CN106248952A - A kind of method of L glutamic acid in quick mensuration food - Google Patents
A kind of method of L glutamic acid in quick mensuration food Download PDFInfo
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- CN106248952A CN106248952A CN201610541904.4A CN201610541904A CN106248952A CN 106248952 A CN106248952 A CN 106248952A CN 201610541904 A CN201610541904 A CN 201610541904A CN 106248952 A CN106248952 A CN 106248952A
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Abstract
The present invention relates to a kind of method of L glutamic acid in quick mensuration food, comprise the following steps: solid or semi-solid sample are pulverized with suitable instrument or minces, weigh the sample having pulverized or having minced in right amount, add suitable solvent and carry out homogenizing, centrifugation, filtration, obtain free L Glutamic acid extract, or the L Glutamic acid extract (fluid sample, without above-mentioned pre-treatment, directly filters) of protein structure is obtained constituting by Proteolytic enzyme；By sample extracting solution through suitably diluting and adjusting pH to 8.6；The L glutamic acid of sample detection liquid under conditions of glutamte dehydrogenase (GIDH) exists by nicotinamide adenine dinucleotide (NAD+) it being reduced into nicotinamide adenine dinucleotide (NADH), NADH reacts generation first a ceremonial jade-ladle, used in libation under the effect of diaphorase with iodine tetrazolium nitroxyl chloride (INT)；Use light to absorb microplate reader at 490nm, measure the absworption peak of first a ceremonial jade-ladle, used in libation, utilize external standard method, calculate L content of glutamic acid in sample according to first a ceremonial jade-ladle, used in libation light absorption value.Detection method of the present invention have easy and simple to handle, accuracy good, quick, high flux, the feature such as easy to spread.
Description
Technical field
The present invention relates to a kind of method of Pidolidone in quick mensuration food, particularly relate to a kind of de-based on glutamic acid
Hydrogen enzyme and the principle of oxidation and reduction of diaphorase double-direction control, absorb microplate reader in conjunction with light and quickly detect the side of Pidolidone
Method.
Background technology
Pidolidone is widely present in vegeto-animal body, is naturally occurring nutrition in food.At present about
The detection method of Pidolidone is a lot, exhales including polarimetry, ultraviolet spectrophotometer method, neutralization titration, ninhydrin method, Wa Shi
Inhale instrument method and aminoacid robot method etc..Wherein polarimetry, ultraviolet spectrometry light photometer measuring method, neutralization titration, ninhydrin method are not
Tool specificity, is only applicable to the detection of the more single monosodium glutamate Glutamic Acid of composition；Wa Shi breath analyzer method because of course of reaction by
Pidolidone decarboxylase controls, and has specificity, but operates more complicated, does not notes, it is easy to introduce error；Aminoacid is certainly
Dynamic analyser method is the ninhydrin method of improvement, it is adaptable to all amino acid analysis, easy and simple to handle, accuracy good, but instrument and consumptive material
Costliness, and a sample can only be detected every time, fail to realize high flux.The most necessary exploitation one species specificity is good, operation
Simplicity and detecting instrument price and consumables cost can be Pidolidone fast high-flux detection method in the most well-established food.
Summary of the invention
It is an object of the invention to overcome above-mentioned deficiency, propose a kind of method of Pidolidone in quick mensuration food.Profit
With Pidolidone under conditions of glutamte dehydrogenase (GIDH) exists by nicotinamide adenine dinucleotide (NAD+) be reduced into
Nicotinamide adenine dinucleotide (NADH), NADH reacts generation with iodine tetrazolium nitroxyl chloride (INT) under the effect of diaphorase
The principle of first a ceremonial jade-ladle, used in libation, absorbs microplate reader in conjunction with light, measures the absworption peak of first a ceremonial jade-ladle, used in libation, utilize external standard method, according to first a ceremonial jade-ladle, used in libation extinction at 490nm
Value calculates Pidolidone content in sample.96 samples can be detected owing to light absorbs microplate reader simultaneously, and detection can be automatically performed
Reading and derivation result, therefore can realize high flux and quickly detect.
