Method for producing purified human milk oligosaccharides derived from a microbial fermentation process

Abstract

A method for producing human milk oligosaccharides (HMOs) obtained by microbial fermentation is disclosed, comprising the steps of: a) providing a fermentation broth containing at least one HMO and biomass; b) adding a polyvalent cation salt to the fermentation broth; and c) subjecting the mixture of step b) to centrifugation.

Description

[Technical Field]

[0001] The present invention relates to a method for producing purified human milk oligosaccharides by microbial fermentation. More specifically, the present invention relates to facilitating the separation of residual biomass and other solid contaminants from the fermentation broth by centrifugation. [Background technology]

[0002] Human breast milk contains a significant amount of carbohydrates. Carbohydrates present in human breast milk include monosaccharides such as L-fucose and N-acetylneuraminic acid, the disaccharide lactose, and up to 20 g / L of oligosaccharides, so-called "human milk oligosaccharides (HMOs)." HMOs represent the third most abundant component of human breast milk. Over 150 structurally distinct oligosaccharides are estimated to be present in human milk. Selected HMOs are listed in Table 1. Approximately 10–13 of these HMOs are present in human milk at concentrations ranging from several hundred milligrams to several hundred grams per liter (Thurl et al., (2017), Nutrition Reviews 75(11) 920–933). Among HMOs, neutral HMOs are known, as are acidic HMOs, which contain at least one N-acetylneuraminic acid (NeuAc) moiety. The structural complexity and abundance of these oligosaccharides is unique to human milk and has not been found in the milk of other mammals, including domesticated dairy animals.

[0003] Because HMOs are not digestible by humans, the physiological role of these sugars has been investigated for decades. The prebiotic effect of HMOs was discovered over 100 years ago. When consumed, HMOs can modulate the composition of the human gut microbiome by supporting the growth of beneficial bacteria.

[0004] [Table 1-1]

[0005] [Table 1-2]

[0006] Several other functional effects of HMOs, particularly their effects on neonatal development, have become clear in recent years. HMOs are known to act as decoys to reduce the risk of infection by bacterial and viral pathogens that attach to human cells by binding to their surface glycoproteins. Furthermore, various HMOs have anti-inflammatory effects and act as immunomodulators. Therefore, HMOs have been proposed to reduce the risk of developing food allergies. The positive effects of sialylated HMOs on the development of the neonatal central nervous system are also extensively discussed (reviewed in "Prebiotics and Probiotics in Human Milk, Origins and Functions of Milk-Borne Oligosaccharides and Bacteria," edited by McGuire M., McGuire M., and Bode L., Academic Press (2017)).

[0007] To take advantage of the beneficial effects of HMOs, efforts have been made to add individual HMOs to nutritional compositions, particularly infant formulas. The limited supply of HMOs for supplementing nutritional compositions has led to the development of procedures for chemically synthesizing HMOs. The shortcomings of such chemical synthesis have led to biocatalytic approaches, which use purified enzymes, such as glycosyltransferases, to synthesize HMOs in vitro. Today, individual HMOs are produced on an industrial scale using fermentation of genetically engineered microbial cells ( WO2015 / 150328A1 , WO2017 / 043382A1 , WO2010 / 070104A1 , WO2012 / 097950A1 ). The HMOs are synthesized by genetically engineered microbial cells and can be recovered from the fermentation medium and / or cell lysate to obtain a substantially pure preparation of the HMO.

[0008] During their recovery from the fermentation broth, HMOs are usually present in the form of a liquid process stream, e.g., an aqueous solution, which contains the HMO of interest and which needs to be separated from the solid residual phase of the fermentation broth during the critical first step of the purification process, which separation is generally achieved in the first step either by filtration or by centrifugation.

[0009] Due to the inevitable clogging of the filtration membrane and the need for repeated filtration steps to ensure that most of the solid phase is captured by the filter without compromising the yield of HMO, filtration alone tends to be complicated in large-scale production. Currently, a relatively good fermentation clarified liquid can be obtained by using a method that combines a depth filtration system with more than five layers of depth filtration membranes for the treatment of cell fermentation liquid, but this method has limited processing capacity and higher costs, which greatly limit the large-scale application of the depth filtration system in production.

[0010] On the other hand, centrifugation is widely known and applied in large-scale industrial production. For example, disc centrifuges are widely used in the fields of fruit juice beverages, tea beverages, winemaking, seasonings, edible oils, plant proteins, animal proteins, fish processing, biopharmaceuticals, starch processing, industrial fermentation, yeast extraction, natural extraction, etc., and are widely used in large-scale industrial production. Continuous processing modes are highly suitable for large-scale cell fermentation production in the field of biopharmaceuticals. However, when centrifugal clarification of fermentation broth using a disc centrifuge at the end of fermentation is performed as the first separation step, the clarified supernatant still contains multiple components, such as a small number of cells, colloidal substances, cell debris, etc. Because the efficiency of the centrifuge separation step is important for all subsequent purification steps required to achieve food-grade products, especially in large-scale production, there is still a need for improved centrifugation of fermentation broth in the production of HMOs. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention aims to provide an improved method for processing HMO-producing microbial fermentation broth using a centrifuge system, addressing the problems of low throughput, high turbidity, large depth filtration membrane dosage, and higher processing costs in the previous fermentation broth pretreatment clarification process, in order to more efficiently remove various impurities in the fermentation broth, such as cell debris, host DNA, proteins, etc., and obtain a clarified fermentation broth containing target HMOs with a smaller remaining solid portion, thus further simplifying the process steps required for downstream HMO purification and reducing process production costs. [Means for solving the problem]

