CN113341059B - Bionic digestion method for growing pig stomach-small intestine-large intestine and application of bionic digestion method to estimation of effective value of feed - Google Patents
Bionic digestion method for growing pig stomach-small intestine-large intestine and application of bionic digestion method to estimation of effective value of feed Download PDFInfo
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Abstract
The invention discloses a method for simulating the stomach-small intestine-large intestine digestion process of a growing pig and applying the method to the determination of the effective energy value of feed. The method comprises the following steps: crushing and sieving a feed sample; preparing gastric buffer, small intestine buffer, large intestine buffer, simulated gastric fluid (containing pepsin), concentrated simulated small intestine fluid (containing amylase, trypsin, chymotrypsin), and concentrated simulated large intestine fluid (containing amylase, trypsin, chymotrypsin and cellulase); loading the crushed feed samples into simulated digestion tubes of a monogastric animal bionic digestion system, and respectively adding simulated gastric juice into the simulated digestion tubes in sequence through computer program control to perform gastric simulated digestion; adding concentrated simulated small intestine liquid to perform small intestine simulated digestion; adding concentrated simulated large intestine liquid to perform large intestine simulated digestion; and after digestion is finished, cleaning is carried out, after undigested residues are obtained, the total energy value of the residues and the feed sample is measured, and the effective energy value of the feed is calculated. The method overcomes the defect that the large intestine digestion stage in the traditional in-vitro simulated digestion method only contains cellulase and lacks amylase, trypsin and chymotrypsin with certain active concentration in large intestine liquid in pigs, so that the measured value is more similar to the digestion energy and metabolic energy measured value of an animal test method, and has the advantages of higher correlation and lower estimation deviation.
Description
Technical Field
The invention belongs to the field of livestock raising, relates to a method for simulating the gastrointestinal digestion process of a growing pig, and in particular relates to a method for rapidly estimating the effective energy value of the feed by simulating the digestion process of the stomach, small intestine and large intestine of the growing pig in vitro.
Background
Over the last 50 years, animal nutritionists have proposed a number of in vitro digestion models in simulating the digestion process of growing pigs' stomach-small intestine-large intestine and have attempted to rapidly estimate the effective value of pig feed by simulated digestion techniques. The pepsin-pancreatic juice-fiber carbohydrate enzyme simulation of growing pigs by danish scholars Boisen and Fernandez in 1997 to estimate the effective value of pig feed has been the most widely used method of pig in vitro digestion in the field of global animal science (Boisen, s., and j.a. fern randez 1997.Prediction of the total tract digestibility of energy in feedstuffs and pig diets by in vitro analyses. Animal Feed Science and Technology 68:277-286). The method is applied to construction of the database of effective energy values of the raw materials of the growing pig feed in Danish and France. However, the absolute value of the energy digestibility of the pig feed raw material estimated by the simulated digestion method and the energy digestibility of the growing pig in vivo can be different by 4.4% -9.7%. Measurement of the Dry matter digestibility ratio of distillers' grains over Dry corn by simulating the growth of pig stomach-small intestine-large intestine digestion Process by pepsin-pancreatic juice-feces extract fermentation at university of Minnesota, huang et al, USA The digestibility of the dry matter in the whole digestive tract of pigs is 7.6% higher, and the correlation is very low (R 2 =0.02; huang Z., P.E.Urriola, I.J.Salfer, M.D.Stern, G.C.Shurson.Differences in in vitro hydrolysis and fermentation among and within high-fiber ingredients using a modified three-step procedure in growing pigs. Journal of Animal Science,2017, 95:5497-5506). The test results of the method show that the in vitro digestion degree in the in vitro simulated growing pig digestion is greatly different from the in vivo digestion degree of the growing pig. The main reason is 1) that the degradation of digestive enzyme activity in the small intestine stage in the simulated digestion process causes the sharp reduction of the digestive enzyme activity, so that the digestive enzyme activity in the small intestine of the growing pig cannot be matched; 2) In the simulated digestion of the large intestine, single cellulase or fiber hydrolase cannot be adopted to represent the activities of digestive enzymes in the blind colon, such as amylase, trypsin and chymotrypsin besides cellulase, and the inoculation fermentation of the fecal extract cannot be adopted to standardize the types and the quantity of the living bacteria, so that the deviation between the simulated digestion process of the large intestine and the digestion process of the growing pig large intestine is larger; 3) Buffer composition, digestion time, and washing process after digestion of in vitro stomach-small intestine-large intestine simulated digestion result in a mismatch between the extent of digestion and the extent of digestion in vivo. The invention patent 'program-controlled pig bionic digestion system and method for rapidly determining pig feed digestion energy by using the same' (Zhao Feng, et al, application number 201910360566.8) disclosed by the team is only applicable to estimation of pig digestion energy values of cereal feed raw materials, and the measurement values of high protein and high fiber raw materials and feeds are obviously lower than those of in-vivo methods because the attenuation of digestive enzymes in the small intestine simulated digestion stage and the concentrated simulated large intestine liquid consisting of low-dose cellulase are not considered in the large intestine simulated digestion stage. Therefore, there are drawbacks in the application range. Next, in operation, the technical scheme provided by the invention patent (Zhang Tieying et al, patent number ZL 200810100818.5) entitled "in vitro digestion simulator for broiler chickens" by the national academy of agricultural sciences of livestock research: the feed is added into a stomach simulated digester of the broiler chickens, and chyme is transferred into a small intestine digestion chamber to continue digestion after digestion is finished. Boisen and Fernandez (1997) simulated in vitro pig feed digestion processes Is characterized by comprising the following technical scheme: 0.5g of feed is added into a triangular flask, pepsin solution is added to simulate gastric digestion, pH is adjusted to 2.0, pancreatic juice solution is added to simulate small intestine digestion, pH is adjusted to 6.8, carbohydrate complex enzyme is added to simulate large intestine digestion, and finally sulfosalicylic acid is used for precipitating undigested protein, and filter paper is used for filtering and separating hydrolysate and unhydrolyzed product. The whole operation process is manual operation, the process is complicated, and human interference factors are large, so that the standardization of the method and the comparability of measured data are not facilitated. In the automation of the in-vitro simulated digestion process, the invention of the national academy of agricultural sciences Beijing livestock veterinarian research institute is named as a monogastric bionic digestion system and a method for simulating the digestion of monogastric animals based on the system (Zhao Feng and the like, and the patent number is ZL 200910078147.1) provides the technical scheme that: the full-automatic simulation of the digestion process of the stomach, the small intestine and the large intestine of the monogastric animal is realized through computer program control, and meanwhile, the commercialized full-automatic bionic digestion system product is formed through patent permission. However, the technical solution provided by the patent is only an automatic control process of gastric-small-large intestine simulated digestion, but lacks basic parameters such as simulated digestion liquid and buffer liquid components for simulating the digestion of the stomach, the small intestine and the large intestine of a growing pig, digestion time of each stage, cleaning of products and the like, and does not have comparison of the simulated digestion measurement result with the measurement result of a pig in-vivo method. Therefore, accurate simulation of the digestion process in pigs cannot be achieved.
