CN109781483A - A kind of safe and efficient measuring method of protein content - Google Patents
A kind of safe and efficient measuring method of protein content Download PDFInfo
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Abstract
The invention discloses a kind of safe and efficient measuring methods of protein content, including Protein Extraction: by rice sample and reagent mixing is extracted, ultrasonic wave extraction 1~3 time, and often inferior to ultrasonic wave extraction 30~40 minutes under 45~55 DEG C, 300~500W of power；After ultrasonic wave extraction, it is centrifuged to obtain supernatant, using constant volume reagent constant volume to 25ml, mixes well, obtains extracting solution of protein.The present invention selects sodium hydroxide ethanol water, starch gelatinization phenomenon is not present in extraction process, protein can be extracted completely, the sample protein matter content measured is substantially identical as standard method, it is a kind of complete extracting method, provides basis with Accurate Determining Protein Content of Rice to be stable.The method of the present invention is reproducible, and measurement result stability is high.The present invention is without using toxic reagents such as the concentrated sulfuric acid, SDS, mercaptoethanols, it is ensured that experimentation safety, it is not damaged to personnel health.
Description
Technical field
The present invention relates to a kind of safe and efficient Accurate Determining methods of protein content, more particularly to rice paddy seed correlation egg
White matter component content Accurate Determining method.
Background technique
Protein content determination is the basic technology in biological study.Method of protein measurement mainly has kjeldahl determination
Method, Coomassie Brilliant Blue, BCA method etc., and have higher accuracy.Kjeldahl's method can be surveyed again to after Protein Extraction
It is fixed, the total nitrogen in raw material can also be measured, but measuring method is complicated, working efficiency is low, using the strong acid of a large amount of danger, and tests
Toxic ammonia is generated in the process, and in addition to sample size requirements height, each sample generally requires several grams.The latter two want sample size
It asks relatively low, is not less than 0.0700g, but be required to that protein is carried out efficiently to extract completely, otherwise measurement result
Inaccuracy.Coomassie Brilliant Blue is swift in response, and reagent is cheap, is suitble to batch to measure, makees standard items to different proteins type,
As a result variant；BCA method is not much different using various criterion product result, but reagent is more expensive, and the test process time is relatively long.
1, Kjeldahl's method: in kelvin bottle, sample and concentrated sulfuric acid heat together, itrogenous organic substance decompose generation ammonia and (disappear
Change), ammonia again and effect of sulfuric acid, become sulfate of ammoniac, this process is generally 2-3 hours.In kelvin distilling apparatus, alkalize through highly basic
Be allowed to decompose to give off ammonia, steam ammonia into acid solution by means of steam, the degree being neutralized according to acid solution can calculate sample nitrogen content.
In order to accelerate to digest, CuSO can be added4Make catalyst, K2SO4To improve the boiling point of solution.Ammonia can be collected with boric acid solution, used
Strong acid titration.
Calculating acquired results are sample nitrogen pool, contain the nitrogen in protein, further comprise inorganic nitrogen and free ammonia
Nitrogen in base acid, theoretically result is higher.Nitrogen pool should be subtracted nonprotein nitrogen to obtain the final product by protein content in sample such as to be acquired.
Such as be intended to further acquire the content of protein in sample, i.e., with proteinic nitrogen in sample multiplied by 6.25 to obtain the final product.
Currently, Kjeldahl's method is the standard method of protein determination in biology, but efficiency is lower, and will be in ventilating kitchen
Interior progress.
2, the acetone method of holoprotein extracts: trichloroacetic acid and acetone extraction gross protein are used, operation sequence is complicated,
It extracts and will lead to protein losses in precipitation process, cause test repeatability bad, in addition using containing β-sulfydryl second in test
The acetone extract of alcohol, it is unpleasant and toxic.
3, it is extracted using protein cleavage liquid, Agricultural University Of Nanjing uses formula as below: 8mol/L urea, 4%SDS, 5%
Mercaptoethanol, 20% glycerol, pH=6.8 50mmol/L Tris-HCl buffer.This method extraction protein is efficient, but its
In contain SDS, be not suitable for Coomassie Brilliant Blue measurement, be not suitable for the measurement of BCA method containing mercaptoethanol, change formula can be bright
Develop and ring recovery rate, in addition SDS is toxic with mercaptoethanol, will affect experiment operator health.
4, point out that all soluble proteins are dissolved in diluted acid or diluted alkaline, generally take 0.2% in Osborne staging
(g/ml) NaOH method extract holoprotein, be verified by experiments, recovery rate is not high, and stability is bad, after use 0.3% (g/ml) instead
NaOH method is extracted, and recovery rate is risen, and still not exclusively, and repeatability is bad, and there are apparent starch gelatinization, severe jammings
Measurement result.
