Purification of oligosaccharides from fermentation broth using filtration

Abstract

A method for purifying oligosaccharides, preferably neutral human milk oligosaccharides (HMO) or sialylated HMO, from a fermentation broth, the method comprising: - providing a fermentation broth containing target oligosaccharides, biomass, microbial cells and other carbohydrates other than the target oligosaccharides; - removing the microbial cells from the fermentation broth to provide a process stream; - subjecting the process stream to a first filtration step using a nanofiltration membrane to provide a filtrate containing the target oligosaccharides; - subjecting the filtrate to a second filtration step using a nanofiltration membrane to provide a retentate containing the target oligosaccharides; - removing salts from the process stream using electrodialysis to provide a purified preparation of the target oligosaccharides.

Description

[0001] The present invention relates to the production of oligosaccharides on an industrial scale by microbial fermentation. More specifically, the present invention relates to the purification of target oligosaccharides from a fermentation broth by using a filtration method. Background Art

[0002] Human milk is a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements. The carbohydrate fraction is the most abundant solid fraction and it can be further divided into lactose and more complex oligosaccharides, the so-called human milk oligosaccharides (HMOs).

[0003] While lactose is used as an energy source, complex oligosaccharides are not metabolized by infants or adults. The fraction of complex oligosaccharides accounts for up to 10% of the total carbohydrate fraction and may consist of more than 150 different oligosaccharides. The presence and concentration of these complex oligosaccharides are unique to humans and therefore are not present in large quantities in the milk of other mammals (e.g. domesticated dairy animals). Although HMOs only account for a small fraction of the total human milk, over the past few decades it has become increasingly clear that they have a very beneficial effect on the development of breastfed infants.

[0004] The most important HMO is 2'-fucosyllactose. Other important HMOs are 3-fucosyllactose, difucosyllactose, lacto-N-tetraose, lacto-N-neotetraose and lacto-N-fucopentose. In addition to these neutral oligosaccharides, acidic HMOs are also present in human milk, including 3'-sialyllactose, 6'-sialyllactose and sialyllactose-N-tetraose (e.g., LST-a, LST-b and LST-c (Table 1)). Due to the presence of at least one sialic acid moiety in HMOs, up to 20% of the total HMO content in human milk is acidic. These structures are closely related to antigenic determinants of glycoconjugates on the surface of epithelial cells (Lewis tissue blood group antigens), and the structural homology between HMOs and epithelial antigenic determinants explains the ability of HMOs to protect against bacterial pathogens (Weichert et al. 2013, Nutr. Res. 33:831; Weichert et al. 2016, J. Virol. 90:4843).

[0005] The presence of complex oligosaccharides in human milk has long been known, and the physiological functions of HMOs have been the subject of medical research for decades (Gura 2014, Science 345:747; Kunz & Egge 2017, In McGuire, McGuire & Bode (eds.) Prebiotics and Probiotics in Human Milk, Elsevier, London, pp. 3-16). For some of the more abundant HMOs, specific functions have been identified (Bode 2012, Glycobiology 22:147; Bode & Jantscher-Krenn 2012, Adv. Nutr. 3S:383; Morrow et al. 2004, J. Pediatr. 145:297). Due to their physiological ability to inhibit infectious agents (bacteria, viruses and bacterial toxins), their positive effects on brain development and their prebiotic function, it is highly desirable to include HMOs in food, especially in infant nutrition products. Table 1 provides an overview of HMOs that can be purified from fermentation broth by the methods disclosed herein.

[0006]

[0007]

[0008] Table 1: List of human milk oligosaccharides that can be purified by the methods disclosed herein.

[0009] The limited supply of individual HMOs and the lack of resources to obtain sufficient quantities of these molecules have prompted the development of processes based on chemical synthesis to generate them. However, the chemical synthesis of HMOs, especially the large-scale production of HMOs of sufficient quality for application in food, has proven to be extremely challenging. In particular, the chemical synthesis of HMOs such as 2'-FL (WO 2010 / 115935 A) requires several hazardous chemicals, which may contaminate the final product.

[0010] The shortcomings of chemical synthesis have prompted the development of several enzymatic and fermentation-based approaches (Miyazaki et al. 2010, Methods Enzymol. 480:511; Murata et al. 1999, Glycoconj. J. 16:189; Baumgartner et al. 2013, Microb. Cell Fact. 12:40; Lee et al. 2012, Microb. Cell Fact. 1:48; Albermann et al. 2001, Carbohydr. Res. 334:97; US 7,521,212 B1; Fierfort & Samain 2008, J. Biotechnol. 134:216). However, these processes produce complex mixtures of oligosaccharides and thus the desired product is contaminated with starting materials (e.g. lactose) as well as intermediates, unwanted by-products (e.g. resulting from the side activities of certain glycosyltransferases) and substrates from the fermentation process (e.g. proteins, polypeptides, organic acids and salts).

[0011] Many of the current methods for purifying individual oligosaccharides from mixtures are technically complex, difficult to scale up, and uneconomical for food applications. Industrial-scale processes have been developed for the purification of the disaccharides lactose and sucrose from whey and molasses, respectively, but these methods include multiple crystallization steps that are complex and produce products in low yields. However, whey and molasses are used as "food grade" products, which are far less complex and demanding (in terms of regulatory requirements) than fermentation broths containing recombinant bacteria or yeast. Gel filtration chromatography is the best method for purifying complex oligosaccharides such as HMOs produced by microbial fermentation, but disadvantages of this method include lack of scalability and unsuitability for continuous processing. Gel filtration chromatography is therefore uneconomical and cannot be used for industrial-scale production of HMOs.

[0012] In theory, gel filtration chromatography is the best method for purifying complex oligosaccharides such as HMOs produced by microbial fermentation, but the disadvantages of gel filtration chromatography include lack of scalability and unsuitability for continuous processing. Therefore, gel filtration chromatography is uneconomical and cannot be used for industrial-scale production of HMOs.

[0013] After developing efficient methods and processes for producing HMOs by microbial fermentation, the next logical step is to develop efficient downstream processes to separate HMOs from the culture broth.

