HMO concentrated solution and preparation method thereof

Abstract

According to the HMO concentrated solution and the preparation method thereof, the mass concentration of HMO in the HMO concentrated solution is 60% or above, the HMO concentrated solution does not contain genetically engineered microorganisms, nucleic acid molecules derived from the genetically engineered microorganisms and protein and organic solvents, the purity of the HMO is at least 95%, and the content of the HMO in the HMO concentrated solution is more than 10%. The grain size of 90% of crystals in the HMO concentrated solution is not greater than 150 microns. The preparation method comprises the following steps: separating and purifying HMO fermentation liquor prepared by a microbial fermentation method, concentrating to 60 wt% or above, cooling to-10-10 DEG C within 0.5-2 hours, keeping stirring in the cooling process, cooling to a target temperature, and continuously keeping stirring for 1-4 hours. According to the invention, the stability of the high-concentration HMO concentrated solution in a refrigeration state can be realized.

Description

Technical Field

[0001] This invention relates to the field of food technology, and in particular to an HMO concentrate and its preparation method. Background Technology

[0002] HMO (human milk oligosaccharides) is the third most abundant solid component in breast milk (after fat and lactose). It has functions such as regulating immunity, helping brain development, and regulating gut microbiota, which are beneficial to the growth and development of infants and young children.

[0003] To incorporate HMOs into nutritional compositions, particularly infant formula, synthetic HMOs are becoming increasingly popular. Currently, HMO products are commonly obtained through microbial conversion of lactose, followed by separation and purification using methods such as chromatography, nanofiltration, and / or electrodialysis. The final product is then dried using solvent crystallization drying, or by direct spray drying of the concentrate, vacuum drum dryer, fluidized bed drying, or other drying methods to obtain various crystalline or amorphous powders.

[0004] For downstream applications of dry-mixed products, powdered HMO has its advantages. However, for more downstream applications where the intermediate state is liquid, concentrated syrup is a more convenient and economical application form. Furthermore, the high concentration of syrup creates osmotic pressure that inhibits microbial growth. With proper sterilization before storage, liquid syrup can be stored for 6 to 12 months, offering convenient storage.

[0005] However, current HMO concentrate syrups, as shown in CN103797021B, are prone to crystallization and lack storage stability due to their high concentration. Stability issues are typically avoided by controlling the syrup concentration to 50% or below. However, the applicant found that even at 50% concentration, microbial growth remains a concern. Furthermore, the applicant's research revealed that while concentrates exceeding 50% can maintain complete dissolution in most processes, crystallization occurs after approximately seven days of refrigeration. Once crystal nuclei appear, the crystallization rate accelerates dramatically, resulting in large crystal clumps at the bottom. This phenomenon not only directly impacts the ease of storage and use of concentrates in northern regions but also places higher demands on transport time and increases costs for shipments traversing significant distances from south to north. Summary of the Invention

[0006] This invention provides an HMO concentrate and its preparation method, which can prevent the concentrated syrup from forming large crystals under long-term refrigeration.

[0007] In a first aspect, the present invention provides an HMO concentrate, wherein the mass concentration of HMO is above 60%, the HMO concentrate does not contain genetically engineered microorganisms, nucleic acid molecules derived from genetically engineered microorganisms and proteins, or organic solvents, the purity of the HMO is at least 95%, and 90% of the crystals in the HMO concentrate have a particle size not greater than 150 micrometers. That is, the crystal particle size D90 in the concentrate is ≤150 micrometers.

[0008] The purity of the HMO is at least 95%, and "purity" refers to the weight of the HMO relative to the dry matter or solute in the HMO concentrate.

[0009] Furthermore, 90% of the crystals in the HMO concentrate have a particle size no larger than 100 micrometers. That is, the crystal particle size D90 in the concentrate is ≤100 micrometers.

[0010] As described in the background art, in the prior art, although HMO concentrates with a concentration of more than 50% can maintain a completely dissolved state in most processes, crystallization will occur after about seven days of storage under refrigeration (around 0°C). Once crystal nuclei appear, the subsequent crystallization rate will accelerate dramatically. After several months, large crystal blocks will form at the bottom, which are difficult to dissolve even by heating. This phenomenon is more pronounced in concentrates with higher concentrations, and neutral oligosaccharides are more prone to this phenomenon than acidic oligosaccharides. Therefore, this limits the application range of HMO concentrates and increases storage and transportation costs.

[0011] To address this issue, the present invention provides an HMO concentrate that maintains good stability under refrigeration even at concentrations of 60% or higher, and even 70% or higher. Specifically, the HMO concentrate, after being stored at 0°C for more than one month, can be completely reconstituted within 20 minutes when the temperature is raised to 30°C without affecting its use; the reconstitution process includes stirring. This discovery fills a gap in the existing market.