In addition, detergent and reducing agent have been substantially all, with the acetone of holoprotein there are also very more cracking formula of liquid
Method is extracted, and classical quantitative approach measurement is not suitable for yet.
Summary of the invention
Inventor long-time test during find, Protein Extraction completeness be influence measurement result it is most important because
Element, and there are apparent starch gelatinization, severe jamming measurement results for 0.3% (g/ml) NaOH method extraction holoprotein.Based on this, originally
Invention is designed to provide a kind of new accurate measuring method of protein content safety and stability.
The object of the invention is achieved through the following technical solutions:
A kind of safe and efficient measuring method of protein content, including Protein Extraction: rice sample and extraction reagent are mixed
It is even, ultrasonic wave extraction 1~3 time, often inferior to ultrasonic wave extraction 30~40 minutes under 45~55 DEG C, 300~500W of power；Ultrasonic wave
After extraction, it is centrifuged to obtain supernatant, using constant volume reagent constant volume to 25ml, mixes well, obtains extracting solution of protein.
Technical staff can different purposes adjust ultrasonic wave extraction numbers, the ultrasonic wave of use for quickly measuring mentions
It takes, is normally applied and is extracted using twice ultrasonic wave, it is demanding to accuracy using ultrasonic wave extraction three times.
Rice protein can be divided into albumin, globulin, alcohol soluble protein and paddy egg by its solubility in different solvents
Bai Si kind component.Wherein rice digestible protein includes albumin, globulin and glutelin.
The safe and efficient measuring method of protein content of the present invention, steps are as follows:
Step (1), Protein Extraction: by rice sample and reagent mixing is extracted, ultrasonic wave extraction 1~3 time, is often inferior to 45
~55 DEG C, ultrasonic wave extraction 30~40 minutes under 300~500W of power；After ultrasonic wave extraction, it is centrifuged to obtain supernatant, using constant volume
Reagent constant volume is mixed well to 25ml, obtains extracting solution of protein；
Step (2), protein content determination: it takes 600 μ l of extracting solution of protein to 10mm glass cuvette, adds and examine horse
2400 μ l of this brilliant blue G250 reagent measures absorbance under 465nm, 600nm after mixing 2min respectively, is denoted as A465, A600 respectively,
A600/A465 is calculated, protein concentration c in extracting solution of protein is calculated according to G250 working curve, is calculated according to formula (1)
The protein content into rice sample；
Wherein, x indicates rice sample protein matter content (%)；C indicates protein concentration (μ g/ml)；V indicates constant volume
(ml), i.e. 25ml；M indicates rice example weight (g)；P indicates to extract amount of reagent (μ l)；
Or, it is such as high to measurement result accuracy requirement, then 200 μ l of extracting solution of protein is taken, 3000 μ l BCA work is added
Liquid, 60 DEG C of water-baths keep the temperature 30min, absorbance A 562 under 562nm are measured in 1 hour, calculate protein according to BCA working curve and mention
Protein concentration in liquid is taken, protein content in rice sample is acquired according to formula (2)；
Wherein, x indicates rice sample protein matter content (%)；C indicates protein concentration (μ g/ml)；V indicates constant volume
(ml)；M indicates rice example weight (g).
In step (1), the rice sample is Brown Rice or polished rice is milled, 80 meshes excessively are made.
The described solid-to-liquid ratio for extracting reagent and rice sample is 1:10~20g/ml or mg/ μ l, preferably 1:14~
15g/ml or mg/ μ l.
30~60v/v% of concentration of alcohol in the extraction reagent, naoh concentration (mass volume ratio concentration) are
0.25~0.4w/v%.The preparation method of the extraction reagent are as follows: weigh 5.000g~8.000gNaOH, be placed in 100ml burning
In cup, with the dissolution of 30~60v/v% ethanol water, pour into 2000ml volumetric flask, then with 30~60v/v% ethanol water
Cleaning, is transferred to volumetric flask together, mixes well, and adds the ethanol water constant volume of 30~60v/v% to 2000ml, sufficiently mixes
It is even.
Preferably, in the extraction reagent concentration of alcohol be 50~60v/v%, naoh concentration be 0.35~
0.4w/v%.The preparation method of the extraction reagent are as follows: 7.000g~8.000gNaOH is weighed, is placed in 100ml beaker, with
The dissolution of 50~60v/v% ethanol water, is poured into 2000ml volumetric flask, then is cleaned with 50~60v/v% ethanol water, one
And it is transferred to volumetric flask, it mixes well, adds the ethanol water constant volume of 50~60v/v% to 2000ml, mix well.
The centrifugal condition are as follows: 5~10min is centrifuged at 8000~12000rpm of revolving speed.
The constant volume reagent is sodium hydrate aqueous solution, the concentration of sodium hydroxide and hydrogen in extraction reagent in constant volume reagent
The concentration of sodium oxide molybdena is identical.Sodium hydroxide can also be by potassium hydroxide generation in extraction reagent and constant volume reagent of the present invention
It replaces.
