Structural ester containing OPL and LPL as well as preparation method and application of structural ester

Abstract

The invention discloses structural ester containing OPL and LPL as well as a preparation method and application thereof, and relates to the technical field of grease. The structural ester containing the OPL and the LPL is prepared from the following raw materials: palmitic acid, glycerol, lipase A, absolute ethyl alcohol, safflower seed oil fully-hydrolyzed oil and lipase B. The structural ester containing the OPL and the LPL is prepared from the following raw materials, the preparation method comprises the following steps: S1, mixing palmitic acid and glycerol, adding lipase A, and reacting to obtain a first material; s2, mixing the first material with absolute ethyl alcohol, then adding lipase A for reaction, and performing separation treatment on a reaction product to obtain a second material; and S3, mixing the second material with the safflower seed oil fully-hydrolyzed oil, then adding lipase B for reaction, and carrying out separation treatment on the reaction product to obtain the product structure ester. The product is rich in OPL and LPL, can effectively improve constipation, and is easy to use.

Description

Technical Field

[0001] The present application relates to the technical field of oils and fats, and in particular to a structural ester containing OPL and LPL, and a preparation method and application thereof. Background Art

[0002] Constipation is common in clinical practice, manifested as difficulty in defecation, reduced bowel movement, dry and hard stools, etc. Due to the irregular rhythm of modern people's lives, unreasonable diet structure and psychological pressure, the incidence of constipation is gradually increasing. Long-term use of laxatives by patients is prone to dependence and damage to the intestines.

[0003] At present, the main products for laxatives include traditional Chinese medicine, dietary fiber and probiotics. Traditional Chinese medicine contains a variety of traditional Chinese medicine ingredients and has complex medicinal properties. Long-term use is prone to side effects. Dietary fiber can only produce certain effects if taken in large doses, and the soluble dietary fiber content is required to account for a higher proportion. It tastes bad and is not easy to take consistently. The types and numbers of intestinal flora in each person are different, and probiotics have limited strains and may not adapt to everyone's intestinal environment. Long-term use can easily change the types of natural flora and affect physical health. In addition, most probiotic products have insufficient or excessive live bacteria, which will have an impact on the body. Therefore, it is necessary to find a new, safe, nutritious and functional substance. Summary of the invention

[0004] The purpose of the present application is to provide a structural ester containing OPL and LPL and a preparation method thereof. The product is rich in OPL (1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride) and LPL (1,3 linoleic acid-2-palmitic acid triglyceride), can effectively improve constipation, and is easy to use.

[0005] Another object of the present application is to provide an application of the above-mentioned structural ester in the preparation of food, health products or medicines that help to moisturize the intestines and promote bowel movements.

[0006] The technical solution of this application is as follows:

[0007] On the one hand, the present invention provides a structural ester containing OPL and LPL, which is prepared from raw materials including the following components:

[0008] Palmitic acid, glycerol, lipase A, anhydrous ethanol, fully hydrolyzed safflower seed oil, and lipase B.

[0009] Furthermore, in some embodiments of the present application, the lipase A is Novozym 435; and the lipase B is Aspergillus niger lipase.

[0010] Furthermore, in some embodiments of the present application, the oleic acid content of the fully hydrolyzed safflower seed oil is 15-25%, and the linoleic acid content is 60-70%.

[0011] Furthermore, in some embodiments of the present application, the total content of OPL and LPL in the above-mentioned structural ester is greater than 60%.

[0012] The present application also provides a method for preparing the above-mentioned structural ester containing OPL and LPL, which comprises the following steps:

[0013] S1: taking palmitic acid and glycerol, mixing them, adding lipase A, and reacting to obtain a first material;

[0014] S2: mixing the first material with anhydrous ethanol, and then adding lipase A to react, and separating the reaction product to obtain the second material;

[0015] S3: mixing the second material with the fully hydrolyzed safflower seed oil, and then adding lipase B to react, and separating and treating the reaction product to obtain a product structure ester.

[0016] Furthermore, in some embodiments of the present application, in the above-mentioned step S1, the molar ratio of palmitic acid to glycerol is 1:(3-8), preferably 1:(3-6); the added amount of lipase A is 3-15% of the total mass of palmitic acid and glycerol, preferably 6-12.

