A method of analysis of intestinal chyme carbohydrate components

Abstract

The application belongs to the technical field of biological analysis, and specifically discloses an analysis method for intestinal chyme carbohydrate components. The intestinal chyme carbohydrate components include monosaccharide components and polysaccharide components; the intestinal chyme to be detected is dried and then re-dissolved in water, and the part dissolved in water is subjected to ion chromatography detection to obtain monosaccharide components that have been degraded by the intestine but not absorbed; the part not dissolved in water is dried and then re-dissolved in acid, and subjected to ion chromatography detection to obtain monosaccharide components that have not been degraded by the intestine; the above two results are integrated to obtain monosaccharide components that are not utilized in the intestinal chyme to be detected; the intestinal chyme to be detected is subjected to enzyme analysis and microbial fermentation analysis respectively, the digestible polysaccharide content in the intestinal chyme is obtained through enzyme analysis, and the indigestible polysaccharide components in the intestinal chyme are obtained through microbial fermentation analysis. The method in the application can realize the analysis of complex intestinal chyme carbohydrate components.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an analytical method for carbohydrate components of intestinal chyme. Background Technology

[0002] Carbohydrate polysaccharides are complex carbohydrate compounds formed by multiple monosaccharide molecules linked by glycosidic bonds. They include digestible and indigestible polysaccharides. Digestible carbohydrate polysaccharides, mainly including starch, can be degraded by animal digestive enzymes. Indigestible carbohydrate polysaccharides, however, are difficult to utilize, primarily due to their complex molecular structure and unique chemical bonding. Compared to digestible carbohydrates such as starch and glycogen, these indigestible polysaccharides possess unique chemical structural characteristics that make them difficult for the body's digestive enzymes to effectively degrade and absorb. First, many indigestible carbohydrate polysaccharides, such as cellulose, hemicellulose, and pectin, have complex molecular chain structures that hinder the hydrolytic action of digestive enzymes. Furthermore, the complexity of the molecular structure and the heterogeneity of the molecular weight of indigestible polysaccharides make their quantitative and qualitative detection challenging.

[0003] The carbohydrate composition of intestinal chyme reflects the body's digestion and absorption of carbohydrates. Analyzing these components allows us to understand the digestion rate and absorption efficiency of different carbohydrates in the intestine, thereby assessing intestinal digestive and absorptive function. Currently, the main analytical method for carbohydrate components is gas chromatography / liquid chromatography. However, gas chromatography requires derivatization to volatilize monosaccharides, a process that may lead to monosaccharide loss or degradation. Furthermore, some monosaccharides, such as uronic acids, are difficult to derivatize, which in some cases hinders the accurate analysis of both polysaccharides and monosaccharides using gas chromatography. In addition, the same monomer may exhibit different derivatized forms, resulting in multiple spectra of the same molecule, reducing analytical accuracy. While high-performance liquid chromatography (HPLC) is relatively simple to operate, polarity is the primary mechanism for separation, which reduces chromatographic resolution. In the detection of complex mixtures like intestinal chyme, only monosaccharide components can be detected, while polysaccharide components are inferred from the monosaccharide components, thus failing to achieve accurate analysis of polysaccharide components. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an analytical method for the carbohydrate components of intestinal chyme. This invention comprehensively analyzes the carbohydrate components in intestinal chyme using ion chromatography, enzymatic analysis, and microbial fermentation analysis techniques.

[0005] A method for analyzing the carbohydrate composition of intestinal chyme, comprising the following steps:

[0006] The carbohydrate components of the intestinal chyme include monosaccharide and polysaccharide components;

[0007] The intestinal chyme to be tested was dried and then reconstituted with water. The water-soluble portion was detected by ion chromatography to obtain the monosaccharide components that had been degraded by the intestine but not absorbed. The water-insoluble portion was dried, reconstituted with acid, and then detected by ion chromatography to obtain the monosaccharide components that had not been degraded by the intestine.

[0008] The intestinal chyme to be tested was subjected to enzyme analysis and microbial fermentation analysis. Enzyme analysis was used to obtain the content of digestible polysaccharides in the intestinal chyme, and microbial fermentation analysis was used to obtain the content of indigestible polysaccharides in the intestinal chyme. The above four results were integrated to determine the specific monosaccharide and polysaccharide components in the intestinal chyme.

