Use of caecal chyme to ferment resistant starch in vitro and method for fermenting resistant starch in vitro

Abstract

The invention belongs to the field of biotechnology, and discloses the use of cecal chyme to ferment resistant starch in vitro, wherein the resistant starch is at least one of corn resistant starch, banana resistant starch, pea resistant starch, rice resistant starch, wheat resistant starch and potato resistant starch; in addition, the application also discloses a method for in vitro fermentation of resistant starch, wherein the resistant starch is first weighed in a centrifuge tube; then fresh cecal chyme is mixed with a diluent, vortexed and filtered to obtain a cecal chyme dilution, which is placed in a 39°C water bath for standby use; then artificial cecal fluid is added to the centrifuge tube in step 1, and then the cecal chyme dilution is added, mixed to obtain a mixed solution; finally, the centrifuge tube containing the mixed solution is placed in a 39°C shaker, incubated at 40rpm for 24h, and the in vitro fermentation of the resistant starch is completed. The method is detailed and feasible, has a simple device, is low in cost, can carry out multiple parallel experiments at the same time, and provides an experimental basis and reference basis for in vitro fermentation experiments of the cecum.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to the use of cecal chyme in in vitro fermentation of resistant starch and a method for in vitro fermentation of resistant starch. Background Art

[0002] The utilization of diet and feed by intestinal flora is of great significance in revealing the health and development of diseases of the body and livestock. The cecum is the most microbially rich area in the gastrointestinal tract and the main site of hindgut fermentation in livestock and rodents. In vitro fermentation technology has the advantages of simple operation, high repeatability, no ethical constraints, and the ability to obtain dynamic data of intestinal flora fermentation at any time. At present, it has become a common technology for the exploration of probiotic functions and digestion of feed raw materials in human and livestock research.

[0003] Prior art 1: Chinese patent application 200610027553.1 discloses a microbial feed additive for promoting the development of goose liver, and its mechanism of action is as follows: using strains screened from the intestines of animals; utilizing a strain composite culture method to culture all strains in the same culture medium; using a composite synchronous solid fermentation method to form a powerful composite microbial flora and form an effective enzyme production capacity, so that the product containing the additive contains a large amount of active enzymes that decompose cellulose and fructose, small peptides, antioxidants, free amino acids, vitamins and other physiological metabolic substances; thereby promoting the digestion and absorption of feed by animals, promoting the synthesis and full utilization of body protein, enhancing the disease resistance and immunity of livestock, poultry and aquatic animals, and improving the feed conversion rate and the production capacity of farmed animals.

[0004] The bacterial sources of all strains in this protocol were derived from the small intestinal chyme of healthy geese.

[0005] Prior art 2: Chinese patent application 201710223245.4 discloses fermentation parameters for in vitro simulated culture of porcine colon microbiota and a culture medium for in vitro simulated culture of porcine intestinal microorganisms. In the culture medium, anaerobic fermentation of isolated porcine colon chyme can produce volatile short-chain fatty acids.

[0006] The problem that this proposal needs to solve: How to propose a method for in vitro fermentation of resistant starch that is easy to operate. Summary of the invention

[0007] To solve the above problems, the present application provides the use of cecal chyme to ferment resistant starch in vitro and a method for fermenting resistant starch in vitro. Based on the principle that intestinal microorganisms can ferment and produce short-chain fatty acids under anaerobic conditions, goose cecal chyme is used for cultivation to promote the enzymatic hydrolysis of resistant starch and increase the production of short-chain fatty acids.

[0008] To achieve the above objectives, the present application discloses the use of cecal chyme to ferment resistant starch in vitro.

[0009] Preferably, the resistant starch is selected from at least one of corn resistant starch, banana resistant starch, pea resistant starch, rice resistant starch, wheat resistant starch and potato resistant starch.

[0010] Further preferably, the resistant starch is selected from at least one of pea resistant starch, rice resistant starch and potato resistant starch.

[0011] Preferably, the cecal chyme is goose cecal chyme

[0012] In addition, the present application also discloses a method for in vitro fermentation of resistant starch, wherein the method for in vitro fermentation of resistant starch is:

[0013] Step 1: Weigh the resistant starch into a centrifuge tube;

[0014] Step 2: Mix the fresh cecal chyme with the diluent, vortex, and filter to obtain the cecal chyme dilution, and place in a 39°C water bath for later use;

[0015] Step 3: Add artificial cecal fluid to the centrifuge tube in step 1, and then add the cecal chyme dilution solution, mix, and obtain a mixed solution;

[0016] Step 4: Place the centrifuge tube containing the mixed solution in a shaker and incubate for at least 24 hours to complete the in vitro fermentation of resistant starch.

