Carboxymethyl lysine remover and application thereof
Technical field
The present invention relates to food processing field, be specifically related to carboxymethyl lysine (CML) and eliminate system
Agent, can reduce the content of the CML generated in food system, as food by capture CML
Additive and extensively utilize.
Background technology
Advanced glycosylation end products (AGEs) is to be produced late period at Maillard reaction by food or organism
A highly oxidized compound of class.Now there are some researches show, AGEs may be with diabetes, chronic heart failure
Exhaust, the generation of the disease such as atherosclerosis relevant to development.Food-borne AGEs is absorbed into via digestive tract
Enter human body, human body AGEs level will be promoted.Long-term food-borne AGEs taking in excess, the most free
State AGEs (glycated amino acid), will cause potential health hazard to human body.
The generation of the means of existing minimizing AGEs content predominantly suppression AGEs: 1) antioxidant, energy
Removing system free radical, the process that suppression is converted to AGEs by the Amadori product of free radical mediated, such as:
VE, rutin, ferulic acid, VC, Quercetin, carnosine etc.;2) dicarbonyl compound trapping agent, can capture
The intermediate product Biformyl of AGEs formation and then the formation of suppression AGEs, such as: aminoguanidine (AG), pyrrole
Tremble amine (VB6) etc.;3) amino competitor, can be with the ammonia on lysine containing amino in its molecular structure
There is Maillard reaction, and then the generation of suppression AGEs in base competition, such as: VB1And derivant (thiamine
Element phosplate, TPP) etc..
As a example by typical case's AGEs carboxymethyl-lysine (CML), existing inhibitor reduces system CML and contains
The possible path of amount, therefore need to be at body as it is shown in figure 1, be the suppression to the part path forming CML
System adds inhibitor before forming CML and can be only achieved the effect reducing CML level.In actual production,
Inhibitor adds the AGEs that front food system has contained certain level, and existing inhibitor cannot be complete
The generation (suppression ratio is about 10%-50%) of full inhibition system AGEs, but, in food system
The AGEs existed, existing means will be unable to eliminate.
Currently there are no and be specifically designed in food the research that existing AGEs eliminates, the most AGEs is not eliminated
Agent is applied to the report in food processing.
Summary of the invention
For solving the problems referred to above, it is an object of the invention to provide a class CML remover, be specifically related to contain
The quinones substance that the polyphenol oxidase of catechol or pyrogallol structure is formed is to CML in simulated foods system
Elimination effect.Remover can be had CML by capture system under certain condition and then be reached CML
The purpose eliminated, its capture principle is as shown in Figure 2.
The purpose of the present invention is achieved through the following technical solutions:
Carboxymethyl lysine remover, described remover is containing catechol or pyrogallol structure list
The quinones substance that the polyphenol oxidase of unit is formed.
Described polyphenol includes 4-methyl pyrocatechol, epicatechin, L-Epicatechin gallate, coffee
Acid, Quercetin, epigallocatechin gallate (EGCG), table GC, luteolin, rutin.
The application in terms of reducing food system carboxymethyl-lysine content of the described remover.
In food system, add described remover, control pH >=7 of system.Preferably, described system
PH is 8.
The reaction temperature of described system is 25-100 DEG C.
The addition of described remover is the 2-3% of system lysine content.
Described remover adds after food system does not generates or generated CML.
Wherein said food system includes milk product, grain and goods thereof, flavoring agent, meat and goods thereof, tank
Head goods.
The present invention, in simulated foods system (glu-lysine system), utilizes liquid chromatography tandem matter
The quinones that polyphenol containing catechol or pyrogallol construction unit is formed by spectrometry (LC-MS/MS)
Material and CML add and thing is identified.Stability according to the quinones substance formed can be divided into:
Stablize quinones substance and be easily polymerized quinones substance two class.
Described stablize quinones substance add with CML and product identify concretely comprise the following steps:
(1) it is 10mM, quinones substance with phosphate buffer (pH 7.0) the preparation CML of 0.2M
Solution for 1mM;
(2) above-mentioned mixing liquid is stirred at room temperature 1min, processes through 0.22 μm micro-filtration membrane, make
With LC-ESI-MS/MS to adding and thing carries out Structural Identification.
