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

Abstract

Disclosed is a method for producing human milk oligosaccharides (HMO) derived from a microbial fermentation process, the method comprising the steps of: a) providing a fermentation broth comprising at least one HMO and a biomass; b) adding a multivalent cation salt to the fermentation broth; and c) centrifuging the mixture of step b).

Description

[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 a fermentation broth by centrifugation. Background Art

[0002] Human breast milk contains a large amount of carbohydrates. The 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, the so-called "human milk oligosaccharides (HMO)". HMOs represent the third most abundant component in human breast milk. It is estimated that there are more than 150 structurally different oligosaccharides in human milk. The selected HMOs are shown in Table 1. About 10 to 13 of these HMOs are present in human milk at concentrations ranging from hundreds of milligrams to several grams per liter (Thurl et al., (2017), Nutrition Reviews 75 (11) 920-933). Among HMOs, neutral HMOs and acidic HMOs containing at least one N-acetylneuraminic acid (NeuAc) moiety are known. The structural complexity and abundance of these oligosaccharides are unique to human milk and have not been found in the milk of other mammals (such as domestic cows).

[0003] Since HMOs cannot be digested by the human body, the physiological effects of these sugars have been studied for decades. The prebiotic effects of HMOs were discovered more than 100 years ago. When consumed, HMOs are able to modulate the composition of the human intestinal microbiota by supporting the growth of beneficial bacteria.

[0004]

[0005]

[0006]

[0007] Table 1: Structures of the main oligosaccharides in human breast milk.

[0008] In recent years, some other functional effects of HMO have been revealed, especially its effects on neonatal development. It is well known that HMO acts as a decoy, reducing the risk of infection by bacterial and viral pathogens adhering to human cells by binding to the surface glycoproteins of human cells. In addition, various HMOs have anti-inflammatory effects and can act as immunomodulators. Therefore, it has been suggested that HMOs can reduce the risk of food allergies. The positive effects of sialylated HMOs on the development of the central nervous system of neonates have also been discussed in depth (for a review, see "Prebiotics and Probiotics in Human Milk, Origins and functions of milk-borne oligosaccharides and bacteria", Academic Press (2017) editors: McGuire M., McGuire M., and Bode L.).

[0009] In order to exploit the beneficial effects of HMOs, efforts are underway to add individual HMOs to nutritional compositions, especially infant formulas.

[0010] Due to the limited supply of HMOs for supplementing nutritional compositions, processes for chemically synthesizing HMOs were developed. The disadvantages of such chemical synthesis led to biocatalytic methods, in which HMOs are synthesized in vitro using purified enzymes (e.g., glycosyltransferases). Today, individual HMOs are produced on an industrial scale using fermentation of genetically engineered microbial cells (WO 2015 / 150328A1, WO 2017 / 043382A1, WO 2010 / 070104 A1, WO 2012 / 097950 A1). 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 HMO preparation.

[0011] During the recovery of HMO from fermentation broth, the HMO is usually present in the form of a liquid process stream, such as an aqueous solution containing the desired HMO, which must be separated from the solid residual phase of the fermentation broth in a crucial first step of the purification process. This separation is usually accomplished in the first step by filtration methods or centrifugation.

[0012] In large-scale production, the filtration method alone is often complicated because the filter membrane may be clogged, and repeated filtration steps are required to ensure that most of the solid phase is captured by the filter medium without affecting the yield of HMO. At present, a method of combining a deep filtration system with more than 5 layers of deep filtration membranes to treat cell fermentation broth can obtain relatively good fermentation clarification broth, but this method has limited processing capacity and high cost, which greatly limits the large-scale application of deep filtration systems in production.

[0013] On the other hand, centrifugation methods are well known and applied in large-scale industrial production. For example, disc centrifuges are widely used in the fields of fruit juice drinks, tea drinks, wine brewing, condiments, edible oils, vegetable proteins, animal proteins, fish processing, biopharmaceuticals, starch processing, industrial fermentation, yeast extraction, natural extraction, etc., and are widely used in large-scale industrial production, and the continuous processing working mode is very suitable for large-scale cell fermentation production in the biopharmaceutical field. However, when the fermentation broth is centrifugally clarified by a disc centrifuge as the first separation step at the end of the fermentation, the clarified supernatant still contains various components such as a small amount of cells, colloidal substances, cell fragments, etc. Since the efficiency of the centrifugation separation step is crucial to all subsequent purification steps required to obtain food-grade products, especially in large-scale production, there is still a need to improve the centrifugation of the fermentation broth in HMO production.

[0014] The present invention aims to solve the problems of low processing capacity, high turbidity, large amount of deep filtration membrane and high processing cost in the existing fermentation broth pretreatment clarification methods, and to provide a convenient method for treating microbial fermentation broth for producing HMO by a centrifugal system, so as to more efficiently remove various impurities in the fermentation broth, such as cell debris, host DNA, protein, etc., to obtain a clarified fermentation broth containing the target HMO and less residual solids, thereby further simplifying the method steps required for downstream HMO purification and reducing the method production cost.

[0015] This object is achieved by a method for producing human milk oligosaccharides (HMO) derived from a microbial fermentation process, the method comprising the steps of:

[0016] a) providing a fermentation broth comprising at least one HMO and biomass;

[0017] b) adding a polyvalent cation salt to the fermentation broth; and

[0018] c) centrifuging the mixture of step b). Summary of the invention

[0019] In a first aspect, a method for producing purified human milk oligosaccharides (HMOs) derived from a microbial fermentation process is provided.