The preparation method of the constant volume reagent: it weighs NaOH and is placed in 100ml beaker, dissolved, poured into distilled water
In 2000ml volumetric flask, about 400ml distilled water is added, mixes well, adds distilled water constant volume to 2000ml, mix well.
In step (2), if protein concentration is more than 300 μ g/ml in extracting solution of protein, appropriate dilution is surveyed again, takes extraction
300 μ l of liquid adds 300 μ l and extracts reagent.
The preparation method of the Coomassie brilliant blue G250 reagent: taking 0.2000g Coomassie brilliant blue G250, with 95% ethyl alcohol
100ml dissolves by several times: first 95% ethyl alcohol about 30ml dissolves, and is cleaned twice with 50ml distilled water later, pours into 2000ml appearance
Measuring bottle, then cleaned with 95% ethyl alcohol of 30ml, volumetric flask is poured into, then cleaned twice with 50ml distilled water, pours into volumetric flask, finally
It is cleaned with 95% ethyl alcohol of residue, all pours into volumetric flask, 85% phosphoric acid of 200ml is added, adds distilled water constant volume, it is sufficiently mixed
It is even, 1 hour is stood, sets in 4 DEG C of refrigerators and saves, it is spare.
The G250 working curve is y=0.0204x+0.8639, R2=0.9971, x indicate protein concentration, y table
Show A600/A465.
The method for drafting of G250 working curve are as follows:
A, 0.1000g BSA (Bovine serum albumin, 98% or more purity) is accurately weighed to be placed in 100ml cleaning beaker,
Completely with distilled water 20ml dissolution, it pours into 100ml volumetric flask, then three times, each 20ml pours into appearance to beaker wash with distilled water
Measuring bottle is mixed into 1000 μ g/ml standard solution with distilled water constant volume, takes 5000 μ l of standard solution, and the 100ml of another cleaning is added
In volumetric flask, distilled water constant volume is added, is mixed into g/ml standard solution of 50 μ；
B, the BSA serial solution of various concentration is prepared in clean tube, (reduction misses each concentration two to repeating three times
Difference), and measured in multi-wavelength spectrophotometer after Coomassie brilliant blue G250 reagent 4000 μ l, 3min is added into test tube
Absorbance under 465nm and 600nm wavelength, is denoted as A465, A600 respectively, is built with linear relationship between A600/A465 and BSA concentration
Vertical G250 working curve: y=0.0204x+0.8639, R2=0.9971, x indicate that protein concentration, y indicate A600/A465.
Table 1: the BSA serial solution that G250 working curve uses is drawn
| Serial number | Concentration (μ g/ml) | Addition standard BSA solution (μ l) | It is added distilled water (μ l) |
| 1 | 300 | Standard solution 300 | 700 |
| 2 | 200 | Standard solution 200 | 800 |
| 3 | 100 | Standard solution 100 | 900 |
| 4 | 50 | Secondary standard solution 1000 | 0 |
| 5 | 10 | Secondary standard solution 200 | 800 |
| 6 | 5 | Secondary standard solution 100 | 900 |
The preparation method of the BCA working solution:
A liquid: BCA-2Na 10.0g, Na are weighed2CO3·H2O 20.0g, sodium tartrate 1.6g, NaOH 4.0g, NaHCO3
9.5g is dissolved in 800ml distilled water, adjusts pH value to 11.25 with 1mol/L NaOH, and constant volume is to 1000ml；
B liquid: CuSO is weighed4·5H2O 4g adds water constant volume to 100ml；
It is mixed into BCA working solution by 50 times of volume A liquid and 1 times of volume B liquid, BCA working solution was suitble to the same day to use.
The BCA working curve is y=0.0009x+0.0261, R2=0.9998, x indicate that protein concentration, y indicate
Absorbance A 562.
The method for drafting of BCA working curve are as follows: the BSA serial solution of various concentration, Mei Genong are prepared in clean tube
Degree two to repeating three times, and the 3000 μ l of BCA working solution into test tube, and 60 DEG C of water-baths keep the temperature 30min, in 1 hour under measurement 562nm
Absorbance is denoted as A562, is modeled with linear relationship between A562 and BSA concentration, establishes BCA working curve: y=0.0009x+
0.0261, R2=0.9998, x indicate that protein concentration, y indicate absorbance A 562.
