CN107782757A - A kind of method using 1H NMR rapid evaluation wheat embryo Lipid Rancidity in Formula Feeds degree - Google Patents
A kind of method using 1H NMR rapid evaluation wheat embryo Lipid Rancidity in Formula Feeds degree Download PDFInfo
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Abstract
The invention discloses a kind of method using 1H NMR rapid evaluation wheat embryo Lipid Rancidity in Formula Feeds degree, belong to grain and oil Quality Detection field.The present invention with nuclear magnetic resonance (1H NMR) technology is as detection means, after being pre-processed to wheat embryo, by FFA, monoglyceride, diglyceride and the content of triglyceride in characteristic peak area situation of change calculating wheat germ lipid on nuclear magnetic spectrum, so as to assess its degree of becoming sour.
Description
Technical field
The present invention relates to a kind of method using 1H-NMR rapid evaluation wheat embryo Lipid Rancidity in Formula Feeds degree, belong to grain and oil product
Quality detection field.
Background technology
China is one of wheat main production country, and wheat is mainly made up of endosperm, wheat bran and plumule three parts, accounts for seed respectively and do
81-84%, 14-16% and 2-3% of weight.Wheat embryo is the byproduct in wheat processing, and it is enriched the big portion of wheat seed
The nutriment divided, is described as " natural nutrition treasure-house " by nutritionist.
Wheat-germ oil (WGO) main component is triglycerides.Wheat embryo lipid total unrighted acid in being rich in
(UFA) and polyunsaturated fatty acid (PUFA) content is respectively up to 80% and more than 60%.In addition, wheat-germ oil is also rich in more
The fat-soluble trace activity substance of kind, such as tocopherol (VE), phytosterol and plant polyphenol etc..Wheat-germ oil has a variety of physiology
Activity, such as reducing blood lipid, anti-oxidant, antifatigue, therefore, wheat-germ oil is described as the preciousness oil of great alimentary health-care function
Fat.
However, China wheat embryo mainly or as feed is sold at present, added value is relatively low.Its main cause is：Embryo
Bud is the part that physiologically active is most strong in wheat seed, and moisture and lipid content are high.However, when wheat embryo and wheat seed point
From rear, under the catalytic action of high vigor fat hydrolase, neutral fats (triglycerides, TAG) hydrolyzes rapidly plumule lipid
A series of products such as reaction, generation diglyceride (DAG), monoglyceride (MAG), free fatty (FFA) and glycerine, cause wheat germ
Lipid acid number rises rapidly.And under the catalysis of cereal oxygenase (lipoxidase) further lipid oxygen can also occur for FFA
Change reaction, be cracked into the products such as aldehyde, ketone, acid, alcohol and the ester of small molecule, cause wheat germ lipid to produce peculiar smell, finally lose edible
Value.
Therefore, in wheat embryo processing and storage, researcher employs measure FFA contents, peroxide value, fennel
The indexs such as fragrant amine value, iodine value assess the degree of becoming sour of lipid, and FFA contents are key indexs in these parameters, FFA too high levels
Cause oil and fat refining cost too high, so as to lose refining value.
At present, cereal and its accessory substance FFA contents of becoming sour are evaluated and typically use titration, more using ethanol, ether conduct
Solvent, alkali blue or phenolphthalein are as titrate indicator, with FFA present in cereal lipid, to be consumed in aqueous slkali according to titration
Alkali lye volume and concentration calculate FFA contents therein.But in actual applications, uranidin in one side grease is deposited
Titration end-point (micro- red) is being caused to be difficult to judge, measurement result error is larger, the professional skill proficiency requirement to determining personnel
It is higher；On the other hand etoh solvent, the ether used in determining is inflammable, explosive organic solvent, and wherein ether belongs to easy
Malicious reagent is made, there is neurotoxicity, while easily cause environmental pollution.Therefore, establish a kind of simple, convenient, high sensitivity new
Method is come to replace traditional titration be extremely urgent.
In recent years, nuclear magnetic resonance technique is widely used in analysis detection field, and the technology is located before having sample
The advantages that reason is simple, favorable reproducibility, high sensitivity, detection rates are fast and data message amount is big, nuclear magnetic resonance technique is main at present
For the detection of aliphatic acid in grease, the completely not detection to wheat embryo Lipid Rancidity in Formula Feeds degree, how nuclear magnetic resonance is utilized
Technology realizes that detection wheat embryo Lipid Rancidity in Formula Feeds degree accurate, that the degree of accuracy is high is technical problem urgently to be resolved hurrily.
