CN102590453B - Phosphate buffer for in-vitro rumen gas production method - Google Patents
Phosphate buffer for in-vitro rumen gas production method Download PDFInfo
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- CN102590453B CN102590453B CN201210035989.0A CN201210035989A CN102590453B CN 102590453 B CN102590453 B CN 102590453B CN 201210035989 A CN201210035989 A CN 201210035989A CN 102590453 B CN102590453 B CN 102590453B
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- phosphate buffer
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- gas production
- production method
- buffer
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- 239000008363 phosphate buffer Substances 0.000 title claims abstract description 47
- 210000004767 rumen Anatomy 0.000 title claims abstract description 14
- 238000000338 in vitro Methods 0.000 title abstract description 22
- 238000004519 manufacturing process Methods 0.000 title abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000012153 distilled water Substances 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 19
- 239000000243 solution Substances 0.000 abstract description 19
- 239000007853 buffer solution Substances 0.000 abstract description 16
- 238000000855 fermentation Methods 0.000 abstract description 16
- 230000004151 fermentation Effects 0.000 abstract description 16
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 abstract description 15
- 230000007547 defect Effects 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 abstract 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 abstract 2
- 235000019270 ammonium chloride Nutrition 0.000 abstract 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 abstract 2
- 229910000397 disodium phosphate Inorganic materials 0.000 abstract 2
- 235000019800 disodium phosphate Nutrition 0.000 abstract 2
- 235000019799 monosodium phosphate Nutrition 0.000 abstract 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 abstract 2
- 229910000162 sodium phosphate Inorganic materials 0.000 abstract 2
- 239000007789 gas Substances 0.000 description 25
- 239000007788 liquid Substances 0.000 description 13
- 239000012530 fluid Substances 0.000 description 12
- 239000000120 Artificial Saliva Substances 0.000 description 8
- 238000013016 damping Methods 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 241000282849 Ruminantia Species 0.000 description 4
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 235000019621 digestibility Nutrition 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 235000013325 dietary fiber Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000021050 feed intake Nutrition 0.000 description 2
- 239000004459 forage Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 235000011960 Brassica ruvo Nutrition 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000000433 anti-nutritional effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 229940099259 vaseline Drugs 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
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- Fodder In General (AREA)
- Feed For Specific Animals (AREA)
Abstract
The invention discloses a phosphate buffer for an in-vitro rumen gas production method. The phosphate buffer consists of 94.2g of Na2HPO4, 5.2g of NaH2PO4.H2O, and 4g of NH4Cl, the 94.2g of Na2HPO4, 5.2g of NaH2PO4.H2O, and 4g of NH4Cl are prepared into 1,000ml of solution by using distilled water, and the pH value of the phosphate buffer is 7.82; CO2 is needed to be introduced into the phosphate buffer for three times before the phosphate buffer is used; and the color of the solution is changed from pink to colorlessness every time, and the CO2 is introduced for one hour every time, the CO2 is introduced for the last time before the phosphate buffer is used to increase the CO2 retaining amount of new phosphate buffer, and then the new phosphate buffer is used. The phosphate buffer avoids the defect that carbonic acid buffer produces gas; by increasing the introducing times and the retaining amount of CO2, the delay time of feed fermentation is reduced, the delay time of the feed fermentation is consistent with that of the carbonic acid buffer, and a formula of the new phosphate buffer and the preparation method for the new phosphate buffer can be used for research and application of the in-vitro gas production method.
Description
Technical field
The present invention relates to feed nutritive value assessment technique field, in particular a kind of phosphate buffer for vitro Rumen aerogenesis method.
Background technology
The feed intake of roughage and digestibility can the production performances of extreme influence ruminant, but measured by intracorporal method waste time and energy, expensive, need a large amount of Feed Sample and can not quantitative determination greatly.So, scientist attempting always by experiment room method to measure roughage feed intake and digestibility.
