CN118120908A - Method for separating and purifying melanoidin based on pH sensitive metal chelate precipitant, melanoidin and application thereof - Google Patents
Method for separating and purifying melanoidin based on pH sensitive metal chelate precipitant, melanoidin and application thereof Download PDFInfo
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Abstract
The invention discloses a method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant, the melanoidin and application thereof, wherein the melanoidin is obtained by purifying the melanoidin synthesized by glucose and glycine through Maillard reaction. The obtained melanoidin has a relatively simple structure and stable property, is favorable for subsequent research of the melanoidin structure, has a lower molecular weight than that of the crude melanoidin, is 6000Da, and is also converted into more small piece-shaped and block-shaped structures by large irregular polygonal blocks and a small amount of granular structures in the crude melanoidin on the microstructure.
Description
Technical Field
The invention belongs to the technical field of separation and purification of melanoidin, and particularly relates to a method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant, melanoidin and application thereof.
Background
Melanoidin (melanoidins) is a high molecular weight Maillard reaction product with complex structure and unequal polymerization degree produced by the reaction of carbohydrate and nitrogen-containing compound with free amino. It is commonly found in food processing and storage processes, such as beer, bread, coffee, vinegar, etc., and contributes to the formation of flavor and color of foods, and has various functional activities such as metal ion chelating property, antioxidation, antibacterial, antihypertensive and antitumor activities, wherein the chelating property of melanoidin plays an important role in binding with metals.
Metal chelate affinity purification is a method for separating and purifying a target product by utilizing the affinity between a transition metal ion and an electron donor group on the target product. The metal affinity purification system generally comprises three components of a medium (or a carrier), a metal ion chelating agent and metal ions, and is widely applied to the separation and purification of various proteins at present.
The intelligent macromolecule is a polymer capable of sensing and receiving information of external environment and making sensitivity response, and environmental triggers behind the transition can be temperature, pH value, electric field, magnetic field, ion intensity increase, existence of certain metabolic chemical substances and formation of polycation-polyanion complex, and the shape, solubility, surface property and the like of the intelligent macromolecule are automatically changed according to environmental changes, so that corresponding reversible changes occur. The pH-sensitive polymer is an intelligent polymer with reversible precipitation-dissolution characteristics, the solubility of which in aqueous solution changes with pH value, and the pH-sensitive polymer is applied to the fields of drug controlled release, material separation, immunoassay and the like, and has been receiving more and more attention in recent years.
Choline-rich foods are often consumed in daily life, such as: red meat (pork, beef, fish) increases the risk of atherosclerosis. There are reports that inhibiting the choline metabolic pathway in vivo can prevent atherosclerosis. It is therefore important to develop an active substance that can inhibit the cholinergic pathway. There is also a current study on the mechanism of atherosclerosis prevention with melanoidin, for example: verzeloniE et al have found that aromatic melanoidins can bind to heme to prevent its absorption, which can act as a catalyst for oxidative damage and cause the occurrence of various cancers and cardiovascular diseases. These outstanding activities make melanoidins a functional ingredient for preventing arteriosclerotic diseases possible.
However, extraction and purification of melanoidin have been a technical problem, and the traditional extraction methods of melanoidin include organic solvent extraction, size exclusion chromatography, water extraction, precipitation, macroporous resin adsorption, enzymolysis and the like. However, the traditional extraction methods have the problems of long reaction time, insufficient extraction and unsatisfactory product yield in the extraction process, and are not beneficial to industrialized mass production. The macroporous resin adsorption method has the advantages of large adsorption capacity, high adsorption speed, short time, good physical and chemical stability and the like, but has certain limitations, for example, after the resin is repeatedly used, a plurality of non-adsorptive components or impurities remain on the surface and inside of the resin to deepen the color of the column, the column effect is reduced, and the resin particles are crushed and the service life is shorter due to the fact that the column bed is extruded too tightly after the repeated use.
Therefore, how to provide an effective separation and purification method of melanoidin is of great significance for subsequent research on the structure of purified melanoidin, the functional activity of preventing atherosclerosis diseases and the like.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The present invention has been made in view of the above and/or problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide a method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant.
