A kit and method for determining the metabolizable energy of feed for fast growing broiler chickens by biomimetic digestion

Abstract

The application provides a kit and a method for determining the metabolic energy of fast-growing broiler feed by biomimetic digestion, which comprises digestive enzyme I, digestive enzyme II, buffer I, buffer II and trichloroacetic acid. The method comprises the following steps: preparation of a simulated digestion solution and a buffer; preheating and sample loading; stomach simulation digestion; small intestine simulation digestion; cleaning of hydrolysis products; treatment of undigested residues; and calculation of the digestion energy value. The kit and the method provided by the application can realize rapid and accurate determination of the metabolic energy value of fast-growing broiler feed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agriculture, and relates to a method for determining metabolizable energy of fast-growing broiler chicken feed, in particular to a biomimetic digestion method and kit for rapidly determining metabolizable energy of broiler chicken feed. BACKGROUND

[0002] The annual output of broiler chickens in China is about 9.82 billion, and about 50 million tons of feed are used for broiler chickens each year. The effective energy supply cost of feed accounts for more than 60% of the formula cost. Therefore, accurately and quickly determining the effective energy value of feed is crucial for fully utilizing unconventional feed resources, reducing the use of corn and soybean meal, achieving low-protein feed, and saving feed formula costs.

[0003] Currently, in the rapid estimation of metabolizable energy of broiler chicken feed raw materials, the Netherlands CVB has established a linear regression relationship between the chemical composition such as crude protein, crude fat, starch, and fiber (crude fiber, neutral detergent fiber, and acid detergent fiber) content and metabolizable energy of about 250 feed raw materials, and developed a special calculation software for dynamic estimation of metabolizable energy. The National Research Council (NRC. 1994. Nutrient requirements of poultry. 8th rev. ed. National Academy Press, Washington, DC), the Chinese feed raw material database (Xiong MB, et al. 2021. China feed ingredients and nutritional value table (32nd edition, 2021) preparation instructions. China Feed 23:97-107), etc. all provide models for estimating metabolizable energy based on chemical composition. However, in this method, the measured values of chemical composition content vary greatly between different laboratories. The deviation usually causes the estimated value of feed metabolizable energy to differ from the true value by more than 200 kcal / kg (error > 5%), which cannot meet the technical requirements of accurate formulation of broiler chicken feed. In addition, different feed raw materials use different models to estimate metabolizable energy, and when there is a lack of a model for estimating the metabolizable energy of a certain raw material, formula personnel will not be able to estimate its metabolizable energy value. Since there are many types of Chinese feed raw materials, many of which are unique to China, such as rough rice (mixed), vinegar residue, soy sauce residue, jujube powder, oil residue cake, etc. Neither the literature nor foreign feed raw material databases nor Chinese feed databases lack models for estimating metabolizable energy, so it is impossible to accurately estimate the metabolizable energy of these unconventional feed raw materials. Due to the lack of a technical platform for estimating the metabolizable energy of all feed raw materials based on chemical composition, the use of chemical composition to estimate the metabolizable energy of broiler chicken feed raw materials in production is only used as a reference based on experience, rather than a quantitative value.

[0004] In the past 30 years, nutritionists around the world have tried to achieve accurate and high-throughput estimation of metabolizable energy values of feedstuffs by simulating the digestion process of feedstuffs in animals. However, the development of simulated digestion methods is mainly aimed at simulating the digestion process of growing pigs (Boisen, S., and J. A. Fernandez. 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; Zhao et al., 2019. Programmed pig bionic digestion system and method for rapidly determining the digestible energy value of pig feed using the system. Invention patent: ZL201910360566.8). Early on, Valdes and Leeson of Guelph University in Canada described a simulated digestion method for poultry in the American Poultry Science Journal (Measurement of metabolizable energy in poultry feeds by an in vitro system. Poultry Science, 1992, 71: 1493-1503). The technical points of the method include: adding 0.5 g of feed sample in a 50 mL triangular flask → adding 10 mL of pepsin solution (concentration: 2 mg / mL 0.075 mol / L HCl solution, pH 4.13) → shaking in a water bath shaker at 37°C for 4 hours to simulate stomach digestion → after completion, adding 4 mL of 0.1 mol / L Tris buffer, adjusting the pH to 7-7.1 with 0.1 mol / L sodium hydroxide → adding 10 mL of digestive enzyme solution (concentration: pancreatin 4 mg / mL, cholate 1.5 mg / mL, enterokinase 0.25 mg / mL, double distilled water) → shaking in a water bath shaker at 37°C for 6 hours to simulate small intestine digestion → after completion, transferring to a centrifuge tube and centrifuging at 1500 g for 15 minutes → discarding the supernatant → adding double distilled water to the precipitate and centrifuging at 1500 g for 15 minutes → discarding the supernatant → drying the residue and measuring the total energy to calculate the in vitro digestible energy.The problems with this method are: 1) The activity and pH of pepsin in the stomach simulated digestion stage, and the activities and pH of amylase, trypsin, and chymotrypsin in the small intestine simulated digestion stage, as well as the enzymatic reaction temperature, are inconsistent with the gastrointestinal digestive environment in chickens; 2) The digestion time in the stomach and small intestine does not match the average residence time of chyme in broilers; 3) Only a 0.075 mol / L HCl solution is used in stomach digestion. Because this solution has no buffering capacity, it is difficult to stabilize the pH value at 4.13 when measuring different feed ingredients; 4) After small intestinal digestion is completed, the digestive enzymes are not inactivated and continue to be active. 5) The process of separating digested and undigested substances using centrifugation followed by discarding the supernatant, double-distilled water dilution, and then centrifugation again resulted in some undigested substances being mistaken for digested substances due to the large water-soluble molecules being mistaken for digested substances. Furthermore, some low-density feed ingredients floated on the water, causing undigested substances to be discarded with the supernatant, making them undetectable. 6) Using Erlenmeyer flasks as a simulated digester resulted in the following: the digestive fluid evaporated and condensed within the flask, causing changes in the volume of the enzymatic reaction and affecting its rate. During vortex mixing, some samples adhered to the flask walls and could not fully contact the digestive fluid, which is inconsistent with the reality of sufficient contact between digestate and digestive fluid in the body. Additionally, there was no exchange of substances between the reaction process and the external environment, and a significant product inhibition of the reaction occurred as the reaction progressed, which is inconsistent with the timely absorption of digestive products by animals.

