Method for extracting epsilon-polylysine and its hydrochloride salt from fermentation broth

Abstract

The present invention provides a method for extracting -polylysine (-PL) and its hydrochloride salt from fermentation broth, which belongs to the field of bio-separation engineering. -PL and its hydrochloride salt are produced from fermentation broth through sequential solid-liquid separation, ultrafiltration, two-stage ion exchange, nanofiltration, evaporation concentration and drying techniques. Technologies of membrane filtration and two-stage ion exchange are applied to the preparation of -PL and its hydrochloride salt in the present invention, and the invention are characterized by reduced cost, improved automation, and increased product yield and purity, and the method of the present invention would be more suitable for industrial production.

Claims

1. A method for extraction of -polylysine (-PL) and its hydrochloride salt from a fermentation broth, comprising the following steps: (1) solid-liquid separating of the fermentation broth: after flocculation or dilution, filtrating or centrifugating the fermentation broth to remove mycelia and water-insoluble impurities, and obtaining a clear fermentation broth supernatant; (2) ultrafiltrating: using an ultrafiltration membrane system to remove the water-soluble macromolecular impurities and water-insoluble impurities in the fermentation broth supernatant, and collecting ultrafiltration permeate; (3) ion-exchanging: pressuring the ultrafiltration permeate into a primary ion exchange column for adsorption until resin is saturated; using a detergent to remove impurities, and using an elution reagent to desorb; collecting eluents from the primary ion exchange column, and then forcing into a secondary ion exchange column, and collecting a final effluent liquid; (4) nanofiltrating: using a nanofiltration membrane system to desalt and concentrate the final effluent liquid obtained from the step (3), and collecting a nanofiltration concentrate; (5) bleaching: decolorating collected nanofiltration concentrate to obtain a decolorated liquid; (6) concentrating: concentrating the decolorated liquid obtained from the step (5) to obtain a concentrate of a -polylysine content of 10-30% as a percent weight/volume; (7) drying: drying the concentrate obtained from the step (6) to obtain -polylysine and its hydrochloride salt product; wherein the ultrafiltration membrane system in step (2) is a tubular membrane or a wound membrane; wherein the molecular weight cutoff of an ultrafiltration membrane in the ultrafiltration membrane system is 50-1000 KDa; wherein the primary ion exchange column in step (3) is a weak or strong acid cation exchange resin, and the secondary ion exchange column in step (3) is a weak or strong base anion exchange resin; wherein the fermentation broth is obtained by fermentation of Streptomyces albulus CGMCC NO. 10480.

2. The method of claim 1, wherein the fermentation broth contains over 50 g.Math.L.sup.1 -PL and over 60 g.Math.L.sup.1 dry-weight cell.

3. The method of claim 1, wherein the fermentation broth is prepared by the following steps: inoculating Streptomyces albulus CGMCC NO. 10480 into a fermentation medium with 6%-8% inoculum volume, controlling fermentation process with a stirring speed 200-800 rpm, a temperature between 28-32 C., ventilation 0.5-2 volume/culture volume/min (vvm), and dissolved oxygen at 28-32%; when pH drops spontaneously to 4.5-5.2 at a first time, maintaining a fermentation pH at 4.8-5.2 for 10-15 h, and adjusting the fermentation pH to 2.8-3.2 and maintaining the fermentation pH for 22-26 h, and finally adjusting the fermentation pH to 4.2-4.8 and maintaining the fermentation pH until; when glycerol or glucose concentration in the fermentation broth drops to less than 10 g.Math.L.sup.1, feeding glycerol or glucose solution to maintain the concentration of glycerol or glucose at 9.5-10.5 g.Math.L.sup.1; when NH.sub.4.sup.+N concentration in the fermentation broth drops to 1 g.Math.L.sup.1 or less, maintaining the NH.sub.4.sup.+N concentration at 0.8-1.2 g.Math.L.sup.1 by feeding ammonium sulfate solution.

4. The method of claim 3, wherein the fermentation medium contains the following components: glucose or glycerol, (NH.sub.4).sub.2SO.sub.4, fish meal, corn steep liquor, KH.sub.2PO.sub.4, MgSO.sub.4.7H.sub.2O.sub.2, FeSO.sub.4.7H.sub.2O, and a pH is at 6.8.