[0014] WO2015 / 049331A discloses a process for purifying neutral HMO. The process uses ion exchange, electrodialysis and simulated moving bed (SMB) chromatography to achieve continuous and efficient purification of large amounts of HMO. Unlike chemical synthesis routes for the production of neutral HMO and their subsequent purification, the fermentation and purification methods described allow for the provision of HMO that is free of harmful chemicals (e.g., trace amounts of heavy metals and organic solvents). The purification method yields a highly purified HMO product in solid form (by spray drying) or as a concentrated syrup, which can be used in food applications.

[0015] WO 2015 / 106943 A describes a simple process for purifying neutral HMO produced by microbial fermentation. The process uses a combination of cation exchange, anion exchange and nanofiltration and / or electrodialysis, which can effectively purify a large amount of neutral HMO. Unlike earlier methods for purifying neutral HMO produced by microbial fermentation, this process does not include a chromatographic separation step. The process produces HMO in solid form, which is a spray-dried material, a crystallized material or a filter-sterilized concentrate. The resulting HMO does not contain proteins and materials produced by the recombinant microbial strain used for production, and is therefore ideal for food, medical food and feed (e.g., pet food) applications.

[0016] WO 2016 / 095924 A describes a method for purifying 2'-FL by crystallization. HMO is produced by microbial fermentation, and after removing the biomass, it is concentrated by nanofiltration and electrodialysis. Finally, acetic acid is used to selectively crystallize 2'-FL from an aqueous solution that also contains 2',3-di-O-fucosyllactose (DFL).

[0017] LNT and LNnT can be separated from carbohydrate byproducts by selective binding to activated carbon, followed by elution with an organic solvent and separation by gel filtration chromatography (Priem et al. 2002, Glycobiology 12:235; Gebus et al. 2012, Carbohydr. Res. 361:83; Baumgartner et al. 2014, ChemBioChem 15:1896). As mentioned above, gel filtration chromatography is a convenient laboratory scale method, but it cannot be effectively scaled up for industrial production.

[0018] WO 2015 / 049331 A discloses that LNT produced by bacterial fermentation is purified to provide a clear, particle-free solution by the following sequence of operations: electrodialysis, nanofiltration, simulated moving bed (SMB) chromatography using a strong cation exchange resin, electrodialysis, ultrafiltration, and a second SMB chromatography.

[0019] Various methods are also described for isolating acidic HMOs, in particular sialyllactose or sialylated oligosaccharides.

[0020] US 2007 / 0020736 A discloses the preparation of 3'-SL, wherein di-sialylated and tri-sialylated lactose are produced as byproducts of a genetically modified strain of the bacterium Escherichia coli. 3'-SL accumulates in the culture broth to a concentration of ∼0.8 mM. 3'-SL is purified using the following steps: centrifugation, adsorption of the supernatant over charcoal and washing away water-soluble salts with distilled water, elution with an aqueous ethanol gradient, and finally separation and desalting on a Biogel column. 49 mg of 3'-SL was obtained from 1 L of culture broth.

[0021] Another disclosed method involves the use of genetically modified strains of E. coli to prepare 3'-SL, and subsequent isolation by heat permeabilization of the cells followed by centrifugation (Priem et al. 2002, Glycobiology 12:235). The material in the supernatant is adsorbed onto charcoal and the water-soluble salts are washed off with distilled water. After elution with an aqueous ethanol gradient, 3'-SL is adsorbed onto HCO3 - The 3′-SL was removed by cation exchange (using the resin in the acidic form) resulting in a 49% recovery efficiency of 3′-SL.

[0022] Another process starting with a fermentation broth includes heat permeabilization, centrifugation, adjusting the pH to 3.0 by adding a strong cation exchange resin in acidic form, and removing precipitated proteins by centrifugation (Fierfort et al. 2008, J. Biotechnol. 134: 261). The pH of the supernatant is then adjusted to 6.0 by adding a weak anion exchanger in alkaline form, and 3'-SL is bonded to the anion exchanger in HCO3-form. After washing with distilled water and then eluting with a continuous gradient of NaHCO3, NaHCO3 is removed by cation exchange (using an acidic form of the resin) until the pH drops to 3.0. Finally, the pH is adjusted to 6.0 with NaOH. This purification strategy achieves a 3'-SL recovery efficiency of 59%.

[0023] Enzymatically produced 3'-SL has also been separated from lactose by nanofiltration (Nordvang et al. 2014, Separ. Purif. Technol. 138:77). The authors showed that two different nanofiltration membranes, one with a molecular weight cutoff (MWCO) of 600-800 Da (sulfonated polyethersulfone (SPES) membrane) and the other with a molecular weight cutoff (MWCO) of 1000-1400 Da (SPES membrane), could separate most of the 3'-SL from lactose after diafiltration. However, the loss of 3'-SL in this process was significant and the purity was low, requiring an additional ion exchange purification step.

[0024] WO 2010 / 106320 A2 describes a method for enriching 3'-SL from whey. First, the protein is removed by ultrafiltration, and then the clarified whey permeate is incubated with an ion exchange resin to capture 3'-SL. After elution from the ion exchange material, the enriched 3'-SL fraction is concentrated by nanofiltration and the concentrate is demineralized. After demineralization, the 3'-SL is concentrated and dried to obtain a final dry product with a 3'-SL content of 20% by weight.

[0025] WO 2018 / 020473 A describes another process for enriching 3'-SL and 6'-SL from a liquid source. The two HMOs are separated from the mother liquor of lactose crystallization by heating the solution, enzymatic treatment and additional ultrafiltration and nanofiltration steps. After enrichment, the content of 3'-SL and 6'-SL is 10-30% by weight (dry mass).

[0026] Starting from the prior art, the object of the present invention is to provide a method for purifying target oligosaccharides, in particular neutral HMO or sialylated HMO, produced by microbial fermentation, wherein the process is easy to scale up and suitable for manufacturing the target oligosaccharides on a commercial or industrial scale, and this can produce a product with a purity that makes the product suitable for human consumption.

