A method for preparing mono-short-chain fatty acid glycerides by utilizing cucurbituril inclusion complexation

Abstract

The present invention discloses a method for preparing mono-short-chain fatty acid glycerides using cucurbituril inclusion complexation. The method utilizes the supramolecular inclusion and volume enlargement effects of fully methoxypolyethylene glycolated cucurbituril [6] on short-chain fatty acid alkane chains to control the reaction of glycerol with the cucurbituril to highly selectively generate mono-short-chain fatty acid glycerides, effectively avoiding the formation of diester and triester by-products. The above method can be used to directly synthesize high-purity mono-short-chain fatty acid glycerides. The product does not require molecular distillation purification, has low process energy consumption, produces few by-products, and has a light product color. When used as a drinking water-based feed antibiotic substitute, the bioavailability is high and the antibiotic substitution efficacy is strong.

Description

Technical Field

[0001] The invention relates to a method for preparing mono-short-chain fatty acid glyceride by utilizing cucurbituril inclusion complexation, and belongs to the field of feed additives. Background Art

[0002] Since the mid-20th century, the addition of antibiotics to feed has been a common practice in the livestock industry to prevent poultry and livestock diseases, promote animal growth, and improve farming efficiency. However, antibiotics in feed have been shown to pose potential teratogenic and carcinogenic risks and may also contribute to the development of drug resistance. Furthermore, livestock often fail to fully absorb the antibiotics they ingest, leading to large amounts of antibiotics being excreted into the environment as metabolites or even in their original form, causing pollution. In the early 21st century, developed countries such as the European Union and the United States took the lead in completely banning the use of antibiotics in feed. On July 9, 2019, my country's Ministry of Agriculture issued "Announcement No. 194 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China," requiring feed manufacturers to cease production of commercial feed containing antibiotic additives, marking the official entry of my country into the era of "antibiotic-free" feed.

[0003] In the era of comprehensive antibiotic bans, researchers in this field are dedicated to finding feed additives that can replace traditional antibiotics. Among them, mono-short-chain fatty acid glycerides (MSTGs) have multiple functions, including repairing intestinal villi, inhibiting harmful bacteria, regulating intestinal microbial balance, and promoting nutrient absorption and utilization. Their complete miscibility with water allows them to be added directly to livestock drinking water, making them convenient to administer and highly bioavailable. They are considered an ideal alternative to feed antibiotics. Currently, the traditional preparation process for MSTGs is direct esterification, which involves selectively producing MSTGs by adjusting the ratio of short-chain fatty acids (typically propionic acid, butyric acid, and valeric acid) to glycerol and optimizing reaction temperature and time. However, in this direct esterification process, the short-chain fatty acids inevitably undergo multiple esterification reactions with glycerol, forming a mixture of mono-, di-, and tri-esters, with the monoester content generally not exceeding 50%. Di- and tri-esters have poor water solubility and low intestinal absorption, which can compromise the product's efficacy as an antibiotic alternative. While molecular distillation can remove diesters and triesters, the equipment required is complex and expensive, and high-temperature distillation can trigger side reactions such as the formation of glycerol hydroxyl groups into ethers, reducing the content of effective components in the product and affecting its color. Therefore, preventing the formation of diesters and triesters during direct esterification and directly obtaining high-purity mono-short-chain fatty acid glycerides is a pressing issue in this field. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing mono-short-chain fatty acid glycerides by utilizing cucurbituril inclusion complexation, characterized in that the synthesis steps and conditions of the method are as follows, and the parts of the following raw materials are all parts by weight:

[0005] 1. A method for preparing mono-short-chain fatty acid glycerides by utilizing cucurbituril inclusion complexation, characterized in that the synthesis steps and conditions of the method are as follows, and the parts of the following raw materials are all parts by weight:

[0006] (1) Disperse 1-5 parts of cucurbituril and 3-15 parts of potassium persulfate in 30-80 parts of distilled water, react at 60-90°C under nitrogen protection for 6-8 hours, cool and filter after the reaction, vacuum dry the filtrate, and then dissolve the resulting solid in 1-5 parts of dimethyl sulfoxide, filter, add 50-100 parts of acetone to the filtrate and collect the precipitate. Finally, wash the precipitate with acetone and vacuum dry to constant weight to obtain fully hydroxylated cucurbituril;