Table 2: the BSA serial solution that BCA working curve uses is drawn
| Serial number | Concentration (μ g/ml) | Addition standard BSA solution (μ l) | It is added distilled water (μ l) |
| 1 | 1000 | Standard solution 200 | 0 |
| 2 | 500 | Standard solution 100 | 100 |
| 3 | 100 | Standard solution 20 | 180 |
| 4 | 50 | Secondary standard solution 200 | 0 |
| 5 | 10 | Secondary standard solution 40 | 160 |
| 6 | 5 | Secondary standard solution 20 | 180 |
It further include the extraction of digestible protein as the safe and efficient measuring method of protein content of the present invention: will
Rice sample and 70v/v% ethyl alcohol mix, and 45~55 DEG C stand 10~18 hours, and 12000rpm is centrifuged 8min, discard supernatant liquid；
Precipitating is mixed with 70v/v% ethyl alcohol, ultrasonic wave extraction 30~40 minutes, 12000rpm at 45~55 DEG C, 300~500W of power
It is centrifuged 8min, discards supernatant liquid, the sample for the alcohol soluble protein that is removed；The sample for removing alcohol soluble protein is mixed with reagent is extracted
It is even, ultrasonic wave extraction 1~3 time, often inferior to ultrasonic wave extraction 30~40 minutes under 45~55 DEG C, 300~500W of power；Ultrasonic wave
After extraction, it is centrifuged to obtain supernatant, using constant volume reagent constant volume to 25ml, is mixed well, digestible protein extracting solution is obtained.
Preferably, the solid-to-liquid ratio of the rice sample and 70v/v% ethyl alcohol is 1:10~20g/ml or mg/ μ l, preferably
For 1:14~15g/ml or mg/ μ l.
Preferably, the solid-to-liquid ratio 1:10~20g/ml or mg/ of the precipitating (in terms of rice sample) and 70v/v% ethyl alcohol
μ l, preferably 1:14~15g/ml or mg/ μ l.
Preferably, the solid-to-liquid ratio 1:10 of the sample (in terms of rice sample) of the described removing alcohol soluble protein and extraction reagent~
20g/ml or mg/ μ l, preferably 1:14~15g/ml or mg/ μ l.
Preferably, the safe and efficient measuring method of protein content of the present invention, steps are as follows:
The extraction of step (1), digestible protein: rice sample and 70v/v% ethyl alcohol are mixed, and 45~55 DEG C of standings 10~
18 hours, centrifugation discarded supernatant liquid；Precipitating is mixed with 70v/v% ethyl alcohol, the ultrasonic wave at 45~55 DEG C, 300~500W of power
It extracts 30~40 minutes, centrifugation discards supernatant liquid, the sample for the alcohol soluble protein that is removed；To remove the sample of alcohol soluble protein with
It extracts reagent to mix, ultrasonic wave extraction 1~3 time, often inferior to ultrasonic wave extraction 30~40 under 45~55 DEG C, 300~500W of power
Minute；After ultrasonic wave extraction, it is centrifuged to obtain supernatant, using constant volume reagent constant volume to 25ml, mixes well, obtains protein extraction
Liquid；
Step (2), protein content determination: it takes 600 μ l of extracting solution of protein to 10mm glass cuvette, adds and examine horse
2400 μ l of this brilliant blue G250 reagent, measures absorbance under 465nm, 600nm respectively, is denoted as A465, A600 respectively after mixing, calculate
A600/A465 calculates protein concentration c in extracting solution of protein according to G250 working curve, water is calculated according to formula (1)
Rice sample protein matter content；
Wherein, x indicates rice sample protein matter content (%)；C indicates protein concentration (μ g/ml)；V indicates constant volume
(ml)；M indicates rice example weight (g)；P indicates to extract amount of reagent (μ l)；
Or 200 μ l of extracting solution of protein is taken, 3000 μ l BCA working solutions are added, 60 DEG C of water-baths keep the temperature 30min, in 1 hour
Absorbance A 562 under 562nm is measured, protein concentration in extracting solution of protein is calculated according to BCA working curve, according to formula (2)
Acquire rice sample protein matter content；
Wherein, x indicates protein content (%) in rice sample；C indicates protein concentration (μ g/ml)；V indicates constant volume body
Product (ml)；M indicates rice example weight (g)
Compared with prior art, the invention has the following advantages:
1, without using toxic reagents such as the concentrated sulfuric acid, SDS, mercaptoethanols, it is ensured that experimentation safety, it is lossless to personnel health
Wound.
2, conventional instrument is only used only, common laboratory is able to achieve；Common agents are used, testing expenses are cheap,
It is easy to carry out.
3, the present invention selects sodium hydroxide ethanol water, and starch gelatinization phenomenon is not present in extraction process, can mention completely
Protein is taken out, the sample protein matter content measured is substantially identical as standard method, is a kind of complete extracting method, for stabilization
Basis is provided with Accurate Determining Protein Content of Rice.
4, the repetition in test is extracted, and residue is easy to dissolve again, increases the stability of measurement result.
5, the method for the present invention is reproducible, and measurement result stability is high.