The present invention solves described technical problem can be by realizing by the following technical solutions:
Comprise the following steps:
(1) solid or semi-solid sample use suitable instrument pulverize or mince；
Weigh the sample having pulverized or having minced in right amount, add the dilute perchloric acid solution of a certain amount of 1.0mol/L or 5-sulfosalisylic
Acid solution carries out homogenizing extraction, and extraction time is 5～10 minutes (notes: this extracting method gained Pidolidone is free L-paddy ammonia
Acid；If wishing, extracting solution includes constituting the Pidolidone of protein structure, then need to increase proteolysis step)；
Utilize centrifuge that sample extracting solution is separated, be centrifuged 5～10 minutes under 1000g centrifugal force；
Take middle level clear liquid, filter with Medium speed filter paper；
Fluid sample, without above-mentioned pre-treatment, directly pipettes appropriate amount of sample, filters with Medium speed filter paper；
(2) take appropriate filtrate, adjust pH to 8.0～9.0, and be diluted to certain volume, as sample detection liquid, sample detection
Liquid Pidolidone concentration controls in the range of 0.006g/L～0.06g/L；
(3) arranging light and absorb microplate reader detection parameter: detection wavelength is 490nm, reading plate mode is hole-specifically to detect by row, reads
The number time is 0.5 second/hole；
(4) draw 50 μ L water respectively, Pidolidone standard serial solution, sample detection liquid detect accordingly to transparent 96 orifice plates
Kong Zhong；
(5) adding 100 μ L detectable A in each corresponding detection hole, composition is the mixture of reagent A 1, A2, A3,
Reagent A 1:pH8～the triethanolamine phosphate buffer of 9, TritonX；Reagent A 2: diaphorase, two nucleoside of nicotinamide adenine
Acid (NAD+)；Reagent A 3: iodine tetrazolium nitroxyl chloride (INT), vibration mixing, use up absorption microplate reader and carry out light absorption value survey for the first time
Fixed；
(6) adding 50 μ L detectable B in each corresponding detection hole, composition is glutamte dehydrogenase (GLDH), vibration
Mixing, after reacting 10～20 minutes, uses up absorption microplate reader and carries out second time light absorption value mensuration；
(7) according to the extinction value difference of twice mensuration front and back, Pidolidone standard curve is set up, by sample detection liquid light absorption value
Difference substitutes into the equation of linear regression of Pidolidone standard curve, tries to achieve sample detection liquid Pidolidone concentration, is multiplied by corresponding dilution
Multiple, is sample Pidolidone content.
Preferred version includes:
Described reagent A 1 is the triethanolamine phosphate buffer of pH8～9, wherein (Triton X-100) Han TritonX
0.5%～1.0%；The enzyme activity unit of reagent A 2 Myocardial yellow enzyme is 1U～3U/mL, NAD+Concentration is 10mg/mL～15mg/
mL；The concentration of reagent A 3 iodine tetrazolium nitroxyl chloride (INT) is 1mg/mL～3mg/mL.
Reagent A 1, reagent A 2, reagent A 3 are mixed by 3:1:1 before using.
Sample detection liquid, the pH to 8.6 of reagent A 1 is adjusted with 2mol/L potassium hydroxide solution.
The enzyme activity unit of described detectable B is >=30U/mL, and the response time is 10～20 minutes.
The method have technical effect that:
1, propose a kind of based on glutamte dehydrogenase with the principle of oxidation and reduction of diaphorase double-direction control, in conjunction with light
Absorb microplate reader and quickly detect the method for Pidolidone in food；
2, there is the feature that specificity is strong, accuracy is high；
3, there is feature easy and simple to handle, quick, high-throughout.
4, instrument price and detectable relative inexpensiveness, it is easy to promote.
Accompanying drawing explanation
Fig. 1 is the Pidolidone canonical plotting of the present invention.
Detailed description of the invention
The present invention is described further below in conjunction with the accompanying drawings:
The present invention comprises the steps of:
1. the preparation of sample detection liquid:
Solid or semi-solid sample crusher or meat grinder pulverize or mince, and take 50g and put into homogenizing cup addition 100mL
Dilute perchloric acid solution of 1.0mol/L carries out homogenizing and extracts 5 minutes (notes: this extracting method gained Pidolidone is free L-paddy ammonia
Acid, if wishing, extracting solution includes constituting the Pidolidone of protein structure, then need to increase proteolysis step), mixture is turned
Move on to 50mL centrifuge tube, be centrifuged 5 minutes under 1000g centrifugal force, carefully push upper strata floating thing aside, take middle level clear liquid, use middling speed
Filter paper filtering (fluid sample, without above-mentioned pre-treatment, directly filters), takes appropriate filtrate, molten with the potassium hydroxide of 2mol/L
Liquid adjusts pH to 8.0～9.0, and is diluted to certain volume, and as sample detection liquid, sample detection liquid Pidolidone concentration controls
In the range of 0.006g/L～0.06g/L.