[0009] This invention, by performing ion chromatography detection on both water-insoluble and water-soluble components, can accurately identify monosaccharide components that have been degraded by the intestines but not absorbed, as well as monosaccharide components that have not been degraded by the intestines. This allows for the identification of unutilized monosaccharide components in the intestinal chyme. Furthermore, through enzyme analysis and microbial fermentation analysis, the digestible and indigestible polysaccharide contents in the intestinal chyme can be determined. Through the step-by-step analysis described above, precise analysis of undigested and degraded carbohydrate components in the intestinal chyme can be achieved.

[0010] In another preferred embodiment, the reagent used for acid redissolution is 2M~2.5M trifluoroacetic acid.

[0011] In another preferred embodiment, the intestinal chyme to be detected is chyme from different intestinal segments; the intestinal segments are the duodenum, jejunum, ileum, cecum, and colon.

[0012] In another preferred embodiment, the conditions for ion chromatography detection are as follows:

[0013] Column: CarboPac™ PA1;

[0014] Mobile phase A is H2O, and mobile phase B is 100mM NaOH. An elution gradient is used, and all mobile phase proportions are volume percentages, as detailed below:

[0015] From 0 min to 9 min, mobile phase A was 95% and mobile phase B was 5%.

[0016] From 9 min to 20 min, mobile phase A was 95% and mobile phase B was 5%.

[0017] 20-30 minutes, mobile phase B is 100%;

[0018] 30-60 min, mobile phase A is 95%, mobile phase B is 5%;

[0019] The injection volume was 5 μL;

[0020] The flow rate was 0.5 mL / min;

[0021] The column temperature is 30℃.

[0022] In another preferred embodiment, the enzyme analysis process is as follows:

[0023] The intestinal chyme to be tested was incubated with α-amylase in an aqueous environment to obtain the content of undigested polysaccharides in the intestinal chyme to be tested; the intestinal chyme to be tested was co-incubated with water to obtain the content of undegraded polysaccharides in the intestinal chyme to be tested.

[0024] The digestible polysaccharide content is obtained by subtracting the undegraded polysaccharide content from the undigested polysaccharide content. In another preferred embodiment, the amount of α-amylase added is 0.01% to 0.02% of the mass of the intestinal chyme to be tested.

[0025] The incubation temperature is 37℃~39℃, and the time is 30min~60min.

[0026] In another preferred embodiment, the specific process of the microbial fermentation is as follows:

[0027] Dissolve the intestinal chyme to be tested in sterile anaerobic saline, centrifuge, and take the supernatant as inoculum;

[0028] The inoculum was inoculated into a culture medium containing polysaccharide substrate and fermented at 37℃~39℃ for 72h~96h.

[0029] The inoculum amount is 8% to 10% of the culture medium volume;

[0030] In the culture medium, the concentration of the polysaccharide substrate is 0.008 g / mL to 0.01 g / mL.

[0031] In another preferred embodiment, the polysaccharide substrate is any one of pectin, xylan, arabinoxylan, β-glucan, and glucomannan.

[0032] In another preferred embodiment, each liter of culture medium contains 0.0156 g calcium chloride dihydrate, 0.0118 g manganese chloride tetrahydrate, 0.0011 g cobalt chloride hexahydrate, 0.0095 g ferric chloride hexahydrate, 0.9511 g ammonium carbonate, 8.3222 g sodium carbonate, 0.3999 g L-cysteine, 1.355 g sodium dihydrogen phosphate, 1.474 g potassium hydrogen phosphate, 0.1426 g magnesium sulfate heptahydrate, 0.2972 g sodium sulfide nonahydrate, 0.0760 g sodium hydroxide, and 0.0011 g resazurite, with water added to make up to 1 L.

[0033] Compared with the prior art, the present invention has the following beneficial effects.

[0034] This invention comprehensively analyzes the carbohydrate components in intestinal chyme using ion chromatography, enzymatic analysis, and microbial fermentation analysis, effectively solving the problem of existing technologies' inability to accurately analyze polysaccharide components. Ion chromatography is used to analyze water-soluble and water-insoluble matter in intestinal chyme separately. The ion chromatography results of water-soluble matter reveal monosaccharides that have been degraded by the intestine but not absorbed; the ion chromatography results of water-insoluble matter reveal monosaccharides that have not been degraded by the intestine. Combining the results of both ion chromatography analyses allows for the accurate identification of unutilized monosaccharides in the intestine. Enzymatic analysis further determines the content of digestible polysaccharides, and microbial fermentation analysis further identifies indigestible polysaccharides, thus achieving a comprehensive analysis of undigested carbohydrates in intestinal chyme.

