A sustained-release butyrate-laurate mixed structure ester and its preparation method and application

Abstract

The invention discloses a sustained-release butyric acid lauric acid mixed structure ester and a preparation method and application thereof, and relates to the technical field of oil synthesis. Specifically, the invention provides a new butyric acid lauric acid mixed structure ester, which mainly includes monobutyric acid dilauric acid glyceryl ester and dibutyric acid monolauric acid glyceryl ester. After the mixed structure ester contacts lipase in the intestine, butyric acid and monolauric acid glyceryl ester can be slowly released, and positive changes in intestinal microorganisms can be induced within 24 hours. Compared with existing feed additives, the mixed structure ester can more effectively improve the intestinal function of animals.

Description

Technical Field

[0001] The invention relates to the technical field of oil synthesis, and in particular to a sustained-release butyrate-laurate mixed structure ester and a preparation method and application thereof. Background Art

[0002] In modern farming, short-chain and medium-chain fatty acids and their glycerides are widely used in feed additives to improve animal health and growth rate, but their rapid digestion and absorption in the body reduces their functional value.

[0003] Butyric acid is a short-chain fatty acid that can be directly absorbed by intestinal cells and is an important energy source for intestinal epithelial cells. Butyric acid has been widely used as a feed additive in the breeding of economic animals such as pigs, cattle, chickens and shrimps. However, orally ingested butyric acid or butyric acid glyceride is easily digested and absorbed in the gastric environment, making it difficult for butyric acid to reach the intestinal area where microorganisms gather, which greatly limits the transmission effect and function of butyric acid.

[0004] Monolaurin is the esterified form of lauric acid glyceride with the best antibacterial and antiviral capabilities, and its biological activity is also better than that of free lauric acid. Currently, monolaurin has been widely used in animal breeding, for example:

[0005] The invention patent application document with application publication number CN110506864A discloses an antibiotic substitute additive for pig feed and its application in pig feed. The combination of monolaurin and formic acid in the additive can significantly increase the height of the jejunal villi of piglets, thereby improving intestinal health and increasing the feed utilization rate of piglets.

[0006] The invention patent application document with the authorization announcement number CN201610247228 discloses a chicken feed additive composed of 10-45% monolaurin, 10-30% dilaurin, 30-50% monostearin and 0-20% carrier. The additive can significantly improve the egg-laying performance and egg quality of laying hens.

[0007] However, current feed additives cannot fully meet the needs of breeding. How to improve the efficiency of feed additives and further effectively improve animal intestinal health is one of the urgent problems to be solved.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The purpose of the present invention is to provide a sustained-release butyrate-laurate mixed structure ester and a preparation method and application thereof.

[0010] The present invention is achieved in that:

[0011] In a first aspect, an embodiment of the present invention provides a butyrate-lauric acid mixed structure ester, which includes the following components: monolaurin monobutyrate and dilaurin monobutyrate.

[0012] In a second aspect, an embodiment of the present invention provides a method for preparing a butyric acid lauric acid mixed structure ester as described in the above embodiment, which comprises: mixing tributyrin and trilaurin to undergo an ester exchange reaction.

[0013] In a third aspect, an embodiment of the present invention provides the use of the mixed structure ester of butyrate and lauric acid as described in the above embodiment or the mixed structure ester of butyrate and lauric acid prepared by the preparation method described in the above embodiment in the preparation of a feed additive for promoting intestinal health.

[0014] In a fourth aspect, embodiments of the present invention provide the use of the mixed structure ester of butyrate and lauric acid as described in the preceding embodiments or the mixed structure ester of butyrate and lauric acid prepared by the preparation method described in the preceding embodiments in the preparation of a feed additive for regulating the intestinal microbial environment.

[0015] In a fifth aspect, an embodiment of the present invention provides a feed additive, which includes the butyrate lauric acid mixed structure ester as described in the above embodiment or the butyrate lauric acid mixed structure ester prepared by the preparation method as described in the above embodiment.

