A method of promoting advanced glycosylation end product formation

Abstract

The application relates to a method for promoting generation of advanced glycation end products, comprising the following steps: preparing a reaction solution by mixing proteins, reducing sugar and riboflavin in a buffer solution; making the reaction solution undergo photo-thermal synergistic reaction to obtain a solution rich in the advanced glycation end products; and monitoring the generation amount of the advanced glycation end products by using fluorescence spectroscopy during the reaction process. The application uses the natural photosensitizer riboflavin to produce active ingredients under light conditions, accelerates the glycosylation reaction of bovine serum albumin and glucose, and effectively solves the problem of slow generation rate of the advanced glycation end products under normal temperature conditions.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology and relates to a method for promoting the formation of advanced glycation end products (AGEs). Specifically, it relates to a method for using riboflavin as a photosensitizer to promote the formation of AGEs. Background Technology

[0002] Advanced glycation end products (AGEs), generated during food heat processing and storage, are a major class of harmful byproducts of the Maillard reaction. They adversely affect the nutritional quality of food and are a primary source of AGEs for the human body. Furthermore, the accumulation of food-derived AGEs in the body can lead to the development and progression of many chronic diseases, including diabetes and its complications, chronic kidney damage, and cardiovascular and cerebrovascular diseases. Given their significant pathophysiological importance, AGEs have become an indispensable basic research reagent in medical, nutritional, and food science research.

[0003] Currently, laboratory and commercial preparation of AGEs mainly involves incubating bovine serum albumin (BSA) with glucose (GLU) under mild conditions (typically 37°C, protected from light), which usually requires four weeks or even longer to obtain a substantial AGEs yield. This lengthy preparation cycle results in low AGEs production efficiency and high costs. Commercially available AGEs reagents are expensive, typically costing tens to hundreds of RMB per milligram, significantly limiting related research. Therefore, there is an urgent need for an efficient and rapid method to prepare AGEs to meet the growing demand for AGEs reagents in scientific research and potential applications.

[0004] Studies have shown that reactive oxygen species (ROS) can significantly promote the formation of advanced AGEs (Advanced Glycation End Products). However, systematic methods and technologies for efficiently and controllably utilizing ROS to significantly shorten the AGE preparation cycle and improve yield are currently lacking. Within food systems, natural components such as riboflavin (RF) can generate highly reactive ROS under light irradiation, such as singlet oxygen (ROS). 1 O2) and triplet excited states with diradical properties ( 3 RF This provides a novel technical approach for introducing external energy (such as light energy) into the AGEs preparation system to generate ROS in situ and efficiently through photosensitization reactions, thereby accelerating the reaction process.

[0005] To address the problems existing in the prior art, this invention provides a method for promoting the formation of advanced glycosylation products (AGEs). By utilizing the natural photosensitizer riboflavin, the method promotes the formation of AGEs through photoexcitation, thus solving the problems of long reaction cycles, slow preparation rates, and low efficiency of AGEs under mild conditions in the prior art. This provides a feasible technical solution for the efficient and rapid preparation of AGEs. Summary of the Invention

[0006] The purpose of this invention is to provide a method for promoting the formation of advanced glycation end products (AGEs), characterized in that, in the advanced glycation reaction system, riboflavin is used as a photosensitizer, and under specific light and temperature conditions, the triplet excited state of riboflavin is generated through photoexcitation. 3 RF It interacts with proteins, accelerates the glycosylation process between proteins and reducing sugars, significantly shortens the formation time of AGEs, and improves efficiency.

