CN112617092A - Acrolein-amino acid adduct and preparation method and application thereof - Google Patents
Acrolein-amino acid adduct and preparation method and application thereof Download PDFInfo
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/68—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
- C07D211/70—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
本发明属于食品加工过程有毒物质控制的技术领域，公开了一类丙烯醛‑氨基酸加合物及其制备方法与应用。本发明提供了六种丙烯醛‑氨基酸加合物的制备方法，产率高，为人体温(37℃)、热加工(160℃)下主要产物，制备得到的加合物的纯度高达95％，其中高效液相色谱图各个显示波长下(200～400nm)的目标单峰的峰纯度高达92％，可利用其作为标准物检测食品中热加工过程是否有该加合物生成，并利用这一结果额外添加氨基酸以控制内源有毒物质的含量。
The invention belongs to the technical field of toxic substance control in food processing, and discloses a class of acrolein-amino acid adducts and a preparation method and application thereof. The invention provides six preparation methods of acrolein-amino acid adducts, which have high yields, are the main products at human body temperature (37° C.) and thermal processing (160° C.), and the purity of the prepared adducts is as high as 95%. Among them, the peak purity of the target single peak at each display wavelength (200-400nm) of the high-performance liquid chromatogram is as high as 92%, which can be used as a standard to detect whether the adduct is formed during the thermal processing in food, and use this Results Additional amino acids were added to control the levels of endogenous toxic substances.
Description
Technical Field
The invention belongs to the technical field of organic compound synthesis, and particularly relates to an acrolein-amino acid adduct and a preparation method and application thereof.
Background
Acrolein is the most reactive aldehyde of the α, β -unsaturated carbonyl structure, widely found in ambient and thermally processed foods, and can also be produced by endogenous metabolism. Studies have shown that acrolein reacts very readily with nucleophilic macromolecules in organisms, interfering with DNA replication and transcription, leading to protein cross-linking and ultimately to various diseases (30 and 50mg/kg body weight for mouse and rat LD 50). In addition, it can also lower the level of intracellular Glutathione (GSH) and interfere with cellular signaling pathways, making the safety problem of acrolein a major concern.
It has been found that the acrolein content of a food product (e.g. potato) after frying is much lower (2-3 orders of magnitude) than the sum of the acrolein content of the frying oil and the acrolein content of the potato, whereas the acrolein content of a digested food product (e.g. potato) is much higher (more than 200 times) than that of the potato chip. Meanwhile, researches show that during the processing of potatoes at 180 ℃, the content of amino acids (amino acid, aspartic acid, glycine, alanine, glutamic acid, valine and the like) in the potatoes is reduced along with the increase of heating time, which is very likely to cause potential food safety hazards because acrolein reacts with food components during frying to cover the actual exposure level of the acrolein in the food.
In addition, more endogenous hazardous substances, such as activated carbonyl compounds, such as acrylamide, acrolein, methylglyoxal and the like, are generated in the food thermal processing process. The amino acid has active group-amino group capable of reacting with active carbonyl group, can react with acrolein easily under heat processing condition, and can be used as acrolein scavenger for controlling generation of endogenous toxicant in high temperature food. The research aims to provide a technology for regulating and controlling the formation of harmful active carbonyl compounds in food by using amino acid, and adds another purpose for the amino acid as a food additive.
Disclosure of Invention
In order to overcome the drawbacks and disadvantages of the prior art, the object of the present invention is to provide the use of a class of acrolein-amino acid adducts as endogenous toxic substance scavengers in the field of food processing;
another object of the present invention is to provide a process for preparing the above acrolein-amino acid adduct, which is simple and gives a product having a high yield and a high purity.
The purpose of the invention is realized by the following technical scheme:
the application of an acrolein-amino acid adduct as an endogenous toxic substance remover in the field of food processing is disclosed, wherein the structural formula of the acrolein-amino acid adduct is at least one of the following six types:
the endogenous toxic substance is preferably at least one of methylglyoxal and glyoxal.
The preparation method of the acrolein-amino acid adduct comprises the following steps: heating acrolein and amino acid in water at constant temperature for reaction, and performing subsequent treatment to obtain the acrolein-amino acid adduct.