[0007] (2) Dissolve 1-3 parts of fully hydroxylated cucurbituril in 10-40 parts of solvent, then add 0.2-0.6 parts of catalyst and 20-60 parts of methoxy polyethylene glycol-carboxylic acid with a number average molecular weight of 200-500 Da, and stir at 70-80°C for 12-14 hours. After the reaction is completed, dialyze the product in deionized water with a dialysis bag with a molecular weight cutoff of 1000-2000 Da for 24-48 hours, and then vacuum dry the product in the dialysis bag to constant weight to obtain fully methoxy polyethylene glycol cucurbituril;

[0008] (3) Dissolve 1-3 parts of short-chain fatty acids in 20-30 parts of solvent, then add 5-15 parts of fully methoxypolyethylene glycol cucurbituril, stir at 20-25°C for 30-60 minutes, then add 0.01-0.03 parts of 4-dimethylaminopyridine and 1-3 parts of glycerol to the above solution, after complete dissolution, add 0.01-0.03 parts of dicyclohexylcarbodiimide, stir at 20-25°C for 12-14 hours, after the reaction is completed, use a dialysis bag with a molecular weight cutoff of 1000-2000 Da to dialyze the product in deionized water for 24-48 hours, then collect the product in the dialysis bag, add 5-10 parts of silica microspheres with competitive guests immobilized on the surface, stir at 20-25°C for 30-60 minutes, and finally filter. The filtrate is heated at 40-50°C and 0.01-0.02 MPa under reduced pressure distillation to obtain single short-chain fatty acid glycerides;

[0009] The cucurbituril in steps (1), (2) and (3) is cucurbituril[6]uril;

[0010] The solvent in steps (2) and (3) is one or more of dimethyl sulfoxide and N,N-dimethylformamide;

[0011] The catalyst in step (2) is one or more of tetrabutyl titanate, monobutyl tin oxide, and p-toluenesulfonic acid;

[0012] The short-chain fatty acid in step (3) is one or more of propionic acid, butyric acid, and valeric acid;

[0013] The competitive guest in step (3) is one or more of spermine and 1,6-hexanediamine.

[0014] The principle of the method involved in the present invention is:

[0015] The present invention adopts a direct oxidation method to prepare fully hydroxylated cucurbit[6]uril, and then utilizes the esterification reaction between the hydroxyl group and the carboxyl group of the product outer wall to graft methoxy polyethylene glycol (PEG) onto the outer wall of cucurbit[6]uril to prepare fully PEGylated cucurbit[6]uril. Due to the perfect match in size and polarity, the fully PEGylated cucurbit[6]uril can utilize its hydrophobic cavity to enclose the alkyl chain of short-chain fatty acid supramolecularly, thereby increasing its molecular volume. At this time, the carboxyl group of the short-chain fatty acid is located outside the cucurbit[6]uril cavity and can still undergo esterification reaction with glycerol. After the enclosed short-chain fatty acid undergoes esterification reaction with one hydroxyl group in glycerol, the space around the remaining two hydroxyl groups in glycerol is aggravated by the steric hindrance effect of the fully PEGylated cucurbit[6]uril, making it difficult to continue esterification to form diesters and triesters. Since the fully PEGylated cucurbit[6]uril binds to short-chain fatty acids only through supramolecular interactions, it can be subsequently stripped from the monoester product using silica microspheres with strong guests immobilized on the surface to obtain high-purity single-short-chain fatty acid glycerides, and the recovered fully PEGylated cucurbit[6]uril can be reused.

