CN109680027A - A kind of grifola frondosus small peptide iron chelate and its preparation method and application - Google Patents
A kind of grifola frondosus small peptide iron chelate and its preparation method and application Download PDFInfo
- Publication number
- CN109680027A CN109680027A CN201811629128.9A CN201811629128A CN109680027A CN 109680027 A CN109680027 A CN 109680027A CN 201811629128 A CN201811629128 A CN 201811629128A CN 109680027 A CN109680027 A CN 109680027A
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- Prior art keywords
- grifola frondosus
- grifola
- small peptide
- iron chelate
- preparation
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- 230000008685 targeting Effects 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000002861 ventricular Effects 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
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Abstract
The invention discloses a kind of grifola frondosus small peptide iron chelate and its preparation method and application, the preparation of the grifola frondosus small peptide iron chelate includes the following steps: (1) Grifola Frondosa sporophore protein extraction；(2) grifola frondosus proteolysis；(3) grifola frondosus protein peptides isolate and purify；(4) preparation of grifola frondosus small peptide iron chelate.The present invention extracts grifola frondosus albumen with Grifola Frondosa sporophore, obtains small peptide by enzymatic hydrolysis, chelates and grifola frondosus peptide iron chelate is made with molysite after isolating and purifying, the iron bioavilability of the peptide iron chelate is better than inorganic salts iron, and has stronger immunocompetence.Grifola frondosus small peptide iron chelate property produced by the present invention is stablized, and soluble easily in water, the small peptide iron chelate is small to the damage of gastrointestinal tract compared with traditional Oral Iron Preparations ferrous sulfate, and can be better absorbed, and can be used as novel iron supplementary.Preparation process of the present invention is simple and efficient, and the polypeptide iron chelate synthesized is high-efficient, and the iron content of chelating is high.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of grifola frondosus small peptide iron chelate and preparation method thereof and answer
With.
Background technique
Grifola frondosus (Grifola frondosa) is one of rare edible and medicinal fungi, belong to Basidiomycotina, Hymenomycetes, without every
Basidiomycetes subclass, Aphyllophorales, Polyporaceae, set flower Pseudomonas fungi also known as polyporus frondosus, thousand Buddhist bacterium, chestnut mushroom, weight mushroom,
Cloud Tan, lotus flower bacterium etc., Japan are referred to as grifola frondosa (Maitake).Wild Grifola frondosa is grown on subtropical zone into temperate forests more, day
Originally, the ground such as Russian, North America and Changbai Mountain in China area, Hebei, Sichuan are widely distributed.Grifola Frondosa sporophore delicious flavour, tool
There is the giving off a strong fragrance of matsutake, while there is very high nutrition and health care to be worth.Contents of saccharide highest in grifola frondosus, protein content therein
It is only second to polysaccharide, and protein content is than higher in the mushrooms such as mushroom, needle mushroom, agaric.Contain 17 kinds of amino acid in fructification,
In 7 kinds of essential amino acids, account for the 40.0% of grifola frondosus total amino acid content.Isoleucine (Ile), day in Grifola Frondosa sporophore
Aspartic acid (Asp) and glutamic acid (Glu) content are higher, and the delicate flavour strong to holding, the chelating of promotion and metal ion, alleviation are tired
Labor, protection human brain nerve etc. play a significant role.
Grifola frondosus is not only full of nutrition, but also has certain healthcare function.But the research of grifola frondosus is stopped more
In terms of Polyose extraction and its biological function, and grifola frondosus polypeptide has the function of that anti-oxidant, Immune-enhancing effect, antitumor etc. are living
Property, the research about grifola frondosus protein and its derivative is rarely reported at present.
Summary of the invention
Goal of the invention: in view of the problems of the existing technology, the present invention provide a kind of grifola frondosus small peptide iron chelate and its
Preparation method, the present invention extract grifola frondosus albumen with Grifola Frondosa sporophore, obtain small peptide by enzymatic hydrolysis, isolate and purify rear and molysite
It chelates and grifola frondosus peptide iron chelate is made, the iron bioavilability of the peptide iron chelate is better than inorganic salts iron, and has stronger
Immunocompetence.
The present invention also provides the applications of grifola frondosus small peptide iron chelate.
Technical solution: to achieve the goals above, a kind of preparation side of grifola frondosus small peptide iron chelate as described herein
Method, which comprises the steps of:
(1) Grifola Frondosa sporophore protein extraction:
Grifola Frondosa sporophore Ultramicro-powder is made in dry Grifola Frondosa sporophore crushing, distilled water is added, after mixing evenly,
Constant temperature stirring is extracted, after, it is cooled to room temperature, is centrifuged, takes supernatant, stand at low temperature is overnight, is centrifuged the supernatant that inclines, precipitating
Washing, vacuum drying crush, and are sieved to get Grifola Frondosa sporophore albumen powder；
(2) grifola frondosus proteolysis:
Grifola Frondosa sporophore albumen powder is weighed, distilled water is added, adds protease, is digested, water after reaction was completed
Liquid centrifugation is solved, supernatant is taken to be dialysed, freeze-drying obtains thick grifola frondosus protein peptides, saves；
(3) grifola frondosus protein peptides isolate and purify:
First step purifying is carried out by ultrafiltration to obtained thick grifola frondosus protein peptides；Pass through sephadex after ultrafiltration purification
Column is isolated and purified to obtain grifola frondosus polypeptide；
(4) preparation of grifola frondosus small peptide iron chelate:
Grifola frondosus polypeptide is weighed, after distilled water dissolution is added, the ascorbic acid solution added adds FeCl2Solution,
Oscillating reactions is centrifuged after reaction, takes supernatant that dehydrated alcohol is added, and overnight precipitation, centrifuging and taking precipitating is dry after washing, obtains
To grifola frondosus small peptide iron chelate.
Wherein, supernatant is first evaporated to the 1/2 of original volume after taking supernatant described in step (1), with tune pH value to 3.5 ashes
Set flower albumen isoelectric point.
Wherein, step (2) protease is food flavor enzyme, compound protease, pepsin, neutral proteinase, alkaline egg
One of white enzyme and trypsase.
Wherein, the additional amount of step (2) described protease is 1600-2100U/g Grifola Frondosa sporophore albumen powder.
Preferably, step (2) enzymolysis time is 1-3.5h.
Most preferably, use hydrolysis by novo grifola frondosus albumen optimum process condition for pH 9, enzyme concentration 1800U/g
Grifola Frondosa sporophore albumen powder, 55 DEG C of hydrolysis temperature, grifola frondosus protein concentration 0.5g/100mL, enzymolysis time 2.5h.The present invention
Under optimum enzymolysis condition, ferrous sequestering power is (1.854 ± 0.025) mg/g, and degree of hydrolysis is (6.14 ± 0.05) %.
