CN116284228A - Method for separating and purifying Sanxia peptide - Google Patents

Abstract

The invention discloses a separation and purification method of Sanxia peptide, which is separated from penicillium oxalicum SG-4 fermentation liquor through steps of fermentation, filtration, ion exchange, rotary evaporation concentration, ethanol desalination and the like, and can be mainly applied to inhibition of post-harvest saprophyticus digitatus of citrus and post-harvest preservation of the citrus. The separation method disclosed by the invention achieves a moderate separation level, removes a large amount of mycelium, pigment and polysaccharide, improves the Sanxia peptide which is only 0.5 g// L in fermentation liquor to 0.897g/L, improves the yield by 79.4%, and can reach 92.9178% at the highest, and the residual sugar content is only 0.28 g/L.

Description

Method for separating and purifying Sanxia peptide

Technical Field

The invention belongs to the technical field of separation and purification of microbial fermentation products, and particularly relates to a separation and purification method of Sanxia peptide.

Background

The Sanxiapeptin is a novel linear antibacterial pentapeptide obtained by separating from a fermentation broth of penicillium oxalicum (Penicillum oxalicum) SG-4, and the specific amino acid composition of the novel linear antibacterial pentapeptide is as follows: abu-N-Me-Thr-Thr-N-Me-Val-Ser molecular formula: c (C) ₂₂ H ₄₁ N ₅ O ₉ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 519.61. the Sanxia peptide can obviously inhibit the growth of post-harvest saprophytic bacteria penicillium digitatum of fruits such as oranges, grapes and the like in vivo or in vitro, has no toxic or side effect on human bodies and environment, has stable effect, can obviously improve the shelf life of citrus fruits, and is a potential fruit preservation bacteriostat. However, in the fermentation process of the penicillium oxalate SG-4, besides the Triisthmus peptide, a large amount of pigments, polysaccharides and other impurities are generated, and if the Triisthmus peptide cannot be separated from the impurities, the popularization and the application of the Triisthmus peptide are severely limited. At present, the physicochemical properties of the Sanxia peptide are not clear, so that the determination of the physicochemical properties of the Sanxia peptide and the selection of a proper separation and purification method become the urgent problem to be solved.

The ion exchange chromatography has the advantages of no toxicity, repeated regeneration and use, simple process, low energy consumption, easy automation, less three wastes generated in the production process, high yield and the like, and is one of the most commonly used separation and purification methods at present. Meanwhile, since biomolecules are almost polar and have different charges at different pH values, ion exchange chromatography is widely used in purification methods of proteins, peptides and other biological macromolecules.

Disclosure of Invention

The invention aims to provide a separation and purification method of Sanxia peptide, which is used for removing impurities such as pigment, polysaccharide and the like in penicillium oxalicum SG-4 fermentation liquor through ion exchange chromatography to obtain the Sanxia peptide with higher content.

The technical scheme adopted by the invention is as follows:

a method for separating and purifying Sanxia peptide comprises the following specific steps:

(1) Activating and fermenting the penicillium oxalicum SG-4 strain to obtain penicillium oxalicum SG-4 fermentation liquor;

(2) Filtering mycelium in the fermentation broth obtained in the step (1) to obtain fermentation filtrate;

(3) Pretreatment of ion exchange resin: filling about 60% of resin into a column by adopting a wet method, washing with water, washing with acid and alkali alternately, keeping the resin neutral by washing with water, and then introducing corresponding buffer solution to obtain a balanced chromatographic column;

(4) Adjusting the pH value of the fermentation filtrate to be consistent with the pH value of the buffer solution, then loading 2-3bv of the fermentation filtrate into the chromatographic column obtained in the step (3) at the flow rate of 3-5bv/h, and stopping loading when the pH value of the effluent at the outlet is consistent with the pH value of the fermentation filtrate;

(5) After the sample is completely loaded, washing with 1-2bv of pure water, eluting with eluent, and collecting the eluent;

(6) Spin-evaporating the eluent to obtain concentrated solution, adding 2-5 times of absolute ethyl alcohol, centrifuging and desalting to obtain supernatant, spin-evaporating and concentrating the supernatant, and lyophilizing to obtain Sanxia peptide;

(7) Resin regeneration: washing the chromatographic column after the elution in the step (5) with 3% -5% HCl solution to be neutral to obtain a regenerated chromatographic column, wherein the regenerated chromatographic column can be reused in the step (3).

Preferably, the filtration in the step (2) is vacuum filtration or plate frame filtration.

Preferably, the ion exchange resin in the step (3) is an anion exchange resin or a cation exchange resin; further preferred anion exchange resins are D201 macroporous strongly basic anion exchange resins and cation exchange resins are 732 strong acid styrene cation exchange resins.

Preferably, the buffer according to step (3)NaAc-HAc solution with pH 3.2 or NaHCO with pH 10.0 ₃ -NaOH solution.

Preferably, the eluent in the step (5) is sodium chloride-buffer solution with the concentration of 0.05-0.9mol/L and the pH of the eluent is 3-10; further preferably, the cation exchange resin is eluted with 0.4-0.9mol/L sodium chloride-NaAc-Hac buffer, pH 7-10, and the anion exchange resin is eluted with 0.05-0.5mol/L sodium chloride-NaHCO ₃ NaOH buffer, pH 3-5.

