CN102590013A - Method for quickly detecting titer of non-starch polysaccharides for feeds - Google Patents
Method for quickly detecting titer of non-starch polysaccharides for feeds Download PDFInfo
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- CN102590013A CN102590013A CN2012100085265A CN201210008526A CN102590013A CN 102590013 A CN102590013 A CN 102590013A CN 2012100085265 A CN2012100085265 A CN 2012100085265A CN 201210008526 A CN201210008526 A CN 201210008526A CN 102590013 A CN102590013 A CN 102590013A
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Abstract
The invention discloses a method for quickly detecting the titer of non-starch polysaccharides for feeds. The method comprises the following steps of: taking the provided starch-free wheat bran as a substrate, uniformly mixing the substrate with the non-starch polysaccharides for feeds to be detected for an enzymatic hydrolysis reaction, measuring the dry weight of the rest substrate after the reaction is completed, calculating the degradation efficiency, and evaluating the titer of the non-starch polysaccharides to be detected according to the degradation efficiency. The method not only can quickly detect the titer of a single enzyme in a buffer solution prepared in a laboratory, but also can quickly detect the titer of a compound enzyme in the buffer solution prepared in the laboratory. Not only can the titer of the single enzyme in an animal digestive juice be quickly detected, but also the titer of the compound enzyme in the animal digestive juice can be quickly detected.
Description
Technical field
The present invention relates to the method that a kind of fast detecting feed is tired with non-starch polysaccharide enzyme.
Background technology
Feed is to use the widest feed enzyme in the present feed industry with non-starch polysaccharide enzyme, mainly comprises zytase, cellulase, glucanase, pectase and mannase, and their few parts are used with the monomeric enzyme form, and major part is used with the complex enzyme form.In China, non-starch polysaccharide enzyme accounts for about 65% of the feed enzyme total market size.In Europe and North America, non-starch polysaccharide enzyme accounts for about 70% of the feed enzyme total market size.
For the assay determination of non-starch polysaccharide enzyme, no matter be monomeric enzyme or complex enzyme, all very loaded down with trivial details, and also the practicality and the repeatability of analysis data are also undesirable.At present, breadboard analysis substrate all is a purity than higher and water-soluble polymkeric substance easily, but their actual natural substrates all are water-insoluble polymer, and these polymkeric substance often are cross-linked with each other together, and constituent is more complicated also.Non-starch polysaccharide enzyme is very poor to the degradation efficiency of natural substrate, often has only about 5～10% of soluble substrate, and is difficult to accurately quantitatively.Though the animal feeding test is directly perceived, reliability is also relatively good, and the time of test oversize (needing 2 months at least), cost is also very high, in actual production process, is difficult to promote, and requires very far away for the express-analysis that requires in the scientific research process.
Summary of the invention
The purpose of this invention is to provide the method that a kind of fast detecting feed is tired with non-starch polysaccharide enzyme.
The method that detection feed provided by the invention is tired with non-starch polysaccharide enzyme; Comprise the steps: that with not amyloid wheat bran provided by the invention be substrate; Itself and feed to be measured are carried out enzyme digestion reaction with the non-starch polysaccharide enzyme mixing; Measure the dry weight of the said substrate of residue after reaction finishes, calculate degradation efficiency, estimate tiring of said non-starch polysaccharide enzyme to be measured according to the gained degradation efficiency according to following formula:
In the said enzyme digestion reaction step of this method, hydrolysis temperature is selected the gastral temperature of letting animals feed.
Wheat bran preparation method provided by the invention; After comprising the steps: the wheat bran raw material soaking, cleaning; Reject wherein complete wheat, carry out the gelatinization reaction again with behind the water mixing, add AMS and react; React with carbohydrase again after reaction finishes, obtain said not amyloid wheat bran after reaction finishes.
In the said gelatinization reactions step of said method, temperature is 95～100 ℃, and the time is 10～15 minutes;
Said adding AMS carries out in the reactions step, and temperature is 65～70 ℃; Time is 25～30 minutes;
Said and carbohydrase carries out in the reactions step, and temperature is 95～100 ℃, and the time is 60 minutes.
The enzyme of said AMS is lived and is 4000u/g; The enzyme of said carbohydrase is lived and is 80000u/g.
Said preparation is the method for amyloid wheat bran not, also comprise the steps: said and carbohydrase react finish after, product is naturally cooled to room temperature after washing and air drying, obtain said not amyloid wheat bran.
Said washing times is 3～4 times, and in the said drying steps, the time is 20～24 hours.
The not amyloid wheat bran for preparing according to the method described above also belongs to protection scope of the present invention.In the said not amyloid wheat bran, the quality percentage composition of starch is no more than 0.01%, and water cut is 8.0%.
