CN108459116A - The method of Neu5Gc in a kind of fluorescence high performance liquid chromatography detection fermentation red meat product - Google Patents
The method of Neu5Gc in a kind of fluorescence high performance liquid chromatography detection fermentation red meat product Download PDFInfo
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- CN108459116A CN108459116A CN201810736939.2A CN201810736939A CN108459116A CN 108459116 A CN108459116 A CN 108459116A CN 201810736939 A CN201810736939 A CN 201810736939A CN 108459116 A CN108459116 A CN 108459116A
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Abstract
The invention discloses the methods of Neu5Gc in a kind of fluorescence high performance liquid chromatography detection fermentation red meat product, include the following steps：Prepare 1mmol/L Neu5Gc standard solutions；Neu5Gc Specification Curve of Increasing；The preparation of fermentation red meat product sample solution；Column front derivation：The accurate sample solution for drawing 900 μ L, is added 100 μ LDMB derivative reagents, is protected from light after derivative 2.5h in water-bath and is put into ice water mixed liquor that be quickly cooled down 0.22 film to be measured for 50 DEG C；Use filler for the C18 reverse chromatograms columns of octadecylsilane, fluorescence detector excitation wavelength 373nm, launch wavelength 448nm, 30 DEG C of column temperature, flow velocity is 0.9m L/min, 10 μ L of sampling volume.Wherein mobile phase A is ultra-pure water, and Mobile phase B is methanol, and gradient elution program is：0‑18min:80%A、20%B；18.1‑21min:100%B;21.1‑28min:80%A、20%B；The assay of Neu5Gc in sample.Testing result of the present invention is accurate, reproducible, the specificity and high sensitivity of detection, can effectively reduce detection time, improves detection efficiency, at low cost, small toxicity.
Description
Technical field
The invention belongs to detection technique fields, and in particular in a kind of fluorescence high performance liquid chromatography detection fermentation red meat product
The method of Neu5Gc.
Background technology
There are a variety of battalion remote, being generated by its unique flavor, microbial fermentation in human history for fermentation meat product
It supports substance and is favored by people convenient for the characteristic of preservation.In general, fermentation meat product is mostly with red meat (beef, mutton, pork
Deng) as fermentation raw material fermented product is made.Here red meat refers generally to animal of the red meat fiber more than white meat fiber
Meat, including (model apricot pellet red meats, diet Neu5Gc intakes and the body anti-such as beef, mutton and pork and its converted products
Neu5Gc antibody relationship research [D] Xiamen University master thesis, 2015.).International cancer research on October 26th, 2015
Red meat is classified as and is potentially carcinogenic object by mechanism, this makes red meat and its red meat product become the much-talked-about topic of health problem.Study table
Bright exogenous NeuGc ALPHA2-3Gal (N-glycolylneuraminic acid, referred to as " Neu5Gc ") is that red meat is carcinogenic
The specific specified risk material of property.Therefore, Neu5Gc contents in fermentation red meat product are detected as fermentation red meat system is weighed
The safety problem leading indicator of product.
Neu5Gc is one of primary structure of sialic acid, and the method for detecting sialic acid at present has very much, and common method has
Resorcinol Method based on colorimetric method, it is thio oneself than appropriate sour (2-Thiobarbituric acid, TBA) method and high-efficient liquid phase color
Spectrometry (RP-HPLC), fluorescence high performance liquid chromatography (HPLC-FLD) etc..Wherein《Chinese Pharmacopoeia》Third portion (2015 editions) includes
Resorcinol-hydrochloric acid method (one kind in spectrophotometry) measures the sialic acid content in capsular polysaccharide, this spectrophotometric
Method can only detect total sialic acid content cannot it is actually detected go out Neu5Gc contents.And existing HPLC detection methods are chiefly used in giving birth to
Neu5Gc is detected in scarlet meat and milk, and Neu5Gc content assaying methods also have not been reported in related fermentation red meat product at present.