The content of the invention
The invention provides a kind of rapid assay methods of wheat embryo Lipid Rancidity in Formula Feeds degree, it be with nuclear magnetic resonance (1H-
NMR) for technology as detection means, FFA, the list calculated by characteristic peak area situation of change on nuclear magnetic spectrum in wheat germ lipid is sweet
Fat, diglyceride and content of triglyceride, so as to assess its degree of becoming sour.
The detection method of the present invention, comprises the following steps：
(1) pre-process：Grain sample is crushed, sieved；
(2) extract：Add solvent to be mixed, concussion extraction, obtain mixed liquor；
(3) concentrate：Mixed liquor is centrifuged, pours out supernatant, distilled water is added in supernatant, is shaken, is stood, after layering,
Lower floor's solution is taken out, drying up lower floor's solution with nitrogen obtains total lipid；
(4) sample introduction：The total lipid that step (3) obtains is put in nuclear magnetic tube, the lipid is carried out1H spectrum analyses；
(5) calculate：According to the relative amount of each lipid composition of the calculated by peak area of each Characteristic chemical shifts.
In one embodiment of the invention, the grain sample is wheat embryo, wheat bran, rice bran or maize
Bud.
In one embodiment of the invention, sieving is 30-120 mesh sieves in the step (1).
In one embodiment of the invention, the solvent in the step (2) is to contain 0.15-0.50% (m:M) it is special
Chloroform/methanol (the 1-3 of butylhydroquinone (TBHQ):1, v/v) solution.
In one embodiment of the invention, step (2) is oscillation extraction 30- under 150-250rpm after adding solvent
60min, extract 3-5 times repeatedly, merge organic phase.
In one embodiment of the invention, step (3) is that mixed liquor 3000-6000rpm is centrifuged into 5-20min, is fallen
Go out supernatant, 10-40mL distilled water is added in supernatant, shake 20-60sec, stand, after layering, lower floor is drawn with dropper
Chloroform layer, it is transferred in teat glass, nitrogen drying, remaining extract remainder is the total lipid in wheat embryo.
In one embodiment of the invention, in step (4) sample introduction method for take the above-mentioned total lipids of 20-100mg in
In 5mm nuclear magnetic tubes, 200-600 μ L (v containing 0.01-0.05% are then added:V) deuterochloroform of trimethyl silane (TMS), capping
Afterwards, the lipid is carried out with 400MHz nuclear magnetic resonance spectrometers1H spectrum analyses.
In one embodiment of the invention, the computational methods of the relative amount of step (5) each lipid composition be must
The peak area for each chemical shift arrived substitutes into following formula and calculated：
MT=M1-MAG+M2-MAG+M1,2-DAG+M1,3-DAG+MTAG+MFFA
Further its content can be calculated according to the molar percentage of lipid each component：
(1)
(2)
(3)
(4)
(5)
(6)
Wherein, k is mole conversion constant (k can be removed in formula 1~6, can be denoted as any real number), A2Represent 1-
The relaxation peak area of the Hydrogen Proton at remote acyl group end, A on MAG carbon skeletons3Represent the Hydrogen Proton at glycerine end on 1,2-DAG carbon skeletons
The area at relaxation peak, A4Represent the Hydrogen Proton relaxation peak area on 2-MAG carbon skeletons 1,3, A5Represent 1-MAG carbon skeletons 2
The relaxation peak area of position Hydrogen Proton, A4.01-4.40Represent wheat-germ oil1On H-NMR collection of illustrative plates between chemical shift 4.01-4.40
The Hydrogen Proton relaxation peak gross area, A4.23-4.40Represent wheat-germ oil1Hydrogen between chemical shift 4.23-4.40 on H-NMR collection of illustrative plates
The proton relaxation peak gross area；M1-MAG、M2-MAG、M1,2-DAG、M1,3-DAG、MTAGAnd MFFARepresent respectively 1-MAG, 2-MAG, 1,2-DAG,
1,3-DAG, TAG and FFA molal quantity, and MTThen represent 1-MAG, 2-MAG, 1,2-DAG, 1,3-DAG, TAG and FFA
Molal quantity sum；W1-MAG(%), W2-MAG(%), W1,2-DAG(%), W1,3-DAG(%), WTAG(%) and WFFA(%) is represented respectively
The percentage composition of 1-MAG, 2-MAG, 1,2-DAG, 1,3-DAG, TAG and FFA mass in wheat-germ oil.
