A method and application of preparing diglyceride for lowering blood lipids by enzymatic hydrolysis

Abstract

The present invention belongs to the technical field of oil and fat chemistry, and specifically relates to a diglyceride for lowering blood lipids by enzymatic hydrolysis, a preparation method and its application. The preparation method comprises the following steps: (1) mixing edible oil, lipase and water, hydrolyzing and dehydrating to obtain a hydrolyzate; (2) mixing the hydrolyzate, glycerol and partial glyceride lipase, reacting and distilling to obtain the diglyceride; the partial glyceride lipase in step (2) is a mixture of lipase Lipase G50 and lipase Novozyme 435. The diglyceride prepared by the present invention has an excellent effect of lowering blood lipids; and the obtained diglyceride has a high yield and a low acid value, and the finished product does not require deacidification treatment, which is suitable for large-scale industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of oil and fat chemistry, and specifically relates to a lipid-lowering diglyceride prepared by enzymatic hydrolysis, a preparation method and an application thereof. Background Art

[0002] Diacylglycerol (DAG), also known as diglyceride, diglyceride, diglyceride, is a type of structural lipid formed by the esterification of one glycerol molecule and two free fatty acid molecules or a fatty acid in triacylglycerol (TAG) replaced by a hydroxyl group. Recent studies have shown that edible oil rich in DAG, as a new type of functional oil, has multiple physiological activities and functions, can prevent and treat fatty liver, cardiovascular and cerebrovascular diseases, does not accumulate in the body after consumption, has important functions such as preventing obesity, and is widely used.

[0003] The proportion of diglycerides in edible oil is over 40%. The main components of edible oil are triglycerides and diglycerides. The triglycerides of traditional vegetable oils are generally around 98%, and diglycerides are around 2%. Diacerides are trace components of natural vegetable oils and endogenous intermediates of fat metabolism in the body. They are natural components present in edible oils. They are different from triglycerides in molecular structure, and therefore have different metabolic methods.

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

[0005] The bio-enzymatic method for preparing diglycerides mainly involves further decomposing daily edible oils with bio-enzymes, decomposing the original triglycerides into diglycerides, and increasing the diglyceride content in edible oils to about 80%. However, the process has the disadvantages of being difficult to control the degree of hydrolysis and prone to over-hydrolysis, and excessive by-products in the product, such as free fatty acids and monoglycerides, affect the quality of the oil.

[0006] Chinese invention patent application CN114540125A discloses a method for preparing unsaturated fatty acids with weight loss and lipid-lowering effects, wherein the main component of the fatty acid obtained by the method is polyunsaturated fatty acid linoleic acid, but the proportion of fatty acid glycerides in the product is not limited and hyperlipidemia cannot be alleviated. Chinese invention patent application CN115433144A discloses a fatty acid ester compound and a method for preparing the same, wherein the structure may include a fatty acyl group formed by removing a hydroxyl group from oleic acid, palmitoleic acid, linoleic acid, linolenic acid, stearic acid, arachidonic acid, eicosatetraenoic acid, eicosapentaenoic acid or docosahexaenoic acid, but there is still no restriction on the proportion of different fatty acid glycerides and the efficacy of reducing hyperlipidemia is not achieved. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides a lipid-lowering diglyceride prepared by enzymatic hydrolysis, a preparation method and application thereof.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for preparing diglyceride for lowering blood lipids by enzymatic hydrolysis comprises the following steps:

[0010] (1) mixing edible oil, lipase and water, hydrolyzing and dehydrating to obtain a hydrolyzate;

[0011] (2) mixing the hydrolyzate, glycerol and partial glyceride lipase, reacting and distilling to obtain the diglyceride;

[0012] In step (2), the partial glycerol ester lipase is a mixture of lipase Lipase G50 and lipase Novozyme 435.

[0013] Preferably, the lipase in step (1) is lipase Lipozyme TL IM, and its addition amount is 0.1%-1.5% of the mass of the edible oil.

[0014] Preferably, in step (1), the mass ratio of the edible oil to water is 4-5:1, the hydrolysis temperature is 50-60° C., and the hydrolysis time is 1-10 h.

[0015] Preferably, in step (2), the mass ratio of lipase Lipase G50 to lipase Novozyme 435 is 1:0.8-1.2.

[0016] Preferably, in step (2), the amount of partial glyceride lipase added is 0.1%-2.0% of the mass of the edible oil, the mass ratio of the hydrolyzate to glycerol is 1:2.5-4, the reaction temperature is 50-60°C, and the reaction time is 1-15h.

