CN112617092B - Acrolein-amino acid adducts, and preparation method and application thereof - Google Patents
Acrolein-amino acid adducts, and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 150000001413 amino acids Chemical class 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 31
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- 229940024606 amino acid Drugs 0.000 claims description 79
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 claims description 54
- 235000001014 amino acid Nutrition 0.000 claims description 39
- 239000012071 phase Substances 0.000 claims description 26
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- 238000006243 chemical reaction Methods 0.000 claims description 18
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- 239000012528 membrane Substances 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 9
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 7
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 7
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 7
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- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 6
- 235000003704 aspartic acid Nutrition 0.000 claims description 6
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 6
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- 230000035484 reaction time Effects 0.000 claims description 6
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- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 5
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 5
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 5
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- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 5
- 235000004554 glutamine Nutrition 0.000 claims description 5
- 235000008521 threonine Nutrition 0.000 claims description 5
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000007791 liquid phase Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical compound NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
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- 239000012264 purified product Substances 0.000 claims description 2
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- 235000013305 food Nutrition 0.000 abstract description 13
- 238000012545 processing Methods 0.000 abstract description 9
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- 231100000614 poison Toxicity 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 4
- 231100000167 toxic agent Toxicity 0.000 abstract description 4
- 230000036760 body temperature Effects 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 16
- 150000002500 ions Chemical class 0.000 description 13
- AIJULSRZWUXGPQ-UHFFFAOYSA-N Methylglyoxal Chemical compound CC(=O)C=O AIJULSRZWUXGPQ-UHFFFAOYSA-N 0.000 description 12
- 235000013606 potato chips Nutrition 0.000 description 12
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 description 10
- 244000061456 Solanum tuberosum Species 0.000 description 9
- 235000002595 Solanum tuberosum Nutrition 0.000 description 9
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- 230000000052 comparative effect Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
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- 238000010828 elution Methods 0.000 description 3
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- 239000000243 solution Substances 0.000 description 3
- 238000004885 tandem mass spectrometry Methods 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 235000019483 Peanut oil Nutrition 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 150000001728 carbonyl compounds Chemical class 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
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- 235000019253 formic acid Nutrition 0.000 description 2
- 239000003517 fume Substances 0.000 description 2
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- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 235000019482 Palm oil Nutrition 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- -1 acrolein-asparagine Chemical compound 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
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- 235000013312 flour Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005417 food ingredient Substances 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 235000021552 granulated sugar Nutrition 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
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- 230000000269 nucleophilic effect Effects 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940120731 pyruvaldehyde Drugs 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
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- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
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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
Landscapes
- 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
The invention belongs to the technical field of toxic substance control in the food processing process, and discloses an acrolein-amino acid adduct, a preparation method and application thereof. 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 working (160 ℃), the purity of the prepared adducts is up to 95%, wherein the peak purity of a target single peak at each display wavelength (200-400 nm) of a high performance liquid chromatogram is up to 92%, the adducts can be used as a standard substance to detect whether the adducts are generated in the hot working process of foods, and amino acid is additionally added to control the content of endogenous toxic substances by utilizing the result.
Description
Technical Field
The invention belongs to the technical field of organic compound synthesis, and particularly relates to an acrolein-amino acid adduct, a preparation method and application thereof.
Background
Acrolein is the most active aldehyde in the α, β -unsaturated carbonyl structure, and is widely found in environmental 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 leading to various diseases (mice and rats with LD50 of 30 and 50mg/kg body weight). In addition, it can also reduce intracellular Glutathione (GSH) levels and interfere with cellular signaling pathways, making the problem of acrolein safety a great concern.
It has been found that after frying food products such as potatoes, the acrolein content of the potato chips 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 potatoes, whereas after digestion, the acrolein content of the potato chips is much higher (200 times) than that of the potato chips. Meanwhile, researches show that the content of amino acids (amino acids, aspartic acid, glycine, alanine, glutamic acid, valine and the like) in the potato is reduced along with the increase of heating time in the processing process of the potato at 180 ℃, and the actual exposure level of acrolein in food is covered up possibly because acrolein reacts with food ingredients in the frying process, so that potential food safety hazards can be generated.
