CN110261332A - A kind of method of simple grain rice measurement content of amylose in rice - Google Patents
A kind of method of simple grain rice measurement content of amylose in rice Download PDFInfo
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Abstract
The invention discloses a kind of simple grain rice measurement content of amylose in rice method, including select amylose content different multiple rice varieties as reference sample；Amylose content is measured respectively；By the rice drying of reference sample and sample to be tested to constant weight, simple grain is chosen respectively, and decladding weighing is added NaOH solution, is shaken；The total volume for adding water to NaOH solution and water is the first volume, and centrifuging and taking supernatant is added isometric glacial acetic acid solution and Wagner's reagent, adds water to the second volume, stands, obtains developing solution；Absorbance value is measured, using the amylose content of reference sample as ordinate, the absorbance value for the 0.025g grain of rice that absorbance value is converted to is abscissa, makes standard curve, or obtain regression equation；Amylose content is calculated in the absorbance value for the 0.025g grain of rice being converted to according to the absorbance value of sample to be tested.It is of the invention unique novel, content of amylose in rice can be measured by simple grain rice, measurement is accurate and reliable.
Description
Technical field
The present invention relates to rice detection technique fields, in particular to rice grain amylose content detection technique field, specifically
Refer to a kind of method of simple grain rice measurement content of amylose in rice.
Background technique
Starch is the main component of rice endosperm, accounts for 90% or so of rice dry weight.Rice starch is mainly by linear straight
Chain starch (0% weight~30% weight) and hyperbranched amylopectin (70% weight~100% weight) composition.Wherein,
The food flavors sensory evaluation value such as Rice Amylose content and the hardness of rice, viscosity, glossiness is closely related.General amylose
The rice of too high levels, the rice being cooked into is hard, and viscosity is low, rice grain is loose, poor taste；And the rice that amylose content is too low,
Rice is too soft, rice grain bonding, and mouthfeel is also bad.Therefore, the content for measuring amylose is evaluation rice cooking and eating quality
One important indicator.
In recent years, with the proposition of the increase of rice yield and agriculture supply side structural reform, people are for high-quality food
The demand of taste rice increasingly increases.Therefore, carry out the survey of Grain Amylose content in advanced lines in rice varieties cultivating process
Fixed, targetedly selecting amylose content is just seemed in the rice material of optimum range with cultivating high-quality flavour rice kind
It is particularly important.
But in conventional breeding, pedigree material for screening and identifying, few then thousands of parts, more parts then up to ten thousand, quantity is huge
Greatly.And the measurement of conventional rice grain amylose content, it needs that paddy sample is first shelled and is milled into polished rice, then will
The sample of 10g or so crushes and crosses 0.25mm sieve, under room temperature referring to national standard GB/T 15683-2008 method after equilibrium water conten
It is measured.Not only have in the process to Rice Samples carry out out it is rough, go out essence, milling sieving etc. cumbersome early-stage preparations process, survey
Timing also needs the rice flour of careful weighing 100mg or so, disappears after boiling in boiling water bath 2 times and is settled to 100ml, expends a large amount of distillation
Water and reagent.Meanwhile for some for the Crop Physiology research that rice quality formation mechenism is carried out, it is possible to which needs are opened
The research and measurement of single spike of rice difference grain position (Superiors grains, Inferior grain) rice grain amylose content difference are opened up, and these are studied
If the measurement of middle rice grain amylose content is using the method for national standard GB/T 15683-2008, often not because of sample size
It is difficult to carry out enough.
Accordingly, it is desirable to provide a kind of for more than sample to be tested quantity or the simple amylose of rice that single sample amount is few contains
The measuring method of amount.
Summary of the invention
In order to overcome the disadvantages of the prior art mentioned above, it is an object of the present invention to provide a kind of simple grain rice to measure water
The method of rice amylose content, it is unique novel, content of amylose in rice can be measured by simple grain rice, measurement accurately may be used
It leans on, is suitable for large-scale promotion application.