Due to microbial fermentation decomposing organic matter, metabolite is generated so that the other impurities in fermentation red meat product are interfered than life
Scarlet meat and milk it is big, this just considerably increases detection difficulty.Common fluorescence high performance liquid chromatography testing conditions：Flowing
It is mutually under the conditions of methanol-acetonitrile-ultra-pure water (7: 8: 85, V/V), the separation of the Neu5Gc appearances such as unleavened red meat and milk is good
It is good, but the red meat product target peak that ferments is overlapped with impurity peaks, can not be efficiently separated.Detection time is longer under isocratic condition,
Influence detection efficiency.Using acetonitrile as mobile phase, there are problems that big cost height of toxicity etc..
Invention content
There is provided that a kind of testing result is accurate, reproducible it is an object of the invention to overcome disadvantages mentioned above, the spy of detection
Anisotropic and high sensitivity can effectively reduce detection time, improve detection efficiency, at low cost, the fluorescence high-efficient liquid phase color of small toxicity
The method of Neu5Gc in spectrum detection fermentation red meat product.
The present invention is achieved through the following technical solutions：
The method of Neu5Gc, includes the following steps in a kind of fluorescence high performance liquid chromatography detection fermentation red meat product of the present invention：
(1) Neu5Gc standard solution is prepared
Prepare 1mmol/L Neu5Gc standard solutions；
(2) Neu5Gc Specification Curve of Increasing
Draw the Neu5Gc marks that 1mmol/LNeu5Gc standard solutions are configured to 10,25,50,100,200,300,400 μm of ol/L
Quasi- solution takes 900 μ L of various concentration Neu5Gc standard solution to be separately added into 100 μ L 4,5- methylene-dioxy -1,2- o-phenylenediamines
Salt (4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride, DMB) derivative reagent, Yu Shui
In bath 50 DEG C be protected from light after derivative 2h be put into ice water mixed liquor be quickly cooled down 0.22 film after sample detection, with standard concentration
For horizontal axis, chromatographic peak area is that linear standard curve is made in the longitudinal axis；
(3) preparation of fermentation red meat product sample solution
Fermentation red meat product (China's classical acid Preserved-fish meat, ferment sausage, ham etc.) is taken, according to the raw material shape of fermentation red meat product
Difference is divided into blocky or meat gruel shape fermentation red meat product；
A, meat gruel shape fermentation red meat product sample solution
The accurate saturated ammonium sulfate solution for weighing 1g meat gruels 10ml30% is homogenized with protein precipitation, and is freeze-dried and is gone sulfuric acid
Ammonium, to make Neu5Gc separate outs, sample addition 10ml2mol/L glacial acetic acids after freeze drying hydrolyze 3h in 80 DEG C of water-baths,
Supernatant is taken with the rotating speed centrifugation 10min of 12000r/min, glacial acetic acid is removed with secondary freeze-drying is carried out after 0.45 μm of membrane filtration, freezes
Dry powder is dissolved in 1ml 0.1mol/LNaOH, 37 DEG C of water-bath 30min, and sample solution is made with 0.45 μm of membrane filtration；
B, massive zymolysis red meat product sample solution
The lean meat part for choosing massive zymolysis red meat product takes 1cm intermediate meat sample 1g below that the saturation of 10ml30% is added
Ammonium sulfate is homogenized with protein precipitation, and is freeze-dried removal ammonium sulfate, and 10ml2mol/L ice is added in the sample after freeze-drying
Acetic acid hydrolyzes 3h in 80 DEG C of water-baths, supernatant is taken with the rotating speed centrifugation 10min of 12000r/min, after 0.45 μm of membrane filtration