In addition, because more endogenous hazardous substances, such as active carbonyl compounds like acrylamide, acrolein, and pyruvaldehyde, are generated during the food heat processing process. The amino acid has an active group-amino group which reacts with active carbonyl, is extremely easy to react with acrolein under the condition of thermal processing, can be used as a scavenger of the acrolein, and can be used for controlling the generation of endogenous toxic substances in high-temperature foods. The present study aims at providing a technology for regulating the formation of harmful active carbonyl compounds in food by amino acids, and adding another use for amino acids as food additives.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the object of the present invention is to provide the use of a class of acrolein-amino acid adducts as an endogenous toxic substance eliminator in the field of food processing;
another object of the present invention is to provide a process for the preparation of the above acrolein-amino acid adduct, which is simple, and the obtained product is high in yield and purity.
The aim of the invention is achieved by the following technical scheme:
use of an acrolein-amino acid adduct of the formula of at least one of the following six types as an endogenous toxic substance eliminator in the field of food processing:
the endogenous toxic substance is preferably at least one of methylglyoxal and glyoxal.
The preparation method of the acrolein-amino acid adducts comprises the following steps: in water, acrolein and amino acid are heated at constant temperature to react, and then the acrolein-amino acid adduct is obtained after subsequent treatment.
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 ℃, preferably 45-65 ℃; the heating reaction time is 1 to 12 hours, preferably 2 to 5 hours.
The heating reaction is carried out under the condition of stirring; the stirring speed is 100-200 rpm.
The subsequent treatment refers to concentration of the reacted system, filtration with a filter membrane, and purification with 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 reduced pressure concentration.
The reverse phase silica gel chromatography is specifically: selecting reverse phase silica gel ODS resin, loading, eluting with 5% methanol water solution, collecting target adduct by 2, 4-dinitrophenylhydrazine solution chromogenic method, detecting purity by high performance liquid phase detection method, collecting eluent, and freeze drying to obtain purified sample. The conditions for freeze-drying are preferably-70 to-40 ℃ and 1 to 100Pa.
According to the high performance liquid phase detection result and the mass spectrogram and nuclear magnetic resonance spectrogram result, 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:
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 working (160 ℃), the purity of the prepared adducts is up to 95%, wherein the peak purity of a target single peak at each display wavelength (200-400 nm) of a high performance liquid chromatogram is up to 92%, the adducts can be used as a standard substance to detect whether the adducts are generated in the hot working process of foods, and amino acid is additionally added to control the content of endogenous toxic substances by utilizing the result.
Drawings
FIG. 1 is a first mass spectrum (positive ion mode) of an acrolein-amino acid adduct prepared in example 1;
FIG. 2 is a first mass spectrum (anion pattern) of an acrolein-amino acid adduct prepared in example 1
FIG. 3 is a secondary mass spectrum (positive ion mode) of the acrolein-amino acid adduct prepared in example 1;
FIG. 4 is a high performance liquid chromatogram of the acrolein-amino acid adduct prepared in example 1;
FIG. 5 is an ultraviolet spectrum of the 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 embodiments of the present invention are not limited thereto.
The conditions for the HPLC method are as follows: chromatographic column Zorbax SB-Aq C18 (4.6 mm. Times.250 mm, 5 μm), mobile phase A pure water, mobile phase B methanol; the chromatographic conditions are as follows: the detection wavelength is 220nm, the flow rate is 0.6mL/min, the column temperature is 40 ℃, and the sample injection amount is 5 mu L; the volume fraction of the mobile phase A is 95%, and the volume fraction of the mobile phase B is 5% and the gradient elution is carried out for 25min.
Under the detection method, the retention time of the target object is 4-10 min, and the target object has the advantages of early peak, repeatability, high separation degree, sharpness and no tailing.