Another object of the present invention is to provide a kind of method of simple grain rice measurement content of amylose in rice, design is skilful
It is wonderful, it is simple to operate, it is at low cost, it is suitable for large-scale promotion application.
To achieve the above objectives, the present invention provides a kind of method of simple grain rice measurement content of amylose in rice, feature
It is, comprising the following steps:
(1) the multiple rice varieties for selecting amylose content different are as reference sample；
(2) amylose content of the reference sample is measured respectively；
(3) by the rice drying of the paddy of the reference sample and sample to be tested to constant weight, the reference is chosen respectively
The paddy of the paddy of sample and the sample to be tested each one, decladding weighing is added NaOH solution, is shaken；
(4) total volume for adding water to the NaOH solution and the water is the first volume, and ice vinegar is added in centrifuging and taking supernatant
Acid solution and Wagner's reagent, the glacial acetic acid solution and the Wagner's reagent are isometric with the supernatant,
The other water is added to the second volume, stands, obtains developing solution；
(5) absorbance value for measuring the developing solution, using the amylose content of the reference sample as ordinate,
For the absorbance value for the 0.025g grain of rice being converted to using the absorbance value of the reference sample as abscissa, production standard is bent
Line, or the regression equation of the standard curve: Y=aX+b is obtained, in formula: Y is the amylose of the reference sample
Content, unit are % weight, and a is the slope of the standard curve, and b is the intercept of the standard curve；X is the reference sample
The absorbance value of the 0.025g grain of rice that is converted to of the absorbance value；
(6) absorbance value for the 0.025g grain of rice being converted to according to the absorbance value of the sample to be tested and described
The amylose content of the sample to be tested is calculated in standard curve or the regression equation.
Preferably, in the step (1), the number of the rice varieties is 4,4 rice varieties it is described
Amylose content is respectively low, partially basic, normal, high.
More preferably, in the step (1), 4 rice varieties are that town is glutinous 19, early No. 1 fragrant, the military fortune round-grained rice of pine respectively
27, Nanjing 11.
Preferably, further comprising the steps of: between the step (1) and the step (2) and using specific gravity for 1.10g/ml-
The salt water of 1.20g/ml chooses seeds to the reference sample.
Preferably, in the step (2), it is described to measure the method for using national standard GB/T 15683-2008.
Preferably, the concentration of the NaOH solution is 1.4M-1.6M, the body of the NaOH solution in the step (3)
50 times -70 times of the weight grams that product ml obtains for the weighing.
Preferably, in the step (3), after the NaOH solution is added and before carrying out the concussion, institute
The method for the simple grain rice measurement content of amylose in rice stated further include: inert glass beads are added.
Preferably, the concussion uses constant-temperature metal bath concussion instrument, and the temperature of the concussion is in the step (3)
80 DEG C -100 DEG C, the speed of the concussion is 800rpm-1200rpm, and the time of the concussion is -30 minutes 20 minutes.
Preferably, first volume is 1.4ml-1.6ml in the step (4), the volume of the supernatant is 50
μ L-70 μ L, second volume are 3.5ml-4.5ml.
Preferably, the time of the standing is -15 minutes 10 minutes in the step (4).
Beneficial effects of the present invention essentially consist in that:
1, the method for simple grain rice of the invention measurement content of amylose in rice includes that (1) selection amylose content is different
Multiple rice varieties as reference sample；(2) amylose content is measured respectively；(3) by reference sample and sample to be tested
Rice drying chooses simple grain, decladding weighing is added NaOH solution, is shaken to constant weight respectively；(4) NaOH solution is added water to
Total volume with water is the first volume, and centrifuging and taking supernatant is added isometric glacial acetic acid solution and Wagner's reagent, adds
Water stands to the second volume, obtains developing solution；(5) absorbance value is measured, is vertical sit with the amylose content of reference sample
Mark, the absorbance value for the 0.025g grain of rice that absorbance value is converted to are abscissa, make standard curve, or obtain regression equation
Formula；(6) absorbance value for the 0.025g grain of rice being converted to according to the absorbance value of sample to be tested and standard curve or recurrence
The amylose content of sample to be tested is calculated in equation, therefore, unique novel, and it is straight can to measure rice by simple grain rice
Chain content of starch, measurement is accurate and reliable, is suitable for large-scale promotion application.