In order to solve the technical problems, the invention provides the following technical scheme: comprising the steps of (a) a step of,
Preparing crude melanoidin from glucose and glycine by Maillard reaction;
Adding an activating agent into the pH sensitive polymer solution for activation, then adding a metal chelating agent solution, and performing coupling reaction to obtain a pH sensitive precipitant solution;
Adding the FeCl 3 solution into the pH sensitive precipitant solution, and chelating to obtain a pH sensitive metal chelating precipitant;
Mixing the crude melanoidin with a pH sensitive metal chelate precipitant, regulating the pH to 4.8-5.2, centrifuging, removing unbound melanoidin from the supernatant, and precipitating to form a compound of the pH sensitive metal chelate precipitant and the melanoidin;
adding the compound into NaOH solution, precipitating, redissolving, continuously adding disodium ethylenediamine tetraacetate solution for water bath stirring reaction, cooling to room temperature after the reaction, regulating the pH of the solution to 4.8-5.2 again, centrifuging, taking supernatant, dialyzing, freeze-drying, and further post-treating to obtain purified melanoidin.
As a preferable scheme of the method for separating and purifying melanoidin based on the pH sensitive metal chelate precipitant, the invention comprises the following steps: the pH sensitive polymer solution is prepared by dissolving pH sensitive polymer Eudragit S-100 in NaOH solution, the concentration is 1%, and the pH is regulated to 6.5-7.5.
As a preferable scheme of the method for separating and purifying melanoidin based on the pH sensitive metal chelate precipitant, the invention comprises the following steps: the metal chelating agent solution is 4- (imidazol-1-yl) aniline solution with the concentration of 10mmol/L, wherein 11mL of the metal chelating agent solution is added to every 1gpH sensitive polymer.
When the addition amount of the metal chelating agent solution is low, chelation is incomplete, the yield of purified melanoidin is reduced, and when the addition amount of the metal chelating agent solution is high, the solution contains redundant unchelated reagent, so that the subsequent experiment is influenced.
As a preferable scheme of the method for separating and purifying melanoidin based on the pH sensitive metal chelate precipitant, the invention comprises the following steps: the activator is 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, and the added pH sensitive polymer has the molar mass of 70-90%.
As a preferable scheme of the method for separating and purifying melanoidin based on the pH sensitive metal chelate precipitant, the invention comprises the following steps: the concentration of the FeCl 3 solution is 60mmol/L, and the addition amount is 1.25mL.
When the addition amount of the FeCl 3 solution is low, chelation is incomplete, the yield of purified melanoidin is reduced, and when the addition amount of the metal chelating agent solution is high, the solution contains redundant unchelated reagent, so that the subsequent experiment is influenced.
As a preferable scheme of the method for separating and purifying melanoidin based on the pH sensitive metal chelate precipitant, the invention comprises the following steps: the crude melanoidin is mixed with a pH sensitive metal chelating precipitant, wherein 0.56mg of crude melanoidin is added per 1gEudragit S-100 of the pH sensitive metal chelating precipitant calculated by Eudragit S-100, the rotating speed is 100r/min, and the oscillation time is 60-80 min.
When the addition amount of the crude melanoidin is low, chelation is incomplete, the yield of the purified melanoidin is reduced, and when the addition amount of the crude melanoidin is high, the solution contains redundant unchelated reagent, so that the subsequent experiment is influenced.
As a preferable scheme of the method for separating and purifying melanoidin based on the pH sensitive metal chelate precipitant, the invention comprises the following steps: the addition amount of the disodium ethylenediamine tetraacetate is 1.5-4 times of the molar amount of Fe 3+ in the FeCl 3 solution.
As a preferable scheme of the method for separating and purifying melanoidin based on the pH sensitive metal chelate precipitant, the invention comprises the following steps: the temperature of the water bath stirring of the compound added into the disodium ethylenediamine tetraacetate solution is 50 ℃, and the stirring time is 3-4 hours.
Still another object of the present invention is to provide melanoidin obtained by separation and purification based on a pH-sensitive metal chelate precipitant.
It is still another object of the present invention to provide the use of melanoidin isolated and purified based on pH sensitive metal chelating precipitants as a functional ingredient for improving or preventing atherosclerosis.
The invention has the beneficial effects that:
(1) The invention takes glucose and glycine as raw materials to carry out Maillard reaction to generate crude melanoidin, and aims to separate and purify the crude melanoidin, and the separation and purification of the melanoidin can be realized by utilizing the metal chelating capability of the melanoidin and the pH sensitivity of pH sensitive polymers, preparing a pH sensitive metal chelating agent to chelate the crude melanoidin and simply regulating the pH.
