CN103760305A - Method for determining true nutritive value of small intestines on nutrients by adopting three-step in-vitro technique - Google Patents
Method for determining true nutritive value of small intestines on nutrients by adopting three-step in-vitro technique Download PDFInfo
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- CN103760305A CN103760305A CN201410016733.4A CN201410016733A CN103760305A CN 103760305 A CN103760305 A CN 103760305A CN 201410016733 A CN201410016733 A CN 201410016733A CN 103760305 A CN103760305 A CN 103760305A
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- 238000000034 method Methods 0.000 title claims abstract description 66
- 210000000813 small intestine Anatomy 0.000 title claims abstract description 34
- 235000015097 nutrients Nutrition 0.000 title claims abstract description 8
- 238000000338 in vitro Methods 0.000 title abstract description 16
- 230000000050 nutritive effect Effects 0.000 title abstract description 8
- 210000001819 pancreatic juice Anatomy 0.000 claims abstract description 38
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 35
- 229920002472 Starch Polymers 0.000 claims abstract description 27
- 235000019698 starch Nutrition 0.000 claims abstract description 27
- 239000008107 starch Substances 0.000 claims abstract description 27
- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 8
- 238000010306 acid treatment Methods 0.000 claims abstract description 4
- 235000019621 digestibility Nutrition 0.000 claims description 38
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000004677 Nylon Substances 0.000 claims description 5
- 229920001778 nylon Polymers 0.000 claims description 5
- 244000309464 bull Species 0.000 claims description 4
- 235000013336 milk Nutrition 0.000 claims description 4
- 239000008267 milk Substances 0.000 claims description 4
- 210000004080 milk Anatomy 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 241001494479 Pecora Species 0.000 claims description 2
- 241000282849 Ruminantia Species 0.000 abstract description 16
- 230000029087 digestion Effects 0.000 abstract description 6
- 238000012258 culturing Methods 0.000 abstract 2
- 210000004767 rumen Anatomy 0.000 abstract 2
- 238000006731 degradation reaction Methods 0.000 abstract 1
- 230000000593 degrading effect Effects 0.000 abstract 1
- 230000002349 favourable effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 9
- 229940055695 pancreatin Drugs 0.000 description 7
- 108010019160 Pancreatin Proteins 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 108090000631 Trypsin Proteins 0.000 description 5
- 102000004142 Trypsin Human genes 0.000 description 5
- 239000012588 trypsin Substances 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 4
- 235000019620 fat digestibility Nutrition 0.000 description 3
- 239000004459 forage Substances 0.000 description 3
- 238000013459 approach Methods 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- MGSRCZKZVOBKFT-UHFFFAOYSA-N thymol Chemical compound CC(C)C1=CC=C(C)C=C1O MGSRCZKZVOBKFT-UHFFFAOYSA-N 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 102000002568 Multienzyme Complexes Human genes 0.000 description 1
- 108010093369 Multienzyme Complexes Proteins 0.000 description 1
- 239000005844 Thymol Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004913 chyme Anatomy 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940116540 protein supplement Drugs 0.000 description 1
- 235000005974 protein supplement Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 229960000790 thymol Drugs 0.000 description 1
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- Fodder In General (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for determining the true nutritive value of small intestines on nutrients by adopting a three-step in-vitro technique. The method comprises the following three steps of culturing rumens, carrying out acid treatment and culturing pancreatic juice. The three-step in-vitro technique provided by the invention can be used for changing the method for feed treatment by utilizing pancreatic enzymes in the prior art, is used for direct treatment by utilizing pancreatic juice, can be used for digesting and degrading RUP (rumen un-degradation feed protein), and also can provide a favorable way for the estimation of ruminant small intestine starch true nutritive value and fat true nutritive value by adopting the pancreatic juice method. By adopting the method, the digestion situation of the ruminant on protein, starch and fat in feed can be simulated once, and three important indexes of the protein, starch and fat small intestine true nutritive value can be determined, and the method is simple to operate, is easy to realize, and saves the cost.
