Phytosterol formula for promoting rumen fermentation and determination method thereof
阅读说明：本技术 一种促进瘤胃发酵的植物甾醇配方及其测定方法 (Phytosterol formula for promoting rumen fermentation and determination method thereof ) 是由 成艳芬 吕东海 吴子辰 朱伟云 朱强 于 2020-08-11 设计创作，主要内容包括：本发明公开了一种促进瘤胃发酵的植物甾醇配方及其测定方法,添加植物甾醇可以提高微生物发酵的产气量及挥发性脂肪酸产量,同时降低乳酸产量。植物甾醇的添加量比甾醇组分对瘤胃发酵影响更大。添加植物甾醇B2μg/瓶时对瘤胃发酵的促进作用最明显。(The invention discloses a phytosterol formula for promoting rumen fermentation and a determination method thereof. The addition amount of phytosterol has a greater effect on rumen fermentation than the sterol component. When the phytosterol B is added into 2 mu g/bottle, the promoting effect on rumen fermentation is most obvious.)
1. A formula of phytosterol for promoting rumen fermentation is characterized by comprising the following raw materials in parts by mass;
91.14-99.18 parts of total sterol, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol and 0.10-95.17 parts of stigmasterol.
2. The ruminal fermentation-promoting phytosterol formulation of claim 1,
the fermentation substrate of the phytosterol formula for promoting rumen fermentation is 600mg of total mixed ration.
3. The ruminal fermentation-promoting phytosterol formulation of claim 2,
the raw material addition amount of the phytosterol formula for promoting rumen fermentation is 2-12ug per bottle.
4. The ruminal fermentation promoting phytosterol formulation as claimed in claim 3, wherein the total sterol is in the range of 0-99.18 parts, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol, 0.10-95.17 parts of stigmasterol:
95.64 parts of total sterols, wherein: 55.19 parts of beta-sitosterol, 29.88 parts of campesterol and 10.08 parts of stigmasterol.
5. The ruminal fermentation promoting phytosterol formulation as claimed in claim 3, wherein the total sterol is in the range of 0-99.18 parts, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol, 0.10-95.17 parts of stigmasterol:
91.14 parts of total sterols, wherein: 44.71 parts of beta-sitosterol, 27.23 parts of campesterol and 16.63 parts of stigmasterol.
6. The ruminal fermentation promoting phytosterol formulation as claimed in claim 3, wherein the total sterol is in the range of 0-99.18 parts, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol, 0.10-95.17 parts of stigmasterol:
95.4 parts of total sterols, wherein: 46.7 parts of beta-sitosterol, 25.1 parts of campesterol and 22.8 parts of stigmasterol.
7. The ruminal fermentation promoting phytosterol formulation as claimed in claim 3, wherein the total sterol is in the range of 0-99.18 parts, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol, 0.10-95.17 parts of stigmasterol:
98.54 parts of total sterols, wherein: beta-sitosterol 0 part, campesterol 0 part and stigmasterol 95.17 parts.
8. The ruminal fermentation promoting phytosterol formulation as claimed in claim 3, wherein the total sterol is in the range of 0-99.18 parts, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol, 0.10-95.17 parts of stigmasterol:
99.18 parts of total sterols, wherein: 79.71 parts of beta-sitosterol, 7.44 parts of campesterol and 0.10 part of stigmasterol.
9. The method for measuring a phytosterol formulation for promoting rumen fermentation according to claim 1, wherein the method for measuring a phytosterol formulation for promoting rumen fermentation comprises the following steps:
heating the fermentation bottle to 39 ℃, and placing the phytosterol and the total mixed ration in the fermentation bottle;
inserting a sensor needle of an air pressure converter into the fermentation bottle, and reading air pressure data;
recording the volume of gas flowing into the syringe cylinder until the pressure in the fermentation bottle is restored to the atmospheric environment pressure;
the gas in the syringe barrel was discarded and the gas production was measured at the same time intervals until the fermentation was terminated.
Technical Field
The invention relates to the field of phytosterol, in particular to a formula of phytosterol for promoting rumen fermentation and a determination method thereof.
Background
At present, along with the gradual stabilization of society in recent years, the living standard of people is further improved, and the center of gravity of the breeding industry gradually changes from the aspects of animal growth performance and production benefit, such as the attention on animal health, feed safety, animal product quality and the like. The plant sterol is a novel functional green feed additive, belongs to a plant steroid compound, and has plant active ingredients. The phytosterol existing in nature is divided into free type and esterified type, the esterified type phytosterol is more soluble in organic solvent, the absorption and utilization rate of the esterified type phytosterol is about 5 times higher than that of the free type phytosterol, and the functional effect of the esterified type phytosterol is wider. It is reported by Mingzhu et al that adding a certain amount of phytosterol into rat diet can effectively reduce cholesterol content in blood, prevent cardiovascular and cerebrovascular diseases, and protect liver and blood vessels. In recent years, phytosterol has been found to have anti-inflammatory, anticancer and antioxidant effects, and also to have high safety. At present, the body shadow of the phytosterol is spread in the industries of food, medicine, cosmetics and the like, and the hair and horn are exposed in agriculture. Researches on aquatic products, poultry and monogastric animals show that the proper addition amount of the phytosterol can reduce the cholesterol content in vivo, improve the physique of the animals, improve the production efficiency and further improve the economic benefit. Researches in dairy cows such as Xinxinmei also find that the addition of 200mg/d phytosterol in daily ration can obviously improve the lactation yield of the dairy cows, shorten the first-time blending time and the non-pregnancy time and improve the physique of the dairy cows.