[0017] Preferably, the artificial cecal fluid is prepared by mixing inorganic salts, urea, peptone, a reducing agent, short-chain fatty acids, casein and pure water;

[0018] The inorganic salt is selected from at least one of sodium salt, calcium salt, potassium salt, magnesium salt, cobalt salt and ammonium salt and at least comprises ammonium salt;

[0019] The reducing agent is selected from at least one of L-cysteine ​​hydrochloride, ferrous sulfate, and ascorbic acid;

[0020] The short-chain fatty acid is selected from at least one of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid.

[0021] Preferably, the concentration of cecal chyme in the cecal chyme dilution solution is 0.04-0.06 g / mL.

[0022] Preferably, steps 1 to 4 are performed in a low oxygen or anaerobic environment, and the oxygen content in the low oxygen environment is not higher than 0.1%.

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides the use of cecal chyme in in vitro fermentation of resistant starch and a method for in vitro fermentation of resistant starch. Goose cecal chyme is used for culture to accelerate the enzymatic hydrolysis of resistant starch and increase the yield of short-chain fatty acids. The method is detailed and feasible, the device is simple, the cost is low, and multiple parallel experiments can be carried out simultaneously, providing an experimental basis and reference basis for cecal in vitro fermentation experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The pH change pattern of cecal chyme fermentation liquid of different groups of geese within 24 hours;

[0026] Figure 2 The changes of acetic acid in the fermented cecal chyme of different groups of geese within 24 hours, * indicates intra-group comparison (compared with the previous time point), # indicates inter-group comparison (compared with Group C);

[0027] Figure 3 The change pattern of propionic acid in goose cecal chyme fermentation liquid in different groups within 24 hours, * indicates intra-group comparison (compared with the previous time point), # indicates inter-group comparison (compared with group C);

[0028] Figure 4 The change pattern of isobutyric acid in the fermented liquid of goose cecal chyme in different groups within 24 hours, * indicates intra-group comparison (compared with the previous time point), # indicates inter-group comparison (compared with group C);

[0029] Figure 5 The change pattern of butyric acid in the fermented liquid of goose cecal chyme in different groups within 24 hours, * indicates intra-group comparison (compared with the previous time point), # indicates inter-group comparison (compared with group C);

[0030] Figure 6 The change pattern of isovaleric acid in goose cecal chyme fermentation liquid in different groups within 24 hours, * indicates intra-group comparison (compared with the previous time point), # indicates inter-group comparison (compared with group C);

[0031] Figure 7 The change pattern of valeric acid in goose cecal chyme fermentation liquid in different groups within 24 hours, * indicates intra-group comparison (compared with the previous time point), # indicates inter-group comparison (compared with group C);

[0032] Figure 8 The figure shows the change pattern of total SCFA in cecal chyme fermentation liquid of geese in different groups within 24 hours. In the figure, * indicates intra-group comparison (compared with the previous time point), and # indicates inter-group comparison (compared with Group C). DETAILED DESCRIPTION

[0033] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0034] Corn resistant starch: LOUISFRANCOIS, France, product code: 2023101101B;

[0035] Banana resistant starch: LOUISFRANCOIS, France, product code: 2023101802A;

[0036] Pea resistant starch: Beijing Jinclone Biotechnology Co., Ltd., product code: CS9142Y-100;

[0037] Rice resistant starch: Shaanxi Panier Biotechnology Co., Ltd., product code: PB240120;

[0038] Wheat resistant starch: Shaanxi Panier Biotechnology Co., Ltd., product code: PB240126;

[0039] Potato resistant starch: Shaanxi Panier Biotechnology Co., Ltd., product code: PB240115.

[0040] Example 1

[0041] 1. Experimental Animals

[0042] The experimental animals were selected from five 95-day-old male Magang geese from Kaiping City, Guangdong Province, and their daily diet was rice.

[0043] 2. Cecal Chyme Processing

[0044] Five Magang geese were killed and dissected immediately. Fresh cecal chyme was taken and placed in five 5 mL cryovials. 2.0 g of cecal chyme was weighed in five 50 mL centrifuge tubes, and 40 mL of chyme diluent was added to each tube. The tubes were vortexed for 3-5 min until there were no large lumps of chyme. The chyme was filtered through a 70 μm cell sieve and stored in a 39°C water bath for later use. The remaining chyme was stored at -80°C.

