CN103472018A - Method for measuring true protein content of corn stalk fermentation feed - Google Patents
Method for measuring true protein content of corn stalk fermentation feed Download PDFInfo
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- CN103472018A CN103472018A CN2013104475359A CN201310447535A CN103472018A CN 103472018 A CN103472018 A CN 103472018A CN 2013104475359 A CN2013104475359 A CN 2013104475359A CN 201310447535 A CN201310447535 A CN 201310447535A CN 103472018 A CN103472018 A CN 103472018A
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Abstract
The invention discloses a method for measuring the true protein content of corn stalk fermentation feed. By utilizing Coomassie brilliant blue, absorbance values under different conditions are measured and recorded, a standard curve is drawn, the protein content of a sample is calculated, and according to changes of absorbance, the digestion effect of the sample is measured. Compared with the prior art, the measurement method has the advantages that an optimized precipitation method is adopted for implementation, the true protein content of the feed can be effectively measured, nonprotein interference is eliminated at the same time, and the effect is good; the measurement method is easy to operate, an expensive large instrument is not needed, needed cost is low, and the method can be widely applied to inspection in actual production of feed factories.
Description
Technical field
The present invention relates to the determination of feeds quality application technology, particularly a kind of method of measuring maize straw fermented feed true protein content.
Background technology
In feed, the content of protein is one of important indicator of estimating the feed nutritive value height.Protein in feed is also referred to as thick protein, it comprises true protein and nonprotein two parts nitrogen substance, yet the nucleic acid in nonprotein, nitrogenous carbohydrates, porphyrin, alkaloid, acid amides, nitrogenous lipoid, acid amides, pigment and nitre state thing etc. also contain nitrogen, usually also be credited in crude protein content, and these materials often can not be absorbed by ox, sheep, bird or utilize fully, therefore caused the very different of the waste of nutrition and feeding quality, nutriment.And true albumen refers to the content of the true protein that can be absorbed, utilize by the fowl poultry, from effect, the content of true protein is more suitable for the index as check feed nutritive value height.Therefore, assay method how to optimize the true albumen of feed seems significant.
The domestic assay method for crude protein content has a lot, such as Kjeldahls method (standard method), biuret method, Coomassie brilliant blue method and forint-phenol law etc., but the assay method for the true albumen of feed is not listed national standard in, but wherein there is a kind of method to be widely used, be the precipitation method, its principle is roughly sample in copper-bath, add sodium hydroxide solution to be alkalescence, protein precipitate and separate with the solubility nitrogen substance of nonprotein, measure the nitrogen content in sediment, be converted into the content of true protein, and the precipitation method also exist many shortcomings, not very perfect, therefore need a kind of fast and convenient, the assay method that degree of accuracy is higher.
Summary of the invention
The objective of the invention is for above-mentioned existing problems, a kind of method of measuring maize straw fermented feed true protein content is provided, the method has provided optimum digestion process condition, choose the maize straw fermented feed as experimental raw, utilize the Coomassie brilliant blue method to probe into the top condition of sample digestion in the precipitation method, because the Coomassie brilliant blue method has highly sensitive, measure fast and convenient, the advantages such as interfering material is few, so used the method to probe into out true protein sample to be measured is carried out pretreated than suitable condition; By using control variate method, change separately respectively the digestion temperature, digestion time and Na0H concentration are probed into the digestion pretreatment condition, further optimize the precipitation method, make it more perfect.
Technical scheme of the present invention:
A kind of method of measuring maize straw fermented feed true protein content, utilize Coomassie brilliant blue, measure and record absorbance under each condition, draw the protein content that typical curve calculates sample, weigh the treatments of the sample effect according to the variation of absorbance, comprise the following steps:
1) get 30 centrifuge tubes, every centrifuge tube adds the sample to be measured after the pulverizing of equal in quality;
2) 30 centrifuge tubes are divided into to five alkali concn groups, every group of six centrifuge tubes, add respectively the distilled water of different volumes in each centrifuge tube and fully shake up, every group adds respectively the NaOH solution of different volumes, variable concentrations and again shakes up, will only add the centrifuge tube of distilled water as the blank group simultaneously;
3) centrifuge tube is put into to water bath, is digested pre-service respectively under 50 ℃ and 40 ℃ of steady temperatures, the digestion time of each alkali concn group be respectively 6,4,3,2,1 and 0.5h digestion process in constantly shake up;
4) again mix after having digested, draw sample, mix with distilled water diluting, then centrifugal 5min under the 6000r/min revolution;
5) draw respectively supernatant, add normal concentration CBB reagent, make blank tube and standard protein pipe simultaneously, the setting wavelength is 595nm, and be 5min action time, and the distilled water zeroing, measure absorbance;
6) record absorbance under each condition, calculate the protein content of each sample by drawing typical curve, determine the digestion effect of each sample according to the variation of absorbance.