The method that quick test feed provided by the invention is tired with non-starch polysaccharide enzyme serves as to analyze substrate with the wheat bran of eliminating starch, and the feed that adding needs to analyze in temperature of setting and solution is used non-starch polysaccharide enzyme.This feed enzyme can be a monomeric enzyme, also can be complex enzyme.After the reaction time of setting, through filtration washing, drying, measure the dry weight of residual substrate, calculate degradation efficiency, with this tiring as leading indicator evaluation comparison institute's enzyme analysis.This test method not only can be prepared tiring in the damping fluid in the laboratory by the single enzyme of fast detecting, also can fast detecting complex enzyme tiring in the damping fluid of laboratory preparation.Not only can fast detecting single enzyme tiring in animal digestion liquid, also can fast detecting complex enzyme tiring in animal digestion liquid.This method is directly perceived, easy, does not need very accurate analytical instrument, and can check the influence of the environment of animal alimentary canal to the role of apoenzyme effect simultaneously, thereby can more objectively reflect all sidedly that the actual of these feed enzyme tire.Feed Manufacturing enterprise and raiser can analyze tiring of non-starch polysaccharide enzyme and compare with reference to said method; Be applicable to research, production and the application of feed, be specially adapted to the fast detecting that non-starch polysaccharide enzyme is tired in livestock and poultry and the aquatic feeds with non-starch polysaccharide enzyme.
Description of drawings
Fig. 1 is the wheat bran degradation effect in the damping fluid.
Embodiment
Below in conjunction with specific embodiment the present invention is done further elaboration, but the present invention is not limited to following examples.Said method is conventional method if no special instructions.Said raw material all can get from open commercial sources if no special instructions.
Embodiment 1:
Take by weighing 10kg general goods wheat bran, soaked 24 hours at normal temperature (20～30 ℃), clean wherein body refuse and other solubility foreign material, robust fibre such as cot that skim is floating and wheat straw with the 60kg clear water.Clean repeatedly 3 times with clear water, and then filter, push, obtain clean wheat bran, its water cut is 65%, and general assembly (TW) is 25kg.Gained wheat bran is divided on pallet in batches, and choosing to remove does not wherein have broken wheat.Though this part does not have broken wheat seldom, and is influential to later experiments result's precision, so must reject.In capacity is 100 liters rustless steel container, add the cleaning wheat bran of above-mentioned acquisition, add 45kg distilled water again, logical then Steam Heating is warming up to 98 ℃, keeps residual starch in the abundant gelatinization wheat bran 12 minutes.Be cooled to 65 ℃ then, (AMS, enzyme are lived and are 4000u/g, and available from the magnificent prosperous and powerful bioengineering in east, Beijing company limited, catalog number is 107658305 to add 10 gram starch liquefacation enzymes.), kept 30 minutes.Progressively be heated to 95 ℃ then; Add 20 gram carbohydrase (enzyme is lived and to be 80000u/g, and available from the magnificent prosperous and powerful bioengineering in east, Beijing company limited, catalog number is 1003301) again; Be incubated 60 minutes, become glucose sugar and the maltose that is dissolved in water with the residual starch of abundant saccharification.Naturally cool to room temperature, filter, extruding obtains the slag filter, cleans filter residue 3 times with distilled water again, with abundant filtering residual glucose and maltose, obtains pure wheat bran, and its water cut is 65%, and weight is 22kg.Is water cut that 65% 22kg wheat bran is divided in batches and on pallet, carried out the normal temperature pneumatic conveying drying, reaches constant weight later on basically in 24 hours, obtains pure wheat bran 7.6kg; Water cut is 8.0%; Remaining content of starch is no more than 0.01%, is divided in to be sealed in the container, in 4 ℃ of refrigerators, preserves.
Get the triangular flask of 12 500ml, (the pH value is 5.5, in 5000ml distilled water, adds 4.5ml glacial acetic acid and 34.85 gram anhydrous sodium acetates in each triangular flask, to add the 250ml damping fluid; Thoroughly dissolving; The concentration of acetate ion is 0.1mol/L), then respectively add the wheat bran of 10 gram embodiment 1 preparation gained again, its water cut is 8.0%, content of starch is no more than 0.01%; Be placed in the water-bath at 37 ℃ of constant temperature, in these 12 triangular flasks, add the feed enzyme A to be analyzed of equivalent then.Whenever extracted a triangular flask at a distance from 30 minutes, filter with 8 layers of hospital gauze, the time is no more than 2 minutes.After filter finishing, clean, got filter residue after 3 times with 250ml distilled water; Divide on the stainless steel pallet; The normal temperature cooling was 2 hours again, weighed then to constant weight in 8 hours for 80 ℃ of pneumatic conveying dryings; Calculate degradation efficiency according to following formula, estimate tiring of feed enzyme A to be analyzed according to the gained degradation efficiency:
Analyze the residual weight of substrate after relatively degrading through different time.The result is as shown in Figure 1.Along with the increase of soaking enzymolysis time, the degradation rate of dry also increases again in the pure wheat bran, but degradation speed is constantly weakening.Through 6 hours enzymolysis, the degradation rate of pure wheat bran dry reached 14.2%.
