Method for detecting net absorption amount of intestinal short-chain fatty acid

Abstract

The invention relates to the technical field of metabonomics, in particular to a method for detecting the net absorption amount of intestinal short-chain fatty acid. The method comprises the following steps: aiming at a to-be-detected organism, obtaining the flow of SCFA in ileum chyme, the amount of SCFA in excrement and the amount of SCFA generated by in-vitro fermentation of ileum chyme; according to the flow of the SCFA in the ileum chyme, the amount of the SCFA in the excrement and the amount of the SCFA generated by in-vitro fermentation of the ileum chyme, the net absorption amount of the SCFA in the intestinal tract is obtained; the amount of SCFA generated by in-vitro fermentation of ileum chyme is obtained based on the following method: taking ileum chyme as a substrate and colon chyme as a microbial inoculation solution, carrying out in-vitro bionic fermentation, and then detecting the amount of SCFA. The invention provides a method for measuring the net absorption amount of SCFA by an in-vivo and in-vitro combined fermentation technology, so that the defect that the intestinal tract health state is evaluated by using intestinal tract chime and excrement SCFA concentration traditionally is overcome, and the potential influence of diet on health can be accurately evaluated.

Description

Technical Field

[0001] The present invention relates to the technical field of metabolomics, and particularly to a method for detecting the net absorption amount of short-chain fatty acids in the intestine. Background Art

[0002] Short-chain fatty acids (SCFAs) are the main end products of anaerobic fermentation of indigestible carbohydrates by intestinal microorganisms in humans and monogastric animals, mainly including acetic acid, propionic acid, and butyric acid, which together account for 90%-95% of SCFAs. The composition of SCFAs is significantly affected by diet, and their molar ratio depends on the fermented substrates. SCFAs play an important role in regulating intestinal energy metabolism and maintaining intestinal environmental homeostasis, and also play an important regulatory role in extra-intestinal organs and tissues. Butyric acid is the preferred substrate for colon epithelial cells and has the functions of maintaining the intestinal barrier, anti-inflammation, and regulating the immune system; propionic acid is metabolized in the liver and has the functions of energy metabolism, anti-inflammation, and enhancing the intestinal mechanical barrier function; acetic acid is an energy source for muscle tissue and has the functions of affecting appetite and satiety and regulating the intestinal barrier.

[0003] Since microbial fermentation mainly occurs in the large intestine of humans and monogastric animals, and it is difficult to directly collect samples from the large intestine region, there are certain difficulties in measuring the production of SCFAs. In addition, the traditional methods for evaluating the probiotic functions of indigestible carbohydrates on the host mainly involve measuring the concentration of microbial metabolite SCFAs in intestinal chyme or feces. However, the above methods have two main drawbacks: one is that the vast majority (more than 85%) of SCFAs are directly utilized after being absorbed by intestinal epithelial cells or enter the body tissues through the blood circulation, and the concentration of SCFAs in intestinal chyme cannot reflect their actual production amount; the other is that the SCFAs absorbed by the intestine are the main substances that play the probiotic roles of regulating the body's glycolipid metabolism and immune function, etc., and the concentration of SCFAs in intestinal chyme cannot accurately reflect the net absorption amount of SCFAs. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a method for detecting the net absorption amount of short-chain fatty acids in the intestine.

[0005] The present invention has developed a method that can accurately measure the net absorption amount of SCFAs after the fermentation of indigestible carbohydrates in the intestine by intestinal microorganisms. It can reflect the quantity, type, proportion, and energy supply situation of SCFA net absorption under specific dietary additions, and further evaluate the potential impact of diet on health and study the interaction between intestinal flora and the host.

[0006] In a first aspect, the present invention provides a method for detecting the net absorption amount of short-chain fatty acids in the intestine, including: For the biological sample to be tested, obtain the flow rate of SCFAs in ileal chyme, the flow rate of SCFAs in feces, and the production rate of SCFAs generated by in vitro fermentation of ileal chyme. Based on the flow rate of SCFAs in ileal chyme, the flow rate of SCFAs in feces, and the production rate of SCFAs generated by in vitro fermentation of ileal chyme, obtain the net absorption amount of intestinal SCFAs. The production rate of SCFAs generated by in vitro fermentation of the ileal chyme is obtained based on the following method: Using ileal chyme as the substrate and colonic chyme as the microbial inoculum, perform in vitro bionic fermentation and then detect the amount of SCFAs.

