Method for measuring fermentation characteristics of fibers in feed raw materials and fiber fermentation energy supply through in-vitro fermentation

Abstract

The invention relates to the field of feed, in particular to a method for measuring fermentation characteristics of fibers in feed raw materials and energy supply of fiber fermentation through in-vitro fermentation. The method can replace animal experiments, quantitatively determine the content and proportion of total posterior intestine fermentable carbohydrates including soluble dietary fibers in the feed raw materials and the compound feed, evaluate the fermentation utilization rate of the dietary fibers in the feed raw materials in the posterior intestines, and measure the energy provided by the fermentation of the dietary fibers in the feed raw materials; energy values in the feed raw material database can be corrected according to animals of different stages and varieties; according to the method, corresponding faeces can be conveniently collected as a bacterial source for in-vitro fermentation for pigs of different varieties and stages, and the fermentation utilization rate of dietary fibers in the feed raw materials by different pigs is measured, so that the energy provided by the fermentation of the dietary fibers in the feed raw materials is measured for the pigs of different varieties and stages, and the utilization rate of the dietary fibers in the feed raw materials is measured. The energy values of all the raw materials in the database are corrected, and the purpose of precise formula is achieved.

Description

Technical Field

[0001] The present invention relates to the field of feed, and in particular to a method for determining the fermentation characteristics of fiber in feed raw materials and the energy supply of fiber fermentation by in vitro fermentation. Background Art

[0002] Dietary fiber is known as the seventh nutrient. Fiber can be divided into soluble fiber (SF) and insoluble fiber (ISF) based on its water solubility; and can be further divided into fermentable fiber and non-fermentable fiber (inert fiber) based on its fermentability. Fermentable fiber can regulate intestinal flora, and fiber fermentation can produce short-chain fatty acids, which not only provide energy but also act on various receptors in the intestine to regulate the animal's endocrine system and various physiological functions. Therefore, fermentable fiber has the function of regulating the sow's endocrine system and is of great significance to improving the production performance of sows. However, the fermentability of fiber cannot be detected by chemical methods, because there are huge differences in the microorganisms in the hindgut of pigs of different breeds and ages, resulting in different fermentation utilization rates of fiber. Therefore, the fermentability of fiber is not a fixed value.

[0003] There are few reports in foreign literature on the fermentable dietary fiber content in raw materials. Although there are in vitro fermentation experiments on some feed raw materials in simulated rumen or hindgut environments in domestic literature, the usual detection indicators only include: gas production, gas production curve, volatile fatty acid production, bacterial flora changes, dry matter digestibility, and neutral detergent fiber digestibility. There are no reports on dietary fiber digestibility (digested dietary fiber is fermentable dietary fiber). The reason may be that the AOAC "enzyme-weight method" cannot achieve accurate weighing of trace dietary fiber in fermentation experiments, and it is difficult to precipitate and separate soluble fiber in the solution by increasing the fermentation volume.

[0004] In some feed ingredient databases (such as the CVB database in the Netherlands), fiber fermentability is characterized by fermentable non-starch polysaccharides (fNSP). The specific value of fNSP is currently determined through animal metabolism experiments and obtained indirectly through calculation. The calculation formula is: Digestible non-starch polysaccharides (fNSP) = total digestible organic matter - digestible crude protein - digestible crude fat - starch - enzymatically digestible carbohydrates - lactic acid - amino acids - ethanol - propionic acid - butyric acid - oligosaccharides. Because multiple calculations are required, errors in determining the content and digestibility of protein, fat, starch, and other ingredients accumulate in the final result, leading to unpredictable deviations. Furthermore, most databases lack complete fNSP data, with many uncommon ingredients lacking fNSP data. Existing databases are only applicable to three-way pigs, and the applicable age range of the data is unclear. Pigs of different ages and breeds have significant differences in their utilization of fiber in feed ingredients, which can lead to errors in calculating feed formulas for different pig breeds.

[0005] Although the classic animal nutritional metabolism experimental method is theoretically accurate for determining the digestible energy and metabolizable energy of pig feed raw materials, it requires a large number of personnel, a long time, and high financial costs to carry out relevant animal experiments. If the fiber digestibility and energy value of different feed raw materials are determined for different breeds of pigs one by one according to the traditional animal experimental method, the workload will be enormous, and the input and output will not be cost-effective.

[0006] Regarding the digestible energy value of feed, in addition to animal experiments, current methods also use the stomach-small intestine-large intestine bionic digestion method to obtain undigested residues. The total energy of the residue is then measured using the oxygen bomb method to calculate the digestible energy. For example, patents "CN110057964A Programmable Pig Bionic Digestion System and Method for Rapidly Determining the Digestible Energy Value of Pig Feed Using the System" and "CN113341059B A Growing Pig Stomach-Small Intestine-Large Intestine Bionic Digestion Method and Its Application to Estimating the Effective Energy Value of Feed" are also complex to operate and require expensive instrumentation and consumables. They are not suitable for large-scale sample measurement and ignore the differences in fiber fermentation among animals of different breeds and ages.