[0012] The object is to provide a method for the production of human milk oligosaccharides (HMOs) obtained by microbial fermentation, comprising: a) providing a fermentation broth comprising at least one HMO and biomass; b) adding a multivalent cation salt to the fermentation broth; and c) subjecting the mixture of step b) to centrifugation. This is achieved by a method comprising:

[0013] In a first aspect, a method for producing purified human milk oligosaccharides (HMOs) obtained by microbial fermentation is provided. In a second aspect, there is provided the use of a polyvalent cation salt as an additive to the fermentation broth at the end of a microbial fermentation process in the production of human milk oligosaccharides. DETAILED DESCRIPTION OF THE INVENTION

[0014] According to a first aspect, there is provided a method for producing human milk oligosaccharides (HMOs) by microbial fermentation, comprising: a) providing a fermentation broth comprising at least one HMO and biomass; b) adding a multivalent cation salt to the fermentation broth; and c) subjecting the mixture of step b) to centrifugation. A method is provided, comprising:

[0015] As currently and generally understood in the related art, "microbial fermentation" should be understood as a generally large-scale, industrial metabolic process in which the enzymatic degradation and utilization of nutrients, particularly carbohydrates, and the conversion of compounds to other compounds occurs during the cultivation of microorganisms, such as bacteria, fungi, and molds. As such, industrial or large-scale microbial fermentation is the process of controlling microorganisms, i.e., bacteria, yeasts, and molds, to modify foods to produce desired products.

[0016] Currently, and generally in the related art, "microorganism" refers to and encompasses any microscopic organism, including either single cells, cell clusters, or multicellular, relatively complex organisms, suitable for use in the methods according to the invention, and includes in particular bacteria and yeast. Microorganisms used according to the invention may be cultured in liquid media and generally require a carbon source in the media in order to grow and replicate.

[0017] Presently, and throughout this invention, "recombinant" refers to genetically engineered DNA prepared by transplanting or splicing a gene from one species into the cells of a host microorganism of a different species, where such DNA becomes part of the host's genetic makeup and is replicated.

[0018] Consequently, "host microorganism" is intended to mean any microorganism containing a nucleic acid sequence or expressed protein foreign to / not naturally occurring in a recombinant host microorganism, wherein the foreign / not naturally occurring nucleic acid sequence in said microorganism is integrated into the genome of the host microbial cell. "Non-naturally occurring" thereby means that the nucleic acid sequence / protein (e.g., enzyme) is foreign to said host microbial cell, i.e., the nucleic acid sequence / protein is heterologous with respect to the microbial host cell. The heterologous sequence may be stably introduced into the genome of the host microbial cell, for example, by transfection, transformation, or transduction; the technique to be applied depends on the host cell into which the sequence is to be introduced. Various techniques are known to those skilled in the art and are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Thus, the host microorganism into which the heterologous sequence has been introduced produces the heterologous protein encoded by the nucleic acid sequence according to the present invention.

[0019] For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof, as well as nucleotide sequences encoding the enzymes required for the metabolic pathway and / or biosynthesis of the human milk oligosaccharide of interest. Introduction of nucleic acid sequences into host microbial cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986), and Sambrook et al., 1989, supra.

[0020] A wide variety of expression systems can be used to produce the HMOs of the present invention. Such vectors include, inter alia, chromosomal, episomal, and virally derived vectors, such as those derived from bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and combinations thereof, such as those derived from plasmids and bacteriophage genetic elements, e.g., cosmids and phagemids. Expression system constructs can contain control regions that regulate, as well as engender, expression. Generally, any system or vector suitable for maintaining, propagating, or expressing polynucleotides and synthesizing HMOs in a host can be used for expression in this context. The appropriate DNA sequence can be inserted into the expression system by any of a variety of well-known and routine techniques, such as those set forth in Sambrook et al., supra.

[0021] Currently, "desired oligosaccharides" designates oligosaccharides that are specifically and intentionally produced in the applied process. Accordingly, "undesired oligosaccharides" refer to oligosaccharides that are not intended to be produced or generated during the production of desired oligosaccharides or oligosaccharides formed by the used organisms unrelated to the desired oligosaccharides. "Metabolic products" or "metabolic sugar products" or "sugar intermediates" refer to sugars, i.e., carbohydrate products, that are generated during the production of desired oligosaccharides, and "unused sugar substrates" refer to starting molecules or moieties that are used for / during the production of desired oligosaccharides.

[0022] As mentioned at the outset, the present invention relates to a method for producing a desired oligosaccharide using a host microorganism, wherein said desired oligosaccharide does not naturally occur in said host cell and is a human milk oligosaccharide. Reference in this context is made to the HMOs listed in Table 1 above.

[0023] Using the method according to the invention, an effective production method is provided whereby the desired oligosaccharides can be produced in a form that is essentially no longer present of unwanted solids or otherwise interfering solid products from the fermentation broth or unused solid substrates.

[0024] As used herein, the term "culturing" means growing a microorganism in a medium and under conditions that are acceptable and suitable for the production of the desired human milk oligosaccharides. Several suitable host microorganisms and media and conditions for their culture will be readily available to one of skill in the art upon reading the present disclosure in conjunction with the art's technical and professional background.

[0025] In step a) of the disclosed method of the present invention, a fermentation broth is provided that contains at least one HMO and biomass from a fermentation process. The desired HMO, such as 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), lacto-N-tetraose (LNT), or 6'-sialyllactose (6'-SL), is produced by fermentation of genetically modified microorganisms. Fermentation can be carried out in any suitable fermentation medium, for example, a chemically defined fermentation medium.