2, the method for simple grain rice of the invention measurement content of amylose in rice includes that (1) selection amylose content is different
Multiple rice varieties as reference sample；(2) amylose content is measured respectively；(3) by reference sample and sample to be tested
Rice drying chooses simple grain, decladding weighing is added NaOH solution, is shaken to constant weight respectively；(4) NaOH solution is added water to
Total volume with water is the first volume, and centrifuging and taking supernatant is added isometric glacial acetic acid solution and Wagner's reagent, adds
Water stands to the second volume, obtains developing solution；(5) absorbance value is measured, is vertical sit with the amylose content of reference sample
Mark, the absorbance value for the 0.025g grain of rice that absorbance value is converted to are abscissa, make standard curve, or obtain regression equation
Formula；(6) absorbance value for the 0.025g grain of rice being converted to according to the absorbance value of sample to be tested and standard curve or recurrence
The amylose content of sample to be tested is calculated in equation, therefore, ingenious in design, simple to operate, at low cost, is suitable for
Large-scale promotion application.
These and other objects of the invention, feature and advantage, are filled by following detailed descriptions and claim
Fission is existing, and can be achieved by means, device and the their combination specially pointed out in appended claims.
Specific embodiment
In order to solve in rice variety selective or physiological Study rice more than the sample to be tested quantity or when single sample amount is few
The problem of amylose content determination, a kind of method that the present inventor proposes simple grain rice measurement content of amylose in rice, including
Following steps:
(1) the multiple rice varieties for selecting amylose content different are as reference sample；
(2) amylose content of the reference sample is measured respectively；
(3) by the rice drying of the paddy of the reference sample and sample to be tested to constant weight, the reference is chosen respectively
The paddy of the paddy of sample and the sample to be tested each one, decladding weighing is added NaOH solution, is shaken；
(4) total volume for adding water to the NaOH solution and the water is the first volume, and ice vinegar is added in centrifuging and taking supernatant
Acid solution and Wagner's reagent, the glacial acetic acid solution and the Wagner's reagent are isometric with the supernatant,
The other water is added to the second volume, stands, obtains developing solution；
(5) absorbance value for measuring the developing solution, using the amylose content of the reference sample as ordinate,
For the absorbance value for the 0.025g grain of rice being converted to using the absorbance value of the reference sample as abscissa, production standard is bent
Line, or the regression equation of the standard curve: Y=aX+b is obtained, in formula: Y is the amylose of the reference sample
Content, unit are % weight, and a is the slope of the standard curve, and b is the intercept of the standard curve；X is the reference sample
The absorbance value of the 0.025g grain of rice that is converted to of the absorbance value；
(6) absorbance value for the 0.025g grain of rice being converted to according to the absorbance value of the sample to be tested and described
The amylose content of the sample to be tested is calculated in standard curve or the regression equation.
In the step (1), the number of the rice varieties can according to need determination, preferably, in the step
(1) in, the numbers of the rice varieties is 4, the amylose content of 4 rice varieties is respectively low, it is relatively low,
It is middle and high.
In the step (1), 4 rice varieties can be any suitable rice varieties, more preferably, described
In step (1), 4 rice varieties be respectively town glutinous 19 (obtained from Jiangsu Hilly Ground Zhenjiang Agriculture Science Research Institute),
Loose early fragrant No. 1 (obtained from Shanghai Songjiang area agricultural technology extension center), military fortune round-grained rice 27 (obtained from Wujin rice research institute),
Nanjing 11 (obtained from Jiangsu Province Agriculture Science Institute).
In order to guarantee that paddy is full, amylose content is relatively stable in reference sample simple grain rice, preferably, in the step
Suddenly between (1) and the step (2), further comprising the steps of: and using specific gravity is the salt water of 1.10g/ml-1.20g/ml to the reference
Sample is chosen seeds.