The lipid composition calculation formula such as FFA employs normalization method in the present invention, i.e. the molar content of one-component accounts for respectively
The percent value sum of component molar total content is 100%, and the relaxation peak face of the molal quantity of each component and its Hydrogen Proton
Product direct ratio, therefore using one-component Hydrogen Proton relaxation peak area account for each component Hydrogen Proton total relaxation peak area ratio
Solve its relative amount.
Table 1
Specifically, in step (4), it is described1H-NMR testing conditions are：Resonant frequency is more than 400MHz；90 ° of pulses；Spectrum
Wide 5-10kHz；Sample 1-6s；Scanning 50-200 times；Trimethyl silane is internal standard (TMS, d=0).
Beneficial effects of the present invention：
(1) present invention uses magnetic resonance detection technology for detection wheat embryo Lipid Rancidity in Formula Feeds degree first.This method has
The advantages that high sensitivity, fast detection rates；, it is necessary to configure NaOH/KOH alcoholic solutions, embryo first in conventional titration method continuous mode
Bud oil solvent (ether/ethanol solution) and phenolphthalein indicator, time-consuming (more than 30min), and titrating solution can not preserve for a long time, portion
Divide solvent such as ether inflammable and harmful, when greatly shortening detection using magnetic nuclear resonance method detection time (10min)
Between, and without any inflammable and explosive reagent, while information content is bigger, can obtain containing for FFA, DAG, MAG and TAG simultaneously
Amount.
(2) in conventional titration method continuous mode, in grease the presence of uranidin substantially increase the judgement of titration end-point
Difficulty, this is easy for causing, and titration measuring result stability is bad, poor reproducibility, will to the professional operation skill for determining personnel
Ask higher.By contrast, magnetic resonance detection technology for detection is simple to operate, favorable reproducibility, and evaluated error is within 2%.
(3) present invention can apply to the detection of other solid matters such as rice bran, wheat bran, corn.This method improves measure
Accuracy, it is practical, detected for wheat quality, food security has positive directive function.
Brief description of the drawings
Influence of Fig. 1 extraction times to wheat germ FFA measurement results
Influence of Fig. 2 mesh sizes to wheat germ FFA measurement results
Influence of Fig. 3 differences extractant to measurement result
Embodiment
The present invention is further elaborated with reference to embodiment.
Embodiment 1
It is sweet accurately to weigh 1- Masine 35-1s, 2- glyceryl monooleates, 1,2- glyceryl dioleates, the oleic acid of 1,3- bis-
Grease, olein and each 30mg of linoleic standard items, in 5mm nuclear magnetic tubes, then add 400 μ L and contain 0.03%
(v:V) the deuterochloroform vibration of trimethyl silane (TMS) is mixed, and after capping, the lipid is entered with 400MHz nuclear magnetic resonance spectrometers
The spectrum analysis of row hydrogen.Testing conditions：90 ° of pulses；Spectrum width 6kHz；Sample 5s；Scanning 60 times；Relaxation delay 3s.Then use
MestReNova softwares are to respectively to each model compound1H-NMR spectrum is handled and analyzed, and records each model compound
Chemical shift, as 1-MAG, 2-MAG, 1,2-DAG, 1 in wheat embryo lipid, 3-DAG, TAG and FFA qualitative foundation.
