CN111961125B - Plukenetia volubilis linneo immune active peptide and preparation method thereof - Google Patents
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Abstract
The invention discloses a plukenetia volubilis linneo immune active peptide and a preparation method thereof, wherein the preparation method of the plukenetia volubilis linneo immune active peptide comprises the following steps: extracting albumin of the plukenetia volubilis linneo; hydrolyzing the albumin to obtain albumin hydrolysate; carrying out ultrafiltration treatment and separation and purification treatment on the albumin hydrolysate; and collecting the polypeptide solution. According to the preparation method of the plukenetia volubilis linneo immunoactive peptide, albumin of plukenetia volubilis linneo is hydrolyzed, and the required peptide segments are separated and enriched in a targeted manner by using ultrafiltration treatment and separation and purification treatment, so that the obtained immunoactive peptide product has stronger functions and higher purity.
Description
Technical Field
The invention relates to the technical field of immunoactive peptides, and particularly relates to an embelia laeta immunoactive peptide and a preparation method thereof.
Background
The immune active peptide refers to an active polypeptide with biological functions of enhancing immune function, transferring immune information, promoting differentiation and maturation of lymphocytes and the like, has the advantages of no toxicity, low allergy, high safety and the like, and has good application prospects in the fields of foods, health-care products, biological medicines and the like. Until now, researchers have separated various peptide fragments with immunological activity from protein zymolytes such as milk protein, soybean protein, rice protein, fish and shellfish protein, collagen and the like, and applied the peptide fragments to clinical research, and remarkable effects are achieved. However, in the existing research, the preparation of the immunoactive peptide by using the plukenetia volubilis linneo protein has not been reported.
The plukenetia volubilis linneo is a new tropical region oil variety, and as fruit oil and protein thereof are listed as new national resource food, the processing and comprehensive utilization of the plukenetia volubilis linneo also gradually draws attention of the food processing industry. The residual plukenetia volubilis linneo meal after oil extraction of the plukenetia volubilis linneo kernel contains abundant protein, but the development and utilization of the protein resource are limited at present. Therefore, the method for separating and preparing the immunocompetent polypeptide by using the plukenetia volubilis linneo meal as the raw material has important significance for the related research of the plant protein source immunoactive peptide, and simultaneously has certain practical significance for improving the added value of the plukenetia volubilis linneo processing by-products and developing products with the function of improving immunity.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides the plukenetia volubilis linneo immune active peptide which can rapidly penetrate through the oral cavity and the stomach of a human body and directly enter the small intestine, is better digested and absorbed by the human body, and has the functions of removing free radicals, improving the immunity of the human body and the like.
The invention also provides a preparation method of the plukenetia volubilis linneo immune active peptide for preparing the plukenetia volubilis linneo immune active peptide.
An immunologically active peptide of embelia according to an embodiment of the first aspect of the invention comprises: 24 peptide segments such as TGGWSLK, WKPW, FLTMEPR, VWLVK, KVVL, MVVKK, LTGLNKL, RLLVWELER, KLSLEWLK, FVKL KL, LGDLGTKL, LTGLDKL, LFAEMDK, EADGTLR, VVFK, TLLNPR, AYLTGLK, WLPDVK, VLWLPR, RQVWEDR, TVLLPR, LVRFPK, TLLFGDK, WSELVK and the like.
The embelia laeta immunoactive peptide provided by the embodiment of the invention has a strong immune function, and the polypeptide is subjected to in vitro immune activity evaluation through a mouse macrophage model RAW264.7, and experiments show that the immunoactive peptide has a promoting effect on the phagocytosis capacity of RAW264.7 macrophages, and has a certain positive effect on the aspects of protecting an organism and strengthening the immunity of the organism.