[0017] Preferably, the distillation in step (2) is molecular distillation, and a centrifugal separation step is also included before the distillation. The rotation speed of the centrifugal separation is 12000-15000 rpm, and the centrifugal separation time is 1-3 min.

[0018] Preferably, the feed temperature of the distillation in step (2) is 100-160° C., the feed flow rate is 14-16 kg / min, and the vacuum degree is 0.1-10 Pa.

[0019] Preferably, the raw materials of the edible oil include, by mass percentage: 60%-99% rapeseed diester oil, 0%-30% peanut oil, 0%-20% DHA algae oil, 0%-20% grape seed oil and 0.01%-0.05% vitamin E.

[0020] More preferably, the raw materials of the edible oil include, by mass percentage: 60%-95% rapeseed diester oil, 1%-20% peanut oil, 0.5%-8% DHA algae oil, 1%-15% grape seed oil and 0.01%-0.05% vitamin E.

[0021] The present invention also relates to diglyceride prepared by the above preparation method.

[0022] The present invention also relates to the use of the diglyceride prepared by the above preparation method in the preparation of a lipid-lowering product.

[0023] The products include, but are not limited to, food.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) In the process of preparing diglyceride of the present invention, lipase and specific partial glyceride lipase are added to catalyze the esterification reaction, and the obtained diglyceride has a high yield and a low acid value. The finished product does not require deacidification treatment and is suitable for large-scale industrial production.

[0026] (2) In the present invention, while using lipase for hydrolysis, a combination of lipase Lipase G50 and lipase Novozyme 435 is also used to hydrolyze the edible oil. The lipase Lipase G50 and lipase Novozyme 435 have obvious synergistic effects. The prepared diglyceride has good lipid-lowering effect and improves the sensory effect. At the same time, it shows good effects in color, aroma, stability and taste. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a comparison of the staining intensity of blood vessels in the tail of zebrafish after treatment with Example 1, the comparative edible oil group, the blank control group and the model control group in the present invention;

[0028] Figure 2 is a comparison chart of the blood vessel staining intensity data of zebrafish tails after treatment with Example 1, the comparative edible oil group, the blank control group and the model control group in the present invention, wherein It means p<0.001 between the two. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0030] Lipase Lipozyme TL IM, 250 IUN / g (ester exchange activity), purchased from Nanjing Chengna Chemical Co., Ltd.;

[0031] Lipase G50 ( Penicillium camembertii free lipase), enzyme activity 50000u / g, purchased from Amano Enzyme Products Group, Japan;

[0032] Lipase Novozyme 435 (Novozym 435), 10000 PLU / g, Shanghai Yuanye Biotechnology Co., Ltd.

[0033] Example 1

[0034] A method for preparing diglyceride for lowering blood lipids, comprising the following steps:

[0035] (1) Mix edible oil (calculated by mass percentage, the raw materials are: 94.47% rapeseed diester oil, 3% peanut oil, 0.5% DHA algae oil, 2% grape seed oil and 0.03% vitamin E), lipase Lipozyme TL IM and water, hydrolyze at 55°C for 6 h, and dehydrate to obtain a hydrolyzate. The mass ratio of edible oil to water is 4.5:1, and the amount of lipase Lipozyme TL IM added is 1.0% of the mass of the edible oil.

[0036] (2) mixing the hydrolyzate, glycerol and partial glycerol lipase (lipase Lipase G50 and lipase Novozyme 435 in a mass ratio of 1:1), reacting at 55° C. for 8 h, then centrifuging at 13,000 rpm for 2 min, and molecularly distilling to obtain the diglyceride;

[0037] Among them, the addition amount of partial glyceride lipase is 1.2% of the mass of edible oil, the mass ratio of the hydrolyzate to glycerol is 1:3.0, the feed temperature of molecular distillation is 130°C, the feed flow rate is 15kg / min, and the vacuum degree of the separation column is 5.0Pa.

[0038] HPLC-ELSD was used to detect the oil composition and content, and the content of diglyceride was 82.3%.

[0039] Example 2

[0040] A method for preparing diglyceride for lowering blood lipids, comprising the following steps:

[0041] (1) Mix edible oil (by mass percentage, the raw materials are: 80% rapeseed diester oil, 9.95% peanut oil, 5% DHA algae oil, 5% grape seed oil and 0.05% vitamin E), lipase Lipozyme TL IM and water, hydrolyze at 50°C for 10 h, and dehydrate to obtain a hydrolyzate. The mass ratio of edible oil to water is 4:1, and the amount of lipase Lipozyme TL IM added is 0.3% of the mass of the edible oil.