At present, the global cultivation industry is developing to the direction of accuracy and high efficiency. Facing the challenge of global resource starvation, how to rapidly and accurately determine the nutrient utilization efficiency, especially the effective value of feed has become a technology of urgent need in industry. The prior art and the prior art are difficult to realize accurate measurement of effective value of pig feed in laboratory, and form a technical proposal with little artificial interference and easy standardization in the measurement process. Therefore, establishing a method for overcoming the above drawbacks, realizing high fidelity and automatic simulation of the stomach-small intestine-large intestine digestion process of growing pigs and applying the method to the estimation of the effective efficiency of feed is a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a method for fully automatically simulating the stomach-small intestine-large intestine digestion process of a growing pig and accurately estimating the effective energy value of feed, which has high fidelity. The method has the advantages that the activity of digestive enzymes after stable attenuation in the gastric-small-large intestine simulated digestion process is approximate to the activity of digestive enzymes in a growing pig body, and especially, the activities of amylase and protease in addition to cellulase in the large intestine simulated digestion also reach the level of the digestive enzymes of large intestine fluid in the body. The method can solve the defects that the activity of digestive enzymes is lower than that of digestive enzymes in the small intestine in vivo due to activity attenuation in simulated digestion of the small intestine and the activities of amylase and protease in simulated digestion of the large intestine are inconsistent with those of the large intestine in vivo, and overcomes the problems of large deviation and low correlation of the measured value of the traditional in vitro digestion method and the measured value of the animal test method.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for estimating the effective energy (digestive energy and metabolic energy) value of a feed by simulating the gastrointestinal tract digestion process of a growing pig, comprising the steps of:
1) Sample treatment and preparation of simulated digestive fluid and buffer solution:
sample treatment: crushing a pig feed sample to pass through a test sieve with the aperture of 0.30mm, sealing in a sample bag, sealing and storing the sample bag for standby;
preparation of simulated gastric fluid: according to the activity of pepsin in gastric juice in pigs, pepsin powder is dissolved in hydrochloric acid solution with pH of 2.0 to prepare simulated gastric juice;
preparation of gastric buffer: preparing a gastric buffer solution by sodium chloride, potassium chloride, dilute hydrochloric acid and deionized water;
preparation of concentrated simulated intestinal fluid: preparing concentrated simulated small intestine solution by using alpha-amylase, trypsin, chymotrypsin and deionized water according to the activity of main digestive enzymes in the pig body empty intestine solution;
preparation of small intestine buffer: dissolving anhydrous disodium hydrogen phosphate, anhydrous sodium dihydrogen phosphate, sodium chloride, potassium chloride and penicillin in deionized water to prepare a small intestine buffer solution;
preparation of concentrated simulated intestinal juice: according to the activities of amylase, trypsin, chymotrypsin and cellulase in the pig body intestinal juice, dissolving the amylase, trypsin, chymotrypsin and cellulase in deionized water to prepare concentrated simulated intestinal juice;
Preparation of large intestine buffer: the large intestine buffer solution is prepared by dissolving anhydrous disodium hydrogen phosphate, anhydrous sodium dihydrogen phosphate, sodium chloride, potassium chloride and penicillin in deionized water.
2) Preparing and loading:
the gastric buffer solution, the small intestine buffer solution and the large intestine buffer solution are put in the corresponding positions of the bionic digestive system of the monogastric animal, and the pipeline of the system is connected with a buffer solution bottle; setting the preheating time of the monogastric animal bionic digestive system to be more than 40 minutes; during preheating of the monogastric bionic digestive system, the following loading operations were performed: and (2) traversing the dialysis bag through a simulated digestion tube in the monogastric animal bionic digestion system, everting two ends of the simulated digestion tube, fastening the dialysis bag by using rubber bands, fixing the dialysis bag on the simulated digestion tube, then tightly plugging one end of the dialysis bag by using a port-turning silica gel plug, weighing the pig feed sample treated in the step (1), placing the pig feed sample in the simulated digestion tube with the dialysis bag, and synchronously measuring the dry matter content of the pig feed sample.
3) Gastric simulated digestion:
adding the simulated gastric fluid in the step 1) into the dialysis bag, and tightly plugging the other end of the simulated digestive tract by using a turnover silica gel plug with a digestive fluid adding pipe; placing the simulated digestive tract in a simulated digestive system of a monogastric animal, and connecting a pipeline according to the principle that water is fed from the lower end of the simulated digestive tract and water is discharged from the upper end of the simulated digestive tract; the simulated gastric digestion parameters are set through the control software of the simulated gastric digestion system of the monogastric animal, and the simulated gastric digestion is started.
4) Simulated digestion of the small intestine:
and after the gastric simulated digestion is finished, the concentrated simulated intestinal fluid is transferred into a digestive fluid storage chamber of the monogastric animal bionic digestion system, small intestine simulated digestion parameters are set through monogastric animal bionic digestion system control software, the concentrated simulated intestinal fluid is automatically pumped into a digestive tube, and small intestine simulated digestion is started.
5) Simulated digestion of large intestine:
and when the simulated digestion of the small intestine is finished, the concentrated simulated large intestine liquid is moved into a digestive liquid storage chamber of the simulated digestion system of the monogastric animal, large intestine simulated digestion parameters are set through control software of the simulated digestion system of the monogastric animal, the concentrated simulated large intestine liquid is automatically pumped into a digestive tube, and the simulated digestion of the large intestine is started.
6) And (3) cleaning hydrolysis products:
and after the simulated digestion of the large intestine is finished, emptying the large intestine buffer solution, and cleaning the obtained hydrolysate.
7) Treatment of undigested residue and calculation of digestibility:
transferring undigested residues in the dialysis bag after the washing in the step 6) into a culture dish, drying, washing the residues with absolute ethyl alcohol, measuring the total energy value of the residues and pig feed samples after absolute drying, and calculating to obtain the enzyme hydrolysate energy value of the feed to be detected.
8) The effective energy (digestion energy, metabolism energy) of the feed to be tested is estimated by the enzyme hydrolysate energy value of the feed to be tested.
In the step 1) of the method, the pig feed is single feed, compound feed or concentrated feed; the single feed is selected from rapeseed meal, cottonseed meal, sunflower meal and peanut meal; the compound feed can be corn-bean pulp type feed, rapeseed pulp type feed, corn-bean pulp type feed, cottonseed pulp type feed, corn-bean pulp type feed, sunflower pulp type feed, corn-bean pulp type feed, peanut pulp type feed, etc. The pulverization is carried out with a plant pulverizer or a mortar.
In the method step 1), the activity of pepsin in the simulated gastric fluid is 890U/mL.
In the step 1), sodium chloride and potassium chloride are dissolved in deionized water, and the pH of the solution is adjusted to 2.0 by using 2mol/L hydrochloric acid at 39 ℃, wherein the concentration of sodium chloride is 80.6mmol/L, and the concentration of potassium chloride is 6mmol/L.
In method step 1) above, the concentrated simulated intestinal fluid contains amylase 4239U/mL, trypsin 1323U/mL, chymotrypsin 166U/mL.
In the step 1) of the method, the small intestine buffer solution contains 30mmol/L disodium hydrogen phosphate, 170mmol/L sodium dihydrogen phosphate, 89.9mmol/L sodium chloride and 15.0mmol/L potassium chloride, and the pH value of the solution is adjusted to 6.44 by 1mol/L sodium hydroxide at 39 ℃.
In the method step 1), the concentrated simulated intestinal fluid contains 1572U/mL amylase, 491U/mL trypsin, 62U/mL chymotrypsin and 0.77U/mL cellulase.
In the step 1) of the method, the large intestine buffer solution contains 30mmol/L disodium hydrogen phosphate, 170mmol/L sodium dihydrogen phosphate, 93.2mmol/L sodium chloride and 11.1mmol/L potassium chloride, and the pH value of the solution is adjusted to 6.42 by 1mol/L sodium hydroxide at 39 ℃.
In the step 2), the monogastric bionic digestive system may be a monogastric bionic digestive system produced by the intelligent technology development limited company in Hunan, and the model number is as follows: SDS-2 or SDS-3.
The molecular weight cut-off of the dialysis bag is 12000-14000 daltons.
The dialysis bag is subjected to the following treatment before use: cutting the dialysis bag into small sections of about 25cm, boiling the dialysis bag in 2000mL of 2% (W/V) sodium bicarbonate and 1mmol/L disodium ethylenediamine tetraacetate solution (pH 8.0) for 10 minutes, and thoroughly cleaning the dialysis bag with distilled water; boiling in 1mmol/L disodium diamine tetraacetate solution (pH 8.0) for 10 min, cooling, storing in refrigerator at 4deg.C for use, filling water in dialysis bag before use, discharging, and thoroughly cleaning the dialysis bag.
In the step 2), the addition amount of the gastric buffer, the small intestine buffer and the large intestine buffer is 1000mL/5 digestive tract.
In the step 2) of the method, the loading amount of the pig feed sample is 1-2 g/root of digestive tract.
In the method step 3), the addition amount of the simulated gastric fluid is 20 mL/root of digestive tract.
In the step 3) of the method, the number of the simulated digestion pipes is 5, the simulated digestion pipes are connected in series, and the digestive juice adding pipes are connected with the system sequentially through quick connectors.