Described LC type is HP 1100, and testing conditions is:
Chromatographic column: ZIC-HILIC chromatographic column 2.1 × 150mm, 3.5 μm
Column temperature: 25 DEG C
Flowing phase: A: ammonium acetate solution (6.5mM)/acetonitrile (10:90, pH 5.5);B: ammonium acetate
Aqueous solution (6.5mM)/acetonitrile (40:60, pH 5.5);Gradient elution
Flow velocity: 0.1mL/min
Sample size: 10 μ L;
Described ESI-MS/MS type is HP 1100, and testing conditions is:
Ion source: ESI+
Dry gas temperature: 200 DEG C
Atomization air pressure: 50psi
Two grades of fragmentation voltage: 1.00V
Sweep limits: m/z 50-750
Described quinones substance of stablizing captures specifically comprising the following steps that of CML ability examination
(1) with 0.2M phosphate buffer (pH 4.5) preparation quinonoid solution so that it is concentration is 0.08mM;
(2) with the solution that phosphate buffered saline CML is 1-10mM of 0.2M;
(3) above-mentioned (1) and (2) solution is after Stopped-flow mixing chamber equal-volume mixes, and pH is
5.0,7.0 or 8.0;
(4) Stopped-flow is used, by reaction system during monitoring 25 DEG C in ultraviolet-visible light district
Absorbance change (200-750nm), measures the kinetics of 4MBQ Yu CML.
Described easy polymerization quinones substance add with CML and product identify concretely comprise the following steps:
(1) with the phosphate buffer (pH 8.0) of 0.2M prepare lysine (Lys) be 0.1M,
Glucose (Glu) is that the solution of 0.1M carrys out simulated foods system;
(2) in (1) described simulated foods system, add 2~3% tie containing catechol or pyrogallol
The quinone that the polyphenol oxidase of structure is formed;
(3) by sample in 80 DEG C or 100 DEG C of heating in water bath 1h;
(4) take sample after 1mL heating, add distilled water and be settled to 5mL;
(5) take 1mL diluent to process through 0.22 μm micro-filtration membrane, use UPLC-ESI-MS/MS to adding
Structural Identification is carried out with thing.
Described UPLC uses Agilent SB1290 type, and testing conditions is:
Chromatographic column: Agilent SB-C18Chromatographic column 2.1 × 50mm, 1.8 μm
Column temperature: 25 DEG C
Flowing phase: A) formic acid: water=1:1000 (v:v), B) acetonitrile
Gradient elution program: 0min (85%A)-4min (15%A)-8min (15%A)-10min (85%
A);
Flow velocity: 0.2mL/min
Sample size: 5 μ L;
Mass spectrum uses type to be maxis Impact, and testing conditions is:
Ion source: ESI+/ESI-;
Capillary voltage: 3.5kV
Charging voltage: 2kV
Dry gas temperature: 180 DEG C
Nebulizer pressure: 0.3bar
Mass-to-charge ratio sweep limits: m/z50-1000
The present invention is to form adjacent benzene diquinone by catechol oxidation, and then adjacent benzene diquinone can directly and CML
The purpose that in reacting thus reaching system, existing CML eliminates.The present invention uses LC-ESI-MS/MS to identify
Quinones adds and product with CML's.As a result, it was confirmed that containing catechol or the polyphenol of pyrogallol structure
The quinones substance that oxidation is formed has capture effect to the CML in simulated foods system.
Compared with prior art, there is advantages that
(1) polyphenol containing catechol or pyrogallol structure is the material of a class Nantural non-toxic side effect,
Wherein, epicatechin, L-Epicatechin gallate, epigallocatechin gallate (EGCG) and table roasting youngster
Theine is the additive tea polyphenols allowing in National Standard of the People's Republic of China GB2760-2014 to use
Main component;Caffeic acid, as one of Semen Vitis viniferae extract main component, is regarded as It is generally accepted by FDA
Security classes additive (GRAS);Rutin and Quercetin are as Citrus reticulata Blanco extract main component, and 4-methyl is adjacent
Benzodiazepines, as the main component of castoreum extract, is regarded as the security classes that It is generally accepted equally by FDA
Additive (GRAS);The AGEs that system has been generated by the quinones substance that above-mentioned substance oxidation is formed has
Good elimination effect, compensate for AGEs inhibitor and system has existed AGEs adiaphorous defect.
(2) quinones substance of the present invention is the disappearing of AGEs in neutral or alkaline food system to pH
Removing solid capacity is higher.
(3) quinones substance of the present invention adds (see embodiment 1) after food system generates CML
Or interpolation (see embodiment 2-4) all can effectively reduce system CML level when not generating CML.
Accompanying drawing explanation
Fig. 1 is the possible path figure that inhibitor suppression CML is formed.
Fig. 2 is the pathway figure that quinones substance reacts with CML.
Fig. 3 is 4-methyl neighbour's benzene diquinone (4MBQ) and CML adds and a of thing) first mass spectrometric figure, b) two
Level mass spectrum and may structure and fracture mode.