Get 3 feeds and use the non-starch polysaccharide enzyme sample, be labeled as respectively: A, B and C.Adopt pure wheat bran edman degradation Edman relatively to tire.
Collect 10 pig stomaches (body weight of live pig is between 95～130kg) in the slaughterhouse, get their content, mix.Use filtered through gauze then, obtain filtered fluid, for use.
Get the triangular flask of 3 500ml, put into A, B and C feed respectively, add 250ml pig stomach filtered fluid more respectively,, zymoprotein is fully dissolved 37 ℃ of waters bath with thermostatic control 2 minutes with each 0.1 gram of non-starch polysaccharide enzyme sample.Add the pure wheat bran of 10 grams (preparation is referring to said method) then simultaneously.After 60 minutes, filter washing simultaneously.Got filter residue, pneumatic conveying drying 12 hours is to constant weight at normal temperatures, and relatively their dry weight calculates degradation efficiency according to following formula, is respectively the tiring of three non-starch polysaccharide enzyme samples of A, B and C according to gained degradation efficiency evaluation mark:
The gained result is referring to table 1.
Table 1, three kinds of feeds are with the result of non-starch polysaccharide enzyme
| Enzyme appearance | Action time | Wheat bran plays starting weight | The degraded back is residual heavy | Degradation rate (%) |
| A | 60 minutes | 10.0 gram | 8.72 gram | 12.8％ |
| B | 60 minutes | 10.0 gram | 9.21 gram | 7.90％ |
| C | 60 minutes | 10.0 gram | 8.34 gram | 16.6％ |
Can know that by table 1 in above-mentioned three kinds of non-starch polysaccharide enzymes, the effect of C enzyme is the most remarkable.In the mensuration process, need not carry out complicated enzymatic analysis and just can obtain comparing result intuitively.And said method has combined multiple composition in the gastric juice to the active influence of zymoprotein.
Sample to be analyzed is non-starch polysaccharide enzyme X, Y and Z, is powdery.The test substrate is the pure wheat bran of self-control (preparation is referring to above-mentioned).
Collect 10 secondary chitterlings (body weight of live pig is between 95～130kg) in the slaughterhouse, get their content, mix.Filter, push with the gauze embedding, obtain 3 liters of filtered fluids.Leave filtered fluid in the carboy in, in 4 ℃ of refrigerators, deposited 24 hours, get the solvent of supernatant as feed enzyme.
Get the triangular flask of 3 500ml, add each 0.1 gram of X, Y and three kinds of feed enzyme samples of Z respectively.Add 250ml chitterlings supernatant more respectively,, zymoprotein is fully dissolved 37 ℃ of water bath heat preservations 5 minutes.And then in X, Y and three enzyme appearance of Z solution, add 10 gram pure wheat bran (preparation method is referring to annex 1) successively respectively, 37 ℃ of water bath heat preservations 60 minutes.
Prepare 3 parallel funnels simultaneously, dispose same gauze (weight, the number of plies and the mode of stacking are all consistent).After water bath heat preservation finishes; Take out X, three treating fluids of Y and Z (enzymolysis time is consistent with the time interval that adds wheat bran as far as possible at interval) successively; Filter, wash (repeat 3 times, use distilled water), extruding and pneumatic conveying drying (all modes of operation are all consistent) respectively.At last, the dry thing that obtains is carried out weighing, calculates degradation efficiency, be respectively the tiring of three feed enzyme samples of X, Y and Z according to gained degradation efficiency evaluation mark according to following formula:
The gained result is referring to table 2.
The effect of three kinds of pure wheat bran of SNSP enzymatic degradation in table 2, the intestinal fluid
| Enzyme appearance | Enzymolysis time | Wheat bran plays starting weight | Wheat bran is residual heavy | Degradation rate |
| X | 60 minutes | 10.0 gram | 8.72 | 12.8％ |
| Y | 60 minutes | 10.0 gram | 8.29 | 17.1％ |
| Z | 60 minutes | 10.0 gram | 9.23 | 7.70％ |
Can know by table 2, more above-mentioned three kinds of non-starch polysaccharide enzymes, in live pig intestinal juice, the degradation effect of sample enzyme Y is best.