[0026] The fermentation medium can vary based on the microorganism used and the desired HMO. The fermentation medium may be supplemented with trace minerals and vitamins, such as calcium pantothenate, biotin, nicotinic acid, myo-inositol, thiamine HCl, and p-aminobenzoic acid. Food-grade processing aids, such as antifoaming agents and pH control agents, may also be used. The temperature of the fermentation step is typically 10-50°C, preferably 25-35°C, and more preferably 28-32°C. The pH of the fermentation step is typically 3-8, preferably 5-7, and more preferably 5.5-6.5. At the end of the fermentation step, the fermentation broth may be subjected to a heat treatment step to inactivate the microorganisms before downstream processing steps.

[0027] Preferably, the microorganism is a genetically modified yeast, such as a Saccharomyces strain, a Candida strain, a Hansenula strain, a Kluyveromyces strain, a Pichia strain, a Schizosaccharomyces strain, a Schwanniomyces strain, a Torulaspora strain, a Yarrowia strain, or a Zygosaccharomyces strain. More preferably, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, and the like. delbrueckii, Yarrowia lipolytica, Zygosaccharomyces rouxii, or Zygosaccharomyces bailii.

[0028] Preferably, the microorganism is also of the genus E. coli or S. cerevisiae, Bifidobacterium, Lactobacillus, Enterococcus, Streptococcus, Staphylococcus, Peptostreptococcus, Leuconostoc, Clostridium, Yersinia, or the like. - Eubacterium, Veilonella, Fusobacterium, Bacterioides, Prevotella, Escherichia, Propionibacterium and Saccharomyces, Bifidobacterium adolescentis adolescentis, B. animalis, B. bifidum, B. breve, B. infantis, B. lactis, B. longum; Enterococcus faecium; Escherichia coli, Klyveromyces marxianus; Lactobacillus acidophilus acidophilus, L. bulgaricus, L. casei, L. crispatus, L. fermentum, L. gasseri, L. helveticus, L. johnsonii, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius, L. sakeisakel, Lactococcus lactis (including but not limited to subspecies lactis, cremoris, and diacetyl actis); Leuconostoc mesenteroides (including but not limited to subspecies mesenteroides); Pediococcus acidilactici, P. pentosaceus; Propionibacterium acidipropionici, P. freudenreichii ssp. shermanii; Staphylococcus carnosus; and Streptococcus thermophilus thermophilus).

[0029] The desired HMO, such as 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), lacto-N-tetraose (LNT), or 6'-sialyllactose (6'-SL), is purified from the fermentation broth, which is the product of the fermentation process. After fermentation, the broth containing the desired HMO is subjected to a separation process as summarized below.

[0030] The term "fermentation broth," as used herein, refers to the product obtained from the fermentation of a microorganism. Thus, the fermentation product includes the biomass (cells), the fermentation medium, the residual substrate material, and any molecules / by-products produced during fermentation, such as the desired HMO. After each step of the purification process, one or more of the components of the fermentation product are removed, resulting in a more purified HMO.

[0031] Presently, and throughout this invention, "biomass" means each and every type of cellular or microbial material, cellular or microbial debris, high molecular weight molecules, and / or residual lysed products, as well as other solid particles attached thereto or present in the fermentation broth to be treated in subsequent separation steps of the method of the invention.

[0032] Optionally, the fermentation broth may be subjected to a heat treatment step to kill bacteria or other undesirable microorganisms that may be present to any significant extent. Typically, the desired HMOs are present in the liquid, or in other words, the supernatant, phase of the fermentation broth, but within the meaning of the present invention, it is also encompassed that the desired HMOs are present or contained in the biomass fraction of the fermentation broth.

[0033] In step b) of the method of the present invention, a polyvalent cation salt is added to the fermentation broth. As used herein, the term "multivalent cation" refers to a cation capable of forming multiple ion pairs, e.g., a multiply charged cation, e.g., a doubly or triply charged cation. Any convenient multivalent cation may find use in the subject salt compositions. In some embodiments, the multivalent cation is divalent. Divalent cations of interest include, but are not limited to, magnesium, zinc, and calcium. In some embodiments, the multivalent cation is trivalent. Trivalent cations of interest include, but are not limited to, aluminum. In certain embodiments of salt compositions, at least one multivalent cation is selected from the group consisting of magnesium, zinc, aluminum, and calcium. In certain embodiments of salt compositions, at least one multivalent cation is magnesium. In certain embodiments of salt compositions, at least one multivalent cation is zinc. In certain embodiments of compositions, at least one multivalent cation is aluminum. In certain embodiments of compositions, at least one multivalent cation is calcium.

[0034] In the sense of the present invention, the term "polyvalent cation salt" covers any polyvalent cation composition containing a suitable counteranion. The counteranion may be monovalent, such as chloride, bromide, fluoride, acetate, or it may be polyvalent, such as divalent or trivalent, such as sulfate, sulfite, phosphate, phosphonate, carbonate.

[0035] The choice of polyvalent cation salt may vary depending on solubility, stability, and possible interactions with other components present in the fermentation broth. The polyvalent cation salt may be added to the fermentation broth at any time before the centrifugation step c) to separate the biomass from the supernatant phase is initiated. Preferably, the polyvalent cation salt is added to the fermentation broth shortly before the subsequent centrifugation step is initiated.

[0036] The polyvalent cation salt may be added in any form suitable, especially for large-scale industrial production, for example as a solid or in the form of an aqueous solution or dispersion. In step c) of the method of the present invention, the mixture of step b), i.e., the fermentation broth containing the desired HMOs and biomass with added polyvalent cation salts, is subjected to centrifugation, thus separating some, most, or all of the solid fraction of the fermentation broth from the liquid supernatant phase.