[0007] To correct the energy values of different pig breeds in the feed raw material database, patent "CN117726476B - Method for Predicting the Effective Energy of Ding'an Black Pig and Ding'an Pig Feed Raw Materials Based on the Effective Energy of Sanyuan Pig Feed Raw Materials" uses animal metabolism tests to measure and compare the differences in digestibility and energy value of specific raw materials in Sanyuan pigs and two other local pig breeds. This directly establishes a correspondence between the digestibility and effective energy value of feed for Sanyuan pigs and local pig breeds, thereby establishing a prediction equation for the effective energy of other pig breeds based on the effective energy of Sanyuan pigs. This method simplifies the workload of conducting animal metabolism tests, but the reliability of the data used to deduce the digestibility of all other raw materials in the database based on metabolism tests of limited raw materials is still controversial. Summary of the Invention

[0008] The purpose of the present invention is to address the deficiencies in the prior art and provide a method for determining the fermentation characteristics of fiber in feed raw materials and the energy supply of fiber fermentation by in vitro fermentation.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] Provided is a method for determining the fermentation characteristics of fiber in feed raw materials and the energy supply of fiber fermentation by in vitro fermentation, the steps comprising:

[0011] S1. Pre-treating the feed raw materials to obtain a sample to be tested, performing enzymatic hydrolysis on the sample to be tested, and then hydrolyzing the residue after enzymatic hydrolysis to determine the total sugar content;

[0012] S2. Collect animal feces to prepare a fecal inoculum, mix the test sample, the fecal inoculum, and the basal culture medium, and then perform anaerobic fermentation. After the anaerobic fermentation is completed, separate the residue, and hydrolyze the residue to determine the total sugar content;

[0013] S3. Calculate the fermentable dietary fiber content in the feed raw material and the energy supply of fiber fermentation by the difference between the total sugar content of the enzymatic hydrolysis residue of the test sample and the total sugar content of the fermentation residue.

[0014] Preferably, in step S1, the pretreatment includes: performing drying treatment, defatting treatment or desugaring treatment on the feed raw material according to its moisture content, fat content and sugar content, and then crushing and screening to obtain the pretreated feed raw material.

[0015] More preferably, the fat content of the pretreated feed raw material is less than 10%, and the sugar content is less than 5%.

[0016] More preferably, the drying process comprises: placing the wet sample in a vacuum drying oven at 70°C±1°C and drying to a constant weight.

[0017] More preferably, the desugaring treatment comprises: washing the feed material with a sugar content ≥5% with 85% ethanol solution for three consecutive times at a ratio of 10 mL per gram of the feed material for desugaring, and then drying in a 40° C. oven.

[0018] Preferably, in step S1, the enzymatic hydrolysis treatment comprises: dispersing the pretreated feed raw material in a buffer solution, and sequentially performing enzymatic hydrolysis with a thermostable α-amylase, enzymatic hydrolysis with a protease, and enzymatic hydrolysis with amyloglucosidase.

[0019] More preferably, the enzymatic hydrolysis with thermostable α-amylase comprises: adding 10 μL of thermostable α-amylase solution and stirring slowly, sealing, placing in a 95° C.-100° C. constant temperature shaking water bath, starting timing when the temperature rises to 95° C., reacting for 35 minutes, and then cooling to 60° C.

[0020] More preferably, the protease hydrolysis comprises: adding 20 μL of protease solution, sealing, continuously shaking, reacting for 30 minutes, adding 0.8 mL of 3 mol / L acetic acid solution while stirring, controlling the sample temperature at 60°C ± 1°C, and adjusting the pH of the sample solution to 4.5 ± 0.2 with 6 mol / L sodium hydroxide solution.

[0021] More preferably, the enzymatic hydrolysis with amyloglucosidase comprises: adding 20 μL of amyloglucosidase solution while stirring, sealing the container, continuously shaking the container in a water bath at 60° C.±1° C., and reacting for 30 minutes before removing the container.

[0022] Preferably, in step S2, the preparation of the fecal inoculum solution comprises: taking fresh feces of pigs of a specific breed and age, filtering the collected feces with sterile 0.9% saline saturated with carbon dioxide at a ratio of 1:5 (W / V) through multiple layers of gauze in an anaerobic glove box to prepare the fecal inoculum solution.

[0023] Preferably, in step S2, the anaerobic fermentation comprises: mixing the sample to be tested, the fecal inoculum and the basal culture medium, and incubating at 39°C for 72 hours; the volume ratio of the fecal inoculum to the basal culture medium is 1:1.