Fig. 4 is EC quinones to be added and a of thing (80 DEG C of heating 1h) with CML) first mass spectrometric figure, b) two
Level mass spectrum.
Fig. 5 is to add the possible molecular structural formula with thing and fracture mode described in Fig. 4.
Fig. 6 is EC quinones to be added and a of thing (100 DEG C of heating 1h) with CML) first mass spectrometric figure, b)
Second order ms figure and may structure and fracture mode.
Fig. 7 is that EGCG quinones adds with CML and a of thing (100 DEG C of heating 1h)) first mass spectrometric figure,
B) second order ms figure.
Fig. 8 is to add the possible molecular structural formula with thing and fracture mode described in Fig. 7.
Detailed description of the invention
Elaborate below in conjunction with specific embodiment.
Embodiment 1
4-methyl neighbour's benzene diquinone (4MBQ) capture CML ability is identified and is added and identifies with product structure,
Specifically include following steps:
(1) 0.2M phosphate buffer (pH 4.5) preparation 4MBQ solution is used so that it is concentration is
0.08mM;
(2) with the solution that phosphate buffered saline CML is 1-10mM of 0.2M, make with above-mentioned
(1), after the mixing of solution equal-volume, pH is 5.0,7.0 or 8.0;
(3) Stopped-flow is used, by reaction system during monitoring 25 DEG C in ultraviolet-visible light district
(200-750nm) absorbance change, measures the kinetics of 4MBQ Yu CML;
(4) preparing CML with the phosphate buffer (pH 7.0) of 0.2M is that 10mM, 4MBQ are
The solution of 1mM;
(5) described to (4) mixing liquid is stirred at room temperature 1min, at 0.22 μm micro-filtration membrane
Reason, prepares liquid to be measured;
LC-ESI-MS/MS is finally used to use following testing conditions that prepared testing sample is detected.
Described LC type is HP 1100, and testing conditions is:
Chromatographic column: ZIC-HILIC chromatographic column 2.1 × 150mm, 3.5 μm
Column temperature: 25 DEG C
Flowing phase: A: ammonium acetate solution (6.5mM)/acetonitrile (10:90, pH 5.5);B: ammonium acetate
Aqueous solution (6.5mM)/acetonitrile (40:60, pH 5.5);Gradient elution
Flow velocity: 0.1mL/min
Sample size: 10 μ L;
Described ESI-MS/MS type is HP 1100, and testing conditions is:
Ion source: ESI+
Dry gas temperature: 200 DEG C
Atomization air pressure: 50psi
Two grades of fragmentation voltage: 1.00V
Sweep limits: m/z 50-750.
Above-mentioned Stopped-flow is used to obtain, the 4MBQ apparent kinetics constant to CML capture effect
(kobs) and second-order kinetics constant (k2) as shown in table 1, show that 4MBQ is to body when pH >=7
In system, the capture ability of CML is strong.When CML be 5mM, 4MBQ be 1mM time, pH 8.0,25
4MBQ in 4MBQ can capture system in 5 minutes at DEG C~at CML, pH7.0,25 DEG C of 36%
In system can being captured in 5 minutes~the CML of 14%.
1:25 DEG C of table, pH 5,7,8 times, the apparent kinetics constant (k that 4MBQ with CML reactsobs)、
Second-order kinetics constant (k2)
Use above-mentioned LC-ESI-MS/MS, use above-mentioned testing conditions that above-mentioned liquid to be measured is detected,
Obtain capturing first mass spectrometric (Fig. 3 a) and second order ms figure (Fig. 3 b) of product, thus can release reaction and produce
The structure chart of thing and possible fracture mode, as shown in Figure 3 b.
Embodiment 2
Epicatechin (EC) quinones capture CML is added the Structural Identification with product, specifically includes following step
Rapid:
(1) with the phosphate buffer (pH 8.0) of 0.2M prepare respectively lysine (Lys) be 0.1M,
Glucose (Glu) is that the solution of 0.1M carrys out simulated foods system;
(2) take the simulated foods system described in 1mL (1), add the EC quinone of 2mM;
(3) by sample in 80 DEG C of heating in water bath 1h;
(4) take sample after 1mL heating, add distilled water and be settled to 5mL;
(5) take 1mL diluent to process through 0.22 μm micro-filtration membrane, use UPLC-ESI-MS/MS pair
Add and carry out Structural Identification with thing.