[0042] (2) mixing the hydrolyzate, glycerol and partial glyceride lipase (lipase Lipase G50 and lipase Novozyme 435 in a mass ratio of 1:0.8), reacting at 50° C. for 15 h, then centrifuging at 12,000 rpm for 3 min, and molecularly distilling to obtain the diglyceride;

[0043] Among them, the addition amount of partial glyceride lipase is 0.6% of the mass of edible oil, the mass ratio of the hydrolyzate to glycerol is 1:2.5, the feed temperature of molecular distillation is 100°C, the feed flow rate is 14kg / min, and the vacuum degree of the separation column is 1.0Pa.

[0044] HPLC-ELSD was used to detect the oil composition and content, and the content of diglyceride was 80.0%.

[0045] Example 3

[0046] A method for preparing diglyceride for lowering blood lipids, comprising the following steps:

[0047] (1) Mix edible oil (by mass percentage, the raw materials are: 60% rapeseed diester oil, 16.96% peanut oil, 8% DHA algae oil, 15% grape seed oil and 0.04% vitamin E), lipase Lipozyme TL IM and water, hydrolyze at 60°C for 1 hour, and dehydrate to obtain a hydrolyzate. The mass ratio of edible oil to water is 5:1, and the amount of lipase Lipozyme TL IM added is 1.5% of the mass of the edible oil.

[0048] (2) mixing the hydrolyzate, glycerol and partial glyceride lipase (lipase Lipase G50 and lipase Novozyme 435 in a mass ratio of 1:1.2), reacting at 60° C. for 2 h, then centrifuging at 15,000 rpm for 1 min, and molecularly distilling to obtain the diglyceride;

[0049] Among them, the addition amount of partial glyceride lipase is 2.0% of the mass of edible oil, the mass ratio of the hydrolyzate to glycerol is 1:4, the feed temperature of molecular distillation is 160°C, the feed flow rate is 16kg / min, and the vacuum degree of the separation column is 8Pa.

[0050] HPLC-ELSD was used to detect the oil composition and content, and the content of diglyceride was 81.2%.

[0051] Comparative Example 1-Comparative Example 4

[0052] The difference between Comparative Examples 1 to 4 and Example 1 is that the added amounts of peanut oil, DHA algae oil and grape seed oil in the edible oil are different. The specific mass percentages are shown in Table 1.

[0053] Table 1 Addition amount of rapeseed diester oil, DHA algae oil and grape seed oil

[0054]

[0055] Comparative Example 5

[0056] The difference between this comparative example and Example 1 is that the partial glyceride lipase is only lipase Lipase G50, and the rest is the same as Example 1.

[0057] Comparative Example 6

[0058] The difference between this comparative example and Example 1 is that the partial glyceride lipase is only lipase Novozyme 435, and the rest is the same as Example 1.

[0059] Comparative Example 7

[0060] The difference between this comparative example and Example 1 is that the raw material ratio of the edible oil is different. In this comparative example, the raw materials of the edible oil are: 50% rapeseed diester oil, 35% peanut oil, 5% DHA algae oil, 9.5% grape seed oil and 0.5% vitamin E by mass percentage.

[0061] Effect test

[0062] Test Example 1

[0063] 1. Sample preparation

[0064] The diglyceride in Example 1 was prepared into a 200 mg / mL stock solution with DMSO and stored at -20°C.

[0065] Comparative edible oils (canola oil 94.47%, peanut oil 3%, DHA algae oil 0.5%, grape seed oil 2% and vitamin E 0.03%) were prepared into 200 mg / mL stock solution with DMSO and stored at -20°C.

[0066] 2. Experimental Animals

[0067] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 3 days post fertilization (3dpf) were used to determine the maximum detection concentration (MTC) of the samples on zebrafish blood lipids and evaluate their effects.

[0068] 3. Detection methods

[0069] (1) MTC determination

[0070] 3dpf wild-type AB strain zebrafish were randomly selected in beakers, and 30 zebrafish were treated in each beaker (experimental group). Except for the normal control group, the other experimental groups were given high-sugar and high-fat feed in water to establish a high-fat model of zebrafish. After 2 days of treatment at 28°C, the high-sugar and high-fat feed was removed, and samples were given in water (concentrations are shown in Table 2), and a normal control group was set up at the same time, with a capacity of 25mL per cup. The liquid was changed every morning and evening. During the sample treatment, the number of zebrafish deaths in each experimental group was counted every day and removed in time. After 3 days of continued treatment at 28°C, the MTC of the sample for high-fat model zebrafish was determined. Example 1 The MTC of diglyceride for high-fat model zebrafish is 2000μg / mL, as shown in Table 2.