All chemical shifts of each model substance hydrogen spectrum and corresponding group are as follows：
1- Masine 35-1s：1.632ppm(-OCO-CH2-CH 2-), 2.353ppm (ROCH2-CHOH-CH 2OH),
3.65ppm(ROCH2-CHOH-CH 2OH), 3.94ppm (ROCH2-CHOH-CH2OH), 4.18ppm (ROCH 2-CHOH-CH2OH)。
2- glyceryl monooleates：1.644ppm(-OCO-CH2-CH 2-), 2.379ppm (ROCH2-CHOH-CH 2OH),
2.38ppm(-OCO-CH 2-), 3.84ppm (HOCH 2-CH(OR)-CH 2OH), 4.93ppm (HOCH2-CH(OR)-CH2OH)。
1,3- glyceryl dioleates：1.630ppm(-OCO-CH2-CH 2-), 2.349ppm (- OCO-CH 2-), 4.05-
4.22ppm(ROCH 2-CHOH-CH 2OR’)。
1,2- glyceryl dioleates：1.62ppm(-OCO-CH2-CH 2-), 2.33ppm (- OCO-CH 2-), 3.73ppm
(ROCH2-CH(OR’)-CH 2OH), 4.28ppm (ROCH 2-CH(OR’)-CH2OH), 5.08ppm (ROCH2-CH(OR’)-
CH2OH)。
Olein：1.610ppm(-OCO-CH2-CH 2-), 2.26-2.36ppm (- OCO-CH 2-), 4.22ppm
(ROCH 2-CH(OR’)-CH 2OR "), 5.27ppm (ROCH2-CH(OR’)-CH2OR”)。
Linoleic acid：0.89ppm(-CH 3), 1.19-1.42ppm (- (CH 2) n-), 1.633ppm (- OCO-CH2-CH 2-),
1.92–2.15ppm(-CH 2- CH=CH-), 2.348ppm (- OCO-CH 2-), 2.77ppm (=HC-CH 2- CH=), 5.28-
5.46ppm(-CH=CH-)。
Embodiment 2
Wheat embryo is taken from Flour production workshop, crushed 100 mesh sieves, extracting screen underflow 4g is placed in conical flask, is then added
Enter 40mL and contain 0.30% (m:M) TBHQ chloroform/methanol (2:1, v/v) solution, oscillation extraction 30min under 200rpm, repeatedly
Extraction 3 times, merge organic phase.Subsequent 3000rpm centrifugations 10min, pours out the settled solution of upper strata chloroform/methanol, adds thereto
Enter the distilled water of 20mL volumes, acutely vibrate 30sec, after stratification, lower floor's chloroform layer is carefully drawn with dropper, is transferred to clean
In net teat glass, nitrogen drying, remaining extract remainder is the total lipid in wheat embryo；Take the above-mentioned total lipids of 50mg in
In 5mm nuclear magnetic tubes, then add 400 μ L and contain 0.03% (v:V) TMS deuterochloroform vibration mixes, after capping, with 400MHz cores
Nuclear magnetic resonance spectrometer carries out hydrogen spectrum analysis to the lipid.Testing conditions：90 ° of pulses；Spectrum width 6kHz；Sample 5s；Scanning 60 times；Relaxation
Postpone 3s.
The peak area of each Hydrogen Proton characteristic peak is respectively A4=0.57, A5=0.73, A2+A3=11.67, A4.01-4.40=
143.25, A4.23-4.40=58.07, A2.25-2.40=214.56, A1=214.19；Wheat embryo fat can then be calculated by substituting into formula
In matter TAG, 1,2-DAG, 1,3-DAG, 1-MAG, 2-MAG and FFA relative amount be respectively 60.08%, 11.58%,
11.64%th, 1.66%, 0.32% and 14.72%.Because FFA contents are more than 10%, show the batch wheat embryo Lipid Rancidity in Formula Feeds
It is strong, it is necessary to extract its lipid rapidly and carry out depickling processing.
Embodiment 3
Wheat embryo is taken to crush 0,30,60,80,100,120 mesh sieves, extracting screen underflow 3g is placed in conical flask, is then added
Enter 30mL and contain 0.50% (m:M) TBHQ chloroform/methanol (2:1, v/v) solution, oscillation extraction 30min under 200rpm, repeatedly
Extraction 3 times, merge organic phase.Organic phase 3000rpm is then centrifuged into 10min, pours out the settled solution of upper strata chloroform/methanol,
The distilled water of 20mL volumes is added thereto, acutely vibrates 30sec, and after stratification, lower floor's chloroform layer is carefully drawn with dropper,
It is transferred in the teat glass of cleaning, nitrogen drying, remaining extract remainder is the total lipid in wheat embryo；Take 50mg above-mentioned total
Lipid then adds 400 μ L and contains 0.03% (v in 5mm nuclear magnetic tubes:V) TMS deuterochloroform vibration mixes, after capping, with
400MHz nuclear magnetic resonance spectrometers carry out hydrogen spectrum analysis to the lipid.Mesh size is to influence Fig. 1 of wheat germ FFA measurement results, in advance
Increasing wheat germ particle diameter in processing to reduce, FFA measured values gradually increase, and after crossing mesh size more than 80 mesh, FFA measurement results are without aobvious
Write difference.