[0005] Referring to the in vitro digestion method for growing pigs established by Boisen and Fernandez (1997), Yegani et al. from the University of Alberta, Canada (Prediction of energetic value of wheat and triticale in broilerchicks: A chick bioassay and an in vitro digestibility technique. Animal Feed Science and Technology, 2013, 183: 40-50) modified some parameters to establish an in vivo simulated digestion method for broilers. The key technical points include: adding 0.5g of feed sample to a 125mL Erlenmeyer flask → adding 3 glass beads and 25mL of 0.1mol / L phosphate buffer → adjusting the pH to 2 with 10mL of 0.2mol / L HCl → adding 1mL of porcine pepsin solution (concentration 25mg / mL) and 0.5mL of chloramphenicol → shaking in a water bath at 41℃ for 2 hours to simulate gastric digestion, and then removing the Erlenmeyer flask from the shaker → adding 10mL of 0.2mol / L phosphate buffer and 5mL of 0.6mol / L sodium hydroxide solution to adjust the pH to 6.8 → adding 1mL of porcine pancreatin solution (concentration 100mg / mL) → shaking in a water bath at 41℃ for 4 hours to simulate small intestinal digestion → adding 5mL of 20% sulfosalicylic acid → precipitation for 30 minutes → filtering with filter paper and drying → determining the total energy and calculating the in vitro digestible energy. The problems with this method are as follows: 1) The activity and pH of pepsin in the simulated gastric digestion stage, the activity and pH of the main digestive enzymes in the simulated small intestinal digestion stage, the volume ratio of sample to digestive fluid, and the digestion time in the stomach and small intestine are all inconsistent with the gastrointestinal digestive conditions in chickens; 2) During digestion, especially in the small intestinal digestion stage, there is a large proliferation of fungi, which leads to changes in the conditions for enzymatic reactions (mainly pH). Chloramphenicol only inhibits bacterial growth and is ineffective against fungi; 3) Sulfosalicylic acid precipitates large molecular weight proteins that are soluble in water and are considered undigested proteins, but the molecular weight range of the corresponding precipitate is unclear and does not correspond to the molecular weight of undigested proteins in vivo; 4) After digestion, medium-speed filter paper is usually used to separate hydrolyzed and unhydrolyzed products. The pore size of medium-speed filter paper is usually 30-50 micrometers. Within this pore size range, many large undigested molecules can pass through the filter paper, resulting in significantly higher measured values. Meanwhile, some feed digestion residues are difficult to filter through filter paper, making this method unsuitable for measurement; 5) In the final weight data, the sample amount is 0.5g, corresponding to approximately 0.1-0.2g of undigested residue (most feed digestion residues fall into this area), and the weight of the filter paper is approximately 0.1-0.2g. Because the filter paper easily absorbs moisture during weighing, and the amount of residue is very small, this significantly amplifies the error in the measurement data.

[0006] Therefore, the main problem with current in vitro simulated digestion methods for broilers is that the in vitro digestion conditions, including digestive enzyme activity, buffer pH, digestion time, and hydrolysis product separation methods, do not match the digestion conditions in the broiler's in vivo gastrointestinal tract. This leads to poor simulated digestion results; for example, the in vitro digestible energy values ​​of corn and barley are 12% and 18% lower than their in vivo metabolizable energy values, respectively, while soybean meal is 34% higher. This significant deviation makes in vitro simulated digestion methods unsuitable for practical application in broiler production.

[0007] Therefore, developing new methods to approximate the in vitro and in vivo digestion processes based on the in vivo digestion conditions of broilers is fundamental to achieving rapid determination of the metabolizable energy of broiler feed and is imperative to address the industry's major technological needs. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide a biomimetic digestion assay kit for the metabolizable energy of fast-growing broiler feed. This kit is compatible with a monogastric animal biomimetic digestive system and can achieve rapid and accurate determination of the metabolizable energy of fast-growing broiler feed.

[0009] Another objective of this invention is to provide a biomimetic digestion method for measuring the metabolizable energy of broiler feed, which enables rapid and accurate measurement of the metabolizable energy of broiler feed.

[0010] To achieve the above objectives, the present invention provides a biomimetic digestion assay kit for metabolizable energy of feed in fast-growing broiler chickens, comprising the following components:

[0011] Digestive enzyme I: Pepsin 60000U;

[0012] Digestive enzymes II: amylase 64170U; trypsin 10120U; chymotrypsin 2990U;

[0013] Buffer I: Sodium dihydrogen phosphate 24.00g; Sodium chloride 3.39g; Potassium chloride 5.07g;

[0014] Buffer II: Sodium chloride 3.86g; Potassium chloride 3.58g; Disodium hydrogen phosphate 4.89g; Sodium dihydrogen phosphate 43.86g; Sodium benzoate 10g; Potassium sorbate 10g;

[0015] 30 mL of 1.25 mol / L trichloroacetic acid.

[0016] The above kit can be used to test 2 samples, with 5 replicates for each sample.

[0017] This invention also provides a biomimetic digestion method for determining the metabolizable energy of feed for fast-growing broiler chickens, comprising the following steps:

[0018] 1) Preparation of simulated digestion fluid and buffer solution

[0019] Preparation of simulated gastric juice: The digestive enzyme I in the biomimetic digestion assay kit as described in claim 1 was dissolved in 250 mL of hydrochloric acid solution at pH 3.66 at 41 °C to prepare simulated gastric juice;

[0020] Preparation of concentrated simulated small intestinal fluid: Dissolve digestive enzyme II in the biomimetic digestion assay kit as described in claim 1 in 30 mL of deionized water and vortex rapidly for 2 minutes to prepare concentrated simulated small intestinal fluid;

[0021] Preparation of gastric buffer solution: Dissolve buffer I in 1800 mL of deionized water in the biomimetic digestion assay kit as described in claim 1, adjust the pH of the solution to 3.66 with 6 mol / L hydrochloric acid at 41 °C, and then make up to 2000 mL to prepare gastric buffer solution.