[0037] Centrifugation techniques, which primarily exploit differences in size, density, specific gravity, and shape, are well known and established in the current field of biomanufacturing. Typically, centrifuges have a rotating mechanism, while filters have no moving parts and are therefore simpler to operate. On the other hand, filters offer slower processing, are prone to clogging, have higher operating costs, and generally require larger surface and floor areas compared to centrifuges.

[0038] Most centrifugation technologies for biomanufacturing are based on cylindrical bowls or disc stacks. Cylindrical bowl technology is primarily used for microbial cultures because it can generate high centrifugal forces (>10,000 g) and produce solid pastes with low supernatant content. Once the solids fill the bowl, the spinning bowl must be stopped to release the solids. Disc stack (Westfalia CSV series) and countercurrent (e.g., kSep, Elutra, and Rotea) centrifuges do not tightly pack cells but form slurries, discharging solids intermittently without the need for stopping during discharging. While countercurrent centrifugation techniques exert low shear forces, other bowl or disc stack centrifugation techniques exert high shear forces and therefore are not used when intact cells are required as a product or intermediate product. Therefore, the type of centrifugation technology can be easily selected by those skilled in the art depending on the desired production parameters.

[0039] It has surprisingly been found that adding polyvalent cation salts to the fermentation broth before the broth is subjected to the centrifugation separation step results in more effective separation. Notably, not only can an increase in the amount of solid phase that accumulates during centrifugation be observed, but also a decrease in the turbidity of the liquid supernatant phase. In this way, the polyvalent cation salts act as a separation-aiding additive, making the subsequent purification steps leading to a pure HMO product easier and faster.

[0040] Without being bound by any theory, it is presumed that the polyvalent cation salts aid in the settling of remaining biomass and other residual solids of the fermentation process, possibly by increasing the size of the precipitate or disrupting the tendency of any residual solids from the fermentation process to become finely dispersed in the fermentation medium, thereby increasing the efficiency of centrifugation overall.

[0041] In other words, the present invention provides a method for producing human milk oligosaccharides (HMOs) obtained by microbial fermentation, the method comprising: a) providing a fermentation broth containing at least one HMO and biomass from a fermentation; and b) subjecting the mixture of step a) to centrifugation wherein a multivalent cation salt is added to the fermentation broth of step a) before the centrifugation of step b) is initiated.

[0042] In another embodiment, the present disclosure provides the aforementioned method, wherein the HMO is selected from the group consisting of 2'-FL, 3-FL, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LNFPV, 3'-SL, 6'-SL, LST-a, LST-b, LST-c, and DSLNT.

[0043] For the named HMOs, good results can be achieved in improved separation quality of the centrifugation separation step. Reference is made to the structures of the HMOs shown in Table 1 presented above.

[0044] In another embodiment, the present disclosure provides the aforementioned method, wherein said polyvalent cation salt is a divalent or trivalent cation salt, preferably selected from the group consisting of Mg, Ca, Sr, Zn, Al, Fe, Mn, and Cu salts and mixtures thereof.

[0045] As counter anion of said divalent or trivalent cation salts there may be present monovalent, divalent, trivalent or tetravalent anions, preferably monovalent or divalent anions. Typically, such salts are selected that are well known in the field of biomolecule production and therefore not only readily available but also certified for use in biomolecule production of food-grade products, such as chlorides, phosphates, phosphonates, sulfites, or sulfates of Mg, Ca, Fe, and Zn.

[0046] Another important aspect in the selection of the polyvalent cation salt is the ease of removing the salt from the HMO-containing phase, either the solid biomass phase separated by the centrifugation step or preferably the liquid supernatant phase. In subsequent further purification steps, the properties of the polyvalent cation salt will best play a role in selecting a well-known, scalable separation method that would be performed anyway, even without the presence of the polyvalent cation salt. Preferably, the polyvalent cation salt can be removed from the HMO-containing phase in a subsequent workup step by ion exchange using ion exchange absorption resins, ion exchange bed filtration, ion exchange column filtration, or related techniques (chromatography).

[0047] In another embodiment, the present disclosure provides the aforementioned method, wherein the multivalent cation salt is selected from the group consisting of MgCl, ZnCl, and CaCl. These salts have good solubility in water and can therefore be easily handled when measuring the amount added to the fermentation broth in the form of an aqueous solution. Furthermore, the cations and anions of the salts can be easily removed in subsequent workup steps by ion exchange methods. In addition, the salts are readily available in high-grade quality and are typically certified safe for use in biomolecule production of food-grade materials.

[0048] In another embodiment, the present disclosure provides the above method, wherein the multivalent cation salt is added to the fermentation broth of step a) in the form of an aqueous solution. In that way, not only can easy handling be achieved in measuring the amount added to the fermentation broth, but continuous large-scale operation of the method of the present invention can be facilitated.

[0049] In another embodiment, the present disclosure provides the above method, wherein the concentration of polyvalent cation salt in the fermentation broth after the addition of polyvalent cations to the fermentation broth (step b) is ≧0.05 wt %, preferably ≧0.1 wt %, more preferably ≧0.15 wt %, and most preferably ≧0.175 wt %, and ≦0.8 wt %, preferably ≦3.0 wt %, ≦2.5 wt %, and most preferably ≦2.0 wt %.