5. The method of claim 1, wherein the nanofiltration membrane system in step (4), is a wound membrane, and the nanofiltration membrane has a molecular weight cutoff of 0.1-1.0 KDa.

6. The method of claim 1, wherein an operation pressure of said nanofiltration in step (4) is 1.0-1.5 MPa.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1. High-pressure liquid chromatograph of the fermentation broth;

(2) FIG. 2. High pressure liquid chromatograph of the final product of -PL obtained by the present invention;

(3) FIG. 3. MALDI-TOF-MS spectra of the final product of -PL hydrochloride salt obtained by the present invention.

EXAMPLES

(4) Materials and Methods:

(5) In the present invention, the ion exchange resin used in the methods is processed by sequential acid washing, alkali washing and acid washing, which is well known by Technical personnel in this field, and the resin is re-usable. In addition, the ultrafiltration membrane and nanofiltration membranes used in the methods is processed by sequential acid washing, alkali washing and acid washing, which is well known by Technical personnel in this field, and the resin is re-usable.

Example 1: Preparation of -PL Fermentation Broth

(6) Streptomyces albulus CGMCC NO. 10480 was inoculated into the 5 L fermentation tank with 6% inoculum volume of fermentation medium, the medium pH was adjusted to 7.5 with ammonia or NaOH. the fermentation process was controlled with the stirring speed 200-800 rpm, temperature 30 C., ventilation 0.5-2 vvm, dissolved oxygen 30%. When pH drops spontaneously to around 5.0 at the first time, the fermentation pH was maintained at around 5.0 for 10 h; then the pH was regulated to 3.0 and maintained for 24 h, finally the pH was adjusted to around 4.5 and maintained until the end of fermentation. When glycerol or glucose concentration in the fermentation broth drops to less than 10 g.Math.L.sup.1, sterilized pure glycerol or 500 g.Math.L.sup.1 glucose solution was fed to maintain the concentration of glycerol or glucose at 10 g.Math.L.sup.1; when NH.sub.4.sup.+-N concentration in the fermentation broth dropped to 1 g.Math.L.sup.1, the concentration was maintained at 1 g.Math.L.sup.1 by feeding the ammonium sulfate solution.

(7) In accordance with the fermentation control method, -PL yield could reach 55.4 g.Math.L.sup.1, dry cell weight was 75.3 g.Math.L.sup.1, the percentage of wet cells accounted for about 40% of the fermentation broth, through 192 h fed-batch fermentation.

Example 2: Preparation of -PL Fermentation Broth

(8) Streptomyces albulus CGMCC NO. 10480 was inoculated into the 5 L fermentation tank with 7% inoculum volume of fermentation medium, the medium pH was adjusted to 7.0 with ammonia or NaOH. The fermentation process was controlled with the stirring speed 200-800 rpm, temperature 32 C., ventilation 0.5 vvm, dissolved oxygen 28%. When pH drops spontaneously to around 4.8 at the first time, the fermentation pH was maintained at around 4.8 for 12 h; then the pH was regulated to 3.2 and maintained for 26 h, finally the pH was adjusted to around 4.8 and maintained until the end of fermentation. When glycerol or glucose concentration in the fermentation broth drops to less than 10 g.Math.L.sup.1, glycerol or glucose solution was fed to maintain the concentration of glycerol or glucose at 9.5 g.Math.L.sup.1; when NH.sub.4.sup.+-N concentration in the fermentation broth dropped to 1.0 g.Math.L.sup.1, the concentration was maintained at 1.2 g.Math.L.sup.1 by feeding the ammonium sulfate solution.

(9) In accordance with the fermentation control method, -PL yield can reach 60 g.Math.L.sup.1, dry cell weight is 85 g.Math.L.sup.1, the percentage of wet cells accounted for about 50% of the fermentation broth, through 192 h fed-batch fermentation.

