A method for separating and purifying melanolike substance based on pH-sensitive metal chelating precipitator, melanolike substance and application thereof

Abstract

The application discloses a method for separating and purifying melanolike substance based on a pH-sensitive metal chelating precipitant, the melanolike substance and application thereof, and the melanolike substance is obtained by purifying melanolike substance synthesized by Maillard reaction of glucose and glycine, and the purification method comprises the following steps: utilizing pH sensitivity of a polymer, synthesizing a pH-sensitive metal chelating precipitant, and combining metal chelating property of the melanolike substance to provide a method for separating and purifying crude melanolike substance by simply adjusting pH. The melanolike substance obtained has relatively simple structure and stable property, is beneficial to subsequent research on melanolike substance structure, and has a lower molecular weight of 6000 Da compared with the crude melanolike substance. In the microstructure, the large irregular polygon block and a small amount of granular structure in the crude melanolike substance are also converted into more small block flaky and block structures. Through further in-vivo animal experiments, it is shown that the MLD can be used as a functional component to inhibit choline metabolism and thus improve arteriosclerosis diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of melanoidin separation and purification, and particularly relates to a method for separating and purifying melanoidin based on a pH-sensitive metal chelating precipitant, melanoidin and application thereof. BACKGROUND

[0002] Melanoidin is a kind of high molecular weight Maillard reaction product with complex structure and different polymerization degrees, which is produced by the reaction of carbohydrates and nitrogen-containing compounds with free amino groups. It is widely present in the process of food processing and storage, such as beer, bread, coffee, vinegar and other foods, and helps to form the flavor and color of foods, and has various functional activities such as metal ion chelation, antioxidant, antibacterial, antihypertensive and antitumor, among which the chelation of melanoidin plays an important role in the combination with metals.

[0003] Metal chelation affinity purification is a method for separating and purifying target products by using the affinity between transition metal ions and electron donor groups on the target products. The metal affinity purification system generally has three components: medium (or carrier), metal ion chelator and metal ion. It is widely used in the separation and purification of various proteins.

[0004] Intelligent polymer is a kind of polymer that can perceive and accept information from external environment and make sensitive response. The environmental triggers behind these transformations can be temperature, pH value, electric field, magnetic field, increase of ionic strength, presence of certain metabolic chemicals and formation of polycation-polyanion complex. According to the changes of environment, the intelligent polymer can automatically change its shape, solubility and surface properties, etc., and undergo corresponding reversible changes. Among them, pH-sensitive polymer is a kind of intelligent polymer with reversible precipitation-dissolution characteristics, which is applied in drug controlled release, material separation, immune analysis and other fields, and has attracted more and more attention in recent years.

[0005] In daily life, people often eat foods rich in choline, such as red meat (pork, beef, fish), which can increase the risk of atherosclerosis. Some documents have reported that inhibiting the choline metabolic pathway in vivo can prevent the occurrence of atherosclerosis. Therefore, it is very important to develop an active substance that can inhibit the choline metabolic pathway. There are also some studies on the mechanism of melanoidin in preventing atherosclerosis, for example, Verzeloni E et al. found that the melanoidin in balsamic vinegar can bind with hemoglobin to prevent its absorption, and hemoglobin can act as a catalyst for oxidative damage and cause various cancers and cardiovascular diseases. These outstanding activities make melanoidin a functional ingredient for preventing atherosclerosis possible.

[0006] However, the extraction and purification of melanoidins has been a technical problem. The traditional methods for extracting melanoidins include organic solvent extraction, size exclusion chromatography, water extraction, precipitation, macroporous resin adsorption, and enzymatic hydrolysis. However, these traditional extraction methods have problems such as long reaction time, insufficient extraction, and unsatisfactory product yield, which are not conducive to large-scale industrial production. Although macroporous resin adsorption has advantages such as large adsorption capacity, fast adsorption speed, short time, and good physical and chemical stability, it also has certain limitations. For example, after repeated use of the resin, non-adsorbed components or impurities may remain on the surface and inside of the resin, causing the column to become darker in color and the column efficiency to decrease. Moreover, the resin particles may break after being squeezed tightly in the column bed for multiple times, resulting in a short service life.

