Method for preparing phosphatidylserine by ultrasonic-assisted enzymatic hydrolysis

Abstract

An ultrasonic-assisted method for preparing phosphatidylserine, comprising the following steps: adding 100-130 parts of phospholipid into a mixture of 150-200 parts of L-serine, 10-20 parts of anhydrous calcium chloride and 400-500 parts of pure water, adding 20-25 parts of phospholipase D for enzymatic hydrolysis reaction, and applying ultrasound in the enzymatic hydrolysis reaction for treatment. The present invention uses an ultrasonic treatment technology to assist phospholipase D to act on phosphatidylcholine and serine to undergo an enzymatic hydrolysis reaction to prepare phosphatidylserine, and at the same time, the ultrasonic frequency, ultrasonic intensity, ultrasonic power, ultrasonic time, ultrasonic temperature, enzyme activity and other parameters are controlled synergistically, so that the enzymatic hydrolysis conversion rate is 98% or higher.

Claims

1. An ultrasound-assisted method for preparing phosphatidylserine characterized by comprising the following steps: (i) adding a phospholipid into a mixture that comprises L-serine, anhydrous calcium chloride and water, and (ii) adding phospholipase D to the mixture to start an enzymatic hydrolysis reaction that would yield the phosphatidylserine, wherein said method requires applying ultrasound waves 30 minutes after starting the enzymatic hydrolysis reaction, wherein the ultrasound waves are applied for 15-20 minutes.

2. The ultrasonic-assisted method for preparing phosphatidylserine of claim 1, wherein the ultrasonic frequency is 20-40 kHz; the ultrasonic intensity is 40-60 W/m.sup.2; and the ultrasonic power is 400-600 W.

3. The ultrasonic-assisted method for preparing phosphatidylserine of claim 2, wherein the ultrasonic frequency is 20-30 kHz; the ultrasonic intensity is 40-50 W/m.sup.2; and the ultrasonic power is 400-500 W.

4. The ultrasonic-assisted method for preparing phosphatidylserine of claim 1, wherein the time of the enzymatic hydrolysis reaction is 2-4 hours.

5. The ultrasonic-assisted method for preparing phosphatidylserine of claim 4, wherein the time of the enzymatic hydrolysis reaction is 2-3 hours.

6. The ultrasonic-assisted method for preparing phosphatidylserine of claim 1, wherein the temperature at the start of enzymatic hydrolysis is 35-45° C.

7. The ultrasonic-assisted method for preparing phosphatidylserine of claim 1, wherein the phospholipid is soybean phospholipid in which the phosphatidylcholine content is 54-55%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the results of the reaction of example 1;

(2) FIG. 2 is a graph showing the results of the reaction of example 2;

(3) FIG. 3 is a graph showing the results of the reaction of example 3.

DETAILED DESCRIPTION OF THE INVENTION

(4) The specific examples of the present invention will be described in detail below with reference to the accompanying drawings.

Example 1

(5) Adding the following substances by weight to a container: 150 g of L-serine, 10 g of anhydrous calcium chloride and 450 ml of pure water at a temperature 35° C.; adding 100 g of phospholipid which is soybean phospholipid in which the phosphatidylcholine content is 55%; performing stirring for 10 minutes; adding 20 ml of phospholipase D with the activity of 20 U/ml; applying ultrasound in an enzymatic hydrolysis process, wherein ultrasonic power is 400 W, ultrasonic intensity is 40 W/cm.sup.2, ultrasonic frequency is 40 kHz, ultrasonic treatment time is 5 minutes, and enzymatic hydrolysis time is 2 hours. When enzymatic hydrolysis ends, the conversion rate of phosphatidylcholine is 90%, and reaction results are shown in FIG. 1.

Example 2

(6) Adding the following substances by weight to a container: 200 g of L-serine, 20 g of anhydrous calcium chloride and 500 ml of pure water at a temperature 45° C.; adding 130 g of phospholipid which is soybean phospholipid in which the phosphatidylcholine content is 54%; performing stirring for 5 minutes; adding 25 ml of phospholipase D with the activity of 60 U/ml; applying ultrasound in an enzymatic hydrolysis process, wherein ultrasonic power is 600 W, ultrasonic intensity is 60 W/cm.sup.2, ultrasonic frequency is 20 kHz, ultrasonic treatment time is 30 minutes, and enzymatic hydrolysis time is 4 hours. When enzymatic hydrolysis ends, the conversion rate of phosphatidylcholine is 93%, and reaction results are shown in FIG. 2.