(2) The model melanoidin prepared by using single amino acid and reducing sugar is relatively simple in melanoidin structure, stable in property and favorable for subsequent research of melanoidin structures, compared with crude melanoidin, the purified melanoidin obtained by separation and purification has a lower molecular weight of 6000Da, and on the microstructure, large irregular polygonal blocks and a small number of granular structures in the crude melanoidin are also converted into more small block sheet-shaped and block-shaped structures.
(3) The melanoidin product can obviously improve the blood lipid level and the levels of TMA and TMAO in blood plasma through long-term administration, can activate an AMPK way to inhibit TMA lyase activity and improve the acidic environment of intestinal tracts, thereby inhibiting the proliferation of harmful bacteria in the intestinal tracts, and plays a role in preventing atherosclerosis diseases through the regulation of the blood lipid level and the levels of TMA and TMAO. Therefore, the melanoidin purified by the pH sensitive metal chelating precipitant is taken as a functional component to inhibit choline metabolism and slow down the risk of arteriosclerosis, thus providing a new strategy for preventing the arteriosclerosis.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a diagram showing the structural characterization of purified melanoidin and crude melanoidin prepared in example 1 of the present invention.
FIG. 2 shows the effect of purified melanoidin prepared in example 1 of the present invention on body weight and food intake of red-meat-induced atherosclerosis mice.
FIG. 3 shows the effect of purified melanoidin prepared in inventive example 1 on blood lipid levels in red-meat induced atherosclerosis mice.
FIG. 4 shows the effect of purified melanoidin prepared in inventive example 1 on Trimethylamine (TMA), trimethylamine oxide (TMAO) and trimethylamine lyase activity (TMA LYASE ACTIVITY) in red-meat-induced atherosclerosis mice.
FIG. 5 shows the effect of purified melanoidin prepared in inventive example 1 on expression of intestinal epithelial cell adenylate activating protein kinase (AMPK) in red meat-induced atherosclerosis mice.
FIG. 6 shows the effect of purified melanoidin prepared in inventive example 1 on pH and short chain fatty acid levels (SCFAs) in red-meat induced atherosclerosis mouse feces.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The raw materials used in the invention are all commonly and commercially available in the field without special description.
Example 1
The embodiment provides a separation and purification method of melanoidin, which specifically comprises the following steps:
1) Preparing coarse melanoidin: 9.0080g of D-anhydrous glucose and 3.7535g of glycine are fully dissolved in distilled water, the solution is fixed in a volumetric flask of 100mL, the solution is poured into a flat plate for freeze drying to constant weight, then the solution is put into a baking oven of 125 ℃ for Maillard reaction for 2 hours, after cooling to room temperature, reactants are ground into fine powder, after being dissolved in distilled water, the solution is subjected to suction filtration by using filter paper No. 1, and dialysis is carried out for 96 hours by using a dialysis bag with the interception amount of 1000Da, and Coarse Melanoidin (CMLD) is obtained through freeze drying;
2) Preparing a pH sensitive precipitant: taking pH sensitive polymer Eudragit S-100 (ES-100), weighing 1g of the pH sensitive polymer Eudragit S-100, dissolving in 2mol/L NaOH solution, regulating the pH value to about 7.2 by using 3mol/L HCl solution, adding 0.4330g of activator 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, stirring at room temperature for 10min, performing an activation reaction, wherein the adding molar ratio of the activator 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide is 80% of the pH sensitive polymer Eudragit S-100, adding 11mL of metal chelating agent 4- (imidazole-1-yl) aniline solution (IA, c=10 mmol/L), stirring in a water bath at 70 ℃ for 2h, and coupling IA to obtain pH sensitive precipitant (ES-IA);
3) Chelating metal ions: adding 1.25mL of FeCl 3 solution (60 mmol/L) into the ES-IA solution, stirring for 1h at room temperature, and chelating Fe 3+ ions to obtain a pH sensitive metal chelating precipitant (ES-IA-Fe 3+);
4) Chelatin: adding 0.56mgCMLD into the pH sensitive metal chelating precipitant solution obtained in the step 3), oscillating for 60min in 100r/min room temperature water bath, combining ES-IA-Fe 3+ with melanoidin, adjusting pH to 5.0, centrifuging, removing unbound melanoidin from supernatant, and precipitating to obtain a pH sensitive metal chelating precipitant and purified melanoidin compound ES-IA-Fe 3+ -MLD;
5) Elution of purified melanoidin: adding a small amount of NaOH solution (c=2mol/L) into the precipitate obtained in the step 4), re-dissolving the precipitate, continuously adding 0.1g of disodium ethylenediamine tetraacetate (EDTA-2 Na) and 200mL of distilled water, and reacting for 3h under the water bath stirring at 50 ℃; cooling to room temperature, regulating pH to 5.0, centrifuging, precipitating to obtain ES-IA, collecting supernatant with EDTA-Fe 3+ and MLD, dialyzing the supernatant with 1000 Da-cut-off dialysis bag for 96 hr, and lyophilizing to obtain purified Melanoidin (MLD).