It carries out secondary freeze-drying and removes glacial acetic acid, freeze-dried powder is dissolved in 1ml 0.1mol/LNaOH, 37 DEG C of water-bath 30min, with 0.45 μm
Sample solution is made in membrane filtration；
(4) column front derivation
Fluorescence high performance liquid chromatography measures the accurate sample solution for drawing 900 μ L, 100 μ LDMB derivative reagents is added, in water-bath
In 50 DEG C be protected from light after derivative 2.5h and be put into ice water mixed liquor that be quickly cooled down 0.22 film to be measured；
(5) fluorescence high-efficient liquid phase chromatogram condition
Use filler for the C18 reverse chromatograms columns of octadecylsilane, fluorescence detector excitation wavelength 373nm, launch wavelength
448nm, 30 DEG C of column temperature, flow velocity are 0.9m L/min, 10 μ L of sampling volume.Wherein mobile phase A is ultra-pure water, and Mobile phase B is first
Alcohol, gradient elution program are：0--18min:80%A, 20%B；18.1--21min:100%B；21.1--28min:80%A,
20%B；
(6) in sample Neu5Gc assay
The sample solution that step (4) obtains is detected by the condition of step (5), measures the peak area size of Neu5Gc, corresponding step
(2) the Neu5Gc standard curves drawn calculate the content of Neu5Gc in sample according to the following formula：
In formula：A is content/(μ g/g or the μ g/mL) of Neu5Gc in sample；m1For Neu5Gc chromatographic peaks in sample and external standard chromatography
The corresponding Neu5Gc mass/μ g of peak area ratio at peak；R is sample extension rate；M is sampling amount/(g or mL).
Compared with prior art, the present invention there is apparent advantageous effect, as can be known from the above technical solutions：It is red according to fermenting
The characteristics of meat products, carries out the pre-treating method of different meat samples, effectively extracts Neu5Gc.By gradient elution, reduce sample inspection
The time is surveyed, detection efficiency is improved.Mobile phase is at low cost using ultra-pure water, methanol, small toxicity.This method can efficiently separate fermentation
Impurity peaks in red meat product and principal component peak, separating degree R>1.5.Testing result is accurate, reproducible, detection specificity and
High sensitivity, 1.6% hereinafter, detection limit (LOD) is 0.001 μm of ol/L, quantitative limit (LOQ) is relative standard deviation (RSD)
0.003μmol/L。
The advantageous effect further illustrated the present invention below by way of specific implementation mode.
Description of the drawings
Fig. 1 is Neu5Gc standard items chromatograms；
Fig. 2 is Neu5Gc standard curves；
Fig. 3 is sample collection of illustrative plates.
Specific implementation mode
Embodiment 1
The method of Neu5Gc in a kind of fluorescence high performance liquid chromatography detection fermentation red meat product, including step once：
(1) Neu5Gc standard solution is prepared
Accurate weighing 0.0825gNeu5Gc standard items, are dissolved in ultra-pure water in 250ml volumetric flasks, are settled to scale, as
1mmol/L Neu5Gc standard solutions.
(2) Neu5Gc Specification Curve of Increasing
Draw the Neu5Gc that 1mmol/L Neu5Gc standard solutions are configured to 10,25,50,100,200,300,400 μm of ol/L
Standard solution takes 900 μ L's of various concentration Neu5Gc standard solution to be separately added into 100 μ LDMB derivative reagents, 50 in water-bath
DEG C be protected from light after derivative 2h be put into ice water mixed liquor be quickly cooled down 0.22 film after sample detection, using standard concentration as horizontal axis,
Chromatographic peak area is that linear standard curve is made in the longitudinal axis；
By shown in Neu5Gc standard items chromatogram (Fig. 1), the appearance time of Neu5Gc is 14.484min, and peak shape is good.With mark
Quasi- a concentration of axis of product, corresponding chromatographic peak area is that linear standard curve is made in axis, such as shown in (Fig. 2), the linear mark of Neu5Gc
Directrix curve equation：Y=3.83964x-16.33805, R2=0.99941.