In the above method step 3), the parameters of the gastric simulated digestion are: the temperature is 39 ℃, the gastric buffer flow rate is 60mL/min, the digestion time is 3h, and the residual liquid is discharged for 3-4 min.
In the method step 4), the addition amount of the concentrated simulated small intestine solution is 2 mL/root of digestive tract.
In the above method step 4), the parameters of the simulated digestion of the small intestine are: the temperature is 39 ℃, the flow rate of the small intestine buffer solution is 60mL/min, the digestion buffer period of the small intestine is 1h, the digestion time of the small intestine is 5h, and the residual liquid is discharged for 3-4 min.
In the method step 5), the addition amount of the concentrated simulated intestinal fluid is 2 mL/root of digestive tract.
In the above method step 5), the parameters of the simulated digestion of the large intestine are: the temperature is 39 ℃, the flow rate of the large intestine buffer solution is 60mL/min, the digestion time of the large intestine is 21h, and the residual liquid is discharged for 3-4 min.
In the method step 6), the washing is performed for 4 hours with 1500mL deionized water each time.
In the step 7), the undigested residues are transferred into a culture dish with constant weight, dried at 65 ℃ until no water mark exists, and then dried at 105 ℃ until the weight is constant; scraping all digestion residues in the culture dish, transferring the digestion residues into an absolute dry glass sand crucible, and flushing the digested residues with 30-40 mL of absolute ethyl alcohol for 3-4 times.
In the step 8), the estimation of the digestion energy of the feed to be tested by the enzyme hydrolysate energy value of the feed to be tested is obtained by substituting the enzyme hydrolysate energy value of the feed to be tested into a regression model of the enzyme hydrolysate energy value of the feed measured by a bionic digestion method and the digestion energy of pigs measured by an in vivo digestion method.
The estimation of the metabolic energy of the feed to be detected through the enzyme hydrolysate energy value of the feed to be detected is calculated by substituting the enzyme hydrolysate energy value of the feed to be detected into a regression model of the feed enzyme hydrolysate energy value measured by a bionic digestion method and the pig metabolic energy measured by an in vivo digestion method.
The "energy value of feed enzyme hydrolysate measured by biomimetic digestion method" mentioned above is the energy value of feed enzyme hydrolysate measured by the method of steps 1) to 7) in the method of the present invention.
The feed is a single feed (such as rapeseed meal, cottonseed meal, sunflower meal and peanut meal), and the Digestion Energy (DE) of the feed to be tested is calculated by bringing the enzyme hydrolysate energy value (EHGE) of the feed to be tested into the following equation: de= 0.9896 ×ehge+160; wherein EHGE represents the enzyme hydrolysate energy value and DE represents the digestion energy; the Metabolic Energy (ME) of the feed to be tested is calculated by taking the enzyme hydrolysate energy value of the feed into the following equation: me= 0.9290 ×ehge+8; wherein EHGE represents enzyme hydrolysate energy value and ME represents metabolic energy;
The feed is compound feed or concentrated feed; the digestion energy is calculated by bringing the enzyme hydrolysate energy value of the feed to be tested into the following equation: de= 0.8851 ×ehge+539; wherein EHGE represents the enzyme hydrolysate energy value and DE represents the digestion energy; the metabolic energy is calculated by bringing the enzyme hydrolysate energy value of the feed to be tested into the following equation: me= 0.9643 ×ehge+113; wherein EHGE represents the enzyme hydrolysate energy value and ME represents the metabolic energy.
According to the variation condition of main digestive enzyme activity of the digestive tract of a growing pig and the attenuation rule of the digestive enzyme activity in-vitro digestion, the invention designs a typical enzyme spectrum and a corresponding buffer system for simulating the hydrolysis environment in the digestive tract of a three-way hybrid growing pig (30-60 kg, dux length x big). According to the design principle of the full-automatic bionic digestive system, simulated gastric juice, concentrated simulated small intestinal juice, concentrated simulated large intestinal juice and corresponding buffer solutions are designed. The feed is subjected to in vivo digestion process of a growth pig of which the simulated size is 30-60 kg in a dialysis tube (the molecular weight cut-off is 12000-14000 daltons) in a digestive tube of a bionic digestive system of a monogastric animal at 39 ℃. During digestion, the hydrolysates are automatically separated by dialysis tubing. Finally, the digested residue is dried and defatted, and the total energy content of the residue is determined. At the same time, the total energy content of the sample is determined. The enzyme hydrolysate energy value of the feed was measured by differential method. The method is suitable for measuring the energy value of the enzyme hydrolysate of the compound feed, the concentrated feed and the feed raw material of the growing pigs.
Compared with the prior art, the invention has the following advantages:
1. in the gastric simulated digestion stage, the absorption effect of the feed sample on pepsin is considered, and the pepsin activity in the gastric simulated digestion stage reaches the pepsin activity value in the growing pig body by improving the initial activity of pepsin in simulated gastric juice.
2. In the simulated small intestine digestion stage, concentrated simulated small intestine liquid is prepared according to the attenuation rule of amylase, trypsin and chymotrypsin activities in bionic digestion. After the concentrated simulated intestinal fluid is automatically injected into the dialysis tube through the peristaltic pump in the initial section of simulated digestion of the small intestine, the variation of the digestive enzyme activity in the simulated digestion process of the small intestine is consistent with the variation of the digestive enzyme activity of the liquid phase digestive enzyme of the jejunum in the body.
3. In the simulated digestion stage of the large intestine, the concentrated simulated large intestine liquid contains amylase, trypsin and chymotrypsin besides cellulase. After the concentrated simulated large intestine liquid is automatically injected into the dialysis tube through the peristaltic pump in the initial section of the simulated large intestine digestion, the activity of digestive enzyme in the simulated large intestine digestion process is equal to the activity of digestive enzyme corresponding to the blind intestine liquid in the growing pig body.
4. The simulated digestion time of the feed in the stomach, the small intestine and the large intestine is within the passing time range of the feed in each section of the stomach, the small intestine and the large intestine of the growing pig of 30-60 kg, and the consistency of the enzymatic reaction time of the bionic digestion and the enzymatic reaction time in vivo is ensured.
5. The specific activity of digestive enzyme is used for forming, the ion concentration of buffer solution and electrolyte is used for defining simulated gastric fluid, concentrated simulated small intestinal fluid and concentrated simulated large intestinal fluid, so that the digestion capability of simulated digestive fluid prepared in different batches is ensured to be highly repeatable, a standardized simulated digestive fluid kit product can be formed, and the comparison of measurement data among users is facilitated.
6. In the bionic digestion process and after the digestion is finished, the hydrolyzed micromolecular product is washed out of the dialysis tube through a molecular sieve (12000-14000 daltons dialysis tube), and residues trapped in the dialysis tube are regarded as excrement similar to the excretion of growing pigs, so that the separation of digestible products and indigestible products is automatically realized.
7. The method for simulating the stomach-small intestine-large intestine digestion of the growing pigs, which is described by the technology, can fully automatically complete the simulated digestion process in the bionic digestion system of the monogastric animals, can reduce errors introduced by manual operation and greatly improves the reliability of the measurement result.
The invention has the beneficial effects that:
the invention provides a method for estimating feed digestive energy and metabolic energy by simulating the stomach-small intestine-large intestine digestion process of a growing pig. According to the attenuation law of pepsin, amylase, trypsin and chymotrypsin activities in bionic digestion and the activities of corresponding digestive enzymes in growing pigs, simulated digestive juice is prepared, so that the digestion capacity of the digestive juice of the feed in the bionic digestion is consistent with that of gastric juice, small intestinal juice and large intestinal juice in the pigs. Meanwhile, the simulated digestion time of the stomach, the small intestine and the large intestine in the bionic digestion is consistent with the residence time of chyme in the stomach, the small intestine and the large intestine of a growing pig, so that the approximation of the enzymatic reaction conditions of the feed in each stage of the simulated digestion in the bionic digestion and the digestion conditions in the growing pig is realized. In particular, in the simulated digestion of the large intestine, besides cellulase, the activities of amylase and protease also reach the level of the digestive enzyme activity of the large intestinal juice in the body. The method solves the problems that the activity of digestive enzymes is lower than that of digestive enzymes in small intestines in vivo due to activity attenuation in simulated digestion of small intestines and the activities of amylase and protease in simulated digestion of large intestines are inconsistent with that of large intestines in vivo, and the problems of large deviation and low correlation between the measured value of the traditional in vitro digestion method and the measured value of an animal test method are solved. Meanwhile, the method has the advantages of time saving, labor saving, rapidness and high repeatability of the measurement result, and can be used for measuring the digestion energy and the metabolic energy value of the pig feed in a large scale.
