Diglyceride for reducing triglyceride, cholesterol and uric acid as well as biological enzyme preparation method and application thereof

Abstract

The invention relates to the technical field of food, in particular to diglyceride for reducing triglyceride, cholesterol and uric acid as well as a biological enzyme preparation method and application of the diglyceride. The preparation method comprises the following steps: (1) adding water and glycerol into vegetable oil, and then adding immobilized lipase to react to obtain a reactant; (2) centrifuging reactants, collecting a light-phase product, carrying out molecular distillation, and collecting a heavy-phase product to obtain diglyceride; the immobilized lipase comprises lipase and a carrier, the carrier comprises loofah sponge, soybean meal and bentonite, and the mass ratio of the loofah sponge to the soybean meal to the bentonite is 1: (2-4): (3-6). The method has the advantages that the effects of reducing triglyceride, total cholesterol and uric acid are relatively good, and the yield and the purity are relatively high.

Description

Technical Field

[0001] The present invention relates to the field of food technology, and particularly to a diglyceride for reducing triglyceride, cholesterol and uric acid, a method for preparing the same by bioenzymatic method and its application. Background Art

[0002] Diacylglycerol (DAG), also known as di-fatty acid glyceride, diglyceride, and diglycerol, is a product synthesized by esterifying one molecule of glycerol and two molecules of free fatty acids or a structural lipid in which one fatty acid in triacylglycerol (TAG) is replaced by a hydroxyl group. Recent studies have shown that edible oils rich in DAG, as a new type of functional oil, have a variety of physiological activities and functions, can prevent and treat fatty liver and cardiovascular and cerebrovascular diseases, do not accumulate in the body after ingestion, and have important functions such as preventing obesity, and are widely used.

[0003] The proportion of diglyceride in edible oil is more than 40%. The main components of edible oil are triglyceride and diglyceride. The triglyceride in traditional vegetable oil is generally about 98%, and the diglyceride is about 2%. Diglyceride is a trace component of natural vegetable oil and an endogenous intermediate product of in vivo fat metabolism, and is a natural component existing in edible oil. It has a different molecular structure from triglyceride, and thus has a different metabolic mode.

[0004] At present, the preparation methods of diglyceride are mainly divided into chemical method and bioenzymatic method. The chemical method requires high temperature conditions, and the finished product has a deep color and poor flavor. The bioenzymatic method does not require high temperature conditions, the reaction conditions are mild, and the energy consumption is small. Therefore, the preparation of diglyceride by bioenzymatic method is a green and environmentally friendly synthesis process.

[0005] The preparation of diglyceride by bioenzymatic method is mainly to further decompose the daily edible oil with bioenzymes, decompose the original triglyceride into diglyceride, and increase the content of diglyceride in edible oil, which can reach about 80%. However, this process has the problems that the degree of hydrolysis is difficult to control, it is easy to over-hydrolyze, and there are excessive by-products in the product, such as free fatty acids and monoglyceride, which affect the quality of the oil.

[0006] Chinese Patent Publication No. CN1544412A discloses a method for producing diglyceride oil, which comprises the following steps: (1) adsorbing glycerol on an adsorbent material; (2) mixing the adsorbent material obtained in (1) with glycerol and triglyceride, using immobilized lipase as a catalyst, and catalyzing the reaction of triglyceride and glycerol at 20-90°C; (3) separating, removing the immobilized lipase and glycerol adsorbent material from the product obtained in (2), and then separating and removing monoglyceride and fatty acid to obtain diglyceride oil. This method uses triglyceride and adsorbed glycerol as raw materials, does not add water, and produces diglyceride oil by catalyzing glycerolysis reaction with immobilized lipase at low temperature. The by-products in the generated product are few and the color is light. However, the yield and purity of diglyceride need to be further improved, and the effects of reducing triglyceride, total cholesterol, and uric acid also need to be further improved.