Example 3

(7) Adding the following substances by weight to a container: 180 g of L-serine, 15 g of anhydrous calcium chloride and 450 ml of pure water; performing stirring for 8 minutes at a temperature 40° C.; adding 120 g of phospholipid which is soybean phospholipid in which the phosphatidylcholine content is 54.23%; performing stirring for 5 minutes; adding 25 ml of phospholipase D with the activity of 50 U/ml; applying ultrasound in an enzymatic hydrolysis process, wherein ultrasonic power is 500 W, ultrasonic intensity is 50 W/cm.sup.2, ultrasonic frequency is 30 kHz, ultrasonic treatment time is 15 minutes, and enzymatic hydrolysis time is 3 hours. When enzymatic hydrolysis ends, the conversion rate of phosphatidylcholine is 98%, and reaction results are shown in FIG. 3.

Comparative Example 1

(8) Five groups of parallel experiments are designed. The intervention time for applying ultrasound is designed respectively as 0, 0.5, 1, 1.5 and 2 hours at the beginning of an enzymatic hydrolysis reaction, other raw materials and preparation steps are the same as those in example 3, and the conversion rate of phosphatidylcholine when enzymatic hydrolysis ends is shown in table 3:

(9) TABLE-US-00001 TABLE 1 Effect of ultrasonic intervention time on conversion rate of phosphatidylcholine number Group Intervention time/h 1 2 3 4 Intervention time/h 0 0.5 1 1.5 Conversion rate/% 98.2 95.1 90.4 80.2

(10) It can be seen from the above table that in the phosphatidylcholine enzymatic hydrolysis reaction, when the intervention time for applying ultrasound is 0-0.5 hour at the beginning of the enzymatic hydrolysis reaction, the conversion rate is 95% or higher. When the ultrasound is intervened later than 0.5 hour, the conversion rate drops to 90% or less.

Comparative Example 2

(11) Seven groups of parallel experiments are designed. The enzymatic hydrolysis reaction time is designed respectively as 1.5, 2, 2.5, 3, 3.5, 4 and 4.5 hours, other raw materials and preparation steps are the same as those in example 3, and the conversion rate of phosphatidylcholine when enzymatic hydrolysis ends is shown in table 3:

(12) TABLE-US-00002 TABLE 2 Effect of enzymatic hydrolysis time on conversion rate of phosphatidylcholine number Enzymatic Group hydrolysis time/h 1 2 3 4 5 6 7 Enzymatic 1.5 2 2.5 3 3.5 4 4.5 hydrolysis time/h Conversion 89.3 95.2 96.6 98.5 93.4 92.7 90.8 rate/%

(13) It can be seen from the above table that in the phosphatidylcholine enzymatic hydrolysis reaction, the conversion rate is 92% or higher when ultrasonic treatment time is 2-4 hours, and the conversion rate is 95% or higher when the ultrasonic treatment time is 2-3 hours. When the enzymatic hydrolysis time is less than 2 hours and greater than 4 hours, the conversion rate is greatly reduced.

Comparative Example 3

(14) The experiment uses an L.sub.25 (5.sup.6) orthogonal table, and with ultrasonic frequency (A), ultrasonic intensity (B), ultrasonic power (C), ultrasonic time (D), enzymatic hydrolysis temperature (E) and enzyme activity (F) as six investigation factors, five levels are respectively selected for the experiment. By calculating the average of the experimental data of the 6 factors and 5 levels, range analysis of the results is carried out, the optimal level is selected, the optimal experimental range is compared, and the phosphatidylcholine conversion rate is used as an evaluation index to evaluate the orthogonal experiment. The results are shown in Table 3.