Advantageous effects

According to the invention, through research on basic properties of the three gorge peptide, the three gorge peptide is found to have good temperature stability and can be kept stable at 90 ℃; at the same time, the Sanxia peptide is slightly soluble in absolute ethanol, and can maintain its biological activity under strong acid (pH 3.2-4) and strong alkali (pH 9-10). Therefore, the high-purity Sanxia peptide finished product with the content of 0.897g/L and the yield of 92.9178% can be obtained by separating and purifying by using strong-alkaline anion exchange resin or strong-acid cation exchange resin and then desalting by using absolute ethyl alcohol. In addition, the separation and purification of the Sanxia peptide is carried out by combining ion exchange chromatography and ethanol desalination, and the method has the advantages of simple operation, low cost, high automation degree, easy popularization and the like.

Drawings

FIG. 1 is a chemical structure of Sanxia peptide;

FIG. 2 is a three isthmus peptide concentrate before and after the alkali treatment in example 3, wherein A is before the alkali addition and B is after the alkali addition;

FIG. 3 is a schematic illustration of the Sanxia peptide concentrate of example 5 before and after centrifugation with ethanol, wherein A is before centrifugation and B is after centrifugation;

FIG. 4 is a plot and bar graph of the effect of pH, elution buffer and sodium chloride concentration on the adsorption and elution rates of Triisthmus peptides for various loading fermentation filtrate pH's in examples 6-8; wherein a is the plot of the effect of pH on the adsorption rate of the loaded fermentation filtrate, B is the plot of the effect of pH on the elution rate of the elution buffer, C is the histogram of the effect of sodium chloride concentration in the buffer on the elution rate, representing a significant difference at p <0.05 level.

FIG. 5 is a plot and bar graph of the effect of different loading fermentation filtrates, elution buffer pH, sodium chloride concentration, and flow rate on the adsorption and elution rates of Triisthmetin in examples 9-12; wherein A is a line graph of the influence of the pH of the sample fermentation filtrate on the adsorption rate, B is a line graph of the influence of the pH of the elution buffer on the elution rate, C is a bar graph of the influence of the concentration of sodium chloride in the buffer on the elution rate, and D is a bar graph of the influence of the sample flow rate and the elution flow rate on the elution rate; * Indicating significant differences, which are shown to be very significant.

FIG. 6 is an ion exchange chromatography diagram in example 13.

Detailed Description

The data in the examples were all detected by hplc, the chromatograph selected was the shimadzu hplc uv-vis (SPD-16), C18 liquid column (neptene C18 x 4.6,5 μm), purchased from shimadzu instruments, inc, under the following chromatographic conditions:

a detector: a DAD detector;

chromatographic column: neptene C18 x 4.6,5 μm;

detection wavelength: 210nm;

sample injection volume: 50. Mu.L;

flow rate: 1.0mL/min;

the detection method comprises the following steps: mobile phase a:850mL of purified water+1.2 g of sodium dihydrogen phosphate+0.1% of phosphoric acid were dissolved and filtered, and 150mL of chromatographic methanol was added and mixed well. Mobile phase B: acetonitrile; the residence time and the proportion of mobile phase are shown in Table 1:

TABLE 1 ratio of mobile phases A and B at different times

EXAMPLE 1 cultivation of Penicillium oxalicum SG-4 fermentation broth

(1) Medium configuration: 200g/L of potato, 20g/L of glucose and 20g/L of agar (agar is not added if the potato is a liquid culture medium), and sterilizing at 121 ℃ for 20min to obtain the PDA culture medium for standby.

(2) Strain activation: activating the original strain of penicillium oxalicum SG-4 on a PDA flat-plate culture medium, and culturing for 3-5d at 28 ℃ to obtain a strain which has proper colony size, single phenotype, white hyphae and shorter filaments and generates a large amount of grayish green spores on the flat-plate for later use.

(3) Seed liquid culture: the activated strain is selected and inoculated in 200mL PDA liquid culture medium, and cultured for 5d at 28 ℃ under the condition of 120rpm/min, thus obtaining seed liquid.

(4) Fermentation culture: and (3) inoculating all the seed liquid obtained in the step (3) into a fermentation tank containing 10L of culture medium, and culturing for 4d at 28 ℃ and 80rpm/min to obtain penicillium oxalicum SG-4 fermentation liquid.

Example 2 Effect of temperature on isolation and purification of Triisthmidin

In order to explore the effect of temperature on the content of Sanxia peptide, the following experiments were designed, the specific steps were as follows:

(1) Filtering the penicillium oxalate SG-4 fermentation liquor by using a small plate frame filter, discarding mycelium, collecting fermentation filtrate, adding equal volume of absolute ethyl alcohol, uniformly mixing, centrifuging at 10000rpm/min for 5 minutes, taking out, removing inactive impurity precipitate, performing rotary evaporation and concentration on supernatant fluid to obtain concentrated solution, and freeze-drying the concentrated solution to obtain a crude product of the Sanxia peptide; wherein the pressure P of the plate-frame filtration is 0.3MPa, and the filter membrane is 0.45 mu m;

(2) 10mg of the crude Sanxia peptide is dissolved in 10mL of pure water, and the mixture is heated to 60 ℃, 70 ℃, 80 ℃ and 90 ℃ respectively to carry out high performance liquid chromatography detection (sample injection amount is 75 mu L).