The HPLC_MS/MS measurement conditions are as follows: chromatographic column: agilent InfinityLab Poroshell 120 EC-C18; mobile phase: 0.1% formic acid water (A), chromatographic grade methanol (B); analysis conditions: mobile phase was 5% b aqueous solution, flow rate: the sample volume was 0.5. Mu.L at 0.4 mL/min. The ion source is an electrospray ion source (ESI), and the scanning range is between 50 and 800 m/z; the source temperature is 300 ℃, the desolvation temperature is 250 ℃, the capillary voltage is 4000V, the scanning speed is 1000Da/sec, and the collision energy is 15-25eV.
The NMR measurement conditions were: 15mg of the sample was dissolved in 550. Mu.L of DMSO and detected under a model 400-600 MHz Avance type III nuclear magnetic resonance apparatus.
Example 1
A process for the preparation of six acrolein-amino acid adducts comprising the steps of: in water, carrying out light-proof heating reaction (reaction at 50 ℃ for 5h and rotation speed of 130 rpm) on acrolein and amino acid according to a molar ratio of 2:1, concentrating under reduced pressure for 1-2 mL, filtering with a 0.45 mu m filter membrane to obtain filtrate, purifying by reverse phase silica gel chromatography to obtain an acrolein-amino acid adduct, wherein the yield is 76-88%, and asparagine, glutamine > valine, threonine > glutamic acid and aspartic acid.
Reversed phase silica gel chromatography: selecting 250mL of reverse phase silica gel ODS resin, performing gradient equilibrium chromatography with the specification of 30X 500mm, loading a sample after evaporation concentration through a 0.45 mu m filter membrane, performing isocratic elution at a full flow rate (1 mL/min) by taking a 5% methanol aqueous solution as an eluent, determining the existence of an adduct by a 2, 4-dinitrophenylhydrazine solution chromogenic method, detecting the purity by a high performance liquid chromatography (HPLC method), collecting a target adduct, and obtaining about 20-60 mg of the high-purity target adduct after freeze drying; the freeze drying condition is-70 to-40 ℃ and 1 to 100Pa,48h.
The characterization of the acrolein-amino acid adducts prepared in this example are shown in FIGS. 1-5. FIG. 1 is a first order 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 adduct after separation and purification; FIG. 5 is a UV spectrum of the adduct. According to the high performance liquid phase detection results shown in fig. 1-5, the purity of the adduct prepared by the method is up to 95%, wherein the peak purity of the target single peak at each display wavelength (200-400 nm) of the high performance liquid chromatogram is up to 92%. Under the detection method, the retention time of the target object is 4-10 min, and the target object has good peak shape with early peak time, repeatability, high separation degree and sharpness without tailing. The molecular formula, molecular weight and structural formula of the identified acrolein-amino acid adduct are shown in table 1:
TABLE 1 Structure identification of several acrolein-amino acid adducts
Example 2
A process for the preparation of an acrolein-amino acid adduct comprising the steps of: in water, acrolein and amino acid are subjected to light-proof heating reaction (reaction at 45 ℃ C. For 5h and rotation speed of 130 rpm) according to a molar ratio of 1:1, 1-2 mL is concentrated under reduced pressure, a filter membrane with 0.45 μm is obtained, and the filtrate is purified by reverse phase silica gel chromatography as in example 1 to obtain acrolein-amino acid adducts with yields of between 55 and 62%, wherein asparagine, glutamine > valine, threonine > glutamic acid and aspartic acid.
The structural formula of the obtained acrolein-amino acid adduct is as in example 1, and the purity is above 90%.
Example 3
A process for the preparation of an acrolein-amino acid adduct comprising the steps of: in water, acrolein and amino acid are subjected to a light-proof heating reaction (reaction at 60 ℃ C. For 5h, rotation speed of 130 rpm) according to a molar ratio of 1:2, and after concentrating under reduced pressure for 1-2 mL and passing through a 0.45 μm filter membrane, filtrate is obtained, and purification is carried out by reverse phase silica gel chromatography as in example 1, to obtain acrolein-amino acid adduct with yield of 38-42%, wherein asparagine, glutamine > valine, threonine > glutamic acid, aspartic acid.
The structural formula of the obtained acrolein-amino acid adduct is as in example 1, and the purity is above 90%.