6,804 parts of materials work that sequencing is completed by International Rice Research Institute (IRRI) can be digested using the method for the present invention
Protein content determination is close with Protein Content of Rice report result.
Specific embodiment
Technical solution of the present invention is described further below by specific embodiment.
0.3%NaOH is added in 70% ethanol solution by the research to biotic component dissolution characteristics, discovery in inventor,
Protein Extraction amount is suitable with the directly extraction of 0.3%NaOH aqueous solution, but starch gelatinization phenomenon is not present during testing.Cause
This, inventor devises the horizontal Orthogonal Optimization Test of four factor four.
Sample preparation: rice varieties more light polished rice 10g or so are milled using laboratory Cyclone mill, are crossed 80 meshes, are placed in close
It is spare in envelope.
It extracts reagent: weighing appropriate NaOH, be placed in 100ml beaker, dissolved, poured into the ethanol water of respective concentration
In 2000ml volumetric flask, then with the cleaning of respective concentration ethanol water, it is transferred to volumetric flask together, mixes well, add 30~
The ethanol water constant volume of 60v/v% is mixed well to 2000ml, obtains different alkali concentrations as shown in table 3, concentration of alcohol mentions
Reagent is taken, is transferred in reagent bottle after standing 1 hour, it is spare.
Constant volume reagent: the concentration of sodium hydroxide is identical with the concentration of sodium hydroxide in reagent is extracted in constant volume reagent；It weighs
NaOH is placed in 100ml beaker, is dissolved with distilled water, is poured into 2000ml volumetric flask, and about 400ml distilled water is added, sufficiently mixed
It is even, distilled water constant volume is added to 2000ml, is mixed well, and is transferred in reagent bottle after standing 1 hour, it is spare.
Protein extracting method: weighing 0.0700 ± 0.0010g sample with a ten thousandth balance, be placed in 2.2ml round bottom from
In heart pipe, and is covered in centrifuge tube and write number exactly；Each sample extraction 2 times is added 1000 μ l in centrifuge tube every time and extracts examination
Agent covers, and mixes well, and is put into organic glass centrifuge tube shelf, is placed in and is preheating in 50 DEG C of ultrasonic cleaners, in power 500W
It is taken out after lower ultrasonic wave extraction, dries centrifuge tube, be placed in supercentrifuge, 12000rpm is centrifuged 8min, and supernatant is poured into
In corresponding number 25ml cleaning volumetric flask；It using constant volume reagent by volumetric flask constant volume to 25ml, and mixes well, stands 30min
It is to be measured.
Various Methods for Determing Different Proteins: taking 600 μ l of extracting solution of protein that can suitably dilute if concentration is more than 300 μ g/ml,
300 μ l of extracting solution is such as taken, adds and extracts 300 μ l of reagent, be directly added into 10mm glass cuvette, add Coomassie brilliant blue
2400 μ l of G250 reagent, 2min measures absorbance under 465nm, 600nm respectively after mixing, is denoted as A465, A600 respectively, calculates
A600/A465, according to G250 working curve (y=0.0204x+0.8639, R2=0.9971, x indicate that protein concentration, y indicate
A600/A465 protein concentration c in extracting solution of protein) is calculated, protein in rice sample is calculated according to formula (1) and is contained
Amount；
Wherein, x indicates rice sample protein matter content (%)；C indicates protein concentration (μ g/ in extracting solution of protein
ml)；V indicates constant volume (ml)；M indicates example weight (g)；P indicates to extract amount of reagent (μ l).
The preparation method of the Coomassie brilliant blue G250 reagent: taking 0.2000g Coomassie brilliant blue G250, with 95% ethyl alcohol
100ml dissolves by several times: first 95% ethyl alcohol about 30ml dissolves, and is cleaned twice with 50ml distilled water later, pours into 2000ml appearance
Measuring bottle, then cleaned with 95% ethyl alcohol of 30ml, volumetric flask is poured into, then cleaned twice with 50ml distilled water, pours into volumetric flask, finally
It is cleaned with 95% ethyl alcohol of residue, all pours into volumetric flask, 85% phosphoric acid of 200ml is added, adds distilled water constant volume, it is sufficiently mixed
It is even, 1 hour is stood, sets in 4 DEG C of refrigerators and saves, it is spare.
Table 3: factor and level design table
| Alkali concentration (%) | Concentration of alcohol (%) | Solid-to-liquid ratio (g/ml) | Ultrasonic time (min) |
| 0.25 | 30 | 1:10 | 30 |
| 0.3 | 40 | 1:15 | 40 |
| 0.35 | 50 | 1:20 | 50 |
| 0.4 | 60 | 1:25 | 60 |
It is designed according to table 1, using L1645Orthogonal arrage repeats three times, and orthogonal test scheme is shown in Table 2.