[0007] Therefore, it is of great significance to provide an effective method for separating and purifying melanoidins for subsequent research on the structure and functional activity of melanoidins in preventing atherosclerotic diseases. SUMMARY

[0008] This section is intended to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, the abstract, and the title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0009] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0010] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art and provide a method for separating and purifying melanoidins based on a pH-sensitive metal chelate precipitant.

[0011] To solve the above technical problems, the present application provides the following technical solutions: comprising,

[0012] Crude melanoidins are prepared by a Maillard reaction using glucose and glycine as raw materials;

[0013] An activator is added to a pH-sensitive polymer solution to activate it, and then a metal chelate solution is added to obtain a pH-sensitive precipitant solution through a coupling reaction;

[0014] FeCl3 solution is added to the pH-sensitive precipitant solution to obtain a pH-sensitive metal chelate precipitant through chelation;

[0015] The crude melanoidins are mixed with the pH-sensitive metal chelate precipitant, the pH is adjusted to 4.8-5.2, and centrifugation is performed to remove unbound melanoidins in the supernatant, and the precipitate is a complex of the pH-sensitive metal chelate precipitant and melanoidins;

[0016] The complex is added into NaOH solution, precipitated and dissolved, and then ethylenediamine tetraacetic acid disodium solution is continuously added to carry out water bath stirring reaction. After reaction, the solution is cooled to room temperature, and then the pH value of the solution is adjusted to 4.8-5.2 and centrifuged. The supernatant is dialyzed, freeze-dried, and further treated to obtain the purified melanoidin.

[0017] As a preferred scheme of the method for separating and purifying melanoidin based on the pH-sensitive metal chelate precipitant according to the application, the pH-sensitive polymer solution is prepared by dissolving pH-sensitive polymer Eudragit S-100 in NaOH solution, and the concentration is 1%, and the pH value is adjusted to 6.5-7.5.

[0018] As a preferred scheme of the method for separating and purifying melanoidin based on the pH-sensitive metal chelate precipitant according to the application, the metal chelate solution is 4-(imidazol-1-yl) aniline solution, and the concentration is 10 mmol / L. In the solution, 11 mL of metal chelate solution is added for every 1 g of pH-sensitive polymer.

[0019] When the amount of metal chelate solution is too low, the chelation is not complete, and the yield of purified melanoidin decreases. When the amount of metal chelate solution is too high, the solution contains excess unchelated reagents, which affects subsequent experiments.

[0020] As a preferred scheme of the method for separating and purifying melanoidin based on the pH-sensitive metal chelate precipitant according to the application, the activator is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the molar mass of the added pH-sensitive polymer is 70-90%.

[0021] As a preferred scheme of the method for separating and purifying melanoidin based on the pH-sensitive metal chelate precipitant according to the application, the concentration of the FeCl3 solution is 60 mmol / L, and the amount of addition is 1.25 mL.

[0022] When the amount of FeCl3 solution is too low, the chelation is not complete, and the yield of purified melanoidin decreases. When the amount of metal chelate solution is too high, the solution contains excess unchelated reagents, which affects subsequent experiments.

[0023] As a preferred scheme of the method for separating and purifying melanoidin based on the pH-sensitive metal chelate precipitant according to the application, the crude melanoidin is mixed with the pH-sensitive metal chelate precipitant. In the pH-sensitive metal chelate precipitant, 0.56 mg of crude melanoidin is added for every 1 g of Eudragit S-100, the rotation speed is 100 r / min, and the shaking time is 60-80 min.

[0024] When the amount of the crude melanoidin is too low, the chelation is incomplete, and the yield of the purified melanoidin is reduced; when the amount of the crude melanoidin is too high, the solution contains excessive unchelated reagents, which affects the subsequent experiment.

[0025] As a preferred scheme of the method for separating and purifying the melanoidin based on the pH-sensitive metal chelate precipitant according to the application, the amount of the ethylenediaminetetraacetic acid disodium salt added is 1.5-4 times the molar amount of FeCl3 in the FeCl3 solution. 3+ 1.5-4 times the molar amount of FeCl3 in the FeCl3 solution.

[0026] As a preferred scheme of the method for separating and purifying the melanoidin based on the pH-sensitive metal chelate precipitant according to the application, the temperature of the water bath stirring when the complex is added to the ethylenediaminetetraacetic acid disodium salt solution is 50℃, and the stirring time is 3-4h.