[0016] In a sixth aspect, an embodiment of the present invention provides a feed, which includes the butyrate lauric acid mixed structure ester as described in the above embodiment or the butyrate lauric acid mixed structure ester prepared by the preparation method as described in the above embodiment.

[0017] The present invention has the following beneficial effects:

[0018] The invention provides a new butyric acid lauric acid mixed structure ester, which comprises monobutyric acid dilauric acid glyceryl and dibutyric acid monolauric acid glyceryl. After the mixed structure ester contacts with lipase in the intestinal tract of an animal, it has the effect of slowly releasing butyric acid and monolauric acid glyceryl. Compared with the existing feed additives, it can more effectively improve the transmission effect of functional factors, can induce positive changes in intestinal microorganisms within 24 hours, and can more effectively improve the intestinal function of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1The change of butyric acid content in static digestion in Example 2; wherein A: the change of butyric acid content in simulated gastric digestion. A`: the change of butyric acid content in simulated intestinal digestion; M BL It represents a physical mixture of tributyrin and trilaurin, S BL represents mixed structure esters of butyric acid and lauric acid; the number of replicates for each time point = 3, and the results are expressed as mean ± standard deviation;

[0021] Figure 2 The change of GML content in static digestion in Example 2; wherein A: the change of GML content in simulated gastric digestion; A`: the change of GML content in simulated intestinal digestion; M BL Indicates that S BL A physical mixture of tributyrin and trilaurin with the same fatty acid composition, S BL represents mixed structure esters of butyric acid and lauric acid; the number of replicates for each time point = 3, and the results are expressed as mean ± standard deviation;

[0022] Figure 3 The changes in butyric acid content in the mouse model of Example 2; A: the changes in butyric acid content in intestinal contents at different intestinal sites and different sampling time points; capital letters show the differences in butyric acid content in intestinal contents at different intestinal sites at a certain sampling time point, and different letters indicate significant differences (P < 0.05); lowercase letters show the differences in butyric acid content in intestinal contents at different gavage time points in a certain intestinal site, and different letters indicate significant differences (P < 0.05); structural esters and physical mixtures are compared separately. B: butyric acid content in colon contents 8 hours after gavage, **** indicates P < 0.0001; M BL It represents a physical mixture of tributyrin and trilaurin, S BL represents mixed structural esters of butyric acid and lauric acid; the numerical subscripts '-1, -2, -4, -8' represent samples 1, 2, 4, and 8 hours after intragastric administration, respectively; the number of replicates for each time point = 6, and the results are expressed as mean ± standard deviation;

[0023] Figure 4The changes of GML content in the mouse model of Example 2; wherein, A: the changes of GML content in intestinal contents at different intestinal parts and different sampling time points; capital letters show the difference of butyric acid content in the contents of different intestinal parts at a certain sampling time point, different letters indicate significant differences (P < 0.05), lowercase letters show the difference of butyric acid content in intestinal contents at different gavage time points in a certain intestinal part, different letters indicate significant differences (P < 0.05), structural esters and physical mixtures are compared separately. B: GML content in jejunal contents 1 hour after gavage, **** indicates P < 0.0001; C: GML content in jejunal contents 2 hours after gavage, * indicates P < 0.05; D: GML content in jejunal contents 8 hours after gavage, * indicates P < 0.05; E: GML content in colonic contents 8 hours after gavage, *** indicates P < 0.001; M BL It represents a physical mixture of tributyrin and trilaurin, S BL represents mixed structural esters of butyric acid and lauric acid; P < 0.05 is considered to be significantly different, and P < 0.1 is considered to be a significant trend; the numerical subscripts '-1, -2, -4, -8' represent samples 1, 2, 4, and 8 hours after gavage, respectively; the number of replicates for each time point = 6, and the results are expressed as mean ± standard deviation;

[0024] Figure 5 The figure shows the changes in intestinal microorganisms in the cecum of mice 24 hours after oral gavage in Example 3; wherein, A: abundance of Bifidobacterium; B: abundance of Allobaculum; C: abundance of unidentified F16; D: abundance of CC-115; * indicates that P < 0.05 is considered to be significantly different, and P < 0.1 is considered to be a significant trend. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] The embodiment of the present invention provides a butyrate-lauric acid mixed structure ester, which comprises the following components: monobutyrate dilaurin and dibutyrate monolaurin.