[0022] Preparation of small intestinal buffer: Dissolve buffer II in the biomimetic digestion assay kit as described in claim 1 in 1800 mL of deionized water and adjust the pH of the solution to 6.18 at 41 °C with 6 mol / L phosphoric acid or 6 mol / L sodium hydroxide. Then, make up to 2000 mL to prepare small intestinal buffer.

[0023] 2) Preheating and sample loading

[0024] A biomimetic digestive system for monogastric animals is adopted, which includes a simulated digestive apparatus, a digestive fluid reagent bottle, a buffer solution reagent bottle, a waste fluid container bottle, a cleaning fluid storage bottle, a cleaning fluid reagent bottle, and a cleaning residue container bottle. The simulated digestive apparatus includes a glass tube and a dialysis bag. The glass tube is a hollow tube with a ground joint at each end. An inlet tube and an outlet tube are provided on the side of the tube. The dialysis bag is placed inside the glass tube. The two ends of the dialysis bag extend outward from the two ground joints of the glass tube. The ends of the dialysis bag exposed outside the ground joints are tied and fixed to the ground joints with rubber strips. The two ground joints after tying the ends of the dialysis bag are respectively plugged with a folded silicone stopper and a silicone stopper with an infusion tube.

[0025] The concentrated simulated small intestinal fluid prepared in step 1) was placed into the digestive fluid reagent bottle of the monogastric animal bionic digestive system; the gastric buffer and small intestinal buffer prepared in step 1) were placed into the buffer control chamber of the monogastric animal bionic digestive system; after the monogastric animal bionic digestive system was preheated at 41°C for 60 minutes, 2g of chicken feed sample that had been pulverized and passed through a 60-mesh standard sieve was accurately weighed and poured into the dialysis bag of the simulated digester of the monogastric animal bionic digestive system, and 20mL of simulated gastric fluid prepared in step 1) was added to the dialysis bag, and the bag was plugged with a silicone stopper with an infusion tube; each sample was measured in 5 replicates, and each replicate used 1 simulated digester;

[0026] 3) Stomach simulates digestion:

[0027] 1000 mL of gastric buffer solution was pumped into the dialysis bag of a 5-unit-connected monogastric animal digestive apparatus and then returned to the gastric buffer solution reagent bottle for repeated circulation. The swirling oscillation provided a hybrid mixture of simulated gastric juice and feed sample to simulate gastric digestion. After digestion, the gastric buffer solution outside the dialysis bag in the simulated digestive apparatus was drained. Then, 1000 mL of small intestinal buffer solution was pumped into the dialysis bag of the monogastric animal digestive apparatus and then returned to the small intestinal buffer solution reagent bottle for repeated circulation, so that the pH of the solution in the dialysis bag automatically switched from the physicochemical environment of the stomach to the digestive environment of the small intestine.

[0028] 4) Small intestine simulates digestion:

[0029] Concentrated simulated small intestinal fluid was injected into the dialysis bag of the simulated digester. The small intestinal buffer solution was then pumped back into the small intestinal buffer solution bottle and continuously circulated. The concentrated simulated small intestinal fluid and the feed sample were mixed by swirling and oscillation to begin the simulated digestion of the small intestine. After digestion, the buffer solution outside the dialysis bag in the simulated digester was drained. Then, 1.25 mol / L trichloroacetic acid solution was pumped in, mixed for 5 minutes, and then digestion was terminated.

[0030] 5) Washing of hydrolysis products:

[0031] Deionized water was pumped into the dialysis bags of the five connected monogastric animal simulated digesters and then returned to the deionized water bottle for repeated circulation. After the end of the process, the cleaning fluid outside the dialysis bags in the simulated digesters was discharged. The cleaning was repeated 6 times to remove the hydrolysis products remaining in the dialysis bags throughout the simulated digestion process.

[0032] 6) Treatment of undigested residue and calculation of in vitro digestible energy

[0033] Transfer the undigested residue from the dialysis bag in step 5) to a petri dish, dry it at 65°C until no water marks remain, then dry it at 105°C to constant weight, cool it, and weigh it. Then, scrape off all the undigested residue from the petri dish and transfer it to a glass sand crucible with a known absolute dry weight. Rinse the undigested residue three times with anhydrous ethanol to ensure that the undigested residue and ethanol are thoroughly mixed until the filtrate is colorless. Then, place the glass sand crucible with the defatted undigested residue in a 105°C constant temperature drying oven to constant weight. At the same time, place the petri dish after scraping off the undigested residue in a 105°C constant temperature oven to constant weight. Calculate the in vitro digestible energy based on the feed sample weight and total energy, as well as the weight and total energy of the defatted undigested residue.

[0034] As described above, in step 3) stomach simulated digestion: mixing frequency, 180 rpm; stomach simulated digestion time, 1 h; gastric buffer flow rate, 120-150 mL / min; gastric buffer emptying time, 3 min; stomach-small intestine simulated digestion switching time, 0.5 h.

[0035] As described above, in step 4) small intestine simulated digestion: mixing frequency, 180 rpm; concentrated simulated small intestinal fluid pumped in volume, 3 mL / simulated digester; small intestinal buffer flow rate, 120-150 mL / min; small intestinal simulated digestion time, 5 h; small intestinal buffer emptying time, 3 min; 1.25 mol / L trichloroacetic acid solution pumped in volume, 2 mL / simulated digester.