[0017] Furthermore, in some embodiments of the present application, in the above step S1, the reaction is carried out under vacuum conditions at a temperature of 40-70°C, preferably 55-65°C, for 12-48 hours, preferably 18-40 hours, most preferably 24-36 hours to obtain the first material.

[0018] Furthermore, in some embodiments of the present application, in the above-mentioned step S2, the mass ratio of the first material to anhydrous ethanol is 1: (2-15), preferably 1: (5-10); the added amount of lipase A is 3-15% of the total mass of the first material and anhydrous ethanol, preferably 6-12.

[0019] Furthermore, in some embodiments of the present application, in the above-mentioned step S2, the reaction is carried out at a temperature of 25-45°C, preferably 35-40°C, under a protective gas atmosphere (preferably nitrogen), for 3-12 hours, preferably 6-10 hours, and most preferably 6-8 hours, and the reaction product is separated to obtain a second material.

[0020] Further, in some embodiments of the present application, in the above step S2, n-hexane and water are added to the reaction product, ultrasonically mixed and centrifuged to separate the layers, the lower ethanol-water phase is collected, washed with n-hexane, and then ethanol and water are removed in vacuo to obtain a second material;

[0021] Among them, n-hexane is at a m / v ratio of 1: (1-10), and water is at a m / v ratio of (10-20): 1; the lower ethanol-water phase is collected, washed three times with n-hexane at a v / v ratio of 1: (1-10), and then ethanol and water are removed under vacuum conditions at 85°C to obtain a second material.

[0022] Furthermore, in some embodiments of the present application, in the above-mentioned step S3, the mass ratio of the second material to the fully hydrolyzed safflower seed oil is 1:(2-8), preferably 1:(2-5); the added amount of lipase B is 3-15% of the total mass of the second material and the fully hydrolyzed safflower seed oil, preferably 6-12.

[0023] Furthermore, in some embodiments of the present application, in the above step S3, the reaction is carried out under vacuum conditions at a temperature of 40 to 60° C., preferably 45 to 55° C., for 3 to 18 hours, preferably 6 to 12 hours, most preferably 6 to 9 hours, and the reaction product is separated to obtain a product structured ester.

[0024] Furthermore, in some embodiments of the present application, in the above step S3, the reaction product is subjected to molecular distillation to remove free fatty acids in the distillate, and the triglycerides in the distillate are collected to obtain product structured esters.

[0025] Furthermore, in some embodiments of the present application, during the molecular distillation process, the evaporation temperature is 170-220° C., the rotor speed is 1000-1100 rpm, and the pressure is 1-3 Pa.

[0026] On the other hand, an embodiment of the present application also provides a use of the above-mentioned structural ester containing OPL and LPL in the preparation of foods, health products or medicines that help to moisturize the intestines and promote bowel movements.

[0027] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0028] In view of the above aspects, the embodiments of the present application provide a structured ester containing OPL and LPL and a preparation method thereof, and the use of the structured fat in the preparation of foods, health products or medicines that help to moisturize the intestines and promote bowel movements. It uses components including palmitic acid, glycerol, anhydrous ethanol, and fully hydrolyzed safflower oil as raw materials, and uses lipase to prepare a structured ester product rich in OPL and LPL through a three-step enzymatic reaction; wherein, OPL and LPL can produce oleic acid, linoleic acid and palmitic acid monoglyceride at the sn-2 position after hydrolysis at the sn-1,3 positions. Palmitic acid is not easy to combine with calcium ions at the sn-2 position to form insoluble saponified salts, which reduces the excretion of calcium saponified fat, softens feces, and prevents constipation. In addition, OPL and LPL can induce the proliferation of bifidobacteria and lactobacilli in the intestines of mice, promote intestinal peristalsis, and regulate intestinal endocrine, thereby improving constipation.

[0029] The structural ester product prepared by the method of the present application can effectively increase the content of OPL and LPL in the product, reduce the content of sn-1,3 palmitic acid, reduce the production of byproducts PPP and OPP, LPP, and help to further improve the product efficacy; at the same time, the preparation process does not use any toxic or harmful solvents, avoiding the pollution of the solvent to the product and the environment, and the preparation method is green, safe and efficient. The structural ester product can be further prepared into soft capsules to prevent oxidation, which is beneficial to quality preservation and storage and transportation, and is more convenient to take.