[0027] After a series of creative work, the inventors have provided a butyric acid lauric acid mixed structure ester, which has the characteristics of slowly releasing butyric acid and monolaurin. Compared with existing feed additives, it can more effectively improve animal intestinal function and maintain intestinal environment homeostasis to facilitate animal growth.

[0028] In some embodiments, the weight ratio of glyceryl monolaurate monobutyrate to glyceryl dilaurate dibutyrate is not limited. Preferably, the weight ratio of glyceryl monolaurate monobutyrate to glyceryl dilaurate dibutyrate is (1-5):1, and the weight ratio can specifically be 1:1, 2:1, 3:1, 4:1 or 5:1.

[0029] Preferably, the monolaurin dibutyric acid glyceryl ester includes: 1.2-dilaurin-3-butyric acid glyceryl ester and 1.3-dilaurin-2-butyric acid glyceryl ester.

[0030] Preferably, the dibutyric acid monolaurin comprises: 1.2-dibutyric acid-3-laurin glyceryl and 1.3-dibutyric acid-2-laurin glyceryl.

[0031] Preferably, the butyric acid lauric acid mixed structure ester further comprises at least one of the following components: lauric acid, monobutyric acid glyceryl, tributyric acid glyceryl, monobutyric acid monolauric acid glyceryl and dilauric acid glyceryl. Within this limited range, the butyric acid lauric acid mixed structure ester has a better effect.

[0032] Preferably, the monobutyric acid monolaurin is selected from at least one of 1-butyric acid-3-lauric acid glyceryl, 3-butyric acid-1-lauric acid glyceryl, 2-butyric acid-1-lauric acid glyceryl, 2-butyric acid-3-lauric acid glyceryl, 2-lauric acid-1-butyric acid glyceryl and 2-lauric acid-3-butyric acid glyceryl. More preferably, the butyric acid lauric acid mixed structure ester comprises the following components by weight: 5 to 20 parts of lauric acid, 1 to 10 parts of monobutyric acid glyceryl, 1 to 10 parts of tributyric acid glyceryl, 10 to 30 parts of monobutyric acid monolauric acid glyceryl, 15 to 40 parts of dibutyric acid monolauric acid glyceryl, 5 to 20 parts of dilauric acid glyceryl, and 20 to 50 parts of monobutyric acid dilauric acid glyceryl.

[0033] Preferably, the butyric acid lauric acid mixed structure ester includes the following components by weight: 5 to 20 parts of lauric acid, 1 to 10 parts of monobutyric acid glyceryl, 1 to 10 parts of tributyric acid glyceryl, 3 to 10 parts of 1-butyric acid-3-lauric acid glyceryl, 7 to 20 parts of 2-butyric acid-1-lauric acid glyceryl, 6 to 15 parts of 1.2-dibutyric acid-3-lauric acid glyceryl, 9 to 25 parts of 1.3-dibutyric acid-2-lauric acid glyceryl, 1 to 7 parts of 1.2-dilauric acid glyceryl, 4 to 13 parts of 1.3-dilauric acid glyceryl, 12 to 30 parts of 1.2-dilauric acid-3-butyric acid glyceryl and 8 to 20 parts of 1.3-dilauric acid-2-butyric acid glyceryl. Under the limited range of this specific component and its ratio, the butyric acid lauric acid mixed structure ester can better exert its effect. Specifically, the weight proportion of lauric acid can be selected from 5 parts, 6 parts, 8 parts, 10 parts, 12 parts, 14 parts, 16 parts, 18 parts or 20 parts; the weight proportion of monobutyric acid glyceride can be selected from 1 part, 2 parts, 4 parts, 6 parts, 8 parts or 10 parts; the weight proportion of tributyrin can be selected from 1 part, 2 parts, 4 parts, 6 parts, 8 parts or 10 parts; the weight proportion of 1-butyric acid-3-lauric acid glyceride can be selected from 3 parts, 4 parts, 6 parts, 8 parts or 10 parts; the weight proportion of 1.2-dibutyric acid-3-lauric acid glyceride can be selected from 6 parts, 9 parts, 10 parts, 12 parts, 14 parts, 16 parts, 18 parts, 20 parts or 22 parts; the weight proportion of 1.3-dibutyric acid-2-lauric acid glyceride can be selected from 1. The number of parts can be selected from 9, 10, 12, 14, 16, 18, 20, 22 or 24 parts; the weight parts of 1.2-dilaurin glyceride can be selected from 1, 2, 4, 6 or 7 parts; the weight parts of 1.3-dilaurin glyceride can be selected from 4, 6, 8, 10, 12 or 13 parts; the weight parts of 1.2-dilaurin-3-butyric acid glyceride can be selected from 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 parts; the weight parts of 1.3-dilaurin-2-butyric acid glyceride can be selected from 8, 10, 12, 14, 16, 18 or 20 parts.

