RU2793915C1 - Method for purification of lacto-n-neotetraose - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the purification of lacto-N-neotetraose to obtain nutritional products for infants. Disclosed is a process for purifying lacto-N-neotetraose (LNnT) from a fermentation broth, which includes the steps of: taking a fermentation broth that contains LNnT, biomass, media components, contaminants, and carbohydrates other than LNnT; subjecting the fermentation broth to at least one stage of membrane filtration using nanofiltration membranes, thereby obtaining a filtered solution containing LNnT; subjecting the filtered solution to at least one stage of pseudo-moving bed chromatography, thereby obtaining a purified solution that contains LNnT; subjecting the purified solution to at least one stage of crystallization from water, thus obtaining a crystalline mass; and subjecting such a crystalline mass to at least one homogenization step, thereby obtaining a homogenized and dried purified LNnT product; wherein the purified preparation contains LNnT with a purity of 80% or more.

EFFECT: invention provides effective purification of lacto-N-neotetraose (LNnT) removing the fermentation broth, thus obtaining highly purified LNnT powder.

14 cl, 27 dwg, 10 tbl, 19 ex

Description

Field of invention

The present invention relates to a method for purifying LNnT (lacto-N-neotetraose-lacto-N-neotetraose, Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc). More specifically, the present invention relates to the separation of by-products, impurities and/or contaminants from LNnT resulting from a fermentation process through a purification process.

Prior Art

Human breast milk is considered the best nutrition for infant development. It consists of fats, proteins, vitamins, minerals, trace elements and complex oligosaccharides. In addition to lactose, human breast milk, as well as the milk of other mammals, contains various structurally distinct oligosaccharides, which are also known as human milk oligosaccharides (HMOs) (Usashima T. et al., (2011) Milk 20 Oligosaccharides, Nova Biomedical Books, New York ISBN 978-1-61122-831-1). Currently, it is believed that there are more than 150 structurally different oligosaccharides found in human breast milk. With very rare exceptions, on the one hand, HMOs are characterized by a disaccharide residue, lactose, at their reducing ends. On the other hand, many HMOs contain a fucose residue, a galactose residue, or an N-acetylneuraminic acid residue at their non-reducing end. In addition, there are linear as well as branched representatives. Typically, the monosaccharide residues of HMO are D-glucose, D-galactose, N-acetylglucosamine, L-fucose, and N-acetylneuraminic acid (the latter is better known as sialic acid or lactamic acid). The importance of HMOs for infant feeding is directly related to their biological activities, including protecting newborns from pathogens, supporting the development of the infant's immune system and cognitive abilities. In addition, HMOs serve as a substrate for beneficial bacteria, similar to bifidobacteria or lactobacilli.

Due to the problems accompanying the chemical synthesis of human milk oligosaccharides, several enzymatic methods and enzymatic approaches have been developed. In particular, the enzymatic approach requires the purification of the desired oligosaccharide from a highly complex fermentation broth containing several hundred different individual compounds. The single carbohydrate fraction of a fermentation broth consists of a complex mixture of mono- and oligosaccharides, as well as their derivatives, including substrates (e.g. lactose, fructose, glucose, sucrose and other sugars used as a carbon source), biosynthetic intermediates, individual monosaccharides ( such as glucose, galactose, N-acetyl glucosemin, fucose and N-acetylneuraminic acid), metabolic by-products and other oligo- and polysaccharides synthesized by the microbe. In addition, the structures of many oligosaccharides found in fermentation broth are difficult to identify (for example, oligosaccharides produced from the synthesis of cell surface glycosylation structures similar to those naturally occurring in the host, or oligosaccharides produced by the body as a result of stress). Thus, in many cases, the purification of a biotechnological product can be much more expensive and time-consuming than its production through fermentation.

In particular, fermentation of lacto-N-neotetraose is often associated with excess synthesis of lacto-N-triose II (LNT II, GlcNAc(β1-3)Gal(β1-4)Glc), a biosynthetic intermediate in the biosynthesis of lacto-N-neotetraose, which is often exported from the cell to the medium before being further converted into the target LNnT. In addition, para-lacto-N-neohexaose (pLNnH, Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc) is often found as a side product when LNnT is further converted to N-acetylglucosamine (GalNAc) and glucose instead of export, and even para-lacto-N-neooctose (pLNnO, Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc (β1-3)Gal(β1-4)Glc) was identified as an additionally extended derivative. The latter side products are represented by glucosyl lactose (GlcLac), galactosyl lactose (CalLac), glucosylated LNnT and galactosylated LNnT, which are formed during fermentation. However, oligosaccharides that carry GalNAc at the non-reducing end, such as lacto-N-triose II or pLNnH or pLNnO intermediates, can be effectively removed by glycosidase treatment. In addition, autoclaving (heat treatment) of carbohydrates (eg sucrose or lactose) can lead to the formation of undesirable by-products such as aldol or Maillard reaction products. Isomerization reactions (eg conversion of lactose to lactulose) can also generally lead to high levels of contamination and create isomer oligosaccharides. In order to avoid the formation of by-products due to heat treatment, substrates and sources of C are often subjected to sterile filtration, which, however, causes the risk of contamination of the growth medium with foreign agents.

To obtain breast milk oligosaccharides through microbial fermentation, recombinant microorganisms (recombinant strains of bacteria or yeast) are used. Thus, these fermentation methods are based on genetically modified organisms (GMOs), which are considered extremely important in the food industry sector. Thus, required products must therefore be purified from GMO process residues such as cells, cell fragments, endotoxins, and excess salts in order to obtain regulatory and consumer approval. Lacto-N-neotetraose thus must be purified from a fermentation broth containing a complex mixture of recombinant nucleic acids such as DNA and RNA, as well as recombinant proteins. In addition, microbial fermentation in particular contains significant amounts of endotoxins when E. coli is used. However, contamination of a product for human consumption with recombinant DNA, endotoxins, or proteins is not considered acceptable by either regulators or consumers. Thus, any nucleic acids and proteins derived from the recombinant microorganism must be removed from the desired human milk oligosaccharide.

Known methods for purifying individual oligosaccharides are technically complex and often uneconomical, especially when these starting compounds are obtained as a mixture of several similarly constructed constructs from a fermentation broth, in particular when said oligosaccharide is intended for food applications. For the industrial purification of disaccharides suitable for use in the food industry - lactose or sucrose from complex mixtures, such as whey or molasses, industrial processes have been developed on a multi-ton scale, which include multiple crystallization stages. However, up to the present time, HMO, in general and more specifically lacto-N-neotetraose, has proved difficult to purify, especially when obtained from a fermentation broth. In the case of LNnT, a method has been developed for the synthetic preparation and subsequent crystallization of the resulting product, making it possible to use it as a food ingredient. However, this process does not contain any by-products from the fermentation, and the process gives an almost pure product, which is then isolated for economic reasons as a crystalline substance for further processing afterwards.

Early work on the isolation, characterization, and crystallization of various HMOs was done by Richard Kuhn et al. in the 1950s, where these compounds were isolated from the carbohydrate fraction of mother's milk by column chromatography on activated carbon/celite. The first HMO to be crystallized from Kuhn et. al., was lacto-N-biose I (Kuhn et al., Chem, Ber. 1954, 87(10), 1553-1560), followed in subsequent years by a huge amount of other carbohydrates: lactose-N-tetraose ( Kuhn et al., Chem Ber 1953, 86(6), 827-830; Chem Ber 1954, 87(3), 289-300; Chem Ber 1956, 89(2), 504-511) , lacto-N-fucopentaose I (Kuhn et al., Chem. Ber. 1956, 89(11), 2514-523), 2'-fucosyllactose (Kuhn et al., Chem. Ber. 1955, 88(8), 1135-1146; 1956, 89(11), 2513), lacto-N-triose I and II (Kuhn et al., Chem. Ber. 1956, 89(4), 1027-1033) and lacto-N-neotetraose ( Kuhnetal., Chem Ber 1962, 95(11), 518-522).

To purify lacto-N-neotetraose and the closely related breast milk oligosaccharide lacto-N-tetraose (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc), chromatographic methods, in particular gel filtration chromatography ( Dumon et al., 2001 Glycoconj. J. 18(6), 465-474; Priem et al., Glycobiology 2002, 12(4), 235-240; Baumgartner et al., Chem. Bio. Chem. 2014 15( 13), 1896-1900, Sprenger et al., 2017, J. Biotechnol. 258, 79-91). In this regard, purification by size exclusion chromatography is not suitable for food products on an industrial scale, however, fluidized bed chromatography can be considered a suitable method for purifying LNnT for food products, in combination with other purification steps, to obtain purified LNnT as an end product. .