[0027] It must be emphasized that microbial fermentation broths, especially those containing recombinant microorganisms (bacteria or eukaryotic microorganisms, such as Pasteurella), are much more complex than dairy product streams. For example, whey is composed of ~94% water, 4-5% lactose, 0.5-1% protein and only a few identified minerals (such as calcium, potassium and phosphorus), as well as some vitamins, a simple matrix that is only concentrated and demineralized in dairy streams. In contrast, the matrix of sugar solutions obtained from recombinant microbial fermentation processes is very complex, first of all, according to government regulations, the recombinant biomass is separated and inactivated in accordance with legal requirements. The clarified broth obtained is an uncertain matrix containing different salts and ions, and also contains heavy metals and trace elements. In addition, the challenge of such liquids is to remove cell debris, membrane fragments such as lipids, proteins, molecules derived from microbial cell metabolism and especially DNA. Therefore, the recovery of oligosaccharides produced by recombinant processing aids (e.g., genetically modified bacteria) is more challenging compared to whey and dairy streams because the contaminants within the broth are very diverse in terms of molecular weight, charged molecules (singly and multiply charged), and colored molecules. Summary of the invention

[0028] The present invention provides a method for purifying the target oligosaccharide produced by fermentation in a culture broth or fermentation broth obtained by fermentation of a microorganism using a recombinant fermentation strain in a batch or continuous manner. The purification strategies previously described generally use expensive ion exchange steps (requiring cation and anion exchangers). Ion exchangers cannot be used continuously because they need to be regenerated. The culture broth contains the target oligosaccharide, biomass, culture medium components, salts and pollutants such as other acids and pigments.

[0029] In the purification process, the culture fluid can be subjected to the following purification steps to obtain the target oligosaccharides:

[0030] 1) Separation of microbial cells from culture medium by microfiltration

[0031] 2) Separation of proteins from the clarified culture broth (= process stream) by ultrafiltration

[0032] 3) The first nanofiltration step removes peptides and high molecular weight (HMW) impurities

[0033] 4) The second nanofiltration step removes water and salt

[0034] 5) Activated carbon treatment removes pigments and other impurities

[0035] 6) Electrodialysis or diafiltration using nanofiltration membranes to completely remove contaminating salts

[0036] 7) Remove water through reverse osmosis

[0037] Furthermore, another electrodialysis step can be introduced before the activated carbon treatment to reduce the amount of contaminating ions. In order to obtain the target oligosaccharides in solid form after purification in aqueous solution, the material can be spray dried or granulated. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A process scheme for an exemplary embodiment of the method according to the invention is shown.

[0039] Figure 2 A process scheme according to another exemplary embodiment of the method of the invention is shown.

[0040] Figure 3 A process scheme according to another exemplary embodiment of the method of the invention is shown.

[0041] Figure 4 A process scheme according to another exemplary embodiment of the method of the invention is shown.

[0042] Figure 5 A process scheme according to another exemplary embodiment of the method of the invention is shown.

[0043] Figure 6 Shown is an HPLC chromatogram of clarified fermentation broth containing 3-fucosyllactose.

[0044] Figure 7 An HPLC chromatogram of 3-fucosyllactose after purification of a fermentation broth by an exemplary embodiment of the method of the present invention is shown.

[0045] Detailed description

[0046] According to the present invention, the term "purity" refers to chemical purity and specifies the extent to which a substance (e.g., 2'-FL, 3-FL, DFL, LNT, 3'-SL, 6'-SL or any other target oligosaccharide (Table 1)) is not diluted or mixed with foreign substances. Therefore, chemical purity is an indicator of the relationship between a single substance and any byproducts / impurities. Chemical purity is expressed as a percentage (%) and is calculated using the following formula:

[0047]

[0048] In the composition comprising the target oligosaccharide, the purity of the compound can be determined by any suitable method known to those skilled in the art, such as high performance liquid chromatography (HPLC). Suitable detectors can be selected from electrochemical detectors, refractive index (RI) detectors, mass spectrometers (MS), diode array detectors (DAD) and nuclear magnetic resonance (NMR) detectors. For example, in HPLC, purity can be determined by calculating the ratio of the area below the target peak (representing the amount of the target oligosaccharide) to the area summation below all peaks (representing the amount of the target oligosaccharide and the compound different from the substance in the same chromatogram). However, this means that all impurities can be analyzed by the selected HPLC method. Otherwise, the mass balance method is necessary, i.e., the absolute quantification of the desired product. In the method, pure substances are used as references to quantify purity, and then the purity is judged according to the dry matter obtained from the product (the desired product plus all impurities). According to the present invention, the mass balance method can also be used to determine purity.

[0049] According to the present invention, "culture liquid" or "fermentation liquid" refers to any liquid containing 2'-FL, 3-FL, DFL, LNT, 3'-SL, 6'-SL or any other target oligosaccharide to be purified (Table 1) after fermentation. The terms "culture liquid", "fermentation liquid" and "culture medium" are used as synonyms in this article. The culture liquid contains the target oligosaccharide to be purified as well as biomass (such as biological cells and cell fragments), culture medium components, salts and other contaminants (such as other acids and pigments). The biological cells contained in the culture liquid are biological cells that produce target oligosaccharides in the cell and secrete the compound into the liquid culture medium. The biological cell may include a genetically modified biological cell, such as a genetically modified Escherichia coli cell, or may be composed thereof. The genetic modification may include modifications (especially during the growth period of the biological cell) for producing the target oligosaccharide, or may be composed thereof.

[0050] As used herein, the term "biomass" refers to all biological cells present in the fermentation broth at the end of the fermentation step. Biomass includes microbial cells that produce target oligosaccharides, progeny cells of the microorganism that may have lost the ability to produce target oligosaccharides during the fermentation step, and any other cells that are accidentally present in the fermentation broth at the end of the fermentation step. Therefore, all biological cells present in the fermentation broth at the end of the fermentation step are substantially separated from the fermentation broth, so that the clarified fermentation broth, referred to as a "process stream" (any solution containing or containing the target oligosaccharide to be purified), is substantially or completely free of cells.

[0051] The biomass preferably comprises biological cells producing the target oligosaccharide, preferably bacterial cells producing the target oligosaccharide, more preferably recombinant bacterial cells producing the target oligosaccharide, and most preferably recombinant Escherichia coli (E.coil) cells producing the target oligosaccharide.

[0052] Biomass and / or microbial cells may be removed from the fermentation broth by centrifugation and / or filtration.