[0016] Experiments have found that when the number average molecular weight of the grafted PEG is in the range of 200-500 Da, the volume of the short-chain fatty acid can be increased to an ideal size, and the purity of the mono-short-chain fatty acid glyceride generated by the reaction with glycerol can reach more than 85% (see Examples 1 and 2); if the number average molecular weight of the grafted PEG is less than 200 Da, the volume of the short-chain fatty acid cannot be effectively increased, and the reaction will still produce a large amount of diester and triester by-products, and the purity of the mono-short-chain fatty acid glyceride can only reach a maximum of 62% (see Comparative Example 1); if the number average molecular weight of the grafted PEG is greater than 500 Da, the volume of the short-chain fatty acid is too large, and there is a large steric hindrance in the esterification reaction with glycerol, making it difficult to generate a monoester (see Comparative Example 2). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the synthesis steps of the fully PEGylated cucurbit[6]uril involved in the present invention.

[0018] Figure 2 It is a schematic diagram of the principle of the present invention. DETAILED DESCRIPTION

[0019] The present invention is specifically described below through examples, which are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Technical engineers in this field can make some non-essential improvements and adjustments to the present invention based on the contents of the above invention. Example 1

[0020] 1. Disperse 1 part of cucurbit[6]uril and 3 parts of potassium persulfate in 30 parts of distilled water, react at 60°C under nitrogen protection for 6 hours, cool and filter after the reaction, vacuum dry the filtrate, dissolve the obtained solid in 1 part of dimethyl sulfoxide, filter, add 50 parts of acetone to the filtrate and collect the precipitate, wash the precipitate with acetone, and vacuum dry to constant weight to obtain fully hydroxylated cucurbit[6]uril;

[0021] 2. Dissolve 1 part of fully hydroxylated cucurbit[6]uril in 10 parts of dimethyl sulfoxide, add 0.2 parts of tetrabutyl titanate and 20 parts of methoxy polyethylene glycol-carboxylic acid with a number average molecular weight of 250 Da, and stir at 80°C for 12 hours. After the reaction is completed, dialyze the product in deionized water with a dialysis bag with a molecular weight cutoff of 1500 Da for 24 hours, and then vacuum dry the product in the dialysis bag to constant weight to obtain fully methoxy polyethylene glycol-cucurbit[6]uril;

[0022] 3. Dissolve 1 part of butyric acid in 20 parts of dimethyl sulfoxide, then add 5 parts of fully methoxypolyethylene glycolated cucurbit[6]uril, stir at 20°C for 30 minutes, then add 0.01 parts of 4-dimethylaminopyridine and 1 part of glycerol to the above solution, and after complete dissolution, add 0.01 parts of dicyclohexylcarbodiimide, and stir at 20°C for 12 hours. After the reaction is completed, use a dialysis bag with a molecular weight cutoff of 1500 Da to dialyze the product in deionized water for 24 hours, then collect the product in the dialysis bag, add 5 parts of silica microspheres with spermine immobilized on the surface, stir at 20°C for 30 minutes, and finally filter. The filtrate is distilled under reduced pressure at 40°C and 0.01 MPa to obtain mono-short-chain fatty acid glycerides;

[0023] The gas chromatography-mass spectrometry test results showed that the content of mono-short-chain fatty acid glycerides in the product was 87%, while the contents of diesters and triesters were 9% and 4% respectively. Example 2

[0024] 1. Disperse 5 parts of cucurbit[6]uril and 15 parts of potassium persulfate in 80 parts of distilled water, react at 90°C under nitrogen protection for 8 hours, cool and filter after the reaction, vacuum dry the filtrate, and then dissolve the obtained solid in 5 parts of dimethyl sulfoxide, filter it, add 100 parts of acetone to the filtrate and collect the precipitate. Finally, wash the precipitate with acetone and vacuum dry it to constant weight to obtain fully hydroxylated cucurbit[6]uril;

[0025] 2. Dissolve 3 parts of fully hydroxylated cucurbit[6]uril in 40 parts of dimethyl sulfoxide, add 0.6 parts of tetrabutyl titanate and 60 parts of methoxy polyethylene glycol-carboxylic acid with a number average molecular weight of 450 Da, and stir at 80°C for 14 hours. After the reaction is completed, dialyze the product in deionized water with a dialysis bag with a molecular weight cutoff of 2000 Da for 48 hours, and then vacuum dry the product in the dialysis bag to constant weight to obtain fully methoxy polyethylene glycol-cucurbit[6]uril;