Wherein, it is thick grifola frondosus protein peptides that step (3) the thick grifola frondosus protein peptides, which carry out first step purifying by ultrafiltration,
It is configured to solution, is filtered, is obtained supernatant, solution is dialysed after ultrafiltration, freeze-drying obtains being greater than 5KDa, 1-5KDa and is less than
The grifola frondosus polypeptide fractions of 1KDa.
Wherein, it is isolated and purified after step (3) described ultrafiltration purification by sephadex column as by by glucan
Gel powder is added in distilled water and is sufficiently swollen, and removal suspended particulate is de-gassed；The sephadex of degassing process is molten
Liquid is added in chromatographic column, uses PBS buffer solution as mobile phase, different molecular weight grifola frondosus polypeptide after ultrafiltration is carried out loading and is washed
It is de-, to obtain purified components grifola frondosus polypeptide.
Wherein, 100-120 μ L ascorbic acid solution is added in every 100mg grifola frondosus polypeptide in step (4)；100-120 is added
μLFeCl2Solution；Oscillating reactions temperature is 20-25 DEG C, reacts 4-5min.
Most preferably, the optimum process condition of chelatropic reaction are as follows: every 100mg grifola frondosus polypeptide, when pH is 5, reaction system
Total volume is 10mL, the 100 μ L frerrous chlorides of 1mol/L is added, temperature is 20 DEG C, reaction 5min, the chela obtained with this condition
Object iron content highest is closed, is (31.51 ± 1.32) mg/g.
Grifola frondosus small peptide iron chelate prepared by the preparation method of grifola frondosus small peptide iron chelate of the present invention.
Grifola frondosus small peptide iron chelate prepared by the preparation method of grifola frondosus small peptide iron chelate of the present invention exists
Prepare the application in bioactivity iron supplementary.
Reagent in the present invention is commercially available, alkali protease (200U/mg), neutral proteinase (14000U/g), stomach
Protease (15000U/g), compound protease (120U/mg), flavor protease (20U/mg) purchase are limited in Shanghai source leaf biology
Company；Trypsase (2500U/g) Aladdin Reagent Company；Sephadex G-25, the Beijing Sephadex G-15 Suo Laibaoke
Skill Co., Ltd.
It is dual anti-: (penicillin 100U/mL, streptomysin 0.1mg/mL)；Nonessential amino acid: (l-Alanine, Pidolidone,
L- days (door) winters amide, L- days (door) aspartic acid, L-PROLINE, Serine and glycine)；Fetal calf serum, non-essential amino
Acid solution, MEM culture solution are purchased from Gibco company, the U.S.；It is limited that Pen .- Strep mixed solution is purchased from green skies biotechnology
Company.
Material therefor of the present invention is grifola frondosus, and protein content is only second to polysaccharide.Contain 17 kinds in Grifola Frondosa sporophore albumen
Amino acid, wherein 7 kinds of essential amino acids, account for the 40.0% of grifola frondosus total amino acid content.Different bright ammonia in Grifola Frondosa sporophore
Sour (Ile), aspartic acid (Asp) and glutamic acid (Glu) content are higher, the delicate flavour strong to holding, promotion and metal ion
It chelates, relieve fatigue, protecting human brain nerve etc. and playing a significant role.The grifola frondosus polypeptide that enzymatic hydrolysis grifola frondosus albumen obtains usually has
There is certain bioactivity, and is easy to be digested, the more low speciality of antigenicity.Utilize the more of grifola frondosus Peptide systhesis
Peptide iron chelate is brown ceramic powder, and free from extraneous odour, no free iron is soluble easily in water, steady insoluble in organic solvents, properties such as ethyl alcohol
It is fixed.The polypeptide iron chelate can be absorbed by animal body with molecular forms, and be targeted to specific organization's release ferro element, mend the same of iron
When decrease free iron generation endogenous free radical to cell membrane cause damage the case where.In addition, human immunity system
It unites closely bound up with asiderosis, which can effectively facilitate the enhancing of body immune system.Prepared by the invention
Grifola frondosus small peptide iron chelate property is stablized, highly-safe, is easy to absorb and have certain bioactivity.
The present invention is not only that the industry of integration of drinking and medicinal herbs mushroom and resources effective utilization is promoted to provide new research direction,
Preparation for peptide iron chelate provides raw material.The present invention has probed into the preparation process, basic of grifola frondosus peptide of rice protein chelate
Structure and its mechanism of absorption, and the immunocompetence of grifola frondosus polypeptide and its iron chelate is had studied, it is grifola frondosus peptide of rice protein
Foundation is provided as the functional food additives safely and effectively with certain bioactivity, while improving grifola frondosus
Economy and society value.
The present invention carries out ultraviolet and IR spectrum scanning to grifola frondosus protein peptides and its iron chelate, and map shows ash tree
Flower protein peptides and its iron chelate all have the characteristic absorption of amino acid.But blue shift occurs for the ultraviolet maximum absorption band of chelate, and
The position of peak number, peak intensity and peak changes and N-Fe absorption peak occurs in infrared spectroscopy, shows to generate grifola frondosus peptide iron
Chelate, while illustrating its chelation mechanism are as follows: iron and amino and carboxyl have combination.
The present invention shows that GFP-Fe can keep higher solubility and stabilization in the gastrointestinal tract by External digestion experiment
Property.And in intestinal environment, GFP-Fe solubility ratio FeSO4It is higher more preferably to promote absorption of the small intestine to GFP-Fe.Cell toxicant
Property the results show, GFP-Fe and FeSO4It is lower compared to toxicity.Grifola frondosus peptide iron is chelated by Caco-2 cell model method
The assimilation effect and mechanism of absorption of object are studies have shown that GFP-Fe absorption is better than FeSO4, peptide transporter (PepT1) mediation
The active transport of GFP-Fe exists simultaneously the protein mediated outlet effect of multidrug resistance.
In immunocompetence experiment, the increasing of CGFP, GFP-2, CGFP-Fe, GFP-Fe to mouse boosting cell and macrophage
It grows with significant humidification, shows grifola frondosus protein peptides and its iron chelate to specific immunity and nospecific immunity mistake
Cheng Junyou facilitation, wherein the facilitation of grifola frondosus peptide iron chelate is stronger.Meanwhile grifola frondosus protein peptides and its iron chelate
Object can promote macrophages secrete NO, can also enhance the release of IL-6 and TNF-α cell factor to a certain extent.
The utility model has the advantages that compared with the prior art, the invention has the following advantages that
1, grifola frondosus small peptide iron chelate property produced by the present invention is stablized, soluble easily in water, which relatively passes
The Oral Iron Preparations ferrous sulfate of system is small to the damage of gastrointestinal tract, and can be better absorbed, and can be used as novel iron supplementary.