Embodiment 4
Wheat embryo was crushed into 80 mesh sieves, takes and crushes sample 2g in conical flask, 20mL is then added and contains 0.15%
(m:M) TBHQ chloroform/methanols (2:1, v/v) solution, oscillation extraction 30min under 200rpm, extract 0,1,2,3,4 and 5 time repeatedly,
Merge organic phase.Organic phase 3000rpm is then centrifuged into 10min, pours out the settled solution of upper strata chloroform/methanol, is added thereto
Enter the distilled water of 20mL volumes, acutely vibrate 30sec, after stratification, lower floor's chloroform layer is carefully drawn with dropper, is transferred to clean
In net teat glass, nitrogen drying, remaining extract remainder is the total lipid in wheat embryo；Take the above-mentioned total lipids of 50mg in
In 5mm nuclear magnetic tubes, then add 400 μ L and contain 0.03% (v:V) TMS deuterochloroform vibration mixes, after capping, with 400MHz cores
Nuclear magnetic resonance spectrometer carries out hydrogen spectrum analysis to the lipid.Extraction times are to shown in influence Fig. 2 of wheat germ FFA measurement results.It can be seen that in advance
Increasing extraction times in processing, FFA measured values gradually increase, and after extracting 3 times repeatedly, FFA measurement results are without significant difference.
Embodiment 5
Wheat embryo was crushed into 80 mesh sieves, takes and crushes tri- parts of sample 3g in three conical flasks, be separately added into containing
0.15% (m:M) TBHQ petroleum ethers, n-hexane or chloroform/methanol (2:1, v/v) each 30Ml is shaken under 200rpm as extractant
Extraction 30min is swung, is extracted 3 times repeatedly, merges organic phase.Organic phase 5000rpm is then centrifuged into 10min, pours out the clear of upper strata
Clear reagent, petroleum ether and n-hexane are transferred in the glass tube of cleaning respectively, and the distillation of 20mL volumes is added into chloroform/methanol
Water, acutely vibrates 30sec, and after stratification, lower floor's chloroform layer is carefully drawn with dropper, is transferred in the teat glass of cleaning,
Organic reagent is dried up using nitrogen, collects the wheat germ lipid of three kinds of reagent extractions；The above-mentioned total lipids of 40mg are taken in 5mm nuclear magnetic tubes
In, then add 400 μ L and contain 0.03% (v:V) TMS deuterochloroform vibration mixes, after capping, with 400MHz nuclear magnetic resoance spectrums
Instrument carries out hydrogen spectrum analysis to the lipid.Extractant is to shown in influence Fig. 3 of wheat germ FFA measurement results.It can be seen that chloroform/methanol
Wheat germ lipid FFA, MAG and DAG content being obtained by extraction are higher than n-hexane and petroleum ether.
Embodiment 6
Certain wheat germ sample comminution is crossed into 80 mesh sieves, extracting screen underflow 3g is placed in conical flask, is then added 30mL and is contained
0.25% (m:M) TBHQ chloroform/methanols (2:1, v/v) solution, oscillation extraction 30min under 200rpm, extract 2 times, merge repeatedly
Organic phase.Subsequent 4000rpm centrifugations 10min, pours out the settled solution of upper strata chloroform/methanol, adds 30mL volumes thereto
Distilled water, acutely vibrates 30sec, and after stratification, lower floor's chloroform layer is carefully drawn with dropper, is transferred to the teat glass of cleaning
In, nitrogen drying, remaining extract remainder is the total lipid in wheat embryo；The above-mentioned total lipids of 30mg are taken in 5mm nuclear magnetic tubes, with
After add 400 μ L and contain 0.03% (v:V) TMS deuterochloroform is mixed, and hydrogen is carried out to the lipid with 400MHz nuclear magnetic resonance spectrometers
Spectrum analysis, replication 5 times.The peak area of each Hydrogen Proton characteristic peaks of wheat germ A is respectively A4=4.09, A5=9.50, A2+A3=
92.39 A4.01-4.40=2263.1, A4.23-4.40=976.67, A2.25-2.40=3377.31, A1=3367.86, substitute into formula then
The relative amount that TAG, 1,2-DAG, 1,3-DAG, 1-MAG, 2-MAG and FFA in wheat germ can be calculated is respectively 71.88%,
5.61%th, 8.89%, 1.45%, 0.16%, 12.00%.Wherein 5 FFA assay results are respectively 12.00%,
11.97%th, 12.03%, 12.02%, 11.98%, average 12.00%, RSD=2%.