Comparative example 1 (reaction time too long or too short)
A process for the preparation of an acrolein-amino acid adduct comprising the steps of: in water, acrolein and amino acid were subjected to a heat reaction (reaction at 50 ℃ C. For 1h, 8h, rotation speed 130rpm, respectively) at a molar ratio of 2:1 in the absence of light, concentrated under reduced pressure by 1 to 2mL, filtered through a 0.45 μm filter to obtain a filtrate, and purified by reverse phase silica gel chromatography as in example 1 to obtain an acrolein-amino acid adduct.
When the reaction time is too short, the reaction is insufficient, the residue of amino acid in the resultant is obviously increased, and the yield of the adduct is obviously reduced (34 percent); when the reaction time is too long, other substances are produced in the product, and the yield of the objective adduct is also significantly reduced (71%).
Comparative example 2 (amino acid excess)
A process for the preparation of an acrolein-amino acid adduct comprising the steps of: acrolein and amino acid were reacted in water at a molar ratio of 1:5 at 60℃for 5 hours at 130 rpm), concentrated under reduced pressure by 1 to 2mL, filtered through a 0.45 μm filter to obtain a filtrate, and purified 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 amino acid of the product is obviously increased, and the yield of the reaction is also obviously reduced; meanwhile, the separation effect of column chromatography can be affected, the mixed amino acid in the adduct is not easy to clean, and the purity of the product can be reduced to 78-85%.
Comparative example 3 (reaction temperature too high)
A process for the preparation of an acrolein-amino acid adduct comprising the steps of: in water, acrolein and amino acid were reacted at a molar ratio of 2:1 under heating away from light (reaction at 80 ℃ C. For 5 hours at 130 rpm), concentrated under reduced pressure to 1 to 2mL, and filtered through a 0.45 μm filter to obtain a filtrate, which was purified by reverse phase silica gel chromatography as in example 1 to obtain an acrolein-amino acid adduct.
When the reaction temperature is too high, the side reaction products will increase and the yield of the target adduct will decrease to 55% -65%. This comparative example was conducted in an optimum ratio of "acrolein to amino acid in a molar ratio of 2:1", although the yield was similar to example 2 and higher than 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 detecting and analyzing whether acrolein produced the substance in fried potatoes, while amino acids were added during food production to control the content of endogenous toxic substances in foods.
(1)
S0. raw material acquisition:
preparing potato chips: fresh potatoes are washed, peeled, cut into 1.5mm potato slices, washed three times with deionized water, and surface starch is washed. Frying peanut oil (165 deg.C, 5 min), draining, and sealing under dry condition
S1, adduct extraction:
taking a proper amount of n-hexane for degreasing, continuously adding n-hexane for three times until the n-hexane is not over the potato chips (placed in a fume hood for 30 min/time and 3 times), stirring at intervals, weighing 1g of sample after the n-hexane is volatilized, sequentially adding 10mL of 50% methanol/water for extraction (three times and 10 min/time) and centrifuging at 10000rpm/min for 30min, taking supernatant, spin-drying, adding 6mL of 50% methanol/water for re-dissolution, and passing through a 0.22 mu m organic filter membrane
S2 HPLC_MS/MS detection of adduct content
Adopts MRM positive ion mode, and the selected ion groups are as follows: 227/168 (quantitative ion pairs, collision energy 20 eV); 227/181 (collision energy 22 eV); 227/122 (collision energy 22 eV), mobile phase: phase a/phase B: water/acetonitrile (0.1% formic acid), elution procedure: the flow rate is 0.5mL/min;0-2min, 2-100% B;2-8min,100% B-100% B;8-8.01min,100% B-2% B; the adduct content in the potato chips was measured to be 1.89.+ -. 0.30. Mu.g/kg at 2% B for 8.01-18 min.
