CN112237844A

CN112237844A - Method for improving high polymerization degree epsilon-polylysine in product

Info

Publication number: CN112237844A
Application number: CN202011169276.4A
Authority: CN
Inventors: 张建华; 陈旭升; 赵星宇; 张宏建
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-10-28
Filing date: 2020-10-28
Publication date: 2021-01-19

Abstract

The invention discloses a method for improving high polymerization degree epsilon-polylysine in a product, belonging to the technical field of fermented food industry. The method for improving the content of high polymerization degree epsilon-PL in the epsilon-PL product comprises the following steps of carrying out ultrafiltration treatment on a crude product of epsilon-polylysine, wherein the pH value of feed liquid in the ultrafiltration treatment is controlled to be 5.5-6.5; the initial feeding concentration is 10-30 g/L; the pressure is 0.2-0.25 MPa; the membrane used for ultrafiltration treatment is a PES ultrafiltration membrane with the molecular weight cutoff of 1000-. The method of the invention improves the proportion of the epsilon-polylysine with high polymerization degree in the product, after ultrafiltration, the epsilon-polylysine with high polymerization degree in the product is improved by more than 8.4 percent compared with the original product, and the antibacterial activity to gram-positive bacteria and gram-negative bacteria is improved.

Description

Method for improving high polymerization degree epsilon-polylysine in product

Technical Field

The invention relates to a method for improving high polymerization degree epsilon-polylysine in a product, belonging to the technical field of fermented food industry.

Background

The influence of chemical additives on human health is highly concerned by people, and biological additives are advocated by people with the advantages of safety, effectiveness and no toxic or side effect on human bodies. Epsilon-polylysine (epsilon-poly-L-lysine, epsilon-PL for short) is a homotypic amino acid polymer formed by connecting a plurality of lysine residues through alpha-carboxyl and epsilon-amino, the polymerization degree is 5-35 at most, the molecular weight range is generally 780-4600Da, and the epsilon-polylysine has obvious inhibition effect on gram-positive bacteria, gram-negative bacteria, yeasts and molds under the acidic, neutral and slightly alkaline environment. Therefore, epsilon-PL is widely used as a green, safe biological food preservative.

The antibacterial performance of the epsilon-PL is influenced by the polymerization degree of the epsilon-PL, when the polymerization degree of the epsilon-PL is less than 9, the antibacterial activity is very low, when the polymerization degree is more than 9, the antibacterial activity is higher, and when the polymerization degree of the epsilon-polylysine is more than 25, the antibacterial performance is optimal. In the existing epsilon-PL research, the research on epsilon-PL polymerization degree control is mostly concentrated on links such as strain modification and fermentation process control, and researchers find the existence of epsilon-PL degrading enzyme (PLD) on Streptomyces albulus cell membranes, the optimum pH of the enzyme is 7.0, the enzyme activity is very low in an acidic environment (pH4.0), and the epsilon-PL can be prevented from being degraded by controlling low pH in the fermentation process, so that the epsilon-PL with high polymerization degree is accumulated. There have also been studies on the simultaneous addition of glycerol and sulfonated beta-cyclodextrin to the medium during the synthesis of epsilon-PL to reduce the molecular weight of epsilon-PL from 3.5-4.5kDa to 2.5kDa, which is complicated to operate. Still other researchers have proposed a method for controlling the amount of lysine residues in epsilon-PL by adjusting the concentration of a polyol in the medium. However, there is little research on controlling the polymerization degree of commercial product epsilon-PL through a downstream separation and extraction process.

Epsilon-poly-L-lysine (epsilon-PL for short) is a polymer of amino acids of the same type formed by connecting a plurality of lysine residues through alpha-carboxyl and epsilon-amino. The epsilon-PL backbone is a long aliphatic hydrocarbon chain, forming little helical structure, and is predominantly in a beta-sheet conformation. The ionization constant of the epsilon-PL side chain amino groups is between 8.0 and 9.0, and at pH values below the ionization constant, the amino groups carry a strong positive charge, which causes the residues in the main chain to repel each other, fail to form hydrogen bonds and expand freely, so that epsilon-PL is predominantly beta-sheet and random coil configurations in retentate and permeate solutions at pH values below 8-9.