[0036] As described above, in step 5) washing of the hydrolysis products, 1400-1600 mL of deionized water is used for 5 simulated digesters, and the washing time is 120 minutes per wash.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] 1. In simulated gastric digestion, the pepsin activity and buffer pH of the simulated gastric juice were kept consistent with the pepsin activity and chyme pH of the broiler gizzard gastric juice. Since gastric juice pH affects the digestion of protein by pepsin, it influences the degree of protein feed digestibility. The relatively high pH of the broiler gizzard is one of the reasons for the low digestibility of protein feed in broilers. Therefore, maintaining the pH of the simulated gastric juice and buffer solution consistent with the pH of the broiler gizzard chyme helps reduce the significant difference between in vitro digestibility measurements and in vivo metabolizable energy values ​​of protein feed.

[0039] 2. In simulated small intestinal digestion, the activities of trypsin and chymotrypsin are 70% of those in broiler jejunal fluid. This is consistent with the 61-64% metabolizable energy of feed, which is caused by the excretion of uric acid into feces and urine in broiler feed. This solves the problem that in in vitro digestion, the protein digestibility of protein feed ingredients is much higher than that measured in broiler metabolic experiments, resulting in in vitro digestion values ​​that are much higher than metabolizable energy.

[0040] 3. In the small intestine simulated digestion, the amylase activity and buffer pH are consistent with those of the jejunal fluid of broilers. This helps ensure the full digestion of starch in high-starch cereal feeds such as corn and barley, making their in vitro digestion values ​​close to their in vivo metabolizable energy values. This achieves the goal of reducing the difference between in vitro digestion values ​​and in vivo metabolizable energy values ​​of cereal feeds.

[0041] 4. After the simulated digestion in the small intestine is completed, 1.25 mol / L trichloroacetic acid is injected to completely denature the digestive enzyme proteins, thereby terminating the enzymatic reaction. As a result, in the subsequent separation of hydrolyzed and unhydrolyzed products, the hydrolysis reaction of the enzyme no longer changes with treatment time. This solves the problem of "over-digestion" of protein feed, resulting in measured values ​​higher than metabolizable energy values, and also improves the stability of the measurement results.

[0042] 5. In the in vitro simulated digestion process, potassium sorbate and sodium benzoate were used to inhibit the growth of fungi in the small intestine digestion stage, which effectively solved the problem of the small intestine pH dropping below 6.0 due to fungal fermentation, causing sourness, odor, and low digestibility.

[0043] 6. During the in vitro simulated digestion process, the temperature, mixing frequency, pumping of concentrated simulated small intestinal fluid, pumping of digestive enzyme inactivation solution, digestion time of the stomach and small intestine, and separation of hydrolyzed and unhydrolyzed products are all automatically controlled by setting corresponding parameters in the monogastric animal biomimetic digestion system, thereby realizing fully automatic simulation of in vivo digestion of broiler chickens to determine the metabolizable energy of feed.

[0044] The beneficial effects of this invention are as follows:

[0045] This invention provides a biomimetic digestion method and kit for determining the metabolizable energy of feed in fast-growing broiler chickens. Based on the composition parameters of digestive fluids in vivo, and simulating digestion conditions and time identical to those in vivo in broiler digestion parameters, the kit uses a monogastric animal biomimetic digestion system to fully automatically simulate the digestion process of the broiler's gastrointestinal tract, achieving accurate determination of feed metabolizable energy. This overcomes the shortcomings of traditional Erlenmeyer flask-based simulated digestion methods, such as unclear digestive enzyme activity, inconsistent digestion conditions with in vivo parameters, and an average difference of over 12% in the measured metabolizable energy of broiler chickens. Furthermore, it achieves standardization and automation of the method. It offers advantages such as cost-saving, high repeatability, accuracy, and speed. Attached Figure Description

[0046] Figure 1 The flowchart shows the biomimetic digestion method for measuring the metabolizable energy of fast-growing broiler feed provided by this invention.

[0047] Figure 2 This is a regression model for apparent metabolizable energy (AME) of feed ingredients against in vitro digestible energy (In vitro DE). Detailed Implementation

[0048] The embodiments of the present invention will now be described in detail and comprehensively so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0049] Existing methods for determining the metabolizable energy of broiler feed through in vitro simulated digestion do not consider the fact that uric acid enters the excrement during poultry metabolic experiments, causing the protein metabolic rate to be lower than the digestibility. Therefore, the digestible energy contributed by in vitro digestion to the protein in the feed is higher than the metabolic energy contributed by protein in animal metabolic experiments, resulting in a large discrepancy between the estimated and actual values. This invention, however, adjusts the activity of proteases during simulated small intestinal digestion, reducing the digestibility of protein in broiler feed, thus maintaining consistency with the protein metabolic rate in the determination of broiler feed metabolizable energy.

[0050] Instruments and equipment

[0051] Plant sample pulverizer or mortar; test sieve: 0.3mm (60 mesh); analytical balance: graduation value 0.0001g; pH meter: graduation value 0.01; glass sand crucible (G4, 30mL); petri dish: diameter 90mm; desiccator: anhydrous calcium chloride or color-changing silica gel as desiccant; vortex shaker; magnetic stirrer; electric heating drying oven; oxygen bomb calorimeter (model: 6400, Parr, USA); monogastric animal biomimetic digestive system (model: SDS-2 or SDS-3, Hunan Zhongben Intelligent Technology Development Co., Ltd.).

[0052] Reagents and Materials

[0053] Unless otherwise specified, all reagents are of analytical grade.

[0054] The laboratory water meets the specifications for Grade III water in GB / T 6682-2008.

[0055] Bionic digestion assay kit for metabolizable energy of feed in fast-growing broiler chickens:

[0056] Digestive enzyme I: 60,000 U pepsin.

[0057] Digestive enzymes II: amylase 64170U, trypsin 10120U, chymotrypsin 2990U.

[0058] Buffer I: 24.00 g sodium dihydrogen phosphate, 3.39 g sodium chloride and 5.07 g potassium chloride.

[0059] Buffer II: 3.86g sodium chloride, 3.58g potassium chloride, 4.89g disodium hydrogen phosphate, 43.86g sodium dihydrogen phosphate, 10g sodium benzoate and 10g potassium sorbate.