Drawings
Fig. 1 is a graph showing the estimation of the digestion energy and metabolic energy of pig feed and diet by the bionic digestion method of pig stomach-small intestine-large intestine.
FIG. 2 is a regression equation for enzyme hydrolysis energy value (EHGE) to Digestion Energy (DE) for 10 diets and 9 feed stocks as sample libraries, respectively.
FIG. 3 shows regression equations of enzyme hydrolysis energy value (EHGE) versus Metabolic Energy (ME) for 10 diets and 9 feedstocks, respectively, as sample libraries.
Detailed Description
The invention will be further illustrated with reference to the following specific examples, but the invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The starting materials are available from published commercial sources unless otherwise specified.
The following terms and definitions apply to the present invention:
1 feed always
GE 1 ＝E 1 ×M 1
Wherein:
GE 1 total energy of sample in card (cal)
E 1 -feed sample dry matter mass energy in cal/g;
M 1 -dry feed mass in grams (g).
2 residues always
GE 2 ＝E 2 ×M 2
Wherein:
GE 2 -total residue in card (cal);
E 2 -dry matter mass value of the defatted residue in cal/g;
M 2 -the dry matter of the defatted undigested residue of the feed to be tested is loaded in grams (g).
3 total dry matter of feed
GDM＝M 3 ×DM
Wherein:
GDM-total dry matter of sample in grams (g);
M 3 -sample air-dried mass in grams (g);
DM-dry matter content of air-dried feed sample in percent (%).
The measurement methods for the parameters involved in the following examples were as follows:
GB 3102.4 thermal quantity and unit
GB/T6682-2008 analysis laboratory water specification and test method
Determination of moisture and other volatile matter content in GB/T6435 feed
GB/T14699.1 feed sampling
Measurement of pepsin Activity, reference is made to Wirnt R.and Wolf-Peter F.Pepsin, measurement with haemoglobin as substrate. In. Bergmeyer H.methods of enzymatic analysis. Weinheinm: verlag chemie.1974,1046-1052
Measurement of amylase Activity, reference is made to Dahlqvist A.Amethod for the determination of amylase in intestinal content [ J ]. Scandinavian Journal of Clinical and Laboratory Investigation,1962,14:145-151
Measurement of trypsin Activity, reference is made to Wirnt R.Trypsin, measurement with n. Alpha. -p-tolutemulfonyl-l-arginine methyl ester as substrate. In. Bergmyer H. Methods of enzymatic analysis. Weinheinm: verlag chemie.1974,1013-1023
Measurement of chymotrypsin Activity, reference wirn R.Chromotyrpsin, measurements with n-benzoyl-l-tyrosin ethyl ester as substrate. In. Bergmeyer H. Methods of enzymatic analysis. Weinheim: verlag chemie.1974,1006-1012
Measurement of cellulase Activity reference NY/T912-2020, spectrophotometrically
ISO 9831:1998 determination of total energy of animal feed, animal products and faeces or urine-oxygen bomb calorimeter (Animal feeding stuffs, animal products, and faeces or urine-Determination of gross calorific value-Bomb calorimeter method)
The abbreviations involved in the examples below are defined as follows:
DM: a dry matter; CP: crude protein; ash: coarse ash; EE: crude fat; NDF: neutral washing the fiber; ADF: acid washing the fiber; GE: always.
The following examples relate to the following instruments and devices:
1. plant sample pulverizer or mortar 2, test screen: aperture 0.30mm (60 mesh)
3. Analytical balance: index value 0.0001g 4, pH meter: index value 0.01
5. Glass sand crucible (G) 4 40 mL) 6, petri dish: diameter of 90mm
7. And (3) a dryer: anhydrous calcium chloride or allochroic silica gel as drier
8. An electric heating constant temperature box 9 and an oxygen bomb calorimeter;
10 monogastric animal bionic digestive system (model: SDS-2 or SDS-3, manufactured by the present intelligent technology development Co., ltd. In Hunan).
The following examples relate to the following reagents and materials:
all reagents were analytically pure, except where noted.
The laboratory water should meet the specification of the tertiary water in GB/T6682-2008.
1. Pepsin was purchased from Sigma, catalog number P7000;
2. alpha-amylase was purchased from Sigma, cat# A3306;
3. trypsin was purchased from BBI company under product catalog number a600626;
4. chymotrypsin is purchased from BBI company under product catalog number a600307;
5. cellulases were purchased from Sigma under product catalog number V2010.
6. Dialysis bag: the molecular weight cut-off is 14000 daltons and is manufactured by Viskase company, USA, model MEMBRA-CEL MD 44-14. The pretreatment is as follows: the dialysis bag was cut into small sections of about 25 cm. The dialysis bag was boiled in 2000mL of 2% (W/V) sodium bicarbonate and 1mmol/L disodium edetate solution (pH 8.0) for 10 minutes. The dialysis bag was thoroughly rinsed with distilled water. Placed in 1mmol/L disodium diamine tetraacetate solution (pH 8.0) and boiled for 10 minutes. Cooling, and storing in a refrigerator at 4 ℃ for standby. (note: it must be ensured that the dialysis bag is always immersed in the solution after this, and that the dialysis bag must be gloved) the dialysis bag is filled with water before use, and then drained, and the dialysis bag is thoroughly cleaned.
The simulated digest and buffer used in the examples below were prepared as follows:
gastric juice (pepsin activity 890U/mL) was simulated: pepsin activity in pepsin (Sigma P7000) was determined according to the pepsin activity assay described by wirn and Wolf-Peter (1974). Then, according to the simulated gastric fluid pepsin concentration of 890U/mL, 222.5KU of pepsin was weighed and dissolved in 250mL of hydrochloric acid solution (pH was calibrated at 39 ℃) and stirred slowly until dissolved. Is prepared before use.
Concentrate simulated intestinal fluid (amylase 4239U/mL, trypsin 1323U/mL, chymotrypsin 166U/mL): the activity of the corresponding digestive enzyme in reagent grade amylase (Sigma A3306), trypsin (BBI A600626) and chymotrypsin (BBI A600307) was determined according to the alpha-amylase activity assay described by Dahlqvist (1962), the trypsin activity assay described by wirt (1974), and the chymotrypsin activity assay described by wirt (1974). Then, according to the activities of these three digestive enzymes in simulated intestinal fluid, amylase 84.779KU, trypsin 26.456KU and chymotrypsin 3.323KU were weighed (in amounts) and dissolved in 20mL deionized water, respectively, and stirred slowly until dissolved. Is prepared before use.
Gastric buffer (pH 2.0): 10.36g of sodium chloride and 0.98g of potassium chloride were weighed into a 2000mL beaker, dissolved with 1800mL of deionized water, and the pH of the solution was adjusted to 2.0 with 2mol/L hydrochloric acid (HCL) at 39 ℃. After cooling, the solution was transferred to a 2000mL volumetric flask and was fixed to volume with deionized water.
Small intestine buffer: 8.32g of anhydrous disodium hydrogen phosphate (Na) 2 HPO 4 ) 40.96g anhydrous sodium dihydrogen phosphate (NaH) 2 PO 4 ) 11.55g of sodium chloride (NaCl) and 2.45g of potassium chloride (KCl), 160 kiloU of penicillin. Put into a 2000mL beaker, added 1800mL deionized water for dissolution, and dissolved with 1mol/L phosphoric acid (H 3 PO 4 ) Or 1mol/L sodium hydroxide (NaOH) at 39℃to adjust the pH of the solution to 6.44. After cooling, the solution was transferred to a 2000mL volumetric flask and was fixed to volume with deionized water.