[0035] The method in this invention provides a comprehensive analysis of carbohydrates in intestinal chyme and accurately assesses the digestion, absorption, and fermentation of different carbohydrates in the intestine, thereby providing a more precise basis for intestinal health research and personalized dietary plans. Attached Figure Description

[0036] Figure 1 This is a graph showing the absolute molecular weight analysis of carbohydrate components in intestinal chyme; in the graph, 1 represents the difference signal; 2 represents the multi-angle laser light scattering signal; and 3 represents the intensity of the light scattering signal.

[0037] Figure 2 A is a linear distribution of the absolute molecular weight of carbohydrate components in intestinal chyme; B is a linear distribution of the absolute molecular weight of carbohydrate components in the range of 0 g / mol to 300,000 g / mol; C is a linear distribution of the absolute molecular weight of carbohydrate components in the range of 0 g / mol to 10,000 g / mol.

[0038] Figure 3 The graphs show the results of all polysaccharide monomers that have been degraded but not absorbed by the animal's intestines, as well as the results of unutilized monosaccharides in each intestinal digesta; A is the result of unabsorbed glucose content; B is the result of unabsorbed xylose content; C is the result of unabsorbed fructose content; D is the result of unabsorbed galactose content; E is the result of unabsorbed arabinose content; F is the result of unabsorbed rhamnose content; G is the result of absorbed mannose content; and H is the result of unabsorbed fucose content.

[0039] Figure 4The graph shows the monosaccharide content of all polysaccharides that were not degraded in the corresponding animal intestines; A represents the content of unused glucose; B represents the content of unused xylose; C represents the content of unused fructose; D represents the content of unused galactose; E represents the content of unused arabinose; F represents the content of unused rhamnose; G represents the content of unused mannose; and H represents the content of unused fucose.

[0040] Figure 5 The graph shows the results of the monosaccharide content of carbohydrates that are not utilized by the corresponding intestinal segments; A is the result of the glucose content in undegraded polysaccharides; B is the result of the xylose content in undegraded polysaccharides; C is the result of the fructose content in undegraded polysaccharides; D is the result of the galactose content in undegraded polysaccharides; E is the result of the arabinose content in undegraded polysaccharides; F is the result of the rhamnose content in undegraded polysaccharides; G is the result of the mannose content in undegraded polysaccharides; and H is the result of the fucose content in undegraded polysaccharides.

[0041] Figure 6 The graph shows the starch content in intestinal chyme.

[0042] Figure 7 Figure A shows the gas production results of polysaccharide substrates fermented by colonic chyme microorganisms; Figure B shows the gas production results of glucomannan; Figure C shows the gas production results of β-glucan; Figure D shows the gas production results of xylan; Figure E shows the gas production results of arabinoxylan; Figure E shows the gas production results of pectin. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0044] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0045] English-Chinese translation: GPC / SEC-MALS indicates high-temperature gel permeation chromatography coupled with an 18-angle laser light scattering system; ICS indicates ion chromatography detection; IUGR indicates intrauterine growth restriction.

[0046] The differential detector was an Optilab T-rEX, and the laser light scattering detector was a DAWN HELEOS II, both purchased from Wyatt Technology, CA, USA. The automated gas production recording system was purchased from AGRS-III, China Agricultural University, Beijing. The ion chromatography system was a Thermo ICS5000 purchased from Dionex, Thermo Scientific, Waltham, US.

[0047] Using pigs as an animal model for intestinal chyme research, a system for analyzing the carbohydrate utilization characteristics of intestinal chyme was established to reveal the characteristics of carbohydrate digestion and absorption in the pig intestine.

[0048] 1. Material preparation.

[0049] 1.1 Collect intestinal chyme.

[0050] Using pigs as a research model, intestinal chyme was collected. The molecular weight distribution of carbohydrates in the intestinal chyme was detected, the utilization characteristics of polysaccharides in the intestinal chyme were analyzed, and the digestible carbohydrate content was detected by enzymatic hydrolysis. The collected intestinal chyme was directly stored in liquid nitrogen for later use. The carbohydrate composition of the chyme was verified by microbial fermentation. To ensure the activity of microorganisms in the intestinal chyme, 20% glycerol by weight of the intestinal chyme was added, mixed well, and stored in liquid nitrogen for later use.