TABLE 2 Effect of different temperatures on Sanxia-peptide

The data in Table 2 shows that as the temperature increases from 60℃ to 90℃, the amount of Triisthmus peptide increases, which may be the result of moisture volatilization during heating, resulting in a slight increase in the concentration of Triisthmus peptide with increasing temperature, but the structure of Triisthmus peptide is not destroyed, indicating that the temperature stability of Triisthmus peptide is better.

EXAMPLE 3 investigation of alkali resistance properties of Triisthmetin

(1) 1mL of the concentrate from example 2 was taken in a 2mL EP tube;

(2) Adding 1mL of 4mol/L sodium hydroxide solution, mixing, centrifuging at 10000rpm/min for 5 min, taking out, and separating to obtain supernatant and the content of Sanxia peptide in precipitate (for use);

(3) The supernatant and the pellet were each suspended in 1mL of pure water, and the content of the Sanxins therein was measured.

TABLE 3 variation of Triisthmidin content before and after alkali addition

Sequence number	Substance (B)	Peak area	Dilution factor	Sample injection amount (mu L)	Content g/L
						1	Adding alkali supernatant into the concentrated solution	16674	1.0	10	2.937
2	Adding alkali to the concentrated solution for precipitation	3829.4	1.0	10	0.674
						3	Concentrated solution	17416.8	1.0	5	6.135

As shown in FIG. 2, a large amount of flocculent precipitate was generated after adding sodium hydroxide to the concentrate.

As shown in table 3, the content of the tricin in the supernatant was significantly more than that in the precipitate, and the content of the tricin in the supernatant was essentially half of the concentrate, which was equivalent to doubling the dilution of the tricin concentrate by adding the same volume of sodium hydroxide during the experiment, thus there was little loss of tricin before and after the addition of base, indicating that the tricin was able to maintain its bioactivity (only content) under strong alkaline conditions; meanwhile, the pH of the fermentation liquor is about 3.98, so that the tricisthmus peptide is relatively stable in a strong acid environment, and has acid and alkali resistance, so that the tricisthmus peptide can be separated and purified by using strong acid cation exchange resin or strong alkali anion exchange resin.

EXAMPLE 4 study of the solubility of Triisthmetin in ethanol

In order to explore the solubility of the three isthmuses peptide in ethanol solution, the following experiments were designed, the specific steps were as follows:

(1) Filtering the penicillium oxalate SG-4 fermentation liquor, and concentrating by using a rotary evaporator to obtain a three isthmus peptide concentrated solution, wherein the volume ratio of the concentrated solution before concentration to the concentrated solution after concentration is 10:1, a step of;

(2) Performing chromatographic detection on the three gorge peptide concentrated solution obtained in the step (1), wherein the detected peak area is 9680, and determining the content of the three gorge peptide to be 4.3g/L;

(3) Taking 1-7mL of the Sanxia peptide concentrated solution, supplementing the concentrated solution to 10mL by using absolute ethyl alcohol, refrigerating the concentrated solution for 24 hours, centrifuging the concentrated solution at 10000rpm/min, centrifuging the concentrated solution for 5 minutes to keep supernatant, measuring the actual content of the Sanxia peptide in the supernatant by using a high performance liquid chromatography, and calculating the theoretical total content and yield of the Sanxia peptide.

Wherein the theoretical total content= [ concentrate volume/(concentrate volume+absolute ethanol volume) ]x4.3;

yield = actual content/theoretical content x 100%.

TABLE 4 solubility, content and yield changes of Sanxia peptide at different ethanol concentrations

	A	B	C	D	E	F	G
								concentrate/mL	7	6	5	4	3	2	1
Absolute ethanol/mL	3	4	5	6	7	8	9
								Ethanol concentration	30％	40％	50％	60％	70％	80％	90％
Actual content/mg	2.923	2.500	1.045	0.543	0.165	0.1	0.041
								Theoretical total content/mg	3.01	2.58	2.15	1.72	1.29	0.86	0.43
Yield is good	97.1％	96.89％	48.6％	31.6％	12.8％	11.6％	9.5％

As can be seen from Table 4, as the concentration of ethanol in the solution gradually increased, the yield of Triisthmuses gradually decreased, with the yield of Triisthmuses being 9.5% at 90% and 97.1% at 30%.

Indicating that the solubility of the three isthmuses peptide is different at different concentrations of ethanol; wherein the solubility of 30% ethanol is highest, the solubility of 90% ethanol is lowest, and the yield of the Sanxia peptide of 30% ethanol is highest. The method shows that the three gorge peptide can be recovered by an ethanol precipitation method according to different solubilities of the three gorge peptide in ethanol with different concentrations, so as to achieve the purpose of desalting.

EXAMPLE 5 desalination Studies Using absolute ethanol

As can be seen from example 4, the solubility of the tricin decreases significantly with the increase of the ethanol concentration, so that the sodium chloride in the eluent can be removed by using absolute ethanol after the process of separating and purifying the tricin by using ion exchange resin, and the purpose of desalting can be achieved as follows:

(1) Adjusting the pH value of the fermentation filtrate of the penicillium oxalate SG-4 to be consistent with the pH value of the buffer solution, and then loading the sample into an ion exchange column which is balanced, wherein the loading flow rate is 4bv/h;

(2) After the sample injection is finished, washing with 1bv pure water, eluting with 0.5mol/L sodium chloride-buffer solution, and collecting the eluting clear liquid;

(3) Collecting 3L of eluent, concentrating to 200mL by using a rotary evaporator to obtain concentrated solution;

(4) Taking 1mL of the concentrated solution obtained in the step (3) into a 4mL EP tube, adding 3mL of absolute ethyl alcohol, uniformly mixing, centrifuging at 10000rpm/min for 5 minutes, taking out to obtain supernatant, and measuring the content of the Sanxia peptide in the supernatant.