Microbial fermentation of lacto-N-neotetraose can produce many other carbohydrates such as trioses (LNT II, GlcLac, GalLac) as well as longer chain oligosaccharides such as hexaoses (pLNnH). In addition, hydrolysis products of the resulting oligosaccharides are formed during the fermentation and processing process. Using a purification method that includes several purification steps, LNnT can be obtained in larger quantities, higher purity and higher yield, making it suitable as an ingredient in a food formula or for cosmetic or medical applications.

The process of the present invention represents a cost-effective alternative to prior art processes resulting in a solid powder product and is particularly relevant for the purification of lacto-N-neotetraose for nutritional applications and in particular for the preparation of nutritional products for infants and children who are starting out. walk, medicinal foods, nutritional supplements, or conventional foods.

To overcome all these shortcomings of the known methods, the present invention proposes a new purification process, simple, cost-effective and scalable, for the purification of lacto-N-neotetraose resulting from a fermentation process.

Brief summary of the invention

The present invention relates to a simple and economical process for purifying lacto-N-neotetraose obtained from microbial fermentation. Through microbial fermentation of lacto-N-neotetraose, several other carbohydrates such as trioses and longer chain oligosaccharides such as hexaoses can also be formed. In addition, hydrolysis products of the produced oligosaccharides are formed during the fermentation and processing process.

The purpose of the present invention is to provide a simple, cost-effective and scalable process for obtaining purified lacto-N-neotetraose obtained from microbial fermentation as the main product, while by-products, impurities and/or other contaminants, such as lacto-N -triose II and/or para-lacto-N-neohexaose and/or para-lacto-N-neooctose and/or glucosyl lactose and/or galactosyl lactose are separated from such a main product.

Another object of the present invention is to provide lacto-N-neotetraose with a purity of 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more and 95% or more.

Brief description of graphic materials

On FIG. 1 shows a general diagram of the steps in the purification process according to the present invention.

On FIG. 2 shows the analysis of the retentate of Example 1 for a retentate volume of 80 liters.

On FIG. 3 shows the analysis of the retentate of Example 1 for a retentate volume of 60 liters.

On FIG. 4 shows the analysis of the retentate of Example 1 for a retentate volume of 40 liters.

On FIG. 5 shows the analysis of the permeate of Example 1 for a volume of permeate retentate of 1-20 liters.

On FIG. 6 shows the analysis of the permeate of Example 1 for a volume of permeate retentate of 2-20 liters.

On FIG. 7 shows the HILIC-CAD (Hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the carbohydrate mixture used in Example 4.

On FIG. 8 shows the spectrum of the HILIC-CAD (Hydrophilic Interaction Liquid Chromatography coupled to a Charged Aerosol Detector) spectrum of the carbohydrate mixture used in Example 4.

On FIG. 9 shows the spectrum of the HILIC-CAD (Hydrophilic Interaction Liquid Chromatography coupled to a Charged Aerosol Detector) spectrum of the carbohydrate mixture used in Example 4.

On FIG. 10 shows the HPAEC-PAD (High performance anion exchange chromatography with pulsed amperometric detection) spectrum of the carbohydrate mixture obtained after crystallization in Example 5.

On FIG. 11 shows the HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection) spectrum of the carbohydrate mixture obtained after the first crystallization in Example 6.

On FIG. 12 shows the HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection) spectrum of the carbohydrate mixture obtained after the second crystallization in Example 6.

On FIG. 13 shows the spectrum of the HILIC-CAD (Hydrophilic Interaction Liquid Chromatography coupled to a Charged Aerosol Detector) spectrum of the carbohydrate mixture obtained after crystallization in Example 7.

On FIG. 14 shows the HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection) spectrum of the seed crystals obtained by gel filtration in Example 8.

On FIG. 15 shows the HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection) spectrum of the LNnT starting material used in Example 9.

On FIG. 16 shows the HPAEC-PAD spectrum of the LNnT product prepared in Example 9.

On FIG. 17 shows the HPAEC-PAD spectrum of the LNnT product prepared in Example 10.

On FIG. 18 shows the HPAEC-PAD spectrum of the LNnT product prepared in Example 11.

On FIG. 19 shows the HPAEC-PAD spectrum of the LNnT product prepared in Example 12.

On FIG. 20 shows the HILIC-CAD spectrum (Hydrophilic Interaction Liquid Chromatography coupled to a Charged Aerosol Detector) of the carbohydrate mixture obtained after homogenization in Example 13.

On FIG. 21 shows the HILIC-CAD (Hydrophilic Interaction Liquid Chromatography coupled to a Charged Aerosol Detector) spectrum of the carbohydrate mixture obtained after homogenization in Example 14.

On FIG. 22 shows the HPAEC-PAD spectrum of the LNnT product prepared in Example 15.

On FIG. 23 shows the HILIC-CAD spectrum (Hydrophilic Interaction Liquid Chromatography coupled to a Charged Aerosol Detector) of the LNnT crystalline starting material used in Examples 16-19.

On FIG. 24 shows the HILIC-CAD spectrum (Hydrophilic Interaction Liquid Chromatography coupled to a Charged Aerosol Detector) of the homogenized LNnT product obtained in Example 17.

On FIG. 25 shows the HILIC-CAD (Hydrophilic Interaction Liquid Chromatography coupled to a Charged Aerosol Detector) spectrum of the homogenized LNnT product prepared in Example 18.

On FIG. 26 shows the HILIC-CAD spectrum (Hydrophilic Interaction Liquid Chromatography coupled to a Charged Aerosol Detector) of the homogenized LNnT product prepared in Example 19.

On FIG. 27 shows the HILIC-CAD spectrum (Hydrophilic Interaction Liquid Chromatography coupled to a Charged Aerosol Detector) of the homogenized LNnT product obtained in Example 20.

Detailed description

A simple, cost-effective and scalable process is proposed to obtain purified lacto-N-neotetraose, obtained from a microbial fermentation process, as the main product, while by-products, impurities and / or other contaminants, such as lacto-N-triose II and/or glucosyllactose and/or galactosyl lactose and/or para-lacto-N-neohexaose and/or para-lacto-N-neo-octaose are separated from such main product.

The present invention provides a process for purifying lacto-N-neotetraose (LNnT), in batches or continuously, from a fermentation broth obtained by a microbial fermentation process, wherein purified LNnT is provided with a purity of greater than 80%, greater than 85%, greater than 90% and/or greater than 95%. %. The fermentation broth contains neutral HMO, biomass, media components, contaminants, and carbohydrates other than LNnT. The purity of LNnT in the fermentation broth is 60% or less.

The method of the invention of the present application uses a fermentation broth containing LNnT and other contaminants such as trioses, hexaose and/or tetraose among others as a starting material, such a fermentation broth obtained from a microbial fermentation process. The purity of LNnT in the fermentation broth is 60% or less. The fermentation broth is subjected to the following purification steps:

1) at least one stage of membrane filtration of the solution, wherein the solution is a fermentation broth containing a mixture of carbohydrates resulting from a microbial fermentation process, and after use in subsequent stages of the standard protocol, it is subjected to nanofiltration to significantly reduce the content of higher saccharides, such as pentoses and/or hexaose, up to less than 10%, and the purity of LNnT in the filtered solution is more than 60%, more than 65% or more than 70%;

2) at least one stage of chromatography with SMB (from the English. simulated moving bed - pseudo-moving bed), setting the remaining amount of hexaose below 5% in order to make suitable crystallization in the next stage, the purity of LNnT in the resulting purified solution is more than 75% or more than 80%;

3) at least one crystallization step from water to obtain a crystalline mass which is treated and washed with alcohol, an alcohol/water mixture or a solvent or solvent/water mixture in order to wash away the remaining smaller saccharides by running off, and setting their concentration below 3 % to obtain a purity of LNnT in the resulting crystals of more than 85%, more than 90% or more than 95%;

4) at least one step of homogenizing the resulting, dried crystalline mass by freeze drying, spray drying, tumble dryer/roller dryer, vacuum tumbler/roller dryer, belt dryer, or vacuum belt dryer;

which provides a product containing purified LNnT with a purity of 90% or more.

Membrane filtration as the first step of the method of the present invention is used to reduce the content of oligosaccharides with a higher molecular weight than the desired LNnT of lower molecular weight. There are many different types of membranes that can be used for membrane filtration, but they all differ in their suitability for carbohydrates. The most common materials used for membranes are polymer materials and ceramic membrane modules. While polymer membranes can be processed as hollow fibers or as blocks due to their inherent flexibility, ceramic membranes are limited to use as hollow fiber blocks. Both techniques can be combined into membranes of combined materials or processed sequentially, however, the industry is mainly focused on obtaining homogeneous materials due to cost and/or efficiency.

In case it is desired to reduce the amount of oligosaccharides having a molecular weight greater than that of lacto-N-neotetraose, a membrane filtration step and more particularly a nanofiltration step are used. Nanofiltration is a pressure separation membrane process based on the retention of dissolved molecules, metal ions and other particles above the molecular weight cutoff (MWCO). The membranes used in nanofiltration have a pore size of 2 nm or less, which distinguishes them from the coarser membranes used in other membrane filtration methods such as ultrafiltration and microfiltration.

