Process for efficient purification of neutral human milk oligosaccharides (HMOs) from microbial fermentation

Abstract

The present application discloses a simple process for the purification of neutral human milk oligosaccharides (HMOs) produced by microbial fermentation. The process uses a combination of cationic ion exchanger treatment, an anionic ion exchanger treatment, and a nanofiltration and/or electrodialysis step, which allows efficient purification of large quantities of neutral HMOs at high purity. Contrary to the purification currently used in fermentative production of neutral HMOs, the presented process allows the provision of HMOs without the need of a chromatographic separation. The so purified HMOs may be obtained in solid form by spray drying, as crystalline material or as sterile filtered concentrate. The provided HMOs are free of proteins and recombinant material originating from the used recombinant microbial strains and thus very well-suited for use in food, medical food and feed (e.g. pet food) applications.

Claims

1. A process for the purification of a neutral human milk oligosaccharide in a batch manner or in a continuous manner from a fermentation broth obtained by microbial fermentation, wherein the neutral human milk oligosaccharide is selected from the group consisting of 3-fucosyllactose, 2′,3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6′-galactosyllactose, 3′-galactosyllactose, lacto-N-hexaose and lacto-N-neohexaose, which process comprises i) separating the microbial biomass from the fermentation broth; ii) subjecting the solution obtained in step i) to nanofiltration; iii) subjecting the separated fermentation broth obtained in step ii) to a cation exchanger or to an anion exchanger to obtain a solution; iv) subjecting the solution obtained in step iii) to the cation or anion exchanger not used in step iii); v) subjecting the solution obtained from step iv) to nanofiltration or reverse osmosis or vacuum evaporation or electrodialysis to obtain a purified solution of the neutral human milk oligosaccharide; vi) optionally treating the purified solution obtained after step iv) or step v) with activated carbon; and vii) spray-drying the purified solution obtained after step v) or vi), wherein the neutral human milk oligosaccharide in the purified solution obtained after step v) or after step vi), has a purity of ≥70% as determined by HPLC.

2. The process of claim 1, wherein the separating step i) is performed using ultrafiltration using a cross-flow filter.

3. The process of claim 1, wherein the separated fermentation broth obtained in step i) is diafiltrated before step ii).

4. The process of claim 1, further comprising adding a β-glucosidase to the fermentation broth prior to step i).

5. The process of claim 1, wherein step vi) treating the purified solution obtained after step iv) or step v) with activated carbon is performed.

Description

(1) FIG. 1 shows a scheme of a preferred embodiment of the process according to the present invention for the purification of 2′-fucosyllactose from a fermentation broth comprising the steps: ultrafiltration, cationic and anionic ion exchanger treatment, activated carbon treatment, nanofiltration, electrodialysis and concentration.

(2) FIG. 2 shows a scheme of another preferred embodiment of the process according to the present invention for the purification of 2′-fucosyllactose from a fermentation broth comprising the steps: ultrafiltration, nanofiltration, cationic and anionic ion exchanger treatment, activated carbon treatment, electrodialysis and concentration.

(3) FIG. 3 shows a scheme of another preferred embodiment of the process according to the present invention for the purification of 2′-fucosyllactose from a fermentation broth comprising the steps: ultrafiltration, cationic and anionic ion exchanger treatment, nanofiltration, activated carbon treatment, electrodialysis and concentration.

EXAMPLE 1

Purification of 2′-fucosyllactose From Fermentation Using a Recombinant Microbial Production Strain I

(4) A 2′-fucosyllactose feed-batch fermentation employing a recombinant 2′-fucosyllactose synthesizing E. coli strain (E. coil BL21(DE3) ΔlacZ), containing a genomic integration 2′-fuosyltranferase, encoded by the wbgL gene (see EP 11 1151 571.4), and having an additional copy of the E. coil lacY, manB, manC, gmd and fcl all under the control of a strong constitutive tetracyclin promoter, containing a functional gal operon comprising the genes galM, galK, galT and galE, was grown in a defined salt medium. The defined salt medium comprised 7 g l.sup.−1 NH.sub.4H.sub.3PO.sub.4, 7 g l.sup.−1 K.sub.2HPO.sub.4, 2 g l.sup.−1 KOH, 0.37 g l.sup.−1 citric acid, 1 ml l.sup.−1 antifoam (Struktol J673, Schill+Seilacher), 1 mM CaCl.sub.2, 4 mM MgSO.sub.4, trace-elements and 2% glycerol as carbon source.