Description
Technical field
The present invention relates to a kind of ruminant small intestine digestibility assay method, particularly a kind of method that adopts three step ex vivo technique of pancreatic juice to measure nutrient small intestine digestibility.
Background technology
In ruminant small intestine, can metabolizable protein be that the non-degraded forage protein of microprotein, cud (RUP) and a small amount of intrinsic protein being synthesized by cud forms.Research shows, microprotein small intestine digestibility is (80%-85%) fixing substantially, but RUP small intestine digestibility is affected by the factors such as feed type, feed resource and processing mode.Feed RUP small intestine digestibility is to affect the output of milk of milk cow, the important parameter of production performance.
At present, albumen evaluation system all adopts fixed value to the RUP small intestine digestibility of DIFFERENT FEED, and in Britain's feed evaluation system, NRC system, the RUP small intestine digestibility of all feeds is respectively 90%, 80%.This situation obviously has very large limitation to DIFFERENT FEED.At present, measure feed RUP small intestine digestibility and mainly contain mobile Nylon Bag, three step in vitro method.Running cost is high, process is complicated owing to existing for mobile Nylon Bag, the shortcoming such as there are differences with actual chyme rate travel is difficult for widespread use.Three step in vitro method are with a kind of enzyme or multienzyme complex, to cultivate Feed Sample under specified temp and pH condition.Three step in vitro method technology 1) physiological condition of the simulation ruminant of trying one's best, comprise the potential impact of lumen fermentation; 2) have quick, reliable, cheap feature; 3) widely applicable protein supplements.4) react accurately the digestion of different proteins.So current three step in vitro method are often used as the method for evaluating ruminant feed RUP small intestine digestibility.
That at present conventional three step in vitro method, use is pancreatin (Sigma P-7545).
Pancreatin (Sigma P-7545) consists predominantly of chymotrypsin, trypsase.
The using method of this pancreatin is: be configured to pH value and be 7.8 phosphate buffer solution, wherein include 50ppm thymol, 3g/L pancreatin (Sigma P-7545).In three step in vitro method, in the 3rd step pancreatin cultivation process, the NaOH solution of getting this solution of 13.5ml and 0.5ml, 1mol/L joins culture and cultivates.
The method can only be used for digesting the non-degraded forage protein of cud (RUP) in degraded feed sample, determines the index of this ruminant feed nutritive value of ruminant Protein in small intestine digestibility.But this method can not be measured small intestine starch and fatty digestibility, nutritive value that cannot certain feed of thoroughly evaluating, and cannot eliminate starch and the impact of fat on protein digestibility in feed.
Summary of the invention
The defect existing for solving above-mentioned prior art, the present invention proposes a kind of method of three step ex vivo technique mensuration nutrient small intestine digestibilities, in the present invention's three step in vitro method technology, directly use pancreatic juice to process, the non-degraded forage protein of cud (RUP) that can not only be used in digestion degraded feed sample, can also digest by the starch in the feed of cud and fat, can determine ruminant Protein in small intestine digestibility, starch digestibility, fat digestibility, evaluate out the important indicator of three ruminant feed nutritive values.
For achieving the above object, a kind of three step ex vivo technique of the present invention are measured the method for nutrient small intestine digestibility, and its concrete steps are:
1), cud is cultivated
Take about 15g feed, pack in the Nylon Bag of 9cm × 15cm, be suspended in cud 16 hours, after cultivation, with tap water, rinse sack to flowing water and clarify, and 55 ℃ are dried 48 hours, take out residual samples in sack, and measure N content, content of starch, fat content;
2), acid treatment
Claim the sample of 0.6～0.7g remnants, put into 50ml centrifuge tube, add the HCl solution of the 0.1mol/L that 10ml, pH value are 1.9,38 ℃ of water-bath shaken cultivation 1 hour;
3), pancreatic juice is cultivated
Utilize pancreatic juice to cultivate, after having cultivated, centrifuge tube adds 3ml, 100% (wt/vol) TCA solution immediately, stops the effect of enzyme and precipitates indigested protein, centrifugal 15 minutes with 10000 revs/min;
Measure N content, content of starch, fat content in sediment;
By step 1) in N content, content of starch, fat content and step 3) in the percent of material content ratio of the same race draw protein, starch, the fatty small intestine digestibility of feed sample.