Compared to other stages of membrane filtration, such as reverse osmosis, nanofiltration uses correspondingly coarser membranes and lower operating pressures. However, membranes used for filtration are usually limited and/or highly dependent on their retention properties and/or resistance to temperature or chemicals, such that the application of this method is significantly limited to the use of water and aqueous mixtures. Lacto-N-neotetraose has a molecular weight of 707.6 Da and larger saccharides increased by one or two monosaccharides, respectively, have a molecular weight of 869.3 Da (LNnT+Glc/Gal), 910.3 Da (LNnT+GlcNAc) and 1072.4 Yes (LNnT+LacNAc). Thus, nanofiltration membranes offering the smallest possible MWCO are the membranes used in the present invention. The membranes used in the present invention have an MWCO of 0.2 to 3.5 kDa, more preferably 0.2 to 2.0 kDa, and even more preferably 0.2 to 1.0 kDa.

In order to achieve a reduction in saccharides larger than LNnT, or typically saccharides larger than triose, the sugar mixture solution is prepared for the filtration process by applying pressure to the membrane. Three sugars are used for separation: LNnT, hexaose and triose, which is also present as a by-product derived from the microbial fermentation process in the fermentation broth mixture.

The goal of this first membrane filtration step in the first approach is to deplete hexaose until the sugar mixture accordingly contains relatively less hexaose in order to carry out subsequent SMB chromatography and crystallization steps as efficiently as possible. Alternatively, this first membrane filtration step can be used in a second approach to relatively deplete tetraose and hexaose compared to triose, ultimately enriching LNnT and pLNnH in the retentate in order to be able to obtain purified LNnT after the subsequent step. SMB chromatography and subsequent crystallization step, thus first removing the lower saccharides and then removing the higher saccharides).

An aqueous solution of at least two, preferably three or more than three oligosaccharides, including LNnT, of which at least two differ in mass and all of which originate from a bacterial fermentation process and which are ready for a purification process down to the sugar component using microfiltration , ultrafiltration, anion and cation exchange, activated carbon treatment and filtration, and/or diafiltration and electrodialysis (ED - from English electrodialysis), subjected to nanofiltration by applying a pressure of 100-5000 kPa, more preferably 200-3000 kPa, more preferably 300 -1000 kPa and more preferably 400-500 kPa. The MWCO threshold of the membrane is 0.2 to 3.5 kDa, more preferably 0.2 to 2.0 kDa, and more preferably 0.2 to 1.0 kDa. The preset sugar concentration of the solution is 0.01 to 70% dry solid content (DSC), more preferably 0.1 to 60% DSC, more preferably 1 to 50% DSC, and more preferably 10 to 40% DSC. The dilution factor is 0.01-1. Accordingly, the retentate must be washed with additional amounts of water, depending on the dilution factor. The DSC of the eluate is thus changed by a factor of 0.01-1. With the membrane filtration step, the hexaose content is reduced below 20% in the retentate, more preferably below 15% and more preferably below 10%.

The purity of LNnT in the filtered solution obtained from the membrane filtration step is greater than 60%, greater than 65%, or greater than 70%.

As a further embodiment, the separation of trioses from a mixture also containing trioses as well as tetraoses as well as hexaoses, where these three types of saccharides can be the main components of the sugar solution, allows further purification by means of a second membrane filtration step, in this case, nanofiltration, with the reduction of hexaoses by selecting the appropriate membrane material and pore size, and ultimately obtaining a purified LNnT solution.

As a further embodiment, the separation of hexaoses from a mixture also containing trioses as well as tetraoses as well as hexaoses, where these three types of saccharides can be the main components of the sugar solution, allows further purification by means of a second membrane filtration step, in this case, nanofiltration, with reducing the remaining trioses by selecting the appropriate membrane material and pore size, and ultimately obtaining a purified LNnT solution.

The filtered solution is now subjected to a second stage of pseudo-moving bed chromatography (SMB chromatography), which is used to adjust the content of a fraction containing a certain sugar, such as a hexaose fraction, as compared to fractions that contain tetraose, through specially adapted parameters. The filtered solution, which is the solution obtained from the 1 stage membrane filtration, has a hexaose content below 20%, more preferably below 15%, and more preferably below 10%.

The SMB chromatography step provides the second step of the present invention for separating a HMO mixture whose extract or raffinate (or lower extract or raffinate or higher saccharide extract or raffinate) contains lacto-N-neotetraose tetrasaccharide in a continuous chromatography (or continuous manner) from a filtered solution containing neutral HMO-tetrasaccharide lacto-N-neotetraose and other contaminants - by-products, where the filtered solution contains a mixture of lacto-N-neotetraose and contaminants, contains or consists of a solution that is obtained in stage 1, membrane filtration, and where the purity of the LNnT solution is less than 90%.

The filtered solution is used in at least one purification step using pseudo moving bed chromatography. Thus, two solutions should be obtained, of which one contains the desired lacto-N-neotetraose. After applying this step 2 SMB chromatography, the LNnT can mainly be found either in the extract or in the raffinate, or as a result of the separation of lower oligosaccharides, such as trioses, or as a result of the separation of higher saccharides, such as hexaoses.

Using this second stage of pseudo-moving bed chromatography, LNnT is obtained with higher purity and a continuous path. Thus, large quantities of high quality HMOs can be obtained in a very convenient and economical way. The fluidized bed chromatography step is also highly stable even without a regeneration step of the column material (eg, cationic column material) used in this step. In a preferred embodiment, the purity of the LNnT in the filtered solution is greater than 60%, greater than 65%, or greater than 70%. The term "filtered solution" refers to the solution containing LNnT before the purification step of the single pseudo moving bed chromatography and obtained in step 1, membrane filtration, while "purified solution" refers to the solution after the pseudo moving bed chromatography step.

Said at least one single pseudo moving bed chromatography step 2 has the following:

i) at least 4 columns, preferably at least 8 columns, more preferably at least 12 columns, where at least one column contains a weak or strong cation exchange resin, preferably a cation exchange resin in H ⁺ -form, Ma ⁺ -form, K ⁺ -form or Ca ²⁺ -form; and/or

ii) four zones I, II, III and IV with different flow rates; and/or

iii) an eluent containing or consisting of water, preferably ethanol and water, more preferably 5-15 vol. % ethanol and 85-95 vol. % water, most preferably 9-11 vol. % ethanol and 89-91 vol. % water,

iv) operating temperature 15°-60°C, preferably 20°-55°C, more preferably 25°-50°C.

Since the HMO to be purified is lacto-N-neotetraose, at least one stage of fluidized bed chromatography has the following:

i) four zones I, II, III and IV with different flow rates, where the flow rates are preferably: 22-32 ml/min in zone 1, 17-23 ml/min in zone II, 18-25 ml/min in zone III and/or 14-20 ml/min in zone IV; and/or

ii) a flow rate of 0.5-4 ml/min, preferably 2 ml/min; and/or

iii) eluent flow rate 6-12 ml/min, preferably 8 ml/min; and/or

iv) switching time 14-20 minutes, preferably 16-18 minutes, more preferably 17 minutes.

Preferably, at least one of the columns contains 0.1-5000 kg of cation exchange resin, preferably 0.2-500 kg of cation exchange resin, more preferably 0.5-50 kg of cation exchange resin, most preferably 1.0-20 kg of cation exchange resin.

Importantly, it is possible to proportionally increase the amount of cation exchange material, the flow rate in different zones, the feed rate, the eluent flow rate and/or the switching time. Proportional increase can be an increase by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or all possible correction factors between the specified values.

In columns, a strong cation exchange resin can be used as the stationary phase. Preferably, the cation exchange resin is a sulfonic acid resin, more preferably Purolite® PCR833H resin (Purolite, Ratingen, Germany), Lewatit MDS 2368 and/or Lewatit MDS 1368. If a cation exchange resin is used in the columns, it can be regenerated with sulfuric acid . Sulfuric acid may be used in the eluent, preferably at a concentration of 10 mM sulfuric acid or less. The (strong) cation exchange resin may be in the H ⁺ form or the Ca ²⁺ form.

Operating temperatures above 60° C. are not preferred in fluid moving bed chromatography. It was found that especially in the presence of a strong cation exchange resin (in H ⁺ form or Ca ²⁺ form) as the stationary phase, the neutral oligosaccharides used were significantly destabilized, namely depolymerized, which was unfavorable with regard to the final yield of LNnT.

In one preferred embodiment of the invention, the purified solution can be used in at least one further purification step using fluidized bed chromatography, wherein the purified solution is provided containing a neutral human milk oligosaccharide with a purity greater than 85%, preferably greater than 90%; more preferably greater than 93%. In particular, according to the invention, a HMO product is provided that does not contain recombinant DNA and does not contain proteins of the host strain.