Concentrate simulated intestinal fluid (amylase 1572U/mL, trypsin 491U/mL, chymotrypsin 62U/mL, cellulase 0.77U/mL): the amylase (Sigma A3306) 31.440KU, trypsin (BBI A600626) 9.816KU, chymotrypsin (BBI A600307) 1.234KU, cellulase (Sigma V2010) 15U were weighed out and dissolved in 20mL deionized water and stirred slowly until dissolved. Do not heat up on the heating plate or overheat during preparation. Is prepared before use.
Large intestine buffer: 7.99g of anhydrous disodium hydrogen phosphate (Na) 2 HPO 4 ) 41.23g anhydrous sodium dihydrogen phosphate (NaH) 2 PO 4 ) 11.98g of sodium chloride (NaCl) and 1.82g of potassium chloride (KCl), 160 kiloU of penicillin. Put into a 2000mL beaker, added 1800mL deionized water for dissolution, and dissolved with 1mol/L phosphoric acid (H 3 PO 4 ) Or 1mol/L sodium hydroxide (NaOH) at 39℃to adjust the pH of the solution to 6.42. After cooling, the solution was transferred to a 2000mL volumetric flask and was fixed to volume with deionized water.
Example 1 evaluation of the effective efficacy value of feed by bionic digestion method of growing pigs
1 materials and methods
1.1 feed Material and test diet
9 miscellaneous meal feed raw materials are selected, wherein 1 rapeseed meal, 4 cottonseed meal, 2 sunflower meal and 2 peanut meal. The chemical compositions are shown in Table 1:
TABLE 1 sample sources and chemical composition contents (air drying basis,%)
In order to ensure that the content of CP (crude protein) in the test diet is not more than 21%, and the proportion of a sample to be tested in the test diet is close to 20%, the corn-soybean meal type basic diet and the test diet are prepared according to the requirement of NRC (2012) for growing pig Ca, P, vitamins and trace elements, and the compositions of the corn-soybean meal type basic diet and the test diet are as follows:
TABLE 2 test of diet composition and chemical composition content (air-dried basis,%)
1.2 design of experiments
The Digestion Energy (DE) and the Metabolism Energy (ME) of 1 basal diet and 9 test diets were measured by the total fecal and urinary method. The 20 growing pigs are divided into 2 groups of 10 pigs according to the body weight, each group is continuously subjected to 3 continuous test periods, 10 diet treatments are randomly distributed to each group in each period, and in the 3-period metabolic test, different diets are distributed to the same pig. A total of 6 observations were obtained for each treatment.
The determination of the simulated digestion dry matter digestibility (IVDMD) and enzyme hydrolysate energy value (EHGE) of 9 miscellaneous meal raw materials and 10 test diets adopts a single-factor completely random design, and each sample is repeated for 5 times, and each sample is repeated for 1 digestive tract.
1.3 determination of pig feed digestive and Metabolic energy
20 Dux long x big ternary hybrid castrated boars with initial weight of 41.7+/-2.6 kg are selected and bred in a single metabolism cage. The metabolic chamber environment is controlled according to the parameter requirements of NY/T388. The test pigs were acclimatized in the metabolism cages for 7d followed by a 3d transition period during which commercial feed was gradually transitioned to the test diet; the adaptation period of 5d is fed with test diet, followed by the fecal collection period of 5-7 d. Daily feed intake is supplied according to 4% of the body weight, and two meals of wet mix (the diet and water are mixed evenly according to the ratio of 7:3) are equally divided for feeding every day of 08:00 and 16:00. The water is freely drunk through the pressure nozzle type water fountain. In the test process, the starting time of the total excrement collection adopts color (indicator: fe 2 O 3 ) Color is determined. The feces are frozen and stored at the temperature of minus 20 ℃. Urine was collected by a time-to-time method, with a time of collection of 6d 8:00 to 11d 8:00 in the pilot stage, and urine was collected by placing a urine collection bucket containing 50mL of 3mol/L hydrochloric acid under the urine collection basin of the metabolic cage every day. Urine collected daily during the test was weighed, 20% of the total weight of urine per day was taken and placed in jars and stored at-20 ℃. After each period of test, the feces and urine are thawed at room temperature, and the feces or urine samples of the same pig in the same period on different collection days are mixed. The feces are dried and weighed at 65 ℃, the total energy is measured after crushing, and the total energy is directly measured by urine.
1.4 measuring enzyme hydrolysate energy value of diet and miscellaneous meal by computer program controlled bionic digestive system
The computer program-controlled bionic digestion system consists of four continuous processes of gastric simulated digestion, small intestine simulated digestion, large intestine simulated digestion and hydrolysate cleaning.
1.4.1 simulated digest and buffer:
the concentration of pepsin in simulated gastric fluid was 890U/mL and pH was 2.0 in hydrochloric acid. Gastric buffer, 80.6mmol/L sodium chloride, 6mmol/L potassium chloride, and pH adjusted to 2.0 with hydrochloric acid at 39 ℃.
The concentrated simulated intestinal fluid contains amylase 4239U/mL, trypsin 1323U/mL, chymotrypsin 166U/mL.
The small intestine buffer solution contains 30mmol/L disodium hydrogen phosphate, 170mmol/L sodium dihydrogen phosphate, 89.9mmol/L sodium chloride and 15.0mmol/L potassium chloride, and the pH of the solution is adjusted to 6.44 with 1mol/L sodium hydroxide at 39 ℃.
The concentrated simulated intestinal fluid contains amylase 1572U/mL, trypsin 491U/mL, chymotrypsin 62U/mL and cellulase 0.77U/mL.
The large intestine buffer solution contains 30mmol/L disodium hydrogen phosphate, 170mmol/L sodium dihydrogen phosphate, 93.2mmol/L sodium chloride and 11.1mmol/L potassium chloride, and the pH of the solution is adjusted to 6.42 with 1mol/L sodium hydroxide at 39 ℃.
1.4.2 sample preparation
1.4.2.1 sample collection samples were taken according to GB/T14699.1.
1.4.2.2 sample treatment
The sampled sample was divided into about 200g by a quarter method, and the sample was crushed by a plant crusher or mortar to pass through a test sieve (60 mesh) having a pore diameter of 0.30mm, and the crushed sample was sealed in a sample bag and stored as a sample.
1.4.3 measurement procedure
1.4.3.1 preparation and Loading
1.4.3.1.1 1000mL of gastric buffer, 1000mL of small intestine buffer and 1000mL of large intestine buffer are placed in the corresponding positions of a bionic digestive system (model: SDS-2 or SDS-3 of the intelligent technology development Co., ltd. In Hunan) of a monogastric animal, and the pipelines of the system are connected with a buffer bottle.
1.4.3.1.2 in the control software, the preheating time of the bionic digestive system of the monogastric animal is set to be 60 minutes. And after the parameters of all digestion stages are input, running a simulated digestion process.
1.4.3.1.3 the following loading was performed during preheating of the monogastric biomimetic digestive system.
1.4.3.1.4 the treated dialysis bag is passed across the simulated digestive tract, turned over at both ends and fastened by rubber bands, and secured to the simulated digestive tract. Then, a plug is plugged with a turned-over silica gel plug.
1.4.3.1.5 1-2 g of a feed sample (2 g of test diet or energy feed or 1g of protein feed, accurate to 0.0002 g) was weighed and placed in a simulated digestive tract fitted with a dialysis bag. The dry matter content of the samples was determined simultaneously.
1.4.3.2 simulation of pig gastrointestinal digestion
1.4.3.2.1 gastric simulated digestion
20mL of simulated gastric fluid was added to the dialysis bag.
The other end of the simulated digester is sealed by a turnover silica gel plug with a digestive juice adding pipe.
The simulated digester is placed in a simulated digestive system of a monogastric animal, and a pipeline is connected according to the principle that water is fed from the lower end of the simulated digester and water is discharged from the upper end of the simulated digester. Each group of 5 simulated digesters is connected in series. The digestive juice adding pipe is connected with the system by a quick connector in sequence.
In monogastric animal biomimetic digestive system control software, parameters of gastric stage simulated digestion are: the temperature is 39 ℃, the buffer solution flow rate is 60mL/min, the digestion time is 3h, and the residual liquid is discharged for 4min. Other control parameters are operated according to the instruction book.