[0012] In some embodiments of the present invention, the HMO is a fucoidan oligosaccharide, a sialic acid oligosaccharide, or an oligosaccharide formed from a core glycan structure that does not contain fucoidan or sialic acid groups.

[0013] In this invention, fucoidan oligosaccharides, also known as fucosylated oligosaccharides, refer to oligosaccharides containing fucose residues. Representative substances include 2′-fucosylated lactose and 3-fucosylated lactose.

[0014] 2′-Fucosyllactose (2′-FL) is a trisaccharide formed from fucose and lactose. Commercially available 2′-FL is typically prepared via microbial fermentation and has the same structure as 2′-fucosyllactose found in human milk.

[0015] 3-Fucosyllactose (3-FL) is a trisaccharide formed from fucose and lactose, and is an isomer of 2′-fucosyllactose. This substance can be prepared by microbial fermentation and has the same structure as 3-fucosyllactose found in human milk.

[0016] Sialoyl oligosaccharides, also known as sialylated oligosaccharides, refer to oligosaccharides containing sialic acid residues. Representative examples include 3′-sialyl lactose and 6′-sialyl lactose.

[0017] 3′-sialyllactose (3′-SL) is a structure formed by sialic acid and lactose, and is an isomer of 6′-sialyllactose. This substance is prepared by microbial fermentation and has the same structure as 3′-sialyllactose found in human milk.

[0018] 6′-sialyllactose (6′-SL) is a structure formed by sialic acid and lactose, and is an isomer of 3′-sialyllactose. This substance can be prepared by microbial fermentation and has the same structure as 6′-sialyllactose found in human milk.

[0019] Oligosaccharides formed without a core sugar chain structure containing fucose or sialic acid groups refer to oligosaccharides other than fucose-based and sialic acid-based oligosaccharides. Representative substances include lactosyl-N-tetrasaccharide or lactosyl-N-butose.

[0020] Lacto-N-tetraose (LNT) is a tetrasaccharide formed from galactose and glucose. It is a representative oligosaccharide with a core sugar chain as its basic structure and does not contain fucose or sialic acid groups. This substance can be prepared by microbial fermentation and has the same structure as lacto-N-tetraose found in human milk.

[0021] During the applicant's research, it was discovered that neutral oligosaccharides are more prone to the crystallization and clumping problems described in the invention compared to acidic oligosaccharides. Therefore, preferably, in some embodiments of the invention, the HMO is a neutral HMO.

[0022] In this invention, neutral HMO, or neutral oligosaccharide, refers to oligosaccharide that does not have a negative charge originating from a carboxylic acid group.

[0023] More preferably, the HMO is selected from one or more of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lactosyl-N-triose II, lactosyl-N-butose, lactosyl-N-neotose, lactosyl-N-fucosylpentose I, lactosyl-N-neotose, lactosyl-N-fucosylpentose II, lactosyl-N-fucosylpentose III, lactosyl-N-fucosylpentose V, lactosyl-N-neotose V, lactose-N-difucosylhexose I, lactose-N-difucosylhexose II, 6'-galactosyllactose, 3'-galactosyllactose, lactosyl-N-hexose, and lactosyl-N-neotose.

[0024] Secondly, the present invention provides a method for preparing the above-mentioned HMO concentrate.

[0025] The preparation method provided by this invention includes:

[0026] The HMO fermentation broth obtained by microbial fermentation was separated, purified, and concentrated to more than 70 wt%. Then, it was cooled to -10 to 10 °C within 0.5-2 h, with stirring maintained during the cooling process. After cooling to the target temperature, stirring was continued for 1-4 h.

[0027] Generally, as the temperature decreases, HMO syrup transitions from a stable state to a metastable state. In this state, the solution is easily stimulated to produce a small number of fine crystals. These crystals then act as nuclei, causing the solute to aggregate and arrange around them. Under relatively stable external conditions, the solution enters a crystallization state, often resulting in clumps of crystals at the bottom of the container. As the volume of the container increases, these clumps of crystals become more numerous and larger, making them increasingly difficult to dissolve. However, this invention rapidly cools the solution during the preparation of the concentrate, causing it to enter an unstable state, resulting in a large amount of crystallization and a transition to a stable state. Simultaneously, the size of the precipitated crystals is controlled through stirring, sonication, and other operations. Due to the concentrate's inherent viscosity and the neutral, uncharged nature of HMO, the concentrate only becomes slightly cloudy during subsequent low-temperature storage, and the crystals remain in a non-clumping state.

[0028] In some embodiments of the present invention, the stirring speed is 100-1500 rpm.

[0029] In some embodiments of the present invention, ultrasound is performed intermittently during the stirring process. Preferably, the number of ultrasound sessions is 1-5, with each session lasting 3-5 minutes. The ultrasound equipment can be conventional industrial distributed, vibrating rod, or focused-energy type devices, such as ultrasonic rods, with a frequency of 15-20 kHz and a power of 500-2000 W.