(5) Taking 2.5L of the eluent obtained in the step (2), concentrating the eluent in a rotating way to 250mL to obtain a solid-liquid mixture, carrying out suction filtration and separation on the solid-liquid mixture to obtain solid and concentrated solution, and weighing the weight of the solid;

(6) Mixing the concentrated solution obtained in the step (5) with absolute ethyl alcohol according to the following ratio of 1:3, mixing evenly, centrifuging, pouring out the upper liquid, weighing the sediment together with the EP pipes (weighing the EP pipes in advance), recording the result, calculating the total sediment net weight, and calculating the desalination rate.

TABLE 5 content variation before and after ethanol addition

Sequence number	Substance (B)	Peak area	Dilution factor	Sample injection amount (mu L)	Content g/L
						1	Concentrated solution	17416.8	1.0	5	6.135
2	Supernatant of the concentrated solution added with absolute ethyl alcohol	9026.7	4.0	10	1.590

Note that: and (3) concentrating: absolute ethanol=1:3, and the content of the three isthmuses was measured with a high performance liquid phase, and from the fact that the liquid phase content of the concentrate was four times that of the supernatant, it was found that the content of the three isthmuses was not lost before and after desalting.

Discussion of results: as can be seen from FIG. 3, the solution turned light pink after the addition of absolute ethanol (FIG. 3A), and the precipitation and supernatant separation after centrifugation (FIG. 3B) demonstrated that the impurities such as sodium chloride in the concentrated Sanxia peptide concentrate after elution could be removed by using absolute ethanol.

As can be seen from Table 5, the content of the Sanxia peptide is about 1.590g/L, which is 1/4 of the content (6.135 g/L) of the concentrated solution, after the absolute ethyl alcohol (concentrated solution: absolute ethyl alcohol=1:3) is added, the salt removal effect is good, and almost no loss is caused, so that the loss of the Sanxia peptide can be reduced to the greatest extent by adopting the absolute ethyl alcohol for salt removal.

Effect of ethanol desalination (precipitation of concentrate plus absolute ethanol): calculated, 2.5L of eluent approximately contains 73.1250g of sodium chloride, and sodium chloride solid is separated out by rotary evaporation operation, and the weight is 22.2535g; the total net weight of the sediment is 39.1483g, and the actual desalination rate of the ethanol sediment is 76.96 percent, so that most sodium chloride in the concentrated solution can be removed by using absolute ethanol, and the loss of the Sanxia peptide is reduced.

Theoretical salt content of eluent m=nm=0.5×58.5×2.5= 73.1250g

Actual desalination rate of ethanol = total net sediment weight/(theoretical salt content of eluent-actual desalination by rotary evaporation)

The method comprises the following steps: 39.1483/(73.1250-22.2535) =76.96%.

EXAMPLE 6 influence of sample pH on adsorption Rate of anion exchange resin

(1) Preparing a buffer solution: preparation of 20mmol/L NaHCO ₃ 1L of the solution to be used, and adjusting the pH to 10.0+/-0.05 by using a small amount of 4% NaOH;

(2) Resin filling: filling the column by adopting a wet method, filling D201 macroporous strong-alkali anion exchange resin into the resin column, wherein the filling amount is about 60 percent of the column, and washing the resin by deionized water until alcohol substances (methanol or ethanol) on the resin are washed clean and have no ethanol taste;

(3) Balance column: the resin layer was passed through with 4% HCl solution, 4% NaOH solution, 4% HCl solution at a rate of 2bv/h, respectively, with a throughput of 2bv, and then washed with deionized water until the pH was nearly neutral; pouring out a large amount of water, sucking out a small amount of water by using a suction pipe, and introducing a proper amount of buffer solution to adjust the pH of the chromatographic column to 6.2;

(4) Pretreatment of fermentation liquor: the fermentation broth of Penicillium oxalate SG-4 obtained in example 1 was passed through a plate and frame filter to obtain a mycelium-free fermentation filtrate, wherein the pressure P was 0.3MPa and the pore size of the filter membrane was 0.45. Mu.m.

(5) Loading: the pH of the fermentation filtrate is respectively regulated to 3.2, 4, 5, 6.2, 7, 8, 9 and 10 by 4 percent sodium hydroxide, and then the fermentation filtrate is pumped into a chromatographic column containing anion exchange resin by a constant flow pump, and the loading flow rate is 4bv/h; and stopping loading when the pH of the liquid flowing out of the outlet is close to the pH of the fermentation filtrate.

(6) Eluting: after the sample introduction, the sample was rinsed with 1bv of pure water, followed by a sodium chloride-NaHCO reaction at pH 10,0.5mol/L ₃ NaOH buffer as eluent (1L of corresponding buffer is formulated, thenAdding proper amount of sodium chloride to make its concentration reach 0.5mol/L for eluting with an eluting volume of 3-4bv (the volume of the mixture of sodium chloride and buffer solution), collecting the eluting clear liquid and measuring the liquid phase content, and calculating the adsorption rate and the eluting rate.