Although the ultrafiltration membrane technology is simple to operate, the biggest defect of the membrane separation technology is membrane pollution, and due to continuous interception of large-particle substances in the separation process, the surface area of an adjacent membrane is increased by the dissolved amount and concentration, so that differential polarization is caused. Under the action of the concentration difference, solute back diffusion will generate fluid resistance and local osmotic pressure increase, which finally results in the reduction of the agent osmotic flux. If the concentration polarization is severe, it may even lead to precipitation of solutes and formation of a resistive layer on the membrane surface, eventually leading to a significant reduction in membrane flux.

Therefore, how to separate epsilon-PL by ultrafiltration and maintain membrane flux is a difficult problem.

Disclosure of Invention

In order to solve at least one problem, the invention provides a method for improving the content of high polymerization degree (polymerization degree is 25-35) epsilon-PL, so as to improve the bacteriostatic activity of the product, increase the proportion of high polymerization degree epsilon-polylysine in the product and improve the preservative effect of the product per unit mass.

The first purpose of the invention is to provide a method for improving the content of high polymerization degree epsilon-PL in epsilon-PL products, which is to carry out ultrafiltration treatment on crude epsilon-polylysine, wherein the pH value of feed liquid in the ultrafiltration treatment is controlled to be 5.5-6.5, and is further preferably 6.

In one embodiment of the present invention, the initial feed concentration in the ultrafiltration treatment is 10 to 30g/L, more preferably 20 g/L.

In one embodiment of the present invention, the pressure in the ultrafiltration treatment is 0.2 to 0.25MPa, more preferably 0.25 MPa.

In one embodiment of the present invention, the membrane used in the ultrafiltration treatment is an ultrafiltration membrane having a molecular weight cut-off of 1000-3000Da, and more preferably a PES ultrafiltration membrane (polyethersulfone ultrafiltration membrane).

In one embodiment of the present invention, the high polymerization degree ε -PL means a polymerization degree of 25 to 35.

In one embodiment of the invention, batch variable volume diafiltration is used during the ultrafiltration process, specifically, when the feed liquid is concentrated to 2/5 of the volume of the raw material liquid, water is added to the original volume, and the process is repeated until the high polymerization degree epsilon-PL is no longer present in the permeate.

The second purpose of the invention is to provide a method for preparing an epsilon-PL product with the polymerization degree of 25-35 and the epsilon-PL content of more than 90 percent, which comprises the following steps:

preparing the crude product of the epsilon-polylysine into initial feed liquid with the concentration of 10-30g/L, controlling the pH value to be 5.5-6.5 and the pressure to be 0.2-0.25MPa, and filtering by using a PES membrane with the molecular weight cutoff of 1000-3000Da, thus obtaining the epsilon-PL product with the high polymerization degree and the epsilon-PL content of more than 90 percent.

In one embodiment of the invention, the feed solution pH is further preferably 6; the initial feed concentration is further preferably 20 g/L; the pressure is more preferably 0.25 MPa.

The third purpose of the invention is that the epsilon-PL product with the polymerization degree of 25-35 and the epsilon-PL content of more than 90 percent is prepared by the method.

The fourth purpose of the invention is the application of ultrafiltration in improving the content of epsilon-PL with polymerization degree of 25-35, and specifically comprises the following steps: preparing the crude product of the epsilon-polylysine into initial feed liquid with the concentration of 10-30g/L, controlling the pH value to be 5.5-6.5, and filtering by using an ultrafiltration membrane with the molecular weight cutoff of 1000-3000Da and the pressure of 0.2-0.25MPa to obtain the epsilon-PL product with the polymerization degree of 25-35 and the epsilon-PL content of more than 90 percent.

The invention has the beneficial effects that:

(1) the invention improves the proportion of the epsilon-polylysine with high polymerization degree in the product by a downstream extraction method, improves the proportion of the epsilon-polylysine with high polymerization degree in the product by more than 8.4 percent compared with the original product after ultrafiltration, and improves the bacteriostatic activity of gram-positive bacteria and gram-negative bacteria.

(2) The membrane used in the invention is simple in process, free of phase change, secondary pollution and large in separation coefficient, does not change the property of the original product, and does not increase the extra extraction process cost.