[0053] In the centrifugal process that is applicable to remove biomass from nutrient solution, biomass is obtained with particle form, and supernatant is as the clarification process flow that will be further processed.In the filtration process that is applicable to remove biomass from nutrient solution, filtrate becomes the process flow of clarification.Preferred filtration process of removing biomass is microfiltration and / or ultrafiltration, and the latter gives the ability of removing smaller particles and macromolecules than microfiltration.Microfiltration and ultrafiltration can be operated under dead-end filtration mode (process flow flows perpendicular to filter) or cross-flow filtration mode (process flow flows parallel to filter).

[0054] Microfiltration is a physical separation process in which a fluid containing particles is passed through a medium that includes a porous material containing torturous channels to retain particles (depth filtration) and / or a membrane with a pore size that allows particles / molecules smaller than a specific pore size to pass through (membrane filtration). As used herein, the term "microfiltration" refers to a physical separation process in which biological cells (and cell debris) are removed from a fermentation broth, leaving behind a (clarified) process stream.

[0055] Suitable membranes for removing biomass by microfiltration may have a pore size of at least 0.2 μm and may be hollow fibers or spiral wound membranes. Alternatively, removal of biomass may be achieved by microfiltration using membranes having a MWCO of 100-1000 kDa, preferably 150-500 kDa, to remove biomass, additional cell debris, and larger proteins. For example, such as Membranes such as DS MVP20 (pore size 0.2 μm) (Microdyn-Nadir GmbH, Wiesbaden, Germany) are spirally wound to provide a compact design and better performance, which can be used to separate cells from culture medium. In addition, spiral wound membranes with pore sizes of 0.05-0.1 μm, such as DS MP005, a module containing a PES membrane with a nominal pore size of 0.05 μm, can be used to separate biomass and proteins.

[0056] In addition, hollow fiber modules, such as FS10-FC FUS1582 (Microdyn-Nadir GmbH), use a PES membrane with a MWCO of 150 kDa (5 m 2 ) hollow fiber filtration components can be used as an alternative.

[0057] In summary, the following possible methods for removing biomass from fermentation broth can be used in the present invention:

[0058] i) Harvesting by centrifugation. Remove insoluble components from culture medium in one step. Advantages: Rapid removal of insoluble components.

[0059] ii) Harvesting by microfiltration . Remove insoluble components and large molecules over a certain size from the culture medium in one step. Spiral wound membranes or hollow fiber cross-flow filters can be used for microfiltration with MWCO ≥ 500 kDa. Advantages: Rapid removal of insoluble components and large soluble molecules over a certain size.

[0060] iii) By Harvesting by combining centrifugation and microfiltration :Remove insoluble components and macromolecules over a certain size from the culture medium in two steps. Spiral wound membranes or hollow fiber cross-flow filters can be used for microfiltration with MWCO ≥ 500 kDa. Advantages: Rapid removal of insoluble components and macromolecules over a certain size without clogging the microfiltration membrane.

[0061] Ultrafiltration is a form of membrane filtration that is not fundamentally different from microfiltration. In ultrafiltration, the forces created by pressure and concentration gradients cause a liquid containing particles and soluble macromolecules to pass through a semipermeable membrane to remove such particles and large soluble molecules. This results in the particles and large soluble molecules being retained in the so-called retentate, while water and low molecular weight (LMW) solutes (such as produced neutral and acidic oligosaccharides) pass through the membrane into the permeate (filtrate). The membrane used for ultrafiltration is defined by its MWCO, which describes the maximum molecular weight of a soluble molecule that can pass through the membrane into the permeate. Any particles and molecules larger than the MWCO cannot pass through the membrane and are retained in the retentate. Ultrafiltration can be applied in cross-flow mode, where the flow of the liquid is parallel to the membrane surface, or in dead-end mode, where the flow of the liquid is perpendicular to the membrane surface.

[0062] The clarified process stream comprising the produced target oligosaccharide typically contains a large amount of undesirable impurities including, but not limited to, monovalent ions, divalent ions, amino acids, polypeptides, proteins, organic acids, nucleic acids, monosaccharides and / or oligosaccharides.

[0063] Suitable membranes for removing most proteins by ultrafiltration have a MWCO of at least 50 kDa, preferably at least 30 kDa, more preferably 10 kDa, and may be hollow fiber or spiral wound membranes. Membranes of DS UP010 (MWCO=10 kDa) or SPIRA-CEL DS UP005 (MWCO=5 kDa) (both sold by Microdyn-Nadir GmbH) are spirally wound to provide a compact structural design and better performance, and these membranes can be used to separate residual proteins from cell-free culture fluids.

[0064] In addition, hollow fiber modules can be used, e.g. HF UF Cartridge PM10 (KochMembranes Systems, Wilmington, USA), which is up to 12m 2 and PES membranes with a MWCO of 10 kDa can be used as an alternative.

[0065] Nanofiltration is a membrane filtration method in which the membrane contains nanometer-scale pores (1-10nm). The pore size of nanofiltration membranes is smaller than that of microfiltration and ultrafiltration membranes, but larger than that of membranes used for reverse osmosis. Nanofiltration membranes are mainly made of thin films of polymers (such as polyethylene terephthalate) or metals (such as aluminum) with a pore density of 1-10 6 Hole / cm 2 .

[0066] Nanofiltration is used in purification methods for target oligosaccharides to remove LMW impurities such as peptides and salts and to increase the concentration of oligosaccharides in the clarified process stream or eluate.

[0067] Most HMOs have a molecular weight range of 400 to 1,200 Da. To remove HMW impurities (>1200 Da) such as peptides and pigments as well as LMW impurities (<400 Da) such as salts from a process stream, at least two nanofiltration steps are required.

[0068] After ultrafiltration, all molecules >5 kDa or >10 kDa (depending on the membrane used) should be removed from the process stream. To remove impurities below this threshold but still larger than the target oligosaccharide (e.g., peptides and pigments), a first nanofiltration step can be performed using a MWCO that allows the oligosaccharides to enter the permeate while the HMW impurities are retained in the retentate.

[0069] In order to separate the HMW impurities from the majority of the desired oligosaccharides and to increase the yield of the desired oligosaccharides, the process should be run until >70%, preferably >80%, more preferably >90% of the product passes through the membrane. In addition, a diafiltration step may be included to increase the yield of oligosaccharides in the clarified process stream.