[0030] The structural ester provided in the present application can be used to prepare foods, health products or medicines that help to moisturize the intestines and promote bowel movements. Its specific efficacy is manifested in promoting small intestinal motility, increasing the number and water content of fecal particles, shortening the time to discharge of the first black stool, promoting the increase of small intestinal goblet cells, and promoting the expression of mucin 2 (Muc2) genes and response proteins in the small intestine and colon, and has a significant improvement effect on reduced bowel movement frequency, dry and hard stools, and difficult defecation. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application 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 paying creative work.

[0032] Figure 1 The product structure ester gas chromatogram provided in Example 1;

[0033] Figure 2 The product structure ester gas chromatogram provided for Example 2;

[0034] Figure 3 The product structure ester gas chromatogram provided in Example 3;

[0035] Figure 4 This is the result diagram of the small intestinal propulsion rate of mice in the preventive drug administration experiment in Experimental Example 2;

[0036] Figure 5 This is a graph showing the results of the small intestinal propulsion rate of mice in the treatment and administration experiment in Experimental Example 2;

[0037] Figure 6 This is the result diagram of the first defecation time of mice in Experimental Example 2;

[0038] Figure 7 This is the result diagram of the number of mouse feces in Experimental Example 2;

[0039] Figure 8 This is the result diagram of the moisture content of mouse feces in Experimental Example 2;

[0040] Fig. 9 This is the result of the number of goblet cells in the small intestine of mice in Experimental Example 2;

[0041] Fig.10 This is the result diagram of the relative expression of Muc2 in mouse colon tissue in Experimental Example 2;

[0042] Fig.11 This is the result diagram of the relative expression of Muc2 protein in mouse colon tissue in Experimental Example 2. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0044] It should be noted that relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprises" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprises..." do not exclude the existence of other identical elements in the process, method, article or device including its elements.

[0045] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other.

[0046] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0047] Example 1

[0048] The present application example provides a structural ester containing OPL and LPL, which is prepared by the following method:

[0049] S1: palmitic acid and glycerol were mixed in a molar ratio of 1:3, and then 6% of the mass of the mixed material Novozym435 was added, and the mixture was reacted at a temperature of 55° C. for 24 hours under a vacuum condition of 100 Pa to obtain a first material;

[0050] S2: The first material and anhydrous ethanol are mixed at a mass ratio of 1:5, and then 6% of the mass of Novozym 435 of the mixed material is added, nitrogen is filled and sealed, and the reaction is carried out at 35°C for 6 hours; the reaction product is collected, n-hexane is added at a m / v ratio of 1:1, and pure water is added at a m / v ratio of 10:1, ultrasonic mixing is performed, centrifugation is performed, and the lower ethanol-water phase is collected, and washed with n-hexane at a v / v ratio of 1:1 for 3 times, and then ethanol and water are removed under vacuum conditions at 85°C to obtain the second material;

[0051] S3: The second material is mixed with the fully hydrolyzed safflower seed oil in a mass ratio of 1:2, and then 6% of the mass of the mixed material Aspergillus niger lipase is added, and the reaction is carried out at a temperature of 45°C for 6 hours under a vacuum condition of 100 Pa. The reaction product is subjected to molecular distillation to remove free fatty acids in the evaporation product, and triglycerides in the evaporation product are collected to obtain product structure esters.

[0052] Example 2

[0053] The present application example provides a structural ester containing OPL and LPL, which is prepared by the following method:

[0054] S1: palmitic acid and glycerol are mixed in a molar ratio of 1:4, and then 6% of the mass of the mixed material is added with Novozym435, and the mixture is reacted at a temperature of 60° C. for 30 hours under a vacuum condition of 50 Pa to obtain a first material;

[0055] S2: The first material and anhydrous ethanol are mixed at a mass ratio of 1:7, and then 8% of the mass of Novozym 435 of the mixed material is added, nitrogen is filled and sealed, and the reaction is carried out at 35°C for 7 hours; the reaction product is collected, n-hexane is added at a m / v ratio of 1:2, and pure water is added at a m / v ratio of 15:1, ultrasonic mixing is performed, centrifugation is performed, and the lower ethanol-water phase is collected, and washed with n-hexane at a v / v ratio of 1:2 for 3 times, and then ethanol and water are removed under vacuum conditions at 85°C to obtain the second material;

[0056] S3: The second material is mixed with the fully hydrolyzed safflower seed oil in a mass ratio of 1:3, and then 8% of the mass of the mixed material Aspergillus niger lipase is added, and the reaction is carried out at a temperature of 50°C for 7 hours under a vacuum condition of 50 Pa; the reaction product is subjected to molecular distillation to remove free fatty acids in the evaporation product, and triglycerides in the evaporation product are collected to obtain product structure esters.