[0007] The objective of this invention is achieved through the following technical solution: A method for promoting the formation of advanced glycation end products (AGEs), characterized in that riboflavin is used as a photosensitizer in the glycation reaction, and the method includes the following steps: S1. Preparation of the reaction system: The protein, reducing sugar, and riboflavin were dissolved in phosphate buffer to form a reaction solution; The protein may be selected from one or more of bovine serum albumin, human serum albumin, collagen, ovalbumin, and lysozyme; bovine serum albumin is preferred. The reducing sugar may be selected from one or more of glucose, fructose, lactose, galactose and maltose; glucose is preferred. S2, photo-thermal synergistic reaction: The solutions prepared in step S1 are placed in temperature-controlled incubation devices and incubated under uniform light and constant temperature conditions to promote the formation of advanced glycosylation products. S3. Monitoring and acquisition of the amount of late-stage glycosylation end products generated during the reaction process: The reaction proceeds for a predetermined time, a sample is taken, and the amount of AGEs generated in the sample is determined by fluorescence spectroscopy. When the fluorescence intensity of the measured AGEs reaches the expected value, the reaction is terminated, and a product solution rich in AGEs is obtained.

[0008] In step S1, the phosphate buffer solution has a pH of 7.0-7.6 and a concentration of 0.4-0.6 M. In the prepared reaction solution, the protein concentration is 10-100 mg / mL, the reducing sugar concentration is 0.1-1 M, and the riboflavin concentration is 0.05-0.25 mM, based on the total volume of the phosphate buffer solution.

[0009] In step S2, the light source providing illumination has a wavelength range of approximately 300-520 nm, meaning its characteristic spectrum covers the absorption spectrum of riboflavin, thereby exciting riboflavin. Illumination is preferably provided by a visible light source. The light source can be, for example, an incandescent lamp or an LED lamp, preferably an incandescent lamp, with a power preferably not less than 40 W, and more preferably 40-70 W.

[0010] The reaction temperature is controlled within the range of 25 °C to 60 °C.

[0011] In step S3, the specific operation of determining the amount of AGEs generated by fluorescence spectroscopy is as follows: at an excitation wavelength of 370 nm, the intensity of the fluorescence emission peak near 440 nm is detected with an emission wavelength scanning range of 375-600 nm to characterize the amount of AGEs generated.

[0012] For example, the reaction can be stopped when the fluorescence intensity of AGEs is measured to be approximately 200,000.

[0013] When proteins, reducing sugars, and riboflavin are present in the reaction system, the characteristic fluorescence peaks of AGEs and the fluorescence peaks of riboflavin need to be separated by a spectral fitting method to accurately calculate the fluorescence intensity of AGEs. The spectral fitting method uses the fluorescence spectrum of the reaction system containing only proteins and reducing sugars (excluding riboflavin) as the first component spectrum (AGEs component spectrum) and the fluorescence spectrum of riboflavin as the second component spectrum (riboflavin component spectrum). The fluorescence spectrum of the reaction solution containing proteins, reducing sugars, and riboflavin (the fluorescence spectrum of the reaction system according to the method of the present invention) is linearly fitted, and the quantitative fluorescence intensity of the advanced glycosylation end products is obtained through the fitting coefficient.

[0014] The present invention further relates to a reaction system for carrying out the method of the present invention, comprising: Temperature-controlled incubation apparatus for containing reaction solution; and A light source system, used to provide uniform illumination, is positioned at a suitable location within the incubation apparatus, such as around, on top of, or inside it; The temperature-controlled incubation device and the light source system are placed in a sealed space.

[0015] The incubation device operates at a set temperature and is simultaneously irradiated by the light source system.

[0016] Preferably, the incubation device is a temperature-controlled shaker; the light source system is an incandescent lamp or an LED lamp.

[0017] The method of the present invention can effectively promote the generation of AGEs in late glycosylation reactions, thereby enabling efficient preparation of AGEs.

[0018] The present invention further relates to the use of riboflavin as a photosensitizer in the preparation of advanced glycation end products (AGEs) to promote the formation of AGEs.