[0024] Preferably, the configuration of the basal culture medium includes: per 1000 mL of culture medium containing 0.2 g of peptone, 0.4 g of NH4HCO3, 35 g of NaHCO3, 9.45 g of Na2HPO412H2O, 6.2 g of K2HPO4, 0.6 g of MgSO47H2O, 13.2 mg of CaCl22H2O, 10 mg of MnCl24H2O, 1 mg of CoCl26H2O, 8 mg of FeCl36H2O, and 1 g of cysteine hydrochloride, and 0.1% resazurin is added as an indicator to indicate the anaerobic condition of the culture medium, and carbon dioxide is introduced into the culture medium for saturation after preparation.

[0025] Preferably, in step S2, the step of determining the total sugar content of the residue includes: mixing anhydrous ethanol with the residue and then precipitating, discarding the supernatant and adding 85% ethanol for rinsing, transferring the mixed solution to a centrifuge tube and centrifuging at 8000 rpm for 10 minutes, discarding the supernatant and placing the precipitate in a vacuum oven for drying; adding concentrated sulfuric acid to the dry residue, shaking and mixing evenly, and then shaking in a 35°C water bath for 1 hour, and finally adding 22 mL of water, keeping in a boiling water bath for 2 hours, and then cooling to room temperature, centrifuging to obtain the supernatant, and determining the total sugar content of the residue by the phenol-sulfuric acid method.

[0026] Preferably, in step S3, the calculation formula for the fermentable dietary fiber content is:

[0027] Fermentable dietary fiber = fermentable carbohydrates in the hindgut = percentage of fermentable carbohydrates in the hindgut * carbohydrates entering the hindgut = percentage of fermentable carbohydrates in the hindgut * (total dietary fiber - lignin);

[0028] Among them, total dietary fiber and lignin are the contents in the feed raw material database, or are determined by the AOAC enzymatic-gravimetric method and the acid detergent lignin (ADL) determination method in feed, respectively;

[0029] Hindgut fermentable carbohydrates ratio = hindgut fermentable carbohydrates / carbohydrates entering the hindgut

[0030] = Carbohydrate reduced by fermentation / (total dietary fiber - lignin)

[0031] = (total sugar content of enzymatic residue - total sugar content of fermentation residue) * conversion factor / total sugar content of enzymatic residue * conversion factor

[0032] =(total sugar content of enzymatic residue - total sugar content of fermentation residue) / total sugar content of enzymatic residue.

[0033] Preferably, in step S3, the calculation formula for the fermentable dietary fiber content is:

[0034] Fermentable dietary fiber ≈ total sugar content of enzymatic residue - total sugar content of fermentation residue.

[0035] More preferably, in step S3, the calculation formula for the energy supply of fiber fermentation is:

[0036] Net energy = 9.74*fermentable dietary fiber;

[0037] Among them, 9.74 is the net energy provided by each kg of carbohydrates fermented and utilized in the feed raw material database.

[0038] Preferably, the energy supply of feed raw materials is calculated as follows:

[0039] Net energy = 11.7*digestible protein content + 35.75*digestible fat content + 14.14*(enzymatically digestible starch + 0.9*enzymatically digestible carbohydrates) + 9.74*proportion of fermentable carbohydrates in the hindgut*(total dietary fiber - lignin); or,

[0040] Net energy = 11.7*digestible protein content + 35.75*digestible fat content + 14.14*(enzymatically digestible starch + 0.9*enzymatically digestible carbohydrates) + 9.74*proportion of fermentable carbohydrates in the hindgut*(NSP+fermentable starch+fermentable carbohydrates); or,

[0041] Net energy = Net energy value from database query - 9.74*(NSP*NSP digestibility+fermentable starch+fermentable sugars)+9.74*proportion of fermentable carbohydrates in hindgut*(total dietary fiber-lignin); or,

[0042] Net energy = Net energy value from database query - 9.74*(NSP*NSP digestibility + fermentable starch + fermentable sugars) + 9.74*proportion of fermentable carbohydrates in the hindgut*(NSP+fermentable starch+fermentable sugars);

[0043] Among them, NSP is non-starch polysaccharide;

[0044] 11.7 is the net energy provided by digestion and utilization of 1 kg of protein in the feed raw material database, in MJ;

[0045] 35.75 is the net energy provided by digesting and utilizing 1 kg of fat in the feed raw material database, in MJ;

[0046] 14.14 is the net energy provided by digestion and utilization of 1 kg of starch in the feed raw material database, in MJ;

[0047] The digestible protein content, digestible fat content, enzymatically digestible starch, enzymatically digestible carbohydrates, total dietary fiber, lignin, fermentation-degradable starch, and fermentable carbohydrates can all refer to the values in the feed raw material database.