The in vitro method set up since the 1950's, comprises the aerogenesis method (The gas production method) of the development such as two-phase method, Raab etc. (1983) that British scholar Tilly-Terry (1963) sets up and German He Enhaimu university animal nutrient research institute Menke (1979), fungin enzyme process and Nylon Bag.
In Vitro gas production method (Invitro gas production) is that the people such as (1983) such as Raab L. and German He Enhaimu university animal nutrient research institute Menke K.H. sets up, the very effective method of one of evaluation ruminant feed nutritive value is that current developed country adopts maximum one of technology evaluating ruminant forage grass and feed feeding value that is used for.In Vitro gas production method once can carry out the mensuration of great amount of samples, compared with intracorporal method, do not need a large amount of animals used as test, and result and intracorporal method has high correlation.In Vitro gas production method not only can be simulated the organic matter digestibility of herbage in cud more truly, be estimated the degradable protein content of feed and the metabolizable energy value of single feed or mixed fodder by gas production in vitro amount, but also whether can grasp certain forage grass containing tannin and anti-nutritional profile thereof on the basis of whether adding PVOH.
Current In Vitro gas production method be carbonic acid buffer, this damping fluid in use also exists certain defect, thus has influence on the accuracy of measurement result.
Current In Vitro gas production method uses carbonic acid buffer to consist of: 35g NaHCO
3+ 4.0g NH
4cO
3+ distilled water is mixed with 1000ml solution, and carbonic acid buffer pH value is 7.86, and described phosphate buffer needs to pass into 3 CO before using
2, till all making solution be become colorless by pink, each 1 hour, pass into before use for the last time, to improve the CO of new phosphate buffer at every turn
2recoverable amount, then uses.
The gas that in In Vitro gas production method, feed fermentation mainly produces is CO
2, CH
4deng.When utilizing carbonate buffer solution to cultivate feed, some CO in the gas of generation
2be then derive from carbonic acid buffer, not feed fermentation produces, and has had a strong impact on the accuracy measured like this.
Summary of the invention
Technical matters to be solved by this invention provides a kind of phosphate buffer for vitro Rumen aerogenesis method for the deficiencies in the prior art.
Technical scheme of the present invention is as follows:
For a phosphate buffer for vitro Rumen aerogenesis method, phosphate buffer consists of: 94.2g Na
2hPO
4, 5.2gNaH
2pO
4h
2o, 1.4g NH
4cl, is mixed with 1000ml solution with distilled water, and phosphate buffer pH value is 7.82, and described phosphate buffer needs to pass into 3 CO before using
2, till all making solution be become colorless by pink, each 1 hour, pass into before use for the last time, to improve the CO of new phosphate buffer at every turn
2recoverable amount, then uses.
Phosphate buffer of the present invention avoids the defect of the aerogenesis of carbonic acid buffer own; CO is passed into by increasing
2number of times in order to increase its recoverable amount, reduce the time delay of feed fermentation, consistent with the time delay of carbonic acid buffer, new phosphoric acid buffer formula of liquid and preparation method thereof can be used for the investigation and application of In Vitro gas production method.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
The present invention is a kind of phosphate buffer, decreases the adverse effect that the output gas of damping fluid own brings measurement result accuracy on the one hand, ensures that damping fluid has good buffering effect, to feed In Vitro Fermentation without negative effect on the other hand.
Phosphate buffer of the present invention consists of: 94.2g Na
2hPO
4, 5.2g NaH
2pO
4h
2o, 1.4g NH
4cl, is mixed with 1000ml solution with distilled water, and phosphate buffer pH value is 7.82.
Phosphate buffer of the present invention needs to pass into 3 CO before using
2, till all making solution be become colorless by pink, each 1 hour, process before use for the last time, to improve the CO of new phosphate buffer at every turn
2recoverable amount, then uses.
