CN116144715A

CN116144715A - Enzymatic method for preparing MLCT-rich grease and product thereof

Info

Publication number: CN116144715A
Application number: CN202211304612.0A
Authority: CN
Inventors: 郑明明; 张羽飞; 赖运动; 龚阳敏; 万楚筠; 张逸
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2022-10-24
Filing date: 2022-10-24
Publication date: 2023-05-23

Abstract

The invention discloses an enzymatic method for preparing MLCT grease and a product thereof, comprising the following steps: (1) In a paraffin-water emulsion template system, an organic hydrophobic silicon source and a pore-forming agent are used for preparing hydrophobic hollow SiO by a one-step method ₂ Mesoporous microspheres (HMSB) as hydrophobic carriers; then the free enzyme is immobilized on the hydrophobic carrier by an adsorption method to prepare the immobilized lipidA lipase; (2) Mixing long-chain triglyceride and medium-carbon chain raw materials, adding immobilized lipase, and carrying out enzymatic transesterification or acidolysis under constant-temperature water bath for a certain time; after the reaction is finished, solid-liquid separation is carried out, the obtained solid product is immobilized lipase, the obtained solution is an MLCT-rich oil crude product, and a small amount of fatty acid is removed through molecular distillation or physical adsorption, so that an MLCT-rich oil finished product can be obtained. The method has the advantages of wide raw material adaptability, high catalytic efficiency, high MCLT content, controllable functional fatty acid content, simplicity in operation, environment friendliness, suitability for large-scale amplification production and the like.

Description

Enzymatic method for preparing MLCT-rich grease and product thereof

Technical Field

The invention belongs to the field of grease modification and deep processing of grease, and particularly relates to a method for synthesizing MLCT (multi-stage hybrid CT) by catalyzing transesterification with immobilized enzyme.

Background

The medium-long carbon chain triglyceride (MLCT) has both medium-chain and long-chain fatty acid, belongs to a structural lipid with special nutrition and physiological functions, has the advantages of long-chain triglyceride and medium-chain triglyceride, improves the nutrition function of natural oil, has different metabolic pathways in human bodies, can rapidly supply energy to human bodies, can provide necessary fatty acid, has positive effects on diseases such as diabetes, heart disease, hyperlipidemia, hypertension and the like, and is an important component of functional food. In addition, the MLCT metabolite can improve the nitrogen balance of human bodies, improve the utilization rate of nutrient substances and reduce the generation of inflammatory mediators, can be applied to clinical fat emulsion for injection, and is an important component of parenteral nutrition by adding natural grease into medicines. However, the types of the MLCT products are few at present, so that the preparation methods of the MLCT products with different fatty acid compositions need to be developed to meet the requirements of different scenes.

The traditional preparation of MLCT is a chemical synthesis method, the selectivity of the used chemical catalyst is low, and the fatty acid has randomness in the position of the glycerol skeleton; the method also has the problems of more byproducts, difficult separation of products, high energy consumption, pollution to the environment and the like. CN 103891920A discloses a method for preparing MLCT by using sodium methoxide as a catalyst to perform chemical transesterification, but the separation steps of the product and the catalyst are complicated, and the steps of centrifugation, water washing, vacuum drying and the like are required, so that the produced wastewater is not friendly to the environment and has poor selectivity. In contrast, the enzymatic catalysis for preparing MLCT can directionally change the position of fatty acid on the glycerol skeleton, and the catalysis process is green, safe and economical, and overcomes a plurality of adverse factors of chemical catalysis. Because free enzyme is sensitive to environment, the free enzyme is easy to deform and deactivate in high temperature, organic reagent and acid-base environment, the application of the enzyme under various conditions is limited, the enzyme is immobilized, the heating property of the enzyme can be improved, the tolerance of the organic reagent is improved, the enzyme and the product are easy to separate, and the enzyme can be recycled. In a comprehensive view, the MLCT grease product has the advantages of diversification, simple post-treatment and the like by using the special immobilized enzyme catalyst.

The immobilized lipase commonly used for catalysis is commercialized enzyme such as NOV435, RMIM, TLIM and the like, the enzymes are expensive, the free enzyme is adsorbed in macroporous acrylic resin and other materials to easily generate swelling effect, the repeated use of the enzyme is limited, and the industrialized production of MLCT is not facilitated. CN 107058413A discloses a method for synthesizing medium-long carbon chain triglyceride by enzymatic transesterification, which takes camphor tree seed oil and soybean oil as raw materials, and the medium-long carbon chain triglyceride is generated by catalyzing transesterification by immobilized lipase NOV435, but the reaction still needs more than 2 hours, and the method does not mention recycling of the enzyme.

Disclosure of Invention

The invention aims to solve the technical problems of providing an enzymatic preparation method for preparing MLCT-rich grease aiming at the defects in the prior art, and has the advantages of high catalytic efficiency, high product nutrition value, simplicity in operation, environmental protection, simplicity and convenience in product separation, suitability for large-scale production and the like.

