AU2021201921B2

AU2021201921B2 - Polyunsaturated fatty acid triglyceride and preparation and uses thereof

Info

Publication number: AU2021201921B2
Application number: AU2021201921A
Authority: AU
Inventors: Sheng CAO; Yun Liang; Shenjian WANG
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-03-31
Filing date: 2021-03-26
Publication date: 2022-08-04
Anticipated expiration: 2041-03-26
Also published as: AU2021201921A1; WO2021196881A1; KR20210122095A; CN111560403A

Abstract

A method for preparing a polyunsaturated fatty acid triglyceride. (1) A raw oil is mixed with water, homogenized and then subjected to lipase-catalyzed hydrolysis to 5 obtain a free fatty acid salt and glycerin. (2) The free fatty acid salt is separated from the glycerin. (3) The free fatty acid salt is acidized to obtain a free fatty acid. (4) The free fatty acid is dried and subjected to molecular distillation to obtain a long-chain polyunsaturated fatty acid. (5) The long-chain polyunsaturated fatty acid is mixed with glycerin and subjected to lipase-catalyzed esterification to obtain the 10 polyunsaturated fatty acid triglyceride. The polyunsaturated fatty acid triglyceride prepared by this method has high purity with no chemical residue and less oxidation loss. The disclosure also provides a polyunsaturated fatty acid triglyceride and uses thereof in the preparation of infant formula foods, health food, functional foods or medical products.

Description

POLYUNSATURATED FATTY ACID TRIGLYCERIDE AND PREPARATION AND USES THEREOF TECHNICAL FIELD

This disclosure relates to microbial oil, and more particularly to a

polyunsaturated fatty acid triglyceride, and a preparation and uses thereof.

BACKGROUND

Polyunsaturated fatty acids carry at least two double bonds in their carbon chain,

and exhibit biological activities of stabilizing cell membrane, regulating gene

expression, maintaining cytokine and lipoprotein balance, reducing the risk of

cardiovascular diseases and promoting the growth and development. Polyunsaturated

fatty acids are dominated by n-3, n-6 and n-9 polyunsaturated fatty acids, where the

n-6 and n-3 polyunsaturated fatty acids are preferable due to their excellent biological

activities.

The polyunsaturated fatty acids are usually obtained by fish oil refining or

oleaginous microorganism-mediated fermentation. The polyunsaturated fatty acid

triglycerides are generally prepared by the fermentation, and usually contain medium

and low-purity polyunsaturated fatty acids, which are mainly used in the formula

foods for infant and the elder and health food. As for the medical product industry,

high-purity polyunsaturated fatty acids are preferred.

Currently, the high-purity polyunsaturated fatty acids are prepared mainly by

purifying the medium- and low-purity polyunsaturated fatty acids by means of

chromatography, distillation, solvent extraction, low-temperature crystallization,

supercritical extraction and urea inclusion. However, most of the products prepared by

this process are fatty acid ethyl esters, which are inferior to the fatty acid triglycerides

in terms of food safety and human absorption (the absorption rate of the fatty acid

ethyl esters is only about 20%).

Recently, a novel method for preparing high-purity polyunsaturated fatty acid

esters has been developed, in which medium- and low-purity polyunsaturated fatty acid esters are hydrolyzed into free fatty acids, from which the medium- and short-chain fatty acids are removed, and then the remaining fatty acids are re-esterified to produce the high-purity polyunsaturated fatty acid esters. However, the short-chain fatty acids are usually removed by reaction with short-chain alcohols, during which new chemicals are introduced, resulting in an increase in the production cost and a risk in food safety. Most of the lipases used in the hydrolysis processes of existing polyunsaturated fatty acid esters are specific enzymes that have different sensitivity to the ester bonds in different positions, leading to low hydrolysis speed and poor production efficiency. SUMMARY