The amino acid is at least one of aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gln), valine (Val) and threonine (Thr).
The molar ratio of the acrolein to the amino acid is 1-3: 1.
the dosage ratio of the acrolein to the water is (1-8) mmol (1-50) mL.
The temperature of the heating reaction is 20-80 ℃, and preferably 45-65 ℃; the heating reaction time is 1-12 h, preferably 2-5 h.
The heating reaction is carried out under the condition of stirring; the stirring speed is 100-200 rpm.
And the subsequent treatment refers to concentrating the reacted system, filtering with a filter membrane, and purifying by a reverse phase silica gel chromatography to obtain a purified product. The filter membrane is preferably a 0.45 μm filter membrane; the concentration is preferably concentration under reduced pressure.
The reverse phase silica gel chromatography specifically comprises the following steps: selecting reverse phase silica gel ODS resin, loading, eluting with 5% methanol water solution, collecting target adduct by 2, 4-dinitrophenylhydrazine solution color development method, detecting purity by high performance liquid detection method, collecting eluent, and freeze drying to obtain purified sample. The freeze drying condition is preferably-70 to-40 ℃ and 1 to 100 Pa.
According to the high performance liquid detection result, the mass spectrogram and the nuclear magnetic resonance spectrogram, the purity of the adduct prepared by the method is up to more than 90%.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention provides a preparation method of six acrolein-amino acid adducts, which has high yield, is a main product at human body temperature (37 ℃) and hot processing (160 ℃), has the purity of the prepared adducts as high as 95%, wherein the peak purity of a target single peak at each display wavelength (200-400 nm) of a high performance liquid chromatogram is as high as 92%, can be used as a standard substance to detect whether the adducts are generated in the hot processing process of food, and can be used for additionally adding amino acid to control the content of endogenous toxic substances.
Drawings
FIG. 1 is a first order mass spectrum (positive ion mode) of an acrolein-amino acid adduct prepared in example 1;
FIG. 2 is a first order mass spectrum (anion mode) of an acrolein-amino acid adduct prepared in example 1
FIG. 3 is a secondary mass spectrum (positive ion mode) of an acrolein-amino acid adduct prepared in example 1;
FIG. 4 is a high performance liquid chromatogram of an acrolein-amino acid adduct prepared in example 1;
FIG. 5 is a UV spectrum of an acrolein-amino acid adduct prepared in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The conditions of HPLC determination are as follows: column Zorbax SB-Aq C18(4.6 mm. times.250 mm, 5 μm), mobile phase A being pure water and mobile phase B being methanol; the chromatographic conditions are as follows: detecting wavelength of 220nm, flow rate of 0.6mL/min, column temperature of 40 ℃, and sample injection amount of 5 muL; the volume fraction of the mobile phase A is 95%, and the volume fraction of the mobile phase B is 5%, and the like, and the elution is carried out for 25min in an equal gradient manner.
Under the detection method, the retention time of the target is 4-10 min, and a good peak type with early peak, repeatability, high separation degree, sharp and no tailing appears.
The HPLC _ MS/MS determination conditions are as follows: a chromatographic column: agilent InfinityLab Poroshell 120 EC-C18; mobile phase: 0.1% formic acid water (A), chromatographic grade methanol (B); analysis conditions were as follows: mobile phase is 5% B aqueous solution, flow rate: 0.4mL/min, and the injection volume is 0.5. mu.L. The ion source is an electrospray ion source (ESI), and the scanning range is m/z 50-800; the source temperature was 300 deg.C, the desolvation temperature was 250 deg.C, the capillary voltage was 4000V, the scanning rate was 1000Da/sec, and the collision energy was 15-25 eV.
The NMR measurement conditions were: 15mg of the sample is dissolved in 550 mu L of DMSO and detected under an Avance III nuclear magnetic resonance instrument with 400-600 MHz.