The invention adopts the technical proposal for solving the problems that:

an enzymatic method for preparing MLCT-rich grease comprises the following steps:

(1) Preparation of immobilized lipase: in a paraffin-water emulsion template system, an organic hydrophobic silicon source and a pore-foaming agent are used for preparing hydrophobic hollow SiO by a one-step method ₂ Mesoporous microspheres (HMSB) as hydrophobic carriers; then fixing the free enzyme on a hydrophobic carrier by an adsorption method to prepare immobilized lipase;

(2) Enzymatic preparation of MLCT grease: mixing long-chain triglyceride and medium-carbon chain raw materials, adding immobilized lipase, and carrying out enzymatic transesterification or acidolysis under constant-temperature water bath for a certain time; after the reaction is finished, solid-liquid separation is carried out, the obtained solid product is immobilized lipase immobilized enzyme, the obtained solution is an MLCT-enriched oil crude product, and a small amount of fatty acid is removed through molecular distillation or physical adsorption, so that an MLCT-enriched oil finished product can be obtained. Wherein the medium carbon chain raw material is medium carbon chain fatty acid triglyceride or medium carbon chain fatty acid; when the medium carbon chain raw material is medium carbon chain fatty acid triglyceride, the step (2) generates enzymatic transesterification reaction; and (3) when the medium carbon chain raw material is medium carbon chain fatty acid, performing enzymolysis reaction in the step (2).

According to the scheme, the hydrophobic hollow SiO ₂ Mesoporous microspheres (HMSB) have a size of 200nm-2 μm, a cavity structure, a contact angle of more than 110 DEG and a pore size distribution of 3-10nm.

According to the scheme, the hydrophobic hollow SiO ₂ The specific preparation process of the mesoporous microsphere comprises the following steps: mixing liquid paraffin and water according to a certain volume ratio, placing the mixture in a container, adding a surfactant and a pore-forming agent, and carrying out ultrasonic homogenization to obtain emulsion; pouring the emulsion into a hydrophobic silicon source solution, placing the solution in a low-temperature water bath, stirring for a certain time, filtering, cleaning isooctane, drying, removing pore-forming agent and surfactant by acid washing, and finally filtering and drying to obtain the HMSB.

Further, hydrophobic hollow SiO ₂ In the specific preparation process of the mesoporous microsphere, the volume ratio of paraffin to water is 2:1-8:1; the surfactant is one or more of glyceryl monostearate, span 80, span 20, polyethylene glycol 200, polyglycerol fatty acid ester, sorbitol monolaurate and other water-in-oil type emulsifiers, and the addition amount is 1.0-2.5% of the water mass; the pore-forming agent comprises but is not limited to block polyether F127, triblock copolymer P123 and the like, and the addition amount is 1-6% of the water mass.

Further, hydrophobic hollow SiO ₂ In the specific preparation process of the mesoporous microsphere, the hydrophobic silicon source can be one or more of Methyltrichlorosilane (MTCS), ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane and the likeThe addition amount of the hydrophobic silicon source is 75-120% of the water mass; the hydrophobic silicon source solution is prepared by dispersing a hydrophobic silicon source in isooctane, and the volume ratio of the hydrophobic silicon source to the isooctane is 3: (7-10); the temperature of the low-temperature water bath is 4-15 ℃; the pickling solution is prepared by mixing water, ethanol and hydrochloric acid according to a volume ratio of 2:1:1; the pickling temperature is 60-100 ℃ and the pickling time is 6-8h.

According to the scheme, the specific process of preparing the immobilized lipase by the adsorption method is as follows: adding free enzyme into phosphate buffer solution to prepare a certain enzyme solution concentration, mixing the solution with a hydrophobic carrier according to a certain solid-to-liquid ratio (the ratio between the mass of HMSB and the volume of enzyme solution), incubating by a shaking table to realize immobilization, centrifuging, filtering, washing with PBS for three times, and freeze-drying to obtain immobilized lipase.

Further, in the specific process of preparing the immobilized lipase by an adsorption method, the free enzyme is one or more of candida rugosa lipase, candida antarctica lipase, NS40086 lipase, thermomyces lanuginosus, candida lipolytica lipase and the like; the pH of the enzyme solution is 7.5-9.5, and the concentration is 10-100mg/mL; the ratio of HMSB mass to enzyme solution volume is 1:100-3.5:100 (m/v, g/mL); the immobilization time is 30-90min, and the temperature is 20-40 ℃; the pH of the phosphate buffer is 7.5-9.5. In the process of enzyme immobilization, the phosphate buffer has a larger influence on enzyme immobilization, so that the pH of the phosphate buffer is set to be neutral to alkaline 7.5-9.5; the temperature influence is small, and the normal temperature is generally adopted.

According to the scheme, the fatty acid chain carbon number of the medium-carbon chain fatty acid triglyceride is 6-12, preferably caprylic capric triglyceride rich in caprylic acid (C8) and capric acid (C10) and the like; the medium chain fatty acid is one or a mixture of a plurality of caprylic acid, capric acid, lauric acid and the like according to any proportion.