[0071] Table 2 Results of the concentration test on the effect of samples on zebrafish blood lipids (n=30)

[0072]

[0073] (2) Effects on zebrafish blood lipids

[0074] 3dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker. Except for the normal control group, all other experimental groups were given a high-sugar and high-fat diet in water to establish a high-fat model of zebrafish. After 2 days of treatment at 28°C, except for the model control group, the high-sugar and high-fat diet was removed from the other experimental groups, and samples were given in water (concentrations are shown in Table 3). At the same time, a normal control group and a model control group were set up. The model control group continued to be given a high-sugar and high-fat diet for 8dpf, and the capacity of each cup was 25mL. The liquid was changed every morning and evening. After continuing to treat at 28°C for 3 days, Oil Red O was given for overall fat staining. After decolorization and bleaching, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software to analyze the staining intensity of the zebrafish tail blood vessels (such as Figure 1 The statistical analysis results of this index were used to evaluate the effect of the sample on zebrafish blood lipids. The statistical processing results were expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant. The results are shown in Table 3 and Figure 2 shown.

[0075] Table 3 Test results of tail blood vessel staining intensity

[0076]

[0077] Note: Compared with the model group, P<0.01, P<0.001; compared with Example 1, # P<0.05.

[0078] Test Example 2

[0079] 100 patients with hyperlipidemia were randomly selected and divided into 4 groups, with 25 hyperlipidemia patients in each group, corresponding to the diglycerides of Examples 1-3 and the comparative edible oils, respectively. All drugs were stopped 3 days before the test and during the test period. Each person in each group consumed 30 mL of the product per day for 30 days. The effective number of people was counted according to the evaluation criteria, and the effective rate was calculated. The results are shown in Table 4 below. Effective rate (%) = effective number of people / total number of people × 100%.

[0080] The evaluation criteria are as follows:

[0081] Effective: Uncomfortable symptoms are alleviated or disappear;

[0082] Ineffective: Symptoms remain the same or become more severe.

[0083] Table 4 Test results

[0084]

[0085] Test Example 3

[0086] 100 subjects aged 50-70 were randomly selected and divided into 10 groups, with 10 people in each group. The diglycerides prepared in Examples 1-3 and the diglycerides in Comparisons 1-6 were subjected to sensory evaluation, with a full score of 10 for each item, and the final score was the average score. The results are shown in Table 5.

[0087] Table 5 Sensory evaluation results

[0088]

[0089] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for preparing diglyceride for lowering blood lipids by enzymatic hydrolysis, characterized in that: The following steps are involved: (1) mixing edible oil, lipase and water, hydrolyzing and dehydrating to obtain a hydrolyzate; (2) mixing the hydrolyzate, glycerol and partial glyceride lipase, reacting and distilling to obtain the diglyceride; The partial glyceride lipase in step (2) is a mixture of lipase Lipase G50 and lipase Novozyme 435; Wherein, the raw materials of the edible oil include, by mass percentage: 60%-95% of rapeseed diester oil, 1%-20% of peanut oil, 0.5%-8% of DHA algae oil, 1%-15% of grape seed oil and 0.01%-0.05% of vitamin E; The lipase in step (1) is lipase Lipozyme TL IM; the amount of the lipase added is 0.1%-1.5% of the mass of the edible oil, the mass ratio of the edible oil to water is 4-5:1, the hydrolysis temperature is 50-60°C, and the hydrolysis time is 1-10h; In step (2), the mass ratio of lipase Lipase G50 to lipase Novozyme 435 is 1:0.8-1.2; the addition amount of the partial glyceride lipase is 0.1%-2.0% of the mass of the edible oil; the mass ratio of the hydrolyzate to glycerol is 1:2.5-4; the reaction temperature is 50-60°C; and the reaction time is 1-15h.

2. The preparation method according to claim 1, characterized in that: The distillation in step (2) is molecular distillation, and a centrifugal separation step is also included before the distillation. The rotation speed of the centrifugal separation is 12000-15000 rpm, and the time of the centrifugal separation is 1-3 minutes.

3. The preparation method according to claim 1, characterized in that: The feed temperature of the distillation in step (2) is 100-160° C., the feed flow rate is 14-16 kg / min, and the vacuum degree is 0.1-10 Pa.

4. A diglyceride prepared by the preparation method according to any one of claims 1 to 3.

5. Use of diglyceride prepared by the preparation method according to any one of claims 1 to 3 in the preparation of lipid-lowering products.

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