Claims (8)
1. one kind is detected the method that feed is tired with non-starch polysaccharide enzyme; Comprise the steps: that with claim 7 or 8 said not amyloid wheat bran be substrate; Itself and feed to be measured are carried out enzyme digestion reaction with the non-starch polysaccharide enzyme mixing; Measure the dry weight of the said substrate of residue after reaction finishes, calculate degradation efficiency, estimate tiring of said non-starch polysaccharide enzyme to be measured according to the gained degradation efficiency according to following formula:
2. one kind prepares the not method of amyloid wheat bran; After comprising the steps: the wheat bran raw material soaking, cleaning; Reject wherein complete wheat, carry out the gelatinization reaction again with behind the water mixing, add AMS and react; React with carbohydrase again after reaction finishes, obtain said not amyloid wheat bran after reaction finishes.
3. method according to claim 2 is characterized in that: in the said gelatinization reactions step, temperature is 95～100 ℃, and the time is 10～15 minutes;
Said adding AMS carries out in the reactions step, and temperature is 65～70 ℃; Time is 25～30 minutes;
Said and carbohydrase carries out in the reactions step, and temperature is 95～100 ℃, and the time is 60 minutes.
4. according to claim 2 or 3 described methods, it is characterized in that: the enzyme of said AMS is lived and is 4000u/g; The enzyme of said carbohydrase is lived and is 80000u/g.
5. according to the arbitrary described method of claim 2-4; It is characterized in that: said preparation is the method for amyloid wheat bran not; Also comprise the steps: said and carbohydrase react finish after; Product is naturally cooled to room temperature after washing and normal temperature pneumatic conveying drying, obtain said not amyloid wheat bran.
6. method according to claim 5 is characterized in that: said washing times is 3～4 times, and in the said drying steps, the time is 20～24 hours.
7. the not amyloid wheat bran for preparing of the arbitrary said method of claim 2-6.
8. wheat bran according to claim 7 is characterized in that: in the said not amyloid wheat bran, the quality percentage composition of starch is no more than 0.01%.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105986005A (en) * | 2015-01-28 | 2016-10-05 | 北京晟亚育达生物科技有限公司 | Method of quickly measuring enzyme activity of feed-use phytase |
| CN105986004A (en) * | 2015-01-28 | 2016-10-05 | 北京晟亚育达生物科技有限公司 | Method of quickly measuring enzyme activity of feed-use cellulase |
| CN105986006A (en) * | 2015-01-28 | 2016-10-05 | 北京晟亚育达生物科技有限公司 | Method of quickly measuring enzyme activity of feed-use xylanase |
| CN108559769A (en) * | 2018-04-12 | 2018-09-21 | 上海欧耐施生物技术有限公司 | A kind of appraisal procedure of fodder enzyme |
| CN109459334A (en) * | 2018-10-25 | 2019-03-12 | 杭州派瑞特包装有限公司 | Degradable environment-friendly package material degradation efficiency detection device and detection control method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102229974A (en) * | 2011-05-20 | 2011-11-02 | 湖南农业大学 | Quality detection and evaluation method for feed complex enzyme |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229974A (en) * | 2011-05-20 | 2011-11-02 | 湖南农业大学 | Quality detection and evaluation method for feed complex enzyme |
Non-Patent Citations (2)
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| CDQ12321: "《纯麦麸制备》", 24 November 2011 * |
| 胡建坤等: "饲用木聚糖酶活力的综合评价", 《中国畜牧杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105986005A (en) * | 2015-01-28 | 2016-10-05 | 北京晟亚育达生物科技有限公司 | Method of quickly measuring enzyme activity of feed-use phytase |
| CN105986004A (en) * | 2015-01-28 | 2016-10-05 | 北京晟亚育达生物科技有限公司 | Method of quickly measuring enzyme activity of feed-use cellulase |
| CN105986006A (en) * | 2015-01-28 | 2016-10-05 | 北京晟亚育达生物科技有限公司 | Method of quickly measuring enzyme activity of feed-use xylanase |
| CN108559769A (en) * | 2018-04-12 | 2018-09-21 | 上海欧耐施生物技术有限公司 | A kind of appraisal procedure of fodder enzyme |
| CN109459334A (en) * | 2018-10-25 | 2019-03-12 | 杭州派瑞特包装有限公司 | Degradable environment-friendly package material degradation efficiency detection device and detection control method |
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Application publication date: 20120718 |