[0030] In some embodiments of the present invention, the concentration is carried out in a falling film evaporator, a rotary evaporator or a multi-effect evaporator, preferably a falling film evaporator.

[0031] In some embodiments of the present invention, the cooling step includes: first, rapidly reducing the temperature of the liquid to room temperature through a heat exchanger, and then cooling it to -10 to 10°C, preferably -10 to 4°C, through a jacketed cooling element.

[0032] In some embodiments of the present invention, the HMO fermentation broth, after separation and purification, has a purity of over 95%; preferably, before the concentration is carried out, the broth is subjected to ultra-high temperature instantaneous sterilization.

[0033] In some embodiments of the present invention, the purified HMO fermentation broth is obtained by the following steps:

[0034] (1) The HMO fermentation broth (HMO mass concentration of about 5%) was filtered through a 200nm filter membrane to remove bacteria, resulting in sterile fermentation broth;

[0035] (2) Heat the sterile fermentation broth obtained in step (1) to 43°C; add activated carbon and stir, filter to remove activated carbon and protein, and obtain decolorized solution;

[0036] (3) The decolorized solution obtained in step (2) is sequentially adsorbed and desalted by cation exchange resin and anion exchange resin to obtain a desalted solution; the desalted solution is concentrated under reduced pressure to obtain a desalted concentrate.

[0037] (4) The desalting concentrate obtained in step (3) is separated by a styrene polymer gel column and eluted with pure water to obtain an eluent with a mass concentration of HMO of 5-20%. The eluent is then filtered and subjected to UHT.

[0038] In the above technical solution, the HMO fermentation broth is preferably 2′-FL fermentation broth.

[0039] Thirdly, the present invention provides a nutritional composition comprising the above-mentioned HMO concentrate.

[0040] In this invention, the term "nutritional composition" refers to a composition that provides nutrition to the object. This nutritional composition is typically taken orally or intravenously, and it generally includes a lipid or fat source and a protein source.

[0041] Fourthly, the present invention provides a food product comprising the above-mentioned HMO concentrate.

[0042] In this invention, the term "food" is intended to cover any consumable substance. Therefore, it can be a product intended for human consumption, particularly infant formula, follow-up formula, and foods for infants or young children such as baby cereals. In particular, the HMO concentrate of this invention can be added to infant formula, dehydrated milk, or cereal mixtures.

[0043] This invention provides an HMO concentrate and its preparation method. Through improvements in the preparation method, the stability of the high-concentration HMO concentrate under refrigeration is achieved. This discovery overcomes the limitations of HMO concentrate in terms of its application range and high storage and transportation costs, fills a market gap, and provides technical support for the development of more downstream products containing HMO. Detailed Implementation

[0044] The terms “comprising” or “including” in this invention are open-ended descriptions that include the specified ingredients or steps described, as well as other specified ingredients or steps that do not materially affect them.

[0045] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0046] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0048] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0049] Example 1

[0050] This embodiment provides a 2′-fucosylated lactose concentrate, the preparation method of which is as follows:

[0051] (1) The 2′-fucosylated lactose fermentation broth (2′-fucosylated lactose content is 5%) was filtered through a 200nm filter membrane to remove bacteria, and sterile fermentation broth was obtained;

[0052] (2) Heat the sterile fermentation broth obtained in step (1) to 43°C; add activated carbon (the mass ratio of the amount of activated carbon added to the mass of the sterile fermentation broth is 6:100), stir, filter to remove activated carbon and protein, and obtain decolorized broth;

[0053] (3) The decolorized solution obtained in step (2) is sequentially adsorbed and desalted by cation exchange resin and anion exchange resin to obtain a desalted solution; the desalted solution is concentrated under reduced pressure to obtain a desalted concentrate.

[0054] The cation exchange resin is a strong acid styrene-based cation exchange resin 001X7 (Anhui Samsung Resin Co., Ltd.), and the anion exchange resin is a macroporous weakly basic anion exchange resin D315 with a polyacrylic acid skeleton (Anhui Samsung Resin Co., Ltd.).

[0055] The conductivity of the desalting solution is 8 μS / cm, and the mass concentration of 2′-fucosylated lactose in the desalting solution is 75 g / L;

[0056] The conductivity of the desalting concentrate is 8 μS / cm;

[0057] (4) The desalted concentrate obtained in step (3) is separated using a styrene polymer gel column and eluted with pure water to obtain an eluent; wherein, the column temperature of the chromatographic column is 55℃; the purity of 2′-fucosylated lactose in the eluent is 97%;

[0058] The eluent contains 10% 2′-fucosylated lactose by mass, and is filtered before UHT treatment.