[0020] In a second aspect, there is provided the use of a multivalent cation salt as an additive to a fermentation broth at the end of a microbial fermentation process in the production of human milk oligosaccharides. DETAILED DESCRIPTION

[0021] According to a first aspect, there is provided a method for producing human milk oligosaccharides (HMO) derived from a microbial fermentation process, the method comprising the steps of:

[0022] a) providing a fermentation broth comprising at least one HMO and biomass;

[0023] b) adding a polyvalent cation salt to the fermentation broth; and

[0024] c) centrifuging the mixture of step b).

[0025] At present and as generally understood in the relevant art, "microbial fermentation process" is understood to be a generally large-scale industrial metabolic process in which, during the cultivation of microorganisms such as bacteria, fungi and molds, enzymatic breakdown and utilization of nutrients (particularly sugars) and conversion of compounds into other compounds occur. Thus, industrial or large-scale microbial fermentation is the process of controlling microorganisms (i.e., bacteria, yeasts and molds) to modify food to produce a desired product.

[0026] Currently and generally in the relevant art, "microorganism" currently designates and encompasses any microorganism comprising a single cell, a cell cluster or a multicellular relatively complex organism, which is suitable for use in the method of the present invention, and particularly includes bacteria and yeast. The microorganisms employed according to the present invention can be cultured in a liquid culture medium, and generally require a carbon source in the culture medium to grow and replicate.

[0027] Currently and throughout the invention, "recombinant" refers to genetically engineered DNA that is prepared by transplanting or splicing a gene from one species into the cells of a host microorganism of a different species. This DNA becomes part of the host's genetic makeup and is replicated.

[0028] Therefore, "host microorganism" refers to any microorganism containing a nucleic acid sequence or expressed protein that is foreign to a recombinant host microorganism, and wherein the nucleic acid sequence described in the microorganism is integrated into the genome of the host microorganism cell. Therefore, "non-naturally occurring" refers to that the nucleic acid sequence / protein (e.g., enzyme) is foreign to the host microorganism cell, i.e., the nucleic acid sequence / protein is heterologous to the microbial host cell. Heterologous sequences can be stably introduced (e.g., by transfection, transformation, or transduction) into the genome of the host microorganism cell, wherein the technology that will depend on the host cell to be introduced into the sequence can be applied. 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). Therefore, the host microorganism into which the heterologous sequence has been introduced will produce heterologous proteins encoded by the nucleic acid sequence of the present invention.

[0029] For recombinant production, the host cell can be genetically engineered to incorporate an expression system or part thereof and nucleotide sequences encoding the metabolic pathways and / or enzymes necessary for the biosynthesis of the target human milk oligosaccharide. The introduction of nucleic acid sequences into host microbial cells can be accomplished 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.

[0030] A variety of expression systems can be used to produce the HMOs of the present invention. Such vectors include, inter alia, chromosomal vectors, episomal vectors, and virally derived vectors, such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and vectors derived from combinations thereof, such as vectors derived from plasmid and bacteriophage genetic elements (e.g., cosmids and phagemids). The expression system constructs can contain control regions that regulate expression as well as regions that cause expression. In general, any system or vector suitable for maintaining, propagating or expressing polynucleotides and synthesizing HMOs in a host can be used for expression in this regard. The appropriate DNA sequence can be inserted into the expression system by any of a variety of well-known conventional techniques, such as, for example, those described in Sambrook et al. (supra).

[0031] At present, "desired oligosaccharides" refer to oligosaccharides that should be specifically and intentionally produced by the applied method. Therefore, "non-desired oligosaccharides" represent oligosaccharides that are not desired to be produced or generated during the production of desired oligosaccharides or oligosaccharides formed by the used organisms that are unrelated to the desired oligosaccharides. "Metabolites" or "metabolic sugar products" or "sugar intermediates" are sugars, i.e., carbohydrate products generated during the production of desired oligosaccharides, and "unused sugar substrates" represent starting molecules or parts used to produce desired oligosaccharides / used during the production of desired oligosaccharides.

[0032] As mentioned at the outset, the present invention relates to a method for producing desired oligosaccharides using a host microorganism, wherein the desired oligosaccharides do not naturally occur in the host cell and are human milk oligosaccharides. In this regard, reference is made to the HMOs listed in Table 1 above.

[0033] By means of the process according to the invention, an efficient production process is provided, by means of which the desired oligosaccharides can be produced in a form in which there are substantially no longer any undesirable solids or other obstructive solid products or unused solid substrates from the fermentation broth.

[0034] As used herein, the term "culture" refers to growing microorganisms in a culture medium and under conditions that allow and are suitable for producing the desired human milk oligosaccharides. After reading the disclosure of the present invention and combining the technical and expert background of those skilled in the art, those skilled in the art can easily obtain a variety of suitable host microorganisms and culture media and conditions for culturing them.

[0035] In step a) of the disclosed inventive method, a fermentation broth is provided comprising at least one HMO and biomass from a fermentation process.

[0036] The desired HMO, such as 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyl lactose (3'-SL), lactose-N-tetraose (LNT) or 6'-sialyl lactose (6'-SL), is produced by fermentation of a genetically modified microorganism. The fermentation can be carried out in any suitable fermentation medium, such as, for example, a chemically defined fermentation medium.