[0007] Chinese Patent Publication No. CN119955870A discloses a method for preparing diglyceride with the effects of reducing blood lipid, total cholesterol, blood sugar, and uric acid by enzymatic hydrolysis. The method comprises the following steps: (1) mixing vegetable oil, glycerol, composite lipase, and water to carry out glycerolysis reaction to obtain a reactant; (2) separating the reactant and collecting the upper layer liquid; (3) distilling, separating, decolorizing, and deodorizing the upper layer liquid to obtain diglyceride; the vegetable oil in step (1) is rapeseed oil with low erucic acid and corn oil; the composite lipase in step (1) is Rhizomucor javanicus lipase, Aspergillus niger lipase, and Aspergillus oryzae lipase with a mass ratio of 2-4:1:5-8. This method reduces the dosage of enzymes, and the prepared diglyceride has certain activities of reducing blood lipid, total cholesterol, blood sugar, and uric acid. However, the yield and purity of the prepared diglyceride are relatively low, and the effects of reducing triglyceride, total cholesterol, and uric acid also need to be further improved.

[0008] Therefore, it is very necessary to develop a diglyceride with the effects of reducing triglyceride, total cholesterol, and uric acid, and its bioenzymatic preparation method and application that can solve the above technical problems. Summary of the Invention

[0009] The object of the present invention is to overcome the deficiencies of the prior art and provide a diglyceride with better effects of reducing triglyceride, total cholesterol, and uric acid, and higher yield and purity, and its bioenzymatic preparation method and application.

[0010] The present invention is realized by the following technical solutions: The first aspect of the present invention provides a bioenzymatic preparation method of diglyceride with the effects of reducing triglyceride, cholesterol, and uric acid, which comprises the following steps: (1) Adding water and glycerol to vegetable oil, and then adding immobilized lipase to react to obtain a reactant; (2)Centrifuge the reactants, collect the light-phase product, conduct molecular distillation, and collect the heavy-phase product to obtain diglyceride. The immobilized lipase includes lipase and a carrier. The carrier includes loofah sponge, soybean meal, and bentonite, and the mass ratio of the three is 1:2-4:3-6.

[0011] As an embodiment of the present invention, the mass ratio of the lipase to the carrier is 1:15-20.

[0012] As an embodiment of the present invention, the lipase includes lipase derived from Aspergillus oryzae and lipase derived from Rhizopus niveus, and the mass ratio of the two is 3-5:1.

[0013] As an embodiment of the present invention, the vegetable oil includes at least one of peanut oil, soybean oil, rapeseed oil, olive oil, and corn oil.

[0014] As an embodiment of the present invention, in step (1), 1-2 times the amount of water and 0.4-0.8 times the amount of glycerol are added to the vegetable oil, and then, based on the mass percentage of the vegetable oil, 4-7% of the immobilized lipase is added for reaction, and the reaction is carried out at 50-70°C for 2-6 h.

[0015] As an embodiment of the present invention, the specific parameters of the molecular distillation in step (2) are: the temperature is 110-150°C, and the vacuum degree is less than 50 Pa.

[0016] As an embodiment of the present invention, the preparation method of the immobilized lipase includes the following steps: S1 Add lipase to a buffer solution to obtain a mixed solution; S2 Add loofah sponge, soybean meal, and bentonite to the mixed solution in sequence, mix evenly, filter, and vacuum dry to obtain the product.

[0017] Preferably, the pH of the buffer solution in step S1 is 6.8-7.5.

[0018] Preferably, the mass concentration of lipase in the mixed solution in step S1 is 6-10%.

[0019] Preferably, the mixing process parameters in step S2 are: mixing at 25-35°C for 1-3 h.

[0020] The second aspect of the present invention provides a diglyceride prepared by the above-mentioned method for biochemically preparing diglyceride.

[0021] The third aspect of the present invention provides the application of the above-mentioned diglyceride in the preparation of products for reducing triglyceride and / or reducing cholesterol and / or reducing uric acid.

[0022] The beneficial effects of the present invention are: By screening the composition and dosage ratio of lipase in immobilized lipase, the purity and yield of diglyceride are improved, and at the same time, the effects of reducing triglyceride, total cholesterol and uric acid are enhanced.

[0023] By optimizing the carrier of immobilized lipase, the purity and yield of diglyceride are further improved, and at the same time, the effects of reducing triglyceride, total cholesterol and uric acid are further enhanced. Detailed implementation manners

[0024] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions fall within the protection scope of the present invention.