[0048] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0049] (1) The in vitro fermentation method provided by the present invention has the advantages of simple operation, low cost, good repeatability, and can be used to evaluate the fermentation characteristics of fiber in feed raw materials on a large scale.

[0050] (2) The present invention can replace animal experiments, quantitatively determine the content and proportion of total hindgut fermentable carbohydrates including soluble dietary fiber in feed raw materials and compound feeds, evaluate the fermentation utilization rate of dietary fiber in feed raw materials in the hindgut, and calculate the energy provided by dietary fiber fermentation in feed raw materials; and based on this, the energy value in the feed raw material database can be corrected for animals of different stages and species.

[0051] (3) The present invention can replace animal experiments and quickly and efficiently determine the fermentation and energy supply of fibers in non-conventional feed raw materials that are missing in the database, thus filling the gaps in the database.

[0052] (4) The present invention can conveniently collect corresponding feces as a source of bacteria for in vitro fermentation for pigs of different breeds and stages, and measure the fermentation utilization rate of dietary fiber in feed raw materials by different pigs. Thus, the energy provided by dietary fiber fermentation in feed raw materials can be calculated for pigs of different breeds and stages, and the energy value of each raw material in the database can be corrected to achieve the purpose of accurate formulation. DETAILED DESCRIPTION

[0053] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0054] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0055] The present invention will be further described below with reference to specific examples, but they are not intended to limit the present invention.

[0056] Example 1

[0057] The present invention provides a method for determining the fermentation characteristics of fiber in feed raw materials and the energy supply of fiber fermentation by in vitro fermentation, the steps comprising:

[0058] 1. Sample pretreatment

[0059] (1) Samples with fat content <10%

[0060] If the sample is dry, it can be directly crushed and sieved (30 mesh) for later use.

[0061] If the sample is wet and cannot be crushed directly, weigh an appropriate amount of sample (not less than 5.0 g), place it in a vacuum drying oven at 70℃±1℃ and dry it to constant weight, then crush and sieve it for use.

[0062] Note: If the sample is not suitable for heating, freeze drying can also be used.

[0063] (2) Samples with fat content ≥10%

[0064] To prevent fat from affecting the enzyme's effect on the substrate in the sample and to prevent residual starch from being counted as dietary fiber and causing an inflated result, samples with a fat content ≥10% must be defatted. The defatted sample is then crushed and sieved for later use.

[0065] (3) Samples with sugar content ≥5%

[0066] To prevent excessive sugar content from affecting enzyme activity and potentially inflating the results due to dietary fiber contamination, the sample must be desugared. Weigh an appropriate amount of sample (minimum 5.0 g) and place it in a funnel. Rinse with 85% ethanol at a ratio of 10 mL per gram of sample, discarding the ethanol solution. Repeat this rinse three times. After desugaring, dry the sample in a 40°C oven overnight. Repeatedly crush the dried sample until it is completely sieved; mix thoroughly and set aside.

[0067] Requirements for samples to be tested: pass through a 30-mesh sieve, fat content <10%, sugar content <5%.

[0068] 2. Determination of total sugar content in enzymatic residue

[0069] (1) Accurately weigh the sample (m), approximately 200 mg (accurate to 0.1 mg). Place the sample in a 50 mL centrifuge tube, add 8 mL of MES-TRIS buffer (0.05 mol / L), and shake thoroughly to disperse the sample in the buffer. Simultaneously prepare a blank sample solution and perform the same operation as the sample solution to correct for the effects of the reagents on the determination. Perform at least two replicates for each sample.

[0070] (2) Enzymatic hydrolysis with thermostable α-amylase: Add 10 μL of thermostable α-amylase solution to each sample solution and stir slowly. Seal the container and place in a 95-100°C constant temperature shaking water bath with continuous shaking. When the temperature reaches 95°C, start the timer and react for 35 minutes. Remove the beaker and cool to 60°C. (If the resistant starch content in the sample is >40%, the enzymatic hydrolysis time can be extended to 90 minutes.)

[0071] (3) Protease hydrolysis: Place the sample solution in a 60℃±1℃ water bath, add 20μL of protease solution to each beaker, seal the beaker, start the timer, shake continuously, and react for 30 minutes. Then, add 0.8mL of 3mol / L acetic acid solution while stirring, and control the sample temperature to maintain at 60℃±1℃. Adjust the pH of the sample solution to 4.5±0.2 with sodium hydroxide solution (6mol / L), and then rinse the sample on the pH probe into the conical flask with 0.9mL of deionized water.

[0072] (4) Amyloglucosidase hydrolysis: add 20 μL amyloglucosidase solution while stirring, seal the container, and continue shaking in a 60°C ± 1°C water bath. After reacting for 30 minutes, remove the container.