Drawings

FIG. 1 is a schematic view of a membrane separation apparatus according to the present invention.

FIG. 2 shows the degree of polymerization of ε -PL in ε -PL solution before and after separation in example 2, wherein a is the original product; b is trapped fluid; c is a permeate.

FIG. 3 shows the high and low polymerization (. epsilon. -PL) contents in the permeate of the membrane of different molecular weight cut-off in example 3.

FIG. 4 shows the contents of high and low polymerization (. epsilon. -PL) in the permeate at different pressures in example 4.

FIG. 5 shows the high and low polymerization (. epsilon. -PL) levels in the permeate at different pH values of the feed solutions of example 5.

FIG. 6 shows the high and low polymerization (. epsilon. -PL) levels in the permeate at different feed concentrations in example 6.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

The test method comprises the following steps:

high degree of polymerization (25-35) ε -PL content test: the degree of polymerization of ε -PL was determined by ion pair chromatography. TSKgel ODS-80Ts column (4.6X 250 mm; Tosoh, Tokyo), mobile phase A: 10mM NaH₂PO₄、100mM NaClO₄·H₂O, 10mM sodium octane sulfonate, and phosphoric acid to adjust the pH to 2.6. Mobile phase B: 20mM NaH₂PO₄，200mM NaClO₄·H₂O, 20mM sodium octane sulfonate, pH adjusted to 2.6 with phosphoric acid, 50% (v/v) acetonitrile. Equilibration of the chromatographic column: 45% (v/v) mobile phase A + 55% mobile phase B. Gradient of mobile phase: mobile phase B changed linearly from 55% to 90% at 50 ℃ over 85 min, mixed with mobile phase a, 0.4 mL/min. Detection wavelength: 215 nm. Under the chromatographic condition, correction factors of epsilon-PL with different polymerization degrees are similar, and the content of the epsilon-PL with different polymerization degrees is quantified by a normalization method

Testing of minimum inhibitory concentration: the bacteria are cultured in broth, and the diluted epsilon-PL solution of 1m L and the diluted bacteria solution of 1m L medium are added into each test tube to make the bacteria concentration about 5X 10⁵cfu/m L, wherein the epsilon-PL concentrations of the bacteria from 1 st to 11 th tubes are 1, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 mu g/mL respectively. Culturing at 37 deg.C for 12 hr, culturing yeast and mold at 37 deg.C for 48 hr, and observing with naked eyeThe minimum concentration of epsilon-PL for inhibiting the growth of the thalli is the minimum inhibitory concentration.

Testing of membrane flux: the membrane flux J was calculated according to formula (1) by measuring the volume of the permeate obtained over a certain period of time.

Wherein V is the volume (L) of the permeate and A is the membrane area (m)²) And t is the period of filtration.

Testing of rejection rate: the rejection was calculated according to equation (2):

in the formula, C₀Is the concentration (g/L) of epsilon-PL in the raw material liquid, C₁Concentration of epsilon-PL in the retentate (g/L), V₀And V₁The volumes (L) of the feed solution and the retentate, respectively.

And (3) testing the transmittance: the transmittance is calculated according to equation (3):

in the formula C₂Is the concentration (g/L) of ε -PL in the permeate, V₂Respectively the volume (L) and C of the permeate₀Is the concentration (g/L) of epsilon-PL in the raw material liquid, V₀Volume of feed solution (L).