The phytosterols comprise beta-sitosterol, campesterol, stigmasterol and the like, and have different components, and the content and the proportion of the sterols are also different, so that the influence of the different components of the phytosterols on rumen fermentation is difficult to judge.
Disclosure of Invention
The invention aims to provide a formula of phytosterol for promoting rumen fermentation and a determination method thereof, and aims to solve the technical problem that in the prior art, the phytosterol comprises beta-sitosterol, campesterol, stigmasterol and the like, the components are different, and the content and the proportion of each sterol are also different, so that the influence of different components of phytosterol on rumen fermentation is difficult to judge.
In order to achieve the purpose, the invention adopts a formula of phytosterol for promoting rumen fermentation, which is prepared from the following raw materials in parts by mass; 91.14-99.18 parts of total sterol, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol and 0.10-95.17 parts of stigmasterol.
Wherein the fermentation substrate of the phytosterol formula for promoting rumen fermentation is 600mg of total mixed ration.
Wherein, the raw material adding amount of the phytosterol formula for promoting rumen fermentation is 2-12ug per bottle.
Wherein, 95.64 parts of total sterol, wherein: 55.19 parts of beta-sitosterol, 29.88 parts of campesterol and 10.08 parts of stigmasterol.
Wherein, 91.14 parts of total sterol, wherein: 44.71 parts of beta-sitosterol, 27.23 parts of campesterol and 16.63 parts of stigmasterol.
Wherein, the total sterol is 95.4 parts, wherein: 46.7 parts of beta-sitosterol, 25.1 parts of campesterol and 22.8 parts of stigmasterol.
Wherein, the total sterol accounts for 98.54 parts, wherein: beta-
Wherein, 99.18 parts of total sterol, wherein: 79.71 parts of beta-sitosterol, 7.44 parts of campesterol and 0.10 part of stigmasterol.
The present invention employs the rumen fermentation-promoting phytosterol formulation as set forth in claim 1, and the method for determining the rumen fermentation-promoting phytosterol formulation comprises the steps of:
heating the fermentation bottle to 39 ℃, and placing the phytosterol and the total mixed ration in the fermentation bottle;
inserting a sensor needle of an air pressure converter into the fermentation bottle, and reading air pressure data;
recording the volume of gas flowing into the syringe cylinder until the pressure in the fermentation bottle is restored to the atmospheric environment pressure;
the gas in the syringe barrel was discarded and the gas production was measured at the same time intervals until the fermentation was terminated.
According to the formula of the phytosterol for promoting rumen fermentation and the determination method thereof, the addition of the phytosterol can improve the gas yield and the yield of volatile fatty acid of microbial fermentation and reduce the yield of lactic acid. The addition amount of phytosterol has a greater effect on rumen fermentation than the sterol component. When the phytosterol B is added into 2 mu g/bottle, the promoting effect on rumen fermentation is most obvious.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph comparing the concentration of lactic acid versus different components of phytosterols according to the present invention.
FIG. 2 is a graph comparing the rumen fermentation gas production with the phytosterols of component A of the present invention.
FIG. 3 is a graph comparing the production of rumen fermentation with phytosterols of component B according to the present invention.
FIG. 4 is a graph comparing the production of rumen fermentation with phytosterols of component C according to the present invention.
FIG. 5 is a graph comparing the rumen fermentation gas production with the phytosterols of component D of the present invention.
FIG. 6 is a graph comparing the rumen fermentation gas production with phytosterols of component E according to the present invention.
Fig. 7 is a flowchart of a method for determining a phytosterol formulation that promotes rumen fermentation in accordance with the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
The first embodiment:
the invention provides a phytosterol formula for promoting rumen fermentation, which is prepared from the following raw materials in parts by mass; 0-99.18 parts of total sterol, wherein: 91.14-99.18 parts of beta-sitosterol, 0-29.88 parts of campesterol and 0.10-95.17 parts of stigmasterol.
Further, the fermentation substrate of the phytosterol formula for promoting rumen fermentation is 600mg of total mixed ration.
Furthermore, the raw material addition amount of the phytosterol formula for promoting rumen fermentation is 2-12ug per bottle.
In the embodiment, each sterol sample is subjected to rumen fluid in-vitro fermentation in 3 gradient concentrations, wherein the 3 concentration gradients are 2 mug, 6 mug and 12 mug per bottle respectively, and the phytosterol is not added as a test control group. The fermentation substrate was 600mg of total mixed ration.