[0025] The lipase derived from Aspergillus oryzae in the following examples was purchased from Hebei Qiansheng Biotechnology Co., Ltd., and the lipase derived from Rhizopus niveus was purchased from Hong Kong Jisenbei International Trade Co., Ltd.

[0026] Example 1 A method for preparing diglyceride by biocatalysis for reducing triglyceride, cholesterol and uric acid, comprising the following steps: (1) Add 1.5 times the amount of water and 0.6 times the amount of glycerol to peanut oil, then add 5% immobilized lipase (calculated based on the amount of peanut oil used) and react at 60 °C for 4 h to obtain a reaction product; (2) Centrifuge the reaction product, collect the light-phase product, perform molecular distillation at a temperature of 130 °C and a vacuum degree of 40 Pa, and collect the heavy-phase product to obtain diglyceride.

[0027] The lipase includes lipase derived from Aspergillus oryzae and lipase derived from Rhizopus niveus, and the mass ratio of the two is 4:1.

[0028] The immobilized lipase includes lipase and a carrier, and the mass ratio of the two is 1:18; the carrier includes loofah sponge, soybean meal and bentonite, and the mass ratio of the three is 1:3:5.

[0029] The preparation method of the immobilized lipase includes the following steps: S1 Add lipase to a sodium phosphate buffer solution (pH = 7.5), and the mass concentration of lipase is 8% to obtain a mixed solution; S2 Add loofah sponge, soybean meal and bentonite to the mixed solution in sequence, mix at 30 °C for 2 h, filter, and vacuum dry to obtain the product.

[0030] The yield of the obtained diglyceride is 83.7%, and the purity is 97.4%.

[0031] Example 2 A method for preparing diglyceride by biocatalysis to reduce triglyceride, cholesterol and uric acid, comprising the following steps: (1) Add 1-fold amount of water and 0.4-fold amount of glycerol to soybean oil, then add 4% of immobilized lipase (calculated based on the amount of soybean oil) and react at 50 °C for 6 h to obtain a reaction product; (2) Centrifuge the reaction product, collect the light-phase product, perform molecular distillation at a temperature of 110 °C and a vacuum degree of 20 Pa, and collect the heavy-phase product to obtain diglyceride.

[0032] The lipase includes lipase from Aspergillus oryzae and lipase from Rhizopus niveus, and the mass ratio of the two is 3:1.

[0033] The immobilized lipase includes lipase and a carrier, and the mass ratio of the two is 1:15; the carrier includes loofah sponge, soybean meal and bentonite, and the mass ratio of the three is 1:2:3.

[0034] The preparation method of the immobilized lipase includes the following steps: S1 Add lipase to a sodium phosphate buffer solution (pH = 7.5), and the mass concentration of lipase is 6% to obtain a mixed solution; S2 Add loofah sponge, soybean meal and bentonite to the mixed solution in sequence, mix at 25 °C for 3 h, filter, and vacuum dry to obtain it.

[0035] The yield of the obtained diglyceride is 81.4%, and the purity is 97.0%.

[0036] Example 3 A method for preparing diglyceride by biocatalysis to reduce triglyceride, cholesterol and uric acid, comprising the following steps: (1) Add 2-fold amount of water and 0.8-fold amount of glycerol to corn oil, then add 7% of immobilized lipase (calculated based on the amount of corn oil) and react at 70 °C for 2 h to obtain a reaction product; (2) Centrifuge the reaction product, collect the light-phase product, perform molecular distillation at a temperature of 150 °C and a vacuum degree of 45 Pa, and collect the heavy-phase product to obtain diglyceride.

[0037] The lipase includes lipase from Aspergillus oryzae and lipase from Rhizopus niveus, and the mass ratio of the two is 5:1.

[0038] The immobilized lipase includes lipase and a carrier, and the mass ratio of the two is 1:20; the carrier includes loofah sponge, soybean meal and bentonite, and the mass ratio of the three is 1:4:6.

[0039] The preparation method of the immobilized lipase comprises the following steps: S1 Add lipase to a sodium phosphate buffer solution (pH = 7.5), and the mass concentration of lipase is 10% to obtain a mixed solution; S2 Sequentially add loofah sponge, soybean meal and bentonite to the mixed solution, mix at 35 °C for 1 h, filter, and vacuum dry to obtain the product.