2. the preparation of detectable:
Reagent A 1: composition is the phosphate/Triethanolamine buffer of pH8.0～9.0, wherein (the Triton X-Han TritonX
100) 0.5%～1.0%.This solution preserves under the conditions of 2 DEG C～8 DEG C, and the pot-life is 2 months.
Reagent A 2: composition is diaphorase and nicotinamide adenine dinucleotide (NAD+), wherein the enzyme of diaphorase is lived
Power is 1U～3U/mL, NAD+Concentration is 10mg/mL～15mg/mL.This solution preserves under the conditions of 2 DEG C～8 DEG C, the pot-life
It it is 1 month.
Reagent A 3: composition is iodine tetrazolium nitroxyl chloride (INT), concentration is 1mg/mL～3mg/mL.This solution is at 2 DEG C～8 DEG C
Under the conditions of keep in Dark Place, the pot-life is 1 month.
Take reagent A 1, reagent A 2, reagent A 3 before using respectively in right amount, mix by 3:1:1, be made into detectable A.Now join existing
With.
Detectable B: composition is glutamte dehydrogenase (GLDH), enzyme activity unit is >=30U/mL.This solution at 2 DEG C～
Preserving under the conditions of 8 DEG C, the pot-life is 1 month.
3. the preparation of detecting instrument
Before detection, about 10 minutes startup instruments, preheat.Enter detection interface, detection parameter is set: detect wavelength
For 490nm, reading plate mode is hole-specifically to detect by row, and the reading duration is 0.5 second/hole.
4. measure
1) draw 50 μ L water respectively, Pidolidone standard serial solution, sample detection liquid detect accordingly to transparent 96 orifice plates
Kong Zhong；
2) in each corresponding detection hole, add 100 μ L detectable A, vibration mixing, carry out light absorption value for the first time and measure；
3) in each corresponding detection hole, add 50 μ L detectable B, vibration mixing, after reacting 10～20 minutes, carry out
Light absorption value measures for the second time；
4) according to the extinction value difference of twice mensuration front and back, Pidolidone standard curve is set up, by sample detection liquid light absorption value
Difference substitutes into the equation of linear regression of Pidolidone standard curve, tries to achieve sample detection liquid Pidolidone concentration, is multiplied by corresponding dilution
Multiple, is sample Pidolidone content.
With specific embodiment, a kind of method of Pidolidone in quick mensuration food is illustrated below.
Embodiment one
1. the preparation of sample detection liquid
Weigh soy sample 2g in beaker, be accurate to 0.01g, dilute with water, adjust with 2.0mol/L potassium hydroxide solution
PH to 8.6, transfers in 500mL volumetric flask, and is settled to scale, filters, and makes soy sample detect liquid Pidolidone concentration control
System is in the range of 0.006g/L～0.06g/L.
2. the preparation of detectable
Reagent A 1: weigh triethanolamine phosphate 1.488g, dipotassium hydrogen phosphate (K2HPO4) 0.172g, potassium dihydrogen phosphate
(KH2PO4) 1.4mg, with water dissolution, regulate pH to 8.6 with 2mol/L potassium hydroxide solution, add TritonX (Triton X-
100) 0.5g, is settled to 100mL, is made into the triethanolamine phosphate buffer that pH is 8.6.This solution is under the conditions of 2 DEG C～8 DEG C
Preserving, the pot-life is 2 months.
Reagent A 2: weigh diaphorase lyophilized powder (enzyme activity unit is 25U/mg) 0.08mg, nicotinamide adenine two core
Thuja acid (NAD+) 20mg, it is dissolved in 2mL water, being made into diaphorase enzyme activity unit is 1U/mL, NAD+Concentration is the molten of 10mg/mL
Liquid.This solution preserves under the conditions of 2 DEG C～8 DEG C, and the pot-life is 1 month.