[0073] (5) Pipette 40 mL of anhydrous ethanol preheated to 60°C into a centrifuge tube, shake well, and allow to precipitate at room temperature for at least 1 hour. Then centrifuge at 8000 rpm for 10 minutes and discard the supernatant (pour out as much supernatant as possible. If there is a lot of precipitate, rinse once with 85% ethanol solution and centrifuge again to remove the precipitate).

[0074] (6) Wash the precipitate with 40 mL of acetone, stir for 5 minutes, and centrifuge at 8000 rpm for 10 minutes. Discard the supernatant and place the precipitate in a centrifuge tube at 65°C in a water bath to dryness to obtain a dry residue. (You can also directly use a vacuum oven to dry the precipitate in step 5.)

[0075] (7) Add 2 mL of 12 mol / L concentrated sulfuric acid, shake and mix evenly, place in a 35°C water bath shaker for 1 h, then quickly add 22 mL of water, place in a boiling water bath for 2 h, then cool to room temperature, centrifuge and collect the supernatant, and determine the total sugar content by the phenol-sulfuric acid method.

[0076] 3. Anaerobic fermentation of samples

[0077] (1) Fecal sample collection and processing: Fresh feces of pigs of specific breed and age were collected, sealed in a ziplock bag, and refrigerated for later use. In an anaerobic glove box, the collected feces were diluted with sterile 0.9% saline saturated with carbon dioxide at a ratio of 1:5 (W / V), and then filtered through 4 layers of gauze to prepare the inoculum.

[0078] (2) Basic culture medium composition and preparation: Each 1000 mL of culture medium contains 0.2 g of peptone, 0.4 g of NH₄HCO₃, 35 g of NaHCO₃, 9.45 g of Na₂HPO₄12H₂O, 6.2 g of K₂HPO₄, 0.6 g of MgSO₄7H₂O, 13.2 mg of CaCl₂2H₂O, 10 mg of MnCl₂4H₂O, 1 mg of CoCl₂6H₂O, 8 mg of FeCl₃6H₂O, and 1 g of cysteine hydrochloride. 0.1% resazurin is added as an indicator to indicate anaerobic conditions. After preparation, the medium is saturated with carbon dioxide and set aside.

[0079] (3) Use a 150 mL syringe with a three-way stop valve as the fermentation container, weigh 200 mg of the sample to be tested and place it into the syringe, then add 20 mL of culture medium and 20 mL of fecal inoculum, and incubate in a 39°C air bath shaker for about 72 h; use the fermentation group without sample addition as a blank control, and perform at least 2 replicates for each group; during this period, observe the syringe scale at regular intervals and record the gas production.

[0080] 4. Determination of total sugar content in fermentation residues

[0081] Inject 4 times the volume of anhydrous ethanol into the syringe through the three-way valve, mix it with the fermented mixture in the syringe, and let it settle at room temperature for at least 1 day. Then carefully discard the supernatant and transfer the precipitate completely to a 50mL centrifuge tube (rinse the syringe twice with 85% ethanol and combine the liquid). Centrifuge at 8000 rpm for 10 minutes and discard the supernatant. If there is a lot of precipitate, wash it once with 85% ethanol and centrifuge it again to get the precipitate. Then put the precipitate and the centrifuge tube into a vacuum oven to dry it to obtain a dry residue. Finally, add 2mL of 12mol / L concentrated sulfuric acid, shake and mix evenly, place it in a 35℃ water bath shaker for 1 hour, then quickly add 22mL of water, place it in a boiling water bath for 2 hours, then cool it to room temperature, centrifuge and get the supernatant. Determine the total sugar content using the phenol-sulfuric acid method.

[0082] 5. Result calculation

[0083] Since the dietary fiber content excluding lignin in the samples before and after fermentation is calculated based on the total sugar results measured by the phenol-sulfuric acid method, there are different conversion factors between the dietary fiber and total sugar determination results in different raw materials. In order to reduce the error caused by the conversion factor, the amount of fermentable dietary fiber in the feed raw materials is expressed as its proportion in the total dietary fiber excluding lignin, that is, the proportion of fermentable carbohydrates entering the hindgut. The calculation formula is as follows:

[0084] Hindgut fermentable carbohydrates ratio = hindgut fermentable carbohydrates / carbohydrates entering the hindgut

[0085] = Carbohydrate reduced by fermentation / (total dietary fiber - lignin)

[0086] = (total sugar content of enzymatic residue - total sugar content of fermentation residue) * conversion factor / total sugar content of enzymatic residue * conversion factor

[0087] =(total sugar content of enzymatic residue - total sugar content of fermentation residue) / total sugar content of enzymatic residue.

[0088] Fermentable dietary fiber = fermentable carbohydrates in the hindgut = percentage of fermentable carbohydrates in the hindgut * carbohydrates entering the hindgut = percentage of fermentable carbohydrates in the hindgut * (total dietary fiber - lignin);

[0089] Note: Total dietary fiber and lignin are the contents in the feed ingredient database, or are determined by the AOAC enzymatic-gravimetric method and the acid detergent lignin (ADL) method in feed, respectively.