[0037] The fermentation medium may 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, niacin, inositol, thiamine HCl, para-aminobenzoic acid. Food grade processing aids, such as defoamers and pH control agents, may also be used. The temperature of the fermentation step is typically 10 to 50° C., preferably 25 to 35° C., more preferably 28 to 32° C. The pH of the fermentation step is typically 3 to 8, preferably 5 to 7, more preferably 5.5 to 6.5. At the end of the fermentation step, the fermentation broth may be subjected to a heat treatment step to inactivate the microorganism prior to downstream processing steps.

[0038] Preferably, the microorganism is a genetically modified yeast, such as a yeast strain of saccharomyces cerevisiae, a strain of Candida, a strain of Hansenula, a strain of Kluyveromyces, a strain of Pichia, a strain of fission yeast, a strain of Schwanniomyces, a strain of spore-forming torula yeast, a strain of Yarrowia or a strain of zygosaccharomyces. More preferably, the yeast is a yeast strain of saccharomyces cerevisiae, a strain of Hansenula polymorpha, a strain of Kluyveromyces lactis, a strain of Kluyveromyces marxianus, a strain of Pichia pastoris, a strain of Pichia methanolica, a strain of Pichia stipites, a strain of Candida boidinica, a strain of Schizosaccharomyces pom be, a strain of Schwanniomyces occidentalis, a strain of Torulaspora delbrueckii, a strain of Yarrowia lipolytica, a strain of Zygosaccharomyces rouxii or a strain of Zygosaccharomyces baishi.

[0039] Preferably, the microorganism can also be selected from the genera of Escherichia coli or Saccharomyces cerevisiae, Bifidobacterium, Lactobacillus, Enterococcus, Streptococcus, Staphylococcus, Peptostreptococcus, Leuconostoc, Clostridium, Eubacterium, Veronella, Fusobacterium, Bacteroides, Prevotella, Escherichia, Propionibacterium and Saccharomyces cerevisiae, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum; Enterococcus faecium; Escherichia coli, Cleaveria marxianus; Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sakei, Lactococcus lactis (including but not limited to lactic acid subspecies, lipid subspecies and diacetyl subspecies); Leuconostoc mesenteroides (including but not limited to enteric subspecies); Pediococcus acidilactici, Pediococcus pentosaceus; Propionibacterium acidipropionici, Propionibacterium freundii subspecies Sherman; Staphylococcus carnosus and Streptococcus thermophilus.

[0040] The desired HMO, such as 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyl lactose (3'-SL), lacto-N-tetraose (LNT) or 6'-sialyl lactose (6'-SL), is purified from the fermentation broth as a product of the fermentation process. After fermentation, the broth containing the desired HMO is applied to the separation process as described below.

[0041] The term "fermentation broth" as used in this specification refers to the product obtained by microbial fermentation. Therefore, the fermentation product contains biomass (cells), fermentation medium, residual substrate material, and any molecules / byproducts produced during the fermentation process, such as the desired HMO. After each step of the purification process, one or more components of the fermentation product are removed to produce a more purified HMO.

[0042] At present and throughout the present invention, "biomass" refers to each and any kind of cell or microbial material, cell or microbial fragments, high molecular weight molecules and / or residual lysis products as well as other solid particles adhering thereto or present in the fermentation broth to be processed in the subsequent separation steps of the process according to the invention.

[0043] Optionally, the fermentation broth may be subjected to a heat treatment step to kill bacteria or other undesirable microorganisms that may be present in any significant degree.

[0044] Typically, the desired HMO is present in the liquid, or in other words, in the supernatant phase of the fermentation broth. However, within the meaning of the present invention, it is also encompassed that the desired HMO is present or contained in the biomass fraction of the fermentation broth.

[0045] In step b) of the process according to the invention, a polyvalent cation salt is added to the fermentation broth.

[0046] The term "polyvalent cation" as used herein refers to a cation capable of forming multiple ion pairs, for example, a multi-charged cation, such as a doubly charged cation or a triply charged cation. Any convenient polyvalent cation can be used in the subject salt composition. In some embodiments, the polyvalent cation is divalent. The intended divalent cations include, but are not limited to, magnesium, zinc, and calcium. In some embodiments, the polyvalent cation is trivalent. The intended trivalent cations include, but are not limited to, aluminum. In certain embodiments of the salt composition, the at least one polyvalent cation is selected from magnesium, zinc, aluminum, and calcium. In certain embodiments of the salt composition, the at least one polyvalent cation is magnesium. In certain embodiments of the salt composition, the at least one polyvalent cation is zinc. In certain embodiments of the composition, the at least one polyvalent cation is aluminum. In certain embodiments of the composition, the at least one polyvalent cation is calcium.

[0047] In the sense of the present invention, the term "polyvalent cation salt" relates to any polyvalent cation composition comprising a suitable counterion. The counterion may be monovalent, such as, for example, chloride, bromide, fluoride, acetate, or may be polyvalent, for example divalent or trivalent, such as sulfate, sulfite, phosphate, phosphonate, carbonate.

[0048] The choice of multivalent cationic salt may vary based on solubility, stability, and possible interactions with other components present in the fermentation broth.

[0049] The multivalent cation salt may be added to the fermentation broth at any time before starting the centrifugation step c) to separate the biomass from the supernatant phase. Preferably, the multivalent cation salt is added to the fermentation broth shortly before starting the subsequent centrifugation step.

[0050] The polyvalent cation salt can be added in any form which is particularly suitable for large-scale industrial production, for example as a solid or in the form of an aqueous solution or dispersion.

