CN109164175A - A kind of feed non-starch polysaccharide component analysis method - Google Patents
A kind of feed non-starch polysaccharide component analysis method Download PDFInfo
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- CN109164175A CN109164175A CN201810721793.4A CN201810721793A CN109164175A CN 109164175 A CN109164175 A CN 109164175A CN 201810721793 A CN201810721793 A CN 201810721793A CN 109164175 A CN109164175 A CN 109164175A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/025—Gas chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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Abstract
The present invention relates to a kind of feed non-starch polysaccharide component analysis methods, include the following steps: Feed Sample preparation, Feed Sample pre-treatment, the dissociation of total non-starch polysaccharide component, the dissociation of insoluble non-starch polysaccharide component, the parsing of non-starch polysaccharide component.The present invention parses feed non-starch polysaccharide component using gas chromatography derived from acetic anhydride, accurate to obtain total non-starch polysaccharide constituent content, insoluble non-starch polysaccharide constituent content, Soluble Non Starch Polysaccharides constituent content in feed.
Description
Technical field
The present invention relates to a kind of feed non-starch polysaccharide component analysis methods, more more particularly to the total non-starch of Feed Sample
The method of saccharic composition dissociation, the dissociation of insoluble non-starch polysaccharide component and the parsing of non-starch polysaccharide component.
Background technique
2017 annual more than 9,560 ten thousand tons of soybean import of China, are sure to occupy global first big importer by more than 1,130 ten thousand tons of cereal,
Wherein main cause is that Production of Livestock and Poultry consumption is increasing.Feed resource shortage, people and animals, which strive grain contradiction, increasingly sharpens.Therefore,
It improves feedstuff utilization rate and the non-grain resource of reasonable development (such as grain processing by-product) will ensure that China's animal husbandry continues
The important outlet to develop in a healthy way.Compared with cereal, processing byproduct (bran, poor slag, grouts etc.) is more containing higher non-starch
Sugar.Non-starch polysaccharide wraps up digestible nutrient, resists the degradation of endogenous digestive ferment, formed screen effect (Anguita et al.,
2006)；Non-starch polysaccharide has important nutrition physiology effect simultaneously, partially generates volatile fatty acid through big intestinal fermentation, mentions
For energy.
Non-starch polysaccharide analytic approach can be used as that disclose dietary fiber strong to one of feed nutrient nutritive value in feed
Tool.However, the maximum challenge of present analysis method is repeatability lower than normal form fibre analysis method (NRC, 2012).Need into
One step improves the indigestible carbohydrate of low molecular weight and is included in non-starch polysaccharide system, and corrects the content of indigestible residue
(Gordon et al.,2007).Therefore, accurately the content of non-starch polysaccharide component has great importance in parsing feed.
Currently, there are the ginsengs such as sample weighting amount, hydrolysis time, reagent concentration for the feed non-starch polysaccharide content method of research report
Number disunities, cause measuring accuracy and repeatability low, be not widely used by laboratory also (Theander, 1995；Bach
Knudsen, 1997；NRC, 2012).The present invention is verified by a large amount of analysis of experiments, overcomes current feed non-starch polysaccharide
The test unstability and result high variability of analysis method are more by Soluble Non Starch Polysaccharides component and insoluble non-starch
Saccharic composition effectively dissociates and the parsing of non-starch polysaccharide component, has achieved the purpose that accurate analysis non-starch polysaccharide content.
Summary of the invention
Technology of the invention solves the problems, such as: in order to overcome the test of non-starch polysaccharide analysis method in current feed unstable
Property and result high variability problem, a kind of feed non-starch polysaccharide component analysis method is provided, the suitable title sample of regulation is passed through
Amount, Soluble Non Starch Polysaccharides component and insoluble non-starch polysaccharide component effectively dissociate and the parsing of non-starch polysaccharide component,
It is accurate to obtain feed non-starch polysaccharide constituent content.