[0050] Within a predetermined range of the concentration of polyvalent cation salts in the fermentation broth to be subsequently centrifuged, not only can improved separation results between the precipitate and the supernatant be achieved, but also easy removal of the polyvalent cation salts can be achieved in subsequent workup. Measurement of electrical conductivity can be carried out as an indicator of the scale of the removal task for removing the polyvalent cation salts. In this way, it has surprisingly been found that within a preferred range of salt concentrations, improved separation of the solid and supernatant liquid phases can be achieved, while at the same time the concentration can be maintained in a range in which easy removal of the salts can be achieved in subsequent workup steps.

[0051] In preferred embodiments, the concentration of polyvalent cation salt in the fermentation broth immediately prior to centrifugation is 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% by weight.

[0052] In one embodiment, the present disclosure provides the aforementioned method, wherein the multivalent cation salt is CaCl. It is known that calcium chloride can be used in microbial fermentation processes, mostly at low concentrations as a salt chemical in the fermentation medium. As such, its use has already been approved for food-grade fermentation-based production. Aside from that, the use of calcium chloride can have the advantage that not only calcium cations but also chloride anions can be very easily removed from the separated liquid phase after centrifugation, for example, by ion exchange. Many ion exchange resins are known that provide optimized removal of calcium cations and chloride anions in downstream workup processes. Another advantage can be seen in the non-toxic behavior of calcium chloride toward microbial cells. In fermentation processes where the desired HMOs are separated from the biomass without lysis or other destructive means, such non-toxicity of centrifugation-assisted polyvalent cation salts is beneficial, potentially for reusing the microbial material.

[0053] In another embodiment, the present disclosure provides the above method, wherein the polyvalent cation salt is added as an aqueous solution to the fermentation broth of step a) in a volumetric amount (v / v) ranging from ≧0.25 or ≧0.5 to ≦2.0 or ≦1.75, preferably in a volume ratio of about 1.0.

[0054] In that way it is advantageously possible to provide an input flow to the preferably continuous centrifugation steps that is well controllable and has good flow behavior. In another embodiment, the present disclosure provides the above method, wherein the fermentation broth of step a) further contains at least one monosaccharide, preferably selected from the group consisting of L-fucose, glucose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid.

[0055] In another embodiment, the present disclosure provides the above method, wherein the fermentation broth of step a) further contains at least one additional component, said at least one additional component preferably selected from the group consisting of lactose, sucrose and glycerol.

[0056] In another embodiment, the present disclosure provides the above method, wherein the centrifugation in step c) is carried out in the range of 2,000 g to 15,000 g and / or for at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes, at a temperature of 2°C to 60°C.

[0057] The minimum duration of the centrifugation process depends on various circumstances, such as the g-force applied, continuous or batch processing, the input and output volumes / speeds of the liquid and solid phases, the temperature, the type of centrifuge used, etc. Those skilled in the art of fermentation processes for food-grade HMOs can easily find the optimal centrifugation period for the particular gears and parameters used. Regardless of this aspect, continuous centrifugation is possible for industrial-scale HMO production, where the biomass is discharged from the centrifuge / rotor after a predetermined interval. The optimal interval for such continuous centrifugation also depends on the particular gears and parameters, and can be determined and applied by those skilled in the art.

[0058] The nature of the centrifugation parameters, such as high or low shear force, to be selected depends, for example, on whether the desired HMOs are contained in the supernatant liquid phase of the fermentation broth or whether the desired HMOs are contained in the (still intact) cells of the fermentation broth. In the first case, a higher shear force and a higher range of g-forces can be selected to keep the cells intact, while in the latter case, a lower shear force or a lower range of g-forces is preferably selected. In a subsequent step, the cells containing the desired HMOs can be lysed or otherwise worked up to release the HMOs from the separated biomass. However, the core improvement of the present invention, improved separation by adding polyvalent cation salts to the fermentation broth before centrifugation, is achieved in both cases.

[0059] In another embodiment, the present disclosure provides a method comprising the steps of: i) adding a solution of a multivalent cation salt to the centrifugation pellet obtained by step b) and centrifuging the resulting mixture to obtain a further supernatant solution; ii) subjecting the supernatant solution of the first and any further optional centrifugation steps to at least one filtration; iii) treating the supernatant solution of the first and any further optional centrifugation steps and / or the filtrate of step ii) at least once with a cation exchange resin and / or at least once with an anion exchange resin; iv) subjecting the supernatant solution of the first and any further optional centrifugation steps and / or the filtrate of step ii) or iii) to at least one electrodialysis; v) treating the supernatant solution of the first and any further optional centrifugation steps and / or the filtrate of steps ii), iii) or iv) with activated carbon at least once; and / or vi) subjecting the liquid process stream obtained during the recovery of human milk oligosaccharides from the fermentation broth to at least one crystallization step and / or precipitation step and / or spray drying step. The method as described above further comprises the additional step of purifying at least one HMO selected from one or more of:

[0060] The additional purification steps to obtain pure HMO from centrifugation can be performed in various orders and / or repetitions suitable for the desired HMO. After the centrifugation step, the isolated stream is subjected to one or more purification steps, which may include ultrafiltration, nanofiltration, anion exchange, cation exchange, and decolorization, to form a purified isolated stream. The purified isolated stream is then processed through a concentration step and a dewatering step. These steps may be performed in any order. In other words, the purified isolated stream may first be processed through a concentration step, and then the product of the concentration may be sent to a dewatering step. Alternatively, the purified isolated stream may first be subjected to a dewatering step, and then the product of the dewatering step may be sent to a concentration step. The concentration step may include evaporation, reverse osmosis filtration, and / or nanofiltration. The evaporation process may include, for example, falling film evaporation, rising film evaporation, and rotary evaporation to form a concentrated, purified isolated stream. The dewatering step may include at least one of crystallization, drying, evaporation, and conventional filtration to form an HMO product.