[0034] The present invention also provides a method for preparing a butyric acid lauric acid mixed structure ester as described in any of the above embodiments, which comprises: mixing tributyrin and trilaurin to undergo an ester exchange reaction. The "ester exchange reaction" herein refers to an ester-ester exchange reaction, a reaction in which an ester and an ester (different esters) generate a new ester and a new alcohol / acid / ester under the catalysis of an acid, a base or an enzyme.

[0035] Preferably, when the transesterification reaction occurs, the mixing molar ratio of tributyrin and trilaurin is (0.5-5): (0.5-5);

[0036] Preferably, the mixing molar ratio is (0.5-1.5): (0.5-1.5). More preferably, the mixing molar ratio is 1:1. Under this definition, the generated butyric acid lauric acid mixed structure ester has a better sustained release effect of butyric acid and monolaurin, and can better exert its effect on the animal intestine.

[0037] Preferably, the steps of the transesterification reaction are as follows: under a nitrogen atmosphere, tributyrin and trilaurin are mixed at 60-70° C., after the mixture is uniform and liquid, immobilized lipase is added, the mixture is kept warm for 2-6 hours, and after the immobilized lipase is removed, the butyric acid lauric acid mixed structure ester is obtained;

[0038] Preferably, the amount of the immobilized lipase added is 5-15% of the total weight of the mixture.

[0039] Preferably, the immobilized lipase is selected from any one of immobilized Aspergillus oryzae lipase, immobilized Rhizopus oryzae lipase and immobilized Rhizopus miehei lipase.

[0040] The embodiments of the present invention also provide the use of the butyrate lauric acid mixed structure ester as described in any of the foregoing embodiments or the butyrate lauric acid mixed structure ester prepared by the preparation method as described in any of the foregoing embodiments in the preparation of a feed additive for promoting intestinal health.

[0041] In some embodiments, promoting intestinal health can be achieved by maintaining at least one of the following functions: maintaining the homeostasis of intestinal microbial flora and improving the structure of the microbial flora.

[0042] The embodiments of the present invention also provide the use of the mixed structure ester of butyrate and lauric acid as described in any of the foregoing embodiments or the mixed structure ester of butyrate and lauric acid prepared by the preparation method described in any of the foregoing embodiments in the preparation of a feed additive for regulating the intestinal microbial environment.

[0043] Preferably, regulating the intestinal microbial environment refers to increasing the abundance of intestinal microorganisms;

[0044] Preferably, regulating the intestinal microbial environment refers to increasing the abundance of at least one genus among Bifidobacterium, Allobaculum, F16 and CC-115.

[0045] The embodiment of the present invention further provides a feed additive, which includes the butyrate lauric acid mixed structure ester as described in any of the above embodiments or the butyrate lauric acid mixed structure ester prepared by the preparation method as described in any of the above embodiments.

[0046] In some embodiments, the feed may also include existing feed additive carriers and / or components used for farmed animals, which can be obtained through existing channels and will not be described in detail.

[0047] The embodiment of the present invention further provides a feed, which comprises the butyrate lauric acid mixed structure ester as described in any of the foregoing embodiments or the butyrate lauric acid mixed structure ester prepared by the preparation method as described in any of the foregoing embodiments.