FIG. 1A is a graph showing the molecular weight distribution of purified melanoidin MLD and crude melanoidin CMLD obtained in this example, showing that both the liquid chromatograms of purified melanoidin MLD and crude melanoidin CMLD show singleness, the crude melanoidin has a molecular weight of 13.8kDa, and the purified melanoidin has a molecular weight of 6.0kDa, with a reduced molecular weight. In the model system, although dialysis removes unreacted free glucose and amino acids, carbohydrate chains remain in the crude melanoidin structure and adsorb to melanoidins via non-covalent interactions to form polymers, resulting in higher molecular weights, while after purification by pH sensitive metal chelate precipitants, degradation of carbohydrate chains occurs, thus reducing the molecular weight of purified melanoidin MLD.
FIG. 1B is a Fourier infrared spectrum of purified melanoidin MLD and crude melanoidin CMLD, showing that the functional groups of the purified melanoidin have been significantly altered after purification with a pH sensitive metal chelate precipitant, the purified melanoidin having an additional absorption band at 1720cm -1, possibly due to-C=O stretching; the absorption band at 1605cm -1, which is enhanced in the purified melanoidin spectra compared to the crude melanoidin, is due to the coupling of-c=o stretching and-NH deformation (amide i band and amide ii band); the absorption band at 1318cm -1-1265cm-1 was attributed to-C-N stretching vibration (amide III band), indicating that purified melanoidin is rich in-C=O, -NH, -C-N groups. Furthermore, it is possible to provide a device for the treatment of a disease. The absorption band of the crude melanoidin at 1076cm -1 appears, which cannot be clearly observed in the purified melanoidin due to the stretching vibration of-C-O, further explaining that the polymer produced by Maillard reaction is purified by the pH sensitive metal chelate precipitant to mainly remove part of sugar in the crude melanoidin.
Fig. 1C to F are scanning electron microscope images of crude melanoidin CMLD (C and D) and purified melanoidin MLD (E and F), respectively, and it can be seen that large irregular polygonal blocks and a small number of granular structures are observed in the crude melanoidin, and more small pieces of sheet-like and block-like structures are included in the purified melanoidin, which indicates that the microstructure change occurs after purification of the crude melanoidin.
Animal test for treating atherosclerosis by purified melanoidin
Experimental animals: the experimental animals are 72 SPF clean C57BL/6J male mice with the age of about 6 weeks and the weight of about 20g, and the animals are adaptively bred for one week in the environment of 12 hours in each of illumination and darkness at the temperature of 25+/-2 ℃ and the humidity of 50-60%, and all animal experimental operations meet the requirements of Chinese and international animal protection guidance.
Experimental medicine: 3, 3-dimethylbutanol (DMB, 97%); choline (90%); trimethylamine hydrochloride (purity greater than 98%); total Cholesterol (TC) kit; a Triglyceride (TG) kit, a Low Density Lipoprotein Cholesterol (LDLC) kit, a High Density Lipoprotein Cholesterol (HDLC) kit (institute of bioengineering, tokyo); TGL-1650 model high-speed refrigerated centrifuge; JD1000-2 type electronic analytical balance; TU-1810 type ultraviolet spectrophotometer.
Construction and administration of an atherosclerosis mouse model:
From 72C 57BL/6J mice, 12 were randomly selected and fed as a blank (NC) diet, and the remaining mice were fed with western diet.