[0090] The conversion factor between the determination results of dietary fiber and total sugar can also be ignored, and the fermentable dietary fiber content can be approximately expressed as the difference between the total sugar content of the enzymatic residue and the total sugar content of the fermentation residue, that is:

[0091] Fermentable dietary fiber ≈ total sugar content of enzymatic residue - total sugar content of fermentation residue.

[0092] The energy supply of dietary fiber fermentation can be calculated according to the following formula:

[0093] Net energy (MJ / kg) = 9.74 * fermentable dietary fiber = 9.74 * hindgut fermentable carbohydrates = 9.74 * hindgut fermentable carbohydrate ratio * (total dietary fiber - lignin);

[0094] Based on the data on the proportion of fermentable carbohydrates in the hindgut measured by in vitro fermentation, the energy value of feed ingredients in the current database can also be corrected; according to the different nutritional parameters of feed ingredients in the current database, the corrected net energy can be calculated by the following formula:

[0095] Net energy (MJ / kg) = 11.7 * digestible protein content + 35.75 * digestible fat content + 14.14 * (enzymatically digestible starch + 0.9 * enzymatically digestible carbohydrates) + 9.74 * hindgut fermentable carbohydrates * (total dietary fiber - lignin)

[0096] Net energy (MJ / kg) = 11.7 * digestible protein content + 35.75 * digestible fat content + 14.14 * (enzymatically digestible starch + 0.9 * enzymatically digestible carbohydrates) + 9.74 * hindgut fermentable carbohydrates proportion * (NSP + fermentable degradable starch + fermentable carbohydrates)

[0097] Net energy (MJ / kg) = Net energy value from database query - 9.74 * (NSP * NSP digestibility + fermentable starch + fermentable sugars) + 9.74 * hindgut fermentable carbohydrates percentage * (total dietary fiber - lignin)

[0098] Net energy (MJ / kg) = Net energy value from database query - 9.74 * (NSP * NSP digestibility + fermentable starch + fermentable sugars) + 9.74 * hindgut fermentable carbohydrates percentage * (NSP + fermentable starch + fermentable sugars)

[0099] NSP: non-starch polysaccharide;

[0100] 11.7: The net energy provided by digesting 1kg of protein is 11.7MJ;

[0101] 35.75: The net energy provided by 1kg of fat digested and utilized is 35.75MJ;

[0102] 14.14: The net energy provided by digesting 1kg of starch is 14.14MJ;

[0103] 9.74: The net energy provided by each kg of carbohydrates used in fermentation is 9.74 MJ.

[0104] The digestible protein content, digestible fat content, enzymatically digestible starch, enzymatically digestible carbohydrates, total dietary fiber, lignin, fermentation-degradable starch, and fermentable carbohydrates can all refer to the values in the feed raw material database.

[0105] Example 2

[0106] Determination of fermentable fiber content in feed ingredients by in vitro fermentation of fecal microorganisms from fattening pigs (Du*Chang*Da)

[0107] Wheat bran, defatted rice bran, soybean hulls, oat bran, beet pulp, linseed meal, and palm kernel meal were selected as the feed ingredients to be tested. Fresh feces from six approximately 120 kg fattening pigs (Du*Chang*Da) at the Anyou Taicang Experimental Pig Farm were mixed and used as a fermentation bacterial source. The proportion of fermentable carbohydrates in the hindgut was determined according to the specific procedures of Example 1. The results are shown in Table 1.

[0108] Table 1: Results of in vitro fermentation test of bacterial sources from fattening pig feces

[0109]

[0110] Query the relevant nutritional indicators of each raw material in the CVB 2018 database, see Table 2;

[0111] Table 2: Nutritional indicators of various raw materials in the CVB 2018 database

[0112]

[0113]

[0114] The net energy provided by fiber fermentation was calculated using the following two formulas:

[0115] Net energy provided by fiber fermentation in the CVB raw material database (MJ / kg) = 9.74*(NSP*NSP digestibility + fermentable starch + fermentable sugars)

[0116] Net energy provided by fiber fermentation (MJ / kg) measured by in vitro fermentation method = 9.74 * proportion of fermentable carbohydrates in the hindgut * (NSP + fermentable degradable starch + fermentable sugars)

[0117] The calculation results are shown in Table 3.

[0118] Table 3: Comparison of net energy provided by in vitro fermentation of fattening pig manure and fiber fermentation in the CVB database

[0119]

[0120] From the results in Table 3, it can be seen that the calculated value of the in vitro fermentation energy supply of dietary fiber in the raw materials is well consistent with the net energy value provided by the fattening pig fiber part in the CVB database, indicating that the in vitro fermentation method for determining the fermentation characteristics and fermentation energy supply of dietary fiber in raw materials provided by the present invention is highly accurate, can be used to measure the energy value provided by the fiber in the raw materials during fermentation in the animal hindgut, and has practical value.