As reference, using the FFA contents of titration measuring wheat germ, wheat germ 50g is taken in conical flask, is adding 500mL just
Hexane or petroleum ether, stirring extraction 30min, are extracted 3 times repeatedly, and subsequent 4000rpm centrifuges 15min, are then evaporated under reduced pressure and are removed
Organic reagent.Wheat-germ oil 1.5g is taken in conical flask, 50mL adds ether/ethanol solution and mixed, and adds 2 drop phenolphthalein indicators,
Then with the titration of 0.01mol/LKOH ethanol solutions to terminal (blush).5 FFA assay results are respectively 11.67%,
11.43%th, 11.84%, 11.35%, 11.58%, average 11.57%, RSD=14.72%.
The good and bad contrast of 2. two methods of table, 5 replication links
From table 2,5 measure of nuclear magnetic resonance method are time-consuming shorter, and consumption solvent is less, and sampling amount is less, while result
Stability and reappearance are significantly better than conventional titration method.
Embodiment 7
Certain oil-fat plant is sampled detection to wheat germ sample, counts 3 batches of samples, is denoted as wheat germ A, wheat germ B and wheat respectively
Embryo C, 30 mesh sieves are crushed, extracting screen underflow 4g is placed in conical flask, is then added 40mL and is contained 0.40% (m:M) TBHQ chloroforms/
Methanol (2:1, v/v) solution, oscillation extraction 45min under 200rpm, extract 2 times repeatedly, merge organic phase.Subsequent 4000rpm from
Heart 10min, the settled solution of upper strata chloroform/methanol is poured out, adds the distilled water of 30mL volumes thereto, acutely vibrate 30sec,
After stratification, lower floor's chloroform layer is carefully drawn with dropper, is transferred in the teat glass of cleaning, nitrogen drying, remaining raffinate
Thing is the total lipid in wheat embryo；The above-mentioned total lipids of 30mg are taken in 5mm nuclear magnetic tubes, 400 μ L is then added and contains 0.03%
(v:V) TMS deuterochloroform vibration mixes, and after capping, carries out hydrogen spectrum analysis to the lipid with 400MHz nuclear magnetic resonance spectrometers；
The peak area of each Hydrogen Proton characteristic peaks of wheat germ A is respectively A4=4.09, A5=9.50, A2+A3=92.39,
A4.01-4.40=2263.1, A4.23-4.40=976.67, A2.25-2.40=3377.31, A1=3367.86；Each Hydrogen Protons of wheat germ B are special
The peak area for levying peak is respectively A4=0.57, A5=0.98, A2+A3=15.59, A4.01-4.40=252.68, A4.23-4.40=
108.81, A2.25-2.40=384.13, A1=383.92；The peak area of each Hydrogen Proton characteristic peaks of wheat germ C is respectively A4=0.05, A5
=0.08, A2+A3=1.07, A4.01-4.40=21.36, A4.23-4.40=9.49, A2.25-2.40=31.56, A1=31.35.Substitute into
Formula can then calculate TAG, 1,2-DAG, 1,3-DAG, 1-MAG, 2-MAG and FFA in wheat germ A, wheat germ B and wheat germ C lipids
Relative amount is respectively 71.88%, 5.61%, 8.89%, 1.45%, 0.16%, 12.00%；66.55%th, 8.89%,
8.64%th, 1.28%, 0.19%, 14.46%；77.28%th, 7.78%, 7.59%, 1.37%, 0.21% and 5.76%.