The common feed comprises 73% of basal feed, 5% of sucrose, 7% of grease and 15% of starch;
the western diet feed comprises 53% of basal feed, 5% of sucrose, 7% of grease, 15% of starch and 20% of red meat.
The 72 mice were randomly divided into 6 groups (n=12) of:
normal group (NC), feeding normal feed;
Positive control group (DMB), 3-dimethylbutanol (1%, v/v) (free drinking water) and fed western diet;
model group control group (RM), feeding western diet;
a low dose group (LMLD) dietary supplements melanoidin 50mg/kg/d and fed western diet;
Medium dose group (MMLD), diet supplemented with melanoidin 100mg/kg/d and fed western diet;
High dose group (HMLD) dietary supplement melanoidin 200mg/kg/d and feeding western diet feed dosing regimen: the stomach was irrigated 1 time a day for 4 weeks.
The blank control group (NC) and the model control group (RM) are respectively infused with equal doses of physiological saline;
All mice were free to drink diet during the experiment. Mice were weighed periodically. After 4 weeks of diet intervention, the diet is broken for 12 hours, the eyes are plucked for blood taking, the eyes are sacrificed, the diet is dissected, and all tissues are obtained and stored in a refrigerator at the temperature of minus 80 ℃.
All experiments were performed after at least 3 independent experiments, and data were expressed as mean ± standard deviation. Analysis of data single factor analysis of variance was performed using SPSS21.0 software, followed by Duncan test, the results are shown below.
1. Influence of purified melanoidin on body weight and food intake in red-meat induced atherosclerosis mice
Figures 2A and B show that there was a significant difference (p < 0.05) between all experimental groups in terms of weight gain in mice. After 4 weeks of feeding, RM group body weight increased significantly (p < 0.05) compared to NC group mice. The weight gain was significantly reduced in LMLD and HMLD groups compared to RM group mice (p < 0.05). Their weight gain was 4.3.+ -. 0.42g and 1.9.+ -. 0.67g, respectively. This suggests that dietary supplementation with melanoidins may inhibit weight gain. Dietary supplementation with melanoidins inhibited food intake (p < 0.05) compared to RM group, the differences being statistically significant. This is because melanoidins have a similar function to dietary fibers, which can increase satiety and reduce food intake by intestinal-brain appetite regulators. This regulation of body weight and food intake may involve intestinal microbiota-intestinal-brain regulation mechanisms. Melanoidins prevent weight gain by altering the intestinal microbiota and intestinal barrier that affect food intake.
2. Effect of purified melanoidin on blood lipid levels in red-meat induced atherosclerosis mice
By measuring serum indicators, the data of fig. 3 shows that the TC, TG, LDLC level of the RM group is significantly increased (p < 0.05) compared to the NC group, while the serum HDLC level of the RM group is significantly decreased (p < 0.05). Elevated levels of TG, TC and LDLC indicate an increased risk of atherosclerotic disease. This demonstrates that long-term consumption of red meat can lead to the development of atherosclerotic disease. The levels of TG, TC and LDLC in diet-supplemented melanoidin mice decreased in a dose-dependent manner, with HMLD mice having 46.04%, 29.71% and 39.39% respectively. The results indicate that dietary supplementation with melanoidin has a significant inhibitory effect on the increase in TG, TC and LDLC levels (p < 0.05).
3. Effect of purified melanoidin on red meat-induced atherosclerosis mice TMA, TMAO and TMA lyase activity
As shown in fig. 4, TMA and TMAO concentrations in plasma were measured in this study, and TMA and TMAO levels were significantly elevated in the RM group (p < 0.05) compared to the NC group. TMA and TMAO levels decreased after dietary supplementation with melanoidin. TMA and TMAO levels were reduced by 21.24% and 50.02% respectively for HMLD groups compared to RM groups. Thus, supplementation of melanoidins in diets can inhibit choline-induced TMA and TMAO formation. To further investigate how melanoidins regulate the mechanism of TMAO levels. The activity of the lyase of the intestinal microorganism was investigated. Because choline in red meat is metabolized to TMA mainly by TMA lyase in the intestinal microorganisms, TMAO is produced by oxidation in the liver (FMOs). Therefore, the size of TMA lyase activity also affects the amount of TMAO produced, and is also an effective target for the action of drugs for arteriosclerosis. Therefore, the level of TMA lyase activity was further examined. As shown in fig. 4, the lyase activity of the dietary supplement melanoidin group was decreased. Group HMLD showed a significant decrease in lyase activity (P < 0.05) compared to group RM, indicating that dietary supplementation of melanoidin had an inhibitory effect on TMA lyase activity, thereby reducing TMA production to alleviate the occurrence of arteriosclerotic disease. In summary, melanoidins alleviate the risk of developing arteriosclerotic diseases by inhibiting TMA lyase activity and thereby reducing TMAO production.