[0051] In step c) of the process of the present invention, the mixture of step b) (i.e. the fermentation broth containing the desired HMO and biomass and to which the multivalent cation salt has been added) is centrifuged so that some, most or all of the solid fraction in the fermentation broth is separated from the liquid supernatant.

[0052] Centrifugal technology primarily exploits differences in size, density, specific gravity, and shape, and is well known and established in the current biomanufacturing field. Filters are typically simpler to operate because they do not have any moving parts, whereas centrifuges have a rotating mechanism. On the other hand, filters have slower processing speeds, are more susceptible to clogging, have higher operating costs, and generally require a larger surface area and floor space than centrifuges.

[0053] Most centrifugal techniques used for biomanufacturing are based on tubular bowls or disc stacking. Tubular bowl technology is mainly used for microbial culture because it can produce high centrifugal forces (>10,000g) and can produce solid pastes with low supernatant content. Once the solid fills the bowl, it is necessary to stop the rotating bowl to discharge the solid. Disc stacking centrifuges (Westfalia CSV series) and countercurrent centrifuges (such as kSep, Elutra and Rotea) do not pack cells tightly, but form slurries and discharge solids intermittently without stopping during discharge. The shear force generated by countercurrent centrifugation technology is relatively low, but the shear force generated by other bowl or disc stacking centrifugation technologies is relatively high, so it is not used when intact cells are needed as products or intermediates. Therefore, those skilled in the art can easily select the type of centrifugal technology according to the required production parameters.

[0054] Surprisingly, it was found that adding a multivalent cation salt to the fermentation broth prior to subjecting the broth to a centrifugation separation step resulted in a more efficient separation. Notably, an increase in the amount of solid phase accumulated during the centrifugation process and a decrease in the turbidity of the liquid supernatant phase were observed. Likewise, the multivalent cation salt acted as a separation aid additive, also making the subsequent purification step easier and faster to produce a pure HMO product.

[0055] Without being bound by any theory, it is speculated that the multivalent cation salt aids in the precipitation of residual biomass and other solid residues from the fermentation process, perhaps by increasing the size of the precipitate or interfering with the tendency of any residual solids from the fermentation process to become finely dispersed in the fermentation medium, thereby improving centrifugation efficiency overall.

[0056] In other words, according to the present invention, there is provided a method for producing human milk oligosaccharides (HMO) derived from a microbial fermentation process, the method comprising the steps of:

[0057] a) providing a fermentation broth from a fermentation comprising at least one HMO and biomass; and

[0058] b) centrifuging the mixture of step a),

[0059] Therein, the multivalent cation salt is added to the fermentation broth of step a) before starting the centrifugation of step b).

[0060] In another embodiment, the present disclosure provides a method as described above, wherein the HMO is selected from 2'-FL, 3-FL, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LNFPV, 3'-SL, 6'-SL, LST-a, LST-b, LST-c and DSLNT.

[0061] For the specified HMO, good results can be achieved in terms of improving the separation quality of the centrifugation step. See the structures of the HMOs shown in Table 1 above.

[0062] In another embodiment, the present disclosure provides a method as described above, wherein the multivalent cation salt is a divalent or trivalent cation salt, preferably selected from Mg salts, Ca salts, Sr salts, Zn salts, Al salts, Fe salts, Mn salts and Cu salts and mixtures thereof.

[0063] As counter ions of the divalent or trivalent cationic salts, monovalent, divalent, trivalent or tetravalent anions may be present, preferably monovalent or divalent anions.

[0064] Typically, such salts are chosen to be well known in the art of biomolecule production and thus 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.

[0065] Another important aspect in the selection of the polyvalent cation salt is the ease of removal of the salt from the HMO-containing phase (either the solid biomass phase or preferably the liquid supernatant phase separated by the centrifugation step). In the subsequent further purification steps, the nature of the polyvalent cation salt plays an important role in the selection of a well-known large-scale enabling separation method, which in the best case will be carried out anyway even in the absence of the polyvalent cation salt. Preferably, the polyvalent cation salt can be removed from the HMO-containing phase by ion exchange in the subsequent processing steps using ion exchange absorption resins, ion exchange bed filtration, ion exchange column filtration or related techniques (chromatography).

[0066] In another embodiment, the present disclosure provides a method as described above, wherein the multivalent cation salt is selected from MgCl 2 、ZnCl 2 and CaCl 2 .

[0067] These salts have good solubility in water, so the amount added to the fermentation broth in the form of an aqueous solution can be easily metered. In addition, the cations and anions in the salts can be easily removed by ion exchange methods in subsequent processing steps. In addition, the salts are easily available in high quality and are generally certified as safe for use in biomolecule production of food grade substances.

[0068] In another embodiment, the present disclosure provides a method as described above, wherein the multivalent cation salt is added to the fermentation broth of step a) in the form of an aqueous solution.

[0069] In this way, the amount added to the fermentation broth can be easily metered and the continuous large-scale operation of the process of the invention can be simplified.

[0070] In another embodiment, the present disclosure provides a method as described above, wherein, after adding the polyvalent cation to the fermentation broth (step b)), the concentration of the polyvalent cation salt in the fermentation broth is ≥0.05 weight-%, preferably ≥0.1 weight-%, more preferably ≥0.15 weight-%, and most preferably ≥0.175 weight-%; and ≤0.8 weight-%, preferably ≤3.0 weight-%, ≤2.5 weight-%, and most preferably ≤2.0 weight-%.