(2)
S0. raw material acquisition:
potato chips purchased from local supermarket (Bengao Sha Wu)
S1, adduct extraction:
taking a proper amount of n-hexane for degreasing, continuously adding n-hexane for three times until the n-hexane is not over the potato chips (placed in a fume hood for 30 min/time and 3 times), stirring at intervals, weighing 1g of sample after the n-hexane is volatilized, sequentially adding 10mL of 50% methanol/water for extraction (three times and 10 min/time) and centrifuging at 10000rpm/min for 30min, taking supernatant, spin-drying, adding 6mL of 50% methanol/water for re-dissolution, and passing through a 0.22 mu m organic filter membrane
S2 HPLC_MS/MS detection of adduct content
Adopts MRM positive ion mode, and the selected ion groups are as follows: 227/168 (quantitative ion pairs, collision energy 20 eV); 227/181 (collision energy 22 eV); 227/122 (collision energy 22 eV), mobile phase: phase a/phase B: water/acetonitrile (0.1% formic acid), elution procedure: the flow rate is 0.5mL/min;0-2min, 2-100% B;2-8min,100% B-100% B;8-8.01min,100% B-2% B; the adduct content in the potato chips was measured to be 1.46.+ -. 0.19. Mu.g/kg at 2% B for 8.01-18 min.
(3)
Biscuit processing:
flour, white granulated sugar, shortening, palm oil, ammonium bicarbonate, baking soda, sodium metabisulfite, edible essence, edible salt and amino acid mixture of 0.1-1.5 g amino acid/1 kg total weight are prepared, wherein the amino acid mixture can be 1g of a single amino acid or added according to amino acid with additional health care function. And then rolling, cutting, forming, baking, cooling, packaging and forming according to the conventional processing flow.
After amino acid is added, the content of methylglyoxal and glyoxal in the biscuit can be reduced by 17-23% and not equal.
TABLE 2 content of methylglyoxal and glyoxal in biscuits before and after addition of amino acids
(4)
Processing potato chips:
fresh potatoes are washed, peeled, cut into 1.5mm potato slices, washed three times with deionized water, soaked (0.1-0.5 g amino acid per 100mL water) after surface starch is washed, fried with peanut oil (165 ℃ for 5 min), and then drained and packaged. Wherein the amino acids may be single amino acids or may be used in accordance with the functional collocation of amino acids.
After the degree of the pickling of the amino acid is increased, the content of methylglyoxal and glyoxal in the potato chips can be reduced by 19-25 percent.
TABLE 3 increasing the levels of methylglyoxal and glyoxal in potato chips before and after the amino acid soaking process
The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.
Claims (8)
1. A process for the preparation of an acrolein-amino acid adduct characterized by the steps of: in water, carrying out constant-temperature heating reaction on acrolein and amino acid, and carrying out subsequent treatment to obtain an acrolein-amino acid adduct;
the structural formula of the acrolein-amino acid adduct is at least one of the following six types:
。
2. the method for preparing the acrolein-amino acid adducts according to claim 1, characterized in that:
the amino acid is at least one of aspartic acid, asparagine, glutamic acid, glutamine, valine and threonine.
3. The method for preparing the acrolein-amino acid adducts according to claim 1, characterized in that: the molar ratio of the acrolein to the amino acid is 1-3: 1, a step of; the dosage ratio of the acrolein to the water is (1-8) mmol (1-50) mL.
4. The method for preparing the acrolein-amino acid adducts according to claim 1, characterized in that: the molar ratio of the acrolein to the amino acid is 2:1.
5. the process for the preparation of an acrolein-amino acid adduct of any one of claims 1 to 4, characterized in that: the temperature of the heating reaction is 20-80 ℃; the heating reaction time is 1-12 h.
6. The method for preparing the acrolein-amino acid adducts according to claim 1, characterized in that: the temperature of the heating reaction is 45-65 ℃; the heating reaction time is 2-5 h.
7. The method for preparing the acrolein-amino acid adducts according to claim 1, characterized in that: the subsequent treatment refers to concentration of the reacted system, filtration with a filter membrane, and purification with reverse phase silica gel chromatography to obtain a purified product.
8. The method for preparing an acrolein-amino acid adduct according to claim 7, wherein: the reverse phase silica gel chromatography is specifically: selecting reverse phase silica gel ODS resin, loading, eluting with 5% methanol water solution, collecting target adduct by 2, 4-dinitrophenylhydrazine solution chromogenic method, detecting purity by high performance liquid phase detection method, collecting eluent, and freeze drying to obtain purified sample.
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