[0019] Beneficial effects Compared with the prior art, the present invention has the following significant advantages: 1. Significantly shortened preparation cycle and improved efficiency: By introducing active components generated by riboflavin photosensitization (such as singlet oxygen or triplet excited state of riboflavin) to promote the Maillard reaction, the generation of AGEs can be accelerated several times at 25 ℃-60 ℃. Especially at 35 ℃, the effective generation time of AGEs (i.e. the time for AGEs to reach the specified fluorescence intensity) is shortened to several days. Compared with the traditional method which takes several weeks, the efficiency can be increased by up to 4 times. This solves the problem of slow preparation rate of AGEs under room temperature conditions, and can prepare AGEs efficiently and time-savingly, providing technical support for AGEs production. At the same time, it provides a fast and time-saving method for AGEs preparation for relevant researchers or enterprises.

[0020] 2. Utilizing food-grade natural photosensitizers, which is safe and inexpensive: Riboflavin, a photosensitizer, is a type of vitamin B2 that is naturally found in a variety of foods. Its wide availability, high safety, and low cost make this method easy to scale up and effectively reduce the production cost of AGEs.

[0021] 3. Photothermal synergistic effect: Compared with the traditional thermally driven Maillard reaction, this invention promotes the generation of AGEs through a photothermal synergistic reaction, providing a method to accelerate the preparation of AGEs. At the same time, according to the fact that most of the coefficients 2 (A2) obtained from the fitting decomposition are less than 1, it is known that the required riboflavin is less than 1 times the actual riboflavin, indicating that the proteins and sugars in the reaction system have a certain protective effect on the photodegradation of riboflavin, thereby further stabilizing the reaction process and improving the reliability of the method.

[0022] 4. Mild and controllable process conditions, easy to implement: The required equipment is simple (temperature-controlled shaker, light source), the reaction conditions (temperature, light) are easy to control precisely, the process flow is clear, the reproducibility is good, and it is easy to promote and implement in laboratory and production environments. Attached Figure Description

[0023] Figure 1A schematic diagram of the reaction system for implementing the method of the present invention, wherein 1 is a light source system and 2 is an incubation device; Figure 2 The fluorescence spectra of BSA solution, BSA+GLU solution, BSA+GLU+RF reaction solution, and RF solution in Example 1 are shown as changes over time at a reaction temperature of 55 °C. Figure 2 A-2I shows the fluorescence spectra at times of 0, 3 hours, 7 hours, 21 hours, 24 hours, 27 hours, 31 hours, 45 hours, and 48 hours, respectively; Figure 3 The fluorescence spectra of BSA solution, BSA+GLU solution, BSA+GLU+RF reaction solution, and RF solution in Example 1 are shown as changes over time at a reaction temperature of 45 °C. Figure 3 A-3J shows fluorescence spectra at 0, 10, 24, 34, 48, 58, 72, 82, 96, and 168 hours, respectively. Figure 4 The fluorescence spectra of BSA solution, BSA+GLU solution, BSA+GLU+RF reaction solution, and RF solution in Example 1 are shown as changes over time at a reaction temperature of 35 °C. Figure 4 A-4I shows the fluorescence spectra at 0, 1 day, 2 days, 3 days, 4 days, 7 days, 10 days, 13 days, and 16 days, respectively; Figure 5 A is the fluorescence spectrum of AGEs generated by the BSA+GLU reaction in Example 1 as a function of time at a reaction temperature of 55 °C. Figure 5 B represents the fluorescence spectrum of AGEs separated by BSA+GLU+RF as a function of time at a reaction temperature of 55 °C. Figure 6 A is the fluorescence spectrum of AGEs generated by the BSA+GLU reaction in Example 1 as a function of time at a reaction temperature of 45 °C. Figure 6 B is the fluorescence spectrum of AGEs separated by BSA+GLU+RF in Example 1 as a function of time at a reaction temperature of 45 °C. Figure 7 A is the fluorescence spectrum of AGEs generated by the reaction of BSA+GLU solution in Example 1 as a function of time at a reaction temperature of 35 °C. Figure 7 B represents the fluorescence spectrum of AGEs separated from the BSA+GLU+RF reaction solution in Example 1 as a function of time at a reaction temperature of 35 °C. Figure 8A-8C are the fluorescence spectra of AGEs generated from BSA+GLU solution in Comparative Example 1 at reaction temperatures of 35 ℃, 45 ℃ and 55 ℃, respectively, as a function of time. Figure 9 This is a comparison of the kinetic curves (i.e., the relationship between fluorescence intensity and reaction time of AGEs) of the BSA+GLU+RF reaction solution fitted spectrum component 1 in Example 1 and sample A (BSA+GLU, no RF) in Comparative Example 1 at a reaction temperature of 55℃. Figure 10 This is a comparison of the kinetic curves (i.e. the relationship between fluorescence intensity and reaction time of AGEs) of the BSA+GLU+RF reaction solution fitted to the spectrum of Example 1 and sample A (BSA+GLU, no RF) in Comparative Example 1 at a reaction temperature of 45℃. Figure 11 This is a comparison of the kinetic curves (i.e. the relationship between fluorescence intensity of AGEs and reaction time) of the BSA+GLU+RF reaction solution fitted to the spectrum of Example 1 and sample A (BSA+GLU, no RF) in Comparative Example 1 at a reaction temperature of 35℃. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments. The embodiments described herein are specific implementations of the present invention, intended to elaborate on the technical solutions of the present invention in detail, and do not constitute any limitation on the present invention. Within the technical concept of the present invention, those skilled in the art can make various modifications. Based on the overall concept of the present invention, any reasonable adjustments, substitutions, or improvements made to the parameters, conditions, steps, and materials in the specific embodiments without departing from the principles of the present invention are all within the protection scope of the present invention.