Described UPLC uses Agilent SB1290 type, and testing conditions is:
Chromatographic column: Agilent SB-C18Chromatographic column 2.1 × 50mm, 1.8 μm
Column temperature: 25 DEG C
Flowing phase: A) formic acid: water=1:1000 (v:v), B) acetonitrile
Gradient elution program: 0min (85%A)-4min (15%A)-8min (15%A)-10min (85%
A);
Flow velocity: 0.2mL/min
Sample size: 5 μ L;
Mass spectrum uses type to be maxis Impact, and testing conditions is:
Ion source: ESI+/ESI-;
Capillary voltage: 3.5kV
Charging voltage: 2kV
Dry gas temperature: 180 DEG C
Nebulizer pressure: 0.3bar
Mass-to-charge ratio sweep limits: m/z 50-1000
Use above-mentioned UPLC-ESI-MS/MS, use above-mentioned testing conditions that above-mentioned sample is detected,
Add to EC quinones and CML and the first mass spectrometric (Fig. 4 a) of thing and second order ms figure (Fig. 4 b), can push away
Go out the structure chart of product and possible fracture mode, as shown in Figure 5.EC quinones and CML are described
Reaction forms new material, thus eliminates CML.
Embodiment 3
Epicatechin (EC) quinones capture CML is added the Structural Identification with product, specifically includes following step
Rapid:
(1) with the phosphate buffer (pH 8.0) of 0.2M prepare respectively lysine (Lys) be 0.1M,
Glucose (Glu) is that the solution of 0.1M carrys out simulated foods system;
(2) take the simulated foods system described in 1mL (1), add the EC quinone of 2mM;
(3) by sample in 100 DEG C of heating in water bath 1h;
(4) take sample after 1mL heating, add distilled water and be settled to 5mL;
(5) take 1mL diluent to process through 0.22 μm micro-filtration membrane, use UPLC-ESI-MS/MS pair
Add and carry out Structural Identification with thing.
Described UPLC uses Agilent SB1290 type, and testing conditions is:
Chromatographic column: Agilent SB-C18Chromatographic column 2.1 × 50mm, 1.8 μm
Column temperature: 25 DEG C
Flowing phase: A) formic acid: water=1:1000 (v:v), B) acetonitrile
Gradient elution program: 0min (85%A)-4min (15%A)-8min (15%A)-10min (85%
A);
Flow velocity: 0.2mL/min
Sample size: 5 μ L;
Mass spectrum uses type to be maxis Impact, and testing conditions is:
Ion source: ESI+/ESI-;
Capillary voltage: 3.5kV
Charging voltage: 2kV
Dry gas temperature: 180 DEG C
Nebulizer pressure: 0.3bar
Mass-to-charge ratio sweep limits: m/z 50-1000
Use above-mentioned UPLC-ESI-MS/MS, use above-mentioned testing conditions that above-mentioned sample is detected,
Add to EC quinones and CML and the first mass spectrometric (Fig. 6 a) of thing and second order ms figure (Fig. 6 b), thus
The structure chart of product and possible fracture mode can be released, as shown in Figure 6 b.
Embodiment 4
Epigallocatechin gallate (EGCG) (EGCG) quinones capture CML is added the structure mirror with product
Fixed, specifically include following steps:
(1) with the phosphate buffer (pH 8.0) of 0.2M prepare lysine (Lys) be 0.1M,
Glucose (Glu) is that the solution of 0.1M carrys out simulated foods system;
(2) take the simulated foods system described in 1mL (1), add the EGCG quinone of 3mM;
(3) by sample in 80 DEG C of heating in water bath 1h;
(4) take sample after 1mL heating, add distilled water and be settled to 5mL;
(5) take 1mL diluent to process through 0.22 μm micro-filtration membrane, use UPLC-ESI-MS/MS pair
Add and carry out Structural Identification with thing.
Described UPLC uses Agilent SB1290 type, and testing conditions is:
Chromatographic column: Agilent SB-C18Chromatographic column 2.1 × 50mm, 1.8 μm
Column temperature: 25 DEG C
Flowing phase: A) formic acid: water=1:1000 (v:v), B) acetonitrile
Gradient elution program: 0min (85%A)-4min (15%A)-8min (15%A)-10min (85%
A);
Flow velocity: 0.2mL/min
Sample size: 5 μ L;
Mass spectrum uses type to be maxis Impact, and testing conditions is:
Ion source: ESI+/ESI-;
Capillary voltage: 3.5kV
Charging voltage: 2kV
Dry gas temperature: 180 DEG C
Nebulizer pressure: 0.3bar
Mass-to-charge ratio sweep limits: m/z 50-1000.
Use above-mentioned UPLC-ESI-MS/MS, use above-mentioned testing conditions that above-mentioned sample is detected,
Obtain EGCG quinones and CML to add and the first mass spectrometric (Fig. 7 a) of thing and second order ms figure (Fig. 7 b),
Thus can release the structure chart of product and possible fracture mode, as shown in Figure 8.