorption mechanism shows that the targeted oligopeptide transporter (PepT1) mediates the active transport of GFP-Fe and the efflux function mediated by the multidrug resistance protein exists.
Test example 5
Effect of Grifola frondosa small peptide iron chelate on mouse splenocyte proliferation and macrophage proliferation
The crude Grifola frondosa protein peptide (CGFP) prepared in step (2) of example 1, the Grifola frondosa polypeptide (GFP-2) prepared in step (4) of example 1, the crude Grifola frondosa protein iron chelate (CGFP-Fe) prepared in step (5) of example 1, and the Grifola frondosa small peptide iron chelate (GFP-Fe) were each prepared as a sample solution of 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL using RPMI-1640 complete medium. Add 5X 10 per well in 96-well plates6Spleen cells at cell concentration of one/mL or 2X 106100 mu L macrophage suspension with cell concentration per mL, and then 100 mu L CGFP, GFP, CGFP-Fe and GFP-Fe samples are respectively added to make the final concentrations of the samples respectively 50, 100 and 200 mu g/mL. 3 replicates were made for each concentration of each sample. The negative control group was supplemented with 100. mu.L of RPMI-1640 complete medium instead of the sample solution. The LPS control group was added with 100. mu.L of LPS solution to a final concentration of 10. mu.g/mL. Canavalid protein A (ConA) control group 100. mu.L of ConA solution prepared in RPMI-1640 complete medium was added to a final concentration of 5. mu.g/mL. The 96-well plates were incubated at 37 ℃ in a 5% CO2 incubator for 44 h. Taking out, adding 20 μ L of 5mg/mL MTT solution into each well, continuously placing into an incubator for continuous culture for 4h, removing supernatant from each well, adding 100 μ L DMSO, shaking for 10min, and collecting the supernatant to form purple knot in each wellThe crystals were dissolved, and the absorbance (OD value) was measured at 490nm with a microplate reader to calculate the proliferation rate. The formula is as follows:
the effect of iron chelate of Grifola frondosa small peptide on mouse splenocyte proliferation and macrophage proliferation is shown in FIGS. 10-11, spleen is an important immune organ, is the second immune barrier of human body, and plays an important role in specific immunity of human body. The increment rates of the ferrous chelate groups are respectively higher than those of the grifola frondosa protein peptide group, and the good immunocompetence of the grifola frondosa protein peptide iron chelate is shown. Macrophages are the most primitive immune cells in mammals and are also the first immune barrier in organisms against pathogens. When the pathogen enters the human body, macrophage phagocytizes the pathogen, and then the macrophage plays the role of antigen presenting cell to activate T lymphocyte, thereby participating in nonspecific immunity and specific immunity process and regulating the immune response activity of organism. The proliferation rates of the CGFP-Fe and GFP-Fe groups are as high as 29.14 percent and 27.75 percent, which are better than those of the LPS control group. Therefore, compared with the grifolan peptide, the grifolan peptide iron chelate has a better promotion effect on the proliferation of mouse macrophages.
Test example 6
Effect of Grifola frondosa small peptide iron chelate on NO, IL-6 and TNF-alpha production by mouse macrophages
The cell and sample addition method was the same as in test example 5. After 48h of cell culture, sucking cell culture supernatant (50 mu L) and adding the cell culture supernatant into a 96-hole culture plate, adding an equal volume of NO detection reagent, reacting for 10min, and detecting OD (optical density) by using a microplate reader540nm. With NaNO2And drawing a standard curve by using the solution as a standard substance, and calculating the NO content corresponding to the sample hole. IL-6 and TNF- α were determined according to the ELISA kit.
The results of the effect of iron chelate of Grifola frondosa small peptide on the generation of NO, IL-6 and TNF-alpha by mouse macrophages are shown in FIGS. 12-14, and the Grifola frondosa protein peptide and the iron chelate thereof can significantly increase the content of NO secreted by the macrophages and can also enhance the release of IL-6 and TNF-alpha cytokines to a certain extent. Therefore, both the grifolan peptide and the grifolan iron chelate have good immune enhancement activity.
The research on the immunocompetence of the grifola frondosa protein peptide and the iron chelate thereof through the tests shows that CGFP, GFP-2, CGFP-Fe and GFP-Fe have obvious enhancement effect on the proliferation of mouse splenocytes and macrophages, and the specific immunity and the non-specific immunity are promoted in the process of regulating immune response. In comparison, the promoting effect of the grifolan iron peptide chelate is stronger. Meanwhile, the grifolan peptide and the iron chelate thereof can obviously increase the content of NO secreted by macrophages and can also enhance the release of IL-6 and TNF-alpha cytokines to a certain extent. Therefore, both the grifolan peptide and the grifolan iron chelate have good immune enhancement activity.