As shown in fig. 4 a, the pH of the loading (i.e., the fermentation filtrate) had a large effect on the elution of the sanxiaopeptide, and the adsorption rate of the anion exchange resin to the sanxiaopeptide was significantly increased when the pH of the fermentation filtrate was only gradually increased from 3.2 to 10 without changing other conditions, wherein the adsorption rate was highest when the pH of the fermentation filtrate was 10.

EXAMPLE 7 Effect of elution pH on anion exchange resin elution Rate

The procedure was as in example 6, the pH of the sample (i.e., fermentation filtrate) was maintained at 10, elution pH was set to 3.2, 4, 5, 6.2, 7, 8, 9, 10, and the content of Sanxia peptide in the eluate was measured after elution, and the adsorption rate was calculated.

As a result, as shown in fig. 4B, the pH of the eluent had a great influence on the elution of the tricin, and when other conditions were unchanged, the elution rate of the tricin was gradually increased and then gradually decreased only when the pH of the eluent was gradually increased from 3.2 to 10, wherein the elution rate was highest when the pH of the eluent was 4.

EXAMPLE 8 Effect of eluent salt concentration on anion exchange resin elution Rate

The procedure was as in example 6, wherein the pH of the sample (i.e., the fermentation filtrate) was maintained at 10, the elution pH was maintained at 4, and the concentration of sodium chloride in the eluate was set to 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9mol/L, respectively, and the content of Sanxins in the eluate was detected after elution, and the elution rate was calculated.

As shown in fig. 4C, the concentration of sodium chloride in the eluate had a greater effect on the elution of sanxiatins; when other conditions are unchanged, the concentration of the eluent is gradually increased from 0.05mol/L to 0.9mol/L, and the elution rate of the Sanxiaopeptide shows a tendency of rising and then falling, wherein the elution rate is the highest when the concentration of sodium chloride in the eluent is 0.1mol/L, and the difference from other concentrations is obvious.

Based on the results of examples 6-8, the separation of the Sanxins was performed using anion exchange resin, the pH of the sample (i.e., the fermentation filtrate) was 10, the pH of the eluate was 4, and the concentration of sodium chloride in the eluate was 0.1mol/L, which gave the best results for the separation and purification of Sanxins. The SG-4 fermentation filtrate is separated and purified under the condition, so that the Sanxia peptide eluent with the content of 0.384g/L can be obtained, and the Sanxia peptide product with the content of 5.892g/L can be obtained through rotary evaporation concentration, ethanol desalination and freeze-drying, and the yield is 92.9178%.

EXAMPLE 9 influence of sample pH on adsorption Rate of cation exchange resin

(1) Preparing a buffer solution: preparing 1L of 20mmol/L NaAc solution for later use, and regulating the pH to 3.2+/-0.05 by using a small amount of dilute HAc solution (1 mL of glacial acetic acid is taken, 9mL of pure water is added and the mixture is uniformly shaken);

(2) Resin filling: filling 60% 732 strong acid styrene cation exchange resin by wet method, washing the resin with deionized water until alcohol substances (methanol or ethanol) on the resin are washed clean and have no ethanol smell, treating the equilibrium column with acid and alkali alternately, and finally regulating the pH of the chromatographic column to 4 with buffer solution;

(3) The pH of the fermentation filtrate is respectively adjusted to 3.2, 4, 5, 6.2, 7, 8, 9 and 10 by 4% hydrochloric acid, and then the fermentation filtrate is loaded at the speed of 4bv/h; and stopping loading when the pH of the liquid flowing out of the outlet is close to the pH of the fermentation filtrate.

(6) After the sample was applied, the sample was rinsed with 1bv of pure water, and eluted with a sodium chloride-buffer solution having a pH of 3.2 and 0.5mol/L as an eluent, and the eluate was collected, and the content of Sanxia peptide was measured and the adsorption rate and elution rate were calculated.

As shown in FIG. 5A, the pH of the loading (i.e., fermentation filtrate) had a large effect on the adsorption rate of the Sanxia peptide on the cation exchange resin. The adsorption rate of the resin column to the Sanxia peptide was significantly reduced when other conditions were unchanged, only when the pH of the fermentation filtrate was gradually increased from 3.2 to 10, with the adsorption rate being highest when the pH of the fermentation filtrate was 3.2.

EXAMPLE 10 Effect of elution pH on elution Rate of cation exchange resin

The procedure was as in example 9, maintaining the pH of the sample (i.e., fermentation filtrate) at 3.2, setting the elution pH at 3.2, 4, 5, 6.2, 7, 8, 9, 10, and detecting the content of Sanxia peptide in the eluate after elution, and calculating the adsorption rate.

As shown in fig. 5B, the pH of the eluate had a greater effect on the elution of the sanxianin. When other conditions are unchanged, only the pH value of the eluent is gradually increased from 3.2 to 10, the elution rate of the Sanxia peptide is obviously increased and then slowly decreased, wherein the elution rate is highest when the pH value of the eluent is 9.

EXAMPLE 11 Effect of eluent salt concentration on cation exchange resin elution Rate

The procedure was as in example 9, wherein the pH of the sample (i.e., the fermentation filtrate) was maintained at 3.2, the elution pH was maintained at 10, and the concentration of sodium chloride in the eluate was set to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9mol/L, respectively, and the content of Sanxia peptide in the eluate was detected after elution, and the elution rate was calculated.