[0060] Other reagents: hydrochloric acid (HCl), phosphoric acid (H3PO4), sodium hydroxide (NaOH), sodium bicarbonate (NaHCO3), disodium ethylenediaminetetraacetate (C6HCO3). 10 H 14N2O8Na2·2H2O, disodium EDTA, anhydrous ethanol (C2H6O), trichloroacetic acid, 1.25 mol / L.

[0061] Dialysis bags: Manufactured by Visakase, USA, model MEMBRA-CEL MD44-14, molecular weight cutoff 14000 Daltons. Pretreatment is as follows: Cut the dialysis bags into small segments of approximately 25 cm. Boil the dialysis bags in 2000 mL of 2% (w / v) sodium bicarbonate and 1 mmol / L disodium EDTA solution (pH 8.0) for 10 minutes. Thoroughly rinse the dialysis bags with distilled water. Boil in 1 mmol / L disodium EDTA solution (pH 8.0) for 10 minutes. After cooling, store at 4°C for later use. (Note: Ensure the dialysis bags are always submerged in the solution after this process, and wear gloves when handling the dialysis bags.) Before use, fill the dialysis bags with water, then drain and thoroughly rinse the bags.

[0062] Gastric buffer solution: Dissolve buffer I in 1800 mL of deionized water in a 2000 mL beaker, and adjust the pH of the solution to 3.66 with 6 mol / L hydrochloric acid (HCl) at 41 °C. After cooling, transfer the solution to a 2000 mL volumetric flask and dilute to volume with deionized water.

[0063] Small intestinal buffer: Dissolve Buffer II in 1800 mL of deionized water in a 2000 mL beaker, and adjust the pH of the solution to 6.18 at 41 °C with 6 mol / L phosphoric acid or 6 mol / L sodium hydroxide. After cooling, transfer the solution to a 2000 mL volumetric flask and dilute to volume with deionized water.

[0064] Simulated gastric juice: Dissolve digestive enzyme I in 250 mL of hydrochloric acid solution with pH 3.66 (pH determined at 41°C), stirring slowly until dissolved. Do not heat on a hot plate or overheat during preparation. Prepare immediately before use.

[0065] Concentrated simulated small intestinal fluid: Dissolve digestive enzyme II in 30 mL of deionized water, vortex rapidly for 2 minutes, and prepare immediately before use.

[0066] Example

[0067] Thirty-four samples of commonly used broiler feed ingredients were collected from China, including 10 samples of cereal feed, 5 samples of cereal processing by-products and oils, and 19 samples of protein feed ingredients. The chemical composition is shown in Table 1.

[0068] Table 1 Feed ingredients and their chemical composition (dry matter basis)

[0069]

[0070]

[0071] A one-way completely randomized design was used to determine the apparent metabolizable energy (AME) of the experimental diet and the basal diet, with 6 replicates per treatment and 4 chickens per replicate. The in vitro digestible energy (Invitro DE) of the experimental diet and the basal diet was determined using a biomimetic digestion method, with 5 replicates per treatment and 1 simulated digester per replicate. The differences between the two measurements and the correlation coefficient and regression model of AME on Invitro DE were compared.

[0072] The experimental diet consisted of a basal diet and the feed ingredients to be tested. The formulation composition is shown in Tables 2-1, 2-2, and 2-3.

[0073] 1. Determination of metabolizable energy value of experimental diets for broilers

[0074] The broiler metabolic trial was conducted in four batches. Each batch was conducted as follows: 0-day-old male AA plus commercial broilers were purchased from the hatchery and housed in three-tiered metabolic cages. During the non-metabolic trial period, broilers were fed commercial broiler feed with free access to feed and water. At 22 days of age, broilers with similar and acceptable weights were selected and placed in the metabolic cages, four chickens per cage. Each experimental diet was randomly assigned to six replicate cages. The metabolizable energy determination trial lasted six days, with the first three days being the diet adaptation period. Feeding was withheld from 16:00 on day 3 to 9:00 on day 4. The experimental diet was fed from 09:00 on day 4 to 16:00 on day 6, and withheld from 16:00 on day 6 to 9:00 on day 7. Feed intake was recorded from 9:00 on day 4 to 17:00 on day 6, and excrement was collected from 9:00 on day 4 to 9:00 on day 7 (collected four times daily) and stored at -20°C. Chickens are allowed free access to water. During periods of fasting, chickens are given supplemental glucose saline solution (5% glucose solution with a NaCl concentration of 0.9%).

[0075] 2. The in vitro digestible energy of broiler experimental diets and basal diets was determined using a biomimetic digestion method, such as... Figure 1 As shown.

[0076] Based on the design principles of a biomimetic digestive system, two 1000mL gastric buffer bottles and two 1000mL small intestinal buffer bottles were placed in the corresponding positions of the monogastric animal biomimetic digestive system, and the system's tubing was connected to the buffer bottles. Two samples were measured per biomimetic digestion test, with five replicates per sample and one simulated digester per replicate. Five simulated digesters were connected in series to measure one sample.

[0077] The biomimetic digestion process consists of three stages: stomach, small intestine, and washing of chicken hydrolysate. The parameters set in the biomimetic digestion system control software are as follows: preheating time: 60 minutes; simulated digestion temperature: 41℃; mixing frequency: 180 rpm; stomach simulated digestion time: 1 hour; stomach buffer flow rate: 120 mL / min; stomach buffer emptying time: 3 minutes; stomach-small intestine simulated digestion switching time: 0.5 hours; concentrated simulated small intestinal fluid pumping volume: 3 mL / simulated digester; small intestinal buffer flow rate: 120 mL / min; small intestinal simulated digestion time: 5 hours; small intestinal buffer emptying time: 3 minutes; trichloroacetic acid solution (1.25 mol / L) injection volume: 2 mL / simulated digester, mixing for 5 minutes; parameters for each washing program are as follows: deionized water volume: 1500 mL / 5 simulated digesters; washing time: 120 minutes.