According to the scheme, the long carbon chain triglyceride is one or a mixture of more of linseed oil, perilla oil, hemp oil, evening primrose oil, peony seed oil, sunflower seed oil, soybean oil, sunflower seed oil, conjugated linoleic acid glyceride, DHA algae oil, fish oil, garlic oil, shinyleaf yellowhorn oil and the like according to any proportion.

According to the scheme, in the step (2), the mass ratio of the medium-carbon chain triglyceride to the long-chain triglyceride is 2:3-2:5 (m/m); alternatively, the molar ratio of long chain triglycerides to medium carbon chain fatty acids is from 1:3 to 1:6.

According to the scheme, in the step (2), the addition amount of the immobilized enzyme is 4-8% of the mass of the substrate.

According to the scheme, in the step (2), the temperature of the constant-temperature water bath is 50-90 DEG C

According to the scheme, in the step (2), the transesterification reaction time is 10-60min; the acidolysis reaction time is 1-3h.

According to the scheme, in the step (2), the molecular distillation is carried out at the feeding speed of 10-50mL/min, the distillation pressure of 30-50Pa, the heating temperature of 210-260 ℃ and the scraper rotating speed of 240-350r/min.

According to the scheme, the immobilized lipase separated in the step (2) can be repeatedly used after PBS (phosphate buffer solution) is washed and freeze-dried; the immobilized lipase is repeatedly used for 10 times, and the MLCT content in the prepared oil product rich in MLCT is still kept above 54%.

The oil product rich in MLCT prepared by the method has the acid value of 0.5-2.0mg KOH/g, and the MLCT accounts for 50-82% of the triglyceride by mass percent; the functional fatty acid contained in different types of MLCTs comprises linolenic acid, nervonic acid, oleic acid, linoleic acid, DHA, EPA, ARA and the like, and the content of the functional fatty acid in the MLCTs is more than 55 percent; the Sn1, 3-position fatty acid can be medium carbon chain fatty acid such as caprylic acid, capric acid, lauric acid, etc.; gan San ester content is more than 90%, diglyceride content is 0.1-2.5%, and each index meets the standard requirement of edible oil.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention prepares the hydrophobic hollow SiO by an emulsion template method ₂ The mesoporous microsphere is used as a carrier, the whole process of carrier preparation and hydrophobic modification is completed in one step, organic solvents such as toluene and the like required by post modification are not needed, the complex steps of calcining to remove a template agent and subsequent grafting of hydrophobic groups in the existing carrier preparation are overcome, the preparation process is simple, raw materials are economical and easy to obtain, the time consumption is short, the yield is high, and the preparation method is easy to ruleThe molding and amplifying are green, safe and pollution-free.

2. The invention adopts the carrier hydrophobic hollow SiO ₂ The activity and stability of the free enzyme immobilized on the mesoporous microspheres are higher than those of the free enzyme and commercial enzyme; in addition, the mesoporous structure of the carrier not only can efficiently load enzyme, but also is beneficial to mass transfer and heat transfer of reaction substrates in a system.

3. On the basis of the above, the immobilized lipase and the preparation method can be widely used for catalyzing exchange or acidolysis reaction of long-chain triglyceride and medium-carbon chain triglyceride or medium-carbon chain fatty acid ester, different raw materials can be selected according to requirements to prepare MLCT structural esters rich in different types of fatty acids, such as algae oil and MCT can prepare structural esters rich in DHA, and further such as linseed oil and MCT can prepare structural esters rich in linolenic acid. In addition, the crude product obtained by the enzymatic transesterification or acidolysis can be prepared into the oil finished product rich in MLCT only by simple deacidification, no complex separation and purification steps are needed, and the MLCT content is high.

4. The whole process adopts solvent-free enzyme catalysis, the reaction condition is mild, the reaction time is short, and the separation is simple; compared with the traditional chemical catalysis method, the method does not generate byproducts, a large amount of waste water and secondary waste, and effectively saves cost; compared with a nonaqueous enzyme catalysis method, the method has the advantages of no need of organic solvents, high raw material concentration and high product yield, and the immobilized enzyme can be regenerated and reused for many times, so that the method has great industrial application potential.

Drawings

FIG. 1 is an SEM image of the support material obtained in example 1.

Fig. 2 shows the contact angle of the support material obtained in example 1 with water.

FIG. 3 is a pore size distribution diagram of the support material obtained in example 1

FIG. 4 is a schematic diagram of an enzymatically catalyzed transesterification reaction of medium carbon chain fatty acid triglycerides with long chain fatty acid triglycerides.

FIG. 5 is a schematic diagram of an acidolysis reaction of medium carbon chain fatty acids and long chain fatty acid triglycerides catalyzed by an enzymatic process.

Fig. 6 is a gas chromatogram of a medium-long carbon chain triglyceride finished product synthesized by transesterification of linseed oil and MCT oil in example 1 and a physical mixed gas chromatogram of linseed oil and MCT oil.

Fig. 7 shows a gas chromatogram of a medium-long carbon chain triglyceride product synthesized by transesterification of algae oil and MCT oil in example 4, and a physical mixed gas chromatogram of algae oil and MCT oil.