[0040] The yield of the prepared diglyceride is 82.6%, and the purity is 96.7%.

[0041] Comparative Example 1 The difference from Example 1 is only that the dosage of lipase remains unchanged, but the composition is different. It is a lipase derived from Aspergillus oryzae, and the other conditions are the same.

[0042] The yield of the prepared diglyceride is 74.3%, and the purity is 92.5%.

[0043] Comparative Example 2 The difference from Example 1 is only that the dosage of lipase remains unchanged, but the composition is different. It is a lipase derived from Rhizopus niveus, and the other conditions are the same.

[0044] The yield of the prepared diglyceride is 72.9%, and the purity is 91.8%.

[0045] Comparative Example 3 The difference from Example 1 is only that the dosage of the carrier in the immobilized lipase remains unchanged, but the composition is different. It is loofah sponge and soybean meal, and the mass ratio of the two is 1:3.

[0046] The yield of the prepared diglyceride is 75.7%, and the purity is 93.4%.

[0047] Comparative Example 4 The difference from Example 1 is only that the dosage of the carrier in the immobilized lipase remains unchanged, but the composition is different. It is soybean meal and bentonite, and the mass ratio of the two is 3:5.

[0048] The yield of the prepared diglyceride is 75.1%, and the purity is 92.7%.

[0049] Comparative Example 5 The difference from Example 1 is only that the dosage of the carrier in the immobilized lipase remains unchanged, but the composition is different. It is loofah sponge and bentonite, and the mass ratio of the two is 1:5.

[0050] The yield of the prepared diglyceride is 74.4%, and the purity is 92.5%.

[0051] Test Example 1 Test on the effects of reducing triglyceride and total cholesterol Healthy male SD rats weighing 200±20 g were selected. The maintenance diet consisted of raw materials such as corn, soybean cake, and fish meal. The high-fat diet was made by adding 15% lard to the maintenance diet. The diglyceride diet was made by adding 15% diglyceride (the diglyceride prepared in each example or comparative example) to the maintenance diet. The temperature in the breeding room was maintained at 22±2 °C, and the humidity was maintained at 55±5%. Darkness and light were adjusted at 12-hour intervals.

[0052] After normal feeding with the maintenance diet for one week, 8 rats were randomly selected as the normal control group and continued to be fed with the maintenance diet. The remaining rats were fed with the high-fat diet as the high-fat diet group. After 15 days of feeding, compared with the normal control group, the total cholesterol (TC) and triglyceride (TG) values in the high-fat diet group increased significantly, indicating successful modeling. The high-fat diet group was randomly divided into 9 groups, with 8 rats in each group. There was no significant difference in the TC and TG values among the rats in each group of the high-fat diet group (p>0.05). One of the groups was used as the model control group and continued to be fed with the high-fat diet, and the remaining 8 groups were fed with the diglyceride diet containing the diglyceride prepared in Examples 1-3 and Comparative Examples 1-5. Subsequent experiments were carried out after 30 days of feeding.

[0053] After the experiment, the rats were fasted for 8.5 h, anesthetized with 3% sodium pentobarbital (30 mg / kg) in each group, and sacrificed by cervical dislocation after blood collection. The blood specimens were centrifuged at 4000 rpm, and the serum was taken for the detection of total cholesterol (TC) and triglyceride (TG) in blood lipids. The detection results are shown in Table 1.

[0054] Table 1 Detection results of various indicators

[0055] Note: Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001; compared with Example 1 group, &p<0.05.

[0056] As can be seen from Table 1, the TC and TG in the model control group were significantly higher than those in the normal control group, showing abnormalities. Under the intervention of diglyceride in each example, the TC and TG indicators decreased significantly, indicating that the diglyceride prepared in each example can reduce triglyceride and total cholesterol. The effects of each indicator in each example were significantly better than those in each comparative example, indicating that the intervention effects of the diglyceride prepared in Examples 1-3 on triglyceride and total cholesterol were significantly better than those in each comparative example, especially the composition of the lipase and the composition of the carrier in the immobilized lipase of the present application had a significant synergistic effect in reducing triglyceride and total cholesterol.