Preparing an artificial rumen culture solution by referring to a method of Theodorou and the like, mixing rumen fluid filtered by four layers of gauze and an artificial rumen nutrient solution in a volume ratio of 1:5, subpackaging the total volume of 60ml in a 120ml fermentation bottle, and culturing at a constant temperature of 39 ℃ for 24 hours. The gas production was measured for 3, 6, 9, 12, 24h, respectively. And (3) immediately measuring the pH value of the fermentation liquor after the fermentation is finished, and freezing and storing the fermentation liquor at-20 ℃ for measuring the contents of microbial protein, lactic acid, ammonia nitrogen and volatile fatty acid. The fermentation substrate is dried at 65 ℃ and weighed for calculating the degradation rate of the dry matter of the substrate.
Second embodiment:
the invention provides a phytosterol formula for promoting rumen fermentation, which is prepared from the following raw materials in parts by mass; 0-99.18 parts of total sterol, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol and 0.10-95.17 parts of stigmasterol.
Further, the fermentation substrate of the phytosterol formula for promoting rumen fermentation is 600mg of total mixed ration.
Furthermore, the raw material addition amount of the phytosterol formula for promoting rumen fermentation is 2-12ug per bottle.
Further, 95.64 parts of total sterols, wherein: 55.19 parts of beta-sitosterol, 29.88 parts of campesterol and 10.08 parts of stigmasterol.
In the embodiment, each sterol sample is subjected to rumen fluid in-vitro fermentation in 3 gradient concentrations, wherein the 3 concentration gradients are 2 mug, 6 mug and 12 mug per bottle respectively, and 95.64 parts of total sterol are contained, wherein: 55.19 parts of beta-sitosterol, 29.88 parts of campesterol and 10.08 parts of stigmasterol are used as test groups. The fermentation substrate was 600mg of total mixed ration.
Preparing an artificial rumen culture solution by referring to a method of Theodorou and the like, mixing rumen fluid filtered by four layers of gauze and an artificial rumen nutrient solution in a volume ratio of 1:5, subpackaging the total volume of 60ml in a 120ml fermentation bottle, and culturing at a constant temperature of 39 ℃ for 24 hours. The gas production was measured for 3, 6, 9, 12, 24h, respectively. And (3) immediately measuring the pH value of the fermentation liquor after the fermentation is finished, and freezing and storing the fermentation liquor at-20 ℃ for measuring the contents of microbial protein, lactic acid, ammonia nitrogen and volatile fatty acid. The fermentation substrate is dried at 65 ℃ and weighed for calculating the degradation rate of the dry matter of the substrate.
The third embodiment:
the invention provides a phytosterol formula for promoting rumen fermentation, which is prepared from the following raw materials in parts by mass; 0-99.18 parts of total sterol, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol and 0.10-95.17 parts of stigmasterol.
Further, the fermentation substrate of the phytosterol formula for promoting rumen fermentation is 600mg of total mixed ration.
Furthermore, the raw material addition amount of the phytosterol formula for promoting rumen fermentation is 2-12ug per bottle.
Further, 91.14 parts of total sterols, wherein: 44.71 parts of beta-sitosterol, 27.23 parts of campesterol and 16.63 parts of stigmasterol.
In the embodiment, each sterol sample is subjected to rumen fluid in-vitro fermentation in 3 gradient concentrations, wherein the 3 concentration gradients are 2 mug, 6 mug and 12 mug per bottle respectively, and 91.14 parts of total sterol are contained, wherein: beta-sitosterol 44.71 parts, campesterol 27.23 parts, stigmasterol 16.63 parts as test groups. The fermentation substrate was 600mg of total mixed ration.
Preparing an artificial rumen culture solution by referring to a method of Theodorou and the like, mixing rumen fluid filtered by four layers of gauze and an artificial rumen nutrient solution in a volume ratio of 1:5, subpackaging the total volume of 60ml in a 120ml fermentation bottle, and culturing at a constant temperature of 39 ℃ for 24 hours. The gas production was measured for 3, 6, 9, 12, 24h, respectively. And (3) immediately measuring the pH value of the fermentation liquor after the fermentation is finished, and freezing and storing the fermentation liquor at-20 ℃ for measuring the contents of microbial protein, lactic acid, ammonia nitrogen and volatile fatty acid. The fermentation substrate is dried at 65 ℃ and weighed for calculating the degradation rate of the dry matter of the substrate.
The fourth embodiment:
the invention provides a phytosterol formula for promoting rumen fermentation, which is prepared from the following raw materials in parts by mass; 0-99.18 parts of total sterol, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol and 0.10-95.17 parts of stigmasterol.
Further, the fermentation substrate of the phytosterol formula for promoting rumen fermentation is 600mg of total mixed ration.
Furthermore, the raw material addition amount of the phytosterol formula for promoting rumen fermentation is 2-12ug per bottle.
Further, 95.4 parts of total sterols, wherein: 46.7 parts of beta-sitosterol, 25.1 parts of campesterol and 22.8 parts of stigmasterol.
In the embodiment, each sterol sample is subjected to rumen fluid in-vitro fermentation in 3 gradient concentrations, wherein the 3 concentration gradients are respectively 2 mug, 6 mug and 12 mug per bottle, and the total sterol is 95.4 parts, wherein: 46.7 parts of beta-sitosterol, 25.1 parts of campesterol and 22.8 parts of stigmasterol are taken as test groups. The fermentation substrate was 600mg of total mixed ration.