According to the second aspect of the invention, the preparation method of the embelia nasuta immune active peptide comprises the following steps:
according to some embodiments of the invention, the albumin extracted from plukenetia volubilis comprises:
crushing the wisteria sinensis pulp to obtain wisteria sinensis pulp powder;
placing the Plukenetia volubilis linneo pulp powder in a 24L extraction kettle of a supercritical extraction device, and extracting at 20MPa and 32 ℃ for 120min, wherein the flow rate of CO2 is 60 mL/min;
discarding the extracted mei teng fruit oil, taking out mei teng fruit cake meal, adding a certain amount of first-grade water, performing ultrasonic treatment for 15min, stirring and extracting at constant temperature for a certain time, and centrifuging at the speed of 4000 revolutions per minute for 10min to obtain a supernatant;
putting the supernatant into distilled water, dialyzing for 72 hours at the temperature of 4 ℃, performing rotary evaporation in a rotary evaporator for a certain time, and performing vacuum freeze drying to obtain freeze-dried albumin;
hydrolyzing the albumin to obtain albumin hydrolysate;
carrying out ultrafiltration treatment and separation and purification treatment on the albumin hydrolysate;
and collecting the polypeptide solution.
According to the preparation method of the embelia laeta immunological active peptide, the albumin of the embelia laeta is hydrolyzed, and the required peptide segments are separated and enriched in a targeted manner by using ultrafiltration treatment and separation and purification treatment, so that the obtained immunological active peptide product has stronger function and higher purity.
In addition, the preparation method of the embelia laeta immunoactive peptide according to the embodiment of the invention also has the following additional technical characteristics:
according to some embodiments of the invention, the albumin is extracted under the following conditions: the material-liquid ratio is 1:25(g/mL), the extraction temperature is 40 ℃, and the extraction time is 5 h.
According to some embodiments of the invention, the albumin is hydrolyzed using a protease.
In some embodiments of the invention, the protease is one or more of a neutral protease, papain, an alkaline protease, trypsin, and pepsin.
In some embodiments of the invention, the hydrolysis conditions for the hydrolysis with trypsin are: the enzyme adding amount is 7000U/g, the reaction pH is 7.0, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 4 h.
According to some embodiments of the invention, the albumin hydrolysate is subjected to ultrafiltration treatment using an ultrafiltration membrane having a molecular weight of 3 kDa;
performing ultrasonic treatment on albumin hydrolysate with molecular weight less than 3kDa after ultrafiltration for 15min, separating and purifying, eluting with distilled water with flow rate of 3mL/min, and collecting polypeptide solution eluted for 13-29 min;
the collected polypeptide solution is concentrated.
According to some embodiments of the invention, the albumin hydrolysate is subjected to separation and purification treatment by using a Sephadex G-25 Sephadex column.
According to some embodiments of the invention, the pressure is maintained at 0.2MPa by pressurizing with nitrogen during the ultrafiltration treatment, and the obtained polypeptide solution is placed on ice to ensure its activity.
The embelia laeta immunoactive peptide provided by the embodiment of the invention has stronger function and higher purity.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
Fig. 1 is a flow diagram of a method of preparing an immunologically active peptide of embelia according to an embodiment of the invention;
FIG. 2 is a graphical representation of the results of the effect of different proteases on the degree of albumin hydrolysis of embelia nasi;
FIG. 3 is a HPGPC chromatogram of albumin hydrolysate of Plukenetia volubilis fruits;
FIG. 4 is a molecular weight distribution spectrum of albumin zymolyte of Plukenetia volubilis fruits;
FIG. 5 is a graph showing the effect of various ultrafiltrate fractions of the Albumin hydrolysate of Plukenetia volubilis on the proliferation of RAW264.7 cells;
FIG. 6 is a graph showing the effect of various ultrafiltrate fractions of the Albumin hydrolysate of Plukenetia volubilis on phagocytosis of RAW264.7 cells;
FIG. 7 is a graph of the elution profile of the less than 3kDa component of the albumin zymolyte of embelia.