[0025] All reagents and materials used in the embodiments of this invention are commercially available. Riboflavin was purchased from Shandong Keyuan Biochemical Co., Ltd. (CAS: 83-88-5); sodium dihydrogen phosphate (CAS: 7558-80-7), disodium hydrogen phosphate (CAS: 7758-79-4), and bovine serum albumin (CAS: 9048-46-8) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; glucose was D-(+)-glucose (CAS: 50-99-7).

[0026] Example 1: (1) Sample preparation: The following four sets of test sample solutions were prepared using a 0.05 M phosphate buffer solution with pH 7.4, where the concentrations are based on the total volume of the phosphate buffer solution: Sample 1: Bovine serum albumin solution (BSA), concentration 50 mg / mL; Sample 2: A mixed solution of bovine serum albumin and glucose (BSA + GLU), wherein the concentration of BSA was 50 mg / mL and the concentration of GLU was 0.50 M; Sample 3 (sample according to the method of the present invention): bovine serum albumin solution, glucose and riboflavin reaction solution (BSA + GLU + RF), wherein the concentration of BSA is 50 mg / mL, the concentration of GLU is 0.50 M, and the concentration of RF is 0.2 mM; Sample 4: Riboflavin solution (RF), concentration 0.2 mM.

[0027] (2) Reaction apparatus: such as Figure 1 As shown, it includes a temperature-controlled shaker placed in a sealed space, the shaker temperature is adjusted and kept constant; four 50 W incandescent lamps are evenly arranged above the shaker to ensure uniform illumination of the reaction area.

[0028] (3) Reaction process: The above test sample solutions are placed in the reaction apparatus and reacted at different temperatures for a certain time, and samples are taken at the specified time: a) The reaction was carried out at 35 °C for 16 days, and samples were taken on days 0, 1, 2, 3, 4, 7, 10, 13, and 16. b) The reaction was carried out at 45 °C for 7 days, and samples were taken at 0, 10, 24, 34, 48, 58, 72, 82, 96 and 168 hours.

[0029] c) The reaction was carried out at 55 °C for 48 hours, and samples were taken at 0, 3, 7, 21, 24, 27, 31, 45 and 48 hours.

[0030] (4) Fluorescence spectroscopy determination: The fluorescence intensity of AGEs in the sampled samples was determined using a fluorescence spectrometer (FS5, Edinburgh Instruments, UK). The samples were diluted 50 times with 0.05 M, pH 7.4 phosphate buffer before measurement to subtract the background value of the Raman scattering peak at 425 nm of the phosphate buffer.