Reagent A 3: weigh iodine tetrazolium nitroxyl chloride (INT) 2mg, be dissolved in 2mL water, be made into the INT that concentration is 1mg/mL molten
Liquid.This solution keeps in Dark Place under the conditions of 2 DEG C～8 DEG C, and the pot-life is 1 month.
Take reagent A 1 750 μ L, reagent A 2 250 μ L, reagent A 3 250 μ L before using respectively, mix by 3:1:1, be made into inspection
Test agent A.Now with the current.
Detectable B: weigh glutamte dehydrogenase lyophilized powder (enzyme activity unit is 35U/mg) 2mg, be dissolved in 2mL water,
It is made into the solution that glutamte dehydrogenase enzyme activity unit is 35U/mL.This solution preserves under the conditions of 2 DEG C～8 DEG C, keeps the time limit
It it is 1 month.
3. the preparation of detecting instrument
Before detection, about 10 minutes startup instruments, preheat.Enter detection interface, detection parameter is set: detect wavelength
For 490nm, reading plate mode is hole-specifically to detect by row, and the reading duration is 0.5 second/hole.
4. measure
Draw respectively 50 μ L water, Pidolidone standard serial solution, soy sample detection liquid examine accordingly to transparent 96 orifice plates
In gaging hole.
In each corresponding detection hole, detectable A 100 μ L is added successively with continuous liquid-moving machine；96 orifice plates are put into light
Absorbing microplate reader to read, on grillage, to vibrate 10 seconds, make solution mixing in detection hole, carry out measuring for the first time, instrument records water automatically
Blank absorbency (A1b), Pidolidone standard serial solution light absorption value (A1s) and soy sample detection liquid light absorption value (A1 soy sauce)。
96 orifice plates are taken out, in each corresponding detection hole, adds detectable B 50 μ L successively with continuous liquid-moving machine, will
96 orifice plates are put back to light and are absorbed on microplate reader reading grillage, vibrate 10 seconds, make solution mixing in detection hole, after reacting 10 minutes, carry out
Second time measures, and instrument records water blank absorbency (A automatically2b), Pidolidone standard serial solution light absorption value (A2s) and soy sauce
Sample detection liquid light absorption value (A2 soy sauce).Detection data are shown in Table 1.
Table 1 Pidolidone standard serial solution and soy sample detection liquid extinction value difference
5. result calculates
With Pidolidone standard serial solution concentration as abscissa, extinction value difference is vertical coordinate mapping, tries to achieve standard curve
Equation of linear regression y=20.016x-0.0027 (sees Fig. 1).
Soy sample is detected liquid extinction value difference and substitutes into equation of linear regression y=20.016x-0.0027, try to achieve soy sauce sample
It is 0.0262g/L that liquid Pidolidone concentration is surveyed in product examine, is multiplied by extension rate 250 (2g is settled to 500mL), tries to achieve soy sample L-
Content of glutamic acid is 6.55g/L.
Embodiment two:
1. the preparation of sample detection liquid
About 100g Carnis Gallus domesticus is cut into small pieces, minces with meat grinder, weigh 50g, be accurate to 0.01g, put in homogenizing cup, past
Adding the dilute perchloric acid solution of 1.0mol/L of 100mL 0 DEG C in homogenizing cup, homogenizing extracts 5 minutes (notes: this extracting method gained L-
Glutamic acid is free Pidolidone, if wishing, extracting solution includes constituting the Pidolidone of protein structure, then need to increase albumen water
Solve step), mixture after homogenizing is transferred in 50mL centrifuge tube, be centrifuged 5 minutes under 2000g centrifugal force, in careful removing
Layer adipose membrane, takes middle level clear liquid, filters.
Draw 10mL filtrate in beaker, dilute with water, adjust pH to 8.6, transfer with the potassium hydroxide solution of 2.0mol/L
In 100mL volumetric flask, and it is settled to scale, makes chicken meat sample detection liquid Pidolidone concentration at 0.006g/L～0.06g/L
In the range of.
2. the preparation of detectable
Reagent A 1: weigh triethanolamine phosphate 1.488g, dipotassium hydrogen phosphate (K2HPO4) 0.172g, potassium dihydrogen phosphate
(KH2PO4) 1.4mg, with water dissolution, regulate pH to 8.6 with 2mol/L potassium hydroxide solution, add TritonX (Triton X-
100) 1g, is settled to 100mL, is made into the triethanolamine phosphate buffer that pH is 8.6.This solution is protected under the conditions of 2 DEG C～8 DEG C
Depositing, the pot-life is 2 months.