A first aspect of the present disclosure provides a method for preparing a polyunsaturated fatty acid triglyceride. The preparation method desirably has high production efficiency and low oxidation loss, and the prepared polyunsaturated fatty acid triglyceride desirably has a high purity and is free of the solvent. A second aspect of the present disclosure provides a method of replacing saturated fatty acids in the triglyceride with polyunsaturated fatty acids to improve the content of the polyunsaturated fatty acids. The prepared polyunsaturated fatty acid triglyceride desirably has a high purity, and the polyunsaturated fatty acid thereof desirably has high biological activity and low safety risk. A third aspect of the present disclosure provides a use of the polyunsaturated fatty acid triglyceride in the preparation of formula foods for infant, health foods, functional foods or medical products. The aspects of the disclosure are specifically described as follows. In a first aspect, the present disclosure provides a method for preparing a polyunsaturated fatty acid triglyceride, comprising: (1) mixing a raw oil containing a polyunsaturated fatty acid with water followed by homogenizing at 45-65°C and 20-100 MPa and hydrolysis in the presence of a first lipase to obtain a free fatty acid salt and glycerin; (2) separating the free fatty acid salt obtained in step (1) from the glycerin to obtain the fatty acid salt;

(3) acidizing the free fatty acid salt obtained in step (2) to obtain a free fatty acid;

(4) drying the free fatty acid obtained in step (3); and subjecting the dried free

fatty acid to molecular distillation to remove a short-chain fatty acid to obtain a

long-chain polyunsaturated fatty acid; and

(5) mixing the long-chain polyunsaturated fatty acid obtained in step (4) with

glycerin followed by esterification in the presence of a second lipase to obtain the

polyunsaturated fatty acid triglyceride.

In some embodiments, in step (1), a weight ratio of the raw oil to the water is

1:(0.7-1.3); and/or the reaction mixture is adjusted to pH 6-7.5 with a 10-15%

aqueous sodium hydroxide solution or a 10-15% aqueous potassium hydroxide

solution to perform the hydrolysis in the presence of the first lipase. When the weight

ratio of the raw oil and the water is 1:(0.7-1.3), an oil-water contact area in an

emulsion formed by the homogenization is larger. The homogenization is performed

at 45-65°C and 20-100 MPa to allow the raw oil to be fully mixed with the water and

form a smaller particle size, increasing the oil-water contact area. The large oil-water

contact area helps the raw oils in better contact with a lipase to accelerate the

hydrolysis and improve the production efficiency. The pH of a hydrolysis system will

be gradually reduced due to the free fatty acid generated in the lipase-catalyzed

hydrolysis, and then will exceed a pH range in which the lipase has a relatively high

catalytic activity. Therefore, it is necessary to add an alkaline substance into the

hydrolysis system in real time to control the pH. In some embodiments, the hydrolysis

system is adjusted to pH 6-7.5 with a 10-15% aqueous sodium hydroxide solution or a

10-15% aqueous potassium hydroxide solution to maintain a catalytic activity of the

lipase. Moreover, the addition of a sodium hydroxide solution or a potassium

hydroxide solution can also make the free fatty acid released from the hydrolysis of

the raw oil into a sodium salt or a potassium salt, inhibiting the occurrence of a

reverse reaction of the hydrolysis.

In some embodiments, in step (1) and step (5), the first lipase and the second

lipase are independently a non-specific lipase selected from the group consisting of

Penicillium cyclopium (PG37 lipase), Candida rugosa lipase (CRL), Aspergillus niger lipase, Novozym 435, Lipozyme RM M, C.rugosa lipase, (CRL) and a combination thereof. In a preferred embodiment, the non-specific lipase can hydrolyze fatty acids in each position of the raw oil at the same time, overcoming the defect that the specific lipase can only hydrolyze the fatty acids in position 1 and position 3 of the raw oil and the fatty acids in position 2 have to be rearranged to position 1 or position

3 to be hydrolyzed, so as to accelerate the hydrolysis.