Because FFA contents are more than 10%, show that the batch wheat embryo Lipid Rancidity in Formula Feeds is strong, it is necessary to extract it rapidly
Lipid simultaneously carries out depickling processing, and wheat germ A and wheat germ B FFA values have been above 10%, it is necessary to carry out depickling processing, wheat germ C's
FFA values are relatively low, and wheat quality is preferable, it is not necessary to are handled by depickling.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (10)
- A kind of 1. detection method using 1H-NMR rapid evaluation wheat embryo Lipid Rancidity in Formula Feeds degree, it is characterised in that including with Lower step：(1) pre-process：Grain sample is crushed, sieved；(2) extract：Sample after sieving adds solvent and mixed, and concussion extraction, obtains mixed liquor；(3) concentrate：Mixed liquor is centrifuged, pours out supernatant, distilled water is added in supernatant, is shaken, is stood, after layering, is taken out Lower floor's solution, dry up lower floor's solution with nitrogen and obtain total lipid；(4) sample introduction：The total lipid that step (3) obtains is put in nuclear magnetic tube, the lipid is carried out1H spectrum analyses；(5) calculate：According to the relative amount of each lipid composition of the calculated by peak area of each Characteristic chemical shifts.
- 2. according to the method for claim 1, it is characterised in that methods described also include using oleic acid, olein, The single standard product such as 1,3- stearic acid DAG, 1,2- stearic acid -3- tripalmitins are distinguished1H-NMR is analyzed, sweet to determine Oily three esters (TAG), 1,2- diglycerides (1,2-DAG), 1,3- triglycerides (1,3-DAG), 1- monoglycerides (1-MAG), 2- are sweet The characteristic of different Hydrogen Protons on the group such as oily diester (2-MAG) and methyl on free fatty (FFA), methylene, carboxyl Chemical shift.
- 3. according to the method for claim 1, it is characterised in that sieving is 30-120 mesh sieves in the step (1).
- 4. according to the method for claim 1, it is characterised in that the solvent in the step (2) is to contain 0.15-0.50% (m:M) chloroform/methanol (1-3 of tert-butylhydroquinone (TBHQ):1, v/v) solution.
- 5. according to the method for claim 1, it is characterised in that the step (2) is after adding solvent, under 150-250rpm Oscillation extraction 30-60min, extract 3-5 times repeatedly, merge organic phase.
- 6. according to the method for claim 1, it is characterised in that the step (3) be by mixed liquor 3000-6000rpm from Heart 5-20min, pours out supernatant, and 10-40mL distilled water is added in supernatant, shakes 20-60sec, stands, and after layering, uses Dropper draws lower floor's chloroform layer, is transferred in teat glass, and nitrogen drying, remaining extract remainder is total fat in wheat embryo Matter.
- 7. according to the method for claim 1, it is characterised in that the method for sample introduction is to take 20-100mg in the step (4) Above-mentioned total lipid then adds 200-600 μ L (v containing 0.01-0.05% in 5mm nuclear magnetic tubes:V) trimethyl silane (TMS) Deuterochloroform, after capping, the lipid is carried out with 400MHz nuclear magnetic resonance spectrometers1H spectrum analyses.
- 8. according to the method for claim 1, it is characterised in that described in the step (4)1H-NMR testing conditions are：Altogether Vibration frequency is more than 400MHz；90 ° of pulses；Spectrum width 5-10kHz；Sample 1-6s；Scanning 50-200 times；Trimethyl silane is internal standard (TMS, d=0).