In yet another stage of pseudo-moving bed chromatography, there is the following:

i) at least 4 columns, preferably at least 8 columns, more preferably at least 12 columns, where at least one column contains a weak or strong cation exchange resin, preferably a cation exchange resin in H ⁺ -form, Na ⁺ -form, K ⁺ -form or Ca ²⁺ -form; and/or

ii) four zones I, II, III and IV with different flow rates, and/or

iii) an eluent containing or consisting of water, preferably ethanol and water, more preferably 5-15 vol. % ethanol and 85-95 vol. % water, most preferably 9-11 vol. % ethanol and 89-91 vol. % water, where the eluent optionally contains sulfuric acid, preferably 10 mm or less sulfuric acid; more preferably 2-5 mM or less sulfuric acid and/or

iv) operating temperature 15°-60°C, preferably 20°-55°C, more preferably 25°-50°C.

If the HMO to be purified is lacto-N-neotetraose, another stage of pseudo-moving bed chromatography has the following:

i) four zones I, II, III and IV with different flow rates, where the flow rates are preferably: 22-32 ml/min in zone 1, 18-23 ml/min in zone II, 19-25 ml/min in zone III and/or 15-20 ml/min in zone IV; and/or

ii) a flow rate of 1-4 ml/min, preferably 2 ml/min; and/or

iii) eluent flow rate 6-12 ml/min, preferably 9 ml/min; and/or

iv) switching time 16-22 minutes, preferably 18-20 minutes, more preferably 19 minutes.

In particular, at least one of the columns contains 0.1-5000 kg of cation exchange resin, preferably 0.2-500 kg of cation exchange resin, more preferably 0.5-50 kg of cation exchange resin, most preferably 1.0-20 kg of cation exchange resin .

Importantly, it is possible to proportionally increase the amount of cation exchange material, the flow rate in different zones, the feed rate, the eluent flow rate and/or the switching time. Proportional increase can be an increase by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or all possible correction factors between the specified values. After the purification step using fluidized bed chromatography, the pH of the purified solution can be adjusted to pH 7, preferably by adding a base, more preferably by adding NaOH (eg 0.2 M NaOH).

The purity of LNnT in the purified solution obtained from the fluid moving bed chromatography step is greater than 75% or greater than 80%.

This purified solution is now subjected to a third crystallization step, which is used to produce highly purified LNnT. The purified solution obtained from step 2 SMB chromatography has a hexaose content below 20%, more preferably below 15% and more preferably below 10%.

The crystallization stage includes:

a) obtaining a mixture of at least two, more preferably at least three or more oligosaccharides, of which one is a trisaccharide or saccharide, less random tetraose, another is lacto-N-neotetraose and one is lara-lacto- N-neohexaose or oligosaccharide, more occasionally tetraose of said oligosaccharides, after steps 1 or 2 a purified solution of oligosaccharides is obtained, of which lacto-N-neotetraose is one, preferably at a concentration of at least about 10% DSC, especially at least about 30% DSC in particular at least about 50% DSC and with an LNnT purity of the mixture of at least 15%, preferably at least about 25%, especially at least about 40%, in particular at least about 60%,

b) crystallizing a mixture of said oligosaccharides at an initial temperature of at least 20-30° C., preferably at least about 30-40° C., preferably at least about 40-50° C. and more preferably at least about 50-60° C. C and allowing the saturated mass to crystallize by cooling to at least 40-50°C, preferably at least about 30-40°C, preferably at least about 20-30°C, preferably at least about 10-20°C and more preferably at least about 0-10°C to obtain a homogeneous crystalline mass,

c) treating the resulting crystalline mass with a strictly mixture of an alcoholic solvent such as methanol, ethanol or isopropanol or glycol or glycerol or any other water-miscible alcohol, or solvent and water, where the alcohol or solvent content of this solution is from 10 to 90 vol. %, preferably from 30 to 80 vol. % and more preferably from 50 to 70 vol. %, to obtain a mixture of a solution containing alcohol or solvent, and a crystalline mass,

d) filtering the fraction containing alcohol or solvent and now containing sugar, of the crystalline mass to reduce the content of lower saccharides in the crystalline mass, preferably the washing method can be repeated at least twice to reduce the content of lower saccharides below a certain threshold, which is less than 10%, preferably less than 5% and more preferably less than 3%, before subjecting said crystalline mass to a fourth homogenization step.

The amount of alcohol or solvent used to wash the crystal mass is not critical, but at least about 40-100 liters, preferably about 30-70 liters of alcohol or solvent is used per 10 kilograms of crystal mass. Preferably, the alcohol or solvent solution is then filtered to remove lower molecular weight oligosaccharide by-products. The alcohol or solvent solution, after being filtered and now containing less lacto-N-neotetraose, can be used in another crystallization cycle, if necessary, to obtain more lacto-N-neotetraose. However, not only lacto-N-neotetraose can be purified in this way, but also trisaccharides derived from said fermentation broth or chemical synthesis or biocatalysis, as well as higher saccharides such as pentaoses, hexaoses as well as octaoses, can be purified by washing out lower oligosaccharides with aqueous mixtures containing alcohol or solvent from their crystallization masses.

The purity of LNnT in the crystals obtained at the crystallization stage is more than 80%, more than 90% and more than 95%.

The crystals are subjected to a fourth homogenization step, which is used to obtain a highly homogeneous and purified LNnT product with a residual water content of not more than 20%.

When the crystals are homogenized, dry lacto-N-neotetraose is obtained from the initial crystalline mass. Typically, through the use of drying, a mass transfer process is carried out, consisting of the removal of water or other solvent by evaporation from a solid, semi-solid, or liquid phase. This method is often used as the final preparation step. Often a heat source and an agent are involved to remove the steam generated by the process. In biological products such as feed, grains, and drugs such as vaccines, the solvent to be removed is almost invariably water. Drying may be synonymous with drying or considered an extreme form of drying.

In the most general case, in a gas flow, such as air, heat is used by convection and steam is carried away in the form of moisture. Other options are vacuum drying, where heat is supplied by conduction or radiation (or microwaves), while the steam thus formed is removed by a vacuum system. Another indirect technique is tumble dryer, in which a heated surface is used to provide energy and aspirators draw steam from outside the room.

In the present case of crystallized lacto-N-neotetraose, several methods can be used to provide a homogenized dried substance with suitable product characteristics. One of the most common methods used is oven drying or vacuum oven drying. However, in the case of a crude crystalline mass, drying in an oven or vacuum drying in an oven can only provide a homogeneous substance through the combination of drying and mixing. The dried crystalline mass obtained from the crystallization purification step should therefore be gently spread over the surface to obtain a maximum surface from which the solvent can then evaporate during the process. When using an oven drying step, the temperature is set at 20-200°C, preferably 25-100°C or even more preferably 30-50°C in order to cause the solvents to evaporate. Solvents are removed by air flow. When using vacuum drying in an oven, the temperature is set at 20-200°C, preferably 25-100°C or even more preferably 30-50°C in order to cause the solvents to evaporate. Solvents are removed by means of a pump set to a vacuum in the range of 0.001 to 100 kPa, preferably 0.01 to 10 kPa, and more preferably 0.1 to 1 kPa. Drying is started in two cases from 1 hour to 7 days. The material is stirred before or after drying.

In the second embodiment, the final homogenization is not necessary since the drying of the material is carried out by using oven drying or vacuum oven drying after the second crystallization cycle, which is started after the first crystallization and subsequent drying process. As in this case, the second and third cycle of crystallization takes place, no washing process is needed, no inhomogeneous substance is formed. Generally, in the second and third crystallization cycles, the crystals grow better due to the higher purity obtained, and thus said crystals are easier to dry after additional crystallization steps.

In a third embodiment, the homogenization process is freeze drying. In this method, the crystals obtained after drying the crystallization mass are dissolved in water before being subjected to a freeze drying process. Thus, the LNnT crystals are dissolved in water at a concentration in the range of 0.1-70% DCS, preferably 1-50% DSC and more preferably 5-25% DSC before freezing and before being exposed to a freeze dryer that releases water. and releases a frothy, dry and homogeneous gel-type sugar product.

Ideally, the use of freeze drying, unlike most other drying methods, results in a completely anhydrous or substantially water-free product that indirectly provides a determination of the water content of the sample. In particular, with carbohydrates such as HMO, of which LNnT is one used, the determination of residual water is difficult because, firstly, they are poorly soluble in anhydrous solvents and, secondly, alcohol functional groups (OH groups) have similar - the same spectroscopic properties as water.

As a fourth embodiment, the homogenization process is spray drying. The spray dryer takes in the liquid stream and separates the solution or suspension into a solid phase and a solvent that turns into vapor. The solid phase is usually collected in a drum or cyclone separator. The incoming liquid stream is sprayed through a nozzle into a hot steam stream and evaporated. The nozzle is usually used to create droplets as small as possible, maximizing heat transfer and the rate of water evaporation. Spray dryers can dry the product very quickly compared to other drying methods. They also convert a solution or suspension into a dried and homogenized powder in a single step.