Example 1
Six methods for preparing acrolein-amino acid adducts, comprising the steps of: heating acrolein and amino acid at a molar ratio of 2:1 in water in the dark for reaction (reaction at 50 ℃ for 5h, rotation speed of 130rpm), concentrating under reduced pressure for 1-2 mL, filtering with a 0.45-micrometer filter membrane to obtain a filtrate, and purifying by reverse phase silica gel chromatography to obtain an acrolein-amino acid adduct with a yield of 76-88%, wherein asparagine, glutamine, threonine, glutamic acid and aspartic acid.
Reversed phase silica gel chromatography: selecting 250mL of reverse phase silica gel ODS resin, enabling the specification of a chromatographic column to be 30 x 500mm, after gradient equilibrium chromatographic column, passing an evaporated and concentrated sample through a 0.45-micrometer filter membrane, then loading the sample, taking 5% methanol water solution as eluent, carrying out isocratic elution at a full flow rate (1mL/min), determining the existence of an adduct by using a 2, 4-dinitrophenylhydrazine solution color development method, detecting the purity by using a high performance liquid chromatography (HPLC method), collecting a target adduct, and freeze-drying to obtain the high-purity target adduct of about 20-60 mg; the freeze drying condition is-70 to-40 ℃, 1 to 100Pa and 48 hours.
The characterization diagrams of the acrolein-amino acid adduct prepared in this example are shown in FIGS. 1 to 5. FIG. 1 is a primary mass spectrum (positive ion mode); FIG. 2 is a primary mass spectrum (negative ion mode); FIG. 3 is a secondary mass spectrum (positive ion mode); FIG. 4 is a high performance liquid chromatogram of the separated and purified adduct; FIG. 5 is a UV spectrum of an adduct. According to the high performance liquid chromatography results shown in fig. 1 to 5, the purity of the adduct prepared by the method of the present invention is as high as 95%, wherein the peak purity of the target single peak under each display wavelength (200 to 400nm) of the high performance liquid chromatogram is as high as 92%. Under the detection method, the retention time of the target is 4-10 min, and a good peak pattern with early peak time, repeatability, high separation degree, sharpness and no tailing is presented. The molecular formula, molecular weight and structural formula of the acrolein-amino acid adduct are identified as shown in the following table 1:
TABLE 1 structural identification of several acrolein-amino acid adducts
Example 2
A method for preparing an acrolein-amino acid adduct comprising the steps of: heating acrolein and amino acid at a molar ratio of 1:1 in water in the absence of light for reaction (reaction at 45 ℃ for 5h, rotation speed 130rpm), concentrating under reduced pressure for 1-2 mL, filtering with a 0.45 μm filter membrane to obtain a filtrate, and purifying by reverse phase silica gel chromatography as in example 1 to obtain an acrolein-amino acid adduct with a yield of 55-62%, wherein asparagine, glutamine, valine, threonine, glutamic acid and aspartic acid.
The structural formula of the obtained acrolein-amino acid adduct is shown as example 1, and the purity is more than 90%.
Example 3
A method for preparing an acrolein-amino acid adduct comprising the steps of: heating acrolein and amino acid at a molar ratio of 1:2 in water in the absence of light for reaction (reaction at 60 ℃ for 5h, rotation speed 130rpm), concentrating under reduced pressure for 1-2 mL, filtering with a 0.45 μm filter membrane to obtain a filtrate, and purifying by reverse phase silica gel chromatography as in example 1 to obtain an acrolein-amino acid adduct with a yield of 38-42%, wherein asparagine, glutamine, valine, threonine, glutamic acid and aspartic acid.
The structural formula of the obtained acrolein-amino acid adduct is shown as example 1, and the purity is more than 90%.
Comparative example 1 (too long or too short reaction time)
A method for preparing an acrolein-amino acid adduct comprising the steps of: heating acrolein and amino acid at a molar ratio of 2:1 in water in the dark (reaction at 50 ℃ for 1h and 8h, rotation speed 130rpm), concentrating under reduced pressure for 1-2 mL, filtering with 0.45 μm filter membrane to obtain filtrate, and purifying with reverse phase silica gel chromatography as in example 1 to obtain acrolein-amino acid adduct.