(3) sample solution is prepared
The lean meat part for choosing fermentation ham takes the saturated ammonium sulfate that 10ml30% is added in 1cm intermediate meat sample 1g below molten
Liquid is homogenized with protein precipitation, and is freeze-dried removal ammonium sulfate.To make Neu5Gc separate outs, the sample after freeze-drying is added
10ml2mol/L glacial acetic acids hydrolyze 3h in 80 DEG C of water-baths, take supernatant with the rotating speed centrifugation 10min of 12000r/min, use
Secondary freeze-drying is carried out after 0.45 μm of membrane filtration removes glacial acetic acid.Freeze-dried powder is dissolved in 1ml 0.1mol/LNaOH, 37 DEG C of water-baths
Sample solution is made with 0.45 μm of membrane filtration in 30min.
(4) column front derivation
The accurate sample solution for drawing 900 μ L, is added 100 μ LDMB derivative reagents, in water-bath 50 DEG C be protected from light derivative 2.5h after
It is put into ice water mixed liquor that be quickly cooled down 0.22 film to be measured.
(5) fluorescence high-efficient liquid phase chromatogram condition
Fluorescence detector is combined using 1260 liquid chromatographs of Agilent, selects Agilent ZOTBAX Eclipse XDB-
C18 reverse chromatograms column (250mm × 4.6mm, 5 μm), fluorescence detector excitation wavelength 373nm, launch wavelength 448nm, column temperature 30
DEG C, flow velocity is 0.9m L/min, 10 μ of sampling volume.Wherein mobile phase A is ultra-pure water, and Mobile phase B is methanol, gradient elution journey
Sequence is：0-18min:80%A, 20%B；18.1-21min:100%B；21.1-28min:80%A, 20%B；
(6) ferment ham in Neu5Gc assay
The peak area average value of Neu5Gc is 286.7 in actually measured traditional zymotic ham, the Neu5Gc drawn according to step (2)
Standard curve and following formula：
In formula：A is content/(μ g/g or the μ g/mL) of Neu5Gc in sample；m1For Neu5Gc chromatographic peaks in sample and external standard chromatography
The corresponding Neu5Gc mass/μ g of peak area ratio at peak；R is sample extension rate；M is sampling amount/(g or mL).
Neu5Gc contents in traditional zymotic ham, which are calculated, is：25.67μg/g.
Embodiment 2
Fluorescence efficient liquid phase detects the content of Neu5G in 30 days acid Preserved-fish meat of traditional zymotic：
(1) sample solution is prepared
The lean meat part for choosing 30 days sour Preserved-fish meat of traditional zymotic takes 1cm intermediate meat sample 1g below to be added 10ml30%'s
Saturated ammonium sulfate solution is homogenized with protein precipitation, and is freeze-dried removal ammonium sulfate.To make Neu5Gc separate outs, after freeze-drying
Sample 10ml2mol/L glacial acetic acids be added hydrolyze 3h in 80 DEG C of water-baths, taken with the rotating speed of 12000r/min centrifugation 10min
Supernatant removes glacial acetic acid with secondary freeze-drying is carried out after 0.45 μm of membrane filtration.Freeze-dried powder is dissolved in 1ml 0.1mol/LNaOH,
Sample solution is made with 0.45 μm of membrane filtration in 37 DEG C of water-bath 30min.
(2) column front derivation
The accurate sample solution for drawing 900 μ L, is added 100 μ LDMB derivative reagents, in water-bath 50 DEG C be protected from light derivative 2.5h after
It is put into ice water mixed liquor that be quickly cooled down 0.22 film to be measured.
(3) fluorescence high-efficient liquid phase chromatogram condition
Fluorescence detector is combined using 1260 liquid chromatographs of Agilent, selects Agilent ZOTBAX Eclipse SB-
C18 reverse chromatograms column (250mm × 4.6mm, 5 μm), fluorescence detector excitation wavelength 373nm, launch wavelength 448nm, column temperature 30
DEG C, flow velocity is 0.9m L/min, 10 μ of sampling volume.Wherein mobile phase A is ultra-pure water, and Mobile phase B is methanol, gradient elution journey
Sequence is：0-18min:80%A, 20%B；18.1-21min:100%B；21.1-28min:80%A, 20%B；
(7) in acid Preserved-fish meat Neu5Gc assay
Fluorescence efficient liquid phase detects shown in the chromatogram (Fig. 3) of acid Preserved-fish meat, and the peak shape of gained is good and has higher point with impurity
From degree (R>1.5).