Spray drying is preferably used by setting the concentration of the sugar solution to 1 to 70% DSC, preferably 10 to 60% DSC, preferably 20 to 50% DSC, and more preferably 30 to 40% DSC. The sugar solution is then passed under pressure through the nozzles of a spray dryer with an inlet temperature of 100 to 160°C, preferably 110 to 150°C and more preferably 120 to 140°C. The flow is adjusted to maintain an outlet temperature of 50-80°C, preferably 60-70°C, more preferably 66°C to 67°C. Using these plants, a homogeneous spray-dried powder can be obtained.

As a fifth embodiment, the homogenization process for producing lacto-N-neotetraose is roller drying or drum drying or vacuum roller drying. Roll dryer or drum dryer is a method used to dry liquids from a crude, inhomogeneous material. In the drying process, the raw ingredients are dried at relatively low temperatures over high power rotating rollers, which produce sheets of dried and homogeneous product. Tumble dryer drying techniques result in a dried substance that recovers immediately and retains much of its original taste, odour, color and nutritional value. Some advantages of roller dryers include the ability to dry viscous inhomogeneous solutions that cannot be easily dried by other means, and drum dryers can be easily cleaned and are easy to handle and maintain.

When roller drying is used, the temperature of the rollers is set at 20-200°C, preferably 50-150°C and more preferably 75-125°C to evaporate the solvents. Solvents are removed by air flow. When using vacuum roller drying, the temperature is set at 20-200°C, more specifically 50-150°C and even more specifically 75-125°C to evaporate the solvents. Solvents are removed by means of a pump set to a vacuum in the range of 0.01-100 kPa, preferably 0.1-50 kPa and more preferably 1-10 kPa. In both cases the rolls are rotated at 0.1-100 rpm, preferably between 1 and 10 rpm.

As a sixth embodiment, the homogenization process for producing lacto-N-neotetraose is belt drying or vacuum belt drying. Belt dryer or vacuum belt dryer is a method used to dry liquids from raw materials. In the drying process, the raw ingredients are dried at relatively low temperatures over high capacity moving belts which produce a dried homogeneous product.

The final powder product has an LNnT purity in the purified formulation of 80% or more, preferably 85% or more, more preferably 90% or more, based on the dry matter of the purified formulation.

The present invention has been described with respect to specific embodiments and with reference to the drawings, but the present invention is not limited thereto, but only by the claims. In addition, the terms first, second, and the like in the specification and claims are used to distinguish between like elements, and not necessarily to describe sequence, time, space, rank, or in any other way. It should be understood that the terms used in this way are interchangeable in appropriate circumstances, and that the embodiments of the invention described herein are capable of operating in sequences other than those described or illustrated herein.

It should be noted that the term "comprising" used in the claims should not be considered limited to the means listed hereinafter; it does not exclude other elements or steps. Thus, it should be considered as defining the presence of the claimed features, integers, steps, or components referred to, but it does not preclude the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Thus, the scope of the expression "device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that, with respect to the present invention, the only relevant components of the device are A and B.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with a given embodiment is included in at least one embodiment of the present invention. Thus, the occurrences of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. In addition, specific features, structures, or characteristics may be combined in any suitable manner, as will be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

Likewise, it should be understood that in the description of illustrative embodiments of the invention, various features of the invention are sometimes grouped together in one single embodiment, figure, or description thereof, for the purpose of simplifying the disclosure and aiding in understanding one or more of the various aspects of the invention. This mode of disclosure, however, should not be taken to reflect the idea that the claimed invention requires more features than are explicitly listed in each claim. Rather, as reflected in the following claims, aspects of the invention are less than all features of one of the above disclosed embodiments. Thus, the claims after the detailed description are hereby expressly incorporated into this detailed description, each claim standing alone as a separate embodiment of the present invention.

Furthermore, while some embodiments described herein include some but not all of the features included in other embodiments, combinations of features of different embodiments are intended to be within the scope of the invention and form different embodiments, as will be appreciated by those skilled in the art. areas. For example, in the claims below, any of the claimed embodiments may be used in any combination.

In addition, some of the embodiments are described herein as a method or combination of method elements that may be implemented by a computer system processor or by other means of performing a function. Thus, the processor, with the necessary instructions for implementing such a method or method element, constitutes a means for implementing the method or method element. In addition, the apparatus embodiment element described herein is an example of a means for carrying out the function performed by the element for carrying out the invention.

Numerous specific details are set forth in the description and graphics provided herein. However, it is understood that embodiments of the invention may be practiced without these specific details. In other examples, well-known methods, structures, and techniques have not been shown in detail in order not to obscure this description.

The invention is described by means of a detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be arranged in accordance with the knowledge of experts in this field, without deviating from the true essence or technical idea of the invention, and the invention is limited only by the terms of the attached claims.

Example 1: Nanofiltration of an 80 L LNnT-containing carbohydrate mixture containing two major separation by-products, thus achieving depletion of higher saccharides such as pLNnH and accumulation of smaller saccharides such as trioses and LNnT.

80 L of an aqueous solution of a mixture of sugars is subjected to 200 Da nanofiltration to deplete pLNnH. The solids content (DSC) of the sugar solution is 5.51% and the ratio of the 3 sugars considered is given: 4.0% triose, 62.0% LNnT and 20.8% pLNnH. This corresponds to the equivalent of 4.41 kg of total sugars and 2.73 kg of LNnT. Using a feed pump, the sugar mixture is fed at a pressure of about 400 kPa into the circulation of the filter unit. With a circulation pump, the sugar solution is pumped into the circle at a pressure of 500 kPa around a 200 Da ceramic membrane for filtration through it. With respect to the permeate, the filtered sugar solution leaves the membrane at a pressure of one hundred kPa. After subsequent filtration, 20 liters of each retentate and permeate are analyzed for sugar composition and sugar concentration.

After 20 liters, the retentate changes as follows. 60 L of retentate now has 6.85% DSC, consisting of 3.9% triose, 61.7% LNnT and 22.3% pLNnH. This corresponds to the equivalent of 4.11 kg of total sugars and 2.54 kg of LNnT. The permeate has the following composition: 1.88% DSC with a composition of 4.5% triose, 67.2% LNnT and 11.7% pLNnH. This corresponds to a total of 380 g of sugar and an amount of LNnT of 253 g.

After another 20 liters, the retentate is changed as follows. The remaining 40 L of retentate now has a dry weight of 9.95% DSC with a composition of 3.9% triose, 62.5% LNnT and 24.6% pLNnH. This corresponds to a total amount of sugars of 3.98 kg and an amount of LNnT of 2.49 kg. 20 liters of permeate have the following composition: 2.44% DSC with a composition of 4.6% triose, 67.5% LNnT and 12.1% pLNnH. Equivalent to 490 g total sugar and 329 g LNnT.

These results make it possible to calculate the penetration coefficients for each component - sugar. Triose penetrates by a factor of about 1.15, LNnT by a factor of 1.09 and pLNnH by a factor of 0.55.

The results are shown in Table 1, Table 2 and Table 3.

Example 2: Nanofiltration of a 35 L LNnT-containing carbohydrate mixture containing two main separation by-products and intermediate addition of another 10 L of water to the feed solution to achieve depletion of higher saccharides such as pLNnH and accumulation of smaller saccharides such as trioses and LNnT

35 L of an aqueous solution of a mixture of Sugars is subjected to 200 Da nanofiltration to deplete pLNnH. The solids content (DSC) of the sugar solution is 7.88% and the ratio of the 3 sugars considered is given: 2.8% triose, 58.0% LNnT and 26.1% pLNnH. This corresponds to the equivalent of 2.76 kg of total sugars and 1.60 kg of LNnT. Using a feed pump, the sugar mixture is fed at a pressure of about 400 kPa into the circulation system of the filter unit. The sugar solution is pumped into the circle at a pressure of 500 kPa around a 200 Da ceramic membrane, through which filtration is carried out. With respect to the permeate, the filtered sugar solution leaves the membrane at a pressure of one hundred kPa. The sugar solution is pumped through the membrane in 5 liter increments and the remaining retentate and resulting permeate are analyzed for sugar composition and sugar concentration. After the first 10 liters, add 5 liters of fresh water twice to the supply.

After the first 5 liters, the retentate changes as follows. Now 30 L of retentate has 8.34% DSC, consisting of 2.2% triose, 57.5% LNnT and 26.1% pLNnH. This corresponds to the equivalent of 2.50 kg of total sugars and 1.44 kg of LNnT. The permeate has the following composition: 2.37% DSC with a composition of 3.5% triose, 59.6% LNnT and 16.5% pLNnH. This corresponds to a total of 120 g of sugar and an amount of LNnT of 71 g.