In some embodiments, in step (1), the hydrolysis is performed by adding an

immobilized first lipase to a reactor or by using packed beds carrying the immobilized

first lipase; and

in step (5), the esterification is performed by adding an immobilized second

lipase to a reactor or by using packed beds carrying the immobilized second lipase;

wherein the packed beds carrying the immobilized first lipase are connected in

series or parallel; and the packed beds carrying the immobilized second lipase are

connected in series or parallel.

The immobilized lipase is added into the hydrolysis kettle or made into packed

beds to perform the lipase-catalyzed hydrolysis. In this way, the lipase can be

immobilized at a position that is in full contact with a hydrolyzed substrate, increasing

a contact area between the lipase and the hydrolyzed substrate and accelerating the

hydrolysis. A loss of the lipase during the hydrolysis is reduced and a utilization rate

of the lipase is improved. The series or parallel packed bed reactors further expand the

contact area in the lipase-catalyzed hydrolysis and enhance the contact between the

lipase and the hydrolysis substrate, further accelerating the hydrolysis.

In some embodiments, in step (1), the first lipase is added in an amount of

100-500 U per gram of the raw oil; and the hydrolysis is performed at 35-65°C for

8-72 h. In a preferred embodiment, an addition amount of the lipase influences a

hydrolysis rate of the raw oil, but an increase in the addition amount of the lipase will

largely increase the production cost. A proper hydrolysis temperature can effectively

increase the hydrolysis rate of the lipase, and proper hydrolysis time can make the raw

oil fully hydrolyzed and improve the production efficiency. In step (5), the

esterification is performed at 35-55°C and 100-180 rpm for 30-50 h; the glycerin is

10-18% by weight of the long-chain polyunsaturated fatty acid; and the second lipase

is 5-20% by weight of the long-chain polyunsaturated fatty acid. In a preferred

embodiment, under an anhydrous environment, the lipase-catalyzed esterification is

better performed under the above reaction conditions.

In some embodiments, in step (4), the molecular distillation is performed at a

temperature of 120-185°C, a pressure of less than 0.5 Pa, a feed rate of 15-20 kg/h

and a film-wiping speed of 110-180 rpm. In a preferred embodiment, the pressure less

than 0.5 Pa reduces a boiling temperature of the free fatty acid, avoiding a thermal

decomposition of the free fatty acid. Meanwhile, the pressure also increases a mean

free path of vapor molecules, helping gas molecules reach a condensing surface for

condensing. The short and medium-chain fatty acids (a chain length of 12-20 carbon

atoms) will be gradually evaporated at 130-185°C and then condensed on the

condensing surface to be separated from the long-chain polyunsaturated fatty acid, so

that the obtained polyunsaturated fatty acid has a higher purity. The feed rate of 15-20

kg/hour and the film-wiping speed of 120-180 rpm allow the free fatty acid to form an

ultra-thin and turbulent liquid film, which is conducive to the increase of temperature

and the evaporation of the free fatty acid.

In some embodiments, steps (1)-(5) are all carried out under an oxygen-isolated

state. The oxygen-isolated state is achieved by vacuuming containers of raw materials,

intermediate substances and reaction products, or filling the containers with an inert

atmosphere such as nitrogen.

In some embodiments, the raw oil is a docosahexaenoic acid (DHA) algae oil, an

arachidonic acid (ARA) algae oil containing an or a combination thereof. DHA and

ARA exhibit strong biological activity, and have wide application range and great

market demand.

In a second aspect, the present disclosure provides a polyunsaturated fatty acid

triglyceride prepared by the method provide in the first aspect, comprising:

15% or less by weight of a monoglyceride;

10% or less by weight of a diglyceride; and

7 5% or more by weight of a triglyceride; wherein a weight percentage of a DHA triglyceride or an ARA triglyceride in the polyunsaturated fatty acid triglyceride is not less than 72%.

In a third aspect, the present disclosure provides a use of the polyunsaturated

fatty acid triglyceride in the preparation of infant formulas, health food, functional

foods or medical products.