[0051] 1.2 Model Establishment.

[0052] Normal pigs and pigs with different growth potentials were used as research models, as detailed below.

[0053] At birth, piglets were classified as IUGR pigs based on their birth weight, with those weighing more than two standard deviations below the normal birth weight (0.8 kg–1.1 kg). At 70 days of age, individuals with an average weight significantly below the normal weight were defined as low-growth-potential IUGR pigs. Low-growth-potential IUGR pigs at 98 days of age were designated as low-growth-potential-time-compensated IUGR pigs. Individuals not significantly below the normal average weight were defined as high-growth-potential IUGR pigs. Intestinal digesta from four different pig groups were collected. The molecular weight distribution of carbohydrates in the digesta was analyzed, the utilization characteristics of polysaccharides in the digesta were analyzed, and the digestible carbohydrate content was determined using enzymatic hydrolysis. The collected intestinal digesta was then stored directly in liquid nitrogen for later use.

[0054] 2. Different analytical methods.

[0055] 2.1 The carbohydrate composition of intestinal chyme was detected using the GPC / SEC-MALS method.

[0056] 1) Sample pretreatment: The intestinal chyme of the above four swine groups was dissolved in 0.1M NaNO3 aqueous solution to a final concentration of 1mg / mL and filtered through a filter with a pore size of 0.45μm.

[0057] 2) Instrument Detection: The chromatographic system used was a high-temperature gel permeation chromatography system coupled with 18-angle laser light scattering. The differential detector was Optilab T-rEX, the laser light scattering detector was DAWN HELEOS Ⅱ, and the gel size exclusion columns were Ohpak SB-805 HQ, 300×8mm, Ohpak SB-804 HQ, 300×8mm, and Ohpak SB-803 HQ, 300×8mm. The column temperature was 45℃, the injection volume was 100μL, the mobile phase A was 0.1M NaNO3, the flow rate was 0.4ml / min, and the elution gradient was isocratic for 100min.

[0058] Qualitative results: Figure 1 The diagram shows the absolute molecular weight analysis of carbohydrate components in chyme: retention time is plotted on the x-axis, and molar mass on the y-axis. The multi-angle laser light scattering signal shows that the intensity of the scattered light is proportional to the molecular size and molecular weight of the substance; 1 represents the difference signal, the response value of which depends on the change in the refractive index of the post-column effluent and is related to the type, concentration, and molecular weight of the substance; 3 represents the molecular weight fitted from the two signals. 2 represents the change in molecular weight, showing the change in the sample's molecular weight, i.e., molar mass, over time. Figure 1 As shown in the figure, the molecular weight fluctuated, possibly indicating that polymerization or depolymerization occurred at that time point. Significant changes in molecular weight suggest structural changes, such as molecular aggregation or degradation, that may have occurred during this period, related to reaction or separation processes. Curve 3 represents the intensity of the light scattering signal, typically used to detect changes in the concentration of particles or macromolecules in a sample. The figure shows a strong light scattering signal between 28 and 30 minutes, indicating aggregation of larger particles or macromolecules in the sample, possibly consistent with the fluctuations in molecular weight. This suggests that polymerization or an increase in the number of macromolecules may have occurred at these time points. Curve 1 shows the change in the refractive index of the sample, reflecting changes in the solute concentration. The figure shows a correlation between the change in refractive index and the fluctuations in molecular weight and light scattering signal, indicating that the concentration of the solute in the sample also changed significantly over time. Specific changes may be related to variations in the quantity and properties of the dissolved substances in the sample.

[0059] Quantitative results: Figure 2The figure shows the molecular weight distribution of carbohydrate components in chyme. The logarithm of molar mass is used as the horizontal axis, and the linear proportion corresponding to the molecular weight is used as the vertical axis, which represents the relative content at different molecular weights. The molecular weight of carbohydrate components in the chyme sample shown in the figure is mainly distributed between 4500 g / mol and 10000 g / mol.

[0060] The results above show that the GPC / SEC-MALS method for detecting carbohydrate components in intestinal chyme can only analyze the molecular weight of carbohydrates in intestinal chyme, but cannot analyze the specific polysaccharide and monosaccharide components, and therefore cannot realize the degree of utilization of polysaccharides and monosaccharides by the intestine.