5. Effect of purified melanoidin on red-meat induced atherosclerosis mouse intestinal epithelial cell adenylate activated protein kinase (AMPK)
Example 1 effect on AMPK kinase levels: in recent years, AMP-activated protein kinase (AMPK) has been found to be an important cell energy sensor for maintaining energy homeostasis. When activated by a decrease in energy status, it promotes ATP production by increasing the activity or expression of proteins involved in catabolism, and protects ATP from consumption by shutting down the biosynthetic pathway. To investigate whether dietary supplementation of melanoidins would modulate the expression of AMP-activated protein kinase (AMPK) in intestinal epithelium, the catalytic activity of mouse intestinal epithelium AMPK was determined. As shown in fig. 5, AMPK levels were significantly reduced in the RM group compared to the NC group, while AMPK levels were significantly increased after the diet supplementation with melanoidin. This suggests that dietary supplementation of melanoidins may activate AMPK, thereby shutting down the ATP consumption pathway. ATP is necessary for lyase activity during TMA metabolism. Thus, dietary supplementation of melanoidins can activate the AMPK pathway, reducing ATP consumption processes, thereby reducing TMA lyase activity, and thus inhibiting TMA and TMAO production.
6. Influence of purified melanoidin on pH and short chain fatty acid levels in red-meat induced atherosclerosis mouse feces
Since in fig. 6A, the mouse fecal pH level was significantly reduced after dietary supplementation with melanoidins, whereas the fecal pH level of the RM group was significantly increased over NC group. Research shows that the acidic environment can inhibit the proliferation of harmful bacteria in intestinal tracts and improve the intestinal tract environment. In addition, short Chain Fatty Acids (SCFAs) have an immunomodulatory function in various tissues and organs and play an important role in inhibiting the occurrence of atherosclerosis. Fig. 6B shows that acetic acid, propionic acid, and butyric acid are the major short chain fatty acids of the present study. After melanoidin supplementation in the diet, SCFAs concentration increased significantly (p < 0.05). The acetic acid concentration of the HMLD diet mice reached 44.90.+ -. 2.53. Mu. Mol/g compared to the RM group acetic acid concentration (20.88.+ -. 0.84. Mu. Mol/g). The concentrations of propionic acid and butyric acid also have the same trend. Therefore, the dietary supplement of melanoidin can increase the content of SCFAs, improve the acidic environment of intestinal tracts, possibly regulate intestinal immunity and protect intestinal barriers.
Example 2
The embodiment is used for exploring the influence of the addition amount of the activating agent on the separation and purification effects of the melanoidin in the separation and purification process of the melanoidin, specifically, the addition amount of the activating agent 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide in the step 2) of the embodiment 1 is adjusted to be 60% -100%, the rest of the steps refer to the embodiment 1, the melanoidin is obtained through separation and purification, the yield of the ES-IA is calculated, and the purification effects of the melanoidin are analyzed. The results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the amount of the activator has a remarkable influence on the separation and purification effects of the melanoidin, the yield of the ES-IA gradually increases along with the increase of the content of the activator, and when the content of the activator is too low, the number of the-COOH participating in the chelating of the IA in the solution is lower, and the obtained product ES-IA is also less, so that the yield of the melanoidin obtained by separation and purification is also lower; when the content of the activator is too high, the content of residual-COOH in the solution becomes low, resulting in a change in pH sensitivity, and at the same pH, the precipitation amount of the product decreases, the yield of ES-IA decreases, and thus the yield of melanoidin obtained by separation and purification also decreases.