2, grifola frondosus polypeptide iron chelate produced by the present invention has certain immunocompetence, strengthens body immune system,
The damage of body immune system is caused to have certain restitution asiderosis.
3, the present invention prepares grifola frondosus polypeptide with enzymatic isolation method, and compared with traditional chemical method, enzymatic isolation method more preferably retains product
Structure and activity, reaction condition is mild, and selectivity is high.
4, for the present invention by ultrafiltration and sephadex method to thick grifola frondosus albumen peptide purification, ultrafiltration is widely used in polypeptide
Liquid isolates and purifies, more traditional separation method, and such as ion-exchange chromatography and high efficiency chromatography analytic approach, low energy consumption, equipment
It is small, selectivity is good, temperature is low and separating rate is fast, separative efficiency is high.
5, the present invention prepares grifola frondosus small peptide iron chelate with chemical method, and the method is simple and efficient, and the polypeptide iron chela synthesized
Conjunction object is high-efficient, and the iron content of chelating is high.
Detailed description of the invention
Fig. 1 is that GFPU-2 passes through Sephadex G-25 elution curve schematic diagram；
Fig. 2 is that GFPU-3 passes through Sephadex G-15 elution curve schematic diagram；
Fig. 3 is that Sephadex G-15 measures standard substance molecular weight schematic diagram；
Fig. 4 is grifola frondosus protein peptides and its iron chelate ultraviolet spectrogram；
Fig. 5 is grifola frondosus protein peptides infrared spectrogram；
Fig. 6 is grifola frondosus small peptide iron chelate infrared spectrogram；
Fig. 7 is digestion situation schematic diagram of the in-vitro simulated stomach environment to grifola frondosus small peptide iron chelate；
Fig. 8 is digestion situation schematic diagram of the in-vitro simulated enteron aisle to grifola frondosus small peptide iron chelate；
Fig. 9 is that mtt assay measures the influence schematic diagram of grifola frondosus small peptide iron chelate and ferrous sulfate to Caco-2 cell；Its
In, A, B, C be respectively intervene 12h, for 24 hours, 48h MTT result.* it is respectively indicated compared to the blank group with * *, P < 0.05 and P <
0.01；
Figure 10 is the influence schematic diagram of grifola frondosus protein peptides and its iron chelate to mouse spleen cell proliferation；
Figure 11 is grifola frondosus protein peptides and its influence schematic diagram that iron chelate is proliferated mouse macrophage；
Figure 12 is the influence schematic diagram that grifola frondosus protein peptides and its iron chelate generate NO to macrophage；
Figure 13 is the influence schematic diagram that grifola frondosus protein peptides and its iron chelate generate IL-6 to macrophage；
Figure 14 is the influence schematic diagram that grifola frondosus protein peptides and its iron chelate generate TNF-α to macrophage.
Specific embodiment
Below in conjunction with drawings and examples, the invention will be further described.
Embodiment 1
(1) through 300 meshes point after crushing dry Grifola Frondosa sporophore with micronizer, Grifola Frondosa sporophore is made
Ultramicro-powder weighs 90g Grifola Frondosa sporophore powder, is that distilled water is added in 1:50 (g/mL) according to solid-liquid ratio, uses after mixing evenly
1mol/L NaOH solution adjusts pH value to 9, is put into stirring in 70 DEG C of thermostat water baths and extracts 3h, during which molten with 1mol/L NaOH
Liquid keeps system pH constant.After, it is cooled to room temperature, 4000r/min is centrifuged 20min.Supernatant rotary evaporation is to substance
Long-pending 1/2, with 2mol/L HCl solution tune pH value to 3.5 (grifola frondosus albumen isoelectric point), stand at low temperature is overnight, 4000r/min
It is centrifuged 25min, incline supernatant, and precipitating washed once with 95% ethanol solution, is dried in vacuo.It crushes, crosses 60 meshes to get ash
Set beggar's entity albumen powder.
(2) a certain amount of Grifola Frondosa sporophore albumen powder is accurately weighed, distilled water is proportionally added into, dissolves Grifola Frondosa sporophore
Albumen makes the mass concentration of solution be 0.5g/100mL, adjusts after the optimum pH 9.0 of alkali protease in optimum temperature 55
DEG C heat preservation 10min, is that alkali protease is added in 1800U/g Grifola Frondosa sporophore albumen powder according to enzyme concentration, digests at 55 DEG C
During which 2.5h keeps system pH constant, reaction was completed by 95 DEG C of enzyme deactivation 10min, and hydrolyzate 4000r/min is centrifuged 25min, supernatant
Liquid is settled to 250mL, obtains 4 DEG C of thick grifola frondosus protein peptides (CGFP) preservations.
(3) purifying for being carried out the first step to obtained thick grifola frondosus protein peptides (CGFP) using ultrafiltration, CGFP is prepared
At the solution of 10mg/mL, and pass through 0.45 μm of water system filter membrane, obtains supernatant.It then, will in the case where room temperature, pressure are 0.3MPa
Solution passes sequentially through the ultrafiltration membrane of 5kDa and 1kDa, obtains>5kDa, and three kinds of components of 5kDa~1kDa and<1kDa are named respectively
For GFPU-1, GFPU-2, GFPU-3, retain GFPU-2, GFPU-3 carries out sephadex column and isolated and purified.First will
20g sephadex powder is added in 500mL distilled water and is sufficiently swollen, and removal suspended particulate is de-gassed；It secondly will degassing
The sephadex solution of processing is added in chromatographic column (1.6 × 60cm), avoids tomography and bubble；Then 0.05mol/L is used
PBS (pH 6.8) buffer as mobile phase, balance 5 column volumes；Loading elution is finally carried out, to obtain purifying group
Point.
(4) different according to GFPU-2 with GFPU-3 molecular weight, Sephadex G-25 and Sephadex G-15 are used respectively
Chromatographic column is further purified, the chromatographic condition of Sephadex G-25 are as follows: applied sample amount 15mg/mL；Mobile phase is 0.05mol/L
PBS (pH 6.8) buffer；Flow velocity is 0.2mL/min；Every pipe collects 5mL.The chromatographic condition of Sephadex G-15 are as follows: on
Sample amount is 25mg/mL；Mobile phase is PBS (pH 6.8) buffer of 0.05mol/L；Flow velocity is 0.4mL/min；Every pipe is collected
6mL respectively obtains GFP-1 and GFP-2 grifola frondosus polypeptide.Effluent measures at 220nm, and collects main component (GFP-1
And GFP-2) carry out protein content and ferrous sequestering power measurement.