[0121] Example 3

[0122] The fermentable fiber content of feed raw materials was determined by in vitro fermentation of lactating sow (Landrace*Largewhite) fecal bacteria, and the energy value provided by fiber fermentation was calculated. The energy value of the raw materials in the database was corrected accordingly.

[0123] Wheat bran, defatted rice bran, soybean hulls, oat bran, beet meal, linseed meal, palm kernel meal, corn husks, 46 soybean meal, citrus pulp, and barley hull powder were selected as the feed ingredients to be tested. Fresh feces from six lactating sows (Landrace and Large White) of parity 2 to 5 at a Jintan pig farm were collected and mixed as a fermentation bacterial source. The proportion of fermentable carbohydrates in the hindgut was determined according to the specific procedures in Example 1. The results are shown in Table 4.

[0124] Table 4: In vitro fermentation test results of lactating sow fecal bacteria

[0125]

[0126]

[0127] Query the relevant nutritional indicators of each raw material in the CVB 2018 database, see Table 5.

[0128] Table 5: Nutritional indicators of various raw materials in the CVB 2018 database

[0129]

[0130] The net energy value provided by fiber fermentation is then calculated according to the following two formulas:

[0131] Net energy provided by fiber fermentation in the CVB raw material database (MJ / kg) = 9.74*(NSP*NSP digestibility + fermentable starch + fermentable sugars)

[0132] Net energy provided by fiber fermentation (MJ / kg) measured by in vitro fermentation method = 9.74 * proportion of fermentable carbohydrates in the hindgut * (NSP + fermentable degradable starch + fermentable sugars)

[0133] The calculation results are shown in Table 6.

[0134] Table 6: Comparison of net energy provided by in vitro fermentation of lactating sow feces and fiber fermentation in the CVB database

[0135]

[0136] Comparing the calculated values of dietary fiber fermentation energy in the raw materials determined by in vitro fermentation of lactating sow feces in Table 6 with the net energy values provided by the fiber part of fattening pigs in the CVB database, it can be found that the values of some raw materials are similar, while the values of some raw materials are quite different, which reflects that the fermentation utilization rate of fiber in lactating sows is quite different from that in fattening pigs.

[0137] Furthermore, the relevant nutritional indicators of protein and fat in the CVB database were queried, see Table 7.

[0138] Table 7: Nutritional indicators of various raw materials in the CVB 2018 database

[0139]

[0140] Then calculate the net energy correction value of the raw material for lactating sows according to the following formula:

[0141] Net energy (MJ / kg) = 11.7 * digestible protein content + 35.75 * digestible fat content + 14.14 * (enzymatically digestible starch + 0.9 * enzymatically digestible carbohydrates) + 9.74 * hindgut fermentable carbohydrates proportion * (NSP + fermentable degradable starch + fermentable carbohydrates)

[0142] The calculation results are shown in Table 8.

[0143] Table 8: Net energy value of raw materials calculated based on in vitro fermentation results of lactating sow feces bacteria (corrected value)

[0144]

[0145] Example 4

[0146] The fermentable fiber content of unconventional feed ingredients was determined by in vitro fermentation of lactating sow (Landrace*Largewhite) fecal bacteria, and the energy value provided by fiber fermentation was calculated.

[0147] Defatted coconut flakes, pea hulls, Wanli fiber, rice husk powder, seaweed residue, sugarcane bagasse, bamboo fiber, and snow swallow shreds were selected as the feed ingredients to be tested. Fresh feces from six lactating sows (Landrace and Large White) of parities 2-5 at a Jintan pig farm were collected and mixed as a fermentation bacterial source. The proportion of fermentable carbohydrates in the hindgut was determined according to the specific procedures in Example 1. The results are shown in Table 9.

[0148] Table 9: In vitro fermentation test results of lactating sow fecal bacteria

[0149]

[0150]

[0151] When the conversion coefficient between the dietary fiber and total sugar determination results is ignored, the energy supply of dietary fiber fermentation in the raw materials can be calculated according to the following formula to obtain an approximate result:

[0152] Net energy (MJ / kg) = 9.74*fermentable dietary fiber ≈ 9.74*(total sugar content of enzymatic residue - total sugar content of fermentation residue)

[0153] Table 10: Fermentation energy value of dietary fiber in raw materials measured by in vitro fermentation of lactating sow feces bacteria

[0154] raw material Net energy provided by dietary fiber fermentation (MJ / kg) defatted coconut flakes 7.46 pea skins 7.82 Wanli Fiber 3.15 Rice husk powder 0.01 Seaweed residue 6.25 Bagasse 2.05 bamboo fiber 1.08 Snow Swallow Broken 0

[0155] In summary, the present invention can replace animal experiments, determine the fermentation utilization rate of dietary fiber in feed raw materials by pigs at different stages, calculate the energy provided by dietary fiber through fermentation, and accordingly correct or calculate the energy value of feed raw materials in the formula database.