[0031] The measurement parameters were set as follows: excitation wavelength 370 nm, emission wavelength scanning range 375-600 nm, scanning step size 1 nm, dwell time 0.5 s, and a total of 2 scans.

[0032] (5) Spectral data analysis: a) Import the raw fluorescence data into Origin software and plot the fluorescence spectra of each group of samples at different reaction time points; b) Use Matlab software to perform fitting analysis on the spectral data. The fitting formula is as follows: Fitted curve = Baseline + A1 × Component 1 (BSA + GLU spectrum) + A2 × Component 2 (RF spectrum) Residual = y(BSA + GLU + RF measured value) - y(fitted curve) Component 1 refers to the portion of the (BSA+GLU+RF) spectral curve corresponding to the (BSA+GLU) spectral curve, and component 2 refers to the portion of the (BSA+GLU+RF) spectral curve corresponding to the RF spectral curve.

[0033] A1 and A2 are the fitting coefficients for component 1 and component 2, respectively.

[0034] Based on the above formula, the fitted spectral curve of (BSA+GLU+RF) is obtained by fitting the spectral curves of (BSA+GLU+RF) and RF.

[0035] (6) Results Analysis: a) such as Figure 2 , Figure 3 and Figure 4 As shown, at reaction temperatures of 55 °C, 45 °C and 35 °C, the fluorescence intensity of the BSA+GLU group (sample 2) increased with increasing reaction time, indicating that AGEs were continuously generated; while in the BSA+GLU+RF group (in this invention, sample 3), the addition of RF further enhanced the fluorescence intensity of the system.

[0036] (b) The A1 values ​​(representing the relative content of AGEs) at each time point were obtained through fitting analysis, and the results are shown in Tables 1, 2, and 3. At 55 ℃, the A1 value peaked at 1.87 at hour 31; at 45 ℃, it peaked at 2.22 at hour 48; and at 35 ℃, it peaked at 4.19 on day 7. This indicates that RF significantly promotes the formation of AGEs, with a more significant promoting effect at 35 ℃, reaching a maximum promoting efficiency of approximately 4 times.

[0037] Table 1. Fitting coefficients of fluorescence spectra for different components at a reaction temperature of 55℃ Table 2. Fitting coefficients of fluorescence spectra for different components at a reaction temperature of 45℃ Table 3. Fitting coefficients of different components in the fluorescence spectra at a reaction temperature of 35 °C. c) Characteristic fluorescence spectra of AGEs obtained from fitting ( Figure 5 , 6As shown in 7), under the condition of RF presence, the intensity of the characteristic fluorescence peak of AGEs at 440 nm is significantly higher than that of the group without RF, further confirming the promoting effect of RF on the formation of AGEs.

[0038] d) It was also observed that the degradation rate of RF (reflected by the change in A2 value) was slower in the presence of BSA+GLU, indicating that the reaction products of BSA and GLU have a certain protective effect on RF.

[0039] As can be seen from this embodiment, under light conditions, riboflavin, as a photosensitizer, can effectively promote the glycosylation reaction between BSA and glucose, significantly shorten the formation time of AGEs and increase the yield, with the promoting effect being particularly significant at 35 °C.

[0040] Comparative Example 1 This comparative example does not contain riboflavin to exclude its photosensitizing effect, and aims to demonstrate the promoting effect of riboflavin on AGEs formation under light conditions by comparing it with Example 1.

[0041] (1) Sample preparation: According to Example 1, a control sample was prepared using a 0.05 M phosphate buffer solution with pH 7.4: Sample A (positive control): a solution of BSA and glucose (BSA + GLU), wherein the concentration of BSA is 50 mg / mL and the concentration of GLU is 0.50 M; Sample B (blank control): Bovine serum albumin solution (BSA) at a concentration of 50 mg / mL.

[0042] (2) Reaction conditions: Sample A and sample B were placed in the reaction apparatus of Example 1: a) The reaction was continued at 35 °C for 16 days; b) The reaction was continued at 45 °C for 7 days; c) The reaction was continued at 55 °C for 48 hours.