FIG. 8 is a graph showing the effect of Sephadex G-25 fraction on the proliferation of RAW264.7 cells;
FIG. 9 is a graph showing the effect of Sephadex G-25 fraction on phagocytosis of RAW264.7 cells;
FIG. 10 is a chromatographic total ion flow diagram of Sephadex G-25 fraction P1;
FIGS. 11-34 correspond to secondary mass spectrograms of different immunoactive peptide fragments respectively according to liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined Denovo sequencing analysis.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
The following describes a method for preparing the embelia nasuta immunoactive peptide according to an embodiment of the present invention with reference to the accompanying drawings.
As shown in fig. 1, the wisteria indica albumin is extracted; hydrolyzing the obtained albumin with protease; performing ultrafiltration on the albumin hydrolysate; further separating and purifying by using a Sephedax G-25 sephadex column; collecting the polypeptide component to obtain the immune active peptide.
Wherein, the best extraction conditions for extracting albumin in the plukenetia volubilis linneo pulp are as follows: the feed-liquid ratio is 1:25(g/mL), the extraction temperature is 40 ℃, and the extraction time is 5 h.
The protease can be one or more of neutral protease, papain, alkaline protease, trypsin and pepsin, preferably trypsin. Wherein, the optimal hydrolysis conditions when trypsin is adopted for hydrolysis are as follows: the enzyme adding amount is 7000U/g, the reaction pH is 7.0, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 4 h.
Preferably, the albumin hydrolysate is ultrafiltered with an ultrafiltration membrane having a molecular weight of 3 kDa. During the ultrafiltration, the pressure was increased with nitrogen gas to maintain the pressure at 0.2 MPa. The obtained polypeptide solution was placed on ice to ensure its activity.
Preferably, the zymolyte is further separated and purified by a Sephadex G-25 Sephadex column. The Sephadex G-25 packing is packed after fully soaking. And (3) carrying out ultrasonic treatment on the zymolyte with the molecular weight of less than 3kDa after ultrafiltration separation for 15min, and eluting with distilled water at the flow rate of 3 mL/min. Preferably, the polypeptide solution eluted at 13min-29min is collected.
After the protein of the embelia laeta is subjected to enzymolysis, the required peptide segments are separated and enriched in a targeted manner by using an ultrafiltration membrane and a Sephadex G-25 Sephadex column, and the obtained immunoactive peptide product has stronger function and higher purity. The in vitro immune activity evaluation of the polypeptide is carried out by a mouse macrophage model RAW264.7, and experiments show that the immune active peptide has a promoting effect on the phagocytosis capacity of RAW264.7 macrophages and also has a certain positive effect on protecting the organism and strengthening the immunity of the organism.
According to the preparation method provided by the embodiment of the invention, the collected high-resolution mass spectrum data set is subjected to de novo analysis on the amino acid sequence of the impalpable fruit immunoactive peptide through LC/MS-MS liquid mass analysis, and 24 peptide segments such as TGGWSPLK, WKPW, FLTMEPR, VVLDVK, KVVL, MVVKK, LTGLNKL, RLLVWELER, KLSLEWLK, FVKLL, LGDLGTKL, LTGLDKL, LFAEMDK, EADGTLR, VVKK, TLLNPR, AYLTGLK, WLPDVK, VLWLPR, RQVEDR, TVLLPR, LVRFPK, TLLFGDK, WSELVK and the like are identified. Through comparison of protein databases BLAST and polypeptide databases PIPOPEP, the polypeptide which is the same as the polypeptide obtained by the invention is not found, so that 24 peptide fragments obtained by the invention are novel polypeptides.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the present invention and are not to be construed as limiting the present invention. The experimental procedures used in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The materials and reagents used in the following examples are as follows:
TABLE 1 preparation of immunologically active peptides as the principal materials
TABLE 2 preparation of Primary Agents by Immunoactive peptides
Example 1 selection of optimal Process conditions for Plukenetia volubilis Albumin
Pulverizing Ampelopsis Grossdentata fruit mealPlacing Plukenetia volubilis linneo meal powder in a 24L extraction kettle of supercritical extraction equipment, extracting at 20MPa and 32 deg.C for 120min with CO2The flow rate was 60 mL/min. Discarding extracted plukenetia volubilis linneo oil, taking out plukenetia volubilis linneo cake meal powder, adding a certain amount of first-grade water, performing ultrasonic treatment for 15min, stirring and extracting at constant temperature for a certain time, centrifuging at the speed of 4000 rpm for 10min to obtain supernatant, dialyzing the supernatant in distilled water at the temperature of 4 ℃ for 72 h, performing rotary evaporation in a rotary evaporator for a certain time, and performing vacuum freeze drying to obtain freeze-dried albumin. According to a simulation experiment, considering the mutual influence of factors such as a material-liquid ratio, an extraction temperature, extraction time and the like, an orthogonal experiment is adopted to determine the optimal extraction condition of albumin, and the experiment passes through L9(34) Orthogonal experiments were performed and the protocol is shown in table 3.