[0061] In step i), repeated rounds of multivalent cation salt-assisted separation by centrifugation are carried out to optimize the overall yield of the desired HMO by washing the centrifugation pellet (slurry) with aqueous solutions of multivalent cation salts.

[0062] In step ii) at least one filtration step is carried out which may preferably be selected from microfiltration, nanofiltration and / or diafiltration and / or reverse osmosis filtration.

[0063] These filtration methods are well known to those skilled in the art and may be selected in relation to the purification task. For example, microfiltration and nanofiltration as workup of process streams from HMO production fermentations is described in European Patent Application EP 3 741 77. For the purposes of this specification, the term filtration also refers to processes using plate and frame filtration, recessed chamber filtration, belt filtration, vacuum filtration, horizontal metal leaf filtration, vertical metal leaf filtration, stacked disc filtration, rotary vacuum filtration and combinations thereof.

[0064] In step iii), at least one treatment with a cation exchange resin and / or with an anion exchange resin is carried out on at least one of the supernatant solutions of the first and further optional centrifugation steps and / or the filtrate of step ii).

[0065] The ion exchange process is advantageously optimized for the removal of polyvalent cation salts and other cations and anions present in the fermentation medium. Specific cation and anion exchange resins are well known to those skilled in the art, and such steps are described, for example, in WO2019063757A1.

[0066] Cation exchange processes can be used to separate charged molecules from HMO-containing streams. Cation exchange (CEX) stationary phases typically contain negatively charged aliphatic sulfonic acid groups in aqueous solution, which tightly bind any strongly basic analytes. This step removes positively charged components, such as residual ammonia, metals, and peptides. The binding capacity of the resins used is generally 1.2-2.2 mmeq / L. Typical resins used for cation exchange include Dow Dowex 88, Resindion JC series (e.g., JC603), and Resindion PK grades (e.g., PK216).

[0067] An anion exchange step can be used to separate charged molecules. In an anion exchange column, the packing material is positively charged and therefore retains negatively charged molecules through Coulomb interaction. This step removes negatively charged components, such as hydrochloride, sulfate, phosphate, organic acids, and negatively charged particles. The binding capacity of the resin used is generally 0.8-2.0 mmeq / L. Typical resins for anion exchange include the Resindion Relite series, such as RAM2, DI 82, and JA100, and the Diaion WA series, such as WA20.

[0068] In step iv), the supernatant solution of the first and further optional centrifugation steps and / or the filtrate of step ii) or iii) is subjected to at least one round of electrodialysis.

[0069] Electrodialysis is a process for the selective removal of ions from a solution. It can be used in conjunction with fermentation to separate ionic fermentation products, such as organic acids, and / or for desalination. Electrodialysis combines dialysis and electrolysis and can be used to separate or concentrate ions in a solution based on the selective electromigration of ions in the solution through a semipermeable membrane.

[0070] The basic principle of electrodialysis consists of an electrolytic cell containing a pair of electrodes connected to a direct current generator and submerged in an electrolyte solution for ion conduction. The electrode connected to the positive pole of the 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 results from the migration of anions and cations toward the anode and cathode, respectively. The membranes used in electrodialysis are essentially sheets of porous ion-exchange material with negatively or positively charged groups and are therefore described as cationic or anionic membranes, respectively. Ion-exchange membranes are typically made of polystyrene bearing suitable functional groups (e.g., sulfonic acid for cationic membranes or quaternary ammonium groups for anionic membranes) crosslinked with divinylbenzene. The electrolyte can be, for example, sodium chloride, sodium acetate, sodium propionate, or sulfamic acid. The electrodialysis stack is then assembled so that the anionic and cationic membranes are parallel, as in a filter press, between two electrode blocks, so that the stream undergoing ion depletion is well separated from the stream undergoing ion enrichment (the two solutions are also called the diluate (subject to ion depletion) and the concentrate (subject to ion enrichment). The heart of the electrodialysis process is the membrane stack, which consists of several anion- and cation-exchange membranes mounted between two electrodes and separated by spacers. By applying a direct current, the anions and cations migrate across the membranes toward the electrodes.

[0071] A decolorization step v) can be performed to remove pigment-containing components. This step can be performed using activated carbon, such as Norit CAI activated carbon, hydrophobic interaction chromatography (HIC), or another adsorption resin that can be functionalized, such as Resindion Relite RAD / F. Decolorization can be performed either before or after the other named purification steps.

[0072] In step vi), the process stream is subjected to at least one crystallization step and / or precipitation step and / or spray drying step. A crystallization process can be used to dehydrate purified HMO material by first forming HMO nuclei in a supersaturated solution and then growing the crystals. Such processes can be either batch or continuous. Typical conditions used for crystallization are set forth in WO2018 / 164937, the disclosure of which is incorporated herein by reference. The method involves concentrating a starting HMO-containing solution to a supersaturated state and then precipitating HMO crystals from the supersaturated solution, preferably by subjecting the supersaturated solution to a temperature of at least 55°C. Preferably, the solution has an HMO content of at least 60% by weight and less than about 98% by weight. Crystallization is typically limited by viscosity as the percentage of HMO crystals grows, preferably with a typical endpoint of about 30% by weight crystals.

[0073] Alternatively, crystallization or precipitation of the at least one HMO from the process stream can be carried out by adding a suitable amount of a water-miscible organic solvent to the process stream containing the at least one HMO. The organic solvent can be selected from the group consisting of C1-C6 alcohols and C1-C4 carboxylic acids. The crystallization or precipitation step of the at least one HMO is not carried out at the end of the recovery process, so that residual amounts of organic solvent or carboxylic acid can be removed by a subsequent process step.