Preparing an artificial rumen culture solution by referring to a method of Theodorou and the like, mixing rumen fluid filtered by four layers of gauze and an artificial rumen nutrient solution in a volume ratio of 1:5, subpackaging the total volume of 60ml in a 120ml fermentation bottle, and culturing at a constant temperature of 39 ℃ for 24 hours. The gas production was measured for 3, 6, 9, 12, 24h, respectively. And (3) immediately measuring the pH value of the fermentation liquor after the fermentation is finished, and freezing and storing the fermentation liquor at-20 ℃ for measuring the contents of microbial protein, lactic acid, ammonia nitrogen and volatile fatty acid. The fermentation substrate is dried at 65 ℃ and weighed for calculating the degradation rate of the dry matter of the substrate.
Fifth embodiment:
the invention provides a phytosterol formula for promoting rumen fermentation, which is prepared from the following raw materials in parts by mass; 0-99.18 parts of total sterol, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol and 0.10-95.17 parts of stigmasterol.
Further, the fermentation substrate of the phytosterol formula for promoting rumen fermentation is 600mg of total mixed ration.
Furthermore, the raw material addition amount of the phytosterol formula for promoting rumen fermentation is 2-12ug per bottle.
Further, total sterols 98.54 parts, wherein: beta-
In the embodiment, each sterol sample is subjected to rumen fluid in-vitro fermentation in 3 gradient concentrations, wherein the 3 concentration gradients are respectively 2 mug, 6 mug and 12 mug per bottle, and the total sterol is 95.4 parts, wherein: 98.54 parts of total sterols, wherein: beta-
Preparing an artificial rumen culture solution by referring to a method of Theodorou and the like, mixing rumen fluid filtered by four layers of gauze and an artificial rumen nutrient solution in a volume ratio of 1:5, subpackaging the total volume of 60ml in a 120ml fermentation bottle, and culturing at a constant temperature of 39 ℃ for 24 hours. The gas production was measured for 3, 6, 9, 12, 24h, respectively. And (3) immediately measuring the pH value of the fermentation liquor after the fermentation is finished, and freezing and storing the fermentation liquor at-20 ℃ for measuring the contents of microbial protein, lactic acid, ammonia nitrogen and volatile fatty acid. The fermentation substrate is dried at 65 ℃ and weighed for calculating the degradation rate of the dry matter of the substrate.
Sixth embodiment:
the invention provides a phytosterol formula for promoting rumen fermentation, which is prepared from the following raw materials in parts by mass; 0-99.18 parts of total sterol, wherein: 0-79.71 parts of beta-sitosterol, 0-29.88 parts of campesterol and 0.10-95.17 parts of stigmasterol.
Further, the fermentation substrate of the phytosterol formula for promoting rumen fermentation is 600mg of total mixed ration.
Furthermore, the raw material addition amount of the phytosterol formula for promoting rumen fermentation is 2-12ug per bottle.
Further, 99.18 parts of total sterols, wherein: 79.71 parts of beta-sitosterol, 7.44 parts of campesterol and 0.10 part of stigmasterol.
Further, referring to fig. 3, the method for determining a phytosterol formulation for promoting rumen fermentation comprises the following steps:
s901: heating the fermentation bottle to 39 ℃, and placing the phytosterol and the total mixed ration in the fermentation bottle;
s902: inserting a sensor needle of an air pressure converter into the fermentation bottle, and reading air pressure data;
s903: recording the volume of gas flowing into the syringe cylinder until the pressure in the fermentation bottle is restored to the atmospheric environment pressure;
s904: the gas in the syringe barrel was discarded and the gas production was measured at the same time intervals until the fermentation was terminated.
In the present embodiment, it is preferred that,
the specific experimental steps are as follows:
1. gas production rate measurement
Before the start of fermentation, the fermentation flasks were heated to a 39 ℃ incubation temperature, and the headspace pressure of each fermentation flask was corrected to atmospheric ambient pressure. During measurement, a sensor needle of the air pressure converter is inserted into a rubber plug above the fermentation bottle, the air pressure of the headspace is read from a display device of the air pressure converter, and the volume of gas flowing into the syringe barrel is recorded to determine the volume of gas generated by fermentation until the pressure of the headspace gas is restored to the atmospheric environmental pressure. After measuring the gas volume, the sensor needle of the gas pressure transducer was pulled out of the cap, the gas in the syringe barrel was discarded and the bottle was returned to the incubator until the next reading, and the gas production was measured at the same time intervals until the end of the fermentation. The time taken to measure the air pressure should be relatively short, not exceeding 10-15 seconds per bottle. Since only a few bottles were removed from the incubator at the same time, it was assumed that the gas temperature in the headspace of the fermentation flask remained constant during the measurement.