[0156] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the present invention specification should be included in the protection scope of the present invention.

Claims

1. A method for determining the fermentation characteristics and energy supply of fiber in feed raw materials by in vitro fermentation, characterized in that the steps include: S1. Pre-treating the feed raw materials to obtain a sample to be tested, performing enzymatic hydrolysis on the sample to be tested, and then hydrolyzing the residue after enzymatic hydrolysis to determine the total sugar content; S2. Collect animal feces to prepare a fecal inoculum, mix the test sample, the fecal inoculum, and the basal culture medium, and then perform anaerobic fermentation. After the anaerobic fermentation is completed, separate the residue, and hydrolyze the residue to determine the total sugar content; S3. Calculate the fermentable dietary fiber content in the feed raw material and the energy supply of fiber fermentation by the difference between the total sugar content of the enzymatic hydrolysis residue of the test sample and the total sugar content of the fermentation residue.

2. The method according to claim 1, characterized in that In step S1, the pretreatment includes: performing drying treatment, defatting treatment or desugaring treatment according to the moisture content, fat content and sugar content of the feed raw material, and then crushing and screening to obtain the pretreated feed raw material.

3. The method according to claim 2, characterized in that The fat content of the pretreated feed raw materials is less than 10%, and the sugar content is less than 5%.

4. The method according to claim 1, wherein In step S1, the enzymatic hydrolysis treatment includes: dispersing the pretreated feed raw material in a buffer solution, and sequentially performing enzymatic hydrolysis with a thermostable α-amylase, enzymatic hydrolysis with a protease, and enzymatic hydrolysis with amyloglucosidase.

5. The method according to claim 1, wherein In step S2, the preparation of the fecal inoculum solution includes: taking fresh feces of pigs of a specific breed and age, filtering the collected feces with sterile 0.9% saline saturated with carbon dioxide at a ratio of 1:5 (W / V) through multiple layers of gauze in an anaerobic glove box to prepare the fecal inoculum solution.

6. The method according to claim 1, characterized in that In step S2, anaerobic fermentation includes: mixing the sample to be tested, the fecal inoculum and the basal culture medium, and incubating at 39° C. for 72 hours; the volume ratio of the fecal inoculum to the basal culture medium is 1:

1.

7. The method according to claim 1, characterized in that In step S2, the step of determining the total sugar content of the residue includes: mixing anhydrous ethanol with the residue and then precipitating it, discarding the supernatant and adding 85% ethanol for rinsing, transferring the mixed solution to a centrifuge tube and centrifuging it at 8000 rpm for 10 minutes, discarding the supernatant and placing the precipitate in a vacuum oven for drying; adding concentrated sulfuric acid to the dry residue, shaking and mixing it evenly, and then shaking it in a water bath at 35°C for 1 hour, and finally adding 22 mL of water, keeping it in a boiling water bath for 2 hours, and then cooling it to room temperature, centrifuging and taking the supernatant, and determining the total sugar content of the residue by the phenol-sulfuric acid method.

8. The method according to claim 1, characterized in that In step S3, the calculation formula for the fermentable dietary fiber content is: Fermentable dietary fiber = fermentable carbohydrates in the hindgut = percentage of fermentable carbohydrates in the hindgut * carbohydrates entering the hindgut = percentage of fermentable carbohydrates in the hindgut * (total dietary fiber - lignin); Among them, total dietary fiber and lignin are the contents in the feed raw material database, or are determined by the AOAC enzymatic-gravimetric method and the acid detergent lignin (ADL) determination method in feed, respectively; Hindgut fermentable carbohydrates ratio = hindgut fermentable carbohydrates / carbohydrates entering the hindgut = Carbohydrates reduced by hindgut fermentation / (total dietary fiber - lignin) = (total sugar content of enzymatic residue - total sugar content of fermentation residue) * conversion factor / total sugar content of enzymatic residue * conversion factor =(total sugar content of enzymatic residue - total sugar content of fermentation residue) / total sugar content of enzymatic residue.

9. The method according to claim 1, characterized in that In step S3, the calculation formula for the fermentable dietary fiber content is: Fermentable dietary fiber ≈ total sugar content of enzymatic residue - total sugar content of fermentation residue.

10. The method according to claim 8 or 9, characterized in that In step S3, the calculation formula for the fiber fermentation energy supply is: Net energy = 9.74*fermentable dietary fiber; Among them, 9.74 is the net energy provided by each kg of carbohydrates fermented and utilized in the feed raw material database.

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