[0027] Another object of the application is to provide the melanoidin separated and purified based on the pH-sensitive metal chelate precipitant.

[0028] Another object of the application is to provide the application of the melanoidin separated and purified based on the pH-sensitive metal chelate precipitant as a functional ingredient for improving or preventing atherosclerosis.

[0029] The application has the following advantages:

[0030] (1) The application uses glucose and glycine as raw materials to generate the crude melanoidin through the Maillard reaction, and separates and purifies the crude melanoidin, uses the metal chelation capacity of the melanoidin and the pH sensitivity of the pH-sensitive polymer, prepares the pH-sensitive metal chelate precipitant, chelates with the crude melanoidin, and then adjusts the pH to separate and purify the melanoidin.

[0031] (2) The model melanoidin prepared by using a single amino acid and a reducing sugar has a relatively simple melanoidin structure and stable properties, which is conducive to the subsequent research on the melanoidin structure, and the purified melanoidin separated and purified has a lower molecular weight of 6000 Da, and the large irregular polygonal block and a small amount of granular structure in the crude melanoidin are also converted into more small block flaky and block structures.

[0032] (3) The melanoidin product of the application can significantly improve the blood lipid level and the level of TMA and TMAO in the blood plasma through long-term consumption, activate the AMPK pathway to inhibit the activity of TMA cleavage enzyme, and improve the intestinal acid environment to inhibit the proliferation of harmful bacteria in the intestine, which together play a role in preventing atherosclerotic diseases. Therefore, the melanoidin purified by the pH-sensitive metal chelate precipitant as a functional ingredient can inhibit the choline metabolism to slow down the risk of atherosclerotic diseases, which provides a new strategy for preventing atherosclerotic diseases. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0034] Figure 1 This is a comparative structural characterization diagram of the purified melanoidin and crude melanoidin prepared in Example 1 of the present invention.

[0035] Figure 2 The results show the effects of purified melanoidins prepared in Example 1 of this invention on body weight and food intake in mice with red meat-induced atherosclerosis.

[0036] Figure 3 The effect of purified melanoidins prepared for Example 1 of the invention on blood lipid levels in mice with red meat-induced atherosclerosis.

[0037] Figure 4 The effects of purified melanoidins prepared for Example 1 of the invention on trimethylamine (TMA), trimethylamine oxide (TMAO), and trimethylamine lyase activity in red meat-induced atherosclerotic mice.

[0038] Figure 5 The effect of purified melanoidins prepared in Example 1 of the invention on the expression of adenosine monophosphate activated protein kinase (AMPK) in intestinal epithelial cells of red meat-induced atherosclerotic mice.

[0039] Figure 6 The effects of purified melanoidins prepared for Example 1 of the invention on the pH value and short-chain fatty acid (SCFA) levels in the feces of mice with red meat-induced atherosclerosis. Detailed Implementation

[0040] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0041] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0042] Second, the "one embodiment" or "an embodiment" referred to herein means a specific feature, structure, or characteristic under discussion. Each of the various embodiments presented in this specification are not necessarily mutually exclusive, but can be selectively implemented in various embodiments of the application.

[0043] The raw materials used in the present application are commercially available in the art.

[0044] Example 1

[0045] The present embodiment provides a method for separating and purifying melanoidins, specifically:

[0046] 1) Preparation of crude melanoidins: 9.0080 g of D-anhydrous glucose and 3.7535 g of glycine were dissolved in distilled water, and the volume was adjusted to 100 mL in a volumetric flask. After freeze-drying to a constant weight on a flat plate, it was placed in a 125°C oven for a Maillard reaction for 2 h. After cooling to room temperature, the reaction was ground into fine powder, dissolved in distilled water, and filtered with No. 1 filter paper. The filtrate was dialyzed for 96 h using a dialysis bag with a cutoff of 1000 Da, and freeze-dried to obtain crude melanoidins (CMLD);

[0047] 2) Preparation of pH-sensitive precipitant: Take pH-sensitive polymer Eudragit S-100 (ES-100), weigh 1 g and dissolve in 2 mol / L NaOH solution. Adjust the pH to about 7.2 with 3 mol / L HCl solution. Add 0.4330 g of activator 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and stir at room temperature for 10 min for activation reaction. The molar ratio of the added activator 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to the pH-sensitive polymer Eudragit S-100 is 80%. Then add 11 mL of metal chelator 4-(imidazol-1-yl) aniline solution (IA, c = 10 mmol / L), and stir at 70°C water bath for 2 h to obtain the pH-sensitive precipitant (ES-IA) coupled with IA;