As shown in FIG. 5C, the concentration of sodium chloride in the eluate had a large effect on the elution of the Sanxia peptide. When other conditions are unchanged, the concentration of the eluent is gradually increased from 0.1mol/L to 0.9mol/L, and the elution rate of the Sanxiaopeptide shows a tendency of rising and then falling, wherein the elution rate is the highest when the concentration of sodium chloride in the eluent is 0.5mol/L, and the difference from other concentrations is obvious.

EXAMPLE 12 Effect of loading flow Rate and elution flow Rate on cation exchange resin elution Rate

The procedure was as in example 9, wherein the pH of the sample (i.e., the fermentation filtrate) was maintained at 3.2, the elution pH was maintained at 10, the sodium chloride concentration in the eluate was maintained at 0.5mol/L, the sample was fed and eluted at 2, 4, 6, 8bv/h, respectively, and the content of Sanxia peptide in the eluate was detected after the elution, and the elution rate was calculated.

As a result, as shown in FIG. 5D, the loading flow rate and the elution flow rate have a large influence on the elution of the Sanxia peptide. When other conditions are kept unchanged and the loading flow rate and the elution flow rate are changed, the adsorption rate and the elution rate of the resin chromatographic column to the Sanxia peptide are changed obviously, when the flow rate is gradually increased from 2bv/h to 8bv/h, the adsorption rate and the elution rate are reduced gradually, wherein when the flow rate is 2bv/h and 4bv/h, no obvious difference exists between the adsorption rate and the elution rate, and when the flow rate is increased to 6bv/h, the adsorption rate and the elution rate are reduced obviously. (D of FIG. 5).

By combining the results of examples 9-12, when the separation of the Sanxia peptide was performed using cation exchange resin, the pH of the fermentation filtrate was 3.2, the pH of the eluent was 9, the sodium chloride concentration of the eluent was 0.5mol/L, and the separation and purification effects on the Sanxia peptide were best when the loading flow rate and the elution flow rate were 2 bv/h. The SG-4 fermentation filtrate is separated and purified under the condition, so that the Sanxia peptide eluent with the content of 0.267g/L can be obtained, and the Sanxia peptide product with the content of 5.628g/L can be obtained through rotary evaporation concentration, ethanol desalination and freeze-drying, and the yield is 88.4182%.

EXAMPLE 13 study of the elution period of Triisthmidin

The method steps are the same as in example 9, wherein the flow rate during loading and elution is 4bv/h, the elution buffer is 0.5mol/L sodium chloride solution, the pH value is 7, the sample injection amount is 1L, and the total elution amount is about 640mL.

As can be seen from FIG. 6, the elution of the Sanxia peptide is mainly divided into three phases, i.e., an earlier elution phase, an intermediate elution phase and a later elution phase, wherein the earlier elution phase has a smaller peak form, the time range is 50-100min, the peak form of the elution phase is larger and bilaterally symmetrical, the time range is 100-150min, the peak form of the absorbance of the later elution phase is continuously reduced, and the time range is 150-190min. Samples were taken during different elution periods and the fermentation and effluent were taken to measure the content of Sanxia peptide, respectively.

TABLE 6 Triisthmetin content and yield during each elution period

Yield = (content of elution period x elution volume)/(fermentation broth content x fermentation broth volume) in the table

=elution phase M/fermentation broth M

The fermentation liquor enters an ion exchange chromatographic column, and the liquid flowing out from the lower part is called effluent liquid, and can be collected for liquid phase detection. As can be seen from Table 8, the content of the three gorge peptides in the different elution periods is inconsistent, wherein the content of the three gorge peptides in the middle elution period is 0.897g/L, which is 5.6 times higher than that in the early elution period and 2 times higher than that in the later elution period, which means that the elution rate of the three gorge peptides in the middle elution period is highest, and the content of the three gorge peptides in the later elution period is obviously reduced because most of the three gorge peptides are eluted in the later elution period, so that the elution time of the three gorge peptides is kept at about 150-190min. In addition, the content of the three gorge peptide is obviously improved compared with the content in the fermentation liquor after elution, which indicates that the ion exchange resin can effectively separate and purify the three gorge peptide, and meanwhile, the content of the three gorge peptide in the effluent is almost 0, which indicates that the ion exchange resin is adopted to separate and purify the three gorge peptide, so that the loss of the three gorge peptide can be reduced to the greatest extent, and the waste is avoided.

Example 14 determination of total sugar content in Triisthmetin

Taking 1.0mL of the original fermentation liquor, 1.0mL of the ion exchange effluent and 1.0mL of the eluent, placing the original fermentation liquor, the ion exchange effluent and the eluent into a 25mL volumetric flask, adding 5.0mL of 4mol/L HCl into a boiling water bath kettle to hydrolyze for 10min, cooling, diluting to a scale with purified water, and shaking uniformly. Then, the DNS reagent (18.2 g of potassium sodium tartrate, which is dissolved in 50mL of distilled water, is added into a test tube with the volume of 0.5mL to 180mm multiplied by 200mm, heated, 0.63g of 3, 5-dinitrosalicylic acid, 2.1g of sodium hydroxide and 0.5g of phenol are sequentially added into the hot solution, stirred until the solution is dissolved, cooled, then the solution is subjected to constant volume to 100mL by distilled water, stored in a brown bottle, stored at room temperature) and 2mL of the solution are taken out, heated in boiling water for 2 to 3min, taken out, cooled, added with 9mL of water, shaken well, and the absorbance A is measured at a wavelength of 540 nm.