Compared to the prior art, this application desirably has the following beneficial

effects.

1. Molecular distillation is employed herein to remove short- and medium-chain

fatty acids from the free fatty acid, so as to obtain a high-purity polyunsaturated fatty

acid. In the traditional separation methods, the short- and medium-chain fatty acids

are removed by esterification with a short-chain alcohol in an organic solvent,

resulting in a loss of the polyunsaturated fatty acid due to undesirable esterification

and organic solvent residue. By contrast, the method provided in this disclosure does

not require chemical solvents and additional chemicals, avoiding the loss of the

polyunsaturated fatty acid caused by esterification as well as a food-safety risk caused

by the residual organic solvent.

2. Before the hydrolysis, the raw oil is mixed with water and then homogenized

to form tiny particles in the water, facilitating the contact of the raw oil with water and

the lipase. Meanwhile, the steric hindrance brought by the distorted molecular

structure of the long-chain polyunsaturated fatty acid is overcome during the

hydrolysis, accelerating the lipase-catalyzed hydrolysis of the raw oil and improving

the reaction efficiency of the hydrolysis.

3. The lipase used herein is a non-specific lipase. The traditionally-used specific

lipase is only sensitive to the fatty acid groups in positions 1 and 3, and can only

directly catalyze the hydrolysis of the fatty acids in position 1 and position 3, and the

fatty acid in position 2 have to be rearranged to position 1 or 3 to be hydrolyzed,

slowing down the hydrolysis of the oil. The non-specific lipase can simultaneously

catalyze the hydrolysis of fatty acids in each position of the raw oil, accelerating the

hydrolysis and improving the reaction efficiency.

4. The preparation process is carried out in an oxygen-isolated environment, which prevents the oxidation of the polyunsaturated fatty acid during the reaction and storage process, and improves the quality of the polyunsaturated fatty acid triglyceride.

DETAILED DESCRIPTION OF EMBODIMENTS It should be noted that the range disclosed herein is not limited to the endpoints and values within the range, and are intended to include values close to the range. Any combination of numerical values made based on the range disclosed herein should be

considered as specifically disclosed in this disclosure.

Unless otherwise specified, the percentages used herein mean weight

percentages. The technical solutions of this disclosure will be described clearly with reference

to the embodiments. As used herein, the DHA algae oil is produced by Linyi Youkang Biology Co.,

Ltd (China) through the fermentation of Schizochytrium sp.; the ARA algae oil is

produced by Linyi Youkang Biology Co., Ltd (China) through the fermentation in the

presence of Mortierella alpine; the water is purified by reverse osmosis; the lipases are produced by Novozymes Inc. (China) or Hangzhou Verychem Science and

Technology Co., Ltd (China); the nitrogen has a purity of 99.99%, and is produced by

the JH-PN49-10 nitrogen generator manufactured by Guangzhou Zhongshan Jigao Science and Technology Co., Ltd (China); and the molecular distillation equipment is a DZ-80 three-stage short-path molecular distillation equipment manufactured by

Sichuan Jiuyuan Chemical Technology Co., Ltd. Unless otherwise specified, other

apparatuses are conventional chemical apparatuses; and reagents are commercially available analytical reagents. The component analysis is performed in accordance

with the national standard.