[0048] In some embodiments, the feed may also include existing feed carriers and / or components used for farmed animals, which can be obtained through existing channels and will not be described in detail.

[0049] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0050] Example 1 Preparation and Composition Determination of Sustained Release Butyric Acid Lauric Acid Mixed Ester

[0051] A certain amount of tributyrin and trilaurin were added to a 100 mL round-bottom flask (molar ratio = 1:1), filled with nitrogen, covered with a turn-down stopper, and placed in a constant temperature water bath oscillator at a temperature of 65°C. After the reaction substrate was mixed evenly and in a liquid state, immobilized lipase (10%, w / w) was added, nitrogen was flushed again for protection, the turn-down stopper was covered, and the reaction was kept warm for 4 hours at 200 rpm. After the reaction was completed, the immobilized enzyme was filtered out and the butyric acid lauric acid mixed structure ester was collected.

[0052] Accurately weigh 10.0 mg of the structural ester and place it in a 10 mL volumetric flask, dilute it to the mark with methanol, and transfer it to a 2 mL injection bottle after complete sonication for GC-MS analysis. The instrument model is 7890A-5975C gas chromatograph (GC-MS), Agilent Technologies, USA. Chromatographic conditions: chromatographic column is HP-5MS (30.0 m × 250 μm, 0.25 μm); the initial temperature of the chromatographic column is 50 ° C for 2 min, then 15 ° C / min to 280 ° C, and keep it for 20 min; the vaporization chamber temperature is 280 ° C; the transmission line temperature is 280 ° C; carrier gas He; carrier gas flow rate is 1.0 mL / min; split ratio is 10: 1; injection volume is 0.5 μ L. Mass spectrometry conditions: EI source; electron energy 70 eV; ion source temperature 300 ° C; quadrupole 150 ° C; scanning mode is Scan; scanning mass range is 20 ~ 800 u.

[0053] After identification, the composition of the mixed structure ester of butyric acid and lauric acid is shown in Table 1.

[0054] Table 1 Composition of mixed structure esters of butyric acid and lauric acid

[0055]

[0056]

[0057] Example 2 In vivo and in vitro digestion characteristics of sustained-release butyrate and laurate mixed structure esters

[0058] The digestion characteristics of butyric acid lauric acid mixed structure esters were studied in an in vitro digestion model and an in vivo mouse model. Specifically, this example uses tributyrin and trilaurin in a molar ratio of 1:1.5 as raw materials, an enzyme addition amount of 7% (w / w), and a reaction at 62°C for 6h to prepare (substantially the same as in Example 1) the butyric acid lauric acid structure ester obtained as the experimental group (S LBL ), with equal molar contents of tributyrin and trilaurin (M LBL ) was used as the control group, and experiments were carried out in an in vitro digestion model and an in vivo mouse model. In the in vitro digestion model, digestion samples were taken at 0min, 30min, 60min, 90min, 120min, 150min, 180min, 210min, and 240min after gavage, of which the first 120min was simulated gastric digestion in vitro, and 120min to 240min was simulated intestinal digestion. In the in vivo mouse model, intestinal content samples (jejunum, ileum, cecum, and colon) were taken at 0min, 60min, 120min, 240min, and 320min after gavage. The collected samples were analyzed by GC-FID, and butyric acid and monolaurin (GML) were monitored as the key objects. The results are shown in the following figure. Figure 1 to Figure 4 shown.

[0059] The results show that in the in vitro digestion model, S BL The results showed that butyrate was released more slowly, and this slow release effect was significant. However, this slow release effect on butyrate was not significant in the simulated intestinal digestion. BL In the in vivo mouse model, except for the colon contents digested for 8 hours, S BL and M BL There were no significant differences in butyrate release.

