Application of angelic acid and / or 4-cyclopentene-1,3-dione in the preparation of choline degradation inhibitors

Abstract

The present invention discloses the use of angelic acid and / or 4-cyclopentene-1,3-dione in the preparation of a choline degradation inhibitor. The present invention firstly finds that angelic acid and 4-cyclopentene-1,3-dione can inhibit the degradation of choline by gastrointestinal microorganisms. As a feed additive, 4-cyclopentene-1,3-dione can inhibit the degradation of choline and improve feed utilization.

Description

Technical Field

[0001] The invention relates to application of angelic acid and / or 4-cyclopentene-1,3-dione in inhibiting gastrointestinal microorganisms from degrading choline, and belongs to the field of compound application. Background Art

[0002] Choline is an essential nutrient for animals that affects a variety of physiological processes in the liver, such as lipid and cholesterol metabolism. Although the liver can produce some choline, choline is mainly obtained from feed. Studies have shown that reduced choline levels can induce fatty liver disease by reducing the outflow of very low-density lipoprotein from hepatocytes. Choline is essential for periparturient dairy cows. Periparturient dairy cows are generally fed rumen-protected choline chloride to reduce the incidence of fatty liver and ketosis. However, gastrointestinal microorganisms can quickly degrade choline in the feed into trimethylamine, resulting in reduced bioavailability of choline in animals, which may be related to the fatty liver or ketosis in some periparturient dairy cows. In addition, trimethylamine and its metabolite trimethylamine oxide in the liver are toxic to animals and endanger animal health. Trimethylamine is also an important substance that causes livestock products to emit fishy odors. However, there are currently no choline degradation inhibitors suitable for animal use. Summary of the invention

[0003] Purpose of the invention: The first purpose of the present invention is to provide the use of angelic acid and / or 4-cyclopentene-1,3-dione in the preparation of a choline degradation inhibitor. The second purpose of the present invention is to provide the use of angelic acid and / or 4-cyclopentene-1,3-dione in the preparation of a feed additive.

[0004] Technical solution: Use of angelic acid and / or 4-cyclopentene-1,3-dione described in the present invention in the preparation of choline degradation inhibitors.

[0005] The choline degradation inhibitor of the present invention comprises angelic acid and / or 4-cyclopentene-1,3-dione. The use of angelic acid and / or 4-cyclopentene-1,3-dione in the preparation of a feed additive is used to inhibit the degradation of choline in the gastrointestinal tract.

[0006] The feed additive is used to reduce the content of trimethylamine oxide and trimethylamine in the blood of livestock.

[0007] The livestock includes one of ruminants, monogastric animals and poultry.

[0008] The feed additive of the present invention comprises angelic acid and / or 4-cyclopentene-1,3-dione as active ingredients.

[0009] The feed additive is used to reduce the fishy smell of livestock products caused by trimethylamine.

[0010] Wherein, the 4-cyclopentene-1,3-dione is used to improve feed utilization.

[0011] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention is the first to discover that angelic acid and 4-cyclopentene-1,3-dione can inhibit the degradation of choline by gastrointestinal microorganisms. As a feed additive, while inhibiting the degradation of choline in the gastrointestinal tract of animals, 4-cyclopentene-1,3-dione can also improve feed utilization. DETAILED DESCRIPTION

[0012] The technical solution of the present invention is further described below in conjunction with specific technical solutions.

[0013] Example 1

[0014] 1. Materials and Methods

[0015] 1.1 Strains and culture media and preparation

[0016] 1.1.1 Strains

[0017] The choline-degrading bacteria used in this study was Escherichia fergusonii ATCC 35469.

[0018] 1.1.2 Culture medium and configuration

[0019] The composition of the culture medium is shown in Table 1.

[0020] Table 1 Culture medium composition (1.0L)

[0021]

[0022]

[0023] Reagent preparation method:

[0024] Preparation of cell-free rumen fluid: Filter the rumen fluid through two layers of gauze to remove large particles and residues. Centrifuge the filtrate at 12,000 rpm / min and 4°C for 20 minutes. Take the supernatant and store it at -20°C for later use.

[0025] Preparation of trace element solution: 1.5 g nitrilotriacetic acid, 1.0 g NaCl, 0.01 g NaMoO4·2H2O, 0.19 g NaSeO4, 0.1 g Na2WO4·2H2O, 3.0 g MgSO4·7H2O, 0.018 g AlK(SO4)2·12H2O, 0.18 g CoSO4·7H2O, 0.18 g CaCl2·2H2O, 0.1 g ZnSO4·7H2O, 0.18 g FeSO 4· 7H2O (0.1 g), MnSO4.H2O (0.45 g), NiSO4·6H2O (0.1 g), CuSO4·5H2O (0.01 g), and H3BO3 (0.01 g) were dissolved in 1 L of deionized water.