Example 3: Preparation of -PL Fermentation Broth

(10) Streptomyces albulus CGMCC NO. 10480 was inoculated into the 5 L fermentation tank with 8% inoculum volume of fermentation medium, the medium pH was adjusted to 6.5 with ammonia or NaOH. The fermentation process was controlled with the stirring speed 200-800 rpm, temperature 30 C., ventilation 2 vvm, dissolved oxygen 32%. When pH drops spontaneously to around 4.5 at the first time, the fermentation pH was maintained at around 4.5 for 15 h; then the pH was regulated to 2.8 and maintained for 22 h, finally the pH was adjusted to around 4.2 and maintained until the end of fermentation. When glycerol or glucose concentration in the fermentation broth drops to less than 10 g.Math.L.sup.1, glycerol or glucose solution was fed to maintain the concentration of glycerol or glucose at 10.5 g.Math.L.sup.1; when NH.sub.4.sup.+-N concentration in the fermentation broth dropped to 1.0 g.Math.L.sup.1, the concentration was maintained at 0.8 g.Math.L.sup.1 by feeding the ammonium sulfate solution.

(11) In accordance with the fermentation control method, -PL yield can reach 68.7 g.Math.L.sup.1, dry cell weight is 63.8 g.Math.L.sup.1, the percentage of wet cells accounted for about 43% of the fermentation broth, through 192 h fed-batch fermentation.

Example 4: Extraction of -PL and its Hydrochloride Salt

(12) According to the following method to extract the -PL and its hydrochloride salt from the fermentation broth of the example 1:

(13) (1) Fermentation broth of example 1 was diluted by adding 4 times the volume of tap water in the fermentation tank, at room temperature, centrifugation of the fermentation broth diluted by disc centrifuge, a clear fermentation broth supernatant was obtained;

(14) (2) The fermentation broth supernatant was concentrated 10 times by subjecting it to an ultrafiltration membrane with MWCO of 1000 KDa, under the operating pressure of 0.3 MPa. The ultrafiltration permeate was collected.

(15) (3) The ultrafiltration permeate pH was adjusted to 5.0 by 6 M NaOH, and then the ultrafiltration permeate was pressured into the primary ion exchange column, which was loaded with strongly acidic cation resin, with the rate of 1.0 BV.Math.h.sup.1 for adsorption. After the saturation of resin, the ion exchange column was washed with deionized water at the rate of 1.0 BV.Math.h.sup.1; and then use 1.0 M HCl to desorb the resin with the rate of 1.0 BV.Math.h.sup.1. The eluents from the primary ion exchange column was collected, and then the eluents were pressured into a secondary ion exchange column, which was loaded with strongly base anion resin, with the rate of 1.0 BV.Math.h.sup.1, and the final eluents was collected;

(16) (4) The ultrafiltration permeate pH was adjust to 5.0 by 6.0 M HCl, and then use nanofiltration membrane MWCO of 100 KDa, under the operating pressure of 1.0 MPa to desalt by adding deionized water with intermittent constant volume mode. After the desalting process, concentrate the eluents 4 times, and then nanofiltration concentrate was collected;

(17) (5) The nanofiltration concentrate was heated to 80 C., and around 1% (w/v) activated carbon was added, the nanofiltration concentrate was decolorized for 20 min under stirring condition, the activated carbon was removed by filtration and the decolorated liquid was collected;

(18) (6) The decolorated liquid obtained from the step (5) was concentrated to -polylysine content of around 10% by evaporation;

(19) (7) The concentrate obtained from the step (6) was dried by spray drying, the -polylysine and its hydrochloride salt product was collected.

(20) According to the above extraction process, the total content of -PL and its hydrochloride in the product was 95.12%, that is, the purity of -PL and its hydrochloride was 95.12%. The yield of -PL and its hydrochloride was 72.3%.

(21) With the same method, the fermentation broth obtained by the examples 2 and 3 was extracted, and the purity of the -PL and its hydrochloride was 96.15% and 95.38%, respectively, and the yield reached 71.2% and 70.6%, respectively.