[0057] Test Example 2 Testing of uric acid lowering effect Male Kunming mice, weighing 18 - 22 g, were adaptively fed with maintenance feed for one week and then randomly divided into 10 groups of 8 mice each. Nine of the groups were gavaged with potassium oxonate at a dose of 100 mg / kg per day for modeling. The remaining 1 group was used as a blank control group and given an equal volume of water for 7 days. During this period, all mice were given maintenance feed. The serum uric acid levels of the modeled mice were significantly higher than those of the blank control group, indicating successful modeling. Moreover, there were no significant differences in the serum uric acid levels among the modeled mice in each group (p > 0.05). Subsequently, eight groups of modeled mice were gavaged with the diglycerides of Examples 1 - 3 and Comparative Examples 1 - 5 at a dose of 0.3 g / kg respectively. One group of modeled mice was used as a model control group and gavaged with an equal volume of water as the blank control group. Ten days later, blood was collected from the eyeballs after the last administration, and serum was obtained by centrifugation to measure the uric acid level. The results are shown in Table 2.

[0058] Table 2 Results of uric acid reduction detection

[0059] Note: Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with Example 1 group, &p < 0.05.

[0060] As can be seen from Table 2, the uric acid level of the model control group was significantly higher than that of the blank control group, showing an abnormality. Under the intervention of diglycerides in each example, the uric acid level decreased significantly, indicating that the diglycerides prepared in each example can reduce uric acid. The uric acid levels of each example were significantly lower than those of each comparative example, indicating that the intervention effect of the diglycerides prepared in Examples 1 - 3 on uric acid was significantly better than that of each comparative example. In particular, the composition of the lipase and the composition of the carrier in the immobilized lipase of the present application had a significant synergistic effect in reducing uric acid.

[0061] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification without departing from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing diglyceride by biocatalysis for reducing triglyceride, cholesterol and uric acid, which is characterized in that, It includes the following steps: (1) Add water and glycerol to vegetable oil, and then add immobilized lipase to react to obtain a reactant; (2) Centrifuge the reactant, collect the light-phase product, perform molecular distillation, and collect the heavy-phase product to obtain diglyceride; The immobilized lipase includes lipase and a carrier. The carrier includes loofah sponge, soybean meal, and bentonite, and the mass ratio of the three is 1:2-4:3-6.

2. The method for preparing diglyceride by biocatalysis according to claim 1, characterized in that, The mass ratio of the lipase to the carrier is 1:15-20.

3. The method for preparing diglyceride by biocatalysis according to claim 1, wherein The lipase includes lipase derived from Aspergillus oryzae and lipase derived from Rhizopus niveus, and the mass ratio of the two is 3-5:

1.

4. The method for preparing diglyceride by biocatalysis according to claim 1, wherein The vegetable oil includes at least one of peanut oil, soybean oil, rapeseed oil, olive oil, and corn oil.

5. The method for preparing diglyceride by biocatalysis according to claim 1, wherein In step (1), add 1-2 times the amount of water and 0.4-0.8 times the amount of glycerol to the vegetable oil, and then add 4-7% of the immobilized lipase based on the mass percentage of the vegetable oil to react, and react at 50-70°C for 2-6 h.

6. The method for preparing diglyceride by biocatalysis according to claim 1, wherein In step (2), the specific parameters of the molecular distillation are: the temperature is 110-150°C, and the vacuum degree is less than 50 Pa.

7. The method for preparing diglyceride by biocatalysis according to claim 1, wherein The preparation method of the immobilized lipase includes the following steps: S1 Add lipase to a buffer solution to obtain a mixed solution; S2 Add loofah sponge, soybean meal, and bentonite to the mixed solution in sequence, mix evenly, filter, and vacuum dry to obtain it.

8. The method for preparing diglyceride by biocatalysis according to claim 7, characterized in that, In step S1, the pH of the buffer solution is 6.8-7.5; the mass concentration of lipase in the mixed solution is 6-10%; in step S2, the mixing process parameters are: mix at 25-35°C for 1-3 h.

9. A diglyceride, characterized in that, Prepared by the method for preparing diglyceride by bioenzymatic method according to any one of claims 1-8.

10. Use of the diglyceride according to claim 9 in the preparation of a product for reducing triglyceride and / or reducing cholesterol and / or reducing uric acid.

Images