(5) Trace elements consisted of 0.101 g l.sup.−1 nitrilotriacetic acid, pH 6.5, 0.056 g l.sup.−1 ammonium ferric citrate, 0.01 g l.sup.−1 MnCl.sub.2×4 H.sub.7O, 0.002 g l.sup.−1 CoCl.sub.2×6 H.sub.2O, 0.001 g l.sup.−1 CuCl.sub.2×2 H.sub.2O, 0.002 g l.sup.−1 boric acid, 0.009 g l.sup.−1 ZnSO.sub.4×7 H.sub.2O, 0.001 g l.sup.−1 Na.sub.2MoO.sub.4×2 H.sub.2O, 0.002 g l.sup.−1 Na.sub.2SeO.sub.3, 0.002 g l.sup.−1 NiSO.sub.4×6 H.sub.2O.

(6) Glycerol-feed consisted of glycerol 800 g l.sup.−1 MgSO.sub.4 2.64 g l.sup.−1 and trace element solution 4 ml l.sup.−1. For 2′-fucosyllactose formation, a lactose feed of 216 g l.sup.−1 was employed. The pH was controlled by using ammonia solution (25% v/v). Feed batch fermentation was cultured at 30° C. under constant aeration and agitation for 90 hours. At 90 hours after the start of the fermentation, most of the added lactose was converted into 2′-fucosyllactose, in order to remove lactose still present in the fermentation supernatant, a second bacterial strain was added to the fermentation vessel 90 hours after the fermentation start.

(7) The added second bacterial strain was genetically identical to the first employed bacteria strain, differing, however, only in the expression of a genome integrated beta-galactosidase. Incubation of the added secondary bacterial strain resulted in the disappearance of the residual lactose within 5 hours. Approximately 10 ml starter culture of the second beta-galactosidase expressing bacterial strain was added per 1 l fermentation broth.

(8) The biomass was then separated from the fermentation medium by ultrafiltration, using a cross-flow filter with a cut-off of 10 kDa (Microdyn Nardir).

(9) An approximately 1 m.sup.3 cell-free fermentation medium was obtained containing 42 g/l 2′-fucosyllactose. The cell-free fermentation medium was then passed over a strong cationic ion exchanger (Lewatit S 6368 A (Lanxess) in H.sup.+ form, size of ion exchanger bed volume was 100 l), in order to remove positive charged contaminants. The obtained solution was then set to pH 7 by the addition of a 2 M sodium hydroxide solution.

(10) The solution was then (without delay) passed over an anionic ion exchanger column (bed volume of ion exchanger was 100 l). The used strong anionic ion exchanger Lewatit S 2568 (Lanxess) was in chloride (Cl.sup.−) form. The obtained solution was again neutralized to pH 7. The thus obtained solution was then diafiltrated using an Alfa-Laval NF99HF nanofiltration membrane and six volumes of sterile deionized water. The solution was further concentrated using the nanofiltration membrane wherein a 2′-fucosyllactose solution of 200 g/l and a conductivity of 7 mS/cm was obtained.

(11) The concentrated 2′-fucosyllactose solution was then treated with activated carbon in order to remove color giving material such as Maillard reaction products and aldol reaction products. As activated carbon 20 g Norit GAC EN per l concentrated 2′-fucosyllactose solution was used, yielding a significantly decolorized solution.

(12) The thus obtained concentrated 2′-fucosyllactose solution was then electrodialysed to 0.3 mS/cm using a PC-Cell BED 1-3 electrodialysis apparatus (PC-Cell, Heusweiler, Germany) equipped with PC-Cell E200 membrane stack. Said stack contained the following membranes: cation exchange membrane CEM: PC S K and the anion exchange membrane AEM:PcAcid60 having a size exclusion limit of 60 Da. A 0.025 M sulfamic acid (amidosulfonic acid) solution was used as an electrolyte in the ED process.