Example 5: Extraction of -PL and its Hydrochloride Salt

(22) According to the following method to extract the -PL and its hydrochloride salt from the fermentation broth of the example 1:

(23) (1) The fermentation broth of example 1 was flocculated by adding a certain concentration of flocculant in the fermentation tank, at room temperature, the fermentation broth was filtrated by plate and frame filter, a clear fermentation broth supernatant was obtained;

(24) (2) The fermentation broth supernatant was concentrated 5 times by subjecting it to an ultrafiltration membrane with MWCO of 10 KDa, under the operating pressure of 0.15 MPa. and the ultrafiltration permeate was collected.

(25) (3) The ultrafiltration permeate pH was adjusted to 8.5 by 6 M NaOH, and then the ultrafiltration permeate was pressured into the primary ion exchange column, which was loaded with macroporous weak acid cation resin, with the rate of 10 BV.Math.h.sup.1 for adsorption. After the saturation of resin, the ion exchange column was washed with deionized water at the rate of 10 BV.Math.h.sup.1; and then use 1 M NaOH to desorb the resin with the rate of 10 BV.Math.h.sup.1. The eluents from the primary ion exchange column was collected, and then the eluents were pressured into a secondary ion exchange column, which was loaded with macroporous weak basic anion resin, with the rate of 10 BV.Math.h.sup.1, and the final eluents was collected;

(26) (4) The ultrafiltration permeate pH was adjust to 8.0 by 6 M HCl, and then use nanofiltration membrane MWCO of 1000 KDa, under the operating pressure of 1.5 MPa to desalt by adding deionized water with continuous constant volume mode. After the desalting process, concentrate the eluents 2 times, and then nanofiltration concentrate was collected;

(27) (5) The nanofiltration concentrate was heated to 100 C., and around 1.5% (m/v) activated carbon was added, the nanofiltration concentrate was decolorized for 30 min under stirring condition, the activated carbon was removed by filtration and the decolorated liquid was collected;

(28) (6) The decolorated liquid obtained from the step (5) was concentrated to -polylysine content of around 30% by evaporation;

(29) (7) The concentrate obtained from the step (6) was dried by spray drying, the -polylysine and its hydrochloride salt product was collected.

(30) According to the above extraction process, the purity of -PL and its hydrochloride was 96.82%. and the yield of -PL and its hydrochloride was 70.5%.

(31) With the same method, the fermentation broth obtained by the examples 2 and 3 was extracted, and the purity of the -PL and its hydrochloride was 97.21% and 96.85%, respectively, and the yield reached 71.5% and 73.6% respectively.

Example 6: Extraction of -PL and its Hydrochloride Salt

(32) According to the following method to extract the -PL and its hydrochloride salt from the fermentation broth of the example 1:

(33) (1) Fermentation broth of example 1 was diluted by adding 8 times the volume of tap water in the fermentation tank, at room temperature, centrifugation of the fermentation broth diluted by disc centrifuge, a clear fermentation broth supernatant was obtained;

(34) (2) The fermentation broth supernatant was concentrated 20 times by subjecting it to an ultrafiltration membrane with MWCO of 800 KDa, under the operating pressure of 0.1 MPa. and the ultrafiltration permeate was collected.

(35) (3) The ultrafiltration permeate pH was adjusted to 7.0 by 6 M NaOH, and then the ultrafiltration permeate was pressured into the primary ion exchange column, which was loaded with macroporous weak acid cation resin, with the rate of 5 BV.Math.h.sup.1 for adsorption. After the saturation of resin, the ion exchange column was washed with deionized water at the rate of 5 BV.Math.h.sup.1; and then use 1 M ammonia to desorb the resin with the rate of 5 BV.Math.h.sup.1. The eluents from the primary ion exchange column was collected, and then the eluents were pressured into a secondary ion exchange column, which was loaded with strongly base anion resin, with the rate of 5 BV.Math.h.sup.1, and the final eluents were collected;

(36) (4) The ultrafiltration permeate pH was adjust to 8.5 by 6 M HCl, and then use nanofiltration membrane MWCO of 500 KDa, under the operating pressure of 1.2 MPa to desalt by adding deionized water with One-time addition mode. After the desalting process, concentrate the eluents 2 times, and then nanofiltration concentrate was collected;

(37) (5) The nanofiltration concentrate was heated to 90 C., and around 2% (m/v) activated carbon was added, the nanofiltration concentrate was decolorized for 15 min under stirring condition, the activated carbon was removed by filtration and the decolorated liquid was collected;

(38) (6) The decolorated liquid obtained from the step (5) was concentrated to -polylysine content of around 20% by evaporation;

(39) (7) The concentrate obtained from the step (6) was dried by spray drying, the -polylysine and its hydrochloride salt product was collected.