[0048] 3) Chelate metal ions: Add 1.25 mL of FeCl3 solution (60 mmol / L) to the ES-IA solution, stir at room temperature for 1 h to chelate Fe 3+ ions, and obtain the pH-sensitive metal chelate precipitant (ES-IA-Fe 3+ );

[0049] 4) Chelate melanoidins: Add 0.56 mg of CMLD to the pH-sensitive metal chelate precipitant solution obtained in step 3), and shake at 100 r / min in a water bath at room temperature for 60 min to obtain the pH-sensitive metal chelate precipitant (ES-IA-Fe 3+ES-IA-Fe 3+ - MLD;

[0050] 5) Elution of purified melanoidin: The precipitate obtained in step 4) was dissolved by adding a small amount of NaOH solution (c = 2 mol / L), and then 0.1 g of disodium ethylenediaminetetraacetate (EDTA-2Na) and 200 mL of distilled water were added. The solution was stirred in a water bath at 50°C for 3 h. After cooling to room temperature, the solution was centrifuged after adjusting the pH to 5.0. The precipitate was ES-IA, and the supernatant was EDTA-Fe. 3+ The supernatant was dialyzed against distilled water for 96 h using a dialysis bag with a molecular weight cut-off of 1000 Da, and then freeze-dried to obtain purified melanoidin (MLD).

[0051] Figure 1 A is the molecular weight distribution graph of purified melanoidin MLD and crude melanoidin CMLD prepared in this example. As can be seen, the liquid chromatogram of purified melanoidin MLD and crude melanoidin CMLD both showed single peaks. The molecular weight of crude melanoidin was 13.8 kDa, while the molecular weight of purified melanoidin was 6.0 kDa, which was reduced. In the model system, although dialysis removed unreacted free glucose and amino acids, the carbohydrate chains in the structure of crude melanoidin were still adsorbed on the melanoidin through non-covalent interactions, forming polymers and resulting in a higher molecular weight. After purification by the pH-sensitive metal chelate precipitant, the carbohydrate chains were degraded, so the molecular weight of purified melanoidin MLD was smaller.

[0052] Figure 1 B is the Fourier infrared spectrum of purified melanoidin MLD and crude melanoidin CMLD. As can be seen, the functional groups of melanoidin changed significantly after purification with the pH-sensitive metal chelate precipitant. Purified melanoidin had an additional absorption band at 1720 cm -1 -1, which was attributed to -C=O stretching; the absorption band at 1605 cm -1 -1 was attributed to the coupling of -C=O stretching and -NH deformation (amide I and amide II bands), which was enhanced in the spectrum of purified melanoidin compared to crude melanoidin; the absorption bands at 1318 cm -1 -1, 1265 cm -1 -1 were attributed to -C-N stretching vibration (amide III band), indicating that the purified melanoidin was rich in -C=O, -NH, and -C-N groups. In addition, the spectrum of crude melanoidin had an absorption band at 1076 cm -1An absorption band due to -C-O stretching vibration was observed in the crude melanoidin, but not in the purified melanoidin, which further indicated that the polymers produced by Maillard reaction were removed from the crude melanoidin by the pH-sensitive metal chelate precipitation.

[0053] Figure 1 C-F are scanning electron micrographs of the crude melanoidin CMLD (C and D) and the purified melanoidin MLD (E and F), respectively. It can be seen that large irregular polygonal block and a small amount of granular structure were observed in the crude melanoidin, and more small block flaky, block structure were contained in the purified melanoidin, which indicated that the microstructure changed after the crude melanoidin was purified.

[0054] Animal experiment of the purified melanoidin in treating atherosclerosis

[0055] Experimental animals: 72 SPF clean grade C57BL / 6J male mice of about 6 weeks old, weighing about 20 g, were first adaptively fed in an environment of temperature 25±2℃, humidity 50-60%, light and darkness for 12 h each for one week, and all animal experimental operations were in accordance with the Chinese and international animal protection guidelines.