In the step (2), the measurement can use any suitable method, preferably, in the step (2)
In, it is described to measure the method for using national standard GB/T 15683-2008.
In the step (3), the drying can use any suitable temperature, preferably, in the step (3)
In, the drying uses 40 DEG C -60 DEG C.
In the step (3), the weighing can use any suitable device, preferably, in the step (3)
In, the weighing uses a ten thousandth balance.
In the step (3), the concentration and dosage of the NaOH solution can according to need determination, preferably, in institute
It states in step (3), the concentration of the NaOH solution is 1.4M-1.6M, and the volume ml of the NaOH solution is the weighing
50 times -70 times of the weight grams of acquisition.
In order to promote amylose in the grain of rice to dissolve out, preferably, the NaOH solution is being added in the step (3)
Later and before carrying out the concussion, the method for the simple grain rice measurement content of amylose in rice further include: be added lazy
Property bead.
In the step (3), the number and diameter of the inert glass beads can according to need determination, more preferably,
In the step (3), the number of the inert glass beads is 2-3, and the diameter of the inert glass beads is 2mm-3mm.
In the step (3), the concussion can use any suitable device, preferably, in the step (3)
In, the concussion uses constant-temperature metal bath concussion instrument, and the temperature of the concussion is 80 DEG C -100 DEG C, and the speed of the concussion is
800rpm-1200rpm, the time of the concussion are -30 minutes 20 minutes.
In the step (4), first volume, the volume of the supernatant and second volume can be according to need
It was determined that the volume of the supernatant is 50 μ preferably, first volume is 1.4ml-1.6ml in the step (4)
L-70 μ L, second volume are 3.5ml-4.5ml.
In the step (4), the concentration of the glacial acetic acid solution can according to need determination, preferably, in the step
Suddenly in (4), the concentration of the glacial acetic acid solution is 30%.
In the step (4), the concentration of the Wagner's reagent can according to need determination, preferably, described
In step (4), the Wagner's reagent contains the iodine of 0.008M/L and the potassium iodide of 0.12M/L.
In the step (4), the time of the standing can according to need determination, preferably, in the step (4)
In, the time of the standing is -15 minutes 10 minutes.
In order to be more clearly understood that technology contents of the invention, spy lifts following embodiment and is described in detail.In the present invention,
Related component or raw material are conventional commercial product, or can be obtained by ordinary skill in the art means.
Embodiment 1
Reference sample selects 4 rice varieties, and respectively (obtained from Hilly area of Jiangsu Province Zhenjiang, agricultural sciences is ground in town glutinous 19
Study carefully institute), pine early fragrant No. 1 (obtained from Shanghai Songjiang area agricultural technology extension center), (obtained from Wujin, rice is ground military fortune round-grained rice 27
Study carefully institute), Nanjing 11 (obtained from Jiangsu Province Agriculture Science Institute).
Sample to be tested selects 8 rice varieties, and respectively (obtained from Xu-Huai area Huaiyin, Jiangsu, agricultural sciences is ground in Huaihe River glutinous 12
Study carefully institute), raise that round-grained rice glutinous No. 2 (obtained from Inst. of Agricultural Science, Lixiahe Prefecture, Jiangsu Prov.), Su Xiang round-grained rice 3 (obtained from Suzhou City's agriculture
The industry academy of sciences), southern round-grained rice 9108 (obtained from academy of agricultural sciences, Jiangsu Province), Soviet Union's round-grained rice 815 is (obtained from the limited public affairs of Jiangsu Zhong Jiang kind industry share
Department), Huaihe River rice No. 5 (obtained from Jiangsu Xuhuai Zone Huaiyin Agricultural Sciences Institute), elegant water 134 is (obtained from Jiaxing City agricultural section
Learn research), raise and educate round-grained rice 2 (obtained from Yancheng City Yandu District Institute of agricultural sciences).