Reagent A 2: weigh diaphorase lyophilized powder (enzyme activity unit is 25U/mg) 0.24mg, nicotinamide adenine two core
Thuja acid (NAD+) 30mg, it is dissolved in 2mL water, being made into diaphorase enzyme activity unit is 3 U/mL, NAD+Concentration is 15mg/mL's
Solution.This solution preserves under the conditions of 2 DEG C～8 DEG C, and the pot-life is 1 month.
Reagent A 3: weigh iodine tetrazolium nitroxyl chloride (INT) 6mg, be dissolved in 2mL water, be made into the INT that concentration is 3mg/mL molten
Liquid.This solution keeps in Dark Place under the conditions of 2 DEG C～8 DEG C, and the pot-life is 1 month.
Take reagent A 1 750 μ L, reagent A 2 250 μ L, reagent A 3 250 μ L before using respectively, mix by 3:1:1, be made into inspection
Test agent A.Now with the current.
Detectable B: weigh glutamte dehydrogenase lyophilized powder (enzyme activity unit is 35U/mg) 2.8mg, be dissolved in 2mL water
In, it is made into the solution that glutamte dehydrogenase enzyme activity unit is 49U/mL.This solution preserves under the conditions of 2 DEG C～8 DEG C, keeps the phase
It is limited to 1 month.
3. the preparation of detecting instrument
Before detection, about 10 minutes startup instruments, preheat.Enter detection interface, detection parameter is set: detect wavelength
For 490nm, reading plate mode is hole-specifically to detect by row, and the reading duration is 0.5 second/hole.
4. measure
Draw respectively 50 μ L water, Pidolidone standard serial solution, chicken meat sample detection liquid examine accordingly to transparent 96 orifice plates
In gaging hole.
In each corresponding detection hole, detectable A 100 μ L is added successively with continuous liquid-moving machine；96 orifice plates are put into light
Absorb microplate reader to read, on grillage, to vibrate 10 seconds, make solution mixing in detection hole.Carrying out measuring for the first time, instrument records water automatically
Blank absorbency (A1b), Pidolidone standard serial solution light absorption value (A1s) and chicken meat sample detection liquid light absorption value (A1 Carnis Gallus domesticus)。
96 orifice plates are taken out, in each corresponding detection hole, adds detectable B 50 μ L successively with continuous liquid-moving machine, will
96 orifice plates are put back to light and are absorbed on microplate reader reading grillage, vibrate 10 seconds, make solution mixing in detection hole.After reacting 15 minutes, carry out
Second time measures, and instrument records water blank absorbency (A automatically2b), Pidolidone standard serial solution light absorption value (A2s) and chicken
Meat sample detection liquid light absorption value (A2 Carnis Gallus domesticus)。
It is the detection of same batch owing to chicken meat sample detection liquid and soy sample detect liquid, so water is blank and Pidolidone
The detection data of standard serial solution and embodiment one identical (being shown in Table 1), chicken meat sample detection liquid measures light absorption value for the first time and is
0.083, the light absorption value that second time measures is 0.305.
5. the mensuration of moisture
Carnis Gallus domesticus moisture presses GB/T9695.15-2008 meat and meat products determination of moisture, and recording moisture is 57ml/100g.
6. result calculates
Chicken meat sample is detected liquid extinction value difference and substitutes into equation of linear regression y=20.016x-0.0027, try to achieve Carnis Gallus domesticus sample
It is 0.0100g/L that liquid Pidolidone concentration is surveyed in product examine, and (50g Carnis Gallus domesticus is dissolved in the dilute perchloric acid solution of 100mL to be multiplied by extension rate 25.7
Plus 28.5mL from the system of the water of Carnis Gallus domesticus, take 10mL filtrate and be settled to 100mL), try to achieve chicken meat sample and dissociate L-paddy ammonia
Acid result is 0.257g/L.
Embodiment described above only have expressed the preferred embodiment of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as, it is noted that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope；Therefore, all impartial changes done with scope of the invention as claimed and modification, the claims in the present invention all should be belonged to
Covering scope.