2. Rate of substrate dry matter degradation
And (3) performing solid-liquid separation on the substances in the fermentation bottle, transferring the solid part into a centrifuge tube with a known weight, washing residues in the fermentation bottle, mixing the residues with a corresponding solid sample, and drying the mixture in an oven at the temperature of (65 +/-2) DEG C under the atmospheric pressure until the weight is constant. The samples were weighed and the dry matter degradation rate was calculated according to the differential method.
3. Lactic acid assay
The determination is carried out by adopting a p-hydroxybiphenyl colorimetric method. The standard lactic acid solution is 1.730g of calcium lactate, dissolved in deionized water, and taken out of a volumetric flask with 1mL and 100mL of constant volume, wherein the volume is 1000 mL. 20 percent copper sulfate solution, 20g of copper sulfate and deionized water are dissolved, and the volume is determined to be 100 ml. 1.5g of p-hydroxybiphenyl, 20ml of 5% sodium hydroxide solution, dissolving in deionized water, fixing the volume to 100ml, and storing in a brown reagent bottle for later use by refrigeration. 5 percent sodium hydroxide, 5g of sodium hydroxide and deionized water are dissolved, and the volume is determined to be 100 ml. Taking 1ml of fermented feed extract liquid and one drop of 20% copper sulfate solution, placing the test tube in ice water, shaking, slowly adding 6ml of concentrated sulfuric acid, mixing uniformly, boiling in a boiling water bath for 5min, cooling, adding 0.2ml of p-hydroxybiphenyl reagent, shaking uniformly, carrying out 30-DEG C constant temperature water bath for 30min, shaking uniformly, taking out, cooling, carrying out color comparison at the wavelength of 560nm, and measuring the OD value.
4. MCP concentration determination
Apparatus, device and reagent preparation
1) Reagent
Coomassie brilliant blue, 0.25N NaOH, Bovine Serum Albumin (BSA), ultrapure water
2) Instrumentation and equipment
5ml centrifuge tube, 7ml centrifuge tube, 4 degree centrifuge, vortex instrument, 5ml, 1ml, 200ul liquid-transfering gun, enzyme-labeling instrument, enzyme-labeling plate
Second, preparation before test
1) A7 ml centrifuge tube was prepared, and 1 tube was prepared for each sample.
2) 5ml centrifuge tubes were prepared, and 1 tube was prepared for each sample.
3) Tips were prepared for 5ml (2/sample), 1ml (3/sample), 200ul (1/sample).
Third, sample MCP determination step
1) Thawing the sample
2) Vortex and shake for 1min to separate microorganisms from chyme
3) Taking 1ml of the mixed solution, centrifuging at 4 ℃ for 8min at 1,000rpm
4) Sucking supernatant 800ul into 5ml centrifuge tube, centrifuging at 14000rpm and 4 deg.C for 20min
5) Removing supernatant, adding 3ml 0.25N NaOH into substrate, and mixing
6) Water bath at 100 deg.C for 10min, centrifugation at 4 deg.C for 14000rpm for 30min
7) And 500. mu.L of the supernatant (depending on the sample concentration, 100. mu.L of the supernatant plus 400. mu.L of water) was transferred to a 7mL centrifuge tube
8) Adding 5ml Coomassie brilliant blue, shaking, mixing, and standing for 2min
9) And 3 samples are paralleled, 200 mu L of the sample is absorbed and added into an enzyme label plate (with the wavelength of 595nm), the color comparison is rapidly completed according to the operation of an enzyme label instrument, and the absorbance is recorded.
Fourth, standard curve making
1) Taking out 0.1mg/mL Bovine Serum Albumin (BSA), and melting at room temperature for later use;
2) and 21 7mL centrifuge tubes are taken, and 3 centrifuge tubes are in a group and are respectively marked with 0ug,5.0ug,10.0ug,20.0ug, 40.0ug and 50.0 ug.
3) The reagents were added to each tube according to the following table:
4) adding Coomassie brilliant blue, shaking, mixing, and standing for 2min
5) One sample is parallel to 3 samples, 200 mu L of the sample is absorbed on an enzyme label plate (with the wavelength of 595nm), the color comparison is rapidly completed according to the operation of an enzyme label instrument, and the absorbance is recorded.
Calculating the MCP concentration: and (4) taking the light absorption value as an independent variable and the MCP content of the standard solution as a dependent variable, and deriving a standard curve formula. And (4) bringing the measured light absorption value into a standard curve and multiplying the standard curve by the dilution multiple to obtain the MCP content in the sample.
Attention points
1) A 4-degree centrifugal machine, the temperature is adjusted to 4 degrees in advance,
2) the water bath kettle is opened to 100 ℃ in advance
Sixth, appendix III
I. Test reagent
A.0.25m NaOH solution: dissolving 10g of sodium hydroxide by using distilled water and fixing the volume to 1000 mL;
B.0.1mg/mL Bovine Serum Albumin (BSA);
C. coomassie brilliant blue solution (G250, 95% ethanol, 85% phosphoric acid solution, double distilled water)
Coomassie brilliant blue solution preparation (avoid light)
1) 100mg Coomassie brilliant blue G250 in 50mL 95% ethanol (ensuring adequate dissolution);
2) then 100mL of 85% phosphoric acid solution (w/v) is added, and the volume is fixed to 1L by double distilled water;
3) filtering, and storing at 4 deg.C in dark for 1 week.