[0056] Experimental drugs: 3,3-dimethylbutanol (DMB, 97%); choline (90%); trimethylamine hydrochloride (purity greater than 98%); total cholesterol (TC) kit; triglyceride (TG) kit, low-density lipoprotein cholesterol (LDLC) kit, high-density lipoprotein cholesterol (HDLC) kit (Nanjing Jiancheng Biological Engineering Institute); TGL-1650 high-speed refrigerated centrifuge; JD1000-2 electronic analytical balance; TU-1810 ultraviolet spectrophotometer.

[0057] Construction of atherosclerotic mouse model and drug administration:

[0058] 12 of the 72 C57BL / 6J mice were randomly selected as a blank control group (NC) and were given ordinary feed, and the rest of the mice were given western diet feed.

[0059] The ordinary feed includes basic feed 73%, sucrose 5%, oil 7%, and starch 15%;

[0060] The western diet feed contains basic feed 53%, sucrose 5%, oil 7%, starch 15%, and red meat 20%.

[0061] The 72 mice were randomly divided into 6 groups (n=12), and the groups were:

[0062] Normal group (NC), fed with ordinary feed;

[0063] Positive control group (DMB), 3,3-dimethylbutanol (1%, v / v) (free drinking water) and fed western diet chow;

[0064] Model control group (RM), fed western diet chow;

[0065] Low-dose group (LMLD), dietary supplementation of melanoidin 50 mg / kg / d and fed western diet chow;

[0066] Medium-dose group (MMLD), dietary supplementation of melanoidin 100 mg / kg / d and fed western diet chow;

[0067] High-dose group (HMLD), dietary supplementation of melanoidin 200 mg / kg / d and fed western diet chow Administration method: once a day by gavage, continuous gavage for 4 weeks.

[0068] Blank control group (NC) and model control group (RM) were gavaged with the same dose of normal saline, respectively;

[0069] All mice were free to drink water and diet during the experiment. The weight of the mice was weighed regularly every week. After 4 weeks of dietary intervention, the mice were fasted for 12 h, and then blood was taken from the eye and the mice were sacrificed, dissected, and the tissues were stored at -80℃.

[0070] All experiments were repeated at least three times independently, and the data were expressed as mean ± standard deviation. Data analysis was performed using SPSS21.0 software for one-way ANOVA, followed by Duncan's test, and the results are shown below.

[0071] 1. The effect of purified melanoidin on the body weight and food intake of mice induced by red meat

[0072] Figure 2 As shown in A and B, there were significant differences between all experimental groups in terms of body weight gain (p<0.05). After 4 weeks of feeding, the body weight of the RM group increased significantly compared with the NC group (p<0.05). Compared with the RM group, the body weight of the LMLD and HMLD groups increased significantly (p<0.05). Their body weight gain was 4.3±0.42 g and 1.9±0.67 g, respectively. This indicates that dietary supplementation of melanoidin can inhibit body weight gain. Compared with the RM group, dietary supplementation of melanoidin can inhibit food intake (p<0.05), and the difference is statistically significant. This is because melanoidin has similar functions to dietary fiber, which can increase satiety and reduce food intake through the gut-brain appetite regulator. This regulation of body weight and food intake may involve the gut microbiota-gut-brain regulation mechanism. Melanoidin prevents weight gain by altering the gut microbiota and intestinal barrier that affects food intake.

[0073] 2. Effect of purified melanoidin on blood lipid levels in red meat-induced atherosclerotic mice

[0074] By determining the serum indicators, Figure 3 The data showed that the levels of TC, TG, and LDLC in the RM group were significantly higher than those in the NC group (p < 0.05), while the serum HDLC level in the RM group was significantly lower (p < 0.05). The increased levels of TG, TC, and LDLC indicated an increased risk of atherosclerotic disease. This proved that long-term consumption of red meat could lead to the occurrence of atherosclerotic disease. The levels of TG, TC, and LDLC in the mice supplemented with melanoidin showed a dose-dependent decrease, and the levels in the HMLD group were reduced by 46.04%, 29.71%, and 39.39%, respectively. The results showed that dietary supplementation with melanoidin had a significant inhibitory effect on the increase in TG, TC, and LDLC levels (p < 0.05).