In described step 1), the centrifuge tube capacity is 50mL, and it is 0.3g that every centrifuge tube adds the amount for the treatment of sample digestion, adds accuracy of measurement for being accurate to 0.1mg.
Described step 2) in, in each alkali concn group, in six centrifuge tubes, add the distilled water volume to be respectively 15,14,12,10 and 8 mL, in every group, add the concentration of NaOH solution to be 4mol/L, the volume that adds NaOH solution is 1,2,4,6 and 8mL, makes concentration of sodium hydroxide solution in these five groups be followed successively by 0.25,0.5,1.0,1.5 and 2mol/L.
In described step 4), the sample taken amount is 1ml, and distilled water volume used is 30.25mL, and the sample solution cumulative volume after dilution is 500ml.
In described step 5), the supernatant uptake is 0.5mL, and the addition of normal concentration CBB reagent is 5mL.
The invention has the beneficial effects as follows: compared with prior art, this measuring method adopts optimizes precipitation method enforcement, can effectively measure the true protein content in feed, has got rid of the interference of nonprotein simultaneously, respond well; This measuring method is simple to operate, does not need expensive large-scale instrument, and required expense is low, can be widely used in the actual production inspection of feed factory.
The accompanying drawing explanation
Fig. 1 is 50 ℃ of sample absorbance curve maps at the digestion temperature.
Fig. 2 is 40 ℃ of sample absorbance curve maps at the digestion temperature.
Embodiment
Below in conjunction with embodiment, specific embodiments of the present invention are further described in detail, but should not limit with this protection domain of invention.
Embodiment:
A kind of method of measuring maize straw fermented feed true protein content, utilize Coomassie brilliant blue, measure and record absorbance under each condition, draw the protein content that typical curve calculates sample, weigh the treatments of the sample effect according to the variation of absorbance, concrete implementation step is:
1) get 30 50mL centrifuge tubes, every centrifuge tube adds respectively the sample after 0.3g pulverizes, and the quality precision is accurate to 0.1mg;
2) 30 centrifuge tubes are divided into to five alkali concn groups, in each alkali concn group, in six centrifuge tubes, add the distilled water volume to be respectively 15,14,12,10 and 8 mL, in every group, add the concentration of NaOH solution to be 4mol/L, the volume that adds NaOH solution is 1,2,4,6 and 8mL, make the concentration of sodium hydroxide solution in these five groups be followed successively by 0.25,0.5,1.0,1.5 and 2 mol/L, sample segment is established the blank group, i.e. adding distil water 16 mL only in sample;
3) centrifuge tube is put into to water bath, is digested pre-service respectively under 50 ℃ and 40 ℃ of steady temperatures, the digestion time of each alkali concn group be respectively 6,4,3,2,1 and 0.5h digestion process in constantly shake up;
4) again mix after having digested, draw the 1ml sample, mix with the 30.25mL distilled water diluting, centrifugal 5min under the 6000r/min revolution then, the sample solution after now diluting is equivalent to be diluted to overall accumulated amount 500mL;
5) draw supernatant 0.5mL, add 5mL normal concentration CBB reagent, make blank tube and standard protein pipe simultaneously, the setting wavelength is 595nm, and be 5min action time, and the distilled water zeroing, measure absorbance.
The typical curve data are in Table 1.
Table 1
| 0 | 1 | 2 | 3 | 4 | 5 | 6 |
| 0.498 | 1.100 | 1.116 | 1.130 | 1.134 | 1.133 | 1.155 |
The sample absorbance data of 50 ℃ of digestion at temperature is in Table 2.
Table 2
40 ℃ of sample absorbance data part tables 3 that digest at temperature.
Table 3
Analytical table 1, table 2 sample absorbance data show: the suitable digestion temperature that Coomassie brilliant blue is measured the true albumen of stalk is 50 ℃, and Optimal digestion time and naoh concentration are respectively 4-6h, 1.5-2mol/L.
6) record absorbance under each condition, calculate the protein content of each sample by drawing typical curve, determine the digestion effect of each sample according to the variation of absorbance.
Fig. 1 is 50 ℃ of sample absorbance curve maps at the digestion temperature, and Fig. 2 is 40 ℃ of sample absorbance curve maps at the digestion temperature.
In analysis chart, curve shows: with alkali concn, increase or digestion time extends, and the absorbance that digestive juice is measured with the CBB method all occur first the raising phenomenon of rear reduction, reason is can not make sample reach catapepsis when the digestion alkali concn hangs down; And during the excessive concentration of alkali, in sample, albumen is discharged fully, but the alkali of high concentration can make protein denaturation, thereby has destroyed the structure of protein, and chromogenic reaction dies down.Under same alkali concn, digestion time is too short can not make the sample catapepsis, when digestion reaches certain hour protein, is discharged fully, now extends action time again and can make protein denaturation equally.