Described step 3) in the pancreatic juice concrete operations of cultivating be: in culture, add the phosphate buffer of 13.5ml, then medium pH value be adjusted to 7.6 with the NaOH solution of 1mol/l, then add pancreatic juice 2.5ml, 38 ℃ of water-bath shaken cultivation 24 hours.
Described pancreatic juice comes from bullock, milk cow or sheep that pancreatic juice collection tube is installed, obtains latter 4 ℃ and is saved to laboratory, standby.
With respect to prior art, beneficial effect of the present invention is: pancreatic juice method of the present invention can replace at present conventional trypsin process for three step in vitro method culture techniques completely, and can evaluate more accurately the Protein in small intestine digestibility of ruminant all feeds.
Meanwhile, pancreatic juice method of the present invention provides a good approach for the evaluation of ruminant small intestine starch digestibility, fat digestibility.By three step in vitro method, just can simulate the digestion situation of ruminant to protein, starch, fat in feed, measure protein, starch, three important indicators of fatty small intestine digestibility.Simple to operate, be easy to realization and cost-saving.
Accompanying drawing explanation
Fig. 1 is pancreatic juice addition and the impact of incubation time in vitro protein digestibility.
Embodiment
Below in conjunction with the drawings and the specific embodiments, the present invention is described in further detail:
A kind of three step ex vivo technique of the present invention are measured the method for nutrient small intestine digestibility, and its concrete steps are:
1), cud is cultivated
Take about 15g feed, pack in the Nylon Bag of 9cm × 15cm, be suspended in cud 16 hours, after cultivation, with tap water, rinse sack to flowing water and clarify, and 55 ℃ are dried 48 hours, take out residual samples in sack, and measure N content, content of starch, fat content;
2), acid treatment
Claim the sample of 0.6～0.7g remnants, put into 50ml centrifuge tube, add the HCl solution of the 0.1mol/L that 10ml, pH value are 1.9,38 ℃ of water-bath shaken cultivation 1 hour;
3), pancreatic juice is cultivated
Utilize pancreatic juice to cultivate, after having cultivated, centrifuge tube adds 3ml, 100% (wt/vol) TCA solution immediately, stops the effect of enzyme and precipitates indigested protein, centrifugal 15 minutes with 10000 revs/min;
Measure N content, content of starch, fat content in sediment;
By step 1) in N content, content of starch, fat content and step 3) in the percent of material content ratio of the same race draw protein, starch, the fatty small intestine digestibility of feed sample.
Described step 3) in the pancreatic juice concrete operations of cultivating be: in culture, add the phosphate buffer of 13.5ml, then medium pH value be adjusted to 7.6 with the NaOH solution of 1mol/l, then add pancreatic juice 2.5ml, 38 ℃ of water-bath shaken cultivation 24 hours.Enteral pH is generally between 7.2-8.0, so use phosphate buffer and NaOH solution that pH is transferred to 7.6 to be conducive to the performance of digestive enzyme activity.According to experimental study, best pancreatic juice addition is 2.5ml (Fig. 1).
Described pancreatic juice come from install pancreatic juice collection tube 2 one full year of life bullock, body weight is 350kg, obtains the pancreatic juice of 2 bullocks, equal proportion is mixed, 4 ℃ are saved to laboratory, standby.
The method of generally acknowledged mensuration protein small intestine digestibility is trypsin process in the world.As shown in Figure 1, the protein small intestine digestibility that pancreatin (Sigma P-7545) method is measured was 91.8% (cross represents); The protein small intestine digestibility that the 2.5ml pancreatic juice cultivation pancreatic juice cultural method of 24 hours adopting in the present invention is measured was 93.2% (square good expression), with not remarkable (P > 0.10) of trypsin process measured value difference.The digestibility and the trypsin process that adopt 1ml (rhombus number) or 5ml (triangle number) pancreatic juice to measure are not inconsistent.