Example 3
The present example was used to investigate the effect of the amount of Fe + on the separation and purification effect of melanoidin in the separation and purification process of melanoidin, specifically, the amount of FeCl 3 added in step 3) of example 1 was adjusted to 500 to 1500 μl, the chelation amount was calculated, the remaining steps were performed according to the process of example 1, and melanoidin was obtained by separation and purification, and the results are shown in table 2.
TABLE 2
As can be seen from Table 2, the addition amount of FeCl 3 solution has a remarkable influence on the separation and purification effect of melanoidin, and when the addition amount of Fe 3+ in the solution is too low, the chelating amount of Fe 3+ on ES-IA is low, and melanoidin cannot be sufficiently adsorbed, so that the yield of melanoidin is low, and as the addition amount of Fe 3+ is increased, the chelating rate is increased, and more melanoidin is further separated and purified. When the addition amount is 1250. Mu.L, the ES-IA-Fe 3+ chelates Fe 3+ ions to the maximum chelating amount. And the metal ions are continuously added, the ES-IA-Fe 3+ does not continuously chelate the metal ions, and more unbound metal ions remain in the solution to influence the subsequent experiments.
Example 4
The present example was used to investigate the influence of the oscillation time of the chelate melanoidin on the melanoidin isolation and purification effect in the melanoidin isolation and purification process, specifically, the oscillation time in step 4) of example 1 was adjusted to be 30 to 70min, the other steps were performed by referring to example 1, and melanoidin was obtained by isolation and purification, and the yield was calculated, and the results are shown in table 3.
TABLE 3 Table 3
As can be seen from Table 3, the shaking time has a significant effect on the separation and purification effects of melanoidin, and when the shaking time is too low in a solution, ES-IA-Fe 3+ cannot fully adsorb melanoidin, the yield is low, and as the shaking time increases, the yield of melanoidin obtained by separation and purification gradually increases, and after 60 minutes of reaction, the adsorption MLD of ES-IA-Fe 3+ basically reaches saturation.
Example 5
The present example was used to investigate the effect of the type of chelate metal ion on the separation and purification effect of melanoidin in the separation and purification process of melanoidin, specifically, the metal ion in step 3) of example 1 was adjusted to be Fe 3+、Ni2+、Cu2+、Fe2+、Co2+、Mn2+, the rest of the steps were described in example 1, and melanoidin was obtained by separation and purification, and the yield was calculated, and the results are shown in table 4.
TABLE 4 Table 4
After the ES-IA chelates Fe 3+、Ni2+、Cu2+ separately, the pH sensitive metal chelate precipitant obtained separated and purified melanoidin, it can be seen from table 4 that the type of chelate metal ion has a significant effect on the separation and purification effect of melanoidin, wherein Fe 3+ ion has the best separation and purification effect on melanoidin and has a higher yield, ni 2+、Cu2+, which is unstable in combination with melanoidin, separated and purified melanoidin, has a too low yield, and in addition, it was found that the combination property of Fe 2+、Co2+、Mn2+ was significantly impaired compared with Fe 3+ and the combination was unstable during the pre-experiment, so that no further experiment was required, and therefore for melanoidin, it was necessary to select Fe 3+ ion with a more stable combination and a higher yield for separation and purification.
Comparative example 2
Comparative example without pH sensitive Polymer added: preparing 0.56mgCMLD to 20mL of solution, adding 1.25mL of FeCl 3 solution (60 mmol/L), regulating the pH to 4.0, oscillating for 60min in a water bath at room temperature of 100r/min, combining Fe 3+ with melanoidin, centrifuging, removing unbound Fe 3+ ions and melanoidin in the supernatant, and precipitating to obtain Fe 3+ -MLD;
Adding a small amount of NaOH solution (c=2mol/L) into the obtained precipitate, re-dissolving the precipitate, continuously adding 0.1g of disodium ethylenediamine tetraacetate (EDTA-2 Na) and 200mL of distilled water, and reacting for 3h under water bath stirring at 50 ℃; after cooling to room temperature, the solution was dialyzed for 96 hours with a dialysis bag having a cutoff of 1000Da, freeze-dried, and purified Melanoidin (MLD) was further obtained, and the yield was calculated.
TABLE 5
As can be seen from Table 5, fe 3+ ions directly react with melanoidin, and metal ions and melanoidin are lost during centrifugation, so that the yield of the obtained melanoidin is low, and Fe 3+ ions are stably combined on ES-IA after the pH sensitive polymer is added, so that the melanoidin can be fully adsorbed, and the yield of the melanoidin is obviously improved.