The paddy of reference sample and sample to be tested passes through specific gravity and sorts seed by salt water for 1.13g/ml, then according to national standard GB/
The method of T15683-2008 measures its amylose content.
Drying to constant weight under the conditions of 55 DEG C for the paddy of reference sample and sample to be tested, selects reference sample and sample to be tested
Paddy each 1, peel off husk, the grain of rice is simultaneously placed in 2ml centrifuge tube by the weight of a ten thousandth balance lower-weighing simple grain grain of rice,
According to grain of rice weight (g): the inertia glass that diameter is 2.5mm is added than adding 1.5MNaOH solution for 1: 60 in liquor capacity (ml)
2, pearl, then centrifuge tube is placed under the conditions of upper 100 DEG C of constant-temperature metal bath concussion instrument (Thermo Shaker) and is shaken 30 minutes,
Speed is 1000rpm.
Centrifuge tube is taken out after 30 minutes, the reaction system for being 1.5ml according to 1.5MNaOH solution+water total volume, supplement
The water of difference is centrifuged 10 minutes under the conditions of 10000r/min.The 50 μ L of supernatant for drawing reference sample and sample to be tested is placed in 5ml
In centrifuge tube, the glacial acetic acid solution of 50 μ L30% is added, 50 μ L iodine (0.008M/L)-potassium iodide (0.12M/L) solution increase
The water of 3.85ml makes reaction system up to 4ml amount of solution, 10 minutes is stood, to color stability.
The developing solution for drawing 200 μ L reference samples and sample to be tested, is placed in 96 hole plates, and quickly measurement is inhaled in microplate reader
Shading value.
The amylose content measured using reference sample according to national standard GB/T15683-2008 method is ordinate, the present invention
The absorbance value for the 0.025g grain of rice that the absorbance value that method measures is converted to (is equal to reference sample simple grain by the method for the invention
The absorbance value measured is multiplied by 0.025 divided by reference sample simple grain grain weight) it is abscissa (the results are shown in Table 1), make standard curve
Go out the regression equation of standard curve: Y=64.943X-5.6877 side by side.
Amylose content that 14 rice varieties of table are measured using GB/T 15683-2008 method, using present invention side
The absorbance value of absorbance value and conversion that method measures
According to above-mentioned regression equation, the 0.025g being converted to sample to be tested according to the absorbance value that the method for the present invention measures
(absorbance value measured by the method for the invention equal to sample to be tested simple grain is multiplied by 0.025 divided by be measured for the absorbance value of the grain of rice
Sample simple grain grain weight) it is X, the amylose content (% weight) of sample to be tested is calculated, as a result see the table below 2:
The amylose content that 28 rice varieties of table use GB/T 15683-2008 method and the method for the present invention to measure
Correlation analysis is shown the amylose content of the rice using method of the invention, measured using simple grain rice and made
With correlation coefficient r=0.9783 between the amylose content numerical value of the rice of national standard GB/T 15683-2008 method measurement, reach
Extremely significant level.
Embodiment 2
Reference sample selects 4 rice varieties, and respectively (obtained from Hilly area of Jiangsu Province Zhenjiang, agricultural sciences is ground in town glutinous 19
Study carefully institute), pine early fragrant No. 1 (obtained from Shanghai Songjiang area agricultural technology extension center), (obtained from Wujin, rice is ground military fortune round-grained rice 27
Study carefully institute), Nanjing 11 (obtained from Jiangsu Province Agriculture Science Institute).
Sample to be tested selects 6 rice varieties, and respectively magnificent round-grained rice 5 is (limited obtained from great Hua Zhong industry group, Jiangsu Province
It is public), raise round-grained rice 5118 (obtained from Inst. of Agricultural Science, Lixiahe Prefecture, Jiangsu Prov.), Su Xiang round-grained rice 100 (obtained from Suzhou City's agricultural
The academy of sciences), southern round-grained rice 5055 (obtained from academy of agricultural sciences, Jiangsu Province), Xu rice No. 9 (obtained from Xuzhou Institute of agricultural sciences), salt round-grained rice 16
(obtained from Yancheng City Yandu District Institute of agricultural sciences).