(5) 0.1g grifola frondosus polypeptide (GFP-2) and thick grifola frondosus protein peptides (CGFP) are accurately weighed respectively, are separately added into
After the dissolution of 10mL distilled water, the ascorbic acid solution of 100 μ L 0.2g/L is added, regulation system pH is 5.0, adds 100 μ L
1mol/L FeCl2Solution, oscillating reactions temperature are 20 DEG C, react 5min.After reaction, 4000r/min is centrifuged
10min takes supernatant that dehydrated alcohol, 4 DEG C of overnight precipitations are added in the ratio of 1:5 (v:v).4000r is centrifuged 15min, takes precipitating, uses
25mL 95% (v/v) ethanol washing is primary, and dry 8h in 50 DEG C of vacuum ovens, obtaining violet solid is grifola frondosus small peptide iron chela
Close object (GFP-Fe) and thick grifola frondosus peptide of rice protein chelate (CGFP-Fe).
Embodiment 2
Embodiment 2 is identical as 1 preparation method of embodiment, the difference is that step (2) is 2100U/g ash according to enzyme concentration
It sets beggar's entity albumen powder and alkali protease is added, digest 1h；120 μ L are added in 0.1g grifola frondosus protein peptides in step (5)
0.2g/L ascorbic acid solution；120 μ L 1mol/L FeCl are added2Solution；Oscillating reactions temperature is 25 DEG C, reacts 4min.
Embodiment 3
Embodiment 3 is identical as 1 preparation method of embodiment, the difference is that step (2) is 1600U/ (g according to enzyme concentration
Grifola Frondosa sporophore albumen powder) alkali protease is added, digest 3.5h.
Embodiment 4
Embodiment 4 is identical as 1 preparation method of embodiment, the difference is that step (2) alkali protease is substituted for respectively
Food flavor enzyme, compound protease, pepsin, neutral proteinase or trypsase, the optimal pH for adjusting each enzyme is respectively 7.0,
7.0,2.0,7.0,8.0, peak enzymolysis-ability temperature 50 C, 45 DEG C, 37 DEG C, 50 DEG C, 37 DEG C.
Embodiment 5
A certain amount of Grifola Frondosa sporophore albumen powder is accurately weighed using (2) the step of embodiment 1, is proportionally added into distillation
Water, dissolution Grifola Frondosa sporophore albumen makes the mass concentration of solution for 0.5g/100mL, after the optimum pH for adjusting each enzyme
10min is kept the temperature in optimum temperature.It is that 1800U/g is separately added into 6 kinds of protease (food flavor enzyme, compound protease, stomaches according to enzyme concentration
Protease, neutral proteinase, alkali protease and trypsase), 0.5,1,2,3,4,5h are digested respectively, during which keep system pH
Be worth (7.0,7.0,2.0,7.0,9.0,8.0) it is constant, each enzyme optimum temperature (50 DEG C, 45 DEG C, 37 DEG C, 50 DEG C, 55 DEG C,
37 DEG C) under hydrolyze, reaction was completed by 95 DEG C of enzyme deactivation 10min.Hydrolyzate 4000r/min is centrifuged 25min, and supernatant is settled to 250mL,
4 DEG C of preservations.Every group of enzyme digestion reaction does 3 parallel tests, and ferrous sequestering power is parallel with degree of hydrolysis measurement experiment to carry out 3 times.
It is optimum process condition pH9, enzyme the result shows that best using hydrolysis by novo Grifola Frondosa sporophore albumen
Measure 1800U/g, 55 DEG C of hydrolysis temperature, substrate mass concentration 0.5%, enzymolysis time 2.5h.Under optimum enzymolysis condition, ferrous chela
Conjunction ability is (1.854 ± 0.025) mg/g, and degree of hydrolysis is (6.14 ± 0.05) %.
The first step is carried out to obtained thick grifola frondosus protein peptides (CGFP) using ultrafiltration using (3) the step of embodiment 1
Purifying, CGFP is configured to 10mg/mL solution, and pass through 0.45 μm of water system filter membrane, obtains supernatant.Then, in room temperature, pressure
Power is that solution is passed sequentially through to the ultrafiltration membrane of 5kDa and 1kDa, obtains>5kDa under 0.3MPa, the three of 5kDa~1kDa and<1kDa
Kind component is respectively designated as GFPU-1, GFPU-2, GFPU-3, and biuret method measures the protein content of each component, while measuring it
Ferrous sequestering power, the results are shown in Table 1.
1 ultrafiltration component protein content of table and ferrous sequestering power measurement
As shown in Table 1, the protein content of the protein content of ultrafiltration three obtained component, > 5kDa component is minimum, and
Generally the protein peptides with preferable sequestering activity are small-molecular peptides, therefore give up the component, so to another two group in embodiment 1
GFPU-2, GFPU-3 is divided further to be studied.
GFPU-2, GFPU-3 are isolated and purified in sephadex column using (3) the step of embodiment 1.First will
20g sephadex powder is added in 500mL distilled water and is sufficiently swollen, and removes suspended particulate；Secondly by the glucan of degassing process
Gel solution is added in chromatographic column (1.6 × 60cm), avoids tomography and bubble；Then the PBS (pH 6.8) of 0.05mol/L is used
Buffer balances 5 column volumes as mobile phase；Loading elution is finally carried out, to obtain purified components.
It is different according to GFPU-2 with GFPU-3 molecular weight, it is chromatographed respectively using Sephadex G-25 and Sephadex G-15
Column is further purified, the chromatographic condition of Sephadex G-25 are as follows: applied sample amount 15mg/mL；Mobile phase is the PBS of 0.05mol/L
(pH 6.8) buffer；Flow velocity is 0.2mL/min；Every pipe collects 5mL.The chromatographic condition of Sephadex G-15 are as follows: applied sample amount is
25mg/mL；Mobile phase is PBS (pH 6.8) buffer of 0.05mol/L；Flow velocity is 0.4mL/min；Every pipe collects 6mL, respectively
Obtain GFP-1 and GFP-2 grifola frondosus polypeptide.Effluent measures at 220nm, and collects main component (GFP-1 and GFP-2)
Carry out protein content and ferrous sequestering power measurement.
GFPU-2 is as shown in Figure 1 by Sephadex G-25 chromatographic column elution curve.As seen from Figure 1, GFPU-2 passes through
After Sephadex G-25, three chromatographic peaks are flowed out, but since second and third peak is much smaller compared with first peak, i.e., more than 20 pipes
Protein content is lower afterwards, therefore only collects the sample solution at first peak.50 DEG C of collection liquid vacuum distillations, are lyophilized after the completion of dialysis
To faint yellow GFP-1 solid powder.
GFPU-3 is as shown in Figure 2 by Sephadex G-15 chromatographic column elution curve.The chromatographic peak obtained as shown in Figure 2
For single symmetrical peak, the grifola frondosus protein peptides illustrated are more uniform small peptide.After Fraction collection concentration dialysis
Freeze-drying, obtains white GFP-2 solid powder.