Determination of ammonia nitrogen concentration
First, the working principle
The ammonia is converted into NH4+ ions in hydrochloric acid solution, and reacts with two molecules of phenol to form a color development reaction of indophenol colored complex (the phenol participating in the reaction can be generated by the action of sodium nitrosoferricyanide and sodium salicylate). The strong reducing substance has interference effect on color reaction and can be removed by oxidation with sodium hypochlorite.
Second, instrument and reagent preparation
1) Reagent (configuration in appendix one)
4.5ml of 0.2M HCl, 2.5ml of phenol solution and 2.0ml of sodium hypochlorite solution
2) Instrumentation and equipment
5ml centrifuge tube, 7ml centrifuge tube, 4 degree centrifuge, water bath, 5ml, 1ml, 200ul liquid-transfering gun, vortex instrument, enzyme-labeling instrument, and enzyme-labeling plate
Third, preparation before test
1) A7 ml centrifuge tube was prepared, and 1 tube was prepared for each sample.
2) 5ml centrifuge tubes were prepared, and 1 tube was prepared for each sample.
3) 1ml (2/sample), 200ul (1-2/sample) tips were prepared.
Four, NH3-N Experimental procedures
One) sample pretreatment
1) And 0.5g rumen fluid sample/chyme/feces sample was acidified by adding 4.5mL of 0.2M HCl.
2) And freezing and storing.
3) Thawed and centrifuged at 15000rpm for 15 minutes at 4 ℃.
Two) color development
1. Taking 0.05mL sample (rumen fluid or fermentation broth 0.01mL) (standard) in 7mL centrifuge (blank is 0.1M hydrochloric acid solution)
2) Adding 2.5ml of phenol solution while mixing; adding 2.0ml sodium hypochlorite solution and shaking up
3) Placing the test tube in a water bath at 95 ℃ for 5 min; 60 deg.C water bath for 10min
4) After taking out and cooling, 3 samples were taken in parallel, and 200. mu.L of the solution was pipetted and added to an ELISA plate (wavelength: 630nm)
5) The color comparison is rapidly completed according to the operation of the enzyme labeling instrument, and the absorbance is recorded
Three) standard curve
1) 0.6607g of ammonium sulfate (dried at 100 ℃ for 12 hours) are weighed out and dissolved in 0.1M hydrochloric acid and the volume is adjusted to 100ml, so as to obtain 100mM standard ammonia solution
2) Diluting with 0.1M hydrochloric acid to obtain 1, 2, 4, 6, 8mM standard ammonia solution
3) And obtaining a standard curve of the absorbance (Y) and the ammonia concentration (X) by colorimetry at 630nm
Attention points
1) A 4-degree centrifugal machine, the temperature is adjusted to 4 degrees in advance,
2) two water baths are needed and are respectively opened to 95 ℃ and 60 ℃ in advance
3) And during color development, the light is shielded as much as possible
Sixth, appendix one
Solution preparation
Phenol solution: 0.0125g of sodium nitroferricyanide is dissolved in 125ml of distilled water, then 33ml of phenol (90% W/V) is added and mixed well, and finally the solution is diluted to 250ml and stored in a brown bottle for later use.
Sodium hypochlorite solution: 1.25g NaOH was dissolved in 150ml distilled water, and 9.5g Na2HPO4 & 7H2O (12.65g Na2HPO4 & 12H2O) were added. After cooling, 12.5ml of sodium hypochlorite solution (5.25%) was added and the solution was made up to 250 ml. Filtered through quick filter paper and stored in brown plastic bottles.
Preparation of hydrochloric acid
0.02mol/L HCL solution: 0.18 ml of hydrochloric acid was measured and 100ml of water was slowly poured.
0.1mol/L HCL solution: 0.9 ml of hydrochloric acid was measured and 100ml of water was slowly poured.
0.2mol/L HCL solution: 1.8 ml of hydrochloric acid was measured and 100ml of water was slowly poured.
0.5mol/L HCL solution: 4.5ml of hydrochloric acid was measured and 100ml of water was slowly poured in.
1.0mol/L HCL solution: 9 ml of hydrochloric acid was measured and 100ml of water was slowly poured in.
Determination of volatile fatty acid concentration
First), GC (gas chromatography) working conditions
The instrument comprises the following steps: GC-14B type gas chromatograph (Shimadzu Japan)
A capillary column: NUKOLTM carpillary Column (Supelco); column No.34292-07B
30m×0.32mm×0.25μm film thickness
Gas chromatograph parameters: the chromatographic column adopts a capillary suction column, the column temperature is 130 ℃, the vaporization temperature is 180 ℃, a hydrogen ion flame detector is adopted, the detection temperature is 180 ℃, the carrier gas is nitrogen, the pressure is 60KPa, the hydrogen pressure is 50KPa, the oxygen pressure is 50KPa, the sensitivity (grade) is 101, and the attenuation is 3.0.