TABLE 3 Albumin extraction test factor level table
The experimental results are shown in table 4, and the factors influencing the extraction rate of the embelia americana albumin obtained by the extreme difference analysis in table 4 are liquid-material ratio > extraction time > extraction temperature, the influence level of the condition is reflected by the size of the mean value, and the maximum mean value is the optimized parameter of the condition. As can be seen from table 4, the maximum mean values among the three conditions are: the material-liquid ratio is 1:25(g/mL), the extraction temperature is 40 ℃, and the extraction time is 5 h. After 3 verification experiments, the average extraction rate under the condition is 52.36 percent, which is equivalent to the orthogonal test result, and the optimized result is proved to be reliable.
TABLE 4 Albumin extraction orthogonal test results
Example 2 selection of Elaeagnus umbellata Albumin hydrolase types and selection of optimal hydrolysis conditions
Mixing the plukenetia volubilis linneo albumin according to a material-liquid ratio of 1:15, adjusting the optimal action temperature and the optimal pH, adding neutral protease, papain, compound protease, trypsin, pepsin and alkaline protease according to an enzyme activity ratio of 6000U/g, carrying out enzymolysis for 3 hours, and screening enzyme types by taking the hydrolysis degree as an index. According to the simulation experiment, considering the mutual influence of factors such as enzymolysis time, enzymolysis temperature, enzyme adding amount, pH and the like, an orthogonal experiment is adopted to determine the optimal enzymolysis condition of albumin, and the experiment passes through L9(34) Orthogonal experiments were performed and the protocol is shown in table 5.
The experimental result is shown in fig. 2, the hydrolysis degrees of the plukenetia volubilis linneo clear enzymolysis solutions of different proteases are significantly different (P <0.05), the hydrolysis degree of the trypsin enzymolysis product of plukenetia volubilis linneo albumin is optimal, and trypsin is selected to be applied to the production of plukenetia volubilis linneo albumin polypeptide in consideration of the process production and the economic benefit. The results of the albumin enzymolysis orthogonal test are shown in table 5, the factors influencing the albumin enzymolysis of the plukenetia volubilis linneo can be obtained by the range analysis of table 5, namely the enzymolysis temperature is greater than the enzymolysis pH is greater than the enzymolysis time is greater than the enzyme addition amount, and the optimal enzymolysis process conditions are as follows: adding 7000U/g enzyme, pH7.0, enzymolysis temperature of 50 deg.C, and enzymolysis time of 4 h. And (3) performing a verification experiment according to the obtained optimal combination to obtain the hydrolysis degree of 28.04% +/-0.07, which proves that the optimization result is more reliable.