Table 4: orthogonal test scheme
| Tested number | Alkali concentration (%) | Concentration of alcohol (%) | Solid-to-liquid ratio (g/ml) | Ultrasonic time min |
| Test 01 | 0.25 | 30 | 1:10 | 30 |
| Test 02 | 0.25 | 40 | 1:15 | 40 |
| Test 03 | 0.25 | 50 | 1:20 | 50 |
| Test 04 | 0.25 | 60 | 1:25 | 60 |
| Test 05 | 0.3 | 30 | 1:20 | 60 |
| Test 06 | 0.3 | 40 | 1:25 | 50 |
| Test 07 | 0.3 | 50 | 1:10 | 40 |
| Test 08 | 0.3 | 60 | 1:15 | 30 |
| Test 09 | 0.35 | 30 | 1:25 | 40 |
| Test 10 | 0.35 | 40 | 1:20 | 30 |
| Test 11 | 0.35 | 50 | 1:15 | 60 |
| Test 12 | 0.35 | 60 | 1:10 | 50 |
| Test 13 | 0.4 | 30 | 1:15 | 50 |
| Test 14 | 0.4 | 40 | 1:10 | 60 |
| Test 15 | 0.4 | 50 | 1:25 | 30 |
| Test 16 | 0.4 | 60 | 1:20 | 40 |
Table 5: orthogonal experiments
Analysis of variance selects the maximum scheme of Protein Extraction amount as test 16.
Table 6: each tested number Statistical Comparison result
Table 7: orthogonal test variance analysis
| Source of variation | Make a variation quadratic sum | Make a variation freedom degree | It makes a variation square | F | P |
| Alkali concentration (%) | 1.446896 | 3 | 0.482299 | 4.228414 | 0.01187 |
| Concentration of alcohol (%) | 40.62117 | 3 | 13.54039 | 118.7114 | 2.09E-18 |
| Liquid-solid ratio | 1.549876 | 3 | 0.516625 | 4.529361 | 0.008729 |
| Ultrasonic time (min) | 0.347197 | 3 | 0.115732 | 1.014649 | 0.39788 |
| Error | 3.992149 | 35 | 0.114061 |
Known by table 7, Protein Extraction biggest impact factor is concentration of alcohol, and solid-to-liquid ratio and alkali concentration are secondly, ultrasonic time
Influence less, but 30min be lower than 40min, best concentration of alcohol 50%~60%, best alkali concentration 0.4%, best solid-to-liquid ratio 1:
10~20g/ml, therefore concentration of alcohol and solid-to-liquid ratio adjustment are remake on the basis of testing 16, three new testing programs are formed, with original
Optimal case is made to extract test again, repeats three times.
Table 8: alkali concentration level compares
Table 9: ethanol concentration level compares
Table 10: solid-to-liquid ratio level compares
Table 11: optimal case re-optimization test
Statistical analysis shows that concentration of alcohol (%) cannot change again, but solid-to-liquid ratio drops to 1:15, not only simplifies test
Operation, and compared with solid-to-liquid ratio 1:20, protein content slightly improves.
Table 12: optimal case re-optimization test statistics result
In conclusion the optimum process of Protein Extraction are as follows: by rice sample and extract reagent mixing, ultrasonic wave extraction 1
~3 times, often inferior to ultrasonic wave extraction 40min under 50 DEG C, 300~500W of power；After ultrasonic wave extraction, it is centrifuged to obtain supernatant, is adopted
It with constant volume reagent constant volume to 25ml, mixes well, obtains extracting solution of protein.Wherein, extracting concentration of alcohol in reagent is 50%
~60%, naoh concentration 0.4%, solid-to-liquid ratio 1:14~15g/ml；Constant volume reagent is the sodium hydroxide that concentration is 0.4%
Aqueous solution.
Embodiment 1
1, sample prepare: with laboratory Cyclone mill resource IRAT 335::C1 introduced to 10g or so International Rice,
ITALPATNA 48::GERVEX 60-C1, KOGONI 91-1::C1 (are respectively derived from Bolivia, Italy, Mali, kind
Kind is sequenced for International Rice, comes from International Rice Research Institute (IRRI)) brown rice milling, 80 meshes are crossed, sets in hermetic bag, writes exactly
Sample ID and number, it is spare.
