CN107167550A - Non-Structural Carbohydrate content assaying method in silage corn - Google Patents
Non-Structural Carbohydrate content assaying method in silage corn Download PDFInfo
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- CN107167550A CN107167550A CN201710552191.6A CN201710552191A CN107167550A CN 107167550 A CN107167550 A CN 107167550A CN 201710552191 A CN201710552191 A CN 201710552191A CN 107167550 A CN107167550 A CN 107167550A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/10—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using catalysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The invention discloses Non-Structural Carbohydrate content assaying method in a kind of silage corn, including：The silage corn complete stool that takes is crushed, fixing, after drying, then crushed 40 mesh sieves, is dried, closed to preserve, and testing sample is made；Quantitatively weigh testing sample, sodium chloride buffer is added to suspend, boiling water bath 10min makes starch gelatinization, less than 60 DEG C are cooled to after gelatinization, alpha amylase liquid, amylopectin enzyme liquid and saccharification enzyme liquid are sequentially added, is incubated hydrolyzes with the optimal reaction environment of each enzyme respectively, constant volume, filtering, take filtrate to survey reduced sugar, Non-Structural Carbohydrate content is calculated according to reduced sugar.By sequentially adding alpha amylase liquid, amylopectin enzyme liquid and saccharification enzyme liquid, it is incubated and is hydrolyzed with the optimum temperature of each enzyme respectively, utilize enzymic catalytic reaction selectivity feature, only starch is thoroughly hydrolyzed, lignin, cellulosic structure are not destroyed, effective exclusive PCR, energy Accurate Determining reduced sugar, and then accurately calculate Non-Structural Carbohydrate content.
Description
Technical field
Contain the present invention relates to Non-Structural Carbohydrate in silage corn detection field, more particularly to a kind of silage corn
Quantity measuring method.
Background technology
Silage corn is a kind of important ensilage, and critical role is occupied in ruminant feed.Silage corn is
Refer to the milk stage in corn to stage of wax ripeness, the whole strain harvest corn by corn stem leaf or with fruit ear can Direct-fed after chopping
The ruminant animals such as ox, sheep, mainly carry out storage fermentation.Silage corn after fermentation have nutritious, palatability it is good, turn
Rate is high, livestock products quality is excellent, the advantages of can storing for a long time, can not only be provided for domestic animal needed for growing and producing
Nutrition, or the winter-spring season of green forage shortage provide feed supplement, are the main forage grass and feeds of livestock breeding industry.
A central file is pointed out within 2015：" careless animal husbandry is accelerated development, the fodder grass such as silage corn and clover is supported
Plantation, carries out ' grain changes feeding ' and breeding binding pattern pilot, promotes grain, industrial crops, fodder grass triple refrigeration to coordinate
Development ".The strategy and policy of China's animal husbandry Long-and Medium-term Development are to greatly develop the production of the ruminant Livestock such as ox, sheep, its
Important basal feed is high-quality roughage, and silage corn is greatly increased as important feed resource, its demand.So,
Plantation silage corn not only becomes the new outlet of grain farmer, while having also conformed to the demand for development in epoch.
The seed selection of Silage Maize varieties is particularly important, and the quality of silage corn is then the important of seed selection Silage Maize varieties
One of condition.In silage corn carbohydrate content number, be evaluate silage corn quality important indicator.Carbon hydrate
Thing is the photosynthetic primary product of silage corn, including structural carbohydrate and Non-Structural Carbohydrate.Structure
Property carbohydrate mainly include cellulose, hemicellulose, lignin, Non-Structural Carbohydrate refers mainly to starch and solvable
Property sugared (maltose, glucose etc.).The existing national standard of the measure of structural carbohydrate and industry standard methods.It is beautiful for ensiling
The measure of Non-Structural Carbohydrate is there is not yet national standard or professional standard issue in rice；Disclosure is also not yet retrieved at present
The method and technology of Non-Structural Carbohydrate content detection in the silage corn delivered.And unstructuredness carbon in silage corn
Hydrate content detection difficult point is the interference of the materials such as the cellulose, hemicellulose, lignin that largely exist, and general ensiling is beautiful
Cellulose, hemicellulose and content of lignin account for 35%~65% in rice, and silage corn unstructuredness carbon is determined with conventional method
During hydrate content, cellulose and hemicellulose are together hydrolyzed into reduced sugar, have a strong impact on Non-Structural Carbohydrate
Assay, it is impossible to obtain accurate result.
The content of the invention
Based on the problems of prior art, it is an object of the invention to provide unstructuredness carbon water in a kind of silage corn
The content of Non-Structural Carbohydrate in compounds content assay method, energy Accurate Determining silage corn.