As shown in table 1, pancreatic juice method of the present invention does not affect (P > 0.10) with at present conventional pancreatin (Sigma P-7545) method in vitro protein digestibility.Various raw material sample standard deviations represent that between two kinds of methods, difference is not remarkable.
Table 1 enzyme process and the comparison of pancreatic juice method to external protein feeds protein digestibility
As shown in table 2, pancreatic juice method and enzyme process there are differences (P < 0.05) to the protein digestibility of external cereal and fatty high feed, and reciprocation is tending towards significantly (P=0.09).This explanation, when containing much starch and when fat in feedstuff, the digestion that starch and fat can interferencing proteins, shows as the protein digestibility of enzymatic assays significantly lower than pancreatic juice method, especially more outstanding in the high feed performance of fat content.
The comparison to external cereal and fatty high feed protein digestibility of table 2 enzyme process and pancreatic juice method
Comprehensive foregoing, pancreatic juice method of the present invention can replace at present conventional trypsin process for three step in vitro method culture techniques completely, and can evaluate more accurately the Protein in small intestine digestibility of ruminant all feeds.
Meanwhile, pancreatic juice method of the present invention provides a good approach for the evaluation of ruminant small intestine starch digestibility, fat digestibility.By three step in vitro method, just can simulate the digestion situation of ruminant to protein, starch, fat in feed, measure protein, starch, three important indicators of fatty small intestine digestibility.
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited to this, and any variation of expecting without creative work or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.
Claims (3)
1. three step ex vivo technique are measured a method for nutrient small intestine digestibility, and its concrete steps are:
1), cud is cultivated
Get appropriate feed, pack Nylon Bag into, be suspended in cud 16 hours, after cultivation, with tap water, rinse sack to flowing water and clarify, and 55 ℃ dry 48 hours, take out residual samples in sack, and measure N content, content of starch, fat content;
2), acid treatment
Claim the sample of 0.6～0.7g remnants, put into 50ml centrifuge tube, add the HCl solution of the 0.1mol/L that 10ml, pH value are 1.9,38 ℃ of water-bath shaken cultivation 1 hour;
3), pancreatic juice is cultivated
Utilize pancreatic juice to cultivate, after having cultivated, centrifuge tube adds 3ml, 100% (wt/vol) TCA solution immediately, stops the effect of enzyme and precipitates indigested protein, centrifugal 15 minutes with 10000 revs/min;
Measure N content, content of starch, fat content in sediment;
By step 1) in N content, content of starch, fat content and step 3) in the percent of material content ratio of the same race draw protein, starch, the fatty small intestine digestibility of feed sample.
2. method according to claim 1, it is characterized in that, described step 3) in pancreatic juice cultivate concrete operations be: the phosphate buffer that adds 13.5ml in culture, with the NaOH solution of 1mol/l, medium pH value is adjusted to 7.6 again, add again pancreatic juice 2.5ml, 38 ℃ of water-bath shaken cultivation 24 hours.
3. method according to claim 1, is characterized in that, described pancreatic juice comes from bullock, milk cow or sheep that pancreatic juice collection tube is installed, obtains latter 4 ℃ and is saved to laboratory, standby.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105136990A (en) * | 2015-08-28 | 2015-12-09 | 石河子大学 | Method for evaluating small intestine digestibility of amino acid in rumen undegradable protein of sheep fattening feed |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102901797A (en) * | 2012-09-20 | 2013-01-30 | 中国农业科学院北京畜牧兽医研究所 | Bionic evaluation method of available phosphorus in pig feed |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105136990A (en) * | 2015-08-28 | 2015-12-09 | 石河子大学 | Method for evaluating small intestine digestibility of amino acid in rumen undegradable protein of sheep fattening feed |
| 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 |
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