After another 5 liters, the retentate changes as follows. The remaining 25 L of retentate now has a dry weight of 9.07% DSC with a composition of 2.3% triose, 57.1% LNnT and 26.9% pLNnH. This corresponds to a total amount of sugars of 2.27 kg in the retentate and an amount of LNnT of 1.30 kg. The combined 10 L permeate has the following composition: 2.21% DSC with a composition of 3.5% triose, 61.1% LNnT and 16.3% pLNnH. Equivalent to 220g total sugar and 135g LNnT.

Now 5 liters of water is added to the feed before additional filtration. After another 5 liters, the retentate is changed as follows. The remaining 25 L of retentate now has a dry weight of 8.95% DSC with a composition of 2.2% triose, 56.1% LNnT and 26.3% pLNnH. This corresponds to a total sugar of 2.24 kg in the retentate and an amount of LNnT of 1.26 kg. The combined 15 L permeate has the following composition: 2.24% DSC with a composition of 3.6% triose, 60.4% LNnT and 16.6% pLNnH. Equivalent to 340g total sugar and 203g LNnT.

Now another 5 liters of water is added to the feed before additional filtration. After another 5 liters, the retentate is changed as follows. The remaining 25 L of retentate now has a dry weight of 7.86% DSC with a composition of 2.2% triose, 56.3% LNnT and 26.9% pLNnH. This corresponds to a total sugar of 1.97 kg in the retentate and an amount of LNnT of 1.11 kg. The combined 20 L permeate has the following composition: 1.94% DSC with a composition of 3.4% triose, 61.3% LNnT and 16.5% pLNnH. Equivalent to 390 g total sugar and 238 g LNnT.

After another 5 liters, the retentate is changed as follows. The remaining 20 L of retentate now has a dry weight of 8.15% DSC with a composition of 2.2% triose, 56.4% LNnT and 27.4% pLNnH. This corresponds to a total amount of sugars of 1.63 kg in the retentate and an amount of LNnT of 919 g. The combined 25 L of permeate has the following composition: 1.94% DSC with a composition of 3.5% triose, 61.9% LNnT and 17.5% pLNnH. Equivalent to 490 g total sugar and 300 g LNnT.

After the last 5 liters, the combined 30 liters of permeate have the following composition: 2.51% DSC with a composition of 3.5% triose, 59.7% LNnT and 16.5% pLNnH. Equivalent to 750 g total sugar and 450 g LNnT. These results lead to penetration coefficients for each sugar component. Triose penetrates at a factor of about 1.55, LNnT at a factor of 1.05 and pLNnH at a factor of 0.62.

The results are shown in Table 4, Table 5 and Table 6.

Example 3: Fluid-bed chromatography of an LNnT-containing mixture of carbohydrates that contains two main by-products to separate, achieving depletion of higher saccharides such as pLNnH below 3% and accumulation of smaller saccharides such as trioses and LNnT

For SMB purification, the clear, particle-free solution containing these 3 oligosaccharides was concentrated to about 300 g/l using a vacuum concentrator at 45°C. For SMB chromatography, a closed loop SMB multi-component system equipped with 12 columns (Prosep® columns with dimensions: 40 mm × 740 mm (Latek, Eppelheim, Germany)) arranged in 2×4 zones was used. Each column contained 760 g of Purolite® PCR833H+ strong cation exchange resin (Purolite, Ratingen, Germany).

The system was operated at 25° C. with the following flow parameters set: zone I flow rate was set to 30.00 ml/min, zone II flow rate was set to 21.00 ml/min, zone III flow rate was set to 21.48 ml/min. min, the zone IV flow rate was set to 18.44 ml/min, the flow was set to 3.00 ml/min, the eluent rate was set to 11.56 ml/min and the switching time was set to 17.92 minutes. The eluent used was water with 10% (v/v) ethanol suitable for use in the food industry. Saccharides less than pentose, such as trioses and LNnT, such as smaller hydrolysis products such as lactose, lacto-N-biose, glucose, galactose and N-acetylgalactosamine, were mainly fractionated into the extract. Higher oligosaccharides, more specifically saccharides larger than tetraoses such as pentaoses, hexaoses such as pLNnH, as well as larger than oligosaccharides such as heptaoses or octaoses, as well as residual salt contamination originating from the eluent or as a result of resin loading, were fractionated into raffinate. With the settings described, the SMB system could operate continuously for at least 3 months.

Using this protocol, the purity of LNnT could be significantly increased. In this first example, LNnT was used with the following specifications: 12.7% DSC with a composition of 3.5% triose, 59.7% LNnT and 16.5% pLNnH. After SMB chromatography, the extract had the following composition: 4.9% triose, 70.4% LNnT and 2.2% pLNnT. The raffinate, in turn, had the following composition: 0.7% triose, 1.1% LNnT and 59.5% pLNnT. The purification yield was approximately 80%.

The results are shown in Table 7.

Example 4: Fluid-bed chromatography of an LNnT-containing mixture of carbohydrates that contains two major by-products to separate, achieving depletion of higher saccharides such as pLNnH below 3% and accumulation of smaller saccharides such as trioses and LNnT

For SMB purification, the clear, particle-free solution containing these 3 oligosaccharides was concentrated to about 300 g/l using a vacuum concentrator at 45°C. For SMB chromatography, a closed loop SMB multi-component system equipped with 12 columns (Prosep® columns with dimensions: 40 mm × 740 mm (Latek, Eppelheim, Germany)) arranged in 2×4 zones was used. Each column contained 760 g of Purolite® PCR833H+ strong cation exchange resin (Purolite, Ratingen, Germany).

The system was operated at 25° C. with the following flow parameters set: zone I flow rate was set to 30.00 ml/min, zone II flow rate was set to 21.00 ml/min, zone III flow rate was set to 21.48 ml/min. min, the zone IV flow rate was set to 18.44 ml/min, the flow was set to 3.00 ml/min, the eluent rate was set to 11.56 ml/min and the switching time was set to 17.92 minutes. The eluent used was water with 10% (v/v) ethanol suitable for use in the food industry. Saccharides less than pentose, such as trioses and LNnT, such as smaller hydrolysis products such as lactose, lacto-N-biose, glucose, galactose and N-acetylgalactosamine, were mainly fractionated into the extract. Higher oligosaccharides, more specifically saccharides larger than tetraose, such as pentaoses, hexaoses, such as pLNnH, as well as large oligosaccharides, such as heptaoses or octaoses, as well as residual salt contaminations originating from the eluent or as a result of resin loading, were fractionated into raffinate. With the setups described, the SMB systems could operate continuously for at least 3 months.

Using this protocol, the purity of LNnT could be significantly increased. In this second example, LNnT was used with the following specifications: 3.7% triose, 67.6% LNnT and 15.4% pLNnH. After SMB chromatography, the extract had the following composition: 4.3% triose, 80.3% LNnT and 0.6% pLNnH. The raffinate, in turn, had the following composition: 5.3% triose, 2.1% LNnT and 47.9% pLNnH. The purification yield was approximately 80%.

The results are shown in Table 8.

Example 5 Crystallization of an LNnT-Containing Carbohydrate Mixture Which Contains One Major Separation By-Product Achieving Below 3% Depletion of Lower Sugars, Such as Trioses

385.3 g of a mixture of carbohydrates with the following composition was subjected to a crystallization process. Triose: 4.9%, LNnT: 70.4%, pLNnH: 2.2. The material was dissolved on a carousel in 0.4 L of deionized water before its DSC was adjusted to 70% using a rotary evaporator under reduced pressure. This mixture of carbohydrates was removed from the rotary evaporator before being stored under ambient conditions that provide a slow carousel cooling rate of 10°/h. Thus, a layer of solid began to form on the surface of the mixture, eventually leading to a fully crystalline mass after 3 days. After complete crystallization, said crystalline mass was vigorously mixed with two parts of 70 vol. % ethanol solution (70 vol.% EtOH/30 vol.% H ₂ O), ultimately removing the resulting wash solution under reduced pressure using a funnel-shaped frit filter. This washing process was repeated twice, resulting in a dried and purified LNnT crystalline mass, which was dried in a vacuum oven at 0.3 kPa and 35°C. The yield of crystallization was determined as 228.0 g (59.2%); the composition of the compounds was determined as follows: triose content: 0.5%, LNnT content: 94.4%, pLNnH content: 1.2%. However, when considering the LNnT purity of the starting material as well as the crystalline product, the yield increases to 79.3% ((94.4%*228 g)/(70.4%*385.3 g)).