The purpose of the present invention is achieved through the following technical solutions：
Embodiment of the present invention provides Non-Structural Carbohydrate content assaying method in a kind of silage corn, including：
After taking detected silage corn complete stool crushed, finished, drying, 40 mesh sieves were crushed again, were dried, it is closed
Preserve, be prepared into testing sample；Testing sample is quantitatively weighed, sodium chloride buffer is added and suspends, boiling water bath 10min makes gelatinized corn starch
Change, less than 60 DEG C are cooled to after gelatinization, sequentially add alphalise starch enzyme liquid, amylopectin enzyme liquid and saccharification enzyme liquid, each enzyme is used respectively
Optimal reaction environment be incubated hydrolysis, constant volume, filtering, take filtrate survey reduced sugar, detected green grass or young crops is calculated according to reduced sugar
Store the Non-Structural Carbohydrate content of corn.
As seen from the above technical solution provided by the invention, non-structural in silage corn provided in an embodiment of the present invention
Property carbohydrate content assay method, its advantage is：
By sequentially adding alphalise starch enzyme liquid, amylopectin enzyme liquid and saccharification enzyme liquid, respectively with the optimal reaction ring of each enzyme
Border is incubated hydrolysis, using enzymic catalytic reaction selectivity feature, and only Non-Structural Carbohydrate is thoroughly hydrolyzed, will not be broken
Bad cellulose, hemicellulose, the structure of lignin, effectively eliminate interference, the reduction contained by energy Accurate Determining silage corn
Sugar, and then the accurate Non-Structural Carbohydrate content for calculating detected silage corn.
Brief description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, being used required in being described below to embodiment
Accompanying drawing be briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for this
For the those of ordinary skill in field, on the premise of not paying creative work, other can also be obtained according to these accompanying drawings
Accompanying drawing.
Fig. 1 is Non-Structural Carbohydrate content assaying method flow in silage corn provided in an embodiment of the present invention
Figure.
Embodiment
With reference to the particular content of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely retouched
State, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based on the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made,
Belong to protection scope of the present invention.
The embodiment of the present invention provides Non-Structural Carbohydrate content assaying method in a kind of silage corn, including (ginseng
See Fig. 1)：
After taking detected silage corn complete stool crushed, finished, drying, 40 mesh sieves were crushed again, were dried, it is closed
Preserve, be prepared into testing sample；Testing sample is quantitatively weighed, sodium chloride buffer is added and suspends, boiling water bath 10min makes gelatinized corn starch
Change, less than 60 DEG C are cooled to after gelatinization, sequentially add alphalise starch enzyme liquid, amylopectin enzyme liquid and saccharification enzyme liquid, each enzyme is used respectively
Optimal reaction environment be incubated hydrolysis, constant volume, filtering, take filtrate survey reduced sugar, detected green grass or young crops is calculated according to reduced sugar
Store the Non-Structural Carbohydrate content of corn.
In the above method, the Non-Structural Carbohydrate content of detected silage corn is calculated according to reduced sugar
Calculation formula be：
In above formula：k：Reductive monosaccharide slope of standard curve；
V1：Sample solution cumulative volume (ml)；
m：Example weight (g)；
V2：Colour developing sample volume (ml)；
0.9：Reductive monosaccharide is converted into the coefficient of Non-Structural Carbohydrate；
OD values：Absorbance
Accuracy：The absolute difference of the measurement result independent twice obtained under the conditions of repeatability must not exceed arithmetic average
The 10% of value.
In said determination method, the addition that sodium chloride buffer is added into the silage corn testing sample is
NaCl mass percentage concentration is 0.3% in 100ml, the sodium chloride buffer；
In said determination method, alphalise starch enzyme liquid, amylopectin enzyme liquid and saccharification enzyme liquid are sequentially added as detection agent, is used
The optimal reaction environment of enzyme, which is incubated hydrolysis, to be included：
First it is gelatinized to the silage corn sample after cooling in thing and adds alphalise starch enzyme liquid, 40min is incubated at 85 DEG C, hydrolysis makes
Silage corn starch gelatinization thing liquid；
Then, amylopectin enzyme liquid is added into silage corn starch gelatinization thing liquefier, carbohydrase is incubated at 60 DEG C
30min, hydrolyzes α -1,6 glycosidic bond amylopectin in the liquefier；
Finally, to adding saccharification enzyme liquid after liquefier, in 60 DEG C of limit dextrins being incubated in 30min, hydrolyzate liquid and
Maltose is into glucose.
In said determination method, alphalise starch enzyme liquid, amylopectin enzyme liquid and saccharification enzyme liquid are sequentially added as in detection agent,
The addition of the alphalise starch enzyme liquid is 10ml, and mass percentage concentration is 1%；
The addition of the amylopectin enzyme liquid is 10ml, and mass percentage concentration is 1%；
The addition of the saccharification enzyme liquid is 10ml, and mass percentage concentration is 10%.