Claims (5)
1. a method of measuring maize straw fermented feed true protein content, it is characterized in that: utilize Coomassie brilliant blue, measure and record absorbance under each condition, draw the protein content that typical curve calculates sample, weigh the treatments of the sample effect according to the variation of absorbance, comprise the following steps:
1) get 30 centrifuge tubes, every centrifuge tube adds the sample to be measured after the pulverizing of equal in quality;
2) 30 centrifuge tubes are divided into to five alkali concn groups, every group of six centrifuge tubes, add respectively the distilled water of different volumes in each centrifuge tube and fully shake up, every group adds respectively the NaOH solution of different volumes, variable concentrations and again shakes up, will only add the centrifuge tube of distilled water as the blank group simultaneously;
3) centrifuge tube is put into to water bath, is digested pre-service respectively under 50 ℃ and 40 ℃ of steady temperatures, the digestion time of each alkali concn group be respectively 6,4,3,2,1 and 0.5h digestion process in constantly shake up;
4) again mix after having digested, draw sample, mix with distilled water diluting, then centrifugal 5min under the 6000r/min revolution;
5) draw respectively supernatant, add normal concentration CBB reagent, make blank tube and standard protein pipe simultaneously, the setting wavelength is 595nm, and be 5min action time, and the distilled water zeroing, measure absorbance;
6) record absorbance under each condition, calculate the protein content of each sample by drawing typical curve, determine the digestion effect of each sample according to the variation of absorbance.
2. measure according to claim 1 the method for maize straw fermented feed true protein content, it is characterized in that: in described step 1), the centrifuge tube capacity is 50mL, and it is 0.3g that every centrifuge tube adds the amount for the treatment of sample digestion, adds accuracy of measurement for being accurate to 0.1mg.
3. measure according to claim 1 the method for maize straw fermented feed true protein content, it is characterized in that: described step 2), in each alkali concn group, in six centrifuge tubes, add the distilled water volume to be respectively 15,14,12,10 and 8 mL, in every group, add the concentration of NaOH solution to be 4mol/L, the volume that adds NaOH solution is 1,2,4,6 and 8mL, makes concentration of sodium hydroxide solution in these five groups be followed successively by 0.25,0.5,1.0,1.5 and 2mol/L.
4. measure according to claim 1 the method for maize straw fermented feed true protein content, it is characterized in that: in described step 4), the sample taken amount is 1ml, and distilled water volume used is 30.25mL, and the sample solution cumulative volume after dilution is 500ml.
5. measure according to claim 1 the method for maize straw fermented feed true protein content, it is characterized in that: in described step 5), the supernatant uptake is 0.5mL, and the addition of normal concentration CBB reagent is 5mL.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104215604A (en) * | 2014-09-16 | 2014-12-17 | 中华人民共和国南通出入境检验检疫局 | Method for measuring content of proteins in grains and feed |
| CN106290204A (en) * | 2016-11-03 | 2017-01-04 | 百奥森（江苏）食品安全科技有限公司 | The detection method of protein in a kind of forage feed |
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| CN101566575A (en) * | 2009-04-29 | 2009-10-28 | 东北制药总厂 | Method for detecting protein content in 2-keto-L-gulonic acid |
| CN201378147Y (en) * | 2009-04-15 | 2010-01-06 | 杭州天辰仪器设备有限公司 | Instrument for the inspection of true protein by Coomassie brilliant blue color development process |
| CN102627694A (en) * | 2012-03-29 | 2012-08-08 | 常熟市珍门麦芽糖厂 | Method for extracting wheat bran protein |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN201378147Y (en) * | 2009-04-15 | 2010-01-06 | 杭州天辰仪器设备有限公司 | Instrument for the inspection of true protein by Coomassie brilliant blue color development process |
| CN101566575A (en) * | 2009-04-29 | 2009-10-28 | 东北制药总厂 | Method for detecting protein content in 2-keto-L-gulonic acid |
| CN102627694A (en) * | 2012-03-29 | 2012-08-08 | 常熟市珍门麦芽糖厂 | Method for extracting wheat bran protein |
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| 曲春香等: "用考马斯亮蓝测定植物提取液中可溶性蛋白质含量方法的研究", 《苏州大学学报》 * |
| 李金霞: "棉秆蛋白的提取及分离纯化", 《万方学位论文数据库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104215604A (en) * | 2014-09-16 | 2014-12-17 | 中华人民共和国南通出入境检验检疫局 | Method for measuring content of proteins in grains and feed |
| CN106290204A (en) * | 2016-11-03 | 2017-01-04 | 百奥森（江苏）食品安全科技有限公司 | The detection method of protein in a kind of forage feed |
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