Example 1

(1) 100 kg of DHA refined oil (DHA: 43%) derived from algae oil was added into a reactor, to which 100 kg of water was added. The reaction mixture was homogenized at 55°C and 60 MPa for 10 min. Packed beds respective carrying immobilized Candida rugosa lipase in an amount of 200 U per gram of the oil and immobilized Crugosa lipase in an amount of 200 U per gram of the oil were connected in series. The homogenized product was filled into the series-connected packed bed, and adjusted to pH 4.8 with an aqueous sodium hydroxide solution. The reactor was vacuumed to a pressure of -0.09 MPa, and then introduced with nitrogen to a pressure of 0.05-0.09 MPa. Then the oil was hydrolyzed at 50°C for 45 h, and a 12% aqueous sodium hydroxide solution was automatically added after 4.5 h of the hydrolysis to maintain the hydrolysis system at a pH of 6.8-7. After the hydrolysis was completed, a mixture containing free fatty acid sodium salt and glycerin was obtained. (2) The mixture obtained in step (1) was transported into a temporary storage tank by nitrogen, and was filtered using a disc-type filter to recover the lipase and obtain a mixture of free fatty acid sodium salt and glycerin, which was allowed to stand for 4-6 h in the temporary storage tank under a nitrogen atmosphere. Then the glycerin and water were removed by separation, and a fatty acid sodium salt was collected.

(3) The fatty acid sodium salt obtained in step (2) was acidized with a 10%

sulfuric acid solution, and then subjected to standing for 2-3 h. The water was

removed, and a free fatty acid was obtained. (4) The free fatty acid obtained in step (3) was heated to 80-90°C, vacuumed to -0.09 MPa, dehydrated for 30-50 min and treated by a three-stage short-path

molecular distillation system, where parameters of the three-stage molecular

distillation system were shown in Table 1. Table 1 Parameters of molecular distillation in Example 1

Primary molecular Secondary molecular Tertiary molecular

distillation distillation distillation

Temperature of the 125-135 150-165 170-180 evaporator, °C

Temperature of the

condensing 50-65 65-75 75--85

surface, °C

Vacuum degree, Pa 0.3-0.4 0.3-0.1 0.1-0.06

Feeding speed, kg/h 16-18 16-18 16-18

Wiping speed, rpm 165 145 125

After the treatment using the three-stage short-path molecular distillation system,

a heavy phase was collected as a high-purity long-chain polyunsaturated fatty acid.

(5) 20 kg of the polyunsaturated fatty acid obtained in step (4) was esterified

with 2.8 kg of glycerin at 45°C and a stirring speed of 140 rpm in the presence of 3 kg of Novozym 435. After being reacted for 40 h, the reaction mixture was washed with

4 kg of 65°C water, and then subjected to standing for 2 h. The excess glycerin and water were removed to obtain a polyunsaturated fatty acid triglyceride.

Example 2

(1) 100 kg ofDHA crude oil (DHA: 45%) derived from an algae oil was added into a reactor, to which 70 kg of water was added. The reaction mixture was homogenized at 45°C and 20 MPa for 10 min. A packed bed was prepared using

immobilized Penicillium cyclopium lipase in an amount of 100 U per gram of the oil.

The homogenized product was filled into the packed bed, and adjusted to pH 4.8 with

an aqueous potassium hydroxide solution. The reactor was vacuumed to a pressure of -0.09 MPa, and then introduced with nitrogen to a pressure of 0.05-0.09 MPa. Then

the oil was hydrolyzed at 35°C for 72 h, and a 10% aqueous potassium hydroxide solution was automatically added after 4.5 h of the hydrolysis to maintain the

hydrolysis system at a pH of 6-6.5. After the hydrolysis was complete, a mixture

containing free fatty acid sodium salt and glycerin was obtained. (2) The mixture obtained in step (1) was transported into a temporary storage

tank by nitrogen, and was filtered using a disc-type filter to recover the lipase and obtain a mixture of free fatty acid potassium salt and glycerin, which was allowed to stand for 4-6 h in the temporary storage tank under a nitrogen atmosphere. Then the glycerin and water were removed by separation, and a fatty acid sodium salt was collected.