II), sample preparation
1. Preparation of reagents
(1) Preparing a 25% w/v metaphosphoric acid solution, dissolving 25g metaphosphoric acid in 100mL double distilled water
(2) Crotonic acid was prepared by adding 0.6464g of crotonic acid to 100mL of metaphosphoric acid solution and making a volume of 100 mL. (80 ml of double distilled water is used firstly, metaphosphoric acid is dissolved by heating, and then the volume is determined to be 100ml)
(3) For a standard sample, 0.9100g of chromatographic standard grade acetic acid, 0.3700g of propionic acid, 0.1765g of butyric acid, 0.1765g of isobutyric acid and 0.1985g of valeric acid and isovaleric acid are accurately weighed, respectively dissolved in double distilled water and then respectively subjected to constant volume to 100 mL.
2. Sample preparation
(1) Taking 1ml sample of fermentation liquor (or filtering rumen fluid) for VFA measurement, adding 0.2ml of crotonic metaphosphate mixed solution, preserving overnight at-20 deg.C in a refrigerator, thawing, centrifuging at 12000rpm for 5min, taking supernatant for preservation, centrifuging at 12000rpm for 5min before measurement (or passing a little through a 0.22 μm needle filter, directly injecting the filtrate into a chromatograph by using a 1.0 microliter microsyringe instantly, and injecting the sample into the chromatograph with the sample amount of 0.2-1.0 μ L.
(2) And (3) measuring chyme and the feces by VFA, putting 1g of chyme and feces into a centrifugal tube, adding 5-10 times of double distilled water, and uniformly mixing. 1mL of the supernatant was added with 0.2mL/mL of crotonic metaphosphate. The other steps are the same as above.
(3) Determination of the Retention time
As with the sample treatment, 0.2mL of a mixture of metaphosphoric acid and crotonic acid was added to 1mL of the standard sample, and the retention times of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid were measured.
(4) Calculation of organic acid concentration
Relative correction factors of organic acids such as acetic acid, propionic acid and butyric acid can be calculated through respective (or concentration) and peak areas of the standard sample and the internal standard crotonic acid, and then the concentrations of the acetic acid, the propionic acid and the butyric acid in each sample can be calculated according to the weight (or concentration) of the acetic acid, the propionic acid and the butyric acid which are in direct proportion to the peak areas of the acetic acid, the propionic acid and the butyric acid.
Mass and converted concentration of volatile acid standard sample
(5) Reproduction ratio of organic acid
Continuously loading an organic acid (such as acetic acid) several times, measuring its peak area value, and calculating its reproduction rate by using the peak area value of the organic acid and the peak area value of the internal standard.
Coefficient of variation of acetic acid reproducibility: 1.2872 + -0.06404
Propionic acid reproduction rate coefficient of variation: 0.5104 + -0.01961
Coefficient of variation of butyric acid reproduction rate: 0.3422 + -0.01037
Coefficient of variation in the reproduction rate of isobutyric acid: 0.4668 + -0.01447
Coefficient of variation of valeric acid reproduction rate: 0.1431 + -0.01754
Coefficient of variation of isovaleric acid reproduction rate: 0.1671 + -0.03496
Molecular weight and time to peak of standards
Boiling point
Molecular weight
Time to peak
Crotonic acid
86.09
1.95min
Acetic acid
118℃
60.05
0.87min
Propionic acid
141℃
74.08
1.0min
Butyric acid
163.5℃
88.11
1.2min
Isobutyric acid
154.5℃
88.11
1.06min
Valeric acid
102.14
1.68min
Isovaleric acid
102.14
1.38min
Gas working conditions for gas chromatography (VFA measurement)
Pressure of pressure reducing valve of steel cylinder
Meteorological chromatographic pressure gauge
Hydrogen gas
0.14MPa
50KPa
AIR (AIR1)
0.4MPa
50KPa
Nitrogen (ARRIER2)
0.4MPa
60KPa
PRIMARYCARRIER(P)
300KPa
The 5 phytosterol formulations used in the experiments, all having a total phytosterol content of > 90%, are shown in table 1 and are labeled as sterols a-E, respectively.
TABLE 1 ingredient Table for phytosterol products of different formulations
Each sterol sample is subjected to rumen fluid in-vitro fermentation in 3 gradient concentrations. The 3 concentration gradients are 2 mug, 6 mug, 12 mug/bottle respectively, 99.18 parts of total sterol, wherein: 79.71 parts of beta-sitosterol, 7.44 parts of campesterol and 0.10 part of stigmasterol are used as test groups. The fermentation substrate was 600mg of total mixed ration.
Preparing an artificial rumen culture solution by referring to a method of Theodorou and the like, mixing rumen fluid filtered by four layers of gauze and an artificial rumen nutrient solution in a volume ratio of 1:5, subpackaging the total volume of 60ml in a 120ml fermentation bottle, and culturing at a constant temperature of 39 ℃ for 24 hours. The gas production was measured for 3, 6, 9, 12, 24h, respectively. And (3) immediately measuring the pH value of the fermentation liquor after the fermentation is finished, and freezing and storing the fermentation liquor at-20 ℃ for measuring the contents of microbial protein, lactic acid, ammonia nitrogen and volatile fatty acid. The fermentation substrate is dried at 65 ℃ and weighed for calculating the degradation rate of the dry matter of the substrate.