FIG. 8 shows the content of medium-long carbon chain triglycerides synthesized 10 times by transesterification of linseed oil and MCT oil catalyzed by immobilized enzymes in example 1.

Wherein MLCT represents medium-long chain triglyceride, LCT represents long chain triglyceride, and MCT represents medium-chain triglyceride.

Detailed Description

For a better understanding of the present invention, the following description will further illustrate the present invention with reference to specific examples, but the present invention is not limited to the following examples.

The detection of the MLCT product generated by catalyzing transesterification with the hollow mesoporous silica sphere immobilized enzyme mainly comprises gas chromatography. The detection conditions were as follows: the chromatographic column was DB-5HT (15 m×0.320mm,0.10 μm) at 380℃inlet temperature, 1. Mu.L inlet volume, split ratio 50:1. high purity helium was used as a carrier gas at a flow rate of 2mL/min. The hydrogen flow rate was 32mL/min and the air flow rate was 200mL/min. The detector was a FID detector with a temperature of 380 ℃. Column box temperature rise program: the initial temperature is kept at 170 ℃ for 2min, and the temperature is raised to 380 ℃ at 5 ℃ per minute for 6min.

The content of glyceride is detected by liquid phase, and the detection conditions are as follows: the column was Venusil XBP Silica um 4.6X1250 mm. ELSD detector, sample 30mg/mL sample injection amount 10uL column temperature 35 ℃; the mobile phase n-hexane/isopropanol/formic acid (15:1:0.03, v: v) was eluted isocratically at a flow rate of 1mL/min.

Glyceride structure determination, chromatographic system: shimadzu UPLC LC-30A; chromatographic column: phenomenex Kinete C18column (100×2.1mm,2.6 μm); sample injection amount: 3 μL; flow rate: 0.4mL/min; column temperature:60 ℃; sample cell temperature: 4 ℃. Phase A: water: methanol: acetonitrile (1:1:1 containing 5mM ammonium acetate); and B phase: isopropyl alcohol: acetonitrile (5:1) (containing 5mM ammonium acetate), gradient elution conditions: 0.5min,20% phase B; 1.5min 40% phase B; 3min 60% phase B; 13min 95% phase B, 7min,20.1min 20% phase B, 25min 20% phase B. Mass spectrometry system: AB Sciex TripleTOF 6600, ESI ion source, positive mode, mass spectrum acquisition mass number range of m/z 100-1200, mass spectrum conditions are as follows: curtainGas, 35.000psi; ion Source Gas1:50.000; ion Source Gas2:50.00; ionSpray Voltage 5500.00V; temperature 600 ℃.

Example 1

An enzymatic method for preparing MLCT grease-enriched oil specifically comprises the following steps:

(1) Preparation of hydrophobic hollow mesoporous silica spheres: 16mL of paraffin and 4mL of water were placed in a beaker, 0.1g of glycerol monostearate was added, 0.1g of F127 was added, and the mixture was homogenized at 5000rpm for 5min with ultrasound to prepare an emulsion. 3mL of MTCS was dissolved in 8mL of isooctane to give an MTCS solution, and the emulsion was poured into the MTCS solution and stirred at 10℃for 3 hours. And (3) carrying out suction filtration on the solution to obtain solid particles, adding isooctane, cleaning the paraffin on the surface of the material, filtering, and drying the obtained material in a 60 ℃ oven overnight. And adding the dried material into a mixed solution containing 200mL of water, 100mL of ethanol and 100mL of hydrochloric acid, stirring for 6h at 60 ℃, and drying again to obtain the carrier-hydrophobic hollow mesoporous silica sphere, wherein the morphology is shown in figure 1, the size is between 200nm and 2 mu m, the contact angle is shown in figure 2, the pore size distribution is shown in figure 3, and the pore size is 3-10nm.

(2) Enzyme immobilization: 3g of free enzymatic hydrolysis candida lipolytica lipase (CSL) is dissolved in 50mL of phosphate buffer solution (50 mM) with pH of 8.5, the carrier and the enzyme solution are mixed according to the solid-to-liquid ratio of 10mg/mL, and the mixture is incubated for 40min at 30 ℃ in a shaking table for centrifugation; then, the precipitate was freeze-dried to obtain an immobilized lipase.

(3) Catalytic transesterification: placing 0.6g of linseed oil and 0.4g of medium carbon chain triglyceride (MCT, caprylic acid 42% and capric acid 58%) into an 8mL reaction bottle, adding 0.06g of immobilized lipase, magnetically stirring and reacting for 15min, and centrifuging to obtain a supernatant which is a crude product rich in MLCT; molecular distillation is carried out on the crude product, the feeding speed is 10mL/min, the distillation pressure is 30Pa, the heating temperature is 220 ℃, the scraper rotating speed is 240r/min, and the delicate MLCT-rich grease is obtained.