1.4.3.2.2 intestinal simulated digestion
At the end of the gastric simulated digestion, 2mL of concentrated simulated intestinal fluid/root canal was accurately transferred into the digestive fluid reservoir of the monogastric animal biomimetic digestive system.
In the control software of the bionic digestive system of the monogastric animals, parameters of simulated digestion at the small intestine stage are as follows: the temperature is 39 ℃, the flow rate of the buffer solution is 60mL/min, the digestion buffer period of the small intestine is 1h, and the digestion time of the small intestine is 5h. The residue was drained for 4min. Other control parameters are operated according to the instruction book.
1.4.3.2.3 large intestine simulated digestion
At the end of the simulated small intestine digestion, 2mL of concentrated simulated large intestine fluid/root canal was accurately transferred into the digestive fluid reservoir of the simulated monogastric animal digestive system.
In monogastric animal bionic digestive system control software, parameters of simulated digestion at the large intestine stage are: the temperature is 39 ℃, the buffer solution flow rate is 60mL/min, the digestion time of the large intestine is 21h, and the residual liquid is discharged for 4min. Washing liquid is 1500 mL/time, each time washing is carried out for 4 hours, and the total washing is carried out for 6 times. Other control parameters are operated according to the instruction book.
1.4.3.3 treatment of digestion residues
After 1.4.3.3.1 digestion, the undigested residue in the dialysis bag was transferred without loss to a 90mm petri dish of known absolute dry weight (this process required removal of the dialysis bag from the simulated digester, and a longitudinal shearing rinse with deionized water).
1.4.3.3.2 after drying the petri dishes containing undigested residues at 65℃until no water mark is present (typically 8-10 h), the dishes are dried to constant weight at 105 ℃.
1.4.3.3.3 the digestate residue in the petri dish was scraped off completely, transferred to a glass sand crucible of known absolute dry weight, and the residue was rinsed 3 times (about 30-40 mL each time) with absolute ethanol, ensuring thorough mixing of the residue with ethanol until the filtrate was colorless. Simultaneously, the petri dish with the residue is placed in a constant temperature oven at 105 ℃ to be baked to constant weight.
1.4.3.3.4 the petri dishes from which undigested residue was scraped and the glass sand crucible with defatted undigested residue were placed in a incubator at 105℃and baked to constant weight.
1.4.3.3.5 the sample of the defatted undigested residue in the glass sand crucible was transferred to a sample weighing paper of an oxygen bomb calorimeter, and the weight of the air-dried sample was weighed. And simultaneously, placing the glass sand crucible with the degreasing residue residues in a constant-temperature oven at 105 ℃ to be baked to constant weight.
The above-mentioned bionic digestion process can be briefly described as follows:
the sample is crushed and sieved by a 0.3mm sieve, and 2g of energy feed or 2g of test diet or 1g of other feed raw materials and 20mL of simulated gastric fluid are added into a digestive tube of a bionic digestive system. Gastric buffer is circulated outside the dialysis tube for 3 hours and the buffer is emptied after the end. Then the small intestine buffer is pumped into the digestion tube and circulated outside the dialysis bag for 6 hours, wherein after the circulation is started for 1 hour, 2mL of concentrated simulated small intestine solution is injected into the dialysis bag of the digestion tube through a peristaltic pump to perform small intestine simulated digestion, and after the completion, the buffer solution is emptied. The large intestine buffer was then pumped into the digestive tract and circulated outside the dialysis bag for 21h. After the large intestine buffer solution is pumped into the digestion tube for 0.1h, 2mL of concentrated simulated large intestine is pumped into a dialysis bag of the digestion tube for large intestine simulated digestion, the buffer solution is emptied after the completion, the automatic cleaning of hydrolysis products is carried out for 6 times, and each time, 1500mL of deionized water is circulated for 4h. After completion of the biomimetic digestion, the residue was defatted 4 times with absolute ethanol 30-40 mL each time, and then dried in a forced air dryer at 105 ℃.
1.5 chemical analysis
Determining the moisture content of the diet and the manure sample by referring to a method of GB/T6435-2006; with reference to the method of ISO9831:1998, parr6400 fully automatic adiabatic oxygen bomb was used to determine total energy of diet, manure, urine samples. Wherein urine energy is measured as follows: 5mL of urine was removed with a pipette to a crucible containing filter paper, dried in a forced air drying oven at 65℃and total energy was measured with a Parr6400 fully automatic adiabatic oxygen bomb meter.
The dry matter content and total energy of the defatted undigested residue were calculated by differential method.
1. The dry matter mass and dry matter mass value of the defatted undigested residue are calculated according to formulas (1) and (2), respectively.
M 2 -the dry matter of undigested residues of the feed to be tested is loaded in grams (g);
W 0 -absolute dry weight of the empty petri dish in grams (g);
W 1 undigested residue + absolute dry weight of the dishThe unit is gram (g);
W 2 -scraping undigested residue and oven-dried weight of the dish in grams (g);
W 3 -oven dry weight of glass sand crucible in grams (g);
W 4 -defatted undigested residue + oven dry weight of glass sand crucible in grams (g);
W 5 -oven dry weight of residue + glass sand crucible in grams (g) after taking a defatted undigested residue sample for determining total energy;
W 6 -the defatted digestion residue air-dries the sample weight in grams (g);
E r -the defatted residue air-dries the sample total energy in calories per gram (cal/g);
2. the feed enzyme hydrolysate energy value (EHGE, in cal/g) basis and the air-dried basis were calculated according to the formulas (3) and (4), respectively.
Wherein:
EHGE-measured feed enzyme hydrolysate energy value in units of cal/g;
GE 1 -total feed loading in calories (cal);
GE 2 -total residue in card (cal);
M 1 -the dry matter of the feed in grams (g);
M 3 the mass of the air-dried feed is expressed in grams (g).
3. Repeatability of
Five replicates were weighed for each sample and the average was taken as the analysis result.
The number of significant digits of the analysis result is two digits after the decimal point.
The relative standard deviation is allowed to be less than or equal to 2%.
1.6 data calculation and statistical analysis
The basic statistics were analyzed using the MEANS module of SAS 9.4 and 75% (Q3) and 25% (Q1) quantiles of 6 replicates per treatment were calculated. iqr=q3-Q1; data outside the range of Q1-1.5 xIQR-Q3 +1.5 xIQR is eliminated as abnormal data. And the TTSET module is adopted to carry out T test on the measured values of the simulated digestion method and the biological method of the experimental diet and the feed raw material, and the P is less than 0.05 and has obvious difference. And (3) analyzing the correlation between the EHGE and the digestive energy and the metabolic energy by using a PROC CORR module, and establishing a regression equation of the digestive energy and the metabolic energy to the EHGE by using a PROC REG module.
2 results
2.1 test diet bionic digestion method measured value and in vivo measured value difference
TABLE 3 bionic digestion and in vivo methods for determining differences in dry matter digestibility and energy efficiency for 10 experimental diets
As can be seen from Table 3, the dry matter digestibility of the simulated digestion method of 10 diets is significantly lower than that of the in vivo method (79.88% -87.69% vs.82.18% -89.32%; P < 0.01). The ratio of the dry matter digestibility of the bionic digestion method to the in-vivo method is between 0.96 and 0.98. EHGE is significantly lower than DE (3497-3820 Kcal/kg DM vs.3623-3923 Kcal/kg DM; P < 0.01), but the EHGE and ME differences of 8 diets are not significant (3497-3792 Kcal/kg DM vs.3500-3781 Kcal/kg DM; P > 0.05). The ratio of EHGE to DE is between 0.96 and 0.98, and the ratio of ME is between 1.00 and 1.01. EHGE/GE was significantly lower than DE/GE (79.25% -87.65% vs.82.09% -89.21%; P < 0.01), but the EHGE/GE was not significantly different from ME/GE for 8 diets (79.25% -87.65% vs.79.31% -85.60%; P > 0.05).