[0078]

[0079]

[0080]

[0081] Clock; emptying time of cleaning fluid, 3 minutes; number of cleaning cycles, 6. After all parameters for the digestion stages have been entered, run the simulated digestion process.

[0082] During the preheating of the monogastric animal biomimetic digestive system, the following sample loading procedure was performed.

[0083] Insert the prepared dialysis bag horizontally through the glass tube, fold both ends outwards, and secure the dialysis bag tightly with rubber bands to the glass tube. Then, seal one end tightly with a silicone stopper.

[0084] Accurately weigh 2g of the pulverized feed sample into a glass test tube (accurate to 0.0001g), and simultaneously determine the dry matter content. Place the sample in a dialysis bag simulating a digester.

[0085] Simulating chicken gastrointestinal digestion

[0086] Stomach simulated digestion

[0087] Add 20 mL of gastric digestive fluid to the dialysis bag.

[0088] The other end of the simulated digester is sealed with a flared silicone plug containing a digestive fluid filling tube.

[0089] After the monogastric animal bionic digestive system has preheated and the control software displays a "ready" message, place the simulated digester into the system. Connect the tubing according to the principle of water inlet at the bottom and water outlet at the top. Connect the five simulated digesters in each group in series. Connect the digestive fluid inlet tubing to the system sequentially using quick-connect couplings. Click "Start Experiment" in the control software.

[0090] Under the control software of the monogastric animal bionic digestive system, the stomach simulates digestion automatically for 1 hour, and then the buffer solution remaining outside the dialysis bag in the simulated digester is discharged.

[0091] Small intestine mimics digestion

[0092] Under the control software of the monogastric animal biomimetic digestive system, after the stomach simulated digestion was completed, the small intestinal buffer solution was automatically pumped into the simulated digester. After circulation for 0.5 hours, 3 mL of concentrated simulated small intestinal fluid was automatically pumped into the dialysis bag of each simulated digester to start the small intestinal simulated digestion for 5 hours. After completion, the buffer solution remaining outside the dialysis bag in the simulated digester was drained. Then, 2 mL of 1.25 mol / L trichloroacetic acid solution was automatically pumped into each simulated digester. After mixing for 5 minutes, 6 washes were performed to remove small molecules remaining in the dialysis bag. A total of 1500 mL of deionized water was pumped into each group of 5 simulated digesters for 120 minutes each time.

[0093] Treatment of digestive residues

[0094] After digestion, the undigested residue in the dialysis bag is transferred without loss to a 90 mm culture dish of known oven-dry weight (this process requires removing the dialysis bag from the simulated digester, cutting it lengthwise, and rinsing it with deionized water).

[0095] After drying the petri dishes containing undigested residue at 65°C until no watermarks remain (usually takes 12–14 hours), they are then dried at 105°C until constant weight is achieved.

[0096] Scrape off all digestion residue from the petri dish and transfer it to a glass frit crucible of known absolute dry weight. Rinse the residue three times with anhydrous ethanol (approximately 30 mL each time), ensuring thorough mixing of the residue and ethanol, until the filtrate is colorless. Simultaneously, place the petri dish containing the residue in a 105°C oven to constant weight.

[0097] The culture dish after scraping off the undigested residue and the glass sand crucible containing the defatted undigested residue were placed in a constant temperature oven at 105℃ and dried to constant weight.

[0098] Transfer the undigested degreased residue from the glass sand crucible to the weighing paper of the bomb calorimeter, and simultaneously place the glass sand crucible containing the degreased residue in a 105°C oven to dry to constant weight.

[0099] When determining the residual total energy of digestive enzymes using the biomimetic digestion method, no feed sample was added to the simulated digester. Other digestion processes and post-digestion treatments were the same as described above. The energy value is GEE.

[0100] 3. Sample preparation and chemical analysis

[0101] Broiler excrement was dried in a 65℃ forced-air drying oven, cooled, and weighed after 24 hours. It was then pulverized and passed through a 0.42mm sieve before sampling. Feed ingredients used for determining fiber content were pulverized and passed through a 0.84mm sieve, while those used for determining other chemical components and GE were passed through a 0.42mm sieve. Dietary samples used for determining in vitro digestible energy were pulverized and passed through a 0.25mm sieve. Moisture (GB / T 6435-2014), CP (GB / T6432-2018), and GE content (ISO 9831:1998) were determined in broiler excrement and experimental diets. The moisture (GB / T 6435-2014), crude protein (GB / T 6432-2018), crude fat (GB / T 6433-2006), crude ash (GB / T 6438-2007), crude fiber (GB / T 6434-2006), acid detergent fiber (NY / T 1459-2007), neutral detergent fiber (GB / T 20806-2006), and GE content (ISO 9831:1998) of feed ingredients were determined.

[0102] 4. Data Processing and Statistical Analysis

[0103] 1) Total energy of feed

[0104] GE1=E1×M1………………(1)

[0105] In the formula:

[0106] GE1—Total energy of the sample, in calories; E1—Dry energy value of the diet sample, in calories per gram (cal / g);

[0107] M1 – Dry weight of feed, in grams (g).

[0108] 2) Residue can always

[0109] GE2=E2×M2………………(2)

[0110] In the formula:

[0111] GE2 – Total residual energy, measured in calories (cal);

[0112] E2 – Energy value of defatted residue dry matter, expressed in calories per gram (cal / g);

[0113] M2 — Dry weight of defatted undigested residue from the sampled feed, in grams (g).

[0114] 3) Total dry matter in feed

[0115] GDM=M3×DM………………(3)

[0116] In the formula:

[0117] GDM – Total Dry Matter of Sample, in grams (g);

[0118] M3 — Sample air-dried mass, in grams (g);

[0119] DM – Dry matter content of air-dried feed samples, expressed as a percentage (%).

[0120] 4) The dry matter mass and dry matter energy value of the defatted undigested residue are calculated according to formulas (4) and (5), respectively.