TABLE 5 results of the proteolytic enzyme orthogonal assay
Example 3 isolation, purification and identification of an immunologically active peptide of embelia nasuta and evaluation of its immunological activity
1. Ultra-filtration separation
In order to purify and enrich active components, the protein enzymolysis product of the embelia meiboma is subjected to ultrafiltration treatment to obtain polypeptide mixed solutions with different molecular weights. Placing the enzymolysis solution in an ultrafiltration cup, sequentially performing ultrafiltration treatment by using ultrafiltration membrane assemblies of 10kDa and 3kDa, sealing the ultrafiltration cup, and pressurizing by using nitrogen to keep the pressure at 0.2 MPa. The obtained polypeptide solution was placed on ice to ensure its activity. Then, the mouse macrophage model RAW264.7 was used to evaluate the in vitro immunological activity of each ultrafiltration fraction at a concentration of 100. mu.g/mL, and compared with a blank control, 1. mu.g/mL Lipopolysaccharide (LPS) and the same concentration of albumin zymolyte (SIP) which had not been ultrafiltered, and the remaining sample was subjected to vacuum freeze-drying at-80 ℃ and stored at-20 ℃ for further use.
As shown in FIGS. 5-6, the in vitro immunocompetence evaluation of each ultrafiltration component shows that the ultrafiltration component with less than 3kDa has the best effect on promoting the proliferation capability of RAW264.7 cells and the capability of phagocytizing neutral red blood. After ultrafiltration separation, the proliferation capacity of the ultrafiltration component with the molecular weight less than 3kDa on RAW264.7 cells is increased by 72.34 percent, 18.09 percent and 85.11 percent respectively compared with that of a blank group, an SIP group and an LPS group; the capacity of the ultrafiltration component with the molecular weight of less than 3kDa to phagocytose neutral red blood for RAW264.7 cells is increased by 85.39%, 30.45% and 19.95% respectively compared with the blank group, the SIP group and the LPS group. The ultrafiltration component with the molecular weight of less than 3kDa can obviously promote the proliferation and activation of RAW264.7 cells and improve the immunity. As shown in Table 6, the ultrafiltration fraction of less than 3kDa had a relatively good amino acid composition and a relatively good immunological activity against the body, and the results of this determination were in accordance with the results of the in vitro immunological activity experiments.
TABLE 6 amino acid composition of the ultrafiltration components of the embelia nasuta albumin zymolyte
2. Sephadex column purification
And filling the Sephadex G-25 filler into a column after fully soaking. Dissolving the zymolyte in distilled water, and performing ultrasonic treatment to obtain a sample. Eluting with distilled water at flow rate of 3mL/min, collecting 3mL per tube, and detecting wavelength of 280 nm. And respectively collecting peak components after elution and separation, and performing in-vitro immune activity evaluation on the components by using a mouse macrophage model RAW264.7 after freeze drying.
The results of the experiments are shown in FIGS. 7-9, where the less than 3kDa ultrafiltration fraction was separated into three major fractions, fractions P1, P2 and P3, on Sephadex G-25 Sephadex columns. All components separated by Sephadex G-25 have no toxic action on RAW264.7 macrophage, can promote activation of RAW264.7 macrophage and phagocytic activity of cells, and improve immunity, and the effect of P1 component is obviously better than that of P2 and P3 components. As can be seen from tables 7 and 8, compared with the LPS group, the P1 component with different concentrations has very significant inhibitory effect on the contents of TNF-alpha and IL-6 secreted by RAW264.7 macrophage after LPS induction, so that the mechanism immune function is regulated, and the P1 component shows better immune activity compared with the P2 and P3 components.
TABLE 7 Effect of Sephadex G-25 fraction on RAW264.7 cytokine TNF- α
TABLE 8 Effect of Sephadex G-25 fraction on RAW264.7 cytokine IL-6
Note: # very significant difference (P < 0.01) compared to blank; there were very significant differences compared to the LPS group (P < 0.01); *. was significantly different from LPS group (P < 0.05).
3. Identification of immunologically active peptide sequences
1) Reductive alkylation
DTT solution was added to the sample to a final concentration of 10mmol/L and reduced in a water bath at 37 ℃ for 4 h. Subsequently, IAA solution was added to a final concentration of 50mmol/L, and the reaction was carried out for 40min with exclusion of light. Desalting with self-packed desalting column, and evaporating solvent in vacuum centrifugal concentrator at 45 deg.C.