[0007] It should be particularly noted that the flow rate of SCFAs in the ileal chyme described in the present invention is actually the SCFAs in the ileal chyme (mmol / kg DMI), which can also be referred to as the SCFA flux in the ileal chyme per unit dry matter intake, that is, the amount of SCFAs in the ileal chyme under the condition of every 1 kg dry matter intake.

[0008] Similarly, the flow rate of SCFAs in the feces described in the present invention is actually the flow rate of SCFAs in the feces (mmol / kg DMI), which can also be referred to as the SCFA flux in the feces per unit dry matter intake, that is, the amount of SCFAs in the feces under the condition of every 1 kg dry matter intake.

[0009] The production rate of SCFAs generated by in vitro fermentation of the ileal chyme described in the present invention is also the amount of SCFAs generated by in vitro fermentation of freeze-dried ileal chyme under the condition of every 1 kg dry matter intake, and the unit can be mmol / kg DMI.

[0010] The present invention finds that the microbial diversity in the pig colon is rich, and the colon length accounts for more than 90% of the posterior intestine of the pig. The indigestible carbohydrates in the feed are mainly metabolized and degraded by microorganisms in the colon. Therefore, an in vitro fermentation technology is introduced to predict the net absorption amount of SCFAs. The fermentation method using ileal chyme as the substrate and colonic chyme as the microbial inoculum enables the prediction of the net absorption amount of SCFAs to have better accuracy.

[0011] Further, the conditions of the in vitro bionic fermentation include: The volume ratio of the inoculum to the culture medium is 1:(10 - 20), the fermentation temperature is 38 - 40 °C, the stirring speed is 60 - 100 rpm, and the fermentation time is 35 - 60 hours.

[0012] Further, the flow rate of SCFAs in the ileal chyme is obtained based on the following method: For the biological sample to be tested, obtain the amount of ileal chyme and the concentration of SCFAs in the ileal chyme, and based on the amount of ileal chyme and the concentration of SCFAs in the ileal chyme, obtain the flow rate of SCFAs in the ileal chyme.

[0013] Further, the flow rate of SCFAs in the feces is obtained based on the following method: For the biological sample to be tested, obtain the fecal excretion amount and the concentration of SCFAs in the feces, and obtain the flow rate of SCFAs in the feces based on the fecal excretion amount and the concentration of SCFAs in the feces.

[0014] Further, chromium sesquioxide is added as an exogenous indicator to the feed for feeding the biological sample to be tested, and the ileal chyme amount and the fecal excretion amount are obtained according to the concentrations of chromium sesquioxide in the feed, in the ileal chyme and in the fecal chyme.

[0015] Further, the yield of SCFAs generated by in vitro fermentation of the ileal chyme is obtained based on the following method: For the biological sample to be tested, obtain the amount of freeze-dried ileal chyme per unit dry matter intake and the yield of in vitro fermented SCFAs, and obtain the yield of SCFAs generated by in vitro fermentation of the ileal chyme based on the amount of freeze-dried ileal chyme per unit dry matter intake and the yield of in vitro fermented SCFAs.

[0016] Further, the yield of in vitro fermented SCFAs is: the amount of SCFAs obtained by fermenting every 1 kg of freeze-dried ileal chyme.

[0017] Further, the ileal chyme is collected by setting a fistula at the terminal of the ileum.

[0018] Further, the SCFAs include one or more of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid or isovaleric acid.

[0019] In a second aspect, the present invention provides a method for evaluating the probiotic function of indigestible carbohydrates on a host, including: Detect the net absorption amount of intestinal short-chain fatty acids by using the foregoing method; obtain the energy provided by SCFAs based on the net absorption amount of intestinal short-chain fatty acids.

[0020] In a third aspect, the present invention provides an in vitro bionic fermentation method for simulating the internal environment of the porcine hindgut, including: using ileal chyme as a substrate and using colonic chyme as a microbial inoculum for in vitro bionic fermentation; the fermentation conditions include: The volume ratio of the inoculum to the culture solution is 1:(10-20), the fermentation temperature is 38-40°C, the stirring speed is 60-100 rpm, and the fermentation time is 35-60 hours.

[0021] In a fourth aspect, the present invention provides an application of the foregoing method in evaluating the potential impact of diet on health, or detecting the interaction between intestinal flora and the host.