(15) TABLE-US-00003 TABLE 3 Orthogonal L.sub.25 (5.sup.6) experimental results and range analysis Factors A B C D F Results Ultrasonic Ultrasonic Ultrasonic Ultrasonic E Enzyme Conversion Serial frequency intensity power time Temperature activity rate number (kHz) (W/m.sup.2) (W) (min) (° C.) (U/ml) (%) 1 20 30 300 5 25 20 70.2 2 20 40 400 10 35 30 99.8 3 20 50 500 15 45 40 98.1 4 20 60 600 20 55 50 99.1 5 20 70 700 30 65 60 88.7 6 30 30 400 15 55 60 96.5 7 30 40 500 20 65 20 77.5 8 30 50 600 30 25 30 94.3 9 30 60 700 5 35 40 94.8 10 30 70 300 10 45 50 92.5 11 40 30 500 30 35 50 95.5 12 40 40 600 5 45 60 96.8 13 40 50 700 10 55 20 72.3 14 40 60 300 15 65 30 94.5 15 40 70 400 20 25 40 95.4 16 50 30 600 10 65 40 87.7 17 50 40 700 15 25 50 90.4 18 50 50 300 20 35 60 92.1 19 50 60 400 30 45 20 67.2 20 50 70 500 5 55 30 85.3 21 60 30 700 20 45 30 90.5 22 60 40 300 30 55 40 89.3 23 60 50 400 5 65 50 90.2 24 60 60 500 10 25 60 88.7 25 60 70 600 15 35 20 65.7 K.sub.1 455.9 440.4 438.6 437.3 439.0 352.9 K.sub.2 455.6 453.8 449.1 441.0 447.9 464.4 K.sub.3 454.5 447.0 445.1 445.2 445.1 465.3 K.sub.4 422.7 444.3 443.6 454.6 442.5 467.7 K.sub.5 424.4 427.6 436.7 435.0 438.6 462.8 K1 91.18 88.08 87.72 87.46 87.80 70.58 K2 91.12 90.76 89.82 88.20 89.58 92.88 K.sub.3 90.90 89.40 89.02 89.04 89.02 93.06 K.sub.4 84.54 88.86 88.72 90.92 88.50 93.54 K.sub.5 84.88 85.52 87.34 87.00 87.72 92.56 Optimal A.sub.1 B.sub.2 C.sub.2 D.sub.4 E.sub.2 F.sub.5 level R.sub.j 91.18 90.76 89.82 90.92 89.58 93.54 Primary FADBCE and secondary order

(16) From the range analysis of the orthogonal experiment, the primary and secondary order of the influences of the six factors on the conversion rate is F>A>D>B>C>E, and based on variance analysis, the optimal experimental conditions are: A.sub.1B.sub.2C.sub.2D.sub.4E.sub.2F.sub.5, that is, the ultrasonic frequency is 20 kHz, the ultrasonic intensity is 40 W/m.sup.2, the ultrasonic power is 400 W, the ultrasonic time is 20 min, the enzymatic hydrolysis temperature is 35° C., and the enzyme activity is 50 U/ml. According to the sum of the same level of experiments of factor A (ultrasonic frequency), it can be seen that K.sub.1>K.sub.2>K.sub.3>K.sub.5>K.sub.4, the preferred ultrasonic frequency range is 20-40 kHz, and the more preferred the ultrasonic frequency range is 20-30 kHz; according to the sum of the same level of experiments of factor B (ultrasonic intensity), it can be seen that K.sub.2>K.sub.3>K.sub.4>K.sub.1>K.sub.5, the preferred ultrasonic intensity range is 40-60 W/m.sup.2, and more preferably the ultrasonic intensity range is 40-50 W/m.sup.2; according to the sum of the same level of experiments of factor C (ultrasonic power), it can be seen that K.sub.2>K.sub.3>K.sub.4>K.sub.1>K.sub.5, the preferred ultrasonic power range is 400-600 W, and the more preferred ultrasonic power range is 400-500 W; according to the sum of the same level of experiments of factor D (ultrasonic time), it can be seen that K.sub.4>K.sub.3>K.sub.2>K.sub.1>K.sub.5, the preferred ultrasonic time range is 10-20 min, and the more preferred ultrasonic time range is 15-20 min; according to the sum of the same level of experiments of factor E (temperature), it can be seen that K.sub.2>K.sub.3>K.sub.4>K.sub.1>K.sub.5, the preferred ultrasonic temperature range is 35-55° C., and the more preferable ultrasonic temperature range is 35-45° C.; and according to the sum of the same level of experiments of factor F (enzyme activity), it can be seen that K.sub.4>K.sub.3>K.sub.2>K.sub.5>K.sub.1, the preferred enzyme activity range is 30-60 U/ml, and the more preferred enzyme activity range is 30-50 U/ml.