(3) The fatty acid sodium salt obtained in step (2) was acidized with a 10%

sulfuric acid solution, and then subjected to standing for 2-3 h. The water was removed, and a free fatty acid was obtained. (4) The free fatty acid obtained in step (3) was heated to 80-90°C, vacuumed to

-0.09 MPa, dehydrated for 30-50 min and treated by a three-stage short-path

molecular distillation system, where parameters of the three-stage molecular

distillation system were shown in Table 2. Table 2 Parameters of molecular distillation in Example 2

Primary molecular Secondary molecular Tertiary molecular

distillation distillation distillation

Temperature of the 120-128 130-155 160-170 evaporator, °C

Temperature of the

condensing 45-55 55-65 65--75

surface, °C

Vacuum degree, Pa 0.3-0.4 0.3-0.1 0.1-0.06

Feeding speed, kg/h 15-17 15-17 15-17

Wiping speed, rpm 155 135 110

a heavy phase was collected as a high-purity long-chain polyunsaturated fatty acid

was obtained.

(5) 20 kg of the polyunsaturated fatty acid obtained in step (4) was esterified

with 2 kg of glycerin at 35°C and a stirring speed of 100 rpm in the presence of 1 kg

of Novozym 435. After being reacted for 50 h of the reaction, the reaction mixture was washed with 4 kg of 65°C water, and then subjected to standing for 2 h. The excess glycerin and water were removed to obtain a polyunsaturated fatty acid triglyceride.

Example 3 100 kg of ARA crude oil (ARA: 46%) derived from Mortierella alpine was added into a reactor, to which 130 kg of water was added. The reaction mixture was homogenized at 65°C and 100 MPa for 10 min. Aspergillus niger lipase in an amount

of 500 U per gram of the oil was added into the reactor. An aqueous sodium hydroxide

solution was added to adjust the mixture to pH 4.8. The reactor was vacuumed to a

pressure of -0.09 MPa, and then introduced with nitrogen to adjust a pressure of 0.05-0.09 MPa. Then the oil was hydrolyzed at 65°C for 8 h, and a 15% aqueous

sodium hydroxide solution was automatically added after 4.5 h of the hydrolysis to maintain the hydrolysis system at a pH of 7-7.5. After the hydrolysis was completed, a

mixture containing free fatty acid sodium salt and glycerin was obtained.

(2) The mixture obtained in step (1) was transported into a temporary storage tank by nitrogen, and was filtered using a disc-type filter to recover the lipase and

obtain a mixture of free fatty acid sodium salt and glycerin, which was allowed to

stood for 4-6 h in the temporary storage tank under a nitrogen atmosphere. Then the

glycerin and water were removed by separation, and a fatty acid sodium salt was collected. (3) The fatty acid sodium salt obtained in step (2) was acidized with a 10%

sulfuric acid solution, and then subjected to standing for 2-3 h. The water was

removed, and a free fatty acid was obtained. (4) The free fatty acid obtained in step (3) was heated to 80-90°C, vacuumed to

-0.09 MPa, dehydrated for 30-50 min and treated by a three-stage short-path

molecular distillation system, where working parameters of the three-stage molecular distillation system were shown in Table 3.

Table 3 Parameters of molecular distillation in Example 3

Primary molecular Secondary molecular Tertiary molecular

distillation distillation distillation

Temperature of the 130-140 155-170 175-185 evaporator, °C

Temperature of the

condensing 50-65 65-75 75--85

surface, °C

Vacuum degree, Pa 0.3-0.4 0.3-0.1 0.1-0.06

Feeding speed, kg/h 18-20 18-20 18-20

Wiping speed, rpm 180 160 135

was obtained.

(5) 20 kg of the polyunsaturated fatty acid obtained in step (4) was esterified

with 3.6 kg of glycerin at 55°C and a stirring speed of 180 rpm in the presence of 4 kg

of Lipozyme RM M. After being reacted for 30 h, the reaction mixture was washed

with 4 kg of 65°C water, and then subjected to standing for 2 h. The excess glycerin

and water were removed to obtain a polyunsaturated fatty acid triglyceride.