Grifola frondosus protein peptides molecular weight determination is carried out to GFP-2 with Sephadex G-15 chromatographic column.With vitamin B12
(1350Da), oxidized form of glutathione (613Da), reduced glutathione (307Da) and hippuric acid (178Da) are used as standard
Product, according to molecular weight logarithm with elution volume is in a linear relationship does standard curve, be measured, tied according to the elution volume of sample
Fruit is as shown in Figure 3.
The elution volume and molar mass linear relationship of standard substance are y=-0.0144x+3.7612, R2=0.9965.Root
It is 963Da according to the molecular size range that GFP-2 is calculated in the elution volume of GFP-2, thus it is speculated that it is by 7~8 Amino acid profiles.
Hydrolysate is purified by ultrafiltration, Sephadex G-25 and Sephadex G-15, obtains ferrous chelating
Strongest group of ability is divided into GFP-2, and sequestering power is (269.7 ± 3.9) μ g/g.Molecule measuring is carried out by exclusion chromatography
Fixed, obtaining GFP-2 relative molecular weight is 963Da.Grifola frondosus Argine Monohydrochloride composition analysis show aspartic acid, glutamic acid and
Histidine content is higher, with ferrous ion chelate in play main effect.
Embodiment 6
Grifola frondosus small peptide iron chelate (GFP-Fe) ultraviolet spectra and infrared spectrum analysis
Using ultraviolet spectra to purifying grifola frondosus albumen peptide composition (GFP-2) and its iron chelate (GFP- in embodiment 1
Fe it) is scanned, qualitative observation sample ultra-violet absorption spectrum situation of change.A certain amount of GFP-2 and GFP-Fe is weighed respectively, is used
Distilled water dissolution is configured to the sample solution that concentration is 0.2mg/mL, and ultraviolet spectra is carried out in the wave-length coverage of 190~400nm
Scanning.
Infrared spectrum analysis is carried out to GFP-2 and GFP-Fe using KBr pressed disc method.KBr is placed in infrared drying oven and is done
It is dry to constant weight, accurately weigh GFP-2 the and GFP-Fe sample of 1.5mg respectively, the powder with 150mg KBr is fully ground respectively.
After grinding uniformly, thin slice is compressed by tablet press machine.The KBr of 150mg is accurately weighed again as ground control.Sample is existed
Fourier Transform Infrared Spectrometer is scanned and measures, 4000~400cm of range of scanning-1, scanning times 32 times, resolution ratio
2cm-1。
Grifola frondosus albumen peptide composition (GFP-2) and its grifola frondosus small peptide iron chelate (GFP-Fe) uv absorption spectra are such as
Shown in Fig. 4, the UV absorption of characteristic amino acid residue is mainly at 280nm, and the purple of peptide bond (amido bond at chopping up proteins)
Outer absorption is mainly at 190~220nm, it is seen that and what is mainly showed in sample is the UV absorption of the peptide bond after protein cleavage,
Accordingly, it can be said that this sample contains more peptide fragment protein.
Compare grifola frondosus albumen peptide composition (GFP-2) and grifola frondosus small peptide iron chelate (GFP-Fe) ultra-violet absorption spectrum can
Know, after chelating, maximum absorption peak becomes 192nm by 194nm, and blue shift occurs for absorption peak, this is because the oxygen atom on carbonyl is joined
With Fe2+Complexing, Fe2+Addition charge migration transition, ligand field transition has occurred, cause the charge of carbonyl to change
Become, absorption band blue shift.Illustrated with this, chelate and be two different substances before not chelating.
Fig. 5 and Fig. 6 is respectively that grifola frondosus albumen peptide composition (GFP-2) and its grifola frondosus small peptide iron chelate (GFP-Fe) are red
External spectrum figure.
3377.97cm in Fig. 5-1The absorption peak at place is the stretching vibration peak of N-H, belongs to the characteristic absorption peak of amino acid.
1654.15cm-1The strong absworption peak of appearance belongs to amide I band, is drawn by the C=O asymmetric stretching vibration of polypeptide skeleton
It rises；1547.95cm-1And 1259.66cm-1The absorption peak at place belongs to II band of amide and Amide Ⅲ band respectively, is N-H respectively
Thus absorption peak caused by bending vibration, the stretching vibration of C-H infers that grifola frondosus protein peptides structure may be β-pleated sheet.
1400.58cm-1Absorption peak, be the symmetrical stretching vibration peak of carboxylate radical.Meanwhile 1068.53cm-1There is (Pt-NH in place2) compared with
Strong absworption peak, in 517.34cm-1(Pr-NH2) characteristic absorption peak it is obvious.
Fig. 5 and Fig. 6 comparison it is found that still remain the characteristic absorption peak of albumen peptide matters in chelate infrared spectroscopy, but
The infrared spectroscopy of grifola frondosus protein peptides and grifola frondosus small peptide iron chelate has apparent on absorption peak number, intensity and position
Difference, both show the difference in structure.The stretching vibration peak of N-H mobile and remitted its fury from lower wave number to high wave number,
Amide I band peak intensity weakens, Pt-NH2Absorption peak appears in 1053.41cm-1Place and be strong absworption peak, may infer that Fe2+With-
NH2There is stronger combination.C=O symmetrical stretching vibration peak and asymmetric stretching vibration peak intensity, which have, largely to die down, explanation
Fe2+Also there is stronger combination with-COOH.800cm after chelating-1~500cm-1Fingerprint region peak disappear, a new peak
526.85cm-1Occur, is because the stretching vibration of N-Fe generates.
Test example 1
The in-vitro simulated stomach environment digestion of grifola frondosus small peptide iron chelate
Simulate the gastric juice: precise 0.2g sodium chloride and 3g pepsin (enzyme activity 15000U/g) measure the water of 80mL, in
In the beaker of 100mL, stirring the mixed liquor dissolves it sufficiently, then adjusts the pH to 2.0 of the mixed liquor simultaneously with 1mol/L HCl
The constant volume in 100mL volumetric flask.CGFP-Fe, GFP-Fe aqueous solution for preparing 5mg/mL respectively, finally obtain for embodiment 1
Thick grifola frondosus peptide of rice protein chelate (CGFP-Fe), grifola frondosus small peptide iron chelate (GFP-Fe) and 0.5mg/mL FeSO4Water
Solution.The above-mentioned solution of 10mL is inhaled respectively into 50mL conical flask, and with 0.5mol/L salt acid for adjusting pH value to 2.0, conical flask is put
Enter in 37 DEG C of constant-temperature shaking incubators and preheat 5min, respectively add simulate the gastric juice 10mL again and mix, in 37 DEG C of constant temperature oscillation casees
150rpm reacts 0,10,30,60,120,180min respectively, takes out 95 DEG C of water-bath enzyme deactivation 10min, stands cooling, 4000r/min
It is centrifuged 10min, supernatant is collected, with the concentration of iron in Phen colorimetric method for determining sample solution.