[0075] 3. Effect of purified melanoidin on TMA, TMAO, and TMA lyase activity in red meat-induced atherosclerotic mice

[0076] As Figure 4 shown, the concentrations of TMA and TMAO in plasma were determined in this study. Compared with the NC group, the levels of TMA and TMAO in the RM group were significantly increased (p < 0.05). After dietary supplementation with melanoidin, the levels of TMA and TMAO decreased. Compared with the RM group, the levels of TMA and TMAO in the HMLD group were reduced by 21.24% and 50.02%, respectively. Therefore, supplementation of melanoidin in the diet could inhibit the formation of TMA and TMAO induced by choline. To further investigate the mechanism by which melanoidin regulates TMAO levels, the lyase activity of intestinal microorganisms was explored. Because choline in red meat is mainly metabolized to TMA by TMA lyase in intestinal microorganisms, and then oxidized to TMAO by (FMOs) in the liver, the size of TMA lyase activity also affects the amount of TMAO generated, which is also an effective target for drugs acting on atherosclerotic disease. Therefore, the level of TMA lyase activity was further explored. As Figure 4 shown, the lyase activity of the melanoidin-supplemented group showed a downward trend. Compared with the RM group, the lyase activity of the HMLD group was significantly reduced (P < 0.05), indicating that dietary supplementation with melanoidin had an inhibitory effect on TMA lyase activity, which in turn reduced the production of TMA to alleviate the occurrence of atherosclerotic disease. In summary, melanoidin reduces the generation of TMAO by inhibiting TMA lyase activity, thereby reducing the risk of atherosclerotic disease.

[0077] 5. Effect of purified melanoidin on intestinal epithelial cell adenosine monophosphate-activated protein kinase (AMPK) in red meat-induced atherosclerotic mice

[0078] Example 1 Effect on AMPK kinase level: In recent years, studies have found that AMP-activated protein kinase (AMPK) is an important cellular energy sensor that maintains 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 being consumed by turning off biosynthetic pathways. To study whether dietary supplementation of melanoidin can regulate the expression of AMP-activated protein kinase (AMPK) in the intestinal epithelium, the catalytic activity of mouse intestinal epithelial AMPK was determined. As shown in Figure 5

[0079] 6. Effect of purified melanoidin on the pH and short-chain fatty acid levels of red meat-induced atherosclerotic mouse feces

[0080] As Figure 6 A, the pH level of mouse feces was significantly reduced after dietary supplementation of melanoidin, while the pH level of feces in the RM group was significantly higher than that in the NC group. Studies have shown that an acidic environment can inhibit the proliferation of harmful bacteria in the gut and improve the intestinal environment. In addition, short-chain fatty acids (SCFAs) have immunomodulatory functions in various tissues and organs and play an important role in inhibiting the occurrence of atherosclerosis. Figure 6 B shows that acetic acid, propionic acid and butyric acid are the main short-chain fatty acids in this study. After supplementing melanoidin in the diet, the concentration of SCFAs increased significantly (p<0.05). Compared with the acetic acid concentration of the RM group (20.88±0.84 μmol / g), the acetic acid concentration of the HMLD group reached 44.90±2.53 μmol / g. The concentrations of propionic acid and butyric acid also showed the same trend. Therefore, the addition of melanoidin in the diet can increase the content of SCFAs, improve the acidic environment of the gut, and possibly regulate the intestinal immune system and protect the intestinal barrier.

[0081] Example 2

[0082] This example is to investigate the effect of the amount of activator added during the separation and purification of melanoidin on the separation and purification of melanoidin. Specifically, the amount of activator 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide added in step 2) of Example 1 is adjusted to 60% to 100%, and the remaining steps are based on Example 1. Melanoidin is separated and purified, the ES-IA yield is calculated, and the purification effect of melanoidin is analyzed. The results are shown in Table 1. ​

[0083] Table 1

[0084]

[0085] As shown in Table 1, the amount of activator has a significant impact on the separation and purification of melanoidins. As the activator content increases, the yield of ES-IA gradually increases. When the activator content is too low, the number of -COOH molecules participating in IA chelation in the solution is low, resulting in less ES-IA product and thus a lower yield of melanoidins obtained through separation and purification. When the activator content is too high, the remaining -COOH content in the solution also decreases, leading to changes in its pH sensitivity. At the same pH, the amount of product precipitation decreases, the yield of ES-IA decreases, and thus the yield of melanoidins obtained through separation and purification also decreases.