2.2 bionic digestion of feed Material and in vivo determination of the energy efficiency differences
TABLE 4 bionic digestion method and in vivo method for determining the difference in energy efficiency of miscellaneous meal
Table 4 shows that EHGE of rapeseed meal, cottonseed meal 3, sunflower meal 1 is significantly lower than DE (P < 0.01), EHGE of cottonseed meal 2 is significantly higher than DE (P < 0.01), and EHGE of cottonseed meal 1, cottonseed meal 4, sunflower meal 2, peanut meal 1, peanut meal 2 is not significantly different from DE (P > 0.05). The EHGE of rapeseed meal, cottonseed meal 1, cottonseed meal 4, sunflower meal 2, peanut meal 2 was not significantly different from ME (P > 0.05), whereas the EHGE of cottonseed meal 2, cottonseed meal 3, sunflower meal 1, peanut meal 1 was significantly higher than ME (P < 0.05). The ratio of EHGE to DE is between 0.86 and 1.05, and the ratio of EHGE to ME is between 0.96 and 1.20.
The EHGE/GE of the rapeseed meal, the cottonseed meal 3 and the sunflower meal 1 is obviously lower than the DE/GE (P < 0.05), the EHGE/GE of the cottonseed meal 2 is obviously higher than the DE/GE (P < 0.01), and the EHGE/GE of the cottonseed meal 1, the cottonseed meal 4, the sunflower meal 2, the peanut meal 1 and the peanut meal 2 have no obvious difference from the DE/GE (P > 0.05). There was no significant difference between EHGE/GE of rapeseed meal, cottonseed meal 1, cottonseed meal 4, sunflower meal 2, peanut meal 2 and ME/GE (P > 0.05), whereas EHGE/GE of cottonseed meal 2, cottonseed meal 3, sunflower meal 1 and peanut meal 1 was significantly higher than ME/GE (P < 0.05).
2.3 bionic digestion method and in vivo method for determining variation coefficient of feed energy value
TABLE 5 coefficient of variation of simulated digestive energy and metabolic energy of EHGE and in vivo
Table 5 shows that the coefficients of variation of the 10 diets EHGE, DE and ME were between 0.22% and 0.65%,0.40% and 1.25% and 0.37% and 1.48%, respectively. The variation coefficients of the 9 miscellaneous meal raw materials EHGE, DE and ME are respectively between 0.51 percent and 1.91 percent, between 2.58 percent and 9.05 percent and between 4.25 percent and 8.62 percent.
2.4 correlation of simulated digestion and in vivo measurement of test diet and miscellaneous meal
Table 6 pearson correlation coefficients of simulated digestion and in vivo measurements of experimental diet and miscellaneous meal
* Representing significant correlation coefficient (P < 0.05)
Table 6 shows that the correlation coefficients of the simulated digestion dry matter digestibility and the in vivo dry matter digestibility, EHGE and DE, and EHGE and ME are 0.96 (P < 0.01), 0.97 (P < 0.01), and 0.98 (P < 0.01), respectively, in 10 diets. The correlation coefficients of EHGE/GE and DE/GE and ME/GE are 0.98 (P < 0.01) and 0.99 (P < 0.01), respectively. Among the 9 miscellaneous meal raw materials, the correlation coefficients of the dry matter digestibility by the bionic digestion method and the dry matter digestibility by the in vivo method, EHGE and DE, and EHGE and ME are 0.91 (P < 0.01), 0.92 (P < 0.01) and 0.91 (P < 0.01). The correlation coefficients of EHGE/GE and DE/GE and ME/GE are 0.93 (P < 0.01) and 0.92 (P < 0.01), respectively. The correlation coefficients of biomimetic and in vivo dry matter digestibility, EHGE and DE, and EHGE and ME were 0.97 (P < 0.01), 0.96 (P < 0.01), and 0.95 (P < 0.01), respectively, for all 19 samples. The correlation coefficients of EHGE/GE and DE/GE and ME/GE are all 0.97 (P < 0.01).
2.5 regression model of in vivo method measurement of test diet and feed raw material to bionic digestion method measurement
Fig. 2 is a regression equation for Digestion Energy (DE) for 9 feed stocks and 10 feed biomimetic digestion values (EHGE). As can be seen from fig. 2, the EHGE-to-DE regression equation of 9 miscellaneous meal-like materials is de= 0.9896 ×ehge+160 (R 2 ＝0.8432，RSD＝182Kcal/kg DM，P<0.01 A) is provided; the EHGE to DE regression equation for 10 diet was de= 0.8851 ×ehge+539 (R 2 ＝0.9411，RSD＝23Kcal/kg DM，P<0.01)。
FIG. 3 is a regression equation of simulated digestion energy (EHGE) versus Metabolic Energy (ME) for 9 feed stocks and 10 diets. As can be seen from fig. 3, the regression equation of the 9 miscellaneous meal-like raw materials EHGE to ME is me= 0.9388 ×ehge+3 (R 2 ＝0.8282，RSD＝182Kcal/kg DM P<0.01 A) is provided; regression equation of 10 diet EHGE versus ME was me= 0.9559 ×ehge+146 (R 2 ＝0.9697，RSD＝18Kcal/kg DM P<0.01)
TABLE 7 evaluation of feed raw materials and deviations of feeds DE and ME by bionic digestion EHGE
From the results, the method provided by the invention has high correlation (the correlation coefficient is more than 0.92) between the enzyme hydrolysate energy values of the feed raw materials and the feeds and the in-vivo digestion energy and the metabolic energy measured based on the simulated growth pig stomach-small intestine-large intestine digestion process of the monogastric animal bionic digestive system. The average value of the relative deviation of DE values of 9 feed raw materials and 10 feeds, which are estimated by a bionic digestion method, and the DE values measured by a pig metabolic test method is 1.9%. The relative deviation of ME values of 9 feed raw materials and 10 feeds estimated by a bionic digestion method and the ME values measured by a pig metabolic test method is 2.0 percent. The digestion energy and the metabolic energy value of the pig feed can be accurately estimated by a regression model through a bionic digestion method.