The technology of the present invention solution: a kind of feed non-starch polysaccharide component analysis method, successively the following steps are included: raising
Expect preparation of samples, Feed Sample pre-treatment, the dissociation of total non-starch polysaccharide component, the dissociation of insoluble non-starch polysaccharide component, non-shallow lake
The parsing of powder polysaccharide component, in which:
(1) Feed Sample prepares: choosing feed and is put into pulverizer, wherein the feed includes mixed feed and composition
The raw material of mixed feed, by the revolving speed control of the pulverizer in 5000~6000r/min, grinding time is 5~10min,
Smashed feed is crossed into 35 meshes；- 18~-22 DEG C are placed in store for future use；
(2) Feed Sample pre-treatment: 45~60mg of reference dietary fiber and 0.15~0.30g of feed is accurately weighed to centrifugation
Guan Zhong is added 20~30mL petroleum ether and is ultrasonically treated 10~15min, and 3000~5000r/min room temperature is centrifuged 5~10min, in abandoning
It is clear and dry.80% EtOH Sonicate processing is added, abandons supernatant and drying.It is added 10~15mL acetate buffer (pH=5.0),
And 60~80 μ L alpha-amylases, boiling water bath is added.Then 60~80 50~60 DEG C of isothermal vibration water of μ L amyloglucosidase are added
Bathe 10~12h.It takes out centrifuge tube to be cooled to room temperature, 3000~5000r/min room temperature is centrifuged 5~10min；
(3) 40~50mL dehydrated alcohol, ice total non-starch polysaccharide component dissociation: is added in (2) treated centrifuge tube
Bath stands 1~2 hour, and 3000~5000r/min room temperature is centrifuged 5~10min, abandons supernatant and drying.10~15mL acetone is added,
3000~5000r/min room temperature is centrifuged 5~10min, discards supernatant liquid and dries.
(4) 50~60mL phosphoric acid buffer insoluble non-starch polysaccharide component dissociation: is added in (2) treated centrifuge tube
Liquid, boiling water bath 1~2 hour, 3000~5000r/min room temperature was centrifuged 5~10min, abandoned supernatant and drying.10~15mL third is added
Ketone, 3000~5000r/min room temperature are centrifuged 5~10min, discard supernatant liquid and dry.
(5) non-starch polysaccharide constituent content parses: it will be added the concentrated sulfuric acid in the non-starch polysaccharide component of dissociation, 30~40
DEG C, 40~50r/min, 1~2h of water bath with thermostatic control, centrifugation is placed in -18~-22 DEG C and stores for future use.Take 200~300 μ L hydrolyzates extremely
In centrifuge tube, the ammonium hydroxide that volume ratio is 1:4:4:4~1:3:3:3: inositol internal standard compound: dehydrated alcohol: sodium borohydride solution is added,
It mixes.Add the glacial acetic acid that volume is 2:5:50~3:6:55: 1- methylimidazole: the solution of acetic anhydride mixes.It is blown through nitrogen
After dry redissolution, the total non-starch polysaccharide component of gas Chromatographic Determination and insoluble non-starch polysaccharide component.
In the step (1), smashed Feed Sample is placed in -18~-22 DEG C and stores for future use.
Soluble Non Starch Polysaccharides group is divided into total non-starch polysaccharide component and insoluble non-starch polysaccharide in the step (5)
The difference of group point.
The advantages of the present invention over the prior art are that:
(1) present invention is according to, it is specified that being suitable for the test such as sample weighting amount, hydrolysis time, reagent concentration with lot of experimental data
Parameter Conditions guarantee the stability of feed non-starch polysaccharide content analysis method.
(2) the present invention overcomes the test unstability of non-starch polysaccharide analysis method in current feed and result height to make a variation
Property, it is effectively dissociated by Soluble Non Starch Polysaccharides component and insoluble non-starch polysaccharide component and non-starch polysaccharide group is decomposed
Analysis, has reached accurate acquisition feed non-starch polysaccharide constituent content purpose.
Detailed description of the invention
Fig. 1 is the flow chart that the present invention realizes；
Fig. 2 is different cultivars wheat samples non-starch polysaccharide component analysis figure；
Fig. 3 is different wheat bran sample non-starch polysaccharide component analysis figures.
Specific embodiment
The present invention parses feed non-starch polysaccharide component using gas chromatography derived from acetic anhydride using the present invention, accurately
Obtain total non-starch polysaccharide constituent content, insoluble non-starch polysaccharide constituent content, Soluble Non Starch Polysaccharides component in feed
Content.
With reference to the accompanying drawing and the present invention is discussed in detail in specific embodiment.But embodiment below is only limitted to explain this hair
Bright, protection scope of the present invention should include the full content of claim, be not limited only to the present embodiment.
Embodiment 1,
As shown in Figure 1, concrete methods of realizing of the present invention can be implemented as follows:
It chooses wheatfeed raw material to be put into pulverizer, then the revolving speed of the pulverizer is controlled into 5000~6000r/
Min, grinding time are 5~10min, and smashed feed is crossed 35 meshes；- 18~-22 DEG C are placed in store for future use；
Feed Sample pre-treatment: 45~60mg of reference dietary fiber and 0.20~0.25g of feed accurately are weighed to centrifuge tube
In, 20~30mL petroleum ether is added and is ultrasonically treated 10~15min, 3000~5000r/min room temperature is centrifuged 5~10min, abandons supernatant
And it is dry.80% EtOH Sonicate processing is added, abandons supernatant and drying.It is added 10~15mL acetate buffer (pH=5.0), and
60~80 μ L alpha-amylases, boiling water bath is added.Then 60~80 50~60 DEG C of isothermal vibration water-baths of μ L amyloglucosidase are added
10~12h.It takes out centrifuge tube to be cooled to room temperature, 3000~5000r/min room temperature is centrifuged 5~10min；
40~50mL dehydrated alcohol is added in centrifuge tube after handling before the Feed Sample, it is small that ice bath stands 1~2
When, 3000~5000r/min room temperature is centrifuged 5~10min, abandons supernatant and drying.10~15mL acetone, 3000~5000r/ is added
Min room temperature is centrifuged 5~10min, discards supernatant liquid and dries.