The acid value of the product is measured to be 0.6mg KOH/g, the content of triglyceride is 96.1 percent, the content of diglyceride is 1.5 percent, and the proportion of MLCT to triglyceride is 77.3 percent; and the linolenic acid content in the product is 33.18%; the total content of caprylic acid and capric acid is 20.83 percent and 16.33 percent respectively; caprylic acid, capric acid sn-1,3 fatty acids were 23.55%,16.44%, respectively (as shown in table 2).

Table 1 shows mass spectrum data of the medium-long carbon chain triglyceride finished product synthesized by transesterification of linseed oil and MCT oil in example 1, wherein the diglyceride is 1.00%, and the MLCT content is 79.71%; the content of caprylic acid, capric acid and linolenic acid triglyceride (8:0-10:0-18:3) reaches 14.57 percent at most.

Table 2 shows the fatty acid content of linseed oil and the fatty acid content of transesterified oil in example 1, which are 20.83% and 16.33% respectively.

TABLE 1

Note that: the statistical data in the table are components with glyceride content of more than 1%

TABLE 2

Note that: the data in Table 2 are in units of%.

Example 2

(1) Preparation of a carrier hydrophobic hollow mesoporous silica sphere: 16mL of paraffin and 4mL of water were placed in a beaker, 0.1g of span 80 was added, 0.1g of F127 was added, and the mixture was homogenized at 5000rpm for 5min with ultrasound to prepare an emulsion. 3mL of ethyl trichlorosilane was dissolved in 15mL of isooctane to obtain ethyl trichlorosilane solution, and the emulsion was poured into the solution and stirred for 3h at 8 ℃. And (3) carrying out suction filtration on the solution to obtain solid particles, adding isooctane, cleaning the paraffin on the surface of the material, filtering, and drying the obtained material in a 60 ℃ oven overnight. The dried material was added to a mixed solution containing 200mL of water, 100mL of ethanol and 100mL of hydrochloric acid, stirred at 60℃for 6 hours, and dried again to obtain a carrier.

(2) Enzyme immobilization: dissolving 2g of free enzyme candida rugosa lipase in 50mL of phosphate buffer solution with pH of 9, mixing the carrier and the enzyme solution according to the solid-to-liquid ratio of 10mg/mL, incubating for 40min at 30 ℃ and centrifuging; then, the precipitate was freeze-dried to obtain an immobilized lipase.

(3) Catalytic transesterification: putting 0.6g of sunflower seed oil and 0.4g of medium carbon chain triglyceride (MCT, caprylic acid 42% and capric acid 58%) into an 8mL reaction bottle, adding 0.06g of immobilized lipase, magnetically stirring and reacting for 15min, centrifuging to obtain supernatant, and carrying out molecular distillation on the obtained crude product, wherein the feeding speed is 20mL/min, the distillation pressure is 40Pa, the heating temperature is 230 ℃, and the scraper rotating speed is 260r/min, so as to obtain the MLCT-enriched grease.

The acid value of the product is measured to be 0.9mgKOH/g, the triglyceride content is 97.3 percent, and the diglyceride is 0.5 percent; MLCT accounts for 77.4% of triglycerides.

Example 3

(1) Preparation of a carrier hydrophobic hollow mesoporous silica sphere: 24mL of paraffin wax and 4mL of water were placed in a beaker, 0.04g of polyglycerin fatty acid ester was added, 0.2g of F127 was added, and homogenization was performed at 8000rpm for 5min to prepare an emulsion. 3mL of octyl trichlorosilane was dissolved in 20mL of isooctane to obtain an octyl trichlorosilane solution, and the emulsion was poured into the solution and stirred at 8℃for 3 hours. Filtering the solid particles, adding isooctane, cleaning redundant paraffin, filtering, and drying the obtained material in a 60 ℃ oven overnight. The dried material was added to a mixed solution containing 200mL of water, 100mL of ethanol and 100mL of hydrochloric acid, stirred at 60℃for 6 hours, and dried again to obtain a carrier.

(2) Enzyme immobilization: 3g of free enzyme Thermomyces lanuginosus lipase is dissolved in 50mL of phosphate buffer solution with pH of 8.5, the carrier and the enzyme solution are mixed according to the solid-to-liquid ratio of 20mg/mL, and the mixture is incubated for 40min at 30 ℃ and centrifuged. And freeze-drying the precipitate to obtain the immobilized lipase.

(3) Catalytic transesterification: placing 0.6g of sunflower seed oil and 0.4g of medium carbon chain triglyceride (MCT, caprylic acid 42% and capric acid 58%) into an 8mL reaction bottle, adding 0.06g of immobilized lipase, magnetically stirring and reacting for 20min, and centrifuging to obtain a supernatant as a crude product; molecular distillation is carried out on the crude product, the feeding speed is 30mL/min, the distillation pressure is 40Pa, the heating temperature is 250 ℃, the scraper rotating speed is 280r/min, and the delicate MLCT-rich grease is obtained.

The acid value of the product is measured to be 0.7mgKOH/g, the triglyceride content is 97.8 percent, and the diglyceride is 1.0 percent; the proportion of MLCT to triglycerides was 80.6%.