As shown in fig. 7, all in all, FeSO4, CGFP-Fe and GFP-Fe can be kept in stomach environment one more
Stable dissolved state.Thus illustrate, grifola frondosus peptide of rice protein chelate can be stabilized in acidic environment, and can be supported
The digestion of pepsin processed.
Test example 2
The in-vitro simulated intestinal environment digestion of grifola frondosus small peptide iron chelate
Simulated intestinal fluid: precise 0.68g potassium dihydrogen phosphate is dissolved in 25mL water, the rear sodium hydroxide that 0.2mol/L is added
Solution 7.7mL and water 50mL uniformly mixes, adds bovine bile 6.0g and trypsase (2500U/mg) 1.0g, stirring makes
It is dissolved completely, and the pH value for finally adjusting the mixed liquor with 0.2mol/L sodium hydroxide solution is settled to 100mL to 7.5.Respectively
Prepare the thick grifola frondosus peptide of rice protein chelate (CGFP-Fe) of 5mg/mL, grifola frondosus small peptide iron chelate (GFP-Fe) aqueous solution and
0.5mg/mL FeSO4Aqueous solution.It takes the above-mentioned three kinds of solution of 10mL into 50mL conical flask respectively, is adjusted with 0.5mol/L hydrochloric acid
Conical flask is put into 37 DEG C of constant-temperature shaking incubators to 2.0 and preheats 5min by pH value, the simulation respectively configured again plus in test example 1
Gastric juice 10mL is simultaneously mixed, and 150rpm takes out after reacting 1h in 37 DEG C of constant temperature oscillation casees, with 0.9mol/L NaHCO3Adjust sample pH value
Value adds simulated intestinal fluid 1mL, places the beaker in 37 DEG C of constant temperature oscillation casees to 5.3, then with 2mol/L NaOH tune pH to 7.5
150rpm reacts 0.5,1,2,4,6h respectively.95 DEG C of enzyme deactivation 10min after reaction, 4000r/min are centrifuged 10min, in collection
Clearly, with the iron content in Phen colorimetric method for determining solution, solubility is obtained.
As shown in figure 8, after the grifola frondosus peptide of rice protein after Gastric juice digestion is digested a period of time via simulated intestinal fluid, GFP-
Fe is compared with FeSO4Compared to higher solubility can be kept, to more preferably small intestine be promoted to absorb GFP-Fe.Due to grifola frondosus protein peptides
The Fe not dissociated in iron2+, and certain protection and buffering are played the role of in the presence of grifola frondosus protein peptides to ferro element, can subtract
Less since the iron insoluble matter that enteron aisle alkaline environment is formed causes the reduction of iron solubility, to guarantee that soluble iron can be by enteron aisle more
It is good to absorb.CGFP-Fe, which changes over time concentration of iron in enteron aisle and is gradually reduced, finally reaches stable state, is due to thick grifola frondosus
The presence of other impurities has an impact to the stability of chelate in protein peptides, to affect the dissolubility of iron in chelate.
The present invention shows that GFP-Fe can keep higher solubility and stabilization in the gastrointestinal tract by External digestion experiment
Property.And in intestinal environment, GFP-Fe solubility ratio FeSO4It is higher more preferably to promote absorption of the small intestine to GFP-Fe.
Test example 3
Grifola frondosus small peptide iron chelate Caco-2 cytotoxicity experiment
Caco-2 cell culture is in containing 20% (v/v) FBS (fetal calf serum), 1% (v/v) dual anti-(Pen .- Strep)
In the MEM complete medium of 1% (v/v) nonessential amino acid, at 37 DEG C, 5%CO2With the cell culture of relative humidity 90%
Culture in case, culture medium were replaced every two days, were tested after cell length is digested to 80% or so with pancreatin.By Caco-2 cell
For inoculation of suspension liquid in 96 orifice plates, every hole is 5 × 103A cell, sets 5%CO2, 72h is cultivated in 37 DEG C of incubators.It is long to cell
To 70~80% or so, it is separately added into the implementation that 150 μ L are 0.1,0.2,0.5,1.0,1.5,2.0,2.5,5.0mg/L containing concentration
The grifola frondosus small peptide iron chelate (GFP-Fe) and FeSO that in example 1 prepared by step (5)4MEM culture medium (10%FBS) do respectively
Pre- 12h, for 24 hours, 48h, each concentration sets 6 multiple holes, blank group of accompanying.5mg/mL MTT solution is added later, continues to be placed in training
It supports in case and cultivates 4h.Cell conditioned medium is discarded, is added in dimethyl sulfoxide DMSO (150 hole μ L/), shakes, in microplate reader at 492nm
Measure the absorbance in every hole.Cell survival rate calculation method is as follows:
Wherein, AExperimental groupFor sample absorbance；AControl groupFor the absorbance of blank group.
MTT experiment result is as shown in figure 9, GFP-Fe and FeSO4Time dependence is presented to the toxicity of cell；Meanwhile it is real
It tests as the result is shown: for 24 hours with the toxicity of 48h GFP-Fe lower than FeSO4, this prompt GFP-Fe can be as the novel benefit iron of safety
Agent.
Test example 4
Measure transepithelial cell resistance
According to the method for secondary culture by cell with 105/ mL is inoculated into the cell Transwell upper chamber side, every in upper chamber side
Hole adds 0.5mL cell suspension, and the every hole in lower room side adds 1.5mL fresh culture, every other day replaces culture solution within first 7 days, often later
It changes liquid, cultivates to 21 days.Cross-film epithelial cell resistance value (TEER) is measured with cell resistance instrument, is greater than 300 Ω cm2It is thin
Born of the same parents' monofilm can be used for transport experiment.Firstly, putting the electrodes into the HBSS for being preheated to 37 DEG C, 20min is balanced；Remove culture plate
In culture medium, the HBSS 0.5mL of preheating is added in upper chamber side, and 1.5mL is added in lower room side, and 37 DEG C of balance 20min wash away simultaneously
The impurity of cell surface；HBSS is removed, the HBSS of preheating is rejoined, measures cross-film resistance value；It is repeated with 1 empty vectors
Step is stated to obtain blank value；The cross-film resistance value of cell is calculated according to following formula
TEER=(measuring resistance value-blank cell resistance value) × culture cell area (1.12cm2)
Choosing Caco-2 cell monolayer cross-film resistance value is more than 300 Ω cm2Cell carry out subsequent experimental.