[0060] In the mouse model, S BL It has a significant effect of delaying the release of GML: after 1 hour of in vivo digestion, S BL and M BL The GML content in the treated groups was significantly higher than that in the blank group. After 2 h of digestion, S BL The jejunal GML content of the treated group was significantly higher than that of M BL The trend of the treatment group, when digestion continued for 8 hours, S BL The content of GML in the jejunum of the treatment group was significantly higher than that of M BL group. Relative to physical mixture, S BLThe sustained release of butyrate during gastric digestion and the sustained high level of release of GML in the intestinal tract in vivo may be beneficial to improve its functional value and maintain the homeostasis of the intestinal environment. BL Treatment group and M BL There was no significant difference in butyrate content between the treatment groups.

[0061] Example 3 The transient effect of sustained-release butyrate and laurate mixed esters on intestinal microorganisms

[0062] Intestinal microorganisms are easily affected by environmental factors such as dietary ingredients. This disturbance of intestinal microorganisms can be manifested in a relatively short period of time (24 to 48 hours). In this example, tributyrin and trilaurin with a molar ratio of 1:2 were used as raw materials, the enzyme addition amount was 12% (w / w), and the reaction was carried out at 67°C for 4 hours to prepare (similar to Example 1) the obtained butyric acid lauric acid structure ester as the experimental group (S LBL ), with the same molar content of tributyrin and trilaurin (M LBL ) was used as the control group, and the experiments were conducted in an in vitro digestion model and an in vivo mouse model. Cecal samples were collected 24 hours after gavage and 16S RNA sequencing was used to analyze the S BL and M BL (Same as the previous example) The disturbance of mouse intestinal microorganisms, the results are as follows Figure 5 shown.

[0063] Bifidobacterium is a recognized probiotic. BL The abundance of Bifidobacterium in the treated group was significantly higher than that in the control group and M BL Group trend.

[0064] In addition, Allobaculum is also a potential intestinal microorganism with health benefits. BL The abundance in the treated group showed a significant increase compared with the blank group, which also proved that S BL Relative to M BL With better potential health benefits.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An application of a butyric acid lauric acid mixed structure ester in the preparation of a feed additive for promoting intestinal health, characterized in that: After the butyric acid and lauric acid mixed structure ester comes into contact with lipase in the animal intestine, it slowly releases butyric acid and monolaurin, inducing positive changes in intestinal microorganisms within 24 hours; The preparation method of butyric acid lauric acid mixed structure ester comprises: mixing tributyrin and trilaurin at 60-70° C. under a nitrogen atmosphere, adding immobilized lipase after the mixture is uniform and liquid, keeping the mixture warm for 2-6 hours, removing the immobilized lipase, and obtaining the butyric acid lauric acid mixed structure ester; The mixing molar ratio of the tributyrin and the trilaurin is (0.5-5): (0.5-5); the addition amount of the immobilized lipase is 5-15% of the total weight of the mixture; The prepared butyric acid lauric acid mixed structure ester comprises the following components: 5-20 parts of lauric acid, 1-10 parts of monobutyric acid glyceryl, 1-10 parts of tributyric acid glyceryl, 3-10 parts of 1-butyric acid-3-lauric acid glyceryl, 7-20 parts of 2-butyric acid-1-lauric acid glyceryl, 6-15 parts of 1.2-dibutyric acid-3-lauric acid glyceryl, 9-25 parts of 1.3-dibutyric acid-2-lauric acid glyceryl, 1-7 parts of 1.2-dilauric acid glyceryl, 4-13 parts of 1.3-dilauric acid glyceryl, 12-30 parts of 1.2- 1,3-dilauryl-2-butyrin and 8 to 20 parts of 1,3-dilauryl-2-butyrin.

2. The use of the butyric acid lauric acid mixed structure ester according to claim 1 in the preparation of a feed additive for promoting intestinal health, characterized in that: The immobilized lipase is selected from any one of immobilized Aspergillus oryzae lipase, immobilized Rhizopus oryzae lipase and immobilized Rhizopus miehei lipase.

3. A use of the butyric acid lauric acid mixed structure ester as claimed in claim 1 in the preparation of a feed additive for regulating the intestinal microbial environment, characterized in that: The regulating the intestinal microbial environment refers to increasing the abundance of at least one genus among Bifidobacterium, Allobaculum and CC-115.

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