Example 4

(1) Preparation of a carrier hydrophobic hollow mesoporous silica sphere: 30mL of paraffin and 4mL of water were placed in a beaker, 0.06g of glycerol monostearate was added, 0.15g of P123 was added, the mixture was sonicated for 5min at 10000rpm, and the mixture was homogenized for 5min to prepare an emulsion. 3mL of MTCS was dissolved in 15mL of isooctane to give an MTCS solution, and the emulsion was poured into the MTCS solution and stirred at 10℃for 3 hours. And (3) carrying out suction filtration on the solution to obtain solid particles, adding isooctane, cleaning the surface of the material, carrying out paraffin filtration, and drying the obtained material in a 60 ℃ oven overnight. The dried material was added to a mixed solution containing 200mL of water, 100mL of ethanol and 100mL of hydrochloric acid, stirred at 60℃for 6 hours, and dried again to obtain a carrier.

(2) Enzyme immobilization: 3g of candida lipase (CSL) as a free enzyme is dissolved in 50mL of phosphate buffer solution with pH of 7, the carrier and the enzyme solution are mixed according to the solid-to-liquid ratio of 12mg/mL, and the mixture is incubated for 30min at 25 ℃ and centrifuged. And freeze-drying the precipitate to obtain the immobilized lipase.

(3) Catalytic transesterification: placing 1.2g of algae oil and 0.8g of medium carbon chain triglyceride (MCT, caprylic acid 42% and capric acid 58%) into a 20mL reaction bottle, adding 0.16g of immobilized lipase, magnetically stirring and reacting for 15min, and centrifuging to obtain a supernatant to obtain a crude product; molecular distillation is carried out on the crude product, the feeding speed is 20mL/min, the distillation pressure is 40Pa, the heating temperature is 260 ℃, the scraper rotating speed is 290r/min, and the delicate MLCT-rich grease is obtained.

The acid value of the product is 2.3mg KOH/g, the triglyceride content is 95.7%, and the diglyceride is 0.4%; MLCT accounts for 72.5% of triglycerides; and the DHA content in the product is 33.43%; the total content of caprylic acid and capric acid is 19.12 percent and 15.18 percent respectively. Caprylic acid, capric acid sn-1,3 fatty acids were 19.05%,12.98%, respectively (Table 4).

Table 3 shows the mass spectrum data of the medium-long carbon chain triglyceride product synthesized by transesterification of algae oil and MCT oil in example 4, showing that the diglyceride content is 1.18% and the MLCT content is 76.99%; caprylic acid, capric acid and DHA triglyceride (8:0-10:0-22:6) content reaches 11.35% at most.

Table 4 shows the fatty acid composition of the algae oil and the transesterified oil in example 4. The DHA content in the ester exchange oil reaches 33.43%, and the caprylic acid and the capric acid are respectively 19.12% and 15.18%.

TABLE 3 Table 3

TABLE 4 Table 4

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Note that: the data in Table 2 are in units of%.

Example 5

(1) Preparation of a carrier hydrophobic hollow mesoporous silica sphere: 16mL of paraffin and 4mL of water were placed in a beaker, 0.1g of span 80 was added, 0.1g of F127 was added, and the mixture was homogenized at 5000rpm for 5min with ultrasound to prepare an emulsion. 3mL of MTCS was dissolved in 10mL of isooctane to give an MTCS solution, and the emulsion was poured into the MTCS solution and stirred at 8℃for 3 hours. Filtering the solution to obtain solid particles, adding isooctane, cleaning the surface of the material, filtering with paraffin, and oven drying at 60deg.C overnight. The dried material was added to a mixed solution containing 200mL of water, 100mL of ethanol and 100mL of hydrochloric acid, stirred at 60℃for 6 hours, and dried again to obtain a carrier.

(2) Enzyme immobilization: 2.5g of free enzyme NS40086 lipase is dissolved in 100mL of phosphate buffer solution with pH of 8.5, the carrier and the enzyme solution are mixed according to the solid-to-liquid ratio of 20mg/mL, and the mixture is incubated for 50min by a shaking table at 25 ℃ for centrifugation. And freeze-drying the precipitate to obtain the immobilized lipase.

(3) Catalytic transesterification: 7g of sunflower seed oil and 4g of medium carbon chain triglyceride (MCT, caprylic acid 42% and capric acid 58%) are taken and placed in a 50mL reaction bottle, 0.8g of the immobilized lipase is added, and after magnetic stirring reaction is carried out for 15min, supernatant is obtained by centrifugation, thus obtaining a crude product; molecular distillation is carried out on the crude product, the feeding speed is 30mL/min, the distillation pressure is 40Pa, the heating temperature is 260 ℃, the scraper rotating speed is 300r/min, and the delicate MLCT-rich grease is obtained.

The acid value of the product is 1.6mgKOH/g, the triglyceride content is 97.8 percent, and the diglyceride is 0.8 percent. MLCT accounts for 70.9% of triglycerides.