[0022] The present invention has the following beneficial effects: The present invention provides a method for measuring the net absorption of short-chain fatty acids (SCFAs) by in vivo-in vitro combined fermentation technology, which improves the accuracy of measuring the net absorption of SCFAs by introducing bionic fermentation technology. The method provided by the present invention can be used to predict the quantity and type of SCFAs net absorbed in the diet and the energy supply to humans (pigs), making up for the shortcomings of traditional analysis techniques that evaluate the intestinal health status based on the SCFA concentrations in intestinal chyme and feces, and having important value for accurately evaluating the potential impact of diet on health and studying the interaction between intestinal flora and the host. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is a schematic diagram of the method for measuring the net absorption of short-chain fatty acids (SCFAs) by in vivo-in vitro combined fermentation technology provided in Embodiment 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0026] The experimental methods involved in the following embodiments, unless otherwise specified, are all conventional methods in the art. For example, reference can be made to the experimental manuals in the art or the conditions recommended in the manufacturer's instructions.

[0027] The experimental materials and reagents involved in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0028] Example 1 Determination of the net absorption of SCFAs in the hindgut of four fiber raw materials by in vivo-in vitro combined fermentation method I. Test method Twenty healthy castrated boars of the (Duroc×Landrace×Yorkshire) three-way cross with an initial body weight (BW) of 60.0 ± 2.1 kg and fitted with a terminal ileal T-cannula were selected and randomly divided into 4 groups with 5 replicates per group and 1 pig per replicate. They were fed wheat bran diet (WB), corn bran diet (CB), sugar beet pulp diet (SBP) and oat bran diet (OB) respectively. The feed formula is shown in Table 1, where chromium sesquioxide was used as an exogenous indicator. According to the Guidelines for Nutrient Requirements of Swine in China (2020), a mixed premix of trace minerals and vitamins was provided for growing pigs to meet the recommended nutritional requirements of pigs.

[0029] Table 1 Composition of the feed formula in the implementation case Note: 1 The premix provided per kg of the complete diet: vitamin A, 5,512 IU; vitamin D3, 2,200 IU; vitamin E, 30 IU; vitamin K3, 2.2 mg; vitamin B 12 , 27.6 μg; riboflavin B2, 4.0 mg; pantothenic acid, 14.0 mg; niacin, 30.0 mg; choline, 400.0 mg; folic acid, 0.7 mg; thiamine, 1.5 mg; vitamin B6, 3.0 mg; biotin, 44.0 μg; manganese, 40.0 mg; iron, 75.0 mg; zinc, 75.0 mg; copper, 20.0 mg; iodine, 0.3 mg; selenium, 0.3 mg.

[0030] Note: 1 The premix provided the following per kg of complete diet for growing pigs: vitamin A, 5,512 IU; vitamin D3, 2,200 IU; vitamin E, 30 IU; vitamin K3, 2.2 mg; vitamin B 12, 27.6 μg; riboflavin, 4.0 mg; pantothenic acid, 14.0 mg; niacin, 30.0 mg; choline chloride, 400.0 mg; folacin, 0.7 mg; thiamine 1.5 mg; pyridoxine 3.0 mg; biotin, 44.0 μg; Mn, 40.0 mg; Fe, 75.0 mg; Zn, 75.0 mg; Cu, 20.0 mg; I, 0.3 mg; Se, 0.3 mg. II. Feeding Management and Sample Collection The experimental fistulated pigs were individually housed in a dedicated stainless-steel pig digestion and metabolism cage (1.40 m × 0.45 m × 0.60 m), and had free access to water during the experiment. Before the formal start of the experiment, each pig was weighed to determine the feed intake. The total daily feed amount for the experimental pigs was 4% of their body weight, and they were fed equally at 09:00 and 15:00 every day. The shed temperature was controlled at 20 - 28 °C. After each feeding, the pen was flushed and cleaned to keep the pig house environment clean and hygienic.

[0031] The diet was fed for 12 days, including a 7-day diet adaptation period. Fecal samples of each pig were collected on days 8 - 10, and ileal chyme samples of each pig were collected on days 11 - 12. The daily feed intake was recorded. The SCFA content in fresh ileal chyme and fecal samples was measured, and the ileal chyme was freeze-dried using a vacuum freeze dryer and the fecal samples were dried in an oven (temperature 65 °C, time 72 h). The chromium and moisture contents in the ileal chyme and fecal samples before and after drying were measured.