When the reaction time is too short, the reaction is insufficient, the residue of amino acids in the resultant is significantly increased, and the yield of the adduct is significantly decreased (to 34%); when the reaction time is too long, the product is accompanied by the generation of other substances, and the yield of the objective adduct is remarkably reduced (71%).
Comparative example 2 (amino acid excess)
A method for preparing an acrolein-amino acid adduct comprising the steps of: reacting acrolein and amino acid at a molar ratio of 1:5 in water at 60 ℃ for 5h at a rotation speed of 130rpm), concentrating under reduced pressure for 1-2 mL, filtering with a 0.45 μm filter membrane to obtain a filtrate, and purifying by reverse phase silica gel chromatography as in example 1 to obtain an acrolein-amino acid adduct.
When the amino acid is excessive, the residual amount of the product amino acid is obviously increased, and the yield of the reaction is also obviously reduced; meanwhile, the separation effect of column chromatography is influenced, amino acid mixed in the adduct is not easy to clean, and the purity of the product is reduced to 78-85%.
Comparative example 3 (reaction temperature too high)
A method for preparing an acrolein-amino acid adduct comprising the steps of: heating acrolein and amino acid at a molar ratio of 2:1 in water in the absence of light (80 ℃ for 5h at 130rpm), concentrating under reduced pressure to 1-2 mL, filtering with a 0.45 μm filter membrane to obtain a filtrate, and purifying by reverse phase silica gel chromatography as in example 1 to obtain the acrolein-amino acid adduct.
When the reaction temperature is too high, the side reaction products are increased, and the yield of the obtained target adduct is reduced to 55-65%. This comparative example carried out the reaction in the optimum ratio of "acrolein to amino acid in a molar ratio of 2: 1", although the yield was similar to that of example 2 and higher than that of example 3. But more by-products are produced at high temperatures.
Application examples
The acrolein-asparagine adduct prepared in example 1 was used as a representative standard for the detection and analysis of whether acrolein produces this substance in fried potatoes, while amino acids can be added during the food preparation process to control the endogenous toxic substance content of food.
(1)
S0. obtaining raw materials:
preparing potato chips: cleaning fresh potato, peeling, cutting into 1.5mm potato slices, cleaning with deionized water for three times, and cleaning starch on the surface. Frying peanut oil (165 deg.C, 5min), draining, and storing under dry condition
S1, adduct extraction:
removing oil with n-hexane, continuously adding n-hexane into potato chips for three times (placing in a fume hood for 30 min/time and 3 times), stirring at intervals, weighing 1g sample after n-hexane is volatilized, sequentially adding 10mL of 50% methanol/water for three times and 10 min/time, centrifuging at 10000rpm/min for 30min, taking supernatant, spin drying, adding 6mL of 50% methanol/water for redissolving, and filtering with 0.22 μm organic filter membrane
S2.HPLC _ MS/MS detection of adduct content
Adopting MRM positive ion mode, and selecting ion pairs as follows: 227/168 (quantitative ion pairs, collision energy 20 eV); 227/181 (collision energy 22 eV); 227/122 (collision energy 22eV), mobile phase: phase A/phase B: water/acetonitrile (0.1% formic acid), elution procedure: the flow rate is 0.5 mL/min; 0-2min, 2% B-100% B; 2-8min, 100% B-100% B; 8-8.01min, 100% B-2% B; 8.01-18min, 2% B, and finally measuring the content of the additive in the potato chip to be 1.89 +/-0.30 mu g/kg.