The peak area average value of Neu5Gc is 303.095 in actually measured acid Preserved-fish meat, according to Neu5Gc standards in embodiment 1
Curve and following formula：
In formula：A is content/(μ g/g or the μ g/mL) of Neu5Gc in sample；m1For Neu5Gc chromatographic peaks in sample and external standard chromatography
The corresponding Neu5Gc mass/μ g of peak area ratio at peak；R is sample extension rate；M is sampling amount/(g or mL).
Neu5Gc contents in 30 days sour Preserved-fish meat of traditional zymotic, which are calculated, is：27.06μg/g.
Embodiment 3
Fluorescence efficient liquid phase detects the content of Neu5G in salami：
(1) sample solution is prepared
Casing is peelled off, the saturated ammonium sulfate solution for accurately weighing 1g meat gruels 10ml30% is homogenized with protein precipitation, and is freezed dry
Dry removal ammonium sulfate.To make Neu5Gc separate outs, 10ml2mol/L glacial acetic acids are added in 80 DEG C of water-baths in the sample after freeze-drying
3h is hydrolyzed in pot, supernatant is taken with the rotating speed centrifugation 10min of 12000r/min, is removed with secondary freeze-drying is carried out after 0.45 μm of membrane filtration
Remove glacial acetic acid.Freeze-dried powder is dissolved in 1ml 0.1mol/LNaOH, 37 DEG C of water-bath 30min, and sample is made with 0.45 μm of membrane filtration
Solution.
(2) column front derivation
The accurate sample solution for drawing 900 μ L, is added 100 μ LDMB derivative reagents, in water-bath 50 DEG C be protected from light derivative 2.5h after
It is put into ice water mixed liquor that be quickly cooled down 0.22 film to be measured.
(3) fluorescence high-efficient liquid phase chromatogram condition
Fluorescence detector is combined using 1260 liquid chromatographs of Agilent, selects Agilent ZOTBAX Eclipse XDB-
C18 reverse chromatograms column (250mm × 4.6mm, 5 μm), fluorescence detector excitation wavelength 373nm, launch wavelength 448nm, column temperature 30
DEG C, flow velocity is 0.9m L/min, 10 μ of sampling volume.Wherein mobile phase A is ultra-pure water, and Mobile phase B is methanol, gradient elution journey
Sequence is：0-18min:80%A, 20%B；18.1-21min:100%B；21.1-28min:80%A, 20%B；
(8) in salami Neu5Gc assay
The peak area average value of actually measured salami Neu5G is 172.6, according to Neu5Gc standard curves in embodiment 1
And following formula：
In formula：A is content/(μ g/g or the μ g/mL) of Neu5Gc in sample；m1For Neu5Gc chromatographic peaks in sample and external standard chromatography
The corresponding Neu5Gc mass/μ g of peak area ratio at peak；R is sample extension rate；M is sampling amount/(g or mL).
Neu5Gc contents in salami, which are calculated, is：16.01μg/g.
The above described is only a preferred embodiment of the present invention, being not intended to limit the present invention in any form, appoint
What is simply repaiied to any made by above example according to the technical essence of the invention without departing from technical solution of the present invention content
Change, equivalent variations and modification, in the range of still falling within technical solution of the present invention.