Claims (10)
1. A method for estimating the effective value of a feed for simulating the gastrointestinal digestion process of growing pigs, comprising the steps of:
1) Sample treatment and preparation of simulated digestive fluid and buffer solution:
sample treatment: crushing a pig feed sample to pass through a test sieve with the aperture of 0.30mm, sealing in a sample bag, sealing and storing the sample bag for standby;
preparation of simulated gastric fluid: according to the activity of pepsin in gastric juice in pigs, pepsin powder is dissolved in hydrochloric acid solution with pH of 2.0 to prepare simulated gastric juice;
preparation of gastric buffer: preparing a gastric buffer solution by sodium chloride, potassium chloride, dilute hydrochloric acid and deionized water;
preparation of concentrated simulated intestinal fluid: preparing concentrated simulated small intestine solution by using alpha-amylase, trypsin, chymotrypsin and deionized water according to the activity of main digestive enzymes in the pig body empty intestine solution;
preparation of small intestine buffer: dissolving anhydrous disodium hydrogen phosphate, anhydrous sodium dihydrogen phosphate, sodium chloride, potassium chloride and penicillin in deionized water to prepare a small intestine buffer solution;
preparation of concentrated simulated intestinal juice: according to the activity of cellulase in the pig body intestinal juice, dissolving amylase, trypsin, chymotrypsin and cellulase in deionized water to prepare concentrated simulated intestinal juice;
Preparation of large intestine buffer: dissolving anhydrous disodium hydrogen phosphate, anhydrous sodium dihydrogen phosphate, sodium chloride, potassium chloride and penicillin in deionized water to prepare a large intestine buffer solution;
the activity of pepsin in the simulated gastric fluid is 890U/mL;
the preparation of the gastric buffer solution comprises the steps of dissolving sodium chloride and potassium chloride in deionized water, and regulating the pH value of the solution to 2.0 by using 2mol/L hydrochloric acid at 39 ℃, wherein the concentration of the sodium chloride is 80.6mmol/L, and the concentration of the potassium chloride is 6mmol/L;
the concentrated simulated intestinal fluid contains amylase 4239U/mL, trypsin 1323U/mL and chymotrypsin 166U/mL;
the small intestine buffer solution contains 30mmol/L disodium hydrogen phosphate, 170mmol/L sodium dihydrogen phosphate, 89.9mmol/L sodium chloride and 15.0mmol/L potassium chloride, and the pH value of the solution is regulated to 6.44 by 1mol/L sodium hydroxide at 39 ℃;
the concentrated simulated intestinal fluid contains amylase 1572U/mL, trypsin 491U/mL, chymotrypsin 62U/mL and cellulase 0.77U/mL;
the large intestine buffer solution contains 30mmol/L disodium hydrogen phosphate, 170mmol/L sodium dihydrogen phosphate, 93.2mmol/L sodium chloride and 11.1mmol/L potassium chloride, and the pH value of the solution is regulated to 6.42 by 1mol/L sodium hydroxide at 39 ℃;
2) Preparing and loading:
The gastric buffer solution, the small intestine buffer solution and the large intestine buffer solution are put in the corresponding positions of the bionic digestive system of the monogastric animal, and the pipeline of the system is connected with a buffer solution bottle; setting the preheating time of the monogastric animal bionic digestive system to be more than 40 minutes; during preheating of the monogastric bionic digestive system, the following loading operations were performed: traversing the dialysis bag through a simulated digestion tube in the monogastric animal bionic digestion system, everting two ends of the dialysis bag, fastening the dialysis bag by using rubber bands, fixing the dialysis bag on the simulated digestion tube, then tightly plugging one end of the dialysis bag by using a port-turning silica gel plug, weighing the pig feed sample treated in the step 1), placing the pig feed sample in the simulated digestion tube provided with the dialysis bag, and synchronously measuring the dry matter content of the pig feed sample;
3) Gastric simulated digestion:
adding the simulated gastric fluid in the step 1) into the dialysis bag, and tightly plugging the other end of the simulated digestive tract by using a turnover silica gel plug with a digestive fluid adding pipe; placing the simulated digestive tract in a simulated digestive system of a monogastric animal, and connecting a pipeline according to the principle that water is fed from the lower end of the simulated digestive tract and water is discharged from the upper end of the simulated digestive tract; setting gastric simulated digestion parameters through control software of a monogastric animal bionic digestion system, and starting gastric simulated digestion;
In the step 3), the parameters of the gastric simulated digestion are as follows: the temperature is 39 ℃, the flow rate of the gastric buffer is 60mL/min, the digestion time is 3 h, and the residual liquid is discharged for 4min;
4) Simulated digestion of the small intestine:
when the gastric simulated digestion is finished, transferring the concentrated simulated intestinal fluid into a small intestine digestive fluid storage chamber of the monogastric animal bionic digestion system, setting small intestine simulated digestion parameters through monogastric animal bionic digestion system control software, automatically pumping the concentrated simulated intestinal fluid into a digestion tube, and starting small intestine simulated digestion;
the parameters of the simulated digestion of the small intestine are as follows: the temperature is 39 ℃, the flow rate of the small intestine buffer solution is 60mL/min, the digestion buffer period of the small intestine is 1h, the digestion time of the small intestine is 5 h, and the residual liquid is discharged for 4min;
5) Simulated digestion of large intestine:
when the simulated digestion of the small intestine is finished, transferring the concentrated simulated large intestine liquid into a digestive liquid storage chamber of the simulated digestion system of the monogastric animal, setting large intestine simulated digestion parameters through control software of the simulated digestion system of the monogastric animal, automatically pumping the concentrated simulated large intestine liquid into a digestive tube, and starting to perform simulated digestion of the large intestine;
the parameters of the simulated digestion of the large intestine are as follows: the temperature is 39 ℃, the flow rate of the large intestine buffer solution is 60mL/min, the digestion time of the large intestine is 21 h, and the residual liquid is discharged for 3-4 min;
6) And (3) cleaning hydrolysis products:
after the simulated digestion of the large intestine is finished, the large intestine buffer solution is emptied, and the obtained hydrolysate is washed,
7) Treatment of undigested residue and calculation of digestibility:
transferring undigested residues in the dialysis bag after the washing in the step 6) into a culture dish, drying, washing the residues with absolute ethyl alcohol, measuring the total energy value of the residues and pig feed samples after absolute drying, and calculating to obtain the enzyme hydrolysate energy value of the feed;
8) Estimating the effective performance of the feed by the enzyme hydrolysate energy value of the feed; the effective energy includes digestive energy and metabolic energy.
2. The method according to claim 1, characterized in that: in the step 1), the pig feed is single feed, compound feed or concentrated feed; the single feed is rapeseed meal, cottonseed meal, sunflower meal or peanut meal; the compound feed is corn-soybean meal type feed, corn-soybean meal type rapeseed meal type feed, corn-soybean meal type cottonseed meal type feed, corn-soybean meal type sunflower meal type feed or corn-soybean meal type peanut meal type feed; the pulverization is carried out with a plant pulverizer or a mortar.
3. The method according to claim 1 or 2, characterized in that: in the step 2), the monogastric bionic digestive system is produced by the intelligent technology development limited company in Hunan, and the model is as follows: SDS-2 or SDS-3;
The molecular weight cut-off of the dialysis bag is 12000-14000 daltons;
the dialysis bag is subjected to the following treatment before use: cutting the dialysis bag into small sections of about 25cm, boiling the dialysis bag in 2000mL of 2% (W/V) sodium bicarbonate and 1mmol/L disodium ethylenediamine tetraacetate solution (pH 8.0) for 10 minutes, and thoroughly cleaning the dialysis bag with distilled water; boiling in 1mmol/L disodium diamine tetraacetate solution (pH 8.0) for 10 min, cooling, storing in a refrigerator at 4deg.C for use, filling water in dialysis bag before use, discharging, and thoroughly cleaning the dialysis bag.
4. The method according to claim 1 or 2, characterized in that: in the step 2), the addition amount of the gastric buffer solution, the small intestine buffer solution and the large intestine buffer solution is 1000mL/5 digestive tracts;
in the step 2) of the method, the loading amount of the pig feed sample is 1-2 g/root of digestive tract.
5. The method according to claim 1 or 2, characterized in that: in the step 3), the addition amount of the simulated gastric fluid is 20 mL per digestive tract;
the number of the simulated digestive tubes is 5, the simulated digestive tubes are connected in series, and the digestive juice adding tube is connected with the system sequentially through a quick connector.
6. The method according to claim 1 or 2, characterized in that: in the step 4), the addition amount of the concentrated simulated small intestine solution is 2 mL per digestive tract.
7. The method according to claim 1 or 2, characterized in that: in the step 5), the addition amount of the concentrated simulated intestinal fluid is 2 mL per digestive tract.
8. The method according to claim 1 or 2, characterized in that: in the step 6), the washing is performed for 6 times, each time, 1500mL of deionized water is used for washing for 4 hours.
9. The method according to claim 1 or 2, characterized in that: in the step 7), the undigested residues are transferred into a culture dish with constant weight, dried at 65 ℃ until no water mark exists, and then dried at 105 ℃ until the weight is constant; scraping all digestion residues in the culture dish, transferring the digestion residues into an absolute dry glass sand crucible, and flushing the digested residues with 30-40 mL of absolute ethyl alcohol for 3-4 times.
10. The method according to claim 1 or 2, characterized in that: in the step 8), the feed is a single feed, and the digestion energy of the feed is calculated by bringing the enzyme hydrolysate energy value of the feed into the following equation: de= 0.9896 ×ehge+160; wherein EHGE represents the enzyme hydrolysate energy value and DE represents the digestion energy;
The feed is compound feed or concentrated feed; the digestion energy is calculated by bringing the enzyme hydrolysate energy value of the feed into the following equation: de= 0.8851 ×ehge+539; wherein EHGE represents the enzyme hydrolysate energy value and DE represents the digestion energy;
the feed is a single feed, and the metabolic energy of the feed is calculated by bringing the energy value of the enzymatic hydrolysate of the feed into the following equation: me= 0.9388 ×ehge+3; wherein EHGE represents enzyme hydrolysate energy value and ME represents metabolic energy;
the feed is compound feed or concentrated feed; the metabolic energy is calculated by bringing the enzyme hydrolysate energy value of the feed into the following equation: me= 0.9559 ×ehge+146; wherein EHGE represents the enzyme hydrolysate energy value and ME represents the metabolic energy.
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