[0032] 300 g of colonic chyme from 5 different pigs (fed the same diet as the fistulated pigs) was obtained by the slaughter method. After mixing, 50 g of the chyme was taken and mixed with 1000 mL of physiological saline to dissolve, and the inoculum was prepared after filtration. Before collecting the colonic chyme samples, all growing pigs were fed the same corn-soybean meal-based diet for 30 consecutive days without adding any antibiotics. All fresh colonic chyme was put into a sample bag, filled with carbon dioxide, immediately placed in a thermos flask and transferred to an in vitro fermentation laboratory. The fresh colonic chyme was mixed evenly, 200 g of the colonic chyme was weighed and diluted with 1200 mL of 39 °C NaCl (9 g / L) solution. The diluted solution was mixed evenly with a stirrer, and the filtrate was obtained by filtering with two layers of sterilized gauze. The above operation procedures were all carried out in a sterile box and ensured to be completed within 1 h.

[0033] The in vitro fermentation medium used Menke medium. The formula of Menke medium: Trace element solution (Solution A): 13.2 g of CaCl2·2H2O, 10.0 g of MnCl2·4H2O, 1.0 g of CoCl2·6H2O, 8.0 g of FeCl3·6H2O. Add deionized water to make up to 100 ml and store in a refrigerator at 4°C; Buffer solution (Solution B): 4.0 g of NH4HCO3, 35.0 g of NaHCO3. Add deionized water to make up to 1000 ml and store in a refrigerator at 4°C; Macroelement solution (Solution C): 9.45 g of Na2HPO4·12H2O, 6.2 g of KH2PO4, 0.6 g of MgSO4·7H2O. Add deionized water to make up to 1000 ml and store in a refrigerator at 4°C; 0.1% Resazurin solution (Solution D): Add 100 mg of resazurin to 100 ml of deionized water to make up the volume, transfer it to a serum bottle, seal it with a rubber stopper and an aluminum cap, wrap it with aluminum foil on the outside to avoid light, and store in a refrigerator at 4°C. When using, draw it with a syringe; Reducing agent solution (Solution E): 625 mg of L-cysteine hydrochloride, 4.0 ml of 1M NaOH, 625 mg of Na2S·9H2O, 95 ml of distilled water. Prepare it immediately before use. The preparation sequence of Menke medium (1L): Add the prepared Solution B (208.1 mL), Solution C (208.1 mL), Solution A (0.1 mL), Solution D (1.0 mL) and distilled water (520.2) into a narrow-necked bottle in sequence and proportion, and gently shake to fully mix the solution. Put the narrow-necked bottle into a microwave oven and boil continuously for 10 min, then put a magnetic bar into the narrow-necked bottle and place it on a magnetic stirrer at 120 rpm for stirring, keep the temperature at 39°C and continuously introduce CO2 gas. After 1 h, use a portable pH meter to measure the pH value of the medium. If the pH is lower than 6.8, adjust the pH to 6.8 by adding NaOH solution (2 mol / L). After adjustment, add Solution E (62.4 mL) to remove the remaining oxygen.

[0034] Inject 450 mL of the in vitro fermentation medium, 30 mL of the microbial inoculum and 5 different fermentation substrates into the fermenter, which are blank control, freeze-dried ileal chyme of the 5 g wheat bran group, freeze-dried ileal chyme of the 5 g corn bran group, freeze-dried ileal chyme of the 5 g beet pulp group and freeze-dried ileal chyme of the 5 g oat bran group respectively. Nitrogen is introduced into the fermenter and the feeding bottle to maintain an anaerobic environment, and the nitrogen bubble generation rate is 1 - 2 per second. The fermentation temperature is maintained at 39 ± 0.5°C, and the stirring speed of the fermentation broth is 80 rpm. The in vitro fermentation lasts for 48 h. Use a disposable syringe to collect 10 mL of the fermentation broth sample to detect acetic acid, propionic acid, butyric acid and valeric acid. Immediately put each collected fermentation broth sample into liquid nitrogen at -80°C for standby.