EXAMPLE 1 preparation of crude epsilon-polylysine

Fed-batch fermentation was carried out using S.albulus M-Z18 as a polylysine-producing strain according to the literature (Pan L, Chen X S, Liu M, et al. efficient production of. epsilon. -poly-L-lysine from glucose by two-stage fermentation, pH shock protocol [ J ]. Process Biochemistry,2017,63, 8-15.). Fermenting for 168h and collecting fermentation liquor; centrifuging the fermentation liquor (rotation speed 8000rpm, time 30min), and removing thallus and other solid; then adsorbing by cation exchange resin D004 (Jiangsu Suqing water treatment engineering group, Inc.) (the bed volume is 60mL, the pH of the sample loading material liquid is 8.5, the concentration of the sample loading epsilon-PL is 35g/L, and the sample loading flow rate is 1BV/h) to remove pigment and foreign protein; removing salt ions through nanofiltration, using a tangential flow nanofiltration system Millipore (Millipore company, USA), wherein the molecular weight cut-off of the membrane is 1KDa, the pH value of the feed liquid is adjusted to 11.0, the pH value in the solution is monitored and adjusted in the whole process to ensure the pH value to be stable in the whole sample loading process, the conductivity of the permeation liquid in each hour is measured, and the concentration of the feed liquid is concentrated to 40% of the original volume when the volume of the solution is reduced to be below 40% of the volume of the feed liquid and the conductivity is between 200 and 300 mu s/cm; and then adjusting the pH value of the obtained concentrated solution to 4, adding 1% (w/v) of activated carbon LT-720 into each 100mL of epsilon-PL concentrated solution, stirring in a water bath at 80 ℃ for 30min, performing suction filtration to separate the activated carbon from the solution, performing rotary evaporation on the solution obtained by suction filtration by using a rotary evaporator in the water bath at 60 ℃ until the volume of the solution is 10% of the original volume, and then putting the solution into a vacuum drying oven (at 60 ℃ for 12-16h) for drying to obtain a crude product of epsilon-polylysine.

The content of high polymerization degree (25-35) epsilon-PL in the prepared crude epsilon-polylysine is 82.84 percent through tests, and the minimum inhibitory concentration MIC of Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8099 are both 12 mu g/mL.

Example 2

A method for improving high polymerization degree epsilon-polylysine in a product comprises the following steps:

the crude product of epsilon-polylysine obtained in example 1 is prepared into 20g/L initial feed liquid, the pH value is controlled to be 6, the temperature of the feed liquid is 25 ℃, a plate type membrane (PES membrane) with the molecular weight cutoff of 3000Da is used for ultrafiltration, and the membrane pressure is 0.25 MPa. Selecting intermittent variable volume percolation in an operation mode, specifically adding water to the original volume when the feed liquid is concentrated to 2/5 of the volume of the raw material liquid, and repeating the steps until the high polymerization degree epsilon-PL does not appear in the permeate; and obtaining an epsilon-polylysine product.

Chromatograms of the trapped solution and the permeated solution are shown in FIG. 2, and the trapped solution and the permeated solution are collected and concentrated to control the concentration to be about 10 g/L. As can be seen from FIG. 2, the epsilon-PL with a degree of polymerization of < 25 in the retentate was substantially not peaked by continuous water addition and was normalized to a degree of polymerization of 25-35 in the retentate of 91.22%. Compared with crude epsilon-polylysine, the proportion of epsilon-PL of 25-35 is improved by 8.38 percent. And the antibacterial performance of the finally obtained epsilon-polylysine product on bacteria is improved, and the minimum inhibitory concentration MIC of Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8099 are respectively reduced to 8 mu g/mL and 10 mu g/mL.

Example 3

Adjusting the molecular weight of the plate-type membrane as shown in Table 1, preparing the crude product of epsilon-polylysine obtained in example 1 into 10g/L initial feed liquid, controlling the pH to be 9 and the temperature of the feed liquid to be 25 ℃, and carrying out ultrafiltration with the membrane pressure of 0.25 MPa; when the feed liquid is concentrated to 2/5 of the volume of the raw material liquid, namely the intercepted liquid volume is concentrated to 2/5 of the volume of the raw material liquid, the ultrafiltration is stopped, and the intercepted liquid and the permeate are collected and detected.

The cut-off and permeate obtained in example 3 were subjected to performance tests, the results of which are shown in table 1 and fig. 3 below:

table 1 test results of example 3

As can be seen from table 1: the overall performance was best for a plate membrane with a molecular weight cut-off of 3000, with a plate membrane with a molecular weight cut-off of 3000 having a permeability of 31.29% for low degrees of polymerization and 8.62% for high degrees of polymerization. As can be seen from fig. 3: compared with membranes with other molecular weights, the plate-type membrane permeation liquid with the molecular weight cutoff of 3000 has the highest content of low polymerization degree epsilon-PL, the content of low polymerization degree (< 25) epsilon-PL is 41.08%, and the content of epsilon-PL with the polymerization degree between 25 and 35 is 58.92%.