[0121]

[0122]

[0123] M2—The dry mass of undigested residue from the sampled feed, expressed in grams (g);

[0124] W0 — Dry weight of empty petri dish, in grams (g);

[0125] W1 — Undigested residue + oven-dry weight of the petri dish, in grams (g);

[0126] W2 — Residue after scraping off undigested residue + oven-dry weight of the petri dish, in grams (g);

[0127] W3 — Dry weight of the glass sand crucible, in grams (g);

[0128] W4 – Undigested defatted residue + oven-dry weight in glass sand crucible, in grams (g);

[0129] W5 — The dry weight of the defatted undigested residue sample used for total energy determination plus the glass sand crucible, in grams (g).

[0130] W6 — Weight of air-dried sample of defatted digestion residue, in grams (g);

[0131] E r —Total energy of the defatted residue air-dried sample, in joules (J / g);

[0132] 5) The in vitro digestible energy (DE, in MJ / kg) dry matter basis of the diet is calculated according to formula (6).

[0133]

[0134] In the formula:

[0135] In vitro DE—the in vitro digestible energy of the tested diet, expressed in kcal / kg;

[0136] GE1 – Total energy of the sampled diet, in calories (cal);

[0137] GE2 – Total residual energy, measured in calories (cal);

[0138] GEe—Residual Total Energy from Digestive Enzymes;

[0139] M1—Dry weight of the sampled feed, in grams (g);

[0140] 6) The apparent metabolizable energy (AME) of the experimental diet was calculated as follows:

[0141] AME (kcal / kg DM) = [Dietary intake (kg) × Total dietary energy (kcal / kg) - Excrement volume (kg) × Total excrement energy (kcal / kg)] / Dietary intake (kg).

[0142] Basic statistical analysis was performed using the MEANS module in SAS 9.4. The 75% (Q3) and 25% (Q1) quantiles for the five replicates under each treatment were calculated. IQR = Q3 - Q1; data outside the range of Q1 - 1.5 × IQR to Q3 + 1.5 × IQR were considered outliers and removed. The PROC TTEST module was used to test the significance of the difference between in vitro DE and AME. The PROC CORR module was used to analyze the correlation between in vitro DE and AME. The PROC REG module was used to build a regression model of AME on in vitro DE. A p-value < 0.05 was considered statistically significant.

[0143] The test results are shown in Table 3.

[0144] Table 3 Comparison of in vitro digestible energy (In vitro DE) and apparent metabolizable energy (AME) of diets

[0145]

[0146]

[0147] 1 Deviation = (In vitro digestible energy - AME) ÷ AME × 100%

[0148] 2* This indicates that P < 0.05

[0149] The t-test results showed that among the 38 diet samples, the in vitro DE value and AME value of 23 diets were not significantly different (P>0.05). The in vitro DE values ​​of wheat flour 1.2, peanut meal 50, cottonseed meal 50, corn gluten meal 60, extruded feather meal, Pingdu wheat, sorghum, brewer's grains 2 and 7 were significantly higher than the AME values ​​(P<0.05), while the in vitro DE values ​​of soybean meal 46, Jilin corn, brewer's grains 8, basal diet 3, bran, and duck fat diets were significantly lower than the AME values ​​(P<0.05). Among the above 15 diets, the absolute values ​​of the relative deviations between the in vitro DE and AME values ​​were all between 0.76% and 2.66%.

[0150] The average ratio of in vitro DE / AME for the 38 diets was 1.00, meaning the value measured by the biomimetic digestion method was close to 100% of the in vivo AME value. This indicates that, overall, the biomimetic digestion method simulated the digestibility of broilers very closely to in vivo digestibility. Furthermore, when the AME of the 38 diet samples varied from 2852 to 3827 kcal / kg DM, the correlation coefficient between in vitro DE and AME measured by the biomimetic digestion method was 0.99 (P < 0.05). In the regression model of AME on in vitro digestibility, such as... Figure 2 As shown, AME = 0.998 × In vitro DE(R) 2 =1, RSD = 44 kcal / kg DM, P < 0.01). This indicates that the regression model has very good simulation performance. The above shows that, when determining the effective energy of raw materials with large differences (different types) using biomimetic digestion, the in vitro DE value still follows the advantage of "one standard for all" in approximating the AME. Therefore, in vitro digestion can accurately and sensitively estimate the AME value of raw materials.

[0151] As can be seen from the above embodiments, the biomimetic digestion assay kit and method for metabolizable energy of fast-growing broiler feed provided by this invention, based on a thorough analysis of broiler in vivo digestion parameters, utilizes a commercially available fully automated monogastric animal biomimetic digestion system and digestive enzyme reagents. Through in vitro simulated digestion, the digestive enzyme activity and buffer ion concentration are made consistent with the in vivo digestive fluid of fast-growing broilers, and the simulated digestive fluid and buffer solution kit product inhibits fungal growth. Furthermore, the biomimetic digestion method closely approximates the in vivo digestion conditions of broilers in terms of sample processing, digestion time, enzymatic reaction termination, and separation of hydrolyzed and unhydrolyzed products, achieving standardization and automation of the assay method. This solves the technical deficiency of in vitro simulated digestion methods for broilers, where the measured results differ from the metabolizable energy values ​​by more than 12%, making standardization and automation difficult.