[0070] After this first nanofiltration step, the only impurities in the process stream containing the target oligosaccharide should be of the same or lower molecular weight than the oligosaccharide and may include monovalent ions, divalent ions, amino acids, organic acids, monosaccharides and / or oligosaccharides.

[0071] Suitable membranes for the first nanofiltration step include polyamide or polypiperazine thin film composite membrane materials that achieve a size exclusion in the range of 400-1,200 Da. Examples include HYDRACoRe70 (MWCO=720 Da) or HYDRACoRe50 (MWCO=1,000 Da) membranes, both from Hydranautics (Oceanside, USA), NP010P (MWCO = 1,000-1,500Da) or NP030P (MWCO = 500-1,000 Da) membrane (Microdyn-Nadir GmbH). Such membranes allow high flux. Other examples of suitable membranes for nanofiltration include SBNF (MWCO = 2000 Da), cellulose acetate nanofiltration membranes (Microdyn-Nadir GmbH) and GE series membranes (MWCO = 1000 Da) from Suez Water Technologies & Solutions (Ratingen, Germany).

[0072] In additional and / or alternative embodiments, the first nanofiltration step achieves the following parameters:

[0073] i) The target oligosaccharide recovery after filtration should be >70%, preferably >80%, more preferably >90%.

[0074] ii) The concentration of target oligosaccharides in the process stream should be <50% (w / v), preferably <40% (w / v), more preferably <30% (w / v), preferably <20% (w / v), more preferably <10% (w / v).

[0075] iii) This step should be carried out at a temperature of <80°C, preferably <50°C, more preferably 4-40°C (particularly suitable for nanofiltration).

[0076] iv) This step should be carried out at a pressure of 5-50 bar, preferably at a pressure of 10-40 bar, more preferably at a pressure of 15-30 bar.

[0077] In a preferred embodiment of the method, after the first nanofiltration step, the process stream containing the target oligosaccharide is concentrated and desalted by applying a second nanofiltration step. The target oligosaccharide can also be concentrated by vacuum evaporation (e.g., using a falling film evaporator or a plate evaporator) or reverse osmosis. The shortcoming of these two technologies is that the process stream is concentrated but without desalination. Therefore, nanofiltration is a preferred method because it can achieve concentration and desalination simultaneously, for example, by using a film with a size exclusion limit of <2nm.

[0078] The method for purifying the target oligosaccharide comprises a step in which the clarified process stream from the first nanofiltration step is subjected to at least one additional nanofiltration step to remove salts, smaller molecules and water. Preferably, the eluate (=filtrate or permeate) from the first nanofiltration is directly subjected to a second nanofiltration step to remove salts and smaller molecules from the clarified process stream.

[0079] Suitable membranes for the first nanofiltration step include polyamide or polypiperazine thin film composite membrane materials that achieve a size exclusion in the range of 150-300 Da, such as Filmtec TM NF270 (Dow Chemical Company, Midland, USA) and XN45 or TS40 membranes (Microdyn Nadir GmbH). Such membranes allow high flux. Other examples include 4040-XN45-TSF (Microdyn-Nadir GmbH) or GE4040F30 and GH4040F50 membranes (SuezWater Technologies & Solutions).

[0080] Prior to electrodialysis, the nanofiltration step effectively removes a large amount of salt and LMW impurities from the process stream containing the target oligosaccharide. After the ultrafiltration step, nanofiltration can also effectively remove LMW contaminants, wherein the removal of such contaminants is beneficial to the concentration and demineralization of the target oligosaccharide solution. Due to more limited thermal contact, the use of nanofiltration to concentrate the target oligosaccharides can reduce energy and processing costs, and provide better product quality.

[0081] The method concentrates target oligosaccharides from an aqueous solution, wherein the concentration of the target oligosaccharides in the aqueous solution is ≤20%, ≤10% or ≤5% (before concentration).

[0082] In additional and / or alternative embodiments, the concentration by nanofiltration should achieve the following parameters:

[0083] i) the oligosaccharide concentration is >100 g / L, preferably >200 g / L, more preferably >300 g / L, most preferably >400 g / L;

[0084] ii) the salt content of the purified solution should be <10 wt%, preferably <5 wt%, more preferably <2 wt%; and / or the conductivity should be 0.5-10.0 mS / cm 2 , preferably 1-8mS / cm 2 , more preferably 1.5-4.0mS / cm 2 .

[0085] iii) This step should be carried out at a temperature of <80°C, preferably <50°C, more preferably 4-40°C (particularly suitable for nanofiltration).

[0086] iv) This step should be carried out at a pressure of 5-50 bar, preferably at a pressure of 10-40 bar, more preferably at a pressure of 15-30 bar.

[0087] Pigments in the clarified solution and / or purified solution can be removed by treatment with activated carbon. The advantage of using activated carbon to remove pigments is that both charged and uncharged (neutral) pigments can be removed.

[0088] Activated carbon, also known as activated charcoal, is a form of carbon that has been treated to create small pores of reduced volume, thereby increasing the surface area available for adsorption. Typically, due to its high degree of microporosity, the surface area of ​​1 gram of activated carbon alone is greater than 30,000 m 2 (determined by gas adsorption).

[0089] Impurities with color, such as Maillard products, riboflavin and other LMW impurities, tend to adsorb to the surface of the carbon particles. Compared to the oligosaccharides produced, the amount of substances with color is much lower and / or in most cases shows hydrophobic behavior. Due to this fact, the removal rate of color contaminants from the process stream is high. Water-soluble materials (such as oligosaccharides) are more weakly bound and can be eluted by washing with water, leaving the color adsorbed on the surface.

[0090] In additional and / or alternative embodiments, the method for purifying a target oligosaccharide further comprises at least one step in which a clarified process stream or eluate from a previous purification step is treated with activated carbon to remove pigments.

[0091] In a preferred embodiment, the activated carbon treatment should be performed after the following steps:

[0092] i) after removal of water and salts in a second nanofiltration step; and / or

[0093] ii) After removal of proteins by ultrafiltration or after removal of remaining salts by electrodialysis (alternatively, a diafiltration step may be introduced).