Embodiment 2
Using method:
1, artificial saliva is prepared
1. after water bath with thermostatic control shaking table, multi-connection water bath being opened; 2. artificial saliva requirement is calculated: during in vitro culture, in every root generating tube, the volume of microbial culture medium is 30-100ml (10-30ml rumen fluid and 20-70ml artificial saliva), generating tube number according to test design calculates the requirement (reserved 20%, cause waste in order to operation technique is improper) of artificial saliva and rumen fluid; 3. damping fluid is prepared; 4. reductant solution (E liquid) is prepared; 5., after each stoste prepared being mixed with ratio in order, insert in shaking bath or water-bath, pass into CO
2gas (air-flow is not easily excessive), makes artificial saliva change pink blueness into by blueness and changes pink into, is finally colourless.
Artificial saliva formula (1000ml): 1000ml artificial saliva contains: distilled water 520.2ml, trace element solution (A liquid) 0.1ml, phosphate buffer (B liquid) 208.1ml, macroelement solution (C liquid) 208.1ml, 0.1% resazurin solution (D liquid) 1.0ml, reductant solution (E liquid) 62.4ml.
A liquid, trace element solution:
13.2g CaCl
22H
2o; 10.0g MnCl
24H
2o; CoCl
21.0g 6H
2o; 8.0g FeCl
36H
2o, adding distil water is to 100ml;
B liquid, phosphate buffer:
94.2g Na
2hPO
4, 5.2g NaH
2pO
4h
2o, 1.4g NH
4cl, adding distil water is to 1000ml, and phosphate buffer pH value is 7.82.Phosphate buffer needs to pass into 3 CO before using
2, till all making solution be become colorless by pink, each 1 hour, process before use for the last time, to improve the CO of new phosphate buffer at every turn
2recoverable amount, then uses.
C liquid, macroelement solution:
9.45g Na
2hPO
412H
2o; 6.2g KH
2pO
4; 0.6g MgSO
47H
2o, adding distil water is to 1000ml;
D liquid, 0.1% resazurin solution:
100mg resazurin is dissolved in 100ml distilled water;
E liquid, reductant solution (matching while using):
1M NaOH 4.0ml; 625mg Na
2s9H2O, adding distil water 95ml.
2. get rumen fluid
Rumen fluid before early raising 1h or after early raising 2h fetch.1. pour hot water into bucket, regulate coolant-temperature gage to 39 ± 0.5 DEG C, for rumen fluid insulation (should regulate through conventional hot water); 2. extract the rumen fluid of three ruminants with vacuum pump, mixing, pours receiving flask into, notes covering bottle cap, in case oxygen is on the impact of microorganism; 3. receiving flask is placed in water-bath, four layers of filtered through gauze (should as quickly as possible, to keep the vigor of microorganism), the filtrate CO of acquisition
2gas is saturated.
3, fermentation substrate is claimed
1. generating tube numbering; 2. with the pan paper of homemade band long handle, accurately take testing sample and be about 200-1000mg, be placed in bottom gas production in vitro pipe, note not allowing sample enter generating tube inlet and be infected with 30ml with upper tube wall; 3., after sample takes, uniform application vaseline on pipe close, gets back into the outer tube of mapping, buckles well clip by pipe close.
4, preparing microorganism nutrient solution
Measure the colourless artificial saliva of aequum and rumen fluid by the volume ratio mixing of 2: 1 ~ 4: 1 with graduated cylinder, pass into CO incessantly
2gas.
5, ferment
Draw microbial culture medium 30 ~ 100ml with the generating tube of sky, pushed the generating tube that sample is housed by three-way pipe, sample is fermented, and collection fermentation generation gas is analyzed.
Embodiment 3
As shown in table 1, phosphate buffer of the present invention is passing into 1 CO before use with carbonic acid buffer
2, when duration of ventilation 1 hour, the final pH of phosphate buffer, methane generating yield and carbonic acid buffer difference are not remarkable.But the feed fermentation response delay time significantly increases, thus extend the time of whole feed fermentation reaction, reduce the efficiency of In Vitro Fermentation, feed In Vitro Fermentation is had a negative impact.