Index measurement:
according to the method of Menke and Steingass (1988) and the like for measuring the gas production rate and the degradation rate of the dry matter of the substrate, according to the method of feed analysis and feed quality detection technology, the solid-liquid separation of the substances in the fermentation bottle is carried out, the residue in the fermentation bottle is washed and mixed with the corresponding sample, then the mixture is dried for 48 hours at 65 ℃, weighed and the degradation rate of the dry matter of the sample is calculated according to the difference method. The method for measuring the lactic acid concentration of the culture solution after the completion of the in vitro fermentation refers to a Barker and Summerson method, the method for measuring the MCP concentration refers to a Makkar method and the like, the method for measuring the NH3-N concentration adopts a Broderick and the like phenol-sodium hypochlorite colorimetric method, and the method for measuring the volatile fatty acid refers to a Qinhelin method.
Experimental results and analysis:
1. effect of different component phytosterols on substrate disappearance
As can be seen from fig. 1, at the end of fermentation, the lactic acid concentration in the test group to which phytosterol was added was decreased compared to the control group, and the concentration had a significant effect on the lactic acid concentration (P < 0.05); when phytosterol was added at 2. mu.g, the lactic acid concentration was significantly lower than 6. mu.g and 12. mu.g (P < 0.05). The sterol component and concentration has no significant interaction with the lactic acid concentration of the fermentation broth (P > 0.05).
2. Effect of different Components phytosterols on fermentation gas production
The effect of different component phytosterols on rumen in vitro fermentation gas production is shown in fig. 2 to 6. The accumulated gas production rate is in a gradually rising trend along with the prolonging of the fermentation time. The influence of sterol concentration on gas production at each time point is significant (P <0.05), wherein 2 mu g of total gas production is the highest, 12 mu g of total gas production is the lowest, and components and concentrations have significant interaction on the total gas production of in vitro fermentation (P < 0.05). The gas production of each group at each time point is higher than that of the control group, when the fermentation is terminated for 24h, the gas production of the B-2 group is the highest and reaches 57.33mL, and the gas production of the control group is the lowest and is 38.07 mL.
3. Effect of different component phytosterols on pH value of fermentation liquor, ammoniacal nitrogen, microbial protein and dry matter degradation rate
Table 2 shows the influence of the phytosterol addition on various indexes of rumen in vitro fermentation, and it can be seen from the table that the pH of the fermentation broth in the test group is lower than that in the control group, and the pH of each group is kept within a normal range. The sterol concentration had a significant effect on pH change (P <0.05), highest in the control group and lowest at 12 μ g. The sterol component and concentration did not interact significantly with the broth pH (P > 0.05). The sterol species has significant influence on the ammonia nitrogen concentration of the fermentation liquor (P <0.05), the D group has the highest concentration, the A group is the second, the B group is the lowest and is significantly lower than the two groups (P < 0.05). The sterol component and concentration has no significant interaction with the ammonia nitrogen concentration of the fermentation liquor (P > 0.05). When the fermentation is carried out for 24 hours, the influence of the sterol concentration on the MCP concentration of the fermentation liquor is obvious (P is less than 0.05); MCP concentration was significantly higher than 2 μ g (P <0.05) with the addition of phytosterols at 6 μ g and 12 μ g. The sterol component and concentration has significant interaction with the fermentation broth MCP concentration (P < 0.05). From the DMD comparison, it can be seen that the sterol component has a significant effect on fermentation broth DMD (P <0.05), with group a being the highest followed by group B. The influence of sterol concentration on fermentation liquor DMD is obvious (P < 0.05). The sterol component and concentration had significant interaction with fermentation broth DMD (P < 0.05).
Table 2 effect of phytosterols on rumen fermentation parameters
4. Effect of different Components phytosterols on microbial metabolites
The effect of different component phytosterols on the concentration of VFA fermented in vitro by rumen is shown in table 3. The sterol concentration has a significant effect on total Volatile Fatty Acids (VFA), acetic acid, butyric acid and the ratio of ethylene to propylene (P <0.05), wherein 2 μ g of the group TVFA and butyric acid are significantly higher than the other concentrations and the control group (P <0.05), and the ratio of ethylene to propylene is significantly higher than the control group (P < 0.05). The sterol component can also significantly affect the ethylene-propylene ratio (P <0.05), with the highest being group D, significantly higher than the lowest group B (P < 0.05). The components and concentrations only had significant interaction with the ethylene-propylene ratio (P < 0.05).
TABLE 3 Effect of phytosterols on rumen in vitro fermentation of volatile fatty acids
5. Conclusion
The addition of phytosterol can improve the gas yield and the yield of volatile fatty acid of microbial fermentation, and simultaneously reduce the yield of lactic acid. The addition amount of phytosterol has a greater effect on rumen fermentation than the sterol component. When the phytosterol B is added into 2 mu g/bottle, the promoting effect on rumen fermentation is most obvious.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.