[0071] In the concentration range of the polyvalent cation salt in the fermentation broth to be subsequently centrifuged, not only an improved separation between precipitate and supernatant can be achieved, but also the polyvalent cation salt can be easily removed in the subsequent treatment. As an indicator of the size of the removal task for removing the polyvalent cation salt, the conductivity can be measured. Thus, it was surprisingly found that in the preferred salt concentration range, an improved separation of solid and supernatant phases can be achieved, while the concentration can be kept in a range that allows easy removal of the salt in the subsequent post-treatment step.

[0072] In a preferred embodiment, the concentration of the polyvalent cation salt in the fermentation broth immediately before centrifugation is 0.2 weight-%, 0.3 weight-%, 0.4 weight-%, 0.5 weight-%, 0.6 weight-%, 0.7 weight-%, 0.8 weight-%, 0.9 weight-%, 1.0 weight-%, 1.1 weight-%, 1.2 weight-%, 1.3 weight-%, 1.4 weight-%, 1.5 weight-%, 1.6 weight-%, 1.7 weight-%, 1.8 weight-%, 1.9 weight-% or 2.0 weight-%.

[0073] In one embodiment, the present disclosure provides a method as described above, wherein the multivalent cation salt is CaCl 2 .

[0074] It is well known that calcium chloride can be used in microbial fermentation processes, mostly in relatively low concentrations as a salt chemical in the fermentation medium. Thus, the use of calcium chloride has been approved for production based on food-grade fermentation. In addition to this, the use of calcium chloride has the advantage that not only calcium cations but also chloride anions can be easily removed from the liquid phase separated after centrifugation, for example by ion exchange methods. Many ion exchange resins are known that are able to optimize the removal of calcium cations and chloride anions during downstream processing. Another advantage is the non-toxic behavior of calcium chloride towards microbial cells. Such non-toxic centrifugation-assisted multivalent cation salts are advantageous in fermentation processes where the desired HMOs are to be separated from the biomass without lysis or other destructive measures, making it possible to reuse the microbial material.

[0075] In another embodiment, the present disclosure provides a method as described above, wherein the multivalent cation salt is added to the fermentation broth of step a) as an aqueous solution in a volumetric amount (v / v) ranging from ≥0.25 or ≥0.5 to ≤2.0 or ≤1.75, preferably at a volume ratio of about 1.0.

[0076] In this way, it is advantageously possible to provide the preferably continuous centrifugation step with an input flow which is easy to control and has a good flow behavior.

[0077] In another embodiment, the present disclosure provides a method as described above, wherein the fermentation broth of step a) further contains at least one monosaccharide, and the at least one monosaccharide is preferably selected from L-fucose, glucose, galactose, N-acetylglucosamine and N-acetylneuraminic acid.

[0078] In another embodiment, the present disclosure provides a method as described above, wherein the fermentation broth of step a) further contains at least one further component, and the at least one further component is preferably selected from lactose, sucrose and glycerol.

[0079] In another embodiment, the present disclosure provides a method as described above, wherein the centrifugation of step c) is carried out at a temperature of 2°C to 60°C in the range of 2,000g to 15,000g and / or lasts for at least 5 min, 10 min, 15 min, 20 min or 30 min.

[0080] The minimum duration of the centrifugation treatment depends on various circumstances, such as the g-forces applied, continuous or batch processing, the input / rates and output / rates of the liquid and solid phases, temperature, the type of centrifuge used, etc. One skilled in the art of food grade HMO fermentation processes can easily find the optimal centrifugation duration and parameters for the individual gears used. In addition to this aspect, continuous centrifugation can be used for industrial scale HMO production, where the biomass is emptied from the centrifuge / rotor after predetermined time intervals. The optimal interval for such continuous centrifugation also depends on the individual circumstances of the specific gears and parameters, and can be determined and applied by a person skilled in the art.

[0081] The nature of the centrifugation parameters to be selected (e.g. high shear or low shear etc.) depends, for example, on the question whether the desired HMO is contained in the supernatant phase of the fermentation broth or whether the desired HMO is contained in the (still intact) cells of the fermentation broth. In the first case, higher shear forces and a higher range of g-forces may be selected, in the latter case, lower shear forces or a lower range of g-forces are preferably selected to keep the cells intact. In a subsequent step, the cells containing the desired HMO may be lysed or otherwise treated to release the HMO from the separated biomass. However, the core improvement of the invention of better separation is achieved in both cases by adding a multivalent cation salt to the fermentation broth prior to centrifugation.

[0082] In another embodiment, the present disclosure provides a method as described above, wherein the method further comprises an additional step of purifying the at least one HMO, wherein the additional step is selected from one or more of the following steps:

[0083] i) adding a solution of a polyvalent cation salt to the centrifuged precipitate obtained from step b) and centrifuging the resulting mixture to obtain a further supernatant;

[0084] ii) filtering the supernatant of the first and optional further centrifugation steps at least once;

[0085] iii) treating the supernatant of the first and optional further centrifugation step 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;

[0086] iv) subjecting the supernatant of the first and optional further centrifugation step and / or the filtrate of step ii) or iii) to at least one electrodialysis;

[0087] v) treating the supernatant of the first and optional further centrifugation step and / or the filtrate of step ii), iii) or iv) at least once with activated charcoal; and / or

[0088] vi) subjecting the liquid process stream obtained during the recovery of human milk oligosaccharides from the fermentation broth to a crystallization step and / or a precipitation step and / or a spray drying step at least once.

[0089] Additional purification steps to obtain pure HMO from the centrifugation may be performed in various orders and / or repetitions as appropriate for the desired HMO.