The results are shown in Table 9:

Example 6 Crystallization of a mixture of carbohydrates containing LNnT, which contains one main separation by-product, to achieve a depletion of lower saccharides, such as trioses, below 3%

462.3 g of a mixture of carbohydrates with the following composition was subjected to a crystallization process. Triose: 4.3%, LNnT: 80.3%, pLNnH: 0.6%. Its amount of LNnT was determined to be 371.1 g. The material was dissolved in a carousel in 0.5 L of deionized water before its DSC was set at 69% using a rotary evaporator under reduced pressure. This mixture of carbohydrates was removed from the rotary evaporator prior to storage at ambient conditions that provide a slow carousel cooling rate of 107 hours. Thus, a layer of solid began to grow on the surface of the mixture, resulting in a fully crystalline mass after 2 days. After complete crystallization, said crystalline mass was vigorously mixed with two parts of 70 vol. % ethanol solution (70 vol. % EtOH/30 vol. % H ₂ O), ultimately removing the resulting wash solution under reduced pressure using a funnel-shaped frit filter. This washing process was repeated twice to obtain a dried and purified LNnT crystalline mass, which was dried in a vacuum oven at 0.3 kPa and 35°C. The crystallization yield was determined as 244.0 g (52.8%); the composition of the compounds was determined as follows: triose content: 6.6%, LNnT content: 89.7%, pLNnH content: less than 0.5%. In this case, purification by crystallization was not successful. However, this product was used in another crystallization.

244.0 g of the carbohydrate mixture was sent to the crystallization process. The amount of LNnT was determined as 218.9 g. The material was dissolved in a carousel in 0.25 L of deionized water before its DSC was set at 70% using a rotary evaporator under reduced pressure. This mixture of carbohydrates was removed from the rotary evaporator prior to storage at ambient conditions that provide a slow carousel cooling rate of 107 hours. Thus, a layer of solid began to grow on the surface of the mixture, eventually leading to a fully crystalline mass after 5 days. After complete crystallization, said crystalline mass was vigorously mixed with two parts of 70 vol. % ethanol solution, eventually removing the resulting wash solution under reduced pressure using a funnel-shaped frit filter. This washing process was repeated twice to obtain a dried and purified LNnT crystalline mass, which was dried in a vacuum oven at 0.3 kPa and 35°C. The yield of crystallization was determined as 204.6 g (83.9%); the composition of the compounds was determined as follows: triose content: 0.7%, LNnT content: 97.4%, pLNnH content: 0.2%. When considering the LNnT purity of the starting material as well as the crystalline product, the yield increases to 91.1% ((97.4%*204.6 g)/(89.7%*244.0 g)). The results are shown in Table 10:

Example 7 Crystallization of a mixture of carbohydrates containing pLNnH, which contains one main separation by-product, to achieve a depletion of lower saccharides such as LNnT below 4%

In a carousel, 250 g of a mixture of carbohydrates with the following composition was subjected to a crystallization process: Triose: less than 0.5%, LNnT: 2.1%, pLNnH: 47.9%. The remaining solids essentially consist of residual salts. The material was dissolved on a carousel in 0.5 L of deionized water before its DSC was adjusted to 55% using a rotary evaporator under reduced pressure. This mixture of carbohydrates was removed from the rotary evaporator prior to storage at ambient conditions that provide a slow carousel cooling rate of 107 hours. Thus, a layer of solid began to form on the surface of the mixture, eventually leading to a fully crystalline mass after 5 days. After complete crystallization, 50 g of the crude indicated crystalline mass was actively mixed with two parts of 70 vol. % ethanol solution (70 vol. % EtOH/30 vol. % H ₂ O), ultimately removing the resulting wash solution under reduced pressure using a funnel-shaped frit filter. This washing process was repeated twice to obtain a dried and purified pLNnT crystalline mass, which was dried in a vacuum oven at 0.3 kPa and 35°C. The yield of crystallization was determined as 13.0 g (26.0%); the composition of the compounds was determined as follows: triose content: 0%, LNnT content: 3.75%, pLNnH content: 80.6%. However, when considering the pLNnT purity of the starting material as well as the crystalline product, the yield increases to 58.3% ((80.6%*13.0 g)/(0.75*47.9%*50 g)) considering humidity of the dried crystalline mass 25%.

Example 8 Preparation of LNnT Seed Crystals from Deionized Water

The 56% mixture of LNnT resulting from the enzymatic process was purified through repeated gel filtration steps with pure water as solvent (Biorad Bio-Gel® P-2, fine purification), finally obtaining LNnT seeds after reduction volume through the use of a rotary evaporator. 10.0 g of spray dried starting material was diluted to 50% DSC with pure water and filtered three times. At each filtration step, the predominantly LNnT containing fractions were pooled, re-concentrated in vacuo and further diluted to 50% DSC and sent to another round of gel filtration. Concentration under reduced pressure gave 2.8 g of a gel which crystallized on standing. The product was analyzed by HPAEC-PAD chromatography; the purity of the unwashed crystals was 83.1% LNnT with 6.3% unseparated triose as the main by-product.

Example 9: Crystallization of 200 g LNnT by addition of NaCl

To prevent gel formation during LNnT crystallization, one weight percent of a food compatible salt such as NaCl is added to the solution. 200 g LNnT (15.0% triose, 65.9% LNnT, 4.4% pLNnH; this is equivalent to 131.8 g pure LNnT) and 2.0 g NaCl are dissolved in 200 ml water. The solution is then set to a DSC value of 70%. Some seed crystals are added to the high viscosity slurry and LNnT is allowed to crystallize at room temperature for one day. After complete crystallization, the crystalline mass is mixed with two parts of 80 vol. % ethanol solution (80 vol. % EtOH/20 vol. % H ₂ O) and centrifuged for 15 min at 6000 rpm. The precipitated mixture of carbohydrates is suspended with another part of 80 vol. % ethanol solution and centrifuged again. This process is repeated. After three cycles of centrifugation, the precipitate is freeze-dried. After the first crystallization, 163.6 g of LNnT are obtained. The purity of LNnT could be increased by about 11% from 65.9% to 76.5% (6.5% triose, 5.2 pLNnH) to give 125.1 g of pure LNnT (94.9% yield).

Example 10: 2nd Crystallization 200 g LNnT

158.6 g of LNnT taken from Example 9 crystallized. Thus, the starting material was dissolved in 159 ml of deionized water and concentrated to 70% DSC under reduced pressure. Seed crystals are added to the high viscosity slurry and LNnT is allowed to crystallize at room temperature for 1 day. After complete crystallization, the crystalline mass is mixed with two parts of 80 vol. % ethanol solution (80 vol. % EtOH/20 vol. % H ₂ O) and centrifuged for 15 min at 6000 rpm. The precipitated mixture of carbohydrates is suspended with another part of 80 vol. % ethanol solution and centrifuged again. This process is repeated. After three cycles of centrifugation, the precipitate is freeze-dried. 115.2 g LNnT is obtained after the second crystallization. The purity of LNnT could be increased by about 6% from 76.5% to 82.4% (2.4% triose, 5.7% pLNnH) to give 94.9 g of pure LNnT (78.2% yield).

Example 11: Crystallization of 100 g LNnT by adding NaCl 100 g LNnT (15.0% triose, 65.9% LNnT, 4.4% pLNnH; equivalent to 131.8 g pure LNnT) and 1.0 g NaCl are dissolved in 100 ml water and then concentrated under reduced pressure to 70% DSC. Seed crystals were added to the high viscosity slurry and LNnT was allowed to crystallize at room temperature for 1 day. After complete crystallization, the crystalline mass is mixed with two parts of 80 vol. % ethanol solution (80 vol. % EtOH/20 vol. % H ₂ O) and centrifuged for 15 min at 6000 rpm. The precipitated product is suspended with another volume ml of 80% EtOH and centrifuged again. This process is repeated. Then, the precipitate is freeze-dried. 67.0 g LNnT is obtained after the first crystallization. The purity of the material could be increased by about 15% from 65.9% to 81.7% (6.2% triose, 6.0% hexaose) to give 54.7 g of pure LNnT (83.1% yield).

Example 12: 2nd Crystallization 100 g LNnT

50.0 g of LNnT, taken from Example 11, crystallize. Thus, the starting material is dissolved in 100 ml of water and concentrated to 70% DSC at 60°C. Seed crystals are added to the high viscosity slurry and LNnT is allowed to crystallize at room temperature for 1 day. After complete crystallization, the crystalline mass is mixed with two volumes of 80 vol. % ethanol solution (80 vol. % EtOH/20 vol. % H ₂ O) and centrifuged for 15 min at 6000 rpm. The precipitated carbohydrate mixture is suspended twice with another volume of 80% EtOH and centrifuged again. This precipitate is then subjected to freeze-drying. 36.0 g LNnT is obtained after the second crystallization. The purity of the material could be increased by about 3% from 81.7% to 84.3% (2.9% triose, 6.4% hexaose) to give 30.3 g of pure LNnT (74.3% yield).