[0094] Activated carbon suitable for removing neutral oligosaccharides, pigments and other contaminants is (but not limited to) granular activated carbon (such as Norit GAC830EN (Cabot Corporation, Boston, USA) and Epibon Y 12x 40 spezial (Donau Carbon, Frankfurt, Germany)) or powdered activated carbon (such as Norit DX1, Norit SA2 (Cabot Corporation) and Carbopal MB4 (Donau Carbon)).

[0095] Electrodialysis combines dialysis and electrolysis and can be used to separate and concentrate ions in solution based on their selective electrical migration through a semipermeable membrane. The use of industrial electrodialysis dates back to the early 1960s, when the process was used to demineralize cheese whey for addition to infant formula. Other applications include adjusting the pH of beverages such as wine, grape juice, apple juice, and orange juice.

[0096] Desalination of brackish water for the production of drinking water and demineralization of milk whey for the production of baby food are the most widespread applications of electrodialysis today. The basic principle of electrodialysis involves an electrolytic cell comprising a pair of electrodes immersed in an electrolyte for conducting ions and connected to a direct current generator. The electrode connected to the positive pole of the direct current generator is the anode and the electrode connected to the negative pole is the cathode. The electrolyte solution then supports the flow of electric current, which is generated by the movement of negative and positive ions towards the anode and cathode respectively. Essentially, the membrane used for electrodialysis is a porous ion exchange resin sheet with negatively or positively charged groups, which are therefore described as cationic or anionic membranes, respectively. Ion exchange membranes usually consist of a polystyrene matrix with suitable functional groups (such as sulfonic acid for cationic membranes or quaternary ammonium groups for anionic membranes) cross-linked with divinylbenzene.

[0097] The electrolyte can be an aqueous solution comprising, for example, sodium chloride, sodium acetate, sodium propionate and / or sulfamic acid. The electrolyte surrounds the cathode and anode and allows electric current to flow in the battery. The electrodialysis stack is then assembled in such a way that anionic membrane and cationic membrane are parallel between two electrode blocks as in a filter press, so that the flow of experiencing ion consumption is well separated from the flow of experiencing ion enrichment. These two solutions are also referred to as diluent (carrying out ion consumption) and concentrated solution (carrying out ion enrichment).

[0098] The core of the electrodialysis process is a membrane stack, which consists of multiple anion exchange membranes and cation exchange membranes installed between two electrodes, separated by spacers. By applying a direct current, anions and cations will migrate through the membranes towards the electrodes, producing a (desalting) dilute stream and a concentrate stream.

[0099] The pore size of the ion exchange membranes used for electrodialysis needs to be small enough to prevent diffusion of product from the dilute stream into the concentrate stream, which is driven by the high concentration difference between the two streams.

[0100] Electrodialysis is used to remove ions from aqueous solutions, while neutral and acidic oligosaccharides remain in the process stream. An important advantage of electrodialysis is that the recombinant DNA molecules can be completely removed from the solution containing the target oligosaccharide. In addition, the amount of salt in the process stream can be significantly reduced by electrodialysis. Indeed, sodium chloride can be completely removed from the process stream. This has the advantage of providing a solution containing the target oligosaccharide that is free of salt (such as sodium chloride), thereby preventing any negative effects of the salt on the final product (e.g., baby food).

[0101] After nanofiltration and activated carbon treatment, most of the salts and smaller organic impurities (such as organic acids) should be removed from the process stream. To ensure effective removal of residual salts and small charged organic species, an electrodialysis step is performed.

[0102] The electrodialysis step can be performed until the process flow reaches 0.05-1.0 mS / cm 2 , preferably 0.1-0.5mS / cm 2 , more preferably 0.2-0.4mS / cm 2 Furthermore, an electrodialysis step may be performed until the salt concentration drops to <10.0 g / L, preferably <5.0 g / L, more preferably <1.0 g / L, most preferably <0.2 g / L.

[0103] For neutral oligosaccharides, the electrodialysis step should be carried out under acidic or neutral pH conditions, preferably pH 3-8, more preferably pH 4-7. For acidic oligosaccharides, since acidic oligosaccharides are unstable under acidic conditions, the electrodialysis step should be carried out under neutral pH conditions, preferably pH 6-8, more preferably pH 6.5-7.5. The pH value of the acidic oligosaccharide solution must be controlled during the electrodialysis process and adjusted with NaOH if necessary.

[0104] A reverse osmosis step can be used instead of nanofiltration to concentrate the target oligosaccharides. Reverse osmosis is a membrane filtration method that concentrates particles larger than 0.1 nm in the retentate of a process stream while removing water. Therefore, reverse osmosis can concentrate a process stream, but it cannot achieve desalination.

[0105] The method can concentrate the target oligosaccharide in an aqueous or organic solvent, wherein the concentration of the target oligosaccharide is ≤20% (w / v), ≤10% (w / v) or ≤5% (w / v) (before concentration).

[0106] In additional and / or alternative embodiments, the concentration step should achieve the following parameters:

[0107] i) The oligosaccharide concentration is >300 g / L, preferably >400 g / L, more preferably >500 g / L, most preferably 600 g / L.

[0108] ii) This step should be carried out at a temperature of <80°C, preferably <50°C, more preferably 4-40°C (particularly suitable for reverse osmosis).

[0109] iii) This step should be carried out at a pressure of 5-50 bar, preferably at a pressure of 10-40 bar, more preferably at a pressure of 15-30 bar.

[0110] In additional and / or alternative embodiments of the process, the solution containing neutral and acidic HMOs is concentrated by vacuum evaporation (e.g. using a rotary evaporator or a plate evaporator) and the following parameters should be achieved:

[0111] i) The oligosaccharide concentration is >300 g / L, preferably >400 g / L, more preferably >500 g / L, most preferably >600 g / L.

[0112] ii) This step should be carried out at a temperature of <80°C, preferably <50°C, more preferably 20-50°C, even more preferably 30-45°C, most preferably 35-45°C (particularly suitable for vacuum evaporation).

[0113] To remove any potential microbial contaminants and endotoxins, the concentrated target oligosaccharides are sterilized by filtration through an ultrafiltration membrane.