The embodiment of the present invention is specifically described in further detail below.
As shown in figure 1, the present embodiment provides Non-Structural Carbohydrate content assaying method, energy in a kind of silage corn
The content of Non-Structural Carbohydrate, comprises the following steps in Accurate Determining silage corn：
Step 1, silage corn complete stool is crushed, finished, dried；
Step 2,40 mesh sieves were crushed again, were dried, it is closed to preserve, it is prepared into testing sample；
Step 3, suspended with sodium chloride buffer；
Step 4, boiling water bath 10min, makes starch gelatinization, is cooled to less than 60 DEG C；
Step 5,85 DEG C of incubation 40min of alphalise starch enzyme liquid are added, hydrolysis makes it liquefy；
Step 6, amylopectin enzyme liquid, 60 DEG C of incubation 30min, hydrolyzing alpha -1,6 glycosidic bond amylopectin are added
Step 7, carbohydrase is added, 60 DEG C of incubation 30min, water hydrolysis limit dextrin and maltose are into glucose
Step 8, constant volume, filter, take filtrate survey reduced sugar；
Step 9, according to the reduced sugar measured, the unstructuredness carbon hydrate for obtaining detected silage corn complete stool is calculated
Thing content.
Embodiment 1
From (the variety name of kind 1 prepared：Northern agriculture ensiling 368, source：Beijing Agricultural College) 3 parts are weighed in sample
3.00g parallel sample, record weighs weight (being accurate to one thousandth gram), plus the sodium chloride buffers of 100ml 0.3% suspend,
Boiling water bath heats 10min, after be cooled to less than 60 DEG C, the addition alphalise starch enzyme liquids of 10ml 1% are cold in being incubated 40min at 85 DEG C
But to room temperature, adjust pH=5.5, add the amylopectases of 10ml 1% in being incubated 30min at 60 DEG C, after be cooled to room temperature, adjust
PH=4.0 is saved, the carbohydrase of 10ml 10% is added, in being incubated 30min, boiling water bath 10min at 60 DEG C, inactivates enzyme, be settled to
250ml, mixes, filters, collect filtrate, filtrate is diluted into 20 times, determine reductive monosaccharide content, non-knot is calculated according to formula
Structure carbohydrate content.Calculation formula is as follows：
Note：k：Reductive monosaccharide slope of standard curve；
V1：Sample solution cumulative volume (ml)；
m：Example weight (g)；
V2：Colour developing sample volume (ml)；
0.9：The conversion coefficient of starch and reductive monosaccharide；
OD values：Light absorption value.
Accuracy：The absolute difference of the measurement result independent twice obtained under the conditions of repeatability must not exceed arithmetic average
The 10% of value.
As a result it is as follows:
Embodiment 2
From (the variety name of kind 2 prepared：Northern agriculture ensiling 3740, source：Beijing Agricultural College) 3 parts are weighed in sample
3.00g parallel sample, record weighs weight (being accurate to one thousandth gram), plus the sodium chloride buffers of 100ml 0.3% suspend,
Boiling water bath heats 10min, after be cooled to less than 60 DEG C, the addition alphalise starch enzyme liquids of 10ml 1% are cold in being incubated 40min at 85 DEG C
But to room temperature, adjust pH=5.5, add the amylopectases of 10ml 1% in being incubated 30min at 60 DEG C, after be cooled to room temperature, adjust
PH=4.0 is saved, the carbohydrase of 10ml 10% is added, in being incubated 30min, boiling water bath 10min at 60 DEG C, inactivates enzyme, be settled to
250ml, mixes, filters, collect filtrate, filtrate is diluted into 20 times, determine reductive monosaccharide content, non-knot is calculated according to formula
Structure carbohydrate content.Calculation formula is as follows：
Note：k：Reductive monosaccharide slope of standard curve；
V1：Sample solution cumulative volume (ml)；
m：Example weight (g)；
V2：Colour developing sample volume (ml)；
0.9：Reductive monosaccharide is converted into the coefficient of Non-Structural Carbohydrate；
OD values：Absorbance.
Accuracy：The absolute difference of the measurement result independent twice obtained under the conditions of repeatability must not exceed arithmetic average
The 10% of value.
As a result it is as follows：
The assay method of the present invention, the characteristics of using enzymic catalytic reaction selectivity, using multi-enzyme system by unstructuredness carbon
Hydrate is thoroughly hydrolyzed into reduced sugar, eliminates the interference of the materials such as cellulose, hemicellulose and lignin, and the rate of recovery is
99%-101%, repeated R2For 0.999.Due in silage corn Non-Structural Carbohydrate content to silage corn new product
It is key index to plant seed selection, new technology spread and basic research, and therefore, assay method of the invention is sustainable to China's animal husbandry
Development serves active promoting function.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art is in the technical scope of present disclosure, the change or replacement that can be readily occurred in,
It should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of claims
Enclose and be defined.