[0152] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A biomimetic digestion method for determining the metabolizable energy of feed for fast-growing broiler chickens, characterized in that, The biomimetic digestion assay kit for metabolizable energy of fast-growing broiler feed consists of the following components: Digestive enzyme I: Pepsin 60000U; Digestive enzymes II: amylase 64170U; trypsin 10120U; chymotrypsin 2990U; Buffer I: Sodium dihydrogen phosphate 24.00g; Sodium chloride 3.39g; Potassium chloride 5.07g; Buffer II: Sodium chloride 3.86g; Potassium chloride 3.58g; Disodium hydrogen phosphate 4.89g; Sodium dihydrogen phosphate 43.86g; Sodium benzoate 10g; Potassium sorbate 10g; 30 mL of 1.25 mol / L trichloroacetic acid; The assay method includes the following steps: 1) Preparation of simulated digestion solution and buffer solution. Preparation of simulated gastric juice: The digestive enzyme I in the biomimetic digestion assay kit was dissolved in 250 mL of hydrochloric acid solution at pH 3.66 at 41 °C to prepare simulated gastric juice; Preparation of concentrated simulated small intestinal fluid: Digestive enzyme II from the biomimetic digestion assay kit was dissolved in 30 mL of deionized water and rapidly vortexed for 2 minutes to prepare concentrated simulated small intestinal fluid; Preparation of gastric buffer solution: Dissolve Buffer I from the biomimetic digestion assay kit in 1800 mL of deionized water, adjust the pH of the solution to 3.66 with 6 mol / L hydrochloric acid at 41 °C, and then bring the volume to 2000 mL to prepare gastric buffer solution. Preparation of small intestinal buffer: Dissolve Buffer II from the biomimetic digestion assay kit in 1800 mL of deionized water and adjust the pH of the solution to 6.18 at 41 °C with 6 mol / L phosphoric acid or 6 mol / L sodium hydroxide. Then, bring the volume to 2000 mL to prepare the small intestinal buffer. 2) Preheating and sample loading A biomimetic digestive system for monogastric animals is adopted, which includes a simulated digestive apparatus, a digestive fluid reagent bottle, a buffer solution reagent bottle, a waste fluid container bottle, a cleaning fluid storage bottle, a cleaning fluid reagent bottle, and a cleaning residue container bottle. The simulated digestive apparatus includes a glass tube and a dialysis bag. The glass tube is a hollow tube with a ground joint at each end. An inlet tube and an outlet tube are provided on the side of the tube. The dialysis bag is placed inside the glass tube. The two ends of the dialysis bag extend outward from the two ground joints of the glass tube. The ends of the dialysis bag exposed outside the ground joints are tied and fixed to the ground joints with rubber strips. The two ground joints after tying the ends of the dialysis bag are respectively plugged with a folded silicone stopper and a silicone stopper with an infusion tube. The concentrated simulated small intestinal fluid prepared in step 1) was placed into the digestive fluid reagent bottle of the monogastric animal bionic digestive system; the gastric buffer and small intestinal buffer prepared in step 1) were placed into the buffer control chamber of the monogastric animal bionic digestive system; after the monogastric animal bionic digestive system was preheated at 41°C for 60 minutes, 2g of chicken feed sample that had been pulverized and passed through a 60-mesh standard sieve was accurately weighed and poured into the dialysis bag of the simulated digester of the monogastric animal bionic digestive system, and 20mL of simulated gastric fluid prepared in step 1) was added to the dialysis bag, and the bag was plugged with a silicone stopper with an infusion tube; each sample was measured in 5 replicates, and each replicate used 1 simulated digester; 3) Stomach simulates digestion: 1000 mL of gastric buffer solution was pumped into the dialysis bag of a 5-unit-connected monogastric animal digestive apparatus and then returned to the gastric buffer solution reagent bottle for repeated circulation. The swirling oscillation provided a hybrid mixture of simulated gastric juice and feed sample to simulate gastric digestion. After digestion, the gastric buffer solution outside the dialysis bag in the simulated digestive apparatus was drained. Then, 1000 mL of small intestinal buffer solution was pumped into the dialysis bag of the monogastric animal digestive apparatus and then returned to the small intestinal buffer solution reagent bottle for repeated circulation, so that the pH of the solution in the dialysis bag automatically switched from the physicochemical environment of the stomach to the digestive environment of the small intestine. 4) Small intestine simulates digestion: Concentrated simulated small intestinal fluid was injected into the dialysis bag of the simulated digester. The small intestinal buffer solution was then pumped back into the small intestinal buffer solution bottle and continuously circulated. The concentrated simulated small intestinal fluid and the feed sample were mixed by swirling and oscillation to begin the simulated digestion of the small intestine. After digestion, the buffer solution outside the dialysis bag in the simulated digester was drained. Then, 1.25 mol / L trichloroacetic acid solution was pumped in, mixed for 5 minutes, and then digestion was terminated. 5) Washing of hydrolysis products: Deionized water was pumped into the dialysis bags of the five connected monogastric animal simulated digesters and then returned to the deionized water bottle for repeated circulation. After the end of the process, the cleaning fluid outside the dialysis bags in the simulated digesters was discharged. The cleaning was repeated 6 times to remove the hydrolysis products remaining in the dialysis bags throughout the simulated digestion process. 6) Treatment of undigested residue and calculation of the energy value of enzyme hydrolysates: Transfer the undigested residue from the dialysis bag in step 5) to a petri dish, dry it at 65°C until there are no water marks, then dry it at 105°C until constant weight, cool it and weigh it. Then, scrape off all the undigested residue from the petri dish and transfer it to a glass sand crucible with a known absolute dry weight. Rinse the undigested residue three times with anhydrous ethanol, ensuring that the undigested residue and ethanol are thoroughly mixed, until the filtrate is colorless. Then, place the glass sand crucible with the defatted undigested residue in a 105°C constant temperature drying oven to constant weight. At the same time, place the petri dish after scraping off the undigested residue in a 105°C constant temperature oven to constant weight. Calculate the in vitro digestible energy based on the feed sample weight and total energy, as well as the weight and total energy of the defatted undigested residue. In step 3) of simulated gastric digestion: mixing frequency, 180 rpm; simulated gastric digestion time, 1 h; gastric buffer flow rate, 120-150 mL / min; gastric buffer emptying time, 3 min; gastric-small intestine simulated digestion switching time, 0.5 h. In step 4) of the simulated small intestine digestion: mixing frequency, 180 rpm; concentrated simulated small intestinal fluid pumping volume, 3 mL / simulated digester; small intestinal buffer flow rate, 120~150 mL / min; small intestinal simulated digestion time, 5 h; small intestinal buffer emptying time, 3 min; 1.25 mol / L trichloroacetic acid solution pumping volume, 2 mL / simulated digester. Step 5) In the cleaning of hydrolysis products, use 1400~1600mL of deionized water for 5 simulated digesters, and clean for 120 minutes per cycle.

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