[0114] In a preferred embodiment of the invention, the purified oligosaccharide solution is passed through a filter assembly of ≤10 kDa, such as a filter of 5 kDa or 3 kDa MWCO. Suitable ultrafiltration membranes for removing endotoxins have a MWCO of at least 10 kDa, preferably at least 5 kDa, and may be spiral wound or hollow fiber ultrafiltration membranes. Examples of spiral wound membranes include DS UP010 (MWCO = 10 kDa) or DS UP005 (MWCO = 5 kDa) membranes (Microdyn-Nadir GmbH) and from Koch Membranes Systems HpHT UF-5K (MWCO = 5 kDa). Examples of hollow fiber modules include the HF UF Cartridge PM5 (MWCO = 5 kDa) or Microzoa from Pall Cooperation (Port Washington, USA) TM Series membranes (MWCO=3 or 6).

[0115] In a preferred embodiment, after filtration sterilization, the purified oligosaccharide solution is spray dried. The solution can be spray dried using hot air to remove preferably at least 85% by weight, or more preferably at least 90% by weight of water. The solution can be spray dried using any conventional spray drying system (preferably with a fluidized bed dryer accessory).

[0116] The purified solution can be spray dried so that the concentration of the target oligosaccharide is 5-60 wt %, preferably 10-50 wt %, more preferably 15-45 wt %. The inlet temperature can be maintained in the range of 110-150° C., preferably 120-140° C., more preferably 125-135° C. The outlet temperature can be maintained in the range of 60-80° C., preferably 65-70° C.

[0117] After spray drying, the purified target oligosaccharides can have the following properties:

[0118] i) in solid particulate form; and / or

[0119] ii) a glass transition temperature of 60-90°C, preferably 62-88°C, more preferably 64-86°C, as determined by differential scanning calorimetry; and / or

[0120] iii) a particle size of 5-500 μm, preferably 10-300 μm, as measured by laser diffraction; and / or

[0121] iv) an average particle size of 10-100 μm, preferably 20-90 μm, more preferably 30-80 μm, most preferably 40-70 μm, as determined by laser diffraction; and / or

[0122] v) an amorphous state is observed by X-ray powder diffraction, preferably an amorphous state without characteristic peaks of crystalline material; and / or

[0123] vi) The moisture content is ≤10 wt. %, preferably ≤8 wt. %, more preferably ≤5 wt. %.

[0124] The purified target oligosaccharide can be used in nutritional applications, preferably medicine or dairy nutrition (eg, cereal products), more preferably infant nutrition or medicine, preferably prevention or treatment of gastrointestinal diseases.

[0125] In one embodiment, the target oligosaccharide is a neutral oligosaccharide or a sialylated oligosaccharide, preferably a human milk oligosaccharide. The target oligosaccharide may be a neutral HMO or a sialylated HMO. In additional and / or alternative embodiments, the target oligosaccharide is selected from the following human milk oligosaccharides: 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose I, lacto-N-neofucopentose, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-neofucopentose Sugar V, lacto-N-difucohexose I, lacto-N-difucohexose II, 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose and lacto-N-neohexose, 3'-sialyl lactose, 6'-sialyl lactose, sialyl lactose-N-tetraose a, sialyl lactose-N-tetraose, sialyl lactose-N-tetraose c, 3-fucosyl-sialyl lactose, disialyl-lacto-N-tetraose and fucosyl-LST b.

[0126] The present invention will be described in conjunction with specific embodiments and with reference to the accompanying drawings, but the invention is not limited thereto and is limited only by the claims. In addition, the terms first, second, etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe order in time, space, by grade, or in any other way. It should be understood that the terms so used are interchangeable where appropriate, and the embodiments of the invention described herein can be operated in other orders than those described or illustrated herein.

[0127] It should be noted that the term "comprising", used in the claims, should not be interpreted as being limited to the means listed thereafter; it does not exclude other elements or steps. It is therefore interpreted as specifying the presence of the mentioned features, integers, steps or components, but does not exclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting only of components A and B. This means that, for the purposes of the present invention, the only relevant components of the device are A and B.

[0128] Throughout this specification, reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner, as will be apparent to one of ordinary skill in the art based on the disclosure herein.

[0129] Similarly, it should be understood that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof in order to simplify the disclosure and facilitate understanding of one or more aspects of the various inventive aspects of the invention. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than those expressly recited in each claim. On the contrary, as reflected in the following claims, inventive aspects lie in less than all features of the individual embodiments disclosed above. Therefore, the claims that follow the detailed description are hereby expressly incorporated into the detailed description, with each claim standing on its own as a separate embodiment of the invention.

[0130] In addition, although some embodiments described herein include some features of other embodiments but not other features, the combination of features of different embodiments is also within the scope of the present invention and forms different embodiments, as understood by those skilled in the art. For example, in the following claims, any claimed embodiments may be used in any combination.

[0131] In addition, some embodiments are described herein as methods or combinations of method elements that can be implemented by a processor of a computer system or by other means of performing the function. Therefore, a processor having the necessary instructions to perform such a method or method element constitutes a means for performing the method or method element. In addition, the elements of the device embodiments described herein are examples of means for performing the functions exercised by the elements for the purpose of implementing the present invention.

[0132] In the description and drawings provided herein, many specific details are set forth. However, it should be understood that embodiments of the present invention can be practiced without these specific details. In other cases, well-known methods, structures and techniques are not shown in detail in order not to obscure the understanding of this specification.

[0133] The present invention will now be described by the specific description of several embodiments of the present invention. Obviously, other embodiments of the present invention can be configured according to the knowledge of those skilled in the art without departing from the true spirit or technical teaching of the present invention, and the present invention is only limited by the terms of the appended claims.

[0134] Example 1: Purification of 3-fucosyllactose from bacterial fermentation

[0135] HMO 3-fucosyllactose is produced by bacterial fermentation, and the bacteria are removed from the fermentation broth by filtration. To remove bacterial cells, the fermentation broth was filtered through a polyethersulfone membrane ( MP005; Microdyn-Nadir, Wiesbaden) was used for microfiltration, followed by ultrafiltration using a hollow fiber module (ULTRADYN FS-10-FS FUS-1582, 150 kDa MWCO; Microdyn-Nadir, Wiesbaden, Germany).