(2)
S0. obtaining raw materials:
potato chips purchased from local supermarket (Bengao sand house)
S1, adduct extraction:
removing oil with n-hexane, continuously adding n-hexane into potato chips for three times (placing in a fume hood for 30 min/time and 3 times), stirring at intervals, weighing 1g sample after n-hexane is volatilized, sequentially adding 10mL of 50% methanol/water for three times and 10 min/time, centrifuging at 10000rpm/min for 30min, taking supernatant, spin drying, adding 6mL of 50% methanol/water for redissolving, and filtering with 0.22 μm organic filter membrane
S2.HPLC _ MS/MS detection of adduct content
Adopting MRM positive ion mode, and selecting ion pairs as follows: 227/168 (quantitative ion pairs, collision energy 20 eV); 227/181 (collision energy 22 eV); 227/122 (collision energy 22eV), mobile phase: phase A/phase B: water/acetonitrile (0.1% formic acid), elution procedure: the flow rate is 0.5 mL/min; 0-2min, 2% B-100% B; 2-8min, 100% B-100% B; 8-8.01min, 100% B-2% B; 8.01-18min, 2% B, and finally measuring the content of the additive in the potato chip to be 1.46 +/-0.19 mu g/kg.
(3)
And (3) biscuit processing:
preparing flour, white granulated sugar, shortening, palm oil, ammonium bicarbonate, baking soda, sodium metabisulfite, edible essence and edible salt, and adding 0.1-1.5 g of amino acid/1 kg of amino acid mixture based on the final total weight, wherein 1g of certain single amino acid can be added, or adding certain amino acid with additional health care function according to certain amino acid collocation. And kneading dough according to the conventional processing flow, rolling and cutting to form, baking, cooling, packaging and forming.
After the amino acid is added, the contents of methylglyoxal and glyoxal in the biscuit can be reduced by 17-23%.
TABLE 2 methylglyoxal and glyoxal content in the biscuit before and after addition of amino acids
(4)
Processing potato chips:
cleaning fresh potato, peeling, cutting into 1.5mm potato slices, cleaning with deionized water for three times, cleaning starch on the surface, soaking (0.1-0.5 g amino acid/100 mL water), frying with peanut oil (165 deg.C, 5min), draining, and packaging. Wherein the amino acid can be a single amino acid or matched according to the functionality of the amino acid.
After the amino acid soaking degree is increased, the contents of methylglyoxal and glyoxal in the potato chips can be reduced by 19-25%.
TABLE 3 increase of methylglyoxal and glyoxal levels in potato chips before and after the amino acid soaking process
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The application of an acrolein-amino acid adduct as an endogenous toxic substance remover in the field of food processing is disclosed, wherein the structural formula of the acrolein-amino acid adduct is at least one of the following six types:
2. use according to claim 1, characterized in that: the endogenous toxic substance is at least one of methylglyoxal and glyoxal.
3. A process for the preparation of acrolein-amino acid adducts of the class described in claim 1 characterized by the steps of: heating acrolein and amino acid in water at constant temperature for reaction, and performing subsequent treatment to obtain the acrolein-amino acid adduct.
4. A process of the class of acrolein-amino acid adducts according to claim 3 characterized in that:
the amino acid is at least one of aspartic acid, asparagine, glutamic acid, glutamine, valine and threonine.
5. A process of the class of acrolein-amino acid adducts according to claim 3 characterized in that: the molar ratio of the acrolein to the amino acid is 1-3: 1; the dosage ratio of the acrolein to the water is (1-8) mmol (1-50) mL.
6. A process of the class of acrolein-amino acid adducts according to claim 3 characterized in that: the molar ratio of acrolein to amino acid is 2: 1.
7. the process of any one of claims 3 to 6 wherein the acrolein-amino acid adduct comprises: the temperature of the heating reaction is 20-80 ℃; the heating reaction time is 1-12 h.
8. A process of the class of acrolein-amino acid adducts according to claim 3 characterized in that: the temperature of the heating reaction is 45-65 ℃; the heating reaction time is 2-5 h.
9. A process of the class of acrolein-amino acid adducts according to claim 3 characterized in that: and the subsequent treatment refers to concentrating the reacted system, filtering with a filter membrane, and purifying by a reverse phase silica gel chromatography to obtain a purified product.
10. The process of claim 9 wherein the acrolein-amino acid adduct comprises: the reverse phase silica gel chromatography specifically comprises the following steps: selecting reverse phase silica gel ODS resin, loading, eluting with 5% methanol water solution, collecting target adduct by 2, 4-dinitrophenylhydrazine solution color development method, detecting purity by high performance liquid detection method, collecting eluent, and freeze drying to obtain purified sample.
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