[0026] 3. Dissolve 3 parts of valeric acid in 30 parts of dimethyl sulfoxide, then add 15 parts of fully methoxypolyethylene glycolated cucurbit[6]uril, stir at 25°C for 60 minutes, then add 0.03 parts of 4-dimethylaminopyridine and 3 parts of glycerol to the above solution, and after complete dissolution, add 0.03 parts of dicyclohexylcarbodiimide, and stir at 25°C for 14 hours. After the reaction is completed, dialyze the product in deionized water with a dialysis bag with a molecular weight cutoff of 2000 Da for 48 hours, then collect the product in the dialysis bag, add 10 parts of silica microspheres with spermine immobilized on the surface, stir at 25°C for 60 minutes, and finally filter. The filtrate is distilled under reduced pressure at 50°C and 0.02 MPa to obtain mono-short-chain fatty acid glycerides;

[0027] The gas chromatography-mass spectrometry test results showed that the content of mono-short-chain fatty acid glycerides in the product was 93%, while the contents of diesters and triesters were 6% and 1% respectively. Comparative Example 1

[0028] 1. Disperse 2 parts of cucurbit[6]uril and 10 parts of potassium persulfate in 50 parts of distilled water, react at 60°C under nitrogen protection for 8 hours, cool and filter after the reaction, vacuum dry the filtrate, and then dissolve the obtained solid in 2 parts of dimethyl sulfoxide, filter it, add 80 parts of acetone to the filtrate and collect the precipitate. Finally, wash the precipitate with acetone and vacuum dry it to constant weight to obtain fully hydroxylated cucurbit[6]uril;

[0029] 2. Dissolve 2 parts of fully hydroxylated cucurbituril in 20 parts of dimethyl sulfoxide, then add 0.4 parts of monobutyltin oxide and 40 parts of methoxypolyethylene glycol-carboxylic acid with a number average molecular weight of 100 Da, and stir at 70°C for 12 hours. After the reaction is completed, dialyze the product in deionized water with a dialysis bag with a molecular weight cutoff of 1000 Da for 24 hours, and then vacuum dry the product in the dialysis bag to constant weight to obtain fully methoxypolyethylene glycol cucurbituril [6];

[0030] 3. Dissolve 2 parts of butyric acid in 30 parts of dimethyl sulfoxide, then add 10 parts of fully methoxypolyethylene glycolated cucurbit[6]uril, stir at 20°C for 30 minutes, then add 0.02 parts of 4-dimethylaminopyridine and 2 parts of glycerol to the above solution, and after complete dissolution, add 0.02 parts of dicyclohexylcarbodiimide, and stir at 20°C for 12 hours. After the reaction is completed, use a dialysis bag with a molecular weight cutoff of 1000 Da to dialyze the product in deionized water for 24 hours, then collect the product in the dialysis bag, add 5 parts of silica microspheres with 1,6-hexanediamine immobilized on the surface, stir at 20°C for 30 minutes, and finally filter. The filtrate is distilled under reduced pressure at 40°C and 0.01 MPa to obtain mono-short-chain fatty acid glycerides;

[0031] The gas chromatography-mass spectrometry test results showed that the content of mono-short-chain fatty acid glycerides in the product was 62%, while the contents of diesters and triesters were 30% and 8% respectively. Comparative Example 2

[0032] 1. Disperse 4 parts of cucurbit[6]uril and 12 parts of potassium persulfate in 70 parts of distilled water, react at 90°C under nitrogen protection for 7 hours, cool and filter after the reaction, vacuum dry the filtrate, dissolve the obtained solid in 4 parts of dimethyl sulfoxide, filter, add 80 parts of acetone to the filtrate and collect the precipitate, wash the precipitate with acetone, and vacuum dry to constant weight to obtain fully hydroxylated cucurbit[6]uril;

[0033] 2. Dissolve 2 parts of fully hydroxylated cucurbit[6]uril in 30 parts of dimethyl sulfoxide, add 0.4 parts of tetrabutyl titanate and 40 parts of methoxy polyethylene glycol-carboxylic acid with a number average molecular weight of 1000 Da, and stir at 80°C for 13 hours. After the reaction is completed, dialyze the product in deionized water with a dialysis bag with a molecular weight cutoff of 2500 Da for 48 hours. Then, vacuum dry the product in the dialysis bag to constant weight to obtain fully methoxypolyethylene glycol cucurbit[6]uril;