Claims (7)
1. A preparation method of a Grifola frondosa small peptide iron chelate is characterized by comprising the following steps:
(1) extracting the protein of the fruit body of the grifola frondosa:
pulverizing dried Maitake Mushroom fruiting body into superfine powder, adding distilled water, stirring, extracting at constant temperature under stirring, cooling to room temperature, centrifuging, collecting supernatant, standing at low temperature overnight, centrifuging, removing supernatant, precipitating, washing, vacuum drying, pulverizing, and sieving to obtain Maitake Mushroom fruiting body protein powder;
(2) carrying out proteolysis on the grifola frondosa:
weighing Grifola frondosa sporophore protein powder, adding distilled water, adding protease, performing enzymolysis, centrifuging hydrolysate after reaction, dialyzing supernatant, and freeze-drying to obtain crude Grifola frondosa protein peptide;
(3) and (3) separating and purifying the grifola frondosa protein peptide:
performing a first-step purification of the obtained crude grifola frondosa protein peptide by ultrafiltration; separating and purifying by Sephadex column to obtain Polyporus frondosus polypeptide;
(4) preparing a grifola frondosa small peptide iron chelate:
weighing Polyporus frondosus polypeptide, dissolving in distilled water, adding ascorbic acid solution, and adding FeCl2Carrying out oscillation reaction on the solution, centrifuging after the reaction is finished, taking the supernatant, adding absolute ethyl alcohol, standing overnight for precipitation, centrifuging, taking the precipitate, washing and drying to obtain the grifola frondosa small peptide iron chelate;
performing ultrafiltration on the crude grifola frondosa protein peptide in the step (3) to purify the crude grifola frondosa protein peptide in the first step to prepare a solution, filtering the solution to obtain a supernatant, performing ultrafiltration and dialysis on the solution, and performing freeze-drying to obtain a grifola frondosa polypeptide component less than 1 KDa; after the ultrafiltration and purification, separating and purifying the mixture by a glucan gel column, namely adding the glucan gel powder into distilled water for full swelling, removing suspended particles and degassing; adding the degassed sephadex solution into a chromatographic column, taking a PBS (phosphate buffer solution) as a mobile phase, and carrying out sample loading elution on the ash tree flower polypeptides with different molecular weights after ultrafiltration to obtain purified components of the ash tree flower polypeptides;
adding 100-120 mu L ascorbic acid solution into every 100mg of grifola frondosa protein peptide in the step (4); adding 100-doped 120 mu LFeCl2A solution; oscillating the reaction temperature to 20-25 ℃, and reacting for 4-5 min.
2. The method for preparing the grifola frondosa small peptide iron chelate complex as claimed in claim 1, wherein the supernatant obtained in step (1) is evaporated to half of the original volume, and the pH is adjusted to the isoelectric point of grifola frondosa protein.
3. The method for preparing the grifola frondosa small peptide iron chelate complex according to claim 1, wherein the protease in the step (2) is one of flavourzyme, complex protease, pepsin, neutral protease, alkaline protease and trypsin.
4. The method for preparing the grifola frondosa small peptide iron chelate complex as claimed in claim 1, wherein the protease in the step (2) is added in an amount of 1600-.
5. The method for preparing the grifola frondosa small peptide iron chelate complex as claimed in claim 1, wherein the enzymolysis time in the step (2) is 1-3.5 h.
6. A Grifola frondosa small peptide iron chelate prepared by the method of claim 1.
7. An application of the Grifola frondosa small peptide iron chelate prepared by the preparation method of the Grifola frondosa small peptide iron chelate of claim 1 in preparing bioactive iron supplement agents.
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| CN105695547A (en) * | 2016-03-30 | 2016-06-22 | 福建农林大学 | Method for preparing grifola frondosa protein peptide-selenium chelate |
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| CN108059652A (en) * | 2018-02-13 | 2018-05-22 | 福建农林大学 | A kind of grifola frondosus selenium chelating peptide prepared using proteolytic cleavage |
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| CN104757252A (en) * | 2015-04-22 | 2015-07-08 | 福建农林大学 | Preparation method of maitake protein zymolyte having antioxidant activity |
| CN105695547A (en) * | 2016-03-30 | 2016-06-22 | 福建农林大学 | Method for preparing grifola frondosa protein peptide-selenium chelate |
| CN105852135A (en) * | 2016-03-30 | 2016-08-17 | 福建农林大学 | Preparation method of edible and medicinal fungus protein peptide-ferrous chelate |
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