Table 1. phosphate buffer and carbonic acid buffer are on impact (logical 1 CO of gas production in vitro characteristic
2gas)
1N＝4；*P＜0.05；NS?P＞0.05
Embodiment 4
As shown in table 2: phosphate buffer of the present invention passes into 3 CO before use
2, when each 1 hour, feed In Vitro Fermentation time delay, final pH, CH
4the concentration of generation rate and VFA and composition difference are all not remarkable, indicate phosphate buffer and have good surge capability, do not have negative effect to feed In Vitro Fermentation.Simultaneously compared with carbonic acid buffer, phosphate buffer significantly reduces the maximum gas production rate of 24 hours gas production rate and model estimation, indicates and uses phosphate buffer to reduce CO like this
2generation rate, reduce the error that damping fluid itself brings measurement result, thus improve the accuracy of mensuration.
Table 2 phosphate buffer and the impact of carbonic acid buffer on gas production in vitro characteristic and lumen fermentation (lead to 3 CO
2gas)
1N＝4；*P＜0.05；NS?P＞0.05
Phosphate buffer of the present invention avoids the defect of the aerogenesis of carbonic acid buffer own; CO is passed into by increasing
2number of times in order to increase its recoverable amount, reduce the time delay of feed fermentation, consistent with the time delay of carbonic acid buffer.Comprehensive foregoing, new phosphoric acid buffer formula of liquid and preparation method thereof can be used for the investigation and application of In Vitro gas production method.
Claims (1)
1. for a phosphate buffer for vitro Rumen aerogenesis method, it is characterized in that, phosphate buffer consists of: 94.2g Na
2hPO
4, 5.2g NaH
2pO
4h
2o, 1.4g NH
4cl, is mixed with 1000ml solution with distilled water, and phosphate buffer pH value is 7.82, and described phosphate buffer needs to pass into 3 CO before using
2, till all making solution be become colorless by pink, each 1 hour, pass into before use for the last time, to improve the CO of new phosphate buffer at every turn
2recoverable amount, then uses.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210035989.0A CN102590453B (en) | 2012-02-17 | 2012-02-17 | Phosphate buffer for in-vitro rumen gas production method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210035989.0A CN102590453B (en) | 2012-02-17 | 2012-02-17 | Phosphate buffer for in-vitro rumen gas production method |
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| Publication Number | Publication Date |
|---|---|
| CN102590453A CN102590453A (en) | 2012-07-18 |
| CN102590453B true CN102590453B (en) | 2015-01-21 |
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|---|---|---|---|
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| CN107942054A (en) * | 2017-11-16 | 2018-04-20 | 北华大学 | A kind of arginyl butanedioic acid cracks enzyme reagent kit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202099277U (en) * | 2011-06-08 | 2012-01-04 | 刘立成 | Simulated rumen in vitro fermentation gas producing device |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6750051B2 (en) * | 2001-04-10 | 2004-06-15 | University Of Kentucky Research Foundation | Compositions and methods for enhancing fiber digestion |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202099277U (en) * | 2011-06-08 | 2012-01-04 | 刘立成 | Simulated rumen in vitro fermentation gas producing device |
Non-Patent Citations (3)
| Title |
|---|
| Comparison of in vitro gas production and nylon bag degradability of roughages in predicting feed intake in cattle;Bhlmmel M et al;《Animal Fred Science and Technology》;19930203;全文 * |
| Interactions between substrate, fermentation end-products, buffering systems and gas production upon fermentation of different carbohydrates by mixed rumen microorganisms in vitro;Beuvink J M W et al;《Applied Microbiology Biotechnology》;19920220;全文 * |
| 利用体外产气法评定反刍动物饲料的营养价值;任莹等;《中国饲料》;20091205(第23期);第17页第2栏第2-6段 * |
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