Example 6

(1) Preparation of hydrophobic hollow mesoporous silica spheres: 16mL of paraffin and 4mL of water were placed in a beaker, 0.1g of glycerol monostearate was added, 0.1g of F127 was added, the mixture was sonicated for 8min, and homogenized at 9000rpm for 5min to prepare an emulsion. 3mL of MTCS was dissolved in 8mL of isooctane to give an MTCS solution, and the emulsion was poured into the MTCS solution and stirred at 15℃for 3 hours. And (3) carrying out suction filtration on the solution to obtain solid particles, adding isooctane, cleaning the surface of the material, carrying out paraffin filtration, and drying the obtained material in a 60 ℃ oven overnight. The dried material was added to a mixed solution containing 200mL of water, 100mL of ethanol and 100mL of hydrochloric acid, stirred at 60℃for 6 hours, and dried again to obtain a carrier.

(2) Enzyme immobilization: 3g of free enzymatic hydrolysis candida lipolytica lipase (CSL) is dissolved in 50mL of phosphate buffer solution with pH of 8.5, the carrier and the enzyme solution are mixed according to the solid-to-liquid ratio of 25mg/mL, and the mixture is incubated for 30min by a shaking table at 30 ℃ for centrifugation. And freeze-drying the precipitate to obtain the immobilized lipase.

(3) Catalytic transesterification: placing 0.6g of garlic fruit oil and 0.4g of medium carbon chain triglyceride (MCT, caprylic acid 42% and capric acid 58%) into an 8mL reaction bottle, adding 0.1g of immobilized lipase, magnetically stirring and reacting for 15min, and centrifuging to obtain a supernatant to obtain a crude product; molecular distillation is carried out on the crude product, the feeding speed is 50mL/min, the distillation pressure is 50Pa, the heating temperature is 250 ℃, the scraper rotating speed is 300r/min, and the delicate MLCT-rich grease is obtained.

The acid value of the product is 0.8mgKOH/g, the triglyceride content is 95.7%, and the diglyceride is 0.4%. MLCT accounts for 69.6% of triglycerides.

Example 7

Substantially the same as in example 1, except that: the immobilized lipase added in the step (3) is 10% (wt%) and the long chain triglyceride used is perilla oil.

The acid value of the product is 1.3mg KOH/g, the triglyceride content is 96.9%, the diglyceride is 1%, and the proportion of MLCT in the triglyceride is 78.0%.

Table 5 shows the MLCT content of the products of examples 1, 2, 4, 6, and 7, and the content of specific fatty acid in MLCT; the MLCT content is 69.6% -78%, and the content of specific fatty acid is above 55%.

TABLE 5

Example 8

Substantially the same as in example 1, except that: the immobilized lipase added in the step (3) is immobilized lipase obtained by centrifugation after the first use, and is obtained by freeze-drying after PBS washing again.

The acid value of the product is 1.8mgKOH/g, the triglyceride content is 96.4%, the diglyceride is 1.3%, and the proportion of MLCT in the triglyceride is 75.1%.

FIG. 8 shows the MLCT content after ten cycles of immobilized enzyme, and the MLCT content after ten uses of immobilized enzyme can still reach 54.2%.

Example 9

Substantially the same as in example 1, except that: in the step (3), 1.74g of linseed oil, 1.2g of caprylic acid and 0.1g of immobilized lipase are taken for reaction for 3 hours at 70 ℃, and the crude product is subjected to molecular distillation to obtain the finished product. The acid value of the finished product is 1.9mgKOH/g, and the insertion rate of octanoic acid is 33.3 percent.

Example 10

Substantially the same as in example 1, except that: in the step (3), 1.74g of linseed oil, 0.6g of caprylic acid, 0.6g of capric acid and 0.1g of immobilized lipase are taken for reaction for 3 hours at 70 ℃, and the crude product is subjected to molecular distillation to obtain a finished product. The acid value of the finished product is 1.7mgKOH/g, and the insertion rate of octanoic acid is 35.9%.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and changes can be made by those skilled in the art without departing from the inventive concept and remain within the scope of the invention.

Claims

1. An enzymatic method for preparing MLCT-rich grease is characterized by comprising the following steps:

(1) Preparation of immobilized lipase: in a paraffin-water emulsion template system, an organic hydrophobic silicon source and a pore-forming agent are used for preparing hydrophobic hollow SiO by a one-step method ₂ Mesoporous microspheres are used as carriers; then fixing the free enzyme on a hydrophobic carrier by an adsorption method to prepare immobilized lipase;

(2) Enzymatic preparation of MLCT grease: mixing long-chain triglyceride and medium-carbon chain raw materials, adding the immobilized lipase obtained in the step (1), and carrying out enzymatic transesterification or acidolysis under constant-temperature water bath; after the reaction is finished, carrying out solid-liquid separation, wherein the obtained solid product is immobilized lipase, the obtained solution is an MLCT-rich oil crude product, and then deacidifying to obtain an MLCT-rich oil finished product; wherein the medium carbon chain raw material is medium carbon chain fatty acid triglyceride or medium carbon chain fatty acid.