[0136] The cell-free liquid was then treated by diafiltration and nanofiltration steps to remove salts and smaller molecules, thereby increasing the purity of 3-FL. A reverse osmosis system model RO40404 (Aqmos GmbH, Rodgau, Germany) was equipped with a Filmtec TM NF270 nanofiltration module. The inlet pressure was set to 8 bar, and the solution was treated 3 times with an equal volume of reverse osmosis water to increase the concentration by halving the starting volume. The 3-FL solution was then passed through a FS-10-FS FUS0181 10 kDa hollow fiber nanofiltration module (Microdyn-Nadir) to remove proteins and peptides.

[0137] Ions of similar size to 3-FL were removed from the process stream by electrodialysis using a PCCell P15 system (PCCell GmbH, Heusweiler, Germany) equipped with a PCCell ED 1000A membrane stack containing a CEM:PCSK cation exchange membrane and a CEM:PcAcid60 anion exchange membrane with a size exclusion limit of 60 Da. The conductivity of the starting solution was 8 to 11 mS / cm 2 Electrodialysis continued until the conductivity dropped to 0.5 mS / cm 2 .

[0138] The 3-FL solution was then treated with activated carbon powder to remove the pigment. The solution was stirred with Norit DX1 activated carbon for 2 hours and then filtered to remove the activated carbon.

[0139] Another round of electrodialysis was performed using the same setup as above. The conductivity of the starting solution was 1.0-1.5 mS / cm 2 Electrodialysis continued until the conductivity dropped to 0.3 mS / cm 2 .

[0140] After electrodialysis, the 3-FL solution was concentrated by reverse osmosis using an Emrich EMRO 1.8 reverse osmosis system (Emrich Edelstahlbau, Polch, Germany) equipped with a CSM RE8040BE reverse osmosis module. The solution was concentrated until the flow rate of the filtration system dropped below 50 L / h. The dry matter after concentration was 20-25 wt %. For spray drying (see Example 2), the 3-FL solution was further concentrated to a dry matter of 45 wt % using a Hei-VAP industrial evaporator (Heidolph Instruments GmbH, Schwabach, Germany). The highly concentrated 3-FL solution was sterilized by filtration through a 5 kDa Spira-Cell WYUP005 2440 C ultrafiltration membrane (Microdyn-Nadir) to remove endotoxins.

[0141] Figure 6 and Figure 7 Shown is the HPLC chromatogram of 3-fucosyllactose purified from clarified fermentation broth by this method. Figure 6 shows the HPLC chromatogram of the clarified fermentation broth, and Figure 7 It is shown that from Figure 6 HPLC chromatogram of the sterile filtered process stream obtained in the clarified fermentation broth of the HPLC chromatogram shown. Comparison of these chromatograms shows that most of the peaks (each peak representing at least one compound) in the HPLC chromatogram of the clarified fermentation broth are absent in the HPLC chromatogram of the sterile filtered process stream.

[0142] Example 2: Obtaining 3-fucosyllactose in solid form by spray drying.

[0143] As described in Example 1, the 3-FL solution obtained by filtration and electrodialysis was concentrated to 45% by weight and filter sterilized to remove any bioburden and endotoxins. The highly concentrated and sterile 3-FL solution was then spray dried using a LTC-GMP spray dryer (Nubilosa, Konstanz, Germany). A 45% by weight 3-FL solution was passed through the spray dryer nozzle at 130°C and 3.5 bar, and the flow rate was adjusted to maintain an exhaust temperature of 66-67°C. Using these settings, a spray-dried powder with a moisture content of less than 5% was obtained. The moisture content was determined by Karl-Fischer titration.

Claims

1. A method for purifying target oligosaccharides from a fermentation broth, the method comprising: - providing a fermentation broth containing target oligosaccharides, biomass, microbial cells and other carbohydrates except the target oligosaccharides; - removing microbial cells from the fermentation broth by microfiltration using a polyethersulfone membrane with a nominal pore size of 0.05 μm and ultrafiltration using a hollow fiber module with a MWCO of 150 kDa to provide a process stream; - Treatment of process streams by diafiltration and nanofiltration steps to remove salts and smaller molecules; - Electrodialysis was used to treat the process stream with a starting solution conductivity of 8 to 11 mS / cm 2 Electrodialysis continued until the conductivity dropped to 0.5 mS / cm 2 ; - treating the process stream with activated carbon to obtain a depigmented process stream; - The process stream was treated again using electrodialysis until the conductivity dropped to 0.3 mS / cm 2 ; - Concentrating the process stream and filter sterilizing to remove endotoxins to obtain a purified preparation of the target oligosaccharide, - wherein the target oligosaccharide is 3-fucosyllactose.

2. The process according to claim 1, wherein the process stream is subjected to a concentration step by reverse osmosis and / or an additional nanofiltration step.

3. The method according to claim 1, wherein the process stream is treated by using a RO40404 type reverse osmosis system equipped with Filmtec TM The concentration step was performed using a NF270 nanofiltration module.

4. The method according to claim 3, wherein the concentration step is carried out under the following conditions - the nanofiltration is performed at a temperature < 80°C; and / or - the reverse osmosis is carried out at a temperature of 20°C to 50°C; and / or - At pressures >5 bar to <50 bar.

5. The method according to any one of claims 2 to 4, wherein the process stream is subjected to a concentration step such that the concentration of the target oligosaccharide is ≥ 100 g / L. The method according to claim 1 , wherein the electrodialysis is electrodialysis under neutral conditions or electrodialysis under acidic conditions.

7. The method according to any one of claims 1 to 4, wherein the process stream is subjected to the removal of proteins and peptides by using a FS-10-FSFUS018 110 kDa hollow fiber nanofiltration module.

8. The method of claim 1, wherein the process stream is spray dried under the following conditions: i) the inlet temperature range is 110-150°C and the outlet temperature range may be 60-80°C; and / or ii) The concentration of the target oligosaccharide in the process stream is 5-60 wt%.

9. The method of claim 1, wherein the purity of the target oligosaccharide in the purified preparation is ≥ 80 wt. %, based on the dry matter of the purified preparation.

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