[0090] After the centrifugation step, the separated stream is subjected to one or more purification steps, which may include ultrafiltration, nanofiltration, anion exchange, cation exchange and decolorization to form a purified separated stream. The purified separated stream is then treated in a concentration step and a dehydration step. These steps may be performed in any order. In other words, the purified separated stream may be first treated in a concentration step, and the concentrated product may then be sent to a dehydration step. Alternatively, the purified separated stream may first undergo a dehydration step, and the product of the dehydration step may then 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, separated stream. The dehydration step may include at least one of crystallization, drying, evaporation and conventional filtration to form an HMO product.

[0091] In step i), the polyvalent cation salt-assisted centrifugation is repeated to optimize the overall yield of the desired HMO by washing the centrifuge precipitate (slurry) with an aqueous solution of a polyvalent cation salt.

[0092] In step ii), at least one filtration step is performed, which filtration step may preferably be selected from microfiltration, nanofiltration and / or diafiltration and / or reverse osmosis filtration.

[0093] These filtration methods are well known to the person skilled in the art and can be selected depending on the purification task. For example, microfiltration and nanofiltration are described in European patent application EP 374177 as post-treatment of process streams from HMO production fermentations. 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.

[0094] In step iii), the supernatant of the first and optional further centrifugation step and / or the filtrate of step ii) is treated at least once with a cation exchange resin and / or with an anion exchange resin.

[0095] The ion exchange treatment is advantageously optimized to remove 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 WO 2019063757 A1.

[0096] Cation exchange treatment can be used to separate charged molecules from HMO-containing streams. Cation exchange (CEX) stationary phases typically contain aliphatic sulfonic acid groups that are negatively charged in aqueous solution and can bind tightly to any strongly basic analyte. This step removes positively charged components such as residual ammonia, metals, and peptides. The binding capacity of the resin used is generally 1.2 to 2.2 mmeq / L. Typical resins used for cation exchange include Dow Dowex 88, Resindion JC series (such as JC603), and Resindion PK grades (such as PK216).

[0097] The anion exchange step can be used to separate charged molecules. In an anion exchange column, the filler is positively charged and therefore retains negatively charged molecules through Coulombic interactions. This step can remove negatively charged components such as chlorides, sulfates, phosphates, organic acids, and negatively charged particles. The binding capacity of the resin used is generally 0.8 to 2.0 mmeq / L. Typical resins for anion exchange include the Resindion Relite series, such as RAM2, DI82, and JA100, and the Diaion WA series, such as WA20.

[0098] In step iv), the supernatant of the first and optional further centrifugation step and / or the filtrate of step ii) or iii) is subjected to at least one electrodialysis.

[0099] Electrodialysis is the process of selectively removing ions from a solution. In combination with fermentation, it can be used 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 electrical migration of ions through a semipermeable membrane.

[0100] The basic principle of electrodialysis is that the electrolytic cell includes a pair of electrodes immersed in an electrolyte for conducting ions and connected to a DC generator. The electrode connected to the positive pole of the DC generator is the anode, and the electrode connected to the negative pole of the DC generator is the cathode. Then, the electrolyte solution supports the flow of current, which is generated by the movement of negative ions and positive ions to the anode and cathode, respectively. The membrane used for electrodialysis is essentially a porous ion exchange material sheet with negative or positive charge groups, and is therefore described as a cationic or anionic membrane, respectively. Ion exchange membranes are usually made of polystyrene cross-linked with divinylbenzene carrying appropriate functional groups (such as sulfonic acid for cationic membranes or quaternary ammonium groups for anionic membranes). The electrolyte can be, for example, sodium chloride, sodium acetate, sodium propionate or aminosulfonic acid. Then, the electrodialysis stack is assembled in such a way that the anionic membrane and the cationic membrane are parallel between the two electrode blocks as in a filter press, so that the material flow undergoing ion depletion is well separated from the material flow undergoing ion enrichment (the two solutions are also referred to as diluent (experience ion depletion) and concentrate (experience ion enrichment)). At the heart of the electrodialysis process is a membrane stack consisting of multiple anion exchange membranes and cation exchange membranes mounted between two electrodes separated by spacers. By applying a direct current, anions and cations will migrate across the membranes towards the electrodes.

[0101] A decolorization step v) may be performed to remove colored components. This step may be performed using activated carbon (such as Norit CAI activated carbon), hydrophobic interaction chromatography (HIC) or another adsorption resin that may be functionalized (such as Resindion Relite RAD / F). Decolorization may be performed before or after other specified purification steps.

[0102] In step vi), the process stream is subjected at least once to a crystallization step and / or a precipitation step and / or a spray-drying step.

[0103] The crystallization process can be used to dehydrate the purified HMO material, first forming HMO nuclei in a supersaturated solution and then growing crystals. This process can be batch or continuous. Typical conditions for crystallization are shown in WO 2018 / 164937, the disclosure of which is incorporated herein by reference. The process includes concentrating the starting HMO-containing solution to a supersaturated state, then precipitating HMO crystals from the supersaturated solution, while preferably keeping the temperature of the supersaturated solution at least 55°C. Preferably, the HMO content of the solution is at least 60wt.% and less than about 98wt.%. As the percentage of HMO crystals increases, crystallization is generally limited by viscosity, preferably, with a typical endpoint of about 30wt.% crystals.