Test example 5
Grifola frondosus small peptide iron chelate transport experiment
Cell culture is as shown in test example 4, when the cell resistance value (TEER) of measurement is greater than 300 Ω cm2When, use HBSS
(pH 7.4) buffer carefully rinses ventricular cell 3 times, gently sucks in hole after being incubated for 30min in 37 DEG C of incubators the 3rd time
HBSS。
Grifola frondosus small peptide iron chelate (GFP-Fe) and FeSO4Drug is from upper chamber to the transhipment of lower room side: upper chamber side difference
The GFP-Fe and FeSO of 0.5mL various concentration (0.5,1.0,2.0mg/L) is added4Solution is added as supply pool in lower room side
1.5mL HBSS is as reception tank.Transhipment of the lower room side to upper chamber side: same concentrations drug solution 1.5mL is added to lower room side and is made
For supply pool, 0.5mL HBSS is added as reception tank in upper chamber side.Having added the Transwell of drug solution and blank to cultivate
Plate is placed in 37 DEG C of incubators, draws 500 μ L of reception tank solution at 30,60,90,120min respectively, while supplying same volume
It is preheated to 37 DEG C of HBSS solution.Using the content of iron in AAS method measurement sample.Drug transport rate uses apparent permeability coefficients
(apparent permeability coefficient, Papp) is indicated.
The GFP-Fe and FeSO of various concentration4Apparent permeability coefficient in the transcellular transport in Caco-2 cell monolayer
It is shown in Table 2.As shown in table 2, GFP-Fe apparent permeability coefficient in upper chamber → lower room under various concentration compares FeSO4Have to a certain degree
Raising, and under the concentration of 2mg/L, the apparent permeability coefficient in upper chamber → lower room is significantly higher than FeSO4, lower room → upper chamber it is apparent
Permeability coefficient is substantially less than FeSO4.It can be seen that GFP-Fe intestinal absorption effect is better than FeSO4.Meanwhile under each concentration
The P of GFP-FeappIt is all larger than the 10 of the defined good drug of absorption in the world-6Cm/s shows that GFP-Fe is imitated with good absorption
Fruit.
On the other hand, the apparent permeability coefficient from upper chamber face to lower room face is significantly greater than from lower room face to above, explanation
GFP-Fe is likely to be absorbed by the transport vehicle of small intestine top side.It simultaneously can by lower room → upper chamber and upper chamber → lower room ratio
Know, the absorption of GFP-Fe is influenced smaller, the further explanation good assimilation effect of GFP-Fe by outlet.
The GFP-Fe and FeSO of 2 various concentration of table4In the apparent permeability coefficient P of Caco-2 cell traffic processapp(×10- 6cm/s)
Note: * indicates the GFP-Fe and FeSO under same experimental conditions, same concentrations4Compare with significant difference (P <
0.05)
By the in-vitro simulated pipe intestinal digesting of above-mentioned test and the transhipment of Caco-2 cell model to grifola frondosus peptide of rice protein chela
The digestion and assimilation effect for closing object are evaluated.Meanwhile by Caco-2 cell model to grifola frondosus peptide of rice protein chelate
Mechanism of absorption is studied.The experimental results showed that the GFP-Fe and FeSO under stomach environment4Preferable stability is all had, and
Under intestinal environment, GFP-Fe ratio FeSO4Solubility is higher.Cytotoxicity experiment shows, GFP-Fe and FeSO4Compared to toxicity compared with
It is low, and the apparent permeability coefficient in upper chamber → lower room by constructing successful Caco-2 cell model transport experiment discovery GFP-Fe
Greater than FeSO4, show that its absorption is better than FeSO4, illustrate that GFP-Fe can be developed into high new of a kind of safety, bioavailability
Type iron supplementary.Studies have shown that targeting peptide transporter (PepT1) of mechanism of absorption mediates the active transport of GFP-Fe, simultaneously
There are the protein mediated outlet effects of multidrug resistance.
Test example 5
Influence of the grifola frondosus small peptide iron chelate to mouse spleen cell proliferation and macrophage proliferation
It is the thick grifola frondosus protein peptides (CGFP) that are prepared step (2) in embodiment 1 with RPMI-1640 complete medium, real
Apply the grifola frondosus polypeptide (GFP-2) of step (4) preparation in example 1, the thick grifola frondosus peptide of rice protein that in embodiment 1 prepared by step (5)
Chelate (CGFP-Fe) and grifola frondosus small peptide iron chelate (GFP-Fe) are respectively configured to 50 μ g/mL, 100 μ g/mL, 200 μ g/
The sample solution of mL.Every hole is added 5 × 10 in 96 orifice plates6The splenocyte or 2 × 10 of a/mL cell concentration6A/mL cell
The 100 μ L of macrophage suspension of concentration, then 100 μ L CGFP, GFP, CGFP-Fe and GFP-Fe sample are separately added into, keep sample whole
Concentration is respectively 50,100,200 μ g/mL.Each concentration of each sample does 3 multiple holes.100 μ L are added in negative control group
RPMI-1640 complete medium replaces sample solution.The LPS solution of the LPS control group addition final concentration of 10 μ g/mL of 100 μ L.Knife
The ConA solution that 100 μ L are prepared with RPMI-1640 complete medium, final concentration of 5 μ g/ is added in legumin A (ConA) control group
mL.96 orifice plates are placed at 37 DEG C in 5%CO2 incubator and cultivate 44h.It takes out, it is molten that 20 μ L 5mg/mL MTT are added in every hole
Liquid continues to be put into incubator and continues to cultivate 4h, and supernatant is removed in every hole, and 100 μ L DMSO are added, and vibrates after 10min into every hole
The purple crystal of formation dissolves, and is measured under 490nm wavelength with microplate reader absorbance (OD value), calculates proliferation rate.Formula is as follows:
Influence result such as Figure 10-11 institute of the grifola frondosus small peptide iron chelate to mouse spleen cell proliferation and macrophage proliferation
Show, spleen is the second immunization barrier of human body, plays in human body specific immunity important as important immune organ
Effect.The appreciation rate of ferrous chelate compound group is respectively higher than grifola frondosus protein peptides group, shows grifola frondosus peptide of rice protein chelate
Good immunocompetence.Macrophage is the immunocyte of most original in the mammalian body, while being also in organism to disease-resistant
First of immunization barrier of substance.When pathogen enters in human body, macrophage is swallowed, and subsequent macrophage will play anti-
Original is in the effect of delivery cell, and T lymphocyte is activated, to participate in nospecific immunity and specific immunity process, adjusts life
The immune response activity of object.The proliferation rate of CGFP-Fe and GFP-Fe group is up to 29.14% and 27.75%, compares better than LPS
Group.It can be seen that being had more for grifola frondosus peptide of rice protein chelate is compared with grifola frondosus protein peptides to the proliferation of mouse macrophage
Facilitation.
Test example 6
Grifola frondosus small peptide iron chelate generates the influence of NO, IL-6 and TNF-α to mouse macrophage
Cell and sample Adding Way are the same as test example 5.Cells and supernatant (50 μ L) is drawn after cell culture 48h is added 96
In well culture plate, then and be added isometric NO detection reagent, react and detect OD with microplate reader after 10min540nm.With NaNO2It is molten
Liquid draws standard curve as standard items, calculates NO content corresponding to sample well.IL-6 is measured according to ELISA kit
And TNF-α.