Claims (7)
1. Non-Structural Carbohydrate content assaying method in a kind of silage corn, it is characterised in that including：
After taking detected silage corn complete stool crushed, finished, drying, 40 mesh sieves were crushed again, were dried, it is closed to protect
Deposit, be prepared into testing sample；The testing sample is quantitatively weighed, sodium chloride buffer is added and suspends, boiling water bath 10min makes starch
Gelatinization, is cooled to less than 60 DEG C after gelatinization, alphalise starch enzyme liquid, amylopectin enzyme liquid and saccharification enzyme liquid are sequentially added, respectively with each
The optimal reaction environment of enzyme is incubated hydrolysis, and constant volume, filtering take filtrate to survey reduced sugar, calculate what is detected according to reduced sugar
The Non-Structural Carbohydrate content of silage corn.
2. Non-Structural Carbohydrate content assaying method in silage corn according to claim 1, it is characterised in that
In methods described, the calculating that the Non-Structural Carbohydrate content of detected silage corn is calculated according to reduced sugar is public
Formula is as follows：
In above formula：k：Reductive monosaccharide slope of standard curve；
V1：Sample solution cumulative volume (ml)；
m：Example weight (g)；
V2：Colour developing sample volume (ml)；
0.9：With the conversion coefficient of reductive monosaccharide；
OD values：Light absorption value；
Accuracy：The absolute difference of the measurement result independent twice obtained under the conditions of repeatability must not exceed arithmetic mean of instantaneous value
10%.
3. Non-Structural Carbohydrate content assaying method in silage corn according to claim 1 or 2, its feature exists
In in methods described, the addition that sodium chloride buffer is added into the silage corn starch is 100ml, and the sodium chloride delays
NaCl mass percentage concentration is 0.3% in fliud flushing.
4. Non-Structural Carbohydrate content assaying method in silage corn according to claim 1, it is characterised in that
In methods described, alphalise starch enzyme liquid, amylopectin enzyme liquid and saccharification enzyme liquid are sequentially added as detection agent, with the optimum temperature of enzyme
Being incubated hydrolysis includes：
First it is gelatinized to the silage corn sample after cooling in thing and adds alphalise starch enzyme liquid, 40min is incubated at 85 DEG C, hydrolysis makes ensiling
Corn starch pasting thing liquid；
Then, amylopectin enzyme liquid is added into silage corn starch gelatinization thing liquefier, carbohydrase 30min is incubated at 60 DEG C,
Hydrolyze the α -1,6 glycosidic bond amylopectin in the liquefier；
Finally, to addition saccharification enzyme liquid after liquefier, in 60 DEG C of limit dextrins and malt being incubated in 30min, hydrolyzate liquid
Sugar is into glucose.
5. Non-Structural Carbohydrate content assaying method in the silage corn according to claim 1 or 4, its feature exists
Alphalise starch enzyme liquid, amylopectin enzyme liquid and saccharification enzyme liquid are sequentially added as in detection agent in, methods described,
The addition of the alphalise starch enzyme liquid is 10ml, and mass percentage concentration is 1%；
The addition of the amylopectin enzyme liquid is 10ml, and mass percentage concentration is 1%；
The addition of the saccharification enzyme liquid is 10ml, and mass percentage concentration is 10%.
6. Non-Structural Carbohydrate content assaying method in silage corn according to claim 1 or 2, its feature exists
In, in methods described, the silage corn complete stool detected crushed, finished, dry after, 40 mesh sieves were crushed again, were dried,
It is closed to preserve, it is prepared into testing sample.
7. Non-Structural Carbohydrate content assaying method in silage corn according to claim 1 or 2, its feature exists
In, in methods described, constant volume, filtering.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110333329A (en) * | 2019-05-08 | 2019-10-15 | 重庆瑞钛科技有限公司 | The rapid detection method of amylopectin content in wine brewing grain |
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| CN104007186A (en) * | 2013-02-26 | 2014-08-27 | 南京林业大学 | Method for analyzing content of starch in OCC raw material papermaking white water |
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|---|---|---|---|---|
| CN1474183A (en) * | 2003-08-01 | 2004-02-11 | 莱阳农学院 | Method for detecting starch content in ash tree flower polysaccharide |
| CN102010888A (en) * | 2010-09-21 | 2011-04-13 | 烟台喜旺科技发展有限公司 | Method for detecting starch |
| CN102156159A (en) * | 2011-05-18 | 2011-08-17 | 云南烟草科学研究院 | Method for detecting starch content in tobacco |
| CN102608258A (en) * | 2012-02-24 | 2012-07-25 | 泸州品创科技有限公司 | Method for measuring starch content in fermented grains |
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Application publication date: 20170915 |