[0104] Alternatively, crystallization or precipitation of at least one HMO from a process stream can be carried out by adding a suitable amount of a water-miscible organic solvent to a process stream containing at least one HMO. The organic solvent can be selected from C1- to C6-alcohols and C1- to C4-carboxylic acids. No crystallization or precipitation step of the at least one HMO is carried out at the end of the recovery process, so that residual amounts of organic solvent or carboxylic acid can be removed by subsequent process steps.

[0105] Suitable spray drying processes for recovering HMO in solid form are described, for example, in EP 3524067 A1, EP 3494804 A1, EP 3494805 A1, EP 3494806 A1 or EP 3494807 A1.

[0106] In another embodiment, the present disclosure provides a method as described above, 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 below 150°C, preferably below 140°C and more preferably below 135°C.

[0107] In another embodiment, the present disclosure provides a method as described above, 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 an additional or alternative embodiment, the solution of any of steps i) to v) or the process stream of step vi) contains at least one HMO in an amount 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) of the total sugars.

[0108] The present invention further relates to the use of a multivalent cation salt in the fermentation production of human milk oligosaccharides, wherein the multivalent cation salt is added to a fermentation broth containing human milk oligosaccharides and biomass, and then the fermentation broth is centrifuged to remove the biomass.

[0109] Addition of the multivalent cation salt resulted in better separation of the biomass and other solid materials from the liquid supernatant phase, and less observable coloration of the liquid phase.

[0110] Preferably, the amount of polyvalent cation salt added before the centrifugation step is determined by optimizing higher separation and decolorization effects and maintaining low conductivity of the liquid phase so that subsequent removal of cations and anions in subsequent purification steps is easier.

[0111] In one embodiment, the present disclosure provides for use of a multivalent cation salt in the above method, wherein the multivalent cation salt is calcium chloride.

[0112] The present invention will be described with respect to specific embodiments, but the invention is not limited thereto, but 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 a sequence in time, space, ranking, or any other manner. It should be understood that the terms so used are interchangeable where appropriate, and the embodiments of the present invention described herein are capable of operating in an order different from the order described or illustrated herein.

[0113] It is worth noting 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. Therefore, it should be interpreted as specifying the presence of the features, integers, steps or components mentioned, but does not exclude the presence or addition of one or more other features, integers, steps or components or groups thereof. Therefore, 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. This means that, for the purposes of the present invention, the only relevant steps of the method are A and B.

[0114] In this specification, reference to "one embodiment" or "an embodiment" means that the particular features, structures, or characteristics described in connection with that embodiment are included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in various places in this specification do not necessarily always refer to the same embodiment. In addition, it will be apparent to one of ordinary skill in the art from this disclosure that the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0115] Similarly, it should be understood that in describing representative 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 of the various inventive aspects. This method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than those expressly listed in each claim. On the contrary, as reflected in the following claims, inventive aspects may require fewer features than all of the features of any of the foregoing disclosed embodiments. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, and each claim stands on its own as a separate embodiment of the invention.

[0116] In addition, although some embodiments described herein include some features included in other embodiments but not other features, the combination of features of different embodiments is intended to fall within the scope of the present invention and form different embodiments, as understood by those skilled in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0117] In the description 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 to facilitate understanding of the description and drawings.

[0118] The invention will now be described by way of a detailed description of several embodiments of the invention. The present invention is limited only by the terms of the appended claims.

[0119] Example

[0120] Example 1: By adding CaCl 2 To purify 2'-FL

[0121] The 2'-FL fermentation broth was taken directly from the fermenter at the end of the fermentation and used in this experiment. Five centrifuge tubes (each containing 5 mL of fermentation broth (fb)) were each washed with 5 mL of water (comparative sample), 0.50% CaCl 2 Aqueous solution (w / w), 1.00% CaCl 2 Aqueous solution (w / w) and 1.50% CaCl 2 The mixture was mixed by vortexing and centrifuged at 1800 g for 10 min at 10 °C. The resulting solution was poured into a new centrifuge tube and the OD was determined. 600 and conductivity. By adding CaCl 2 , can obtain a lower OD 600 This clearly indicates a better separation of the solids contained in the fermentation broth from the liquid supernatant.

[0122] sample Conductivity [μSi] <![CDATA[OD 600 ]]> 5mL fb+5mL water 2.97 0.91 <![CDATA[5mL fb+5mL 0.5%CaCl 2 Aqueous solution (w / w)]]> 6.23 0.43 <![CDATA[5mL fb+5mL 1%CaCl 2 Aqueous solution (w / w)]]> 9.52 0.32 <![CDATA[5mL fb+5mL 1.5%CaCl 2 Aqueous solution (w / w)]]> 12.80 0.34

[0123] Table 2: Containing 2'-FL and supplemented with CaCl 2 The conductivity and OD of the supernatant of the fermentation broth 600 .

[0124] Example 2: By adding CaCl 2 To purify 6'-SL

[0125] The 6'-SL fermentation broth was taken directly from the fermenter at the end of the fermentation and used in this experiment. Five centrifuge tubes (each containing 5.5 mL of fermentation broth (fb)) were each washed with 5 mL of water (comparative sample), 5 mL of 0.50% CaCl 2 Aqueous solution (w / w), 5 mL 1.00% CaCl 2 Aqueous solution (w / w) and 5 mL 1.50% CaCl 2 The mixture was mixed by vortexing and centrifuged at 1800 g for 10 min at 10 °C. The resulting solution was poured into a new centrifuge tube and the OD was determined. 600 and conductivity. By adding CaCl 2 , can obtain a lower OD 600 This clearly indicates a better separation of the solids contained in the fermentation broth from the liquid supernatant.