The paddy of reference sample and sample to be tested passes through specific gravity and sorts seed by salt water for 1.20g/ml, then according to national standard GB/T
The method of 15683-2008 measures its amylose content.
Drying to constant weight under the conditions of 50 DEG C for the paddy of reference sample and sample to be tested, selects reference sample and sample to be tested
Paddy each 1, peel off husk, the grain of rice is simultaneously placed in 2ml centrifuge tube by the weight of a ten thousandth balance lower-weighing simple grain grain of rice,
According to grain of rice weight (g): the inertia glass that diameter is 2.5mm is added than adding 1.6MNaOH solution for 1: 50 in liquor capacity (ml)
3, pearl, then centrifuge tube is placed under the conditions of upper 90 DEG C of constant-temperature metal bath concussion instrument (Thermo Shaker) and is shaken 20 minutes,
Speed is 800rpm.
Centrifuge tube is taken out after 25 minutes, the reaction system for being 1.6ml according to 1.5MNaOH solution+water total volume, supplement
The water of difference is centrifuged 10 minutes under the conditions of 10000r/min.The 60 μ L of supernatant for drawing reference sample and sample to be tested is placed in 5ml
In centrifuge tube, the glacial acetic acid solution of 60 μ L 30% is added, 60 μ L iodine (0.008M/L)-potassium iodide (0.12M/L) solution increase
The water of 3.32ml makes reaction system up to 3.5ml amount of solution, 12 minutes is stood, to color stability.
The developing solution for drawing 200 μ L reference samples and sample to be tested, is placed in 96 hole plates, and quickly measurement is inhaled in microplate reader
Shading value.
The amylose content measured using reference sample according to national standard GB/T 15683-2008 method is ordinate, this hair
The absorbance value for the 0.025g grain of rice that the absorbance value that bright method measures is converted to (is equal to reference sample simple grain side through the invention
The absorbance value that method measures is multiplied by 0.025 divided by reference sample simple grain grain weight) it is abscissa (the results are shown in Table 3), production standard is bent
Line goes out the regression equation of standard curve: Y=55.868X-1.1139 side by side.
Amylose content that 34 reference samples of table are measured using GB/T 15683-2008 method, using present invention side
The absorbance value of absorbance value and conversion that method measures
According to above-mentioned regression equation, the 0.025g being converted to sample to be tested according to the absorbance value that the method for the present invention measures
(absorbance value measured by the method for the invention equal to sample to be tested simple grain is multiplied by 0.025 divided by be measured for the absorbance value of the grain of rice
Sample simple grain grain weight) it is X, the amylose content (% weight) of sample to be tested is calculated, as a result see the table below 4:
The amylose content that 46 rice varieties of table use GB/T 15683-2008 method and the method for the present invention to measure
Correlation analysis is shown the amylose content of the rice using method of the invention, measured using simple grain rice and made
With correlation coefficient r=0.9950 between the amylose content numerical value of the rice of national standard GB/T 15683-2008 method measurement, reach
Extremely significant level.
Embodiment 3
Reference sample selects 4 rice varieties, and respectively (obtained from Hilly area of Jiangsu Province Zhenjiang, agricultural sciences is ground in town glutinous 19
Study carefully institute), pine early fragrant No. 1 (obtained from Shanghai Songjiang area agricultural technology extension center), (obtained from Wujin, rice is ground military fortune round-grained rice 27
Study carefully institute), Nanjing 11 (obtained from Jiangsu Province Agriculture Science Institute).
Sample to be tested selects 8 rice varieties, and respectively (obtained from Jiangsu, legendary god of farming's great Feng kind industry science and technology is limited for good fortune round-grained rice 1606
Company), Shanghai early fragrant soft No. 2 (obtained from Academy of Agricultural Sciences, Shanghai City), even round-grained rice 15 (obtained from Lianyungang Academy of Agricultural Science), holy
Rice 18 (obtained from Shandong Rice Research Institute), nasal mucus rice No. 15 (obtained from the Jiangsu Province Agriculture Science Institute Suqian institute of agricultural sciences), Wu Yun
Round-grained rice 32 (obtained from Jiangsu Wujin rice research institute), new rice 22 (obtained from Henan Xinxiang Academy of Agricultural Science), river in Zhejiang Province are excellent
1540 (obtained from academies of agricultural sciences, Ningbo City).