(40) According to the above extraction process, the purity of -PL and its hydrochloride was 97.10%. The yield of -PL and its hydrochloride was 75.2%.

(41) With the same method, the fermentation broth obtained by the examples 2 and 3 was extracted, and the purity of the -PL and its hydrochloride was 95.13% and 96.51%, respectively, and the yield reached 70.2% and 74.1% respectively.

(42) Contrast 1:

(43) The fermentation broth obtained from Example 1 was extracted by using the method described in CN201110053004.2, and the following was a specific implementation process.

(44) The pH of the fermentation broth was adjusted to 5.0 with 2 mol/L hydrochloric acid in the acidification tank, and the acidified solution was heated to 100 C., and was incubated for 10 minutes. The acidified solution was cooled down to room temperature, then it was filtered through the plate frame to get clear filtrate. 6 mol/L sodium hydroxide solution was added to the clear filtrate, the pH of filtrate was adjusted to 7.2 to obtain pretreatment liquid.

(45) The pretreatment liquid was pumped into the adsorption column filled with macroporous strong acidic ion exchange resin (D001) with height to diameter ratio of 10:1, and the adsorption process should be controlled at a rate of 4 ml/min (about 4 BV.Math.h.sup.1), until the resin saturated. Wash the saturated resin with 5 column volumes of deionized water at a flow rate of 5 ml/min (about 5 BV.Math.h.sup.1). 1 mol/L acetic acid solution was used to desorb the resin, and the flow rate was controlled at 1 ml/min (about 1 BV.Math.h.sup.1). Desorbing solution containing -PL was collected at different retention times by Itzhaki methods or HPLC. 2.0% (w/w) of activated carbon was added to the desorbing solution containing -PL, and the mixture was heated to 100 C., and was stirred for 60 minutes for decolorization, and then cooled it down to room temperature. Finally, the mixture was filtered to yield a decolorized solution.

(46) Decolorized solution containing -PL was forced into an evaporation concentration device to conduct cyclic concentration, When the -PL content reached 20% (w/w), the concentration process was stopped.

(47) -PL product can be obtained by freeze-drying the concentrated liquid. The recovery and purity of -PL and its hydrochloride salt were 62.1% and 52.6%, respectively.

(48) Contrast 2:

(49) Omit step (2) in Example 4, other conditions were in consistent with Example 4, and treated the same fermentation broth as in Example 1. The results show that the purity and the yield of -PL and its hydrochloride salt obtained by this method are 92.10% and 77.2%, respectively.

(50) Contrast 3:

(51) The step (3) in Example 4 were adjusted, and other treatment conditions were in consistent with Example 4, and treated the same fermentation broth as in Example 1.

(52) The specific adjusted step (3) was that the pH of the permeate was adjusted to 7.0 by 6 M NaOH, then the permeate was forced into the primary ion exchange column packed with macroporous weak acidic cation exchange resin at the rate of 5 BV.Math.h.sup.1 for ion exchange; when the resin was saturated, it was washed by deionized water at the rate of 5 BV.Math.h.sup.1; after washing, the resin was eluted with 1 M ammonia water at the rate of 5 BV.Math.h.sup.1, and the eluents were collected.

(53) The results show that the purity and the yield of -PL and its hydrochloride salt are 93.40% and 80.31%, respectively.

(54) Contrast 4:

(55) Omit step (4) in Example 4, and other conditions were in consistent with Example 4, and treated the same fermentation broth as in Example 1. The results show that the purity and the yield of -PL and its hydrochloride salt obtained by this method are 73.65% and 76.86%, respectively.

(56) While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.