2. The method for preparing MLCT grease-enriched enzyme method according to claim 1 is characterized in that the hydrophobic hollow SiO ₂ The mesoporous microsphere has a size of 200nm-2 μm, a mesoporous aperture of 3-10nm and a contact angle of more than 110 degrees.

3. The method for preparing the MLCT grease-rich enzymatic process according to claim 1 is characterized in that the hydrophobic hollow SiO ₂ The specific preparation process of the mesoporous microsphere comprises the following steps: mixing liquid paraffin with water, adding a surfactant and a pore-forming agent, and carrying out ultrasonic homogenization to obtain emulsion; pouring the emulsion into a hydrophobic silicon source solution, placing the solution in a low-temperature water bath for stirring reaction, filtering, washing, drying, removing pore-forming agent and surfactant by acid washing, filtering, and drying to obtain the hydrophobic hollow SiO ₂ Mesoporous microspheres.

4. The enzymatic method for preparing MLCT fat-rich oil according to claim 3, wherein the volume ratio of liquid paraffin to water is 2:1-8:1; the surfactant is one or more of glyceryl monostearate, span 80, span 20, polyethylene glycol 200, polyglycerol fatty acid ester and sorbitol monolaurate, and the addition amount is 1.0-2.5% of the water mass; the pore-forming agent comprises one or more of block polyether F127 and triblock copolymer P123, and the addition amount is 1-6% of the water mass; the hydrophobic silicon source is one or more of methyltrichlorosilane, ethyltrichlosilane, butyltrichlosilane and octyltrichlosilane, and the addition amount of the hydrophobic silicon source is 75-120% of the water mass; the hydrophobic silicon source solution is prepared by dispersing a hydrophobic silicon source in isooctane, and the volume ratio of the hydrophobic silicon source to the isooctane is 3: (7-10); the temperature of the low-temperature water bath is 4-15 ℃, and the stirring reaction time is 2-4h; the pickling solution is prepared by mixing water, ethanol and hydrochloric acid; the pickling temperature is 60-100 ℃ and the pickling time is 6-8h.

5. The enzymatic method for preparing the MLCT-enriched grease according to claim 1, which is characterized in that the specific process for preparing the immobilized lipase by an adsorption method is as follows: adding free enzyme into phosphate buffer solution to prepare enzyme solution, and then mixing the enzyme solution with a hydrophobic carrier to immobilize so as to obtain immobilized lipase.

6. The method for preparing the MLCT grease-enriched enzymatic method according to claim 5 is characterized in that the free enzyme is one or more of candida rugosa lipase, candida antarctica lipase, NS40086 lipase, thermomyces lanuginosus and candida lipolytica lipase; the pH of the enzyme solution is 7.5-9.5, and the concentration is 10-100mg/mL; the ratio of the mass of the hydrophobic carrier to the volume of the enzyme solution is 1:100-3.5:100 (m/v, g/mL); the immobilization time is 30-90min, and the temperature is 20-40 ℃; the pH of the phosphate buffer is 7.5-9.5.

7. The enzymatic method for preparing the MLCT fat rich in the oil, according to claim 1, wherein the fatty acid chain carbon number of the medium-carbon chain fatty acid triglyceride is 6-12; the medium chain fatty acid is one or a mixture of several fatty acids with 6-12 carbon atoms according to any proportion; the long carbon chain triglyceride is one or more of linseed oil, perilla oil, hemp oil, evening primrose oil, peony seed oil, sunflower seed oil, soybean oil, sunflower seed oil, conjugated linoleic acid glyceride, DHA algae oil, fish oil, garlic oil and shinyleaf yellowhorn oil according to any proportion.

8. The enzymatic method for preparing the MLCT fat rich in the oil, according to claim 1, characterized in that in the step (2), the mass ratio of the medium-carbon chain fatty acid triglyceride to the long-chain triglyceride is 2:3-2:5, or the molar ratio of the long-chain triglyceride to the medium-carbon chain fatty acid is 1:3-1:6; the addition amount of the immobilized lipase is 4% -8% of the mass of the substrate; the temperature of the constant-temperature water bath is 50-90 ℃; the transesterification reaction time is 10-60min, or the acidolysis reaction time is 1-3h.

9. The method for preparing the MLCT fat-enriched enzymatic method according to claim 1, wherein in the step (2), the deacidification adopts molecular distillation or physical adsorption; the molecular distillation has a feeding speed of 10-50mL/min, a distillation pressure of 30-50Pa, a heating temperature of 210-260 ℃ and a scraper rotating speed of 240-350r/min.

10. The MLCT enriched fat product prepared by the method of any one of claims 1 to 9, wherein the acid value is between 0.5 and 2.0mg KOH/g, and the mass percentage of MLCT in triglycerides is between 50% and 82%; the functional fatty acid comprises one or more of linolenic acid, nervonic acid, oleic acid, linoleic acid, DHA, EPA and ARA, and the content of the functional fatty acid in MLCT is above 55%; the Sn1 and 3-site fatty acid is a medium carbon chain fatty acid; gan San ester content is greater than 90%, and diglyceride content is 0.1-5%.