[0057] Example 3

[0058] The present application example provides a structural ester containing OPL and LPL, which is prepared by the following method:

[0059] S1: palmitic acid and glycerol were mixed in a molar ratio of 1:6, and then 12% of the mass of the mixed material Novozym435 was added, and the mixture was reacted at a temperature of 65° C. for 36 hours under a vacuum condition of 10 Pa to obtain a first material;

[0060] S2: The first material is mixed with anhydrous ethanol at a mass ratio of 1:10, and then Novozym 435 of 12% by mass of the mixed material is added, nitrogen is filled and sealed, and the reaction is carried out at 40°C for 8 hours; the reaction product is collected, n-hexane is added at a m / v ratio of 1:3, and pure water is added at a m / v ratio of 20:1, ultrasonically mixed evenly, centrifuged and separated, the lower ethanol-water phase is collected, and washed with n-hexane at a v / v ratio of 1:3 for 3 times, and then ethanol and water are removed under vacuum conditions at 85°C to obtain the second material;

[0061] S3: The second material is mixed with the fully hydrolyzed safflower oil in a mass ratio of 1:5, and then 12% of the mass of the mixed material Aspergillus niger lipase is added, and the reaction is carried out at a temperature of 55°C for 9 hours under a vacuum condition of 10 Pa; the reaction product is subjected to molecular distillation to remove free fatty acids in the evaporation product, and triglycerides in the evaporation product are collected to obtain product structure esters.

[0062] Test Example 1

[0063] The structured ester products provided in Examples 1 to 3 were subjected to gas chromatography detection, and the results were as follows: Figure 1 to Figure 3 As shown; the two peaks on the right side of the figure represent OPL and LPL, indicating that the product structure ester provided in the embodiment of the present application is rich in OPL and LPL.

[0064] Test Example 2

[0065] The purpose of this test example is to verify the laxative effect of the structural ester product of the present application on the constipation model KM mice and its effect on the expression of mucin 2 (Muc2) in the small intestine and colon.

[0066] The structured ester product provided in Example 3 was used to establish a mouse constipation model using loperamide hydrochloride. A blank control group, a positive control group, a model group, and low-, medium-, and high-dose groups were set up. The effects of the structured esters rich in OPL and LPL on the small intestinal ink propulsion rate, the time to the first black stool, the number of stool particles within 6 to 24 hours, the water content of stool, and small intestinal pathology of mice were observed through small intestinal propulsion experiments and defecation experiments. At the same time, the effects of the Muc2 gene and its expression in the small intestine and colon tissues were determined.

[0067] The experiment showed that there were significant differences between each dose group and the model group (P<0.05). The structural esters rich in OPL and LPL were beneficial to the defecation of mice, and increased the content of Muc2 in small intestinal tissue and promoted the expression of Muc2 and its gene in colon tissue, indicating that the structural esters rich in OPL and LPL have a good laxative effect. The details are as follows:

[0068] (1) Animal safety pathology experiments

[0069] The mice in the experiment were half male and half female, 10 mice in each group, and weighed 25 to 35g. The experiment used 10mg / kg of loperamide hydrochloride to create constipation models in the model group, positive control group, and low, medium, and high dose groups. The blank control group was gavaged with sterile saline, the positive control group was gavaged with Megastory (a commercially available structural ester, the main component of which is OPO (1,3-oleic acid-2-palmitoylglycerol triester) used as a nutritional enhancer for infant formula, which has a laxative effect, and its OPO content is ≥43g / 100g), and the dose group was gavaged with the structural ester of Example 3. The administration time was continuous for 14 days, during which the weight changes of the mice were monitored, and finally the mice were dissected for small intestinal tissue pathology.

[0070] Experimental results:

[0071] 1. During the experiment, the weight of mice in each group was stable: s<1.5 (standard deviation), their appetite was good, their coat color was normal, their activities were normal, and their growth and development were good.