[0045] 3. Reagent Preparation

[0046] Resazurin indicator: Weigh 0.1 g of resazurin indicator powder, add 100 mL of distilled water, and stir until dissolved to obtain 0.1% resazurin indicator.

[0047] Chyme diluent: disodium hydrogen phosphate (Na2HPO4) 2.4g, potassium dihydrogen phosphate (KH2PO4) 1.8g, L-cysteine ​​0.2g, agar 0.2g, Tween-80 0.2g, add distilled water 400mL, stir until dissolved, sterilize by high pressure to deoxygenate, keep in a 39℃ water bath before use, and use within 48h after preparation.

[0048] Artificial cecal fluid: weigh 27.84g Na2HPO4 into a beaker, add to 2L distilled water, heat to 85℃, then add 20.0g casein, and stir at 85℃ until dissolved, NaH2PO4 24.48g, CaCl2·2H2O 0.5g, NaHCO3 27.2g, KCl1.32g, MgSO4·7H2O 0.4g, L-cysteine ​​hydrochloride 0.001g, urea 1.5g, CoCl·6H2O 0.003g, (NH4)2SO4 2.0g, peptone 2.0g, acetic acid, propionic acid, butyric acid, and valeric acid are 6.0mg, 2.0mg, 120.0mg, and 200.0mg respectively. The solution is milky white. If there is an insoluble precipitate in the solution, filter it with 200-mesh silk cloth. Add 2 mL of 0.1% resazurin indicator to the prepared artificial cecal fluid, and the solution will be light blue. Before use, add CO2 to deoxygenate the solution, so that the solution changes from light blue to light pink. Place it in a 39°C water bath before use and use it within 48 hours after preparation.

[0049] 4. Experimental Procedure

[0050] Step 1: Place 0.2 g of resistant starch (RS3) in a 15 ml centrifuge tube;

[0051] Step 2: Take fresh cecal chyme and mix with the diluent, vortex, and filter through a 70 μm cell sieve to obtain a cecal chyme dilution with a cecal chyme concentration of 0.05±0.002 g / mL, and place in a 39°C water bath for later use;

[0052] Step 3: Add 10 mL of artificial cecal fluid to the centrifuge tube in step 1, and then add 1 mL of cecal chyme dilution solution, mix, and obtain a mixed solution;

[0053] Step 4: Place the centrifuge tube containing the mixed solution in a 39°C shaker and incubate at 40 rpm for 24 hours to complete the in vitro fermentation of resistant starch.

[0054] 5. Experimental Design

[0055] The experimental design groups are shown in Table 1:

[0056] Table 1

[0057]

[0058]

[0059] The experiment was divided into 8 groups (n=5), namely, negative control group (N), blank control group (C), and 6 resistant starch groups. The fermentation system of each group was prepared according to Table 1, mixed evenly and placed in a shaker at 39°C and 40rpm for fermentation. The corresponding centrifuge tubes were taken out at 0, 3, 6, 12, and 24h, respectively, immediately placed on ice, centrifuged at 4000rpm for 5min, and the supernatant was dispensed into 4 2mL centrifuge tubes, 1.5mL per tube, and the remaining supernatant was dispensed into 5mL centrifuge tubes for subsequent pH determination (samples need to be stored at 4°C and the determination must be completed within 48h), short-chain fatty acid content determination (samples need to be stored at -20°C), and the determination results were used to evaluate whether the fermentation method is feasible.

[0060] 6. Determination of pH of fermentation broth

[0061] The samples were taken out in advance, equilibrated to a temperature of about 25°C, measured after the pH meter was calibrated, and the pH of each group was recorded for subsequent analysis.

[0062] 7. Determination of short-chain fatty acids in fermentation broth by internal standard method

[0063] 7.1 Sample preparation

[0064] Deproteinized solution: Accurately weigh 25 g of metaphosphoric acid and 0.217 mL of 2-ethylbutyric acid (2EB), and dilute to a 100 mL volumetric flask to prepare a 25% (w / v) metaphosphoric acid deproteinized solution containing 2 g / L of internal standard 2EB, and store at -20°C.

[0065] Mixed standard stock solution: according to Table 2, take 6 SCFAs into a 100 mL volumetric flask, add deionized water to the mark, mix well, and store at -20°C.