[0035] Methods for measuring SCFAs in ileal chyme, feces, and in vitro bionic fermentation broth are as follows: Take 1 g of the sample and place it in a 10 mL centrifuge tube. Add 2 mL of 0.1% hydrochloric acid (v / v), place it on ice for 25 min, mix well, centrifuge at 15,000 r / min for 15 min, take the supernatant, filter it through a 0.22 μm filter membrane (Millipore, Bedford, OH, USA), and inject it into a gas chromatograph (Agilent HP 6890 series, Santa Clara, CA, USA) to detect the content of volatile fatty acids in the fecal sample.

[0036] III. Calculation methods 1. Fresh ileal chyme amount per 1 kg dry matter intake (g / kg DMI) = concentration of chromium in feed (g / kg) / Cr concentration in fresh ileal chyme (g / kg).

[0037] 2. Fresh fecal excretion amount per 1 kg dry matter intake (g / kg DMI) = concentration of chromium in feed (g / kg) / Cr concentration in fresh fecal sample (g / kg).

[0038] 3. Amount of SCFAs in ileal chyme per 1 kg dry matter intake (mmol / kg DMI) = fresh ileal chyme amount per 1 kg dry matter intake (g / kg DMI) × SCFA concentration in fresh ileal chyme (mg / kg) / molar mass of SCFA / 1000.

[0039] 4. Amount of SCFAs in feces per 1 kg dry matter intake (mmol / kg DMI) = fresh fecal excretion amount per 1 kg dry matter intake (g / kg DMI) × SCFA concentration in fresh feces (mg / kg) / molar mass of SCFA / 1000.

[0040] 5. Amount of freeze-dried ileal chyme per 1 kg dry matter intake (g / kg DMI) = concentration of chromium in feed (g / kg) / Cr concentration in freeze-dried ileal chyme (g / kg).

[0041] 6. Amount of SCFAs produced by in vitro fermentation of freeze-dried ileal chyme per 1 kg dry matter intake (mmol / kg DMI) = amount of freeze-dried ileal chyme per 1 kg dry matter intake (g / kg DMI) / 10 × amount of SCFAs in in vitro fermentation (mmol / kg DMI) / 1000.

[0042] 7. The amount of SCFA synthesized in the intestine per 1 kg of dry matter intake (mmol / kg DMI) = the flow rate of SCFA in the ileal chyme per 1 kg of dry matter intake (mmol / kg DMI) + the amount of SCFA generated by in vitro fermentation of freeze-dried ileal chyme produced per 1 kg of dry matter intake (mmol / kg DMI).

[0043] 8. The net absorption amount of SCFA in the hindgut per 1 kg of dry matter intake (mmol / kg DMI) = the amount of SCFA synthesized in the intestine per 1 kg of dry matter intake (mmol / kg DMI) - the amount of SCFA in the feces per 1 kg of dry matter intake (mmol / kg DMI).

[0044] 9. The energy provided by the net absorption of SCFA in the hindgut per 1 kg of dry matter intake = the net absorption amount of different types of SCFA (acetic acid, propionic acid, butyric acid, valeric acid) in the hindgut per 1 kg of dry matter intake (mmol / kg DMI) × the calorific value of combustion of SCFA (kJ / mol) / 1000.

[0045] The aforementioned test procedures are as Figure 1 shown.

[0046] IV. Test Results 1. Net absorption amounts of SCFA of four fiber raw materials: Table 2 shows the outflow mass of ileal chyme, fecal mass, the amount of SCFA in ileal chyme, the amount of SCFA in feces, the amount of SCFA generated by in vitro fermentation, the net absorption amount of SCFA in the hindgut, and the energy generated by the net absorption of SCFA in the hindgut under the conditions of 4 fiber raw materials. The net absorption amount of SCFA in the oat bran test group was significantly higher than that in the 3 test groups of beet pulp, wheat bran, and corn bran. Among them, the net absorption amount of SCFA in the corn bran test group was the lowest. Similarly, the results of the ileal terminal digestibility and total tract digestibility of the 4 fiber raw materials of wheat bran, corn bran, beet pulp, and oat bran showed that the total dietary fiber digestibility in the oat bran test group was significantly higher than that in the 3 test groups of beet pulp, wheat bran, and corn bran. Among them, the total dietary fiber digestibility in the corn bran test group was the lowest.