[0034] 3. Dissolve 2 parts of valeric acid in 20 parts of dimethyl sulfoxide, then add 10 parts of fully methoxypolyethylene glycolated cucurbit[6]uril, stir at 25°C for 80 minutes, then add 0.02 parts of 4-dimethylaminopyridine and 2 parts of glycerol to the above solution, and after complete dissolution, add 0.02 parts of dicyclohexylcarbodiimide, and stir at 25°C for 14 hours. After the reaction is completed, use a dialysis bag with a molecular weight cutoff of 2500 Da to dialyze the product in deionized water for 48 hours, then collect the product in the dialysis bag, add 10 parts of silica microspheres with 1,6-hexanediamine immobilized on the surface, stir at 25°C for 60 minutes, and finally filter. The filtrate is distilled under reduced pressure at 50°C and 0.01 MPa to obtain mono-short-chain fatty acid glycerides;

[0035] No chromatographic peaks related to short-chain fatty acid glycerides appeared in the gas chromatography-mass spectrometry test results, which means that the esterification reaction did not occur.

Claims

1. A method for preparing mono-short-chain fatty acid glycerides by utilizing cucurbituril inclusion complexation, characterized in that The synthesis steps and conditions of the method are as follows, and the parts of the following raw materials are all parts by weight: (1) Disperse 1-5 parts of cucurbituril and 3-15 parts of potassium persulfate in 30-80 parts of distilled water, react at 60-90°C under nitrogen protection for 6-8 hours, cool and filter after the reaction, vacuum dry the filtrate, and then dissolve the resulting solid in 1-5 parts of dimethyl sulfoxide, filter, add 50-100 parts of acetone to the filtrate and collect the precipitate. Finally, wash the precipitate with acetone and vacuum dry to constant weight to obtain fully hydroxylated cucurbituril; (2) Dissolve 1-3 parts of fully hydroxylated cucurbituril in 10-40 parts of solvent, then add 0.2-0.6 parts of catalyst and 20-60 parts of methoxy polyethylene glycol-carboxylic acid with a number average molecular weight of 200-500 Da, and stir at 70-80°C for 12-14 hours. After the reaction is completed, dialyze the product in deionized water with a dialysis bag with a molecular weight cutoff of 1000-2000 Da for 24-48 hours, and then vacuum dry the product in the dialysis bag to constant weight to obtain fully methoxy polyethylene glycol cucurbituril; (3) Dissolve 1-3 parts of short-chain fatty acids in 20-30 parts of solvent, then add 5-15 parts of fully methoxypolyethylene glycol cucurbituril, stir at 20-25°C for 30-60 minutes, then add 0.01-0.03 parts of 4-dimethylaminopyridine and 1-3 parts of glycerol to the above solution, after complete dissolution, add 0.01-0.03 parts of dicyclohexylcarbodiimide, stir at 20-25°C for 12-14 hours, after the reaction is completed, use a dialysis bag with a molecular weight cutoff of 1000-2000 Da to dialyze the product in deionized water for 24-48 hours, then collect the product in the dialysis bag, add 5-10 parts of silica microspheres with competitive guests immobilized on the surface, stir at 20-25°C for 30-60 minutes, and finally filter. The filtrate is heated at 40-50°C and 0.01-0.02 MPa under reduced pressure distillation to obtain single short-chain fatty acid glycerides; The cucurbituril in steps (1), (2) and (3) is cucurbituril[6]uril; The solvent in steps (2) and (3) is one or more of dimethyl sulfoxide and N,N-dimethylformamide; The catalyst in step (2) is one or more of tetrabutyl titanate, monobutyl tin oxide, and p-toluenesulfonic acid; The short-chain fatty acid in step (3) is one or more of propionic acid, butyric acid, and valeric acid; The competitive guest in step (3) is one or more of spermine and 1,6-hexanediamine.

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