The paddy of reference sample and sample to be tested passes through specific gravity and sorts seed by salt water for 1.10g/ml, then according to national standard GB/T
The method of 15683-2008 measures its amylose content.
Drying to constant weight under the conditions of 60 DEG C for the paddy of reference sample and sample to be tested, selects reference sample and sample to be tested
Paddy each 1, peel off husk, the grain of rice is simultaneously placed in 2ml centrifuge tube by the weight of a ten thousandth balance lower-weighing simple grain grain of rice,
According to grain of rice weight (g): the inert glass beads 2 that diameter is 3mm are added than adding 1.4MNaOH solution for 1: 70 in liquor capacity (ml)
, then centrifuge tube is placed under the conditions of upper 80 DEG C of constant-temperature metal bath concussion instrument (Thermo Shaker) and is shaken 25 minutes, speed
For 1200rpm.
Centrifuge tube is taken out after 30 minutes, the reaction system for being 1.4ml according to 1.5MNaOH solution+water total volume, supplement
The water of difference is centrifuged 10 minutes under the conditions of 10000r/min.The 70 μ L of supernatant for drawing reference sample and sample to be tested is placed in 5ml
In centrifuge tube, the glacial acetic acid solution of 70 μ L 30% is added, 70 μ L iodine (0.008M/L)-potassium iodide (0.12M/L) solution increase
The water of 4.29ml makes reaction system up to 4.5ml amount of solution, 15 minutes is stood, to color stability.
The developing solution for drawing 200 μ L reference samples and sample to be tested, is placed in 96 hole plates, and quickly measurement is inhaled in microplate reader
Shading value.
The amylose content measured using reference sample according to national standard GB/T 15683-2008 method is ordinate, this hair
The absorbance value for the 0.025g grain of rice that the absorbance value that bright method measures is converted to (is equal to reference sample simple grain side through the invention
The absorbance value that method measures is multiplied by 0.025 divided by reference sample simple grain grain weight) it is abscissa (the results are shown in Table 5), production standard is bent
Line goes out the regression equation of standard curve: Y=55.681X-1.2608 side by side.
Amylose content that 54 reference samples of table are measured using GB/T 15683-2008 method, using present invention side
The absorbance value of absorbance value and conversion that method measures
According to above-mentioned regression equation, the 0.025g being converted to sample to be tested according to the absorbance value that the method for the present invention measures
(absorbance value measured by the method for the invention equal to sample to be tested simple grain is multiplied by 0.025 divided by be measured for the absorbance value of the grain of rice
Sample simple grain grain weight) it is X, the amylose content (% weight) of sample to be tested is calculated, as a result see the table below 6:
The amylose content that 68 rice varieties of table use GB/T 15683-2008 method and the method for the present invention to measure
Correlation analysis is shown the amylose content of the rice using method of the invention, measured using simple grain rice and made
With correlation coefficient r=0.9952 between the amylose content numerical value of the rice of national standard GB/T 15683-2008 method measurement, reach
Extremely significant level.
Therefore, the present invention is a kind of method using simple grain rice measurement content of amylose in rice, can solve rice varieties
The problem of rice grain amylose content measures more than the sample to be tested quantity or when single sample amount is few in breeding or physiological Study, surveys
Amount is accurate and reliable.
To sum up, the method for simple grain rice of the invention measurement content of amylose in rice is unique novel, can pass through simple grain rice
Content of amylose in rice is measured, measurement is accurate and reliable, and it is ingenious in design, it is simple to operate, it is at low cost, it is suitable for large-scale promotion
Using.