[0047] The production of SCFA mainly comes from the microbial metabolism process of total dietary fiber. Therefore, it can be seen that the results of the net absorption amounts of SCFA of the four fiber raw materials of wheat bran, corn bran, beet pulp, and oat bran in Table 2 are consistent with the results of the total tract digestibility of total dietary fiber, indicating that the measurement of the net absorption amount of SCFA in the pig intestine in this technical system has good accuracy.

[0048] Table 2 Net absorption amounts of SCFA of four fiber raw materials per kg of dry matter intake 2. Types and proportions of SCFA net absorption in the hindgut of pigs by four fiber raw materials: Table 3 shows the net absorption amounts of acetic acid, propionic acid, butyric acid, and valeric acid and the proportion of the total net absorption of SCFA after microbial metabolism of four different fiber raw materials in the hindgut of pigs. The results show that the proportion of acetic acid net absorption in the corn bran group in the total net absorption of SCFA is significantly higher than that in the three experimental groups of wheat bran, beet pulp, and oat bran; the proportion of butyric acid net absorption in the wheat bran and beet pulp groups in the total net absorption of SCFA is significantly higher than that in the corn bran and oat bran experimental groups.

[0049] Table 3 Proportion of net absorption of SCFA by four fiber raw materials per kg of dry matter intake Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments or equivalently replace some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the net absorption amount of short-chain fatty acids in the intestine, characterized in that, Comprising: For the biological sample to be measured, obtaining the flow rate of SCFAs in ileal chyme, the flow rate of SCFAs in feces, and the production rate of SCFAs generated by in vitro fermentation of ileal chyme; Based on the flow rate of SCFAs in ileal chyme, the flow rate of SCFAs in feces, and the production rate of SCFAs generated by in vitro fermentation of ileal chyme, obtaining the net absorption amount of intestinal SCFAs; The production rate of SCFAs generated by in vitro fermentation of ileal chyme is obtained based on the following method: Using ileal chyme as the substrate and colonic chyme as the microbial inoculum, performing in vitro bionic fermentation and then detecting the amount of SCFAs.

2. The method according to claim 1, characterized in that The conditions of the in vitro bionic fermentation include: The volume ratio of the inoculum to the culture medium is 1:(10 - 20), the fermentation temperature is 38 - 40 °C, the stirring speed is 60 - 100 rpm, and the fermentation time is 35 - 60 hours.

3. The method according to claim 1, wherein The flow rate of SCFAs in ileal chyme is obtained based on the following method: For the biological sample to be measured, obtaining the amount of ileal chyme and the concentration of SCFAs in ileal chyme, and based on the amount of ileal chyme and the concentration of SCFAs in ileal chyme, obtaining the flow rate of SCFAs in ileal chyme.

4. The method according to claim 1, wherein The flow rate of SCFAs in feces is obtained based on the following method: For the biological sample to be measured, obtaining the fecal excretion amount and the concentration of SCFAs in feces, and based on the fecal excretion amount and the concentration of SCFAs in feces, obtaining the flow rate of SCFAs in feces.

5. The method according to claim 3 or 4, characterized in that, Adding chromium sesquioxide as an exogenous indicator to the feed fed to the biological sample to be measured, and obtaining the amount of ileal chyme and the fecal excretion amount according to the concentration of chromium sesquioxide in the feed, ileal chyme, and fecal chyme.

6. The method according to any one of claims 1-5, characterized in that, The ileal chyme is collected by setting a fistula at the terminal of the ileum.

7. The method according to any one of claims 1-6, characterized in that, The SCFAs include one or more of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, or isovaleric acid.

8. A method for evaluating the probiotic function of indigestible carbohydrates on the host, characterized in that, Comprising: Detecting the net absorption amount of intestinal short-chain fatty acids by the method according to any one of claims 1 - 7; obtaining the energy provided by SCFAs based on the net absorption amount of intestinal short-chain fatty acids.

9. An in vitro bionic fermentation method for simulating the internal environment of the porcine hindgut, characterized in that, Comprising: Using ileal chyme as the substrate and colonic chyme as the microbial inoculum for in vitro bionic fermentation; the fermentation conditions include: The volume ratio of the inoculum to the culture medium is 1:(10 - 20), the fermentation temperature is 38 - 40 °C, the stirring speed is 60 - 100 rpm, and the fermentation time is 35 - 60 hours.

10. Use of the method according to any one of claims 1 - 7 in evaluating the potential impact of diet on health, or detecting the interaction between intestinal flora and the host.

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