Example 13 Homogenization of LNnT by using spray drying

228.0 g LNnT with the following formulation was homogenized by spray drying the indicated product on a Buchi B-290 sampler: (0.5% triose, 94.4% LNnT, 1.2% pLNnH). Thus, the substance was dissolved in 1.5 l (15.2% DSC) of water, then subjected to sterile filtration (pore size 45 mm) and subjected to spray drying at an inlet temperature of 130°C and an outlet of 66°C. 178.0 g (78.1%) of a white homogeneous powder was obtained with a residual water content of 8.6% determined by Karl Fischer titration. Purity was determined as 0.4% triose, 94.4% LNnT, and 1.1% pLNnH to give 168.0 g of pure, homogeneously spray-dried LNnT.

Example 14 Homogenization of LNnT Using Spray Drying

204.0 g LNnT with the following formulation was homogenized by spray drying the indicated product on a Buchi B-290 sampler: (0.7% triose, 97.4% LNnT, 0.2% pLNnH). Thus, the substance was dissolved in 1.2 L (14.6% DSC) of water, then sterile filtered (pore size 45 mm) and spray dried at an inlet temperature of 130°C and an outlet of 66°C. 129.0 g (63.2%) of a white homogeneous powder was obtained with a residual water content of 7.1% determined by Karl Fischer titration. The purity of the material was determined as 0.3% triose, 96.43% LNnT and less than 0.25% pLNnH to give 124.2 g of pure, homogeneous spray dried LNnT.

Example 15 Homogenization of LNnT by using spray drying

110.7 g LNnT with the following formulation was homogenized by spray drying the indicated product on a Buchi B-290 sampler: (2.4% triose, 82.4% LNnT, 5.7% pLNnH). Thus, the substance was dissolved in 300 ml (27% DSC) of water, then subjected to sterile filtration (pore size 45 mm) and subjected to spray drying at an inlet temperature of 130°C and an outlet temperature of 66°C. 71.9 g (65.0%) of a white homogeneous powder was obtained, which showed approximately the same spectrum as the starting material from Example 10, as evidence of the compatibility of spray drying of the obtained material.

Example 16: Drying LNnT by using an oven dryer

9.629 g of crystalline LNnT was dried in an oven. After 7 days at 35°C, its weight decreased to 9.612 g, which corresponds to 99.8%. The purity of LNnT was determined as 88.7% before and 88.2% after drying, proving the suitability of oven drying at 35°C.

Example 17: Drying LNnT by using vacuum oven drying

10.307 g of crystalline LNnT was dried in an oven. After 7 days at 35°C, its weight decreased to 10.219 g, which corresponds to 99.8%. The purity of LNnT was determined as 88.7% before and 90.3% after drying, proving the suitability of oven drying at 35°C.

Example 18 Homogenization of LNnT Using Freeze Drying

10.265 g of crystalline LNnT was freeze-dried on a Christ BETA 2-8 LD plus part. Thus, this HMO was dissolved in a carousel in 90 ml of deionized water and frozen at -80°C before being subjected to a freeze drying process, set at 0.2*10 ^-3 kPa and cooled to -85°C. After 7 days, the weight decreased to 9.668 g, which corresponds to 94.2%. The purity of LNnT was determined to be 88.7% before and 90.2% after drying, making freeze drying a suitable homogenization method.

Example 19 Homogenization of LNnT Using Freeze Drying

15.404 g of crystalline LNnT was freeze-dried on a Christ BETA 2-8 LD plus part. Thus, this HMO was dissolved in a carousel in 60 ml of deionized water and frozen at -80°C before being subjected to a freeze drying process, set at 0.2*10 ^-3 kPa and cooled to -85°C. After 7 days, the weight decreased to 14.566 g, which corresponds to 94.2%. The purity of LNnT was determined to be 88.7% before and 89.5% after drying, making freeze drying a suitable homogenization method.

Claims (34)

1. A method for purifying lacto-N-neotetraose (LNnT) from a fermentation broth, which includes the steps of: - take the fermentation broth, which contains LNnT, biomass, components of the environment, contaminants and carbohydrates other than LNnT; - subjecting the fermentation broth to at least one stage of membrane filtration using nanofiltration membranes, thus obtaining a filtered solution containing LNnT; - subjecting the filtered solution to at least one stage of pseudo-moving bed chromatography, thus obtaining a purified solution that contains LNnT; - subjecting the purified solution to at least one stage of crystallization from water, thus obtaining a crystalline mass; And subjecting such a crystalline mass to at least one homogenization step, thereby obtaining a homogenized and dried purified LNnT preparation; wherein the purified preparation contains LNnT with a purity of 80% or more. 2. The method of claim 1, wherein the membrane filtration step is nanofiltration using a membrane that has a molecular weight cutoff of 0.2 to 3.5 kDa. 3. The method according to any one of paragraphs. 1, 2, in which the membrane has a cut-off threshold for molecular weight from 0.2 to 2.0 kDa. 4. The method according to any one of paragraphs. 1-3, in which the membrane has a molecular weight cut-off from 0.2 to 1.0 kDa. 5. The method according to any one of paragraphs. 1-4, in which the filtered solution obtained from the membrane filtration step contains LNnT with a purity of more than 60%, more than 65% or more than 70%. 6. The method of claim 1, wherein the pseudo moving bed chromatography step comprises the following: i) at least 4 columns, preferably at least 8 columns, more preferably at least 12 columns, where at least one column contains a weak or strong cation exchange resin, preferably a cation exchange resin in H ⁺ -form, Na ⁺ -form, K ⁺ -form or Ca ²⁺ -form; and/or ii) four zones I, II, III and IV with different flow rates; and/or iii) an eluent containing or consisting of water, preferably ethanol and water, more preferably from 5 to 15 vol. % ethanol and from 85 to 95 vol. % water, most preferably 9 to 11 vol. % ethanol and from 89 to 91 vol. % water, iv) operating temperature 15 to 60°C, preferably 20 to 55°C, more preferably 25 to 50°C. 7. The method according to claim 1 or 6, wherein the pseudo moving bed chromatography step comprises the following: i) four zones I, II, III and IV with different flow rates, where the flow rates are preferably 22 to 32 ml/min in zone 1, 17 to 23 ml/min in zone II, 18 to 25 ml/min in zone III and/or 14 to 20 ml/min in zone IV; and/or ii) a flow rate of 0.5 to 4 ml/min, preferably 2 ml/min; and/or iii) an eluent flow rate of 6 to 12 ml/min, preferably 8 ml/min; and/or iv) switching time 14 to 20 minutes, preferably 16 to 18 minutes, more preferably 17 minutes. 8. The method according to any one of paragraphs. 1-7, in which the purified solution obtained by chromatography with fluidized bed contains LNnT with a purity of more than 75% or more than 80%. 9. The method according to any one of paragraphs. 1, 6-8, in which the purified solution obtained as a result of at least one stage of chromatography with fluidized bed, can be subjected to at least one additional stage of purification using chromatography with pseudomoving layer, thus obtaining a purified solution containing LNnT with a purity greater than 85%, preferably greater than 90%; more preferably more than 93%. 10. The method according to p. 1, in which the stage of crystallization includes: - obtaining a mixture of at least two oligosaccharides containing at least LNnT at a concentration of at least about 1% DSC, preferably at least 10% DSC, preferably at least 30% DSC and more preferably at least about 50% DSC, and the purity of such LNnT in the mixture is at least about 15%, preferably at least about 25%, preferably about 40%, and more preferably at least about 60%; - crystallizing a mixture of at least two oligosaccharides obtained from the stage of pseudo-moving bed chromatography, at an initial temperature of at least about 20 to 30°C, preferably at least about 30 to 40°C, preferably at least about 40 up to 50°C and more preferably at least about 50 to 60°C; - allowing the saturated mass to crystallize by cooling to at least 40-50°C, preferably at least about 30-40°C, preferably at least about 20-30°C, preferably at least about 10-20°C and more preferably at least about 0-10°C to obtain a homogeneous crystalline mass, - treatment of the crystalline mass with a mixture of an alcoholic solvent or a water-miscible alcohol or solvent and water, where the content of alcohol or solvent in the solution is from 10 to 90 vol. %, preferably from 30 to 80 vol. % and more preferably from 50 to 70 vol. % to obtain a mixture of a solution containing alcohol or solvent, and a crystalline mass; And - filtering the fraction containing alcohol or solvent and now containing sugar, the crystalline mass to reduce the content of lower saccharides in the crystalline mass. 11. The method according to claim 1 or 10, wherein the content of lower saccharides is less than 10%, preferably less than 5% and more preferably less than 3%. 12. The method according to any one of paragraphs. 1-11, in which the crystals obtained from the crystallization stage contain LNnT with a purity of more than 85%, more than 90% and more than 95%. 13. The process of claim 1, wherein the homogenizing step can be selected from freeze drying, spray drying, roller drying or drum drying and/or belt drying or vacuum belt drying. 14. The method according to any one of paragraphs. 1-13, in which the purity of LNnT in the purified preparation is 85% or more, more preferably 90% or more, based on the dry matter of the purified preparation.

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