Blank control: 1.0mL of purified water was taken in a 25mL volumetric flask, and the absorbance A was measured after the treatment by the above-mentioned method.

Standard solution: the absorbance A was measured after the treatment by the above method in a 25mL volumetric flask containing 1.0mL of 3.00% glucose standard solution.

And (3) calculating:

wherein: 3.00% -concentration of standard glucose solution for total sugar determination;

sample A, sample liquid absorbance;

standard a-standard liquid absorption.

TABLE 7 comparison of absorbance of different solutions

Sequence number	Substance name	Corresponding absorbance
			1	Blank control	Zero return after initial 0.222
2	3% standard glucose solution	0.320
			3	The fermentation broth is as such	0.285
4	Ion exchange effluent	0.274
			5	Eluent (eluent)	0.003

Discussion of results:

total sugar in broth as received = 3% × (0.285/0.320) = 0.0267, i.e. 2.67% (26.7 g/L);

total sugar in the eluate = 3% × (0.003/0.320) = 0.00028, i.e. 0.028% (0.28 g/L).

After ion exchange, the total sugar in the solution is obviously reduced from 26.7g/L to 0.28g/L, which indicates that the ion exchange method can effectively reduce the residual sugar in the fermentation broth, is convenient for separating the Sanxia peptide and improves the purity of the Sanxia peptide.

Comparative example 1 Ultrafiltration to obtain Triisthmetin

(1) Filtering the penicillium oxalate SG-4 fermentation liquor to remove most of thalli, and then performing filter pressing by using a plate-frame filter to obtain fermentation filtrate without mycelium, namely a standard substance, wherein the pressure of plate-frame filtration is 0.3MPa, and the aperture of a filter membrane is 0.45 mu m; sampling to obtain SG-4 fermentation stock solution, SG-4 filtrate and SG-4 pressure filtrate respectively;

(2) Centrifuging the fermentation filtrate obtained in the step (1) at 25 ℃ for 30min after passing through an ultrafiltration column, wherein the centrifugation speed is 5000rpm/min, concentrating and sampling to obtain ultrafiltration permeate, and performing rotary evaporation and concentration on the ultrafiltration permeate to obtain concentrated solution-1; wherein the ultrafiltration column adopts 2540 multifunctional roll type membrane, the equipment model is equipment model RNF-2500, the membrane core is SMU-110, and Milli-Q water is used for pre-cleaning;

(3) Adding Tris-HCl buffer solution, repeatedly performing for 3 times in an ultrafiltration column, and respectively sampling to obtain an ultrafiltration liquid primary water adding sample and an ultrafiltration liquid secondary water adding sample;

(5) Pouring out the water in the ultrafiltration tube, slightly rinsing the tube with milliQ water for several times, if the tube bottom has visible protein sediment, adding the water, then slightly blowing with a gun head, and then pouring out. Then adding 0.2M NaOH solution, standing at room temperature for 20min, washing with MilliQ water, and preserving at 4deg.C until the next use;

(6) Respectively carrying out rotary evaporation concentration on the obtained ultrafiltration primary water adding sample and ultrafiltration secondary water adding sample until the volume is 1/10 of the original volume, and collecting to obtain concentrated solution-2 and concentrated solution-3;

(7) All the samples were subjected to high performance liquid chromatography and the content of the Sanxia peptide was measured, and the results are shown in Table 8:

table 8 isolation and purification of Triisthmetin by ultrafiltration

Type of solution	Peak of liquid phase	Dilution factor	Sample injection amount (mu L)	Content (g/L)
					SG-4 fermentation stock solution	10795.7	1.0	75	0.267
SG-4 filtrate	8763	1.0	30	0.542
					SG-4 filter pressing liquid	9380.7	1.0	75	0.232
SG-4 ultrafiltration permeate	5229.1	1.0	75	0.129
					Concentrating-1	17257	1.0	75	0.427
SG-4 ultrafiltration once water addition	7657.1	1.0	75	0.189
					SG-4 ultrafiltration secondary water addition	6370.8	1.0	75	0.157
SG-4 concentrated solution-2	10093.5	1.0	75	0.250
					SG-4 concentrated solution-3	3106	1.0	30	0.192

The results show (Table 8) that the content of Triisthmus peptide in SG-4 fermentation stock solution was about 0.267g/L, and that the filter press had little effect on the content of Triisthmus peptide in the fermentation solution, but that filtration could increase the content thereof, which may be a result of filtering out more water and large impurities, so that the content of Triisthmus peptide therein was increased. The content of the three gorge peptide is reduced to 0.129g/L after ultrafiltration, which is probably because the ultrafiltration cut-off molecular weight is above 1000, the molecular weight of the three gorge peptide is smaller, the ultrafiltration effect is poor, but the content of the three gorge peptide is obviously increased after concentration; the sample after ultrafiltration primary water addition and secondary water addition also contains a certain amount of the three gorge peptide, but the content of the three gorge peptide after concentration is still about 0.2g/L, the concentration is lower, and compared with the three gorge peptide with the content of 0.897g/L obtained by an ion exchange method, the three gorge peptide is low in content and large in loss when the separation and purification of the three gorge peptide are carried out by adopting the ultrafiltration method, and the industrial requirement is not met.