[0043] (3) Measurement and Analysis: After the reaction, the sample was subjected to fluorescence spectroscopy and data analysis according to the method described in Example 1, and the characteristic fluorescence intensity and generation curve of AGEs were obtained respectively. Figure 8 ).

[0044] (4) Results and Comparison The AGE generation kinetics data (i.e., fluorescence intensity versus reaction time curves) of sample A (BSA+GLU, no RF) at different temperatures in this comparative example were compared with the corresponding data of the fitted spectrum component 1 of sample 3 (BSA+GLU+RF) in Example 1. The results show: a) At the same reaction temperature and time point, the characteristic fluorescence intensity (at 440 nm) of AGEs in Sample 3 (containing RF) of Example 1 was significantly higher than that of Sample A.

[0045] b) At 55 °C, to achieve a similar amount of AGEs (with fluorescence intensity reaching 200,000 as the indicator), the reaction time required for sample 3 (containing RF) was approximately 40% shorter than that for sample A. Figure 9 ).

[0046] c) At 45 °C, to achieve similar AGEs formation levels (based on a fluorescence intensity of 200,000), the reaction time required for sample 3 (containing RF) was approximately 50% shorter than that for sample A. Figure 10 ).

[0047] d) At 35 °C, to achieve similar AGEs formation levels (based on a fluorescence intensity of 200,000), the reaction time required for sample 3 (containing RF) was approximately 80% shorter than that for sample A. Figure 11 )above.

[0048] The comparative results show that the AGEs formation rate under conventional heating reaction without the addition of riboflavin is significantly lower than that of the system with the addition of riboflavin and light irradiation under the same conditions according to the method of the present invention, which proves that riboflavin, as a photosensitizer, has a significant promoting effect on AGEs formation.

Claims

1. A method for promoting the formation of advanced glycation end products (AGEs), comprising the following steps: S1. Dissolve the protein, reducing sugar, and riboflavin in phosphate buffer to prepare a reaction solution; S2. Place the reaction solution from step S1 in an incubation device and carry out the incubation reaction in a reaction environment with uniform light and constant temperature; S3. The reaction proceeds for a predetermined time, a sample is taken, and the amount of advanced glycosylation end products generated in the sample is determined by fluorescence spectroscopy; when the fluorescence intensity of the advanced glycosylation end products reaches the expected value, the reaction is terminated, and a solution rich in the end products is obtained.

2. The method according to claim 1, wherein in step S1, the protein is selected from one or more of bovine serum albumin, human serum albumin, collagen, ovalbumin and lysozyme; and the reducing sugar is selected from one or more of glucose, fructose, lactose, galactose and maltose.

3. The method according to claim 1, wherein in step S1, the concentration of the protein is 10-100 mg / mL, the concentration of the reducing sugar is 0.1-1.0 M, and the concentration of the riboflavin is 0.05-0.25 mM, based on the total volume of the phosphate buffer solution.

4. The method according to claim 1, wherein in step S2, the temperature is 25°C to 60°C; and the light source providing illumination comprises a light source in the 300-520 nm wavelength band.

5. The method according to claim 1, wherein in step S3, the amount of late glycosylation end products generated is determined as follows: the intensity of the fluorescence emission peak near 440 nm is detected at a fluorescence spectral excitation wavelength of 370 nm, and the emission wavelength scan range is 375-600 nm.

6. A reaction system for carrying out the method according to any one of claims 1 to 5, comprising: Temperature-controlled incubation apparatus for containing mixed reaction solutions; and A light source system used to provide uniform illumination; The temperature-controlled incubation device and the light source system are placed in a sealed space.

7. The reaction system according to claim 6, wherein the incubation device operates at a constant temperature while being irradiated by the light source system.

8. The reaction system according to claim 7, wherein the light source system is an incandescent lamp or an LED lamp.

9. The use of riboflavin as a photosensitizer to promote the formation of advanced glycation end products (AGEs) in the preparation of AGEs.

Images