50~60mL phosphate buffer is added in centrifuge tube after handling before the Feed Sample, boiling water bath 1~2 is small
When, 3000~5000r/min room temperature is centrifuged 5~10min, abandons supernatant and drying.10~15mL acetone, 3000~5000r/ is added
Min room temperature is centrifuged 5~10min, discards supernatant liquid and dries.
The middle addition concentrated sulfuric acid after the Feed Sample non-starch polysaccharide component dissociation, 30~40 DEG C, 40~50r/min
1~2h of water bath with thermostatic control, centrifugation, is placed in -18~-22 DEG C and stores for future use.It takes 200~300 μ L hydrolyzates into centrifuge tube, body is added
Product is than the ammonium hydroxide for 1:4:4:4~1:3:3:3: inositol internal standard compound: dehydrated alcohol: sodium borohydride solution mixes.Add volume
For the glacial acetic acid of 2:5:50~3:6:55: 1- methylimidazole: the solution of acetic anhydride mixes.After being dried with nitrogen redissolution, gas phase color
Spectrum measures total non-starch polysaccharide component and insoluble non-starch polysaccharide component.
The non-starch polysaccharide of eight kinds of wheats is measured using " feed non-starch polysaccharide component analysis method " disclosed by the invention
Constituent content is as shown in Figure 2.Total non-starch polysaccharide content range of wheat are as follows: 7.82~10.24, the interracial coefficient of variation
9.28%；The insoluble non-starch polysaccharide content range of wheat are as follows: 6.66~8.73, the interracial coefficient of variation 9.71%；It is small
The Soluble Non Starch Polysaccharides content range of wheat are as follows: 1.15~1.57, the interracial coefficient of variation 9.44%.
Embodiment 2,
As shown in Figure 1, concrete methods of realizing of the present invention can be implemented as follows:
It chooses wheat bran feedstuff to be put into pulverizer, then the revolving speed of the pulverizer is controlled into 5000~6000r/
Min, grinding time are 5~10min, and smashed feed is crossed 35 meshes；- 18~-22 DEG C are placed in store for future use；
Feed Sample pre-treatment: 45~60mg of reference dietary fiber and 0.15~0.20g of feed accurately are weighed to centrifuge tube
In, 20~30mL petroleum ether is added and is ultrasonically treated 10~15min, 3000~5000r/min room temperature is centrifuged 5~10min, abandons supernatant
And it is dry.80% EtOH Sonicate processing is added, abandons supernatant and drying.It is added 10~15mL acetate buffer (pH=5.0), and
60~80 μ L alpha-amylases, boiling water bath is added.Then 60~80 50~60 DEG C of isothermal vibration water-baths of μ L amyloglucosidase are added
10~12h.It takes out centrifuge tube to be cooled to room temperature, 3000~5000r/min room temperature is centrifuged 5~10min；
40~50mL dehydrated alcohol is added in centrifuge tube after handling before the Feed Sample, it is small that ice bath stands 1~2
When, 3000~5000r/min room temperature is centrifuged 5~10min, abandons supernatant and drying.10~15mL acetone, 3000~5000r/ is added
Min room temperature is centrifuged 5~10min, discards supernatant liquid and dries.
50~60mL phosphate buffer is added in centrifuge tube after handling before the Feed Sample, boiling water bath 1~2 is small
When, 3000~5000r/min room temperature is centrifuged 5~10min, abandons supernatant and drying.10~15mL acetone, 3000~5000r/ is added
Min room temperature is centrifuged 5~10min, discards supernatant liquid and dries.
The middle addition concentrated sulfuric acid after the Feed Sample non-starch polysaccharide component dissociation, 30~40 DEG C, 40~50r/min
1~2h of water bath with thermostatic control, centrifugation, is placed in -18~-22 DEG C and stores for future use.It takes 200~300 μ L hydrolyzates into centrifuge tube, body is added
Product is than the ammonium hydroxide for 1:4:4:4~1:3:3:3: inositol internal standard compound: dehydrated alcohol: sodium borohydride solution mixes.Add volume
For the glacial acetic acid of 2:5:50~3:6:55: 1- methylimidazole: the solution of acetic anhydride mixes.After being dried with nitrogen redissolution, gas phase color
Spectrum measures total non-starch polysaccharide component and insoluble non-starch polysaccharide component.