- 9. according to the method for claim 1, it is characterised in that the meter of the relative amount of each lipid composition of the step (5) Calculation method is that the peak area for each chemical shift that will be obtained substitutes into following formula calculating：M1-MAG=k × A5,<mrow> <msub> <mi>M</mi> <mrow> <mn>1</mn> <mo>,</mo> <mn>3</mn> <mo>-</mo> <mi>D</mi> <mi>A</mi> <mi>G</mi> </mrow> </msub> <mo>=</mo> <mfrac> <mrow> <mi>k</mi> <mo>&times;</mo> <mrow> <mo>(</mo> <mrow> <msub> <mi>A</mi> <mrow> <mn>4.01</mn> <mo>-</mo> <mn>4.40</mn> </mrow> </msub> <mo>-</mo> <mn>2</mn> <msub> <mi>A</mi> <mrow> <mn>4.23</mn> <mo>-</mo> <mn>4.40</mn> </mrow> </msub> <mo>-</mo> <mn>2</mn> <msub> <mi>A</mi> <mn>5</mn> </msub> </mrow> <mo>)</mo> </mrow> </mrow> <mn>5</mn> </mfrac> <mo>,</mo> <msub> <mi>M</mi> <mrow> <mi>T</mi> <mi>A</mi> <mi>G</mi> </mrow> </msub> <mo>=</mo> <mfrac> <mrow> <mi>k</mi> <mo>&times;</mo> <mrow> <mo>(</mo> <mrow> <mn>2</mn> <msub> <mi>A</mi> <mrow> <mn>4.23</mn> <mo>-</mo> <mn>4.40</mn> </mrow> </msub> <mo>+</mo> <mn>2</mn> <msub> <mi>A</mi> <mn>5</mn> </msub> <mo>-</mo> <msub> <mi>A</mi> <mn>2</mn> </msub> <mo>-</mo> <msub> <mi>A</mi> <mn>3</mn> </msub> </mrow> <mo>)</mo> </mrow> </mrow> <mn>4</mn> </mfrac> </mrow><mrow> <msub> <mi>M</mi> <mrow> <mi>F</mi> <mi>F</mi> <mi>A</mi> </mrow> </msub> <mo>=</mo> <mfrac> <mrow> <mi>k</mi> <mo>&times;</mo> <mrow> <mo>(</mo> <msub> <mi>A</mi> <mrow> <mn>2.25</mn> <mo>-</mo> <mn>2.40</mn> </mrow> </msub> <mo>)</mo> </mrow> </mrow> <mn>2</mn> </mfrac> <mo>-</mo> <mn>3</mn> <msub> <mi>M</mi> <mrow> <mi>T</mi> <mi>A</mi> <mi>G</mi> </mrow> </msub> <mo>-</mo> <mn>2</mn> <mrow> <mo>(</mo> <msub> <mi>A</mi> <mrow> <mn>1</mn> <mo>,</mo> <mn>2</mn> <mo>-</mo> <mi>D</mi> <mi>A</mi> <mi>G</mi> </mrow> </msub> <mo>+</mo> <msub> <mi>A</mi> <mrow> <mn>1</mn> <mo>,</mo> <mn>3</mn> <mo>-</mo> <mi>D</mi> <mi>A</mi> <mi>G</mi> </mrow> </msub> <mo>)</mo> </mrow> <mo>-</mo> <mrow> <mo>(</mo> <msub> <mi>A</mi> <mrow> <mn>1</mn> <mo>-</mo> <mi>M</mi> <mi>A</mi> <mi>G</mi> </mrow> </msub> <mo>+</mo> <msub> <mi>A</mi> <mrow> <mn>2</mn> <mo>-</mo> <mi>M</mi> <mi>A</mi> <mi>G</mi> </mrow> </msub> <mo>)</mo> </mrow> <mo>,</mo> </mrow>MT=M1-MAG+M2-MAG+M1,2-DAG+M1,3-DAG+MTAG+MFFAFurther its content can be calculated according to the molar percentage of lipid each component：(1)(2)(3)(4)(5)(6)Wherein, k is mole conversion constant (k can be removed in formula 1~6, can be denoted as any real number), A2Represent 1-MAG carbon bones The relaxation peak area of the Hydrogen Proton at remote acyl group end, A on frame3Represent the Hydrogen Proton relaxation peak at glycerine end on 1,2-DAG carbon skeletons Area, A4Represent the Hydrogen Proton relaxation peak area on 2-MAG carbon skeletons 1,3, A5Represent 2 hydrogen matter of 1-MAG carbon skeletons The relaxation peak area of son, A4.01-4.40Represent wheat-germ oil1Hydrogen Proton between chemical shift 4.01-4.40 on H-NMR collection of illustrative plates The relaxation peak gross area, A4.23-4.40Represent wheat-germ oil1Hydrogen Proton relaxes between chemical shift 4.23-4.40 on H-NMR collection of illustrative plates The Henan peak gross area；M1-MAG、M2-MAG、M1,2-DAG、M1,3-DAG、MTAGAnd MFFA1-MAG, 2-MAG, 1,2-DAG, 1,3- are represented respectively DAG, TAG and FFA molal quantity, and MTThen represent 1-MAG, 2-MAG, 1,2-DAG, 1,3-DAG, TAG and FFA mole Quantity sum；W1-MAG(%), W2-MAG(%), W1,2-DAG(%), W1,3-DAG(%), WTAG(%) and WFFA(%) represents wheat respectively The percentage composition of 1-MAG, 2-MAG, 1,2-DAG, 1,3-DAG, TAG and FFA mass in embryo oil.
- 10. according to the method for claim 1, it is characterised in that the grain sample is wheat embryo, wheat bran, rice Chaff or maize germ.
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