2, digestible protein extracting method:
A, 0.0700 ± 0.0010g sample is weighed, is set in 2.2ml round bottom centrifuge tube, and is covered in centrifuge tube and writes number exactly；
1000 μ l, 70% ethyl alcohol is added in each centrifuge tube, covers, mixes well, be put into organic glass centrifuge tube shelf, be placed in advance
Heat by baking oven 16 hours of 50 DEG C, set in supercentrifuge by morning next day, and 12000rpm is centrifuged 8min, discards supernatant liquid；
B, 1000 μ l, 70% ethyl alcohol is added in each centrifuge tube, covers, mixes well, and is put into organic glass centrifuge tube
Frame is set in the solid spy's P9 ultrasonic cleaning machine for having been preheated with 50 DEG C, ultrasonic wave extraction 40 minutes at power 500W, is taken out, and is wiped
Dry centrifuge tube, sets in supercentrifuge, and 12000rpm is centrifuged 8min, discards supernatant liquid；
C, 1000 μ l are added in each centrifuge tube and extract reagent (ethanol water of the extraction reagent for sodium hydroxide, ethyl alcohol
Concentration is 60%, naoh concentration 0.4%), it covers, mixes well, be put into organic glass centrifuge tube shelf, be placed in advance
In heat to 50 DEG C of solid spy's P9 ultrasonic cleaning machine, ultrasonic wave extraction 40 minutes at power 500W take out, dry centrifuge tube, set
In supercentrifuge, 12000rpm is centrifuged 8min, and supernatant is poured into corresponding number 25ml cleaning volumetric flask；
D, residue, which adds, extracts 1000 μ l of reagent, and acutely shaking is mixed to complete, repeats C step operation；
E, it is primary to repeat D step.
F, using constant volume reagent (sodium hydrate aqueous solution that constant volume reagent is 0.4% as concentration) constant volume to 25ml, and sufficiently
It mixes to be measured.
3, Various Methods for Determing Different Proteins
600 μ l of extracting solution of protein is taken, if measuring concentration is more than 300 μ g/ml, can suitably dilute, such as take 300 μ of extracting solution
L is added and is extracted 300 μ l of reagent, is directly added into 10mm glass cuvette, adds 2400 μ l of Coomassie brilliant blue G250 reagent,
2min measures absorbance under 465nm and 600nm after mixing, is denoted as A465 and A600 respectively, A600/A465 is calculated, with the side of calibration
The working curve of method acquisition seeks protein concentration c, with formula
Wherein, x indicates protein content (%)；C indicates protein concentration (μ g/ml)；V indicates constant volume (ml), i.e.,
25ml；M indicates example weight (g)；P indicates to extract amount of reagent (μ l)；
Measuring three kind digestible protein contents is respectively 6.1540%, 6.5296%, 6.0986%.
Claims (10)
1. a kind of safe and efficient measuring method of protein content, it is characterised in that including Protein Extraction: by rice sample and mentioning
Reagent is taken to mix, ultrasonic wave extraction 1~3 time, often inferior to 30~40 points of ultrasonic wave extraction under 45~55 DEG C, 300~500W of power
Clock；After ultrasonic wave extraction, it is centrifuged to obtain supernatant, using constant volume reagent constant volume to 25ml, mixes well, obtains Protein Extraction
Liquid.
2. the safe and efficient measuring method of protein content according to claim 1, it is characterised in that steps are as follows:
Step (1), Protein Extraction: by rice sample and extract reagent mix, ultrasonic wave extraction 1~3 time, often inferior to 50 DEG C,
Ultrasonic wave extraction 30~40 minutes under 300~500W of power；After ultrasonic wave extraction, it is centrifuged to obtain supernatant, it is fixed using constant volume reagent
Hold 25ml, mix well, obtains extracting solution of protein；
Step (2), protein content determination: it takes 600 μ l of extracting solution of protein to 10mm glass cuvette, it is bright to add coomassie
Blue 2400 μ l of G250 reagent, measures absorbance under 465nm, 600nm respectively, is denoted as A465, A600 respectively after mixing, calculate
A600/A465 calculates protein concentration c in extracting solution of protein according to G250 working curve, water is calculated according to formula (1)
Rice sample protein matter content；
Wherein, x indicates rice sample protein matter content (%)；C indicates protein concentration (μ g/ml)；V indicates constant volume
(ml)；M indicates rice example weight (g)；P indicates to extract amount of reagent (μ l)；
Or 200 μ l of extracting solution of protein is taken, 3000 μ l BCA working solutions are added, 60 DEG C of water-baths keep the temperature 30min, measure in 1 hour
Absorbance A 562 under 562nm calculate protein concentration in extracting solution of protein according to BCA working curve, are acquired according to formula (2)
Rice sample protein matter content；
Wherein, x indicates protein content (%) in rice sample；C indicates protein concentration (μ g/ml)；V indicates constant volume
(ml)；M indicates rice example weight (g).
3. according to claim 1 or the safe and efficient measuring method of protein content, it is characterised in that the rice sample
Product are Brown Rice or polished rice is milled, 80 meshes excessively are made.
4. according to claim 1 or the safe and efficient measuring method of protein content, it is characterised in that the extraction examination
The solid-to-liquid ratio of agent and rice sample is 1:10~20g/ml or mg/ μ l, preferably 1:14~15g/ml or mg/ μ l.