Example 4

Adjusting the operation pressure as shown in Table 2, preparing the crude product of epsilon-polylysine obtained in example 1 into 10g/L initial feed liquid, controlling the pH to be 9 and the temperature of the feed liquid to be 25 ℃, and performing ultrafiltration by using a plate type membrane with the molecular weight cutoff of 3000 Da; when the feed liquid is concentrated to 2/5 of the volume of the raw material liquid, namely the intercepted liquid volume is concentrated to 2/5 of the volume of the raw material liquid, the ultrafiltration is stopped, and the intercepted liquid and the permeate are collected and detected.

The cut-off and permeate obtained in example 4 were subjected to performance tests, the results of which are shown in table 2 below and fig. 4:

table 2 test results of example 4

As can be seen from table 2 and fig. 4: the retention rate of the plate-type membrane on epsilon-PL solution and the membrane flux increase along with the increase of the pressure under different operating pressures, and the retention rate on epsilon-PL slightly decreases along with the increase of the pressure; the pressure does not substantially affect the permeation of a plate membrane with a molecular weight cut-off of 3000 to epsilon-PL with a high degree of polymerization, but at lower pressures the permeation of epsilon-PL with a low degree of polymerization is relatively high. It can be seen that epsilon-PL of low degree of polymerization passes through the membrane relatively more easily during the separation process. However, the separation effect was not significant because the content of low polymerization degree ε -PL in the retentate was very low (17.73%), and the difference in molecular weight between high and low polymerization degree ε -PL was not significant. Therefore, the membrane flux at the operating pressure of 0.25MPa is relatively large, and the operating pressure of 0.25MPa is selected as the optimal operating pressure.

Example 5

The pH value is adjusted as shown in Table 3, the crude product of epsilon-polylysine obtained in example 1 is prepared into 10g/L initial feed liquid, the temperature of the feed liquid is 25 ℃, a plate type membrane with the molecular weight cutoff of 3000Da is used, and the pressure of the ultrafiltration membrane is 0.25 MPa; when the feed liquid is concentrated to 2/5 of the volume of the raw material liquid, namely the intercepted liquid volume is concentrated to 2/5 of the volume of the raw material liquid, the ultrafiltration is stopped, and the intercepted liquid and the permeate are collected and detected.

The cut-off liquid and the permeate obtained in example 5 were subjected to performance tests, and the test results are shown in table 3 below and fig. 5:

table 3 test results of example 5

As can be seen from table 3: the retention rate of a plate type membrane with the molecular weight cutoff of 3000Da on an epsilon-PL solution is reduced along with the increase of pH, and the membrane flux is basically stable; as can be seen from fig. 5: as the pH increases, the low polymerization degree polysomy ratio in the permeate increases first and then decreases; the content of low polymerization degree e-PL was the highest at pH 6 and 75.32%, and at around the isoelectric point at pH 9 and the content of low polymerization degree e-PL was the lowest and 22.08%; the epsilon-PL permeability increases with increasing pH, the epsilon-PL permeability is very low under acidic conditions, and the plate-type membrane with a molecular weight cut-off of 3000Da at pH 3 has a low degree of polymerization epsilon-PL permeability of 2.89%; when the pH of the feed solution was 10, the transmittance for low polymerization degree e-PL was 37.51%, and the transmittance for high polymerization degree e-PL was 21.67%; at pH 6, the ratio of low polymerization degree e-PL in the permeate is high, and at this pH, the permeation rate of low polymerization degree e-PL is more different from that of high polymerization degree e-PL, and separation of high polymerization degree e-PL is facilitated.

Example 6

The initial feed concentration was adjusted as shown in Table 4, the pH was controlled to 6, the feed temperature was controlled to 25 ℃, ultrafiltration was carried out using a plate-type membrane having a molecular weight cut-off of 3000Da, and the membrane pressure was 0.25 MPa. When the feed liquid is concentrated to 2/5 of the volume of the raw material liquid, namely the intercepted liquid volume is concentrated to 2/5 of the volume of the raw material liquid, the ultrafiltration is stopped, and the intercepted liquid and the permeate are collected and detected.