[0072] 2. HE staining of mouse small intestinal tissue showed under an optical microscope that the model group had broken and shed mucosal layers, degeneration, necrosis and shedding of some mucosal epithelial cells, and infiltration of inflammatory cells; the blank control group had a normal mucosal layer structure, no epithelial shedding, no obvious inflammatory reaction in the lamina propria, and normal intestinal glands. Semi-quantitative analysis of small intestinal pathological changes showed that the differences between each group and the model group were statistically significant (P<0.05); there was no significant difference between the dose group and the blank control group (P>0.05), indicating that in the above pathological changes, the structural esters rich in OPL and LPL had no significant effect on each dose group.

[0073] 3. After PAS staining of mouse small intestinal tissue, image acquisition and analysis were performed. The results showed that the difference was statistically significant (P<0.05), and there was no abnormal change in the number of goblet cells in each dose group. The villus height and crypt depth were measured and the ratio was calculated. The results are shown in Table 1:

[0074] Table 1

[0075]

[0076] Note: Compared with the model group, a :p<0.01, b :p<0.05; compared with the blank group, c :p<0.01,

[0077] d :p>0.05

[0078] The results show that the structural esters rich in OPL and LPL have a mitigating effect on the modeling agent loperamide hydrochloride. There was no significant difference between the dose group and the blank control group (P>0.05), indicating that there was no obvious adverse effect on the small intestine of mice. In summary, the structural esters rich in OPL and LPL have good safety.

[0079] (2) Experiment on the efficacy of laxative

[0080] 1. Small Intestinal Motility Experiment - Preventive Administration

[0081] Each group of mice was fed with daily feed, the dosage group was gavaged with the structural ester provided in Example 3, the positive control group was gavaged with Megastory, and the blank control group and the model control group were gavaged with sterile distilled water, and the administration time was continuous for 7 days. After the last gavage of the test sample, each group of mice fasted but could not be watered for 20 hours. Except for the blank control group, the other groups were given 10 mg / kg loperamide hydrochloride. After 0.5 hours, the dosage group was gavaged with ink containing the structural ester rich in OPL and LPL at the corresponding concentration. The blank control group and the model control group were only gavaged with ink, and the positive control group was gavaged with ink containing Megastory. After 25 minutes, the cervical vertebra was immediately dislocated and killed, the intestinal cavity was opened to separate the mesentery, the intestinal tube from the pylorus at the upper end and the lower end to the ileocecal part was cut, and the small intestine was pulled into a straight line. The measured length was the "total length of the small intestine", and the length from the pylorus to the front of the ink was the "ink advancement length".

[0082] 2. Small Intestinal Motility Test - Therapeutic Drug Administration

[0083] The model control group, the dose group and the positive control group were gavaged with 10 mg / kg loperamide hydrochloride. 0.5 hours later, the dose group was gavaged with structural esters rich in OPL and LPL, the positive control group was gavaged with Megastory, and the blank control group and the model control group were gavaged with distilled water. During this period, the mice in each group were fed with daily feed, and the food intake and water intake of the mice in each group were recorded and the status of the mice was observed.

[0084] The administration time was 7 days. After the last gavage, mice in each group were fasted but not watered for 20 hours. Except for the blank control group, other groups were given 10 mg / kg loperamide hydrochloride. 0.5 hours later, the dose group was gavaged with ink containing structural esters rich in OPL and LPL at corresponding concentrations. The blank control group and the model control group were only gavaged with ink, and the positive control group was gavaged with ink containing Megastory. After 25 minutes, the mice were immediately killed by dislocation of the cervical vertebra, the intestinal cavity was opened to separate the mesentery, and the intestinal tube from the pylorus at the upper end and the ileocecal part at the lower end was cut. The small intestine was pulled into a straight line, and the measured length was the "total length of the small intestine", and the length from the pylorus to the front of the ink was the "ink advancement length".

[0085] The results of small intestinal propulsion rate in the preventive and therapeutic drug administration experiments are as follows: Figure 4 , Figure 5 shown.