[0074] Suitable spray drying processes for the recovery of HMOs in solid form are described, for example, in EP 3524067 A1, EP 3494804 A1, EP 3494805 A1, EP 3494806 A1 or EP 3494807 A1.

[0075] In another embodiment, the present disclosure provides a method as described, wherein an aqueous solution containing at least one HMO and optionally at least one monosaccharide is spray dried at a nozzle temperature of at least 110°C, preferably at least 120°C, more preferably at least 125°C, and less than 150°C, preferably less than 140°C, more preferably less than 135°C.

[0076] In another embodiment, the present disclosure provides the above method, wherein the fermentation broth contains at least one HMO in an amount of less than 30% (w / v), less than 25% (w / v), or less than 20% (w / v). In additional or alternative embodiments, any of the solutions of steps i) through v) or the process stream of step vi) contains at least one HMO in an amount that provides a total amount of sugars of at least 20% (w / v), 30% (w / v), or 35% (w / v), and up to 45% (w / v), 50% (w / v), or 60% (w / v).

[0077] The present invention is further directed to the use of polyvalent cation salts in the fermentative production of human milk oligosaccharides, wherein the polyvalent cation salts are added to a fermentation broth containing human milk oligosaccharides and biomass before the fermentation broth is subjected to centrifugation to remove the biomass.

[0078] The addition of multivalent cation salts resulted in better separation of biomass and other solid materials from the liquid supernatant phase, and less observable coloration of the liquid phase. Preferably, the amount of multivalent cation salt added before the centrifugation step is determined by optimizing the trade-off between higher separation and decolorization action and keeping the conductivity of the liquid phase low to facilitate subsequent removal of cations and anions in later purification steps.

[0079] In one embodiment, the present disclosure provides the use of a multivalent cation salt in the above method, wherein the multivalent cation salt is calcium chloride. While the present invention will be described with reference to specific embodiments, the present invention is not limited thereto, but only by the scope of the claims. Furthermore, terms such as "first," "second," etc. in the description and claims are used to distinguish between similar elements, whether temporally, spatially, sequentially, or in any other manner, and are not necessarily used to describe an order. Terms so used are interchangeable under appropriate circumstances, and it should be understood that the embodiments of the present invention described herein can operate in orders other than those described or illustrated herein.

[0080] It should be noted that the term "comprising", when used in the claims, should not be interpreted as being restricted to the means listed thereafter, nor should it exclude other elements or steps. Thus, it should be interpreted as specifying the presence of the mentioned stated feature, integer, step or component, but without precluding the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression "a method comprising steps A and B" should not be limited to a method consisting only of steps A and B. It means that, in the context of the present invention, the only relevant steps of the method are A and B.

[0081] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily always refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[0082] Similarly, in describing representative embodiments of the invention, it should be understood that various features of the invention are sometimes grouped together in a single embodiment, drawing, or description thereof for the purpose of streamlining the disclosure and facilitating understanding of one or more of the various inventive aspects. This method of disclosure is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, aspects of the invention may require fewer than all features of any preceding disclosed embodiment. Thus, the claims following the Detailed Description are hereby expressly incorporated into this Detailed Description, with each claim standing on its own as a separate embodiment of this invention.

[0083] Furthermore, although some embodiments described herein include some features but not other features included in other embodiments, combinations of features from different embodiments are intended to form different embodiments within the scope of the present invention, as would be understood by one of ordinary skill in the art. For example, in the following claims, any of the claimed embodiments may be used in any combination.

[0084] In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the present invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order to facilitate understanding of the description and drawings.

[0085] The present invention is now described by a detailed description of several embodiments thereof. The present invention is limited only by the terms of the appended claims. [Example]

[0086] Example 1: Purification of 2'-FL by adding CaCl 2'-FL fermentation broth was collected directly from the fermenter at the end of fermentation and used in this experiment. Five centrifuge tubes, each containing 5 mL of fermentation broth (fb), were diluted with 5 mL of water (control sample), 0.50% (w / w) CaCl in water, 1.00% (w / w) CaCl in water, and 1.50% (w / w) CaCl in water, respectively. The mixtures were mixed by vortexing and centrifuged at 1800 g for 10 minutes at 10°C. The resulting solutions were transferred to new centrifuge tubes and analyzed by OD. 600 and conductivity were determined. By adding CaCl2, a lower OD 600 It was possible to obtain a 2'-FL solution having a pH of 10.0 or higher, which clearly indicates a better separation of the solids contained in the fermentation broth from the liquid supernatant.

[0087] [Table 2]

[0088] Example 2: Purification of 6'-SL by adding CaCl The 6'-SL fermentation broth was collected directly from the fermenter at the end of fermentation and used in this experiment. Five centrifuge tubes, each containing 5.5 mL of fermentation broth (fb), were diluted with 5 mL of water (control sample), 5 mL of 0.50% (w / w) CaCl in water, 5 mL of 1.00% (w / w) CaCl in water, and 5 mL of 1.50% (w / w) CaCl in water, respectively. The mixtures were mixed using a vortexer and centrifuged at 1800 g for 10 minutes at 10°C. The resulting solutions were transferred to new centrifuge tubes and analyzed by OD. 600 and conductivity were determined. By adding CaCl2, a lower OD 600 It was possible to obtain a 6'-SL solution having a pH of 10.0 or higher, which clearly indicates a better separation of the solids contained in the fermentation broth from the liquid supernatant.