The non-starch for measuring eight kinds of wheat brans using " feed non-starch polysaccharide component analysis method " disclosed by the invention is more
Saccharic composition content is as shown in Figure 3.Total non-starch polysaccharide content range of wheat bran are as follows: 30.41~38.25, interracial variation
Coefficient 6.37%；The insoluble non-starch polysaccharide content range of wheat bran are as follows: 28.42~36.23, the interracial coefficient of variation
6.73%；The Soluble Non Starch Polysaccharides content range of wheat bran are as follows: 1.78~2.42, the interracial coefficient of variation 10.42%.
It should be noted that those skilled in the art are that this hair may be implemented completely according to the various embodiments described above of the present invention
Bright independent claims and the full scope of appurtenance, realize process and the same the various embodiments described above of method；And the present invention is not
It elaborates and partly belongs to techniques well known.
The above, part specific embodiment only of the present invention, but scope of protection of the present invention is not limited thereto, appoints
In the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of, should all cover by what those skilled in the art
Within protection scope of the present invention.
Claims (3)
1. a kind of feed non-starch polysaccharide component analysis method, which comprises the steps of: Feed Sample prepares, raises
Expect that sample pre-treatments, the dissociation of total non-starch polysaccharide component, the dissociation of insoluble non-starch polysaccharide component, non-starch polysaccharide group are decomposed
Analysis, in which:
(1) Feed Sample prepares: choosing feed and is put into pulverizer, the feed includes mixed feed and composition mixed feed
Raw material, by the revolving speed control of the pulverizer in 5000~6000r/min, grinding time is 5~10min, will be smashed
Feed crosses 35 meshes, stores for future use；
(2) Feed Sample pre-treatment: 45~60mg of reference dietary fiber and 0.15~0.30g of feed are weighed into centrifuge tube, is added
Enter 20~30mL petroleum ether and be ultrasonically treated 10~15min, 3000~5000r/min room temperature is centrifuged 5~10min, abandons supernatant and does
It is dry；80% EtOH Sonicate processing is added, abandons supernatant and drying；10~15mL acetate buffer, the pH=of acetate buffer is added
5.0, and 60~80 μ L alpha-amylases, boiling water bath is added；Then 60~80 μ L amyloglucosidase, 50~60 DEG C of constant temperature shakes are added
10~12h of water-bath is swung, centrifuge tube is taken out and is cooled to room temperature, 3000~5000r/min room temperature is centrifuged 5~10min；
(3) total non-starch polysaccharide component dissociation: 40~50mL dehydrated alcohol is added in (2) treated centrifuge tube, ice bath is quiet
It sets 1~2 hour, 3000~5000r/min room temperature is centrifuged 5~10min, abandons supernatant and drying；Addition 10~15mL acetone, 3000
~5000r/min room temperature is centrifuged 5~10min, discards supernatant liquid and dries；
(4) insoluble non-starch polysaccharide component dissociation: being added 50~60mL phosphate buffer in (2) treated centrifuge tube,
Boiling water bath 1~2 hour, 3000~5000r/min room temperature was centrifuged 5~10min, abandoned supernatant and drying；10~15mL acetone is added,
3000~5000r/min room temperature is centrifuged 5~10min, discards supernatant liquid and dries；
(5) non-starch polysaccharide constituent content parses: it will be added the concentrated sulfuric acid in the non-starch polysaccharide component of dissociation, 30~40 DEG C, and 40
~50r/min 1~2h of water bath with thermostatic control, centrifugation, is placed in -18~-22 DEG C and stores for future use, take 200~300 μ L hydrolyzates to centrifuge tube
In, the ammonium hydroxide that volume ratio is 1:4:4:4~1:3:3:3: inositol internal standard compound: dehydrated alcohol is added: sodium borohydride solution mixes；
Add the glacial acetic acid that volume is 2:5:50~3:6:55: 1- methylimidazole: the solution of acetic anhydride mixes；It is dried with nitrogen multiple
After molten, the total non-starch polysaccharide component of gas Chromatographic Determination and insoluble non-starch polysaccharide component.
2. feed non-starch polysaccharide component analysis method according to claim 1, it is characterised in that: in the step (1),
Smashed Feed Sample is placed in -18~-22 DEG C and stores for future use.
3. feed non-starch polysaccharide component analysis method according to claim 1, it is characterised in that: in the step (5)
Soluble Non Starch Polysaccharides group is divided into the difference of total non-starch polysaccharide component and insoluble non-starch polysaccharide group point.
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Cited By (1)
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