[0126] sample Conductivity [μSi] <![CDATA[OD 600 ]]> 5mL fb+5mL water 7.8 17.94 <![CDATA[5mL fb+5mL 0.5%CaCl 2 Aqueous solution (w / w)]]> 11.49 0.42 <![CDATA[5mL fb+5mL 1%CaCl 2 Aqueous solution (w / w)]]> 14.77 0.43 <![CDATA[5mL fb+5mL 1.5%CaCl 2 Aqueous solution (w / w)]]> 18.04 0.56

[0127] Table 3: Containing 6'-SL and supplemented with CaCl 2 The conductivity and OD of the supernatant of the fermentation broth 600 .

[0128] Example 3: By adding FeCl 3 To purify 2'-FL

[0129] The 2'-FL fermentation broth was taken directly from the fermenter at the end of the fermentation and used in this experiment. Five centrifuge tubes (each containing 5 mL of fermentation broth (fb)) were each washed with 5 mL of water (comparative sample), 5 mL of 0.73% FeCl 3 Aqueous solution (w / w), 5 mL 1.46% FeCl 3 Aqueous solution (w / w) and 5 mL 2.19% FeCl 3 The solution was diluted with water (w / w). In the previous experiments, iron (III) chloride was used in an equimolar amount with calcium chloride. The mixture was mixed by vortexing and centrifuged at 2700 g for 10 minutes at room temperature. The resulting solution was poured into a new centrifuge tube and the OD was determined. 600 and conductivity. By adding FeCl 3 , can obtain a lower OD 600 This clearly indicates a better separation of the solids contained in the fermentation broth from the liquid supernatant.

[0130] sample Conductivity [μSi] <![CDATA[OD 600 ]]> 5mL fb+5mL water 3.12 2.05 <![CDATA[5mL fb+5mL 0.73FeCl 3 Aqueous solution (w / w)]]> 7.39 0.58 <![CDATA[5mL fb+5mL 1.46%FeCl 3 Aqueous solution (w / w)]]> 12.08 1.58 <![CDATA[5mL fb+5mL 2.19%FeCl 3 Aqueous solution (w / w)]]> 17.55 1.63

[0131] Table 4: Conductivity and OD of the supernatant of fermentation broth containing 2'-FL and supplemented with FeCl600 。

Claims

1. A method for producing human milk oligosaccharides (HMO) derived from a microbial fermentation process, the method comprising the steps of: a) providing a fermentation broth comprising at least one human milk oligosaccharide and biomass; b) adding a polyvalent cation salt to the fermentation broth; and c) centrifuging the mixture of step b).

2. The method according to claim 1, wherein: The human milk oligosaccharide is selected from 2'-FL, 3-FL, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LNFPV, 3'-SL, 6'-SL, LST-a, LST-b, LST-c and DSLNT.

3. The method according to claim 1 or 2, wherein: The multivalent cation salt is a divalent or trivalent cation salt, preferably selected from Mg salts, Ca salts, Sr salts, Zn salts, Al salts, Fe salts, Mn salts, Cu salts and mixtures thereof.

4. The method according to any one of claims 1 to 3, wherein: The multivalent cation salt is selected from MgCl2, ZnCl2 and CaCl2.

5. The method according to any one of claims 1 to 4, wherein: The multivalent cation salt is added to the fermentation broth in step a) in the form of an aqueous solution.

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

7. The method according to any one of claims 1 to 6, wherein: The multivalent cation salt is CaCl2.

8. The method according to any one of claims 1 to 7, wherein: The multivalent cation salt is added to the fermentation broth of step a) as an aqueous solution, and the volume amount (v / v) of the multivalent cation salt ranges from ≥0.25 or ≥0.5 to ≤2.0 or ≤1.75, preferably about 1.0 volume amount.

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, and the at least one monosaccharide is preferably selected from L-fucose, glucose, galactose, N-acetylglucosamine and N-acetylneuraminic acid.

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, which is preferably selected from lactose, sucrose and glycerol.

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

12. The method according to any one of claims 1 to 11, wherein: The method further comprises an additional step of purifying the at least one human milk oligosaccharide, the additional step being selected from one or more of the following steps: i) adding the solution of the polyvalent cation salt to the centrifuged precipitate obtained from step b), and centrifuging the resulting mixture to obtain a further supernatant; ii) filtering the supernatant of the first and optional further centrifugation steps at least once; iii) treating said supernatant of said first and optional further centrifugation step 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 said supernatant of said first and optional further centrifugation step and / or the filtrate of step ii) or iii) to at least one electrodialysis; v) treating said supernatant of said first and optional further centrifugation step and / or the filtrate of step ii), iii) or iv) at least once with activated charcoal; and / or vi) subjecting a liquid process stream obtained during the recovery of said human milk oligosaccharides from said fermentation broth to a crystallization step and / or a precipitation step and / or a spray drying step at least once.

13. The method according to any one of claims 1 to 12, wherein: The fermentation broth contains the at least one human milk oligosaccharide 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 the at least one human milk oligosaccharide, the amount of the at least one human milk oligosaccharide being 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) of the total sugars.

14. The method according to any one of claims 10 to 13, wherein: The aqueous solution containing the at least one human milk oligosaccharide and optionally the 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 and more preferably less than 135°C.

15. Use of a multivalent cation salt in the fermentation production of human milk oligosaccharides, wherein: The multivalent cation salt is added to the fermentation broth before centrifuging the fermentation broth containing human milk oligosaccharides and biomass to remove the biomass.

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

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