[0086] Example 3

[0087] This embodiment is used to investigate the Fe process during the separation and purification of melanoidins. + The effect of dosage on the separation and purification of melanoidins was specifically investigated by adjusting the amount of FeCl3 solution added in step 3) of Example 1 to 500-1500 μL, calculating the chelation amount, and referring to Example 1 for the remaining steps. The melanoidins were then separated and purified, and the results are shown in Table 2.

[0088] Table 2

[0089]

[0090]

[0091] As shown in Table 2, the amount of FeCl3 solution added has a significant impact on the separation and purification of melanoidins. In the solution, Fe... 3+ When the addition amount is too low, Fe on ES-IA 3+ The chelation amount is low, and the adsorption of melanoidins is insufficient, resulting in a low yield of melanoidins. With the decrease of Fe... 3+ Increasing the dosage led to a higher chelation rate, resulting in the further separation and purification of more melanoidins. At a dosage of 1250 μL, ES-IA-Fe... 3+ Chelating Fe 3+ The ions have reached maximum chelation. Continue adding metal ions, ES-IA-Fe. 3+ The chelation of metal ions ceases, leaving more unbound metal ions in the solution, which affects subsequent experiments.

[0092] Example 4

[0093] This example is to explore the effect of the shock time of chelated melanoidin on the separation and purification of melanoidin. Specifically, the shock time in step 4) of Example 1 is adjusted to 30-70 min, and the rest of the process is referred to Example 1. The melanoidin is separated and purified, and the yield is calculated. The results are shown in Table 3.

[0094] Table 3

[0095]

[0096] As can be seen from Table 3, the shock time has a significant effect on the separation and purification of melanoidin. In the solution, when the shock time is too low, ES-IA-Fe 3+ cannot fully adsorb melanoidin, and the yield is low. As the shock time increases, the yield of melanoidin obtained by separation and purification gradually increases. After 60 min of reaction, ES-IA-Fe 3+ adsorbs MLD to saturation.

[0097] Example 5

[0098] This example is to explore the effect of the type of chelated metal ions on the separation and purification of melanoidin. Specifically, the metal ions in step 3) of Example 1 are adjusted to Fe 3+ , Ni 2+ , Cu 2+ , Fe 2+ , Co 2+ , and Mn 2+ , respectively. The rest of the process is referred to Example 1. The melanoidin is separated and purified, and the yield is calculated. The results are shown in Table 4.

[0099] Table 4

[0100]

[0101] After ES-IA chelates Fe 3+ , Ni 2+ , and Cu 2+ , respectively, the pH-sensitive metal chelated precipitant obtained is used to separate and purify melanoidin. As can be seen from Table 4, the type of chelated metal ions has a significant effect on the separation and purification of melanoidin. Among them, Fe 3+ ion has the best effect on the separation and purification of melanoidin, and has a higher yield. Ni 2+ , Cu 2+ , and melanoidin are not stable, and the yield of melanoidin obtained by separation and purification is too low. In addition, it is found in the pre-experiment that the combination performance of Fe 2+ , Co 2+ , and Mn 2+ is better than that of Fe 3+The Fe ions are not further experimented because of the discount and unstable combination, so for melanoidins, Fe ions with more stable combination and higher yield need to be selected 3+ The Fe ions are separated and purified.

[0102] Comparative Example 2

[0103] Comparative Example without adding pH-sensitive polymer: 0.56 mg of CMLD is configured into a 20 mL solution, 1.25 mL of FeCl3 solution (60 mmol / L) is added, the pH is adjusted to 4.0, and the solution is shaken at 100 r / min in a room temperature water bath for 60 min. The Fe 3+ The Fe ions are combined with melanoidins, centrifuged, and the uncombined Fe ions in the supernatant are removed 3+ The Fe ions and melanoidins are precipitated 3+ - MLD;

[0104] The obtained precipitate is added with a small amount of NaOH solution (c = 2 mol / L), the precipitate is redissolved, and 0.1 g of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) and 200 mL of distilled water are continuously added and reacted at 50°C in a water bath for 3 h. After cooling to room temperature, the solution is dialyzed in a dialysis bag with a cut-off of 1000 Da for 96 h, and freeze-dried to further obtain purified melanoidins (MLD) with a calculated yield.