[0026] Preparation of fatty acid solution: weigh 8.0 g NaOH and dissolve it in 800 mL deionized water, then add 6.85 mL acetic acid, 3.00 mL propionic acid, 1.84 mL butyric acid and 0.55 mL valeric acid, add appropriate amount of deionized water to make up to 1 L, and store at 4°C for later use.

[0027] Preparation of vitamin solution: weigh 54.7g KH2PO4 and dissolve it in 1L deionized water, then add 20.4mg biotin, 20.4mg folic acid, 164mg calcium pantothenate, 164mg niacinamide, 164mg riboflavin, 164mg thiamine hydrochloride, 164mg vitamin B6 hydrochloride, 20.4mg p-aminobenzoic acid and 20.5mg cobalamin in sequence, mix thoroughly, filter and sterilize, dispense into anaerobic tubes, and store at 4℃ for later use.

[0028] 1.1.3 Culture medium preparation

[0029] Preparation of anaerobic culture medium (1L): According to the above culture medium formula, accurately measure each component into a wide-bottomed narrow-necked bottle, add deionized water to make up to 1L, heat and boil in a microwave oven until the liquid changes from blue to red, quickly take it out, immediately pass carbon dioxide for about 2 hours, and then add L-cysteine ​​hydrochloride until the liquid becomes clear and then start to package. Pass carbon dioxide into the fermentation tube in advance to exclude air, then add 9.6mL of culture medium, seal the bottle mouth with a rubber stopper and cover it with an aluminum cap, put it in an autoclave for sterilization, and set aside after cooling.

[0030] 1.2 Experimental design

[0031] Angelica acid solution or 4-cyclopentene-1,3-dione was added to the fermentation tubes respectively, and the addition amount was shown in Table 2, with 4 replicates in each group. 20.0mmol / L choline was added as a substrate to each fermentation tube, and the bacterial solution cultured to the logarithmic phase was inoculated into the culture medium that had been injected with 0.1mL of vitamin solution and angelica acid solution / 4-cyclopentene-1,3-dione in advance, with an inoculation amount of 3.0%, and cultured at 39°C and 50rpm / min for 60h.

[0032] Table 2 Inhibitor addition amount (final concentration)

[0033]

[0034] 1.3 Sample collection and index determination

[0035] 1.3.1 Growth curve drawing

[0036] At the end of the fermentation, the absorbance (OD value) of the bacterial solution in the shaken fermentation tube was detected at a wavelength of 600 nm using a spectrophotometer.

[0037] 1.3.2 Choline concentration determination

[0038] 1.0 mL of fermentation broth was centrifuged at 13000×g and 4°C for 2 min, and the supernatant was collected. The concentration was determined using a bovine choline ELISA test kit (Coab, China), and the test steps were referred to the instruction manual.

[0039] 1.4 Data Statistical Processing

[0040] The data were analyzed using One-way ANOVA (Tukey) in SPSS20.0 software, and P<0.05 indicated a significant difference.

[0041] 2. Results

[0042] As shown in Tables 3 and 4, angelic acid and 4-cyclopentene-1,3-dione can specifically inhibit the degradation of choline by Escherichiafergusonii and can inhibit choline degradation under certain concentration conditions, but have little effect on microbial growth.

[0043] Table 3. Effect of angelic acid on choline degradation by Escherichia fergusonii

[0044]

[0045] Note: Data without letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05); the initial concentration of choline was 20.0mmol / L; without the addition of Escherichia fergusonii, four concentrations of angelic acid had no effect on the degradation of choline (19.6, 19.8, 19.9, 19.5mmol / L).

[0046] Table 4. Effect of 4-cyclopentene-1,3-dione on choline degradation by Escherichia fergusonii

[0047]

[0048]

[0049] Note: Data without letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05); the initial concentration of choline was 20.0mmol / L; without the addition of Escherichia fergusonii, four concentrations of 4-cyclopentene-1,3-dione had no effect on the degradation of choline (19.5, 19.8, 19.7, 19.5mmol / L).

[0050] Example 2

[0051] 1 Materials and methods

[0052] 1.1 Experimental Materials

[0053] 1.1.1 Experimental animals and rumen fluid collection

[0054] The experimental animals were raised at the Dairy Cow Experimental Base of the College of Animal Science and Technology of Nanjing Agricultural University (Nanjing, Jiangsu Province). Three multiparous Holstein cows were equipped with permanent rumen fistulas, with an average weight of 613±35kg. On the day of the experiment, rumen fluid was collected half an hour before morning feeding, quickly put into a thermos bottle pre-filled with carbon dioxide and brought back to the laboratory.