In conclusion, the invention takes glucose and glycine as raw materials to carry out Maillard reaction to generate crude melanoidin, the crude melanoidin is separated and purified, the metal chelating ability of the melanoidin and the pH sensitivity of pH sensitive polymers are utilized, the pH sensitive metal chelating agent is prepared to chelate with the crude melanoidin, and then the separation and purification of the melanoidin can be realized by simply adjusting the pH, so that the molecular weight of the obtained purified melanoidin is lower than that of the crude melanoidin, and the molecular weight of the purified melanoidin is 6000Da. In microstructure, large irregular polygonal blocks and a small amount of granular structures in the coarse melanoidin are also converted into more small sheet-shaped and block-shaped structures.
The melanoidin product obtained by the invention can obviously improve the blood lipid level and the levels of TMA and TMAO in blood plasma by long-term administration, can activate the AMPK way to inhibit the activity of TMA lyase and improve the acidic environment of intestinal tracts, thereby inhibiting the proliferation of harmful bacteria in the intestinal tracts, and has the effect of preventing atherosclerosis diseases by regulating the blood lipid level and the levels of TMA and TMAO together. Therefore, the melanoidin purified by the pH sensitive metal chelating precipitant is taken as a functional component to inhibit choline metabolism and slow down the risk of arteriosclerosis, thus providing a new strategy for preventing the arteriosclerosis.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (10)
1. A method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant is characterized by comprising the following steps of: comprising the steps of (a) a step of,
Preparing crude melanoidin from glucose and glycine by Maillard reaction;
Adding an activating agent into the pH sensitive polymer solution for activation, then adding a metal chelating agent solution, and performing coupling reaction to obtain a pH sensitive precipitant solution;
Adding the FeCl 3 solution into the pH sensitive precipitant solution, and chelating to obtain a pH sensitive metal chelating precipitant;
Mixing the crude melanoidin with a pH sensitive metal chelate precipitant, regulating the pH to 4.8-5.2, centrifuging, removing unbound melanoidin from the supernatant, and precipitating to form a compound of the pH sensitive metal chelate precipitant and the melanoidin;
adding the compound into NaOH solution, precipitating, redissolving, continuously adding disodium ethylenediamine tetraacetate solution for water bath stirring reaction, cooling to room temperature after the reaction, regulating the pH of the solution to 4.8-5.2 again, centrifuging, taking supernatant, dialyzing, freeze-drying, and further post-treating to obtain purified melanoidin.
2. The method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant according to claim 1 wherein: the pH sensitive polymer solution is prepared by dissolving pH sensitive polymer Eudragit S-100 in NaOH solution, the concentration is 1%, and the pH is regulated to 6.5-7.5.
3. The method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant according to claim 1 wherein: the metal chelating agent solution is 4- (imidazol-1-yl) aniline solution with the concentration of 10mmol/L, wherein 11mL of the metal chelating agent solution is added to every 1gpH sensitive polymer.
4. The method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant according to claim 1 wherein: the activator is 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, and the added pH sensitive polymer has the molar mass of 70-90%.
5. The method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant according to claim 1 wherein: the concentration of the FeCl 3 solution is 60mmol/L, and the addition amount is 1.25mL.
6. The method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant according to claim 1 wherein: the crude melanoidin is mixed with a pH sensitive metal chelating precipitant, wherein 0.56mg of crude melanoidin is added per 1gEudragit S-100 of the pH sensitive metal chelating precipitant calculated by Eudragit S-100, the rotating speed is 100r/min, and the oscillation time is 60-80 min.
7. The method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant according to claim 1 wherein: the addition amount of the disodium ethylenediamine tetraacetate is 1.5-4 times of the molar amount of Fe 3+ in the FeCl 3 solution.
8. The method for separating and purifying melanoidin based on a pH sensitive metal chelating precipitant according to claim 1 wherein: the temperature of the water bath stirring of the compound added into the disodium ethylenediamine tetraacetate solution is 50 ℃, and the stirring time is 3-4 hours.
9. The melanoidin obtained by the separation and purification of any one of claims 1 to 8.
10. Use of melanoidin according to claim 9 as a functional ingredient for the amelioration or prophylaxis of atherosclerosis.
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