(13) Then, the obtained solution was then concentrated under vacuum at 40° C. to obtain a 45% 2′-fucosyllactose solution. The concentrated solution was then again treated with ion exchangers, Lewatit S 5368 A (Lanxess) in Na.sup.+ form (bed volume of the used ion exchanger was 10 l) and after neutralization with the anionic ion exchanger Lewatit S 2568 (Lanxess) in Cl.sup.− form (bed volume of the employed ion exchanger was 10 l).

(14) The obtained 2′-fucosyllactose solution was then treated with activated carbon (Norit DX1 Ultra). For 1 l of a 45% 2′-fucosyllactose solution 30 g activated carbon were employed.

(15) The solution was then again subjected to electrodialysis until a conductivity of less than 0.3 mSi/cm was obtained.

(16) Subsequently, the solution was subjected to sterile filtration by passing the solution through a 3 kDa filter (Pall Microza ultrafiltration hollow fiber module SEP-2013, Pall Corporation, Dreieich).

(17) Part of the obtained 2′-fucosyllactose solution was then spray dried for analysis.

(18) For NMR spectra recording the spray-dried product was dissolved in hexadeuterodimethyl sulfoxide (DMSO-d.sub.6). For the proton and .sup.13C analysis the following characteristic chemical shifts were observed:

(19) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 6.63 (d, J=6.5 Hz, 1H), 6.28 (d, =4.7 Hz, 1H), 5.21 (d, J=2.4 Hz, 1H), 5.19 (d, J=2.4 Hz, 1H), 5.01 (d, J=2.2, 2H), 4.92 (d, J=5.0 Hz, 1H), 4.89 (dd, J=4.6, 1.3 Hz, 2H), 4.78 (d, J=5.3 Hz, 1H), 4.74 (d,J=5.1 Hz, 1H), 4.63 (m, 6H), 4.53 (t, d, J=5.5, 1H), 4.46 (d, J=5.2 Hz, 1H), 4.44 (d, J=5.0 Hz, 1H), 4.38-4.26 (m, 5H), 4.23 (d, J=0.9, 1H), 4.05 (d, J=0.9, 1H), 4.00 (quin, J=3.3, 1H), 3.68-3.50 (m, 7H), 3.59-3.50 (m, 13H), 3.50-3.37 (m, 6H), 3.24 (dt,=8.8, 2.2 Hz, 1H), 3.14 (m, 2H), 2.96 (td, J=8.4, 4.7 Hz, 1H), 1.04 (d, J=6.1 Hz, 3H), 1.03 (d, J=6.1 Hz, 3H).

(20) .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 100.99, 100.85, 100.35, 100.25, 96.59, 92.02, 78.13, 77.78, 77.16, 77.01, 75.27 75.05, 74.67, 73.70, 72.33, 71.62, 71.56, 70.91, 69.90, 69,64, 68.75, 68.16, 66.33, 60.17, 59.82, 59.67, 16.37, 16.36.

(21) Chemicals shifts were found to be consistent with the 2′-fucosyllactose structure.

(22) Using this protocol a 45% 2′-fucosyllactose concentrate with a purity of 94.5% could be obtained (determined by HPLC analysis). Major contaminants were 3′-fucosyllactose (1.8%), difucosyllactose (2.9%), and lactose (0.3%).

(23) The yield of the purification was approximately 70%.

(24) Importantly, no recombinant material could be determined in 10 g of freeze material using 50 cycles of OCR. Protein amount of the obtained material was determined as <50 μg/g freeze dried material by using a nano-Bradford assay (Roth, Karlsruhe Germany). Total amount of ash was determined with 0.19%. Concentration of heavy metals was (arsenic cadmium, lead and mercury) below 0.1 μg/g material. Endotoxin levels were determined to be <0.005 EU/ml 2′-fucosyllactose concentrate.