[0066] Gradient mixed standard solution: prepare 5 1.5mL centrifuge tubes according to Table 3, add 0.2mL of deproteinized solution to each tube, add a certain amount of mixed standard stock solution according to Table 2, make up to 0.8mL with deionized water, and the total liquid volume in each tube is 1mL. After mixing evenly, filter through a 0.22μm microporous membrane into a liquid phase vial and store at 4℃ for testing.

[0067] Table 2

[0068]

[0069]

[0070] Table 3

[0071]

[0072] After the sample was thawed on ice, it was centrifuged at 12000rpm at 4℃ for 5min. 1mL of supernatant was taken and 200μL of deproteinized solution was added. Obvious white turbidity was visible. It was incubated on ice for 30min, mixed every 10min, and centrifuged at 12000rpm at 4℃ for 5min. The supernatant was filtered through a 0.22μm microporous membrane into a liquid phase vial and stored at 4℃ for testing.

[0073] 7.2 Gas chromatography conditions

[0074] Agilent 7890B gas chromatograph, chromatographic column DB-wax capillary column (20℃-250℃: 30m×320μm×0.25μm), FID detector, detector temperature 250℃, air flow 450mL / min, hydrogen gas flow 40mL / min, tail gas flow 43mL / min, chromatographic column flow (N2) 2mL / min, 10μL injection needle, injection volume 4.5μL, injection port temperature 250℃, pressure 10.5psi. Initial column temperature 80℃, maintained for 3min, 10℃ / min to 160℃, maintained for 1min, method running time total 12min.

[0075] Result analysis:

[0076] 1.Reference Figure 1 , the pH of the negative control group tended to be stable as the fermentation time prolonged, indicating that the negative control had a reference value. The intra-group comparison showed that the pH of pea RS3 (G group) decreased significantly after 3h of fermentation (p = 0.0046), the pH of potato RS3 (P group) decreased significantly after 6h of fermentation (p < 0.0001), and the pH of rice RS3 (R group) decreased significantly after 12h of fermentation (p < 0.0001). The pH of wheat RS3 (W group) did not change significantly during the fermentation period. The pH change trend of corn (M group) and banana RS3 (B group) was similar to that of the blank control group (C group).

[0077] 2.Reference Figure 2 Intra-group comparison showed that within 24 hours of fermentation, the acetic acid content in different treatment groups increased with the extension of fermentation time. The acetic acid content of pea and rice resistant starch increased significantly after 3 hours of fermentation, the acetic acid content of wheat and potato resistant starch increased significantly after 6 hours of fermentation, and the acetic acid content of corn and banana resistant starch increased significantly after 12 hours of fermentation. After 24 hours of fermentation, the acetic acid content in pea resistant starch and rice resistant starch was the highest, followed by potato resistant starch. There was also a certain increase in other treatment groups compared with the control group.

[0078] 3.Reference Figure 3Intra-group comparison showed that within 24 hours of fermentation, the propionic acid content of different treatment groups increased with the extension of fermentation time. The propionic acid content of pea resistant starch increased significantly after 3 hours of fermentation, and the propionic acid content of wheat and potato resistant starch increased significantly after 6 hours of fermentation. After 24 hours of fermentation, the content of acetic acid in pea resistant starch and rice resistant starch was the highest. The content of acetic acid in other treatment groups was relatively close, but all higher than that in the control group.

[0079] 4.Reference Figure 4 Intra-group comparison shows that the isobutyric acid content is very low or even below the detection limit within 0-3 hours of fermentation. The isobutyric acid content in different treatment groups increases with the fermentation time within 6-24 hours of fermentation. The isobutyric acid content in pea resistant starch increases significantly after 0 hours of fermentation, and the isobutyric acid content in rice resistant starch increases significantly after 6 hours of fermentation. After 24 hours of fermentation, the isobutyric acid content in pea resistant starch is the highest, followed by rice resistant starch. The isobutyric acid content in other treatment groups increases accordingly, but the content after 24 hours of fermentation is lower than that in the control group.

[0080] 5.Reference Figure 5 Intra-group comparison showed that within 24 hours of fermentation, the butyric acid content in different treatment groups increased with the extension of fermentation time. The butyric acid content in potato resistant starch increased significantly after 0 hours of fermentation, and the butyric acid content in pea, rice and wheat resistant starch increased significantly after 3 hours of fermentation. After 24 hours of fermentation, the butyric acid content in pea resistant starch and rice resistant starch was the highest, followed by the butyric acid content in potato resistant starch. The butyric acid content in other treatment groups was higher than that in the control group.