[0055] 1.1.2 Preparation of artificial rumen fermentation broth

[0056] The collected fresh rumen fluid was filtered through 4 layers of gauze, and the rumen fluid and buffer solution (specific components are shown in Table 5) were mixed at a ratio of 1:9 (v / v) to prepare a mixed artificial rumen culture solution. The mixed fermentation solution was dispensed into 180 mL fermentation bottles. The temperature of the whole process was maintained at 39 ° C, and pure carbon dioxide was continuously introduced to maintain an anaerobic environment. A magnetic stirrer was used to stir to keep the rumen fluid and buffer solution mixed evenly. 1 g of rice straw powder and inhibitors (angelic acid or 4-cyclopentene-1,3-dione) were pre-added to the fermentation bottle and placed in a constant temperature incubator at 39 ° C for 30 minutes. After the dispensing was completed, the culture was carried out for 72 hours at a constant temperature oscillator at 39 ° C and 80 r / min.

[0057] Table 5 Buffer composition (1L)

[0058]

[0059] Preparation of specific solution:

[0060] (1) Trace element solution (Solution A)

[0061]

[0062]

[0063] Add deionized water to 100 mL

[0064] (2) Buffer solution (Solution B)

[0065] NH4HCO3 4.0g

[0066] NaHCO3 35.0g

[0067] Add deionized water to 1000 mL

[0068] (3) Constant element solution (Solution C)

[0069] Na2HPO4·12H2O 9.45g

[0070] KH2PO4 6.2g

[0071] MgSO4·7H2O 0.6g

[0072] Add deionized water to 1000 mL

[0073] (4) 0.1% Resazurin solution (Solution D)

[0074] 100 mg of resazurin was added into 100 mL of deionized water.

[0075] (5) Reducing agent solution (liquid E)

[0076]

[0077] 1.2 Test methods

[0078] 1.2.1 Experimental design

[0079] Embodiment 1: The experiment sets up 4 groups.

[0080] 1. Treatment group 1: no drugs were added.

[0081] 2. Treatment group 2: angelic acid 1.8mmol / L.

[0082] 3. Treatment group 3: angelic acid 3.6mmol / L.

[0083] 4. Treatment group 4: angelic acid 7.2mmol / L.

[0084] 5. Treatment group 5: 4-cyclopentene-1,3-dione 1.8 mmol / L.

[0085] 6. Treatment group 6: 4-cyclopentene-1,3-dione 3.6mmol / L.

[0086] 7. Treatment group 7: 4-cyclopentene-1,3-dione 7.2mmol / L

[0087] 1.2.2 Chemical analysis

[0088] Determination of total gas production: Use a barometric pressure converter to measure the gas production in the fermentation bottle.

[0089] Choline concentration determination: 1.0 mL of fermentation broth was centrifuged at 13000×g and 4°C for 2 min, and the supernatant was collected. The concentration was determined using a bovine choline ELISA test kit (Coab, China), and the test steps were referred to the instruction manual.

[0090] 1.3 Data Statistical Processing

[0091] The data were analyzed using One-way ANOVA (Tukey) in SPSS20.0 software, and P<0.05 indicated a significant difference.

[0092] 2. Results

[0093] As shown in Tables 6 and 7, in the simulated fermentation in vitro in the rumen, angelic acid (7.2 mmol / L) and 4-cyclopentene-1,3-dione (1.8-7.2 mmol / L) were both able to inhibit the degradation of choline. The difference was that angelic acid reduced the gas production, while 4-cyclopentene-1,3-dione increased the gas production.

[0094] Table 6. Effect of angelic acid on choline degradation by rumen fermentation in vitro

[0095]

[0096] Note: Data without letters in the same column indicate no significant difference (P>0.05), and data with different letters indicate significant difference (P<0.05).

[0097] Table 7. Effects of 4-cyclopentene-1,3-dione on choline degradation by rumen fermentation in vitro

[0098]

[0099] Note: Data without letters in the same column indicate no significant difference (P>0.05), and data with different letters indicate significant difference (P<0.05).

[0100] The results showed that angelic acid and 4-cyclopentene-1,3-dione could protect choline in the diet from being degraded by rumen flora, and 4-cyclopentene-1,3-dione could also improve the utilization of substrate by rumen flora.

Claims

1. Application of angelic acid and / or 4-cyclopentene-1,3-dione in the preparation of choline degradation inhibitors.

2. Use of angelic acid and / or 4-cyclopentene-1,3-dione in the preparation of feed additives, characterized in that: The feed additive is used for inhibiting the degradation of choline in the gastrointestinal tract.

3. The use according to claim 2, characterized in that: The feed additive is used for reducing the content of trimethylamine oxide and trimethylamine in the blood of livestock.

4. The use according to claim 3, characterized in that: The livestock includes one of ruminants and monogastric animals.

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