Claims (2)
1. the method for Neu5Gc, includes the following steps in a kind of fluorescence high performance liquid chromatography detection fermentation red meat product：
（1）Neu5Gc standard solution is prepared
Prepare 1mmol/L Neu5Gc standard solutions；
（2）Neu5Gc Specification Curve of Increasing
Draw the Neu5Gc marks that 1mmol/LNeu5Gc standard solutions are configured to 10,25,50,100,200,300,400 μm of ol/L
Quasi- solution takes 900 μ L of various concentration Neu5Gc standard solution to be separately added into 100 μ LDMB derivative reagents, 50 DEG C in water-bath
Be protected from light after derivative 2.5h be put into ice water mixed liquor be quickly cooled down 0.22 film after sample detection, using standard concentration as horizontal axis,
Chromatographic peak area is that linear standard curve is made in the longitudinal axis；
（3）The preparation of fermentation red meat product sample solution
Fermentation red meat product is taken, the raw material shape according to fermentation red meat product is different, is divided into blocky or meat gruel shape fermentation red meat system
Product；
A, meat gruel shape fermentation red meat product sample solution
The accurate saturated ammonium sulfate solution for weighing 1g meat gruels 10ml30% is homogenized with protein precipitation, and is freeze-dried and is gone sulfuric acid
Ammonium, to make Neu5Gc separate outs, sample addition 10ml2mol/L glacial acetic acids after freeze drying hydrolyze 3h in 80 DEG C of water-baths,
Supernatant is taken with the rotating speed centrifugation 10min of 12000r/min, glacial acetic acid is removed with secondary freeze-drying is carried out after 0.45 μm of membrane filtration,
Freeze-dried powder is dissolved in 1ml 0.1mol/LNaOH, 37 DEG C of water-bath 30min, and sample solution is made with 0.45 μm of membrane filtration；
B, massive zymolysis red meat product sample solution
The lean meat part for choosing massive zymolysis red meat product takes 1cm intermediate meat sample 1g below that the saturation sulphur of 10ml30% is added
Acid ammonium solution is homogenized with protein precipitation, and is freeze-dried removal ammonium sulfate, and 10ml2mol/L ice second is added in the sample after freeze-drying
Acid hydrolyzes 3h in 80 DEG C of water-baths, supernatant is taken with the rotating speed centrifugation 10min of 12000r/min, after 0.45 μm of membrane filtration
It carries out secondary freeze-drying and removes glacial acetic acid, freeze-dried powder is dissolved in 1ml 0.1mol/LNaOH, 37 DEG C of water-bath 30min, with 0.45 μm
Sample solution is made in membrane filtration；
（4）Column front derivation
Fluorescence high performance liquid chromatography measures the accurate sample solution for drawing 900 μ L, 100 μ LDMB derivative reagents is added, in water-bath
In 50 DEG C be protected from light after derivative 2.5h and be put into ice water mixed liquor that be quickly cooled down 0.22 film to be measured；
（5）Fluorescence high-efficient liquid phase chromatogram condition
Use filler for the C18 reverse chromatograms columns of octadecylsilane, fluorescence detector excitation wavelength 373nm, launch wavelength
448nm, 30 DEG C of column temperature, flow velocity are 0.9m L/min, 10 μ L of sampling volume；
Wherein mobile phase A is ultra-pure water, and Mobile phase B is methanol, and gradient elution program is：0--18min:80%A、20%B；
18.1--21min:100%B;21.1--28min:80%A、20%B；
（6）The assay of Neu5Gc in sample
Step（4）Obtained sample solution is by step（5）Condition detection, measure the peak area size of Neu5Gc, corresponding step
（2）The Neu5Gc standard curves of drafting calculate the content of Neu5Gc in sample according to the following formula：
In formula：For Neu5Gc in sample content/（μ g/g or μ g/mL）；For Neu5Gc chromatographic peaks in sample with
The corresponding Neu5Gc mass/μ g of peak area ratio of external standard chromatographic peak；For sample extension rate；For sampling amount/（G or
mL）.
2. the method for Neu5Gc in a kind of fluorescence high performance liquid chromatography detection fermentation red meat product as described in claim 1,
In：Step（3）Middle fermentation red meat product is acid Preserved-fish meat, ferment sausage, ham.
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