The cut-off liquid and the permeate obtained in example 6 were subjected to performance tests, and the test results are shown in the following table 4 and fig. 6:

table 4 test results of example 6

As can be seen from table 4: the sieving effect of a plate type membrane with the molecular weight cutoff of 3000Da on epsilon-PL is influenced by the concentration of feed liquid. The membrane flux is reduced along with the increase of the concentration of the feed liquid, the permeation rate of the thinner solution on the plate type membrane with the molecular weight cutoff of 3000Da is faster, and the rate of the plate type membrane with the molecular weight cutoff of 3000Da to epsilon-PL is reduced along with the increase of the concentration of the feed liquid. As can be seen from fig. 6: when the feed solution concentration is 5g/L, the content of the low-polymerization-degree epsilon-PL in the plate type membrane permeation solution with the molecular weight cutoff of 3000Da is only 17.74 percent, and the plate type membrane with the molecular weight cutoff of 3000Da has basically no selectivity to the high-polymerization-degree epsilon-PL in the solution. On the other hand, when the concentration of the feed solution was 20g/L, the content of low polymerization degree ε -PL was 48.04%, and then the ratio of ε -PL having a low polymerization degree in the permeate decreased as the concentration increased. The low degree of polymerization ε -PL was found to have a transmittance of 25.25% at an initial feed concentration of 30 g/L. The feed concentration affects the permeation of epsilon-PL, the low polymerization degree epsilon-PL permeability is very low at low concentration, the low polymerization degree epsilon-PL permeability is improved with the increase of concentration, but the high polymerization degree epsilon-PL permeability is increased with the increase of initial feed liquid concentration, and the selectivity of the membrane to the high and low polymerization degree epsilon-PL is reduced when the concentration is increased to a certain value. Therefore, the concentration of the feed liquid is 20g/L, the proportion of low polymerization degree epsilon-PL in the permeate liquid is the highest, and the comprehensive separation effect is better.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for improving the content of high polymerization degree epsilon-PL in an epsilon-PL product is characterized in that the method is used for carrying out ultrafiltration treatment on a crude epsilon-polylysine product, wherein the pH value of feed liquid in the ultrafiltration treatment is controlled to be 5.5-6.5.

2. The method according to claim 1, wherein the initial feed concentration in the ultrafiltration treatment is 10 to 30 g/L.

3. The method according to claim 1 or 2, wherein the pressure in the ultrafiltration treatment is 0.2 to 0.25 MPa.

4. The method according to any one of claims 1 to 3, wherein the ultrafiltration membrane used in the ultrafiltration treatment is an ultrafiltration membrane having a molecular weight cut-off of 1000 and 3000 Da.

5. The method according to any one of claims 1 to 4, wherein the high degree of polymerization ε -PL means a degree of polymerization of 25 to 35.

6. The process according to any of claims 1 to 5, wherein the ultrafiltration treatment is carried out by batch variable volume diafiltration, in particular by adding water to the initial volume when the feed solution is concentrated to 2/5% of the volume of the feed solution, and repeating this until no higher degree of polymerization ε -PL is observed in the permeate.

7. A method for preparing an epsilon-PL product with a polymerization degree of 25-35 and an epsilon-PL content of more than 90%, characterized by comprising the following steps:

preparing the crude product of the epsilon-polylysine into initial feed liquid with the concentration of 10-30g/L, controlling the pH value to be 5.5-6.5, and filtering by using an ultrafiltration membrane with the molecular weight cutoff of 1000-3000Da and the pressure of 0.2-0.25MPa to obtain the epsilon-PL product with the polymerization degree of 25-35 and the epsilon-PL content of more than 90 percent.

8. The method of claim 7, wherein the feed solution has a pH of 6; the initial feed concentration was 20 g/L.

9. An epsilon-PL product having a degree of polymerization of 25 to 35 and an epsilon-PL content of 90% or more, produced by the method of claim 7 or 8.

10. The application of ultrafiltration in improving the content of epsilon-PL with the polymerization degree of 25-35 is characterized in that the crude epsilon-polylysine is prepared into initial feed liquid with the concentration of 10-30g/L, the pH value is controlled to be 5.5-6.5, the pressure is 0.2-0.25MPa, and ultrafiltration membrane with the cutoff molecular weight of 1000-3000Da is used for filtration.