EXAMPLE 2

Purification of 2′-fucosyllactose From Fermentation Using a Recombinant Microbial Production Strain II

(25) A 1 m.sup.3 microbial fermentation comprising 2′-fucosyllactose at a concentration of 47 g/L was filtered through a cross flow filter with a cut off of 100 kDa (Microdyn Nadir) to obtain a cell free fermentation medium.

(26) As a fermentation medium the following medium was employed: Major medium components; glycerol 30 g/l, NR.sub.4H.sub.2PO.sub.47 g/l, K.sub.2HPO.sub.47 g/l, citrate 0.3 g/l, KOH 2 g/l, MgSO.sub.4.7H.sub.2O 2 g/l; trace elements: CaCl.sub.2.6H.sub.2O 20 mg/l, nitrilotriacetic acid 101 mg/l, ammonium ferric citrate 56 mg/l, MnCl.sub.2.4H.sub.2O 9.8 mg/l, CoCl.sub.2.6H.sub.2O 1.6 mg/l, CuCl.sub.2.2H.sub.2O 1 mg/l, H.sub.3BO.sub.3 1.6 mg/l, ZnSO.sub.4.7H.sub.2O 9 mg/l, Na.sub.2MoO.sub.4.2H.sub.2O 1.2 mg/l, Na.sub.2SeO.sub.3 1.2 mg/l; feed substances; glycerol and lactose.

(27) The cell free fermentation medium was then passed over a cationic ion exchanger (Lewatit S 6368 A (Lanxess) in H.sup.+ form (volume of ion exchanger bed was 100 l) in order to remove positive charged contaminants. The obtained solution was then set to pH 7 by the addition of a 2 M sodium hydroxide solution. The solution was then, without delay passed over an anionic ion exchanger column (ion exchanger bed volume used was 100 l) comprising the strong anionic ion exchanger Lewatit S 2568 (Lanxess) in chloride (Cl.sup.−) form. The obtained solution was again neutralized to pH 7. The so obtained solution was then diafitrated (using 10 volumes of sterile deionized water) and concentrated using a nanofiltration membrane (Alfa-Laval NF99HF) to obtain a 2′-fucosyllactose solution of 200 g/l and a conductivity of approx. 7 mSi/cm.

(28) The concentrated 2′-fucosyllactose solution was then treated with activated carbon, using g Norit GAC EN per l concentrated 2′-fucosyllactose solution. To the filtered 2′-fucosyllactose solution 40 g/l Norit DX1 Ultra activated carbon was added. The solution was then exposed to the activated carbon at 4° C. for approximately 18 h. After 18 h, the activated carbon was removed from the 2′-fucosyllactose solution by filtration.

(29) The solution was then electrodialysed to a conductivity of <0.3 mS/cm using a PC-Cell BED 1-3 electrodialysis apparatus (PC-Cell, Heusweiler, Germany) equipped with PC-Cell E200 membrane stack. Said stack contained the following membranes: cation exchange membrane CEM: PC SK and the anion exchange membrane AEM:PcAcid60 having a size exclusion limit of 60 Da. A 0.025 M sulfamic acid (amidosulfonic acid) solution was used as an electrolyte in the ED process.

(30) The obtained solution was then concentrated to obtain a 40% 2′-fucosyllactose solution. The obtained 2′-fucosyllactose solution was then passed over a Lewatit S 2568 (Lanxess) Cl.sup.− form (bed volume 10 l) and treated with activated carbon (Norit DX1 Ultra) at 8° C. for 18 h. The solution was then subjected to sterile filtration by passing the solution through a 3 kDa filter (Pall Microza ultrafiltration hollow fiber module SEP-2013, Pall Corporation, Dreieich) and spray-dried using a NUBILOSA LTC-GMP spray dryer (NUBILOSA, Konstanz, Germany).

(31) Using this protocol, 2′-fucosyllactose with a purity of 94% could be obtained (determined by HPLC analysis). Major contaminants were 3′-fucosyllactose (1.8%), difucosyllactose (3.2%) and lactose (0.2%). The yield of the purification was approximately 70%.