Comparative Example

(1) 100 kg of DHA refined oil (DHA: 43%) derived from an algae oil was added into a reactor, to which 100 kg of water was added. The reaction mixture was

homogenized at 55°C and 60 MPa for 10 min. A specific enzyme, Lipozyme RM M

(RML), was added into the reactor in an amount of 200 U per gram of the oil. An

aqueous sodium hydroxide solution was added to adjust the mixture to pH 4.8. The

reactor was vacuumed to a pressure of -0.09 MPa, and then introduced with nitrogen

to a pressure of 0.05-0.09 MPa. Then the oil was hydrolyzed at 50°C for 82 h, and a

12% aqueous sodium hydroxide solution was automatically added during the hydrolysis to maintain the hydrolysis system at a pH of 6.8-7. After the hydrolysis was completed, a mixture containing free fatty acid sodium salt and glycerin was obtained.

(2) The mixture obtained in step (1) was transported into a temporary storage

tank by nitrogen, and was filtered using a disc-type filter to recover the lipase and

obtain a mixture of free fatty acid sodium salt and glycerin, which was allowed to stand for 4-6 h in the temporary storage tank under a nitrogen atmosphere. Then the glycerin and water were removed by separation, and a fatty acid sodium salt was

collected.

(3) The fatty acid sodium salt obtained in step (2) was acidized with a 10%

sulfuric acid solution, and then subjected to standing for 2-3 h. The water was removed, and a free fatty acid was obtained.

(4) The free fatty acid obtained in step (3) was heated to 80-90°C, vacuumed to -0.09 MPa, and then dehydrated for 30-50 min. The temperature was slowly lowered

according to the parameters shown in Table 4, and the free fatty acid was winterized

to make a saturated fatty acid crystallize out. The crystallized saturated fatty acid was removed using a plate-frame filter press, and a polyunsaturated fatty acid was

obtained.

Table 4 Winterization parameters of free fatty acid

Rotation speed Cooling Holding time Step Temperature (°C) Time (min) (Hz) amplitude (h)

1 70 30 15 0 0.5

2 60 30 15 10 0.5

3 50 30 15 10 0.5

4 44 60 15 6 1

5 41 60 15 3 1

6 38 60 15 3 1

7 35 60 15 3 1

8 32 90 15 3 1.5

9 28 90 15 3 1.5

10 25 60 15 3 1

11 21 120 15 4 2

12 19 60 15 2 1

13 17 60 15 2 1

14 16 60 15 1 1

15 15 60 15 1 1

16 14 60 15 1 1

17 13 60 15 1 1

18 12.8 60 15 0.2 1

19 12.6 60 15 0.2 1

20 13 60 15 -0.4 1

21 14 60 15 -1 1

22 12 60 15 2 1

23 10 60 15 2 1

24 8 60 15 2 1

25 6 60 15 2 1

26 6 60 15 0 20

After the winterization, a filtrate was collected, so as to obtain a high-purity

long-chain polyunsaturated fatty acid.

(5) 20 kg of the polyunsaturated fatty acid obtained in step (4) was esterified

with 2.3 kg of glycerin at 45°C and a stirring speed of 140 rpm in a presence of 3.5 kg

of Lipozyme RM IM. After being reacted for 40 h, the reaction mixture was washed

and water were removed to obtain a polyunsaturated fatty acid triglyceride.

Contents of DHA or ARA, monoglyceride, diglyceride and triglyceride in the

polyunsaturated fatty acid triglyceride in each example were measured and shown in

Table 5.

Table 5 Polyunsaturated fatty acid components in Examples and Comparative

Example

DHA/ARA Monoglyceride Example number Diglyceride (%) Triglyceride (%) (0) (0)

Example 1 87.1 8.5 6.2 85.3

Example 2 85.8 11.3 6.6 82.1

Example 3 84.3 14.3 6.9 78.8

Comparative 62.3 17.9 11.3 70.8 example

Table 5 showed that the content of DHA/ARA in the polyunsaturated fatty acid

triglycerides obtained in Examples 1-3 using the method of disclosure were

significantly higher than that obtained using the method of the comparative example.