2) LC-MS/MS detection
Capillary liquid chromatography conditions were as follows:
a. pre-column: 300 μm i.d.. times.5 mm, packed with Acclaim PepMap RPLC C18, 5 μm,
b. and (3) analyzing the column: 150 μm i.d.. times.150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm,
c. mobile phase A: 0.1% formic acid, 2% ACN;
d. mobile phase B: 0.1% formic acid, 80% ACN;
e. flow rate: 600 nL/min;
f. analysis time for each component: 78 min;
the mass spectrometry conditions were as follows:
A. primary mass spectrum parameters:
Resolution：70，000
AGCtarget：3e6
MaximumIT：40ms
Scanrange：350to1800m/z
B. secondary mass spectrum parameters:
Resolution：75，000
AGCtarget：1e5
MaximumIT：60ms
TopN：20
NCE/steppedNCE：27
the results are shown in Table 9, and 24 novel peptide fragments were identified from the P1 fraction. No peptide fragment identical to the peptide fragment of the present invention was found by comparison of protein database BLAST and polypeptide database PIPOPEP.
TABLE 9 immunoactive peptide sequences identified by liquid chromatography-tandem mass spectrometry combined with De novo sequencing analysis
In the description of the present invention, it is to be understood that the terms "first", "second", and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to imply that the number of technical features indicated are in fact significant. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless otherwise specified.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "a specific embodiment," "an example" or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (1)
1. The embelia laeta immunoactive peptide is characterized by comprising: TGGWSLK, WKPW, FLTMEPR, VWLVK, KVVL, MVVKK, LTGLNKL, RLLVWELER, KLSLEWLK, FVKLL, LGDLGTKL, LTGLDKL, LFAEMDK, EADGTLR, VVFK, TLLNPR, AYLTGLK, WLPDVK, VLWLPR, RQVWEDR, TVLLPR, LVRFPK, TLLFGDK, WSELVK24 peptide segments;
the preparation method of the plukenetia volubilis linneo immune active peptide comprises the following steps:
crushing the wisteria sinensis pulp to obtain wisteria sinensis pulp powder;
placing the Plukenetia volubilis linneo pulp powder in a 24L extraction kettle of supercritical extraction equipment, and extracting at 20MPa and 32 deg.C for 120min, wherein the extraction time is CO2The flow rate of (2) is 60 mL/min;
discarding the extracted mei teng fruit oil, taking out mei teng fruit cake meal, adding a certain amount of first-grade water, performing ultrasonic treatment for 15min, stirring and extracting at constant temperature for a certain time, and centrifuging at the speed of 4000 revolutions per minute for 10min to obtain a supernatant;
putting the supernatant into distilled water, dialyzing for 72 hours at the temperature of 4 ℃, performing rotary evaporation in a rotary evaporator for a certain time, and performing vacuum freeze drying to obtain freeze-dried albumin;
hydrolyzing the albumin to obtain albumin hydrolysate;
carrying out ultrafiltration treatment and separation and purification treatment on the albumin hydrolysate;
collecting the polypeptide solution;
the extraction conditions of the albumin are as follows: the material-liquid ratio is 1:25g/mL, the extraction temperature is 40 ℃, and the extraction time is 5 h;
(ii) hydrolyzing the albumin with trypsin;
the hydrolysis conditions when trypsin is used for hydrolysis are as follows: the enzyme adding amount is 7000U/g, the reaction pH is 7.0, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 4 h;
carrying out ultrafiltration treatment on the albumin hydrolysate by adopting an ultrafiltration membrane with the molecular weight of 3 kDa;
performing ultrasonic treatment on albumin hydrolysate with molecular weight less than 3kDa after ultrafiltration for 15min, separating and purifying, eluting with distilled water with flow rate of 3mL/min, and collecting polypeptide solution eluted for 13-29 min;
concentrating the collected polypeptide solution;
separating and purifying the albumin hydrolysate by using a Sephadex G-25 Sephadex column;
during the ultrafiltration treatment, the pressure was increased with nitrogen gas to maintain the pressure at 0.2MPa, and the obtained polypeptide solution was placed on ice to secure its activity.
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