[0061] 2.2. Ion chromatography, enzyme analysis, and microbial fermentation analysis were used to detect carbohydrates in intestinal chyme.

[0062] As can be seen from the results in section 2.1, the GPC / SEC-MALS method alone is insufficient to determine the polysaccharide and monosaccharide composition of intestinal chyme. Therefore, to accurately determine the polysaccharide and monosaccharide composition of intestinal chyme and the degree of intestinal utilization of polysaccharides and monosaccharides, this embodiment combines ion chromatography detection, enzyme analysis, and microbial fermentation analysis to comprehensively analyze the polysaccharide and monosaccharide composition of intestinal chyme, thereby determining the utilization of polysaccharides and monosaccharides in different intestinal segments. The different intestinal segments refer to the duodenum, jejunum, ileum, cecum, and colon. The specific methods are as follows.

[0063] 2.2.1 Determination of monosaccharide composition and content in intestinal chyme using ICS.

[0064] 1) Sample pretreatment: Different intestinal segments of chyme were weighed, freeze-dried, and 200 mg of dried samples of different intestinal segments were obtained. The weight of the freeze-dried samples was used as the total weight for calculating the detection concentration. The dried samples of different intestinal segments were reconstituted with ultrapure water. The water-soluble portion was detected by ion chromatography to obtain the monosaccharide components that were degraded but not absorbed in different intestinal segments. The water-insoluble portion was dried with nitrogen and then reconstituted with 2M trifluoroacetic acid and detected by ion chromatography to obtain the monosaccharide components that were not degraded by the intestine. The two portions were combined to obtain all carbohydrates that were not utilized by the corresponding intestinal segments.

[0065] 2) Detection method: Ion chromatography was used for detection, and an electrochemical detector was employed to analyze and detect the monosaccharide components. The monosaccharides detected mainly included those commonly found in the diet, as well as polysaccharide monomers: glucose, xylose, fructose, galactose, arabinose, rhamnose, mannose, and fucose.

[0066] The CarboPac™ PA 150*4.0mm liquid chromatography column was used; mobile phase A was H2O, mobile phase B was 100mM NaOH; the injection volume was 5uL, the flow rate was 0.5mL / min, and the column temperature was 30℃.

[0067] Elution using a gradient elution method, where all mobile phase proportions are volume percentages, as detailed below:

[0068] From 0 min to 9 min, mobile phase A was 95% and mobile phase B was 5%.

[0069] 9 min to 20 min, mobile phase A is 95% and mobile phase B is 5%.

[0070] 20-30 minutes, mobile phase B is 100%.

[0071] 30-60 minutes, mobile phase A is 95% and mobile phase B is 5%.

[0072] Figure 3 The image shows the results of all monosaccharides that have been degraded but not absorbed by the animal's intestines in the chyme of each intestinal segment, and their content; from Figure 3 It can be seen that there was no difference in the content of free polysaccharide monomers glucose, xylose, fructose, galactose, arabinose, rhamnose, mannose and fucose in the intestines among the four groups. This indicates that there is no difference in the absorption of degraded monosaccharides in the intestines of normal pigs, low growth potential IUGR pigs, low growth potential time-compensated IUGR pigs and high growth potential IUGR pigs.

[0073] Figure 4 The figure shows the monosaccharides that were not degraded in each intestinal segment and their content. Figure 4 It can be seen that, compared with the normal pig group and the high growth potential IUGR pig group, the low growth potential IUGR pig group and the low growth potential time-compensated IUGR pig group had increased levels of undegraded glucose, xylose, and arabinose in the cecum and colon. Furthermore, compared with the normal pig group and the high growth potential IUGR pig group, the low growth potential IUGR pig group had increased levels of undegraded mannose and rhamnose in the cecum and increased levels of undegraded galactose in the colon.

[0074] Figure 5 The figure shows the sum of all monosaccharides that were not utilized by each segment of the intestine, i.e. Figure 3 and Figure 4 The result after superposition. From Figure 5It can be seen that, compared with the normal pig group and the high growth potential IUGR pig group, the low growth potential IUGR pig group and the low growth potential time-compensated IUGR pig group had increased levels of unutilized glucose, xylose, and arabinose in the cecum and colon. Furthermore, compared with the normal pig group and the high growth potential IUGR pig group, the low growth potential IUGR pig group had increased levels of unutilized mannose and rhamnose in the cecum and increased levels of unutilized galactose in the colon.