Comparative example 2 nanofiltration separation to obtain Triisthmidin

(1) Removing most of thalli from the penicillium oxalicum SG-4 fermentation liquor by suction filtration, and then performing filter pressing by using a plate-frame filter to obtain fermentation filtrate without mycelium, and sampling to obtain SG-4 fermentation stock solution, SG-4 filtrate and SG-4 pressure filtrate, wherein the pressure of the plate-frame filtration is 0.3MPa, and the pore diameter of a filter membrane is 0.45 mu m;

(2) The fermented filtrate obtained in the step (1) is put on a nanofiltration column and then subjected to interception treatment, and the sample is taken to obtain SG-4 nanofiltration permeate, wherein the nanofiltration column adopts 2540 multifunctional roll type membranes, the equipment model is RNF-2500, the membrane core is SMR-230 model, and MilliQ is used for pretreatment before use;

(3) Pouring out the water in the nanofiltration tube, slightly rinsing the tube with milliQ water for several times, if the tube bottom has visible protein sediment, adding water, then slightly blowing with a gun head, and then pouring out. Then adding 0.2M NaOH solution, standing at room temperature for 20min, washing with MilliQ water, and preserving at 4deg.C until the next use;

(4) Carrying out rotary evaporation concentration on the obtained SG-4 nanofiltration permeate until the volume is 1/10 of the original volume, and collecting the SG-4 nanofiltration concentrate;

the nanofiltration process mainly comprises the steps of treating fermentation liquor filtrate, and simultaneously, penetrating out small molecules with the molecular weight of less than 100 and other small molecular substances through active ingredients with the molecular weight of more than 100;

table 9 separation and purification of Triisthmidin by nanofiltration

Type of solution	Peak of liquid phase	Dilution factor	Sample injection amount (mu L)	Content (g/L)
					SG-4 fermentation stock solution	10795.7	1.0	75	0.267
SG-4 filtrate	8763	1.0	30	0.542
					SG-4 filter pressing liquid	9380.7	1.0	75	0.232
SG-4 nanofiltration permeate	61.7	1.0	75	0.002
					SG-4 concentrated solution	23419	1.0	75	0.579

The data in Table 9 show that the content of the Sanxia peptide in the permeate obtained after nanofiltration is extremely low and is only 0.002g/L, and 0.579g/L can be achieved after concentration, so that great pressure is brought to the subsequent concentration treatment, and the difference between the concentrated permeate and 0.897g/L of the purified Sanxia peptide is very obvious, so that the concentrated permeate is not suitable for separating and purifying the Sanxia peptide and is not suitable for popularization into industrial production.

Claims (10)

1. A method for separating and purifying Sanxia peptide is characterized in that: the method comprises the following steps:

(1) Fermenting with penicillium oxalicum SG-4, and filtering to obtain a fermentation filtrate containing Sanxia peptide;

(2) Loading the fermentation filtrate obtained in the step (1) to ion exchange resin, and eluting to obtain an eluate;

(3) Concentrating the eluate by rotary evaporation to obtain concentrated solution;

(4) Desalting the concentrated solution with ethanol, concentrating, and lyophilizing to obtain Sanxia peptide;

(5) And (3) regenerating the resin, wherein the regenerated resin is reused in the step (2).

2. The method for separating and purifying the three gorges peptide according to claim 1, wherein the method comprises the steps of: the filtering in the step (1) is vacuum filtration or plate frame filtration.

3. The method for separating and purifying the three gorges peptide according to claim 1, wherein the method comprises the steps of: the ion exchange resin in the step (2) is anion exchange resin or cation exchange resin; wherein the anion exchange resin is D201 macroporous strong alkaline anion exchange resin, and the cation exchange resin is 732 strong acid styrene cation exchange resin.

4. The method for separating and purifying sanxia peptide according to claim 3, wherein: the buffer solution used by the cation exchange resin is NaAc-Hac buffer solution with the pH value of 3.2; the buffer used for the anion exchange resin was NaHCO with a pH of 10.0 ₃ -NaOH buffer.

5. The method for separating and purifying the three gorges peptide according to claim 1, wherein the method comprises the steps of: and (3) before the fermentation filtrate in the step (2) is sampled, the pH value is adjusted to be consistent with the pH value of the buffer solution.

6. The method for separating and purifying the three gorges peptide according to claim 1, wherein the method comprises the steps of: the sample loading flow rate in the step (2) is 3-5bv/h, and the sample loading amount is 2-3bv; the loading was stopped when the pH at the ion exchange resin outlet was near the pH of the fermentation filtrate.

7. The method for separating and purifying the three gorges peptide according to claim 1, wherein the method comprises the steps of: the elution in the step (2) is firstly washed by 1-2bv of pure water, and then is eluted by 3-4bv of eluent.

8. The method for isolating and purifying sanxia peptide according to claim 7, wherein: the eluent is sodium chloride-buffer solution with the concentration of 0.05-0.9mol/L, and the pH value of the eluent is 3-10.

9. According to claim 8The method for separating and purifying the Sanxia peptide is characterized by comprising the following steps of: the eluent of the cation exchange resin is 0.4-0.9mol/L sodium chloride-NaAc-Hac buffer solution, and the pH value is 7-10; the eluent of the anion exchange resin is sodium chloride-NaHCO with the concentration of 0.05-0.5mol/L ₃ NaOH buffer, pH 3-5.

10. The method for separating and purifying the three gorges peptide according to claim 1, wherein the method comprises the steps of: the ethanol in the step (4) is absolute ethanol, and the volume ratio of the concentrated solution to the absolute ethanol is 1:2-5.

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