Moreover, compared with the comparative example, the polyunsaturated fatty acid

obtained in Examples 1-3 had a higher proportion of the polyunsaturated fatty acid

triglyceride, and a lower proportion of the polyunsaturated fatty acid monoglyceride

or the polyunsaturated fatty acid diglyceride. Meanwhile, the method using in

Examples 1-3 has a shorter production time and a higher efficiency. The method for

preparing a polyunsaturated fatty acid triglyceride provided herein is more beneficial.

The obtained polyunsaturated fatty acid triglyceride has a higher purity

polyunsaturated fatty acid, and has higher medicinal and edible value.

The above-mentioned embodiments are only preferred embodiments, and not

intend to limit the scope of the disclosure. It should be noted that various variations

and modifications made by those skilled in the art without departing from the spirit of

the invention should fall within the scope of the disclosure defined by the appended

claims.

The reference to any prior art in this specification is not, and should not be taken

as, an acknowledgement or any form of suggestion that such prior art forms part of

the common general knowledge.

It will be understood that the terms "comprise" and "include" and any of their

derivatives (e.g. comprises, comprising, includes, including) as used in this specification, and the claims that follow, is to be taken to be inclusive of features to which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for preparing a polyunsaturated fatty acid triglyceride, comprising:

(1) mixing a raw oil containing a polyunsaturated fatty acid with water followed

by homogenizing at 45-65°C and 20-100 MPa and hydrolysis in the presence of a first

lipase to obtain a free fatty acid salt and glycerin;

(2) separating the free fatty acid salt obtained in step (1) from the glycerin to

obtain the fatty acid salt;

long-chain polyunsaturated fatty acid; and

(5) mixing the long-chain polyunsaturated fatty acid obtained in step (4) with

polyunsaturated fatty acid triglyceride.

2. The method according to claim 1, wherein in step (1), a weight ratio of the raw

oil to the water is 1:(0.7-1.3); and/or

the reaction mixture is adjusted to pH 6-7.5 with a 10-15% aqueous sodium

hydroxide solution or a 10-15% aqueous potassium hydroxide solution to perform the

hydrolysis in the presence of the first lipase.

3. The method according to claim 1 or 2, wherein the first lipase and the second

Penicillium cyclopium lipase, Candida rugosa lipase, Aspergillus niger lipase,

Novozym 435, Lipozyme RM M, C.rugosa lipase and a combination thereof.

4. The method according to any one of claims 1-3, wherein in step (1), the

hydrolysis is performed by adding an immobilized first lipase to a reactor or by using

packed beds carrying the immobilized first lipase; and in step (5), the esterification is performed by adding an immobilized second lipase to a reactor or by using packed beds carrying the immobilized second lipase; wherein the packed beds carrying the immobilized first lipase are connected in series or parallel; and the packed beds carrying the immobilized second lipase are connected in series or parallel.

5. The method according to any one of claims 1-4, wherein in step (1), the first lipase is added in an amount of 100-500 U per gram of the raw oil; and the hydrolysis is performed at 35-65°C for 8-72 h; and in step (5), the esterification is performed at 35-55°C and 100-180 rpm for 30-50 h; the glycerin is 10-18% by weight of the long-chain polyunsaturated fatty acid; and the second lipase is 5-20% by weight of the long-chain polyunsaturated fatty acid.

6. The method according to any one of claims 1-5, wherein in step (4), the molecular distillation is performed at a temperature of 120-185°C, a pressure of less than 0.5 Pa, a feed rate of 15-20 kg/h and a film-wiping speed of 110-180 rpm.

7. The method according to any one of claims 1-6, wherein steps (1)-(5) are all carried out under an oxygen-isolated state.

8. The method according to any one of claims 1-7, wherein the raw oil is a docosahexaenoic acid (DHA) algae oil, an arachidonic acid (ARA) algae oil containing an or a combination thereof.