[0075] 2.2.2 Utilize enzyme analysis and microbial fermentation analysis.

[0076] 1) Digestive enzyme analysis method for detecting digestible starch content in intestinal chyme.

[0077] 100 mg of chyme sample was weighed and 125 μL of 72% α-amylase was added. The mixture was incubated in ultrapure water at 39°C for 60 min. The undigested starch content was determined by iodometric titration. 100 mg of intestinal chyme was also incubated in ultrapure water at 39°C for 60 min, and the undegraded starch content was determined by iodometric titration. The digestible starch content was obtained by subtracting the undegraded starch content from the undigested starch content. Results are as follows: Figure 6 As shown. From Figure 6 It can be seen that the starch content in the duodenum and jejunum was relatively high in all groups, and the differences between different groups were not significant. These data indicate that starch digestion was relatively consistent in the duodenum, jejunum, and ileum. Significant differences were found in the cecum and colon between the normal pig group and the low growth potential IUGR pig group, indicating that the low growth potential IUGR pig group had a higher content of undegraded starch compared to the normal pig group.

[0078] 2) Microbial fermentation of carbohydrate substrates to verify indigestible polysaccharide components.

[0079] Sample pretreatment: Intestinal chyme supplemented with 20 wt% glycerol was preheated at 39°C for 1 hour, diluted 4-fold with sterile anaerobic saline, and centrifuged at 1000 rpm for 10 min. The supernatant was used as the intestinal chyme inoculum. Common dietary polysaccharides—pectin, xylan, arabinoxylan, β-glucan, and glucomannan—were used as polysaccharide substrates. The culture system consisted of 0.2 g of polysaccharide substrate, 25 mL of culture medium, and 2 mL of intestinal chyme inoculum.

[0080] Each liter of culture medium contains 0.0156g calcium chloride dihydrate, 0.0118g manganese chloride tetrahydrate, 0.0011g cobalt chloride hexahydrate, 0.0095g ferric chloride hexahydrate, 0.9511g ammonium carbonate, 8.3222g sodium carbonate, 0.3999g L-cysteine, 1.355g sodium dihydrogen phosphate, 1.474g potassium hydrogen phosphate, 0.1426g magnesium sulfate heptahydrate, 0.2972g sodium sulfide nonahydrate, 0.0760g sodium hydroxide, and 0.0011g resazurite, with water added to make up to 1L.

[0081] Fermentation system: Carbon dioxide gas was introduced into the culture medium to remove oxygen, the pH was adjusted to 6.86, and sterilization was performed. 0.2g of polysaccharide substrate was weighed and sterilized by ultraviolet irradiation for later use. In the anaerobic workstation, the above five polysaccharide substrates were added to 25 mL of cooled culture medium, placed in fermentation flasks, and 2 mL of intestinal chyme inoculum was added. After sealing, the flasks were preheated to 39°C in an incubator. Then, a cumulative gas production experiment was conducted using an automated gas production recording system, and the cumulative gas production was continuously recorded for 72 hours. Figure 7 The gas production curves for colonic chyme microorganisms fermenting polysaccharide substrates are shown. In the colon, the bacterial strain from high-growth-potential IUGR pigs exhibited higher gas production for xylan and β-glucan compared to low-growth-potential IUGR pigs.

[0082] The results obtained from the ion chromatography analysis include monosaccharides that have been degraded but not absorbed by the intestines, as well as monosaccharides that have not been degraded by the intestines; these constitute the monosaccharide analysis results. The enzyme analysis results and microbial fermentation analysis results constitute the polysaccharide analysis results. Comparing the monosaccharide and polysaccharide analysis results allows for the assessment of their accuracy. If a monosaccharide in the monosaccharide analysis results does not match the polysaccharide analysis results, that monosaccharide can be removed, thereby effectively improving the accuracy of the analysis of monosaccharide and polysaccharide components.

[0083] Furthermore, the above results reveal the following polysaccharide utilization characteristics in IUGR pigs with different growth potentials: compared to high-growth-potential IUGR pigs, low-growth-potential IUGR pigs exhibit poor utilization of undigested polysaccharides in the cecum and colon, rather than differences in digestible starch utilization. Only low-growth-potential IUGR pigs show incomplete starch digestion, with some polysaccharides flowing into the hindgut. Moreover, the poor polysaccharide utilization is primarily due to the degradation of polysaccharide monomers rather than the glycolysis of monomeric sugars.