It can be seen that the purpose of the present invention completely and is effectively achieved.Function and structural principle of the invention
It is shown and is illustrated in embodiment, under without departing substantially from the principle, embodiment can make any modification.So this hair
Bright includes all variant embodiments based on claim spirit and scope of the claims.
Claims (10)
1. a kind of method of simple grain rice measurement content of amylose in rice, which comprises the following steps:
(1) the multiple rice varieties for selecting amylose content different are as reference sample；
(2) amylose content of the reference sample is measured respectively；
(3) by the rice drying of the paddy of the reference sample and sample to be tested to constant weight, the reference sample is chosen respectively
Paddy and paddy each one of the sample to be tested, decladding weighing is added NaOH solution, is shaken；
(4) total volume for adding water to the NaOH solution and the water is the first volume, and it is molten that glacial acetic acid is added in centrifuging and taking supernatant
Liquid and Wagner's reagent, the glacial acetic acid solution and the Wagner's reagent are isometric with the supernatant, are added
In addition the water stands to the second volume, obtains developing solution；
(5) absorbance value for measuring the developing solution, using the amylose content of the reference sample as ordinate, with institute
The absorbance value for stating the 0.025g grain of rice that the absorbance value of reference sample is converted to is abscissa, makes standard curve, or
Obtaining the regression equation of the standard curve: Y=aX+b, in formula: Y is the amylose content of the reference sample,
Unit is % weight, and a is the slope of the standard curve, and b is the intercept of the standard curve；X for the reference sample institute
State the absorbance value for the 0.025g grain of rice that absorbance value is converted to；
(6) absorbance value and the standard for the 0.025g grain of rice being converted to according to the absorbance value of the sample to be tested
The amylose content of the sample to be tested is calculated in curve or the regression equation.
2. the method for simple grain rice measurement content of amylose in rice as described in claim 1, which is characterized in that in the step
(1) in, the numbers of the rice varieties is 4, the amylose content of 4 rice varieties is respectively low, it is relatively low,
It is middle and high.
3. the method for simple grain rice measurement content of amylose in rice as claimed in claim 2, which is characterized in that in the step
(1) in, 4 rice varieties are that town is glutinous 19, pine early No. 1 fragrant, military fortune round-grained rice 27, Nanjing 11 respectively.
4. the method for simple grain rice measurement content of amylose in rice as described in claim 1, which is characterized in that in the step
(1) between the step (2), further comprising the steps of: and using specific gravity is the salt water of 1.10g/ml-1.20g/ml to the reference sample
Product are chosen seeds.
5. the method for simple grain rice measurement content of amylose in rice as described in claim 1, which is characterized in that in the step
(2) described to measure the method for using national standard GB/T 15683-2008 in.
6. the method for simple grain rice measurement content of amylose in rice as described in claim 1, which is characterized in that in the step
(3) in, the concentration of the NaOH solution is 1.4M-1.6M, and the volume ml of the NaOH solution is that the weighing obtains
50 times -70 times of weight grams.
7. the method for simple grain rice measurement content of amylose in rice as described in claim 1, which is characterized in that in the step
(3) in, after the NaOH solution is added and before carrying out the concussion, the simple grain rice measures amylose in rice
The method of content further include: inert glass beads are added.
8. the method for simple grain rice measurement content of amylose in rice as described in claim 1, which is characterized in that in the step
(3) in, the concussion uses constant-temperature metal bath concussion instrument, and the temperature of the concussion is 80 DEG C -100 DEG C, the speed of the concussion
For 800rpm-1200rpm, the time of the concussion is -30 minutes 20 minutes.
9. the method for simple grain rice measurement content of amylose in rice as described in claim 1, which is characterized in that in the step
(4) in, first volume is 1.4ml-1.6ml, and the volume of the supernatant is 50 μ L-70 μ L, and second volume is
3.5ml-4.5ml。
10. the method for simple grain rice measurement content of amylose in rice as described in claim 1, which is characterized in that in the step
Suddenly in (4), the time of the standing is -15 minutes 10 minutes.
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