[0089] [Table 3]

[0090] Example 3: Purification of 2'-FL by adding FeCl3 2'-FL fermentation broth was collected directly from the fermenter at the end of fermentation and used in this experiment. Five centrifuge tubes, each containing 5 mL of fermentation broth (fb), were diluted with 5 mL of water (control sample), 5 mL of 0.73% (w / w) FeCl3 in water, 5 mL of 1.46% (w / w) FeCl3 in water, and 5 mL of 2.19% (w / w) FeCl3 in water, respectively. Iron(III) chloride was used equimolarly with calcium chloride in the previous experiment. The mixture was mixed by vortexing and centrifuged at 2700 g for 10 minutes at room temperature. The resulting solution was transferred to a new centrifuge tube and analyzed by OD. 600 The OD and conductivity were determined. By adding FeCl3, a lower OD 600 It was possible to obtain a 2'-FL solution having a pH of 10.0 or higher, which clearly indicates a better separation of the solids contained in the fermentation broth from the liquid supernatant.

[0091] [Table 4]

Claims

1. 1. A method for producing human milk oligosaccharides (HMOs) by microbial fermentation, comprising: a) providing a fermentation broth comprising at least one HMO and biomass; b) adding a multivalent cation salt to the fermentation broth; and c) subjecting the mixture of step b) to centrifugation A method comprising:

2. 2. The method of claim 1, wherein the HMO is selected from the group consisting of 2'-FL, 3-FL, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LNFPV, 3'-SL, 6'-SL, LST-a, LST-b, LST-c, and DSLNT.

3. 3. The method of claim 1 or 2, wherein the polyvalent cation salt is a divalent or trivalent cation salt, preferably selected from the group consisting of Mg-, Ca-, Sr-, Zn-, Al-, Fe-, Mn-, and Cu-salts and mixtures thereof.

4. The polyvalent cation salt is MgCl ２ , ZnCl ２ , and CaCl ２ 4. The method of claim 1, wherein the compound is selected from the group consisting of:

5. 5. The method of claim 1, wherein the polyvalent cation salt is added to the fermentation broth of step a) in the form of an aqueous solution.

6. 6. The method according to any one of claims 1 to 5, wherein the concentration of polyvalent cation salts in the fermentation broth after step b) is ≥ 0.05 wt.%, preferably ≥ 0.1 wt.%, more preferably ≥ 0.15 wt.%, most preferably ≥ 0.175 wt.%, and ≤ 0.8 wt.%, preferably ≤ 1.0 wt.%, ≤ 2.0 wt.%, ≤ 2.5 wt.%, most preferably ≤ 2.0 wt.%.

7. The polyvalent cation salt is CaCl ２ 7. The method according to claim 1, wherein

8. 8. The method of any one of claims 1 to 7, wherein the polyvalent cation salt is added as an aqueous solution to the fermentation broth of step a) in a volumetric amount (v / v) ranging from ≥ 0.25 or ≥ 0.5 to ≤ 2.0 or ≤ 1.75, preferably in a volumetric amount of about 1.

0.

9. 9. The method according to any one of claims 1 to 8, wherein the fermentation broth of step a) further contains at least one monosaccharide, said at least one monosaccharide preferably being selected from the group consisting of L-fucose, glucose, galactose, N-acetylglucosamine and N-acetylneuraminic acid.

10. 10. The method according to any one of claims 1 to 9, wherein the fermentation broth of step a) further contains at least one further component, said at least one further component preferably being selected from the group consisting of lactose, sucrose and glycerol.

11. 11. The method of any one of claims 1 to 10, wherein the centrifugation in step c) is carried out in the range of 2,000 g to 15,000 g and / or for at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes at a temperature of 2°C to 60°C.

12. Steps below: i) adding a solution of a multivalent cation salt to the centrifugation pellet obtained by step b) and centrifuging the resulting mixture to obtain a further supernatant solution; ii) subjecting the supernatant solution of the first and any further optional centrifugation steps to at least one filtration; iii) treating the supernatant solution of the first and optional further centrifugation steps and / or the filtrate of step ii) at least once with a cation exchange resin and / or at least once with an anion exchange resin; iv) subjecting the supernatant solution of the first and any further optional centrifugation steps and / or the filtrate of step ii) or iii) to at least one electrodialysis; v) treating the supernatant solution of the first and any further optional centrifugation steps and / or the filtrate of steps ii), iii) or iv) with activated carbon at least once; and / or vi) subjecting the liquid process stream obtained during the recovery of human milk oligosaccharides from the fermentation broth to at least one crystallization step and / or precipitation step and / or spray drying step.

12. The method of claim 1, further comprising the additional step of purifying at least one HMO selected from one or more of:

13. 13. The method of any one of claims 1 to 12, wherein the fermentation broth contains at least one HMO in an amount of less than 30% (w / v), less than 25% (w / v), or less than 20% (w / v), and / or any of the solutions of steps i) to v) or the process stream of step vi) contains at least one HMO in an amount that results in a total amount of sugars of at least 20% (w / v), 30% (w / v), or 35% (w / v), and up to 45% (w / v), 50% (w / v), or 60% (w / v).

14. 14. The method according to any one of claims 10 to 13, wherein the aqueous solution containing at least one HMO and optionally at least one monosaccharide is spray dried at a nozzle temperature of at least 110°C, preferably at least 120°C, more preferably at least 125°C, and less than 150°C, preferably less than 140°C, more preferably less than 135°C.

15. 1. Use of a polyvalent cation salt in the fermentative production of human milk oligosaccharides, wherein the polyvalent cation salt is added to a fermentation broth containing human milk oligosaccharides and biomass before the fermentation broth is subjected to centrifugation to remove the biomass.

16. 16. The use according to claim 15, wherein the polyvalent cation salt is calcium chloride.

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