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for analyzing the carbohydrate components of intestinal chyme, characterized in that, Includes the following steps: The carbohydrate components of the intestinal chyme include monosaccharide and polysaccharide components; The intestinal chyme to be tested was dried and then reconstituted with water. The water-soluble portion was detected by ion chromatography to obtain the monosaccharide components that had been degraded by the intestine but not absorbed. The water-insoluble portion was dried, reconstituted with acid, and then detected by ion chromatography to obtain the monosaccharide components that had not been degraded by the intestine. The intestinal chyme to be tested was subjected to enzyme analysis and microbial fermentation analysis. The digestible polysaccharide content in the intestinal chyme was obtained by enzyme analysis, and the indigestible polysaccharide components in the intestinal chyme were obtained by microbial fermentation analysis. The above four results were integrated to determine the specific monosaccharide and polysaccharide components in the intestinal chyme. The conditions for the ion chromatography detection are as follows: Column: CarboPac™ PA1; Mobile phase A is H2O, and mobile phase B is 100mM NaOH. An elution gradient is used, and all mobile phase proportions are volume percentages, as detailed below: From 0 min to 9 min, mobile phase A was 95% and mobile phase B was 5%. From 9 min to 20 min, mobile phase A was 95% and mobile phase B was 5%. 20-30 minutes, mobile phase B is 100%; 30-60 min, mobile phase A is 95%, mobile phase B is 5%; The injection volume was 5 μL; The flow rate was 0.5 mL / min; The column temperature is 30℃.

2. The method for analyzing the carbohydrate components of intestinal chyme according to claim 1, characterized in that, The reagent used for acid resolution is 2M~2.5M trifluoroacetic acid.

3. The method for analyzing the carbohydrate components of intestinal chyme according to claim 1, characterized in that, The intestinal chyme to be tested is chyme from different segments of the intestine; The intestinal segments are the duodenum, jejunum, ileum, cecum, and colon.

4. The method for analyzing the carbohydrate components of intestinal chyme according to claim 1, characterized in that, The specific process of enzyme analysis is as follows: The intestinal chyme to be tested was incubated with α-amylase in an aqueous environment to obtain the content of undigested polysaccharides in the intestinal chyme to be tested; the intestinal chyme to be tested was co-incubated with water to obtain the content of undegraded polysaccharides in the intestinal chyme to be tested. The digestible polysaccharide content is obtained by subtracting the undegraded polysaccharide content from the undigested polysaccharide content.

5. The method for analyzing the carbohydrate components of intestinal chyme according to claim 4, characterized in that, The amount of α-amylase added is 0.01% to 0.02% of the mass of the intestinal chyme to be tested; The incubation temperature is 37℃~39℃, and the time is 30min~60min.

6. The method for analyzing the carbohydrate components of intestinal chyme according to claim 1, characterized in that, The specific process of the microbial fermentation is as follows: Dissolve the intestinal chyme to be tested in sterile anaerobic saline, centrifuge, and take the supernatant as inoculum; The inoculum was inoculated into a culture medium containing polysaccharide substrate and fermented at 37℃~39℃ for 72h~96h. The inoculum amount is 8% to 10% of the culture medium volume; In the culture medium, the concentration of the polysaccharide substrate is 0.008 g / mL to 0.01 g / mL.

7. The method for analyzing the carbohydrate components of intestinal chyme according to claim 6, characterized in that, The polysaccharide substrate is any one of pectin, xylan, arabinoxylan, β-glucan, and glucomannan.

8. The method for analyzing the carbohydrate components of intestinal chyme according to claim 7, characterized in that, Each liter of culture medium contains 0.0156g calcium chloride dihydrate, 0.0118g manganese chloride tetrahydrate, 0.0011g cobalt chloride hexahydrate, 0.0095g ferric chloride hexahydrate, 0.9511g ammonium carbonate, 8.3222g sodium carbonate, 0.3999g L-cysteine, 1.355g sodium dihydrogen phosphate, 1.474g potassium hydrogen phosphate, 0.1426g magnesium sulfate heptahydrate, 0.2972g sodium sulfide nonahydrate, 0.0760g sodium hydroxide, and 0.0011g resazurite, with water added to make up to 1L.

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