[0086] 3. Defecation experiment

[0087] The dose group was gavaged with structural esters rich in OPL and LPL at corresponding concentrations, the positive control group was gavaged with Megastory, and the blank control group and the model control group were only given distilled water. The above operations were continued for 7 days. During this period, the mice in each group were fed with daily feed, and the food intake and water intake of each group of mice were recorded, and the status of the mice was observed.

[0088] After the last oral gavage, mice in each group were fasted but not watered for 20 hours. Except for the blank control group, other groups were given 10 mg / kg loperamide hydrochloride. 0.5 hours later, the dose group was gavaged with ink containing structural esters rich in OPL and LPL at corresponding concentrations, the blank control group and the model control group were only gavaged with ink, and the positive control group was gavaged with ink containing Megastory. From the beginning of the ink gavage, the mice were kept in separate cages and had a normal diet during the period. The time when the mice in each experimental group first excreted black stool was observed and recorded, and the mouse feces within 6 hours and 6 to 24 hours were collected, and the number of particles was recorded. Finally, the collected feces were weighed and recorded as the wet weight of feces, and then the feces were placed in a 65°C oven for drying for 24 hours, weighed and recorded as the dry weight of feces, and the moisture content of mouse feces was calculated.

[0089] Moisture content = (wet mass of feces - dry mass of feces) / wet mass of feces.

[0090] Defecation time results Figure 6 The feces count results are shown in Figure 7 The feces moisture content results are as shown in Figure 8 shown.

[0091] 4. PAS staining of small intestinal goblet cells

[0092] The paraffin-sectioned mouse small intestinal tissue samples were dewaxed with xylene, stained with periodic acid, Schiff, and hematoxylin in sequence, and finally dehydrated and sealed for image acquisition to observe the small intestinal goblet cells, and the number of goblet cells was analyzed using Image pro plus6.0 software.

[0093] Goblet cell count results Fig. 9 shown.

[0094] 5. Immunohistochemical staining

[0095] The paraffin sections of mouse small intestine tissue were dewaxed to water, and the tissue sections were placed in citric acid buffer (PH = 6.0) for antigen repair, and then placed in 3% hydrogen peroxide solution for incubation at room temperature in the dark for 25 minutes, and then blocked with 3% BSA (bovine serum albumin) at room temperature for 30 minutes, and then incubated with primary antibody at 4°C overnight, and covered with secondary antibody (HRP labeled) of the corresponding species of the primary antibody, and then incubated at room temperature for 50 minutes and DAB (diaminobenzidine) was used for color development. Harris hematoxylin counterstained the cell nucleus for 3 minutes, and finally dehydrated and sealed, and photographed under a microscope, and three fields of view were randomly selected, and the average optical density was calculated by Image J 1.54f software to reflect the relative expression of Muc2 protein.

[0096] The average optical density of Muc2 The results are shown in Table 2.

[0097] Table 2

[0098] Group n mean value Blank group 10 184.87±7.11 Model Group 10 <![CDATA[177.89±6.82 c ]]> Positive group 10 <![CDATA[186.50±6.16 a ]]> Low dose group 10 <![CDATA[184.04±7.58 b ]]> Medium dose group 10 <![CDATA[185.54±8.40 a ]]> High dose group 10 <![CDATA[188.09±8.07 a ]]>

[0099] Note: Compared with the model group, a :p<0.01, b :p<0.05; compared with the blank group, c :p<0.01

[0100] 6. Q-PCR determination of Muc2 gene expression

[0101] RNA from mouse colon tissue was extracted according to the kit instructions. After the purity of RNA was detected by electrophoresis, it was reverse transcribed and synthesized into cDNA, which was then used as a template for PCR amplification of β-actin and Muc2 mRNA. The primer sequence of Muc2 was: F-CCACCATTACCACCACTAC; R-TCAGGAGCACTACAGACAT; the primer sequence of β-actin was: F-GCACCACACCTTCTACAA; R-TACGACCAGAGGCATACA. The CT values ​​(cycle numbers) of Muc2 and β-actin were detected, and the △△Ct was calculated with β-actin and the blank group as controls, and the relative expression of the Muc2 gene was calculated using the 2–△△Ct method.

[0102] The relative expression results of Mu2 are shown in Figure 2. Fig.10 shown.