[0081] 6.Reference Figure 6 From the intra-group comparison, it can be seen that the isovaleric acid content was very low or even below the detection limit at 0h of fermentation. After 3h of fermentation, the isovaleric acid content in different treatment groups increased with the extension of fermentation time. Among them, the isovaleric acid content in banana resistant starch increased significantly after 12h of fermentation. After 24h of fermentation, only the isovaleric acid content in banana resistant starch was higher than that in the control group. The isovaleric acid content in other treatment groups was lower than that in the control group.

[0082] 7.Reference Figure 7 Intra-group comparison showed that within 24 hours of fermentation, the valeric acid content in different treatment groups increased with the extension of fermentation time. The valeric acid content in pea resistant starch increased significantly after 6 hours of fermentation, and the valeric acid content in corn and banana resistant starch increased significantly after 12 hours of fermentation. The valeric acid content in the three groups was higher than that in the control group after 24 hours of fermentation, and the valeric acid content in the other treatment groups was lower than that in the control group.

[0083] 8.Reference Figure 8Intra-group comparison showed that within 24 hours of fermentation, the total SCFA content of different treatment groups increased with the extension of fermentation time, among which the content of pea resistant starch increased significantly after 3 hours of fermentation, the content of rice, wheat and potato resistant starch increased significantly after 6 hours of fermentation, and the content of corn and banana increased significantly after 12 hours of fermentation. Moreover, after 24 hours of fermentation, the total SCFA content in the fermentation liquid was higher than that in the control group.

[0084] 9. From the above results, it can be seen that during the 24-hour in vitro fermentation of the six resistant starches, the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid all increased, among which the total content of the six short-chain fatty acids in pea resistant starch increased the most, the total content of the six short-chain fatty acids in rice and potato resistant starch increased more, the total content of the six short-chain fatty acids in banana and wheat resistant starch increased less, and the total content of the six short-chain fatty acids in corn resistant starch increased the least; and in the in vitro fermentation process of the six resistant starches, short-chain fatty acids such as isovaleric acid and isobutyric acid appeared in the content lower than that in the control group after 24 hours of fermentation. Since microorganisms can decompose proteins to produce branched-chain fatty acids, it is speculated that the cecal microorganisms may use nutrients such as nitrogen sources in artificial cecal fluid to produce isovaleric acid and isobutyric acid, and the addition of corresponding resistant starch may inhibit the process of microbial production of branched-chain fatty acids.

[0085] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Use of resistant starch fermented from cecal chyme in vitro; The resistant starch is selected from at least one of pea resistant starch, rice resistant starch and potato resistant starch; The resistant starch is RS3 type resistant starch; and the cecal chyme is goose cecal chyme.

2. A method for in vitro fermentation of resistant starch, characterized in that: The method for in vitro fermentation of resistant starch is: Step 1: Weigh RS3 resistant starch in a centrifuge tube; Step 2: Mix the fresh cecal chyme with the diluent, vortex, and filter to obtain the cecal chyme dilution, and place in a 39°C water bath for later use; Step 3: Add artificial cecal fluid to the centrifuge tube in step 1, and then add the cecal chyme dilution solution, mix, and obtain a mixed solution; Step 4: Place the centrifuge tube containing the mixed solution in a shaker and incubate for at least 24 hours to complete the in vitro fermentation of the resistant starch; Wherein, the RS3 resistant starch is selected from at least one of pea resistant starch, rice resistant starch and potato resistant starch; The cecal chyme in step 2 is goose cecal chyme; The artificial cecal fluid is prepared by mixing inorganic salts, urea, peptone, a reducing agent, short-chain fatty acids, casein and pure water; The inorganic salt is selected from at least one of sodium salt, calcium salt, potassium salt, magnesium salt, cobalt salt and ammonium salt and at least contains ammonium salt; The reducing agent is selected from at least one of L-cysteine ​​hydrochloride, ferrous sulfate, and ascorbic acid; The short-chain fatty acid is selected from at least one of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid.

3. The method for in vitro fermentation of resistant starch according to claim 2, characterized in that: The concentration of cecal chyme in the cecal chyme dilution solution is 0.04-0.06 g / mL.

4. The method for in vitro fermentation of resistant starch according to claim 2, characterized in that: Steps 1 to 4 are performed in a low-oxygen or oxygen-free environment, and the concentration of oxygen in the atmosphere of the low-oxygen environment is not higher than 0.1%.

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