5. according to claim 1 or the safe and efficient measuring method of protein content, it is characterised in that the extraction examination
30~60v/v% of concentration of alcohol in agent, naoh concentration are 0.25~0.4w/v%；Preferably, in the extraction reagent
Concentration of alcohol is 50~60v/v%, and naoh concentration is 0.35~0.4w/v%；
The constant volume reagent is sodium hydrate aqueous solution, the concentration of sodium hydroxide and hydroxide in extraction reagent in constant volume reagent
The concentration of sodium is identical.
6. according to claim 2 or the safe and efficient measuring method of the protein content, it is characterised in that in step (2), institute
When protein concentration is more than 300 μ g/ml in the extracting solution of protein stated, 300 μ l of extracting solution is taken, 300 μ l is added and extracts reagent.
7. according to claim 2 or the safe and efficient measuring method of the protein content, it is characterised in that in step (2), institute
The G250 working curve stated is y=0.0204x+0.8639, R2=0.9971, x indicate that protein concentration, y indicate A600/
A465；
The BCA working curve is y=0.0009x+0.0261, R2=0.9998, x indicate that protein concentration, y indicate extinction
Spend A562.
8. the safe and efficient measuring method of protein content according to claim 1 or 2, it is characterised in that further include that can digest
The extraction of albumen: rice sample and 70v/v% ethyl alcohol are mixed, and 45~55 DEG C stand 10~18 hours, and centrifugation discards supernatant
Liquid；Precipitating is mixed with 70v/v% ethyl alcohol, the ultrasonic wave extraction 30~40 minutes at 45~55 DEG C, 300~500W of power, centrifugation,
Liquid is discarded supernatant, the sample for the alcohol soluble protein that is removed；By the sample for removing alcohol soluble protein and reagent mixing is extracted, ultrasonic wave mentions
It takes 1~3 time, often inferior to ultrasonic wave extraction 30~40 minutes under 45~55 DEG C, 300~500W of power；After ultrasonic wave extraction, centrifugation
It obtains supernatant to mix well using constant volume reagent constant volume to 25ml, obtains digestible protein extracting solution.
9. the safe and efficient measuring method of protein content according to claim 8, it is characterised in that the removing alcohol is molten
The sample (in terms of rice sample) of albumen and extract solid-to-liquid ratio 1:10~20g/ml or mg/ the μ l, preferably 1:14 of reagent~
15g/ml or mg/ μ l.
10. the safe and efficient measuring method of protein content according to claim 8, it is characterised in that steps are as follows:
The extraction of step (1), digestible protein: rice sample and 70v/v% ethyl alcohol are mixed, and 45~55 DEG C of standings 10~18 are small
When, centrifugation discards supernatant liquid；Precipitating is mixed with 70v/v% ethyl alcohol, the ultrasonic wave extraction at 45~55 DEG C, 300~500W of power
30~40 minutes, centrifugation discarded supernatant liquid, the sample for the alcohol soluble protein that is removed；Sample and the extraction of alcohol soluble protein will be removed
Reagent mixes, and ultrasonic wave extraction 1~3 time, often inferior to ultrasonic wave extraction 30~40 minutes under 45~55 DEG C, 300~500W of power；
After ultrasonic wave extraction, it is centrifuged to obtain supernatant, using constant volume reagent constant volume to 25ml, mixes well, obtains protein extract；
Step (2), protein content determination: it takes 600 μ l of extracting solution of protein to 10mm glass cuvette, it is bright to add coomassie
Blue 2400 μ l of G250 reagent, measures absorbance under 465nm, 600nm respectively, is denoted as A465, A600 respectively after mixing, calculate
A600/A465 calculates protein concentration c in extracting solution of protein according to G250 working curve, water is calculated according to formula (1)
Rice sample protein matter content；
Wherein, x indicates rice sample protein matter content (%)；C indicates protein concentration (μ g/ml)；V indicates constant volume
(ml)；M indicates rice example weight (g)；P indicates to extract amount of reagent (μ l)；
Or 200 μ l of extracting solution of protein is taken, 3000 μ l BCA working solutions are added, 60 DEG C of water-baths keep the temperature 30min, measure in 1 hour
Absorbance A 562 under 562nm calculate protein concentration in extracting solution of protein according to BCA working curve, are acquired according to formula (2)
Rice sample protein matter content；
Wherein, x indicates protein content (%) in rice sample；C indicates protein concentration (μ g/ml)；V indicates constant volume
(ml)；M indicates rice example weight (g).
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| CN111505197A (en) * | 2020-05-28 | 2020-08-07 | 河南三方元泰检测技术有限公司 | Method for detecting protein content in food |
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| CN111060468A (en) * | 2020-01-19 | 2020-04-24 | 日照健安检测技术服务有限公司 | Method for rapidly detecting protein content in chitin |
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