[0103] 7. Determination of Muc2 protein expression by Western Blot

[0104] The mouse colon tissue was added with protein extraction reagent, homogenized at 4℃, lysed and centrifuged, and the supernatant was taken. The protein concentration of each sample was adjusted by normalization method, and the protein concentration was determined by microplate reader. After adding protein loading buffer to the sample, it was boiled and denatured for 15 minutes. After separating the protein by polyacrylamide gel electrophoresis, it was transferred to PVDF (polyvinylidene fluoride) membrane. According to the instructions of the immunoblotting solution set, blocking solution was added in sequence, and primary and secondary antibodies were incubated. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the internal reference for luminescence detection. Finally, the target band was analyzed by Image J 1.54f software to calculate the relative expression of Muc2 protein.

[0105] The relative expression of Mu2 protein Fig.11 shown.

[0106] In summary, the embodiment of the present application provides a structural ester containing OPL and LPL, and its preparation method and application. The structural ester of the product is prepared by the method of the present application, which can effectively increase the content of OPL and LPL in the product, reduce the content of palmitic acid at the sn-1,3 position, and reduce the production of byproducts PPP, OPP, and LPP, which helps to further improve the efficacy of the product; at the same time, its preparation process does not use any toxic and harmful solvents, avoids the pollution of the solvent to the product and the environment, and the preparation method is green, safe, and efficient. Its structural ester product can be further prepared into a soft capsule to prevent oxidation, which is beneficial to quality preservation and storage and transportation, and it is more convenient to take. The structural ester provided in the present application can be used to prepare foods, health products, or medicines that help to moisturize the intestines and relieve constipation. Its specific efficacy is manifested in promoting small intestinal motility, increasing the number of fecal particles and water content, shortening the time of the first black stool, promoting the increase of small intestinal goblet cells, and promoting the expression of mucin 2 (Muc2) genes and response proteins in the small intestine and colon, and has a significant improvement effect on the reduction of bowel movements, dry and hard stools, and poor bowel movements.

[0107] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

Claims

1. A structural ester containing OPL and LPL, characterized in that: It is prepared from raw materials including the following components: Palmitic acid, glycerol, lipase A, anhydrous ethanol, fully hydrolyzed safflower seed oil, and lipase B.

2. The structural ester containing OPL and LPL according to claim 1, characterized in that The lipase A is Novozym435; the lipase B is Aspergillus niger lipase.

3. A method for preparing a structural ester containing OPL and LPL as claimed in any one of claims 1 to 2, characterized in that: It includes the following steps: S1: taking palmitic acid and glycerol, mixing them, adding lipase A, and reacting to obtain a first material; S2: mixing the first material with anhydrous ethanol, and then adding lipase A to react, and separating the reaction product to obtain the second material; S3: mixing the second material with the fully hydrolyzed safflower seed oil, and then adding lipase B to react, and separating and treating the reaction product to obtain a product structure ester.

4. The preparation method according to claim 3, characterized in that: In the step S1, the molar ratio of palmitic acid to glycerol is 1:(3-8); and the added amount of lipase A is 3-15% of the total mass of palmitic acid and glycerol.

5. The preparation method according to claim 3, characterized in that: In the step S1, the reaction is carried out at a temperature of 40 to 70° C. under vacuum conditions for 12 to 48 hours to obtain a first material.

6. The preparation method according to claim 3, characterized in that: In the step S2, the mass ratio of the first material to anhydrous ethanol is 1:(2-15); the added amount of lipase A is 3-15% of the total mass of the first material and anhydrous ethanol.

7. The preparation method according to claim 3, characterized in that: In the step S2, the reaction is carried out at a temperature of 25 to 45° C. for 3 to 12 hours under a protective gas atmosphere, and the reaction product is separated to obtain a second material.

8. The preparation method according to claim 3, characterized in that: In the step S3, the mass ratio of the second material to the fully hydrolyzed safflower seed oil is 1:(2-8); and the added amount of lipase B is 3-15% of the total mass of the second material and the fully hydrolyzed safflower seed oil.

9. The preparation method according to claim 3, characterized in that: In the step S3, the reaction is carried out at a temperature of 40 to 60° C. under vacuum conditions for 3 to 18 hours, and the reaction product is separated to obtain a product structure ester.

10. Use of the structural ester containing OPL and LPL as claimed in any one of claims 1 to 2 in the preparation of foods, health products or medicines that help to moisturize the intestines and promote bowel movements.

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