Grifola frondosus small peptide iron chelate generates result such as Figure 12-of the influence of NO, IL-6 and TNF-α to mouse macrophage
Shown in 14, grifola frondosus protein peptides and its iron chelate can dramatically increase the content of macrophages secrete NO, also can be in certain journey
Enhance the release of IL-6 and TNF-α cell factor on degree.Therefore, grifola frondosus protein peptides and grifola frondosus peptide of rice protein chelate have
There is good immune-enhancing activity.
By above-mentioned test to the immunocompetences of grifola frondosus protein peptides and its iron chelate studies have shown that CGFP, GFP-2,
CGFP-Fe, GFP-Fe have significant humidification to the proliferation of mouse boosting cell and macrophage, illustrate adjusting immune answer
There is promotion to specific immunity and nospecific immunity process during answering.In comparison, grifola frondosus peptide of rice protein chelates
The facilitation of object is stronger.Meanwhile grifola frondosus protein peptides and its iron chelate can dramatically increase containing for macrophages secrete NO
Amount, can also enhance the release of IL-6 and TNF-α cell factor to a certain extent.Therefore, grifola frondosus protein peptides and grifola frondosus egg
White peptide iron chelate all has good immune-enhancing activity.
Claims (10)
1. a kind of preparation method of grifola frondosus small peptide iron chelate, which comprises the steps of:
(1) Grifola Frondosa sporophore protein extraction:
Grifola Frondosa sporophore Ultramicro-powder is made in dry Grifola Frondosa sporophore crushing, distilled water, after mixing evenly, constant temperature is added
Stirring is extracted, after, it is cooled to room temperature, is centrifuged, takes supernatant, stand at low temperature is overnight, is centrifuged the supernatant that inclines, and precipitating is washed
It washs, vacuum drying crushes, and is sieved to get Grifola Frondosa sporophore albumen powder；
(2) grifola frondosus proteolysis:
Grifola Frondosa sporophore albumen powder is weighed, distilled water is added, adds protease, is digested, hydrolyzate after reaction was completed
Centrifugation, takes supernatant through dialysing, thick grifola frondosus protein peptides is obtained after freeze-drying, saves；
(3) grifola frondosus protein peptides isolate and purify:
First step purifying is carried out by ultrafiltration to obtained thick grifola frondosus protein peptides；After ultrafiltration purification by sephadex column into
Row isolates and purifies to obtain grifola frondosus polypeptide；
(4) preparation of grifola frondosus small peptide iron chelate:
Grifola frondosus polypeptide is weighed, after distilled water dissolution is added, the ascorbic acid solution added adds FeCl2Solution, oscillation
Reaction, is centrifuged after reaction, takes supernatant that dehydrated alcohol is added, and overnight precipitation, centrifuging and taking precipitating is dry after washing, obtains ash
Set flower small peptide iron chelate.
2. the preparation method of grifola frondosus small peptide iron chelate according to claim 1, which is characterized in that step (1) is described
The half that supernatant is first evaporated to after supernatant original volume is taken, adjusts pH value to grifola frondosus albumen isoelectric point.
3. the preparation method of grifola frondosus small peptide iron chelate according to claim 1, which is characterized in that step (2) is described
Protease is one in flavor protease, compound protease, pepsin, neutral proteinase, alkali protease and trypsase
Kind.
4. the preparation method of grifola frondosus small peptide iron chelate according to claim 1, which is characterized in that step (2) is described
The additional amount of protease is 1600-2100U/g Grifola Frondosa sporophore albumen powder.
5. the preparation method of grifola frondosus small peptide iron chelate according to claim 1, which is characterized in that step (2) is described
Enzymolysis time is preferably 1-3.5h.
6. the preparation method of grifola frondosus small peptide iron chelate according to claim 1, which is characterized in that step (3) is described
It is that thick grifola frondosus protein peptides are configured to solution that thick grifola frondosus protein peptides, which carry out first step purifying by ultrafiltration, filters, obtains supernatant
Liquid, solution are dialysed after ultrafiltration, and freeze-drying obtains being greater than 5KDa, 1-5KDa and the grifola frondosus polypeptide fractions less than 1KDa.
7. the preparation method of grifola frondosus small peptide iron chelate according to claim 1, which is characterized in that step (3) is described
It is sufficiently molten in distilled water by the way that sephadex powder to be added for being isolated and purified after ultrafiltration purification by sephadex column
Swollen, removal suspended particulate is de-gassed；The sephadex solution of degassing process is added in chromatographic column, it is slow with PBS
Fliud flushing carries out loading elution as mobile phase, by different molecular weight grifola frondosus polypeptide after ultrafiltration, to obtain purified components ash tree
Flower polypeptide.
8. the preparation method of grifola frondosus small peptide iron chelate according to claim 1, which is characterized in that every in step (4)
100-120 μ L ascorbic acid solution is added in 100mg grifola frondosus protein peptides；100-120 μ LFeCl is added2Solution；Oscillating reactions temperature
Degree is 20-25 DEG C, reacts 4-5min.
9. grifola frondosus small peptide iron prepared by a kind of preparation method of grifola frondosus small peptide iron chelate described in claim 1 chelates
Object.
10. grifola frondosus small peptide iron chela prepared by a kind of preparation method of grifola frondosus small peptide iron chelate described in claim 1
Close application of the object in preparation bioactivity iron supplementary.
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| CN111018949A (en) * | 2020-01-06 | 2020-04-17 | 上海应用技术大学 | Preparation method and application of grifola frondosa flavor-developing peptide |
| CN111838498A (en) * | 2020-08-19 | 2020-10-30 | 福建农林大学 | Grifola frondosa polypeptide solid beverage with calcium absorption promoting effect and preparation method thereof |
| CN113831421A (en) * | 2021-10-14 | 2021-12-24 | 黄河三角洲京博化工研究院有限公司 | Combined preparation method of grifola frondosa mycelium polypeptide and beta-glucan |
| CN118633745A (en) * | 2024-08-12 | 2024-09-13 | 浙江方格药业有限公司 | Grifola frondosa active polypeptide beverage and preparation method thereof |
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| CN111018949A (en) * | 2020-01-06 | 2020-04-17 | 上海应用技术大学 | Preparation method and application of grifola frondosa flavor-developing peptide |
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| CN113831421B (en) * | 2021-10-14 | 2023-03-17 | 黄河三角洲京博化工研究院有限公司 | Combined preparation method of grifola frondosa mycelium polypeptide and beta-glucan |
| CN118633745A (en) * | 2024-08-12 | 2024-09-13 | 浙江方格药业有限公司 | Grifola frondosa active polypeptide beverage and preparation method thereof |
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