[0105] Table 5

[0106]

[0107] As can be seen from Table 5, the Fe 3+ The Fe ions are directly reacted with melanoidins, and the metal ions are lost in the centrifugation process, so the yield of the obtained melanoidins is low, and after adding the pH-sensitive polymer, the Fe 3+ The Fe ions are stably combined on the ES-IA, and can fully adsorb melanoidins, so the yield of the melanoidins is obviously improved.

[0108] In summary, the present application uses glucose and glycine as raw materials to generate crude melanoidins through Maillard reaction, and uses the metal chelating ability of melanoidins and the pH sensitivity of pH-sensitive polymers to prepare a pH-sensitive metal chelator, which is chelated with crude melanoidins, and then the separation and purification of melanoidins can be realized by simply adjusting the pH. The purified melanoidins have a lower molecular weight of 6000 Da compared with the crude melanoidins. In the microstructure, the large irregular polygonal block and a small amount of granular structure in the crude melanoidins are also converted into more small block flaky and block structures.

[0109] The obtained black-like product can significantly improve blood lipid level and TMA and TMAO levels in plasma through long-term taking, and can also activate the AMPK pathway to inhibit TMA lyase activity and improve the intestinal acid environment, thereby inhibiting the proliferation of harmful bacteria in the intestinal tract, and the two jointly regulate to prevent atherosclerotic diseases. Therefore, the black-like product purified by the pH-sensitive metal chelating precipitant as a functional ingredient can inhibit choline metabolism to slow down the risk of arteriosclerosis disease, and a new strategy is provided for preventing arteriosclerosis disease.

[0110] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A method for separating and purifying melanoidins based on pH-sensitive metal chelate precipitants, characterized by: The application relates to a pH-sensitive metal chelate precipitant for purifying crude melanoidin. The crude melanoidin is prepared by a Maillard reaction with glucose and glycine as raw materials; An activating agent is added to a pH-sensitive polymer solution to activate the solution, and then a metal chelate solution is added to the activated solution to obtain a pH-sensitive precipitant solution through a coupling reaction; The pH-sensitive polymer solution is prepared by dissolving pH-sensitive polymer Eudragit S-100 in a NaOH solution, and the concentration is 1%, and the pH is adjusted to 6.5-7.5; The activating agent is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the molar mass of the added pH-sensitive polymer is 70-90%; The metal chelate solution is a 4-(imidazol-1-yl) aniline solution, and the concentration is 10 mmol / L, wherein 11 mL of the metal chelate solution is added to 1 g of the pH-sensitive polymer; The FeCl3 solution is added to the pH-sensitive precipitant solution to obtain a pH-sensitive metal chelate precipitant through chelation; The concentration of the FeCl3 solution is 60 mmol / L, and the added amount is 1.25 mL; The crude melanoidin is mixed with the pH-sensitive metal chelate precipitant, the pH is adjusted to 4.8-5.2, and centrifugation is performed to remove the unbound melanoidin in the supernatant, and the precipitate is a complex of the pH-sensitive metal chelate precipitant and the melanoidin; The complex is added to a NaOH solution, and the precipitate is redissolved, then disodium ethylenediaminetetraacetate solution is continuously added to perform water bath stirring reaction, after the reaction, the solution is cooled to room temperature, the pH of the solution is adjusted to 4.8-5.2 again, and centrifugation is performed, the supernatant is taken out to perform dialysis, freeze-drying and further post-treatment, and thus, the purified melanoidin is obtained.

2. The method for separating and purifying the melanoidins based on pH-sensitive metal chelate precipitator according to claim 1, characterized in that: The crude melanoidin is mixed with the pH-sensitive metal chelate precipitant, wherein 0.56 mg of the crude melanoidin is added to 1 g of Eudragit S-100 in the pH-sensitive metal chelate precipitant, the rotating speed is 100 r / min, and the oscillation time is 60-80 min.

3. The method for separating and purifying the melanoidins based on pH-sensitive metal chelate precipitator according to claim 1, characterized in that: The amount of the ethylenediaminetetraacetic acid disodium salt added is 1.5 to 4 times the molar amount of Fe in the FeCl3 solution. 3+ molar amount of Fe in the FeCl3 solution.

4. The method for separating and purifying the melanoidins based on pH-sensitive metal chelate precipitator according to claim 1, characterized in that: The temperature of the water bath stirring of the complex in the disodium ethylenediaminetetraacetate solution is 50 DEG C, and the stirring time is 3-4 h.

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