Claims (7)
1. the method for Pidolidone in a quick mensuration food, it is characterised in that: comprise the following steps,
(1) sample is carried out pre-treatment, extract the Pidolidone in sample, centrifugal filtration；
(2) take appropriate filtrate, adjust pH to 8.0～9.0, and be diluted to certain volume, as sample detection liquid, sample detection liquid L-
Aminoglutaric acid concentration controls in the range of 0.006g/L～0.06g/L；
(3) light is set and absorbs microplate reader detection parameter: detection wavelength is 490nm, read plate mode for hole-specifically detecting, during reading by row
Between be 0.5 second/hole；
(4) draw the Pidolidone standard serial solution of 50 μ L respectively, sample detection liquid, water detect hole accordingly to transparent 96 orifice plates
In；
(5) 100 μ L detectable A are added toward each corresponding detection hole, mixing, use light to absorb microplate reader and carry out extinction for the first time
PH-value determination pH；
The composition of described detectable A is the mixture of reagent A 1, A2, A3, reagent A 1: triethanolamine phosphate buffer, song
Draw logical；Reagent A 2: diaphorase, nicotinamide adenine dinucleotide (NAD+)；Reagent A 3: iodine tetrazolium nitroxyl chloride (INT)；
(6) 50 μ L detectable B are added toward each corresponding detection hole, mixing, after reacting 10～20 minutes, use light to absorb enzyme mark
Instrument carries out second time light absorption value and measures；
The component of described detectable B is glutamte dehydrogenase (GLDH)；
(7) according to the extinction value difference of twice mensuration front and back, Pidolidone standard curve is set up, by sample detection liquid extinction value difference generation
Enter the equation of linear regression of Pidolidone standard curve, try to achieve sample detection liquid Pidolidone concentration, be multiplied by corresponding extension rate,
It is sample Pidolidone content.
2. the method for Pidolidone in quickly mensuration food as claimed in claim 1, it is characterised in that described reagent A 1 is
Phosphate/the Triethanolamine buffer of pH8.0～9.0, wherein containing TritonX (Triton X-100) 0.5%～1.0%；Reagent
The enzyme activity unit of A2 Myocardial yellow enzyme is 1U～3U/mL, NAD+Concentration is 10mg/mL～15mg/mL；Iodine tetrazolium in reagent A 3
The concentration of nitroxyl chloride (INT) is 1mg/mL～3mg/mL.
3. as claimed in claim 2 quickly measure the method for Pidolidone in food, it is characterised in that reagent A 1, reagent A 2,
Reagent A 3 is mixed by 3:1:1 before using.
4. the method for Pidolidone in quickly mensuration food as claimed in claim 2, it is characterised in that sample detection liquid, examination
Agent A1 2mol/L potassium hydroxide solution regulates pH to 8.6.
5. the method for Pidolidone in quickly mensuration food as claimed in claim 1, it is characterised in that described detectable B
Enzyme activity unit be >=30U/mL.
6. the method for Pidolidone in quickly mensuration food as claimed in claim 1, it is characterised in that pre-treatment includes following
Step:
Solid or semi-solid sample use suitable instrument pulverize or mince；
Weigh the sample having pulverized or having minced in right amount, add the dilute perchloric acid solution of a certain amount of 1.0mol/L or 5-sulphosalicylic acid is molten
Liquid carries out homogenizing extraction, and extraction time is 5～10 minutes, and this extracting method gained Pidolidone is free Pidolidone；Utilize
Sample extracting solution is separated by centrifuge, is centrifuged 5～10 minutes under 1000g centrifugal force；
Take middle level clear liquid, filter with Medium speed filter paper；
Fluid sample, without above-mentioned pre-treatment, directly pipettes appropriate amount of sample, filters with Medium speed filter paper.
7. the method for Pidolidone in quickly mensuration food as claimed in claim 1, it is characterised in that include that Proteolytic enzyme walks
Suddenly, for extracting the Pidolidone constituting protein structure.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108132213A (en) * | 2017-12-05 | 2018-06-08 | 深圳市博锐德生物科技有限公司 | Spermatozoon activity oxygen class substance quantitative determination reagent kit and application thereof and application method |
| CN109142567A (en) * | 2018-08-06 | 2019-01-04 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method of free amino acid in feed added with free amino acid |
| CN110923129A (en) * | 2019-04-25 | 2020-03-27 | 苏州格锐思生物科技有限公司 | Fd-glutamic acid synthetase activity determination kit and detection method thereof |
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