[0059] (5) The eluent obtained in step (4) is concentrated in a falling film evaporator at 65°C under negative pressure until the mass content of 2′-fucosylated lactose is 70%.

[0060] (6) Cool the concentrated liquid to 4°C within 2 hours. Specifically, first, the liquid temperature is rapidly reduced to room temperature through a heat exchanger, and then cooled to 4°C through a jacketed cooling element (coolant temperature is -15°C). At the same time, the stirring speed is 200 rpm during the cooling process.

[0061] (7) After cooling to 4℃, continue stirring for 2 hours. During the stirring process, use an ultrasonic probe to perform ultrasonication 3 times, each time for 5 minutes. Stop stirring during ultrasonication, that is, ultrasonication and stirring are alternated. The time interval between the 3 ultrasonications within 2 hours is 30 minutes to obtain 2′-fucosylated lactose concentrate. The crystal particle size D90 is measured to be 98 micrometers.

[0062] Example 2

[0063] This embodiment provides a 2′-fucosylated lactose concentrate, the preparation method of which is as follows:

[0064] (1) The 2′-fucosylated lactose fermentation broth (2′-fucosylated lactose content is 5%) was filtered through a 200nm filter membrane to remove bacteria, and sterile fermentation broth was obtained;

[0065] (2) Heat the sterile fermentation broth obtained in step (1) to 43°C; add activated carbon (the mass ratio of the amount of activated carbon added to the mass of the sterile fermentation broth is 6:100), stir, filter to remove activated carbon and protein, and obtain decolorized broth;

[0066] (3) The decolorized solution obtained in step (2) is sequentially adsorbed and desalted by cation exchange resin and anion exchange resin to obtain a desalted solution; the desalted solution is concentrated under reduced pressure to obtain a desalted concentrate.

[0067] The cation exchange resin is a strong acid styrene-based cation exchange resin 001X7 (Anhui Samsung Resin Co., Ltd.), and the anion exchange resin is a macroporous weakly basic anion exchange resin D315 with a polyacrylic acid skeleton (Anhui Samsung Resin Co., Ltd.).

[0068] The conductivity of the desalting solution is 8 μS / cm, and the mass concentration of 2′-fucosylated lactose in the desalting solution is 75 g / L;

[0069] The conductivity of the desalting concentrate is 8 μS / cm;

[0070] (4) The desalted concentrate obtained in step (3) is separated using a styrene polymer gel column and eluted with pure water to obtain an eluent; wherein, the column temperature of the chromatographic column is 55℃; the purity of 2′-fucosylated lactose in the eluent is 97%;

[0071] The eluent contains 10% 2′-fucosylated lactose by mass, and is filtered before UHT treatment.

[0072] (5) The eluent obtained in step (4) is concentrated in a falling film evaporator at 65°C under negative pressure until the mass content of 2′-fucosylated lactose is 70%.

[0073] (6) Cool the concentrated liquid to 4°C within 2 hours. Specifically, first, the liquid temperature is rapidly reduced to room temperature through a heat exchanger, and then cooled to 4°C through a jacketed cooling element (coolant temperature is -15°C). At the same time, the stirring speed is 200 rpm during the cooling process.

[0074] (7) After cooling to 4℃, continue stirring for 2h to obtain 2′-fucosylated lactose concentrate, and the crystal particle size D90 was measured to be 140 micrometers.

[0075] Example 3

[0076] This embodiment provides a 2′-fucosylated lactose concentrate, the preparation method of which is as follows:

[0077] (1) The 2′-fucosylated lactose fermentation broth (2′-fucosylated lactose content is 5%) was filtered through a 200nm filter membrane to remove bacteria, and sterile fermentation broth was obtained;

[0078] (2) Heat the sterile fermentation broth obtained in step (1) to 43°C; add activated carbon (the mass ratio of the amount of activated carbon added to the mass of the sterile fermentation broth is 6:100), stir, filter to remove activated carbon and protein, and obtain decolorized broth;

[0079] (3) The decolorized solution obtained in step (2) is sequentially adsorbed and desalted by cation exchange resin and anion exchange resin to obtain a desalted solution; the desalted solution is concentrated under reduced pressure to obtain a desalted concentrate.

[0080] The cation exchange resin is a strong acid styrene-based cation exchange resin 001X7 (Anhui Samsung Resin Co., Ltd.), and the anion exchange resin is a macroporous weakly basic anion exchange resin D315 with a polyacrylic acid skeleton (Anhui Samsung Resin Co., Ltd.).

[0081] The conductivity of the desalting solution is 8 μS / cm, and the mass concentration of 2′-fucosylated lactose in the desalting solution is 75 g / L;

[0082] The conductivity of the desalting concentrate is 8 μS / cm;

[0083] (4) The desalted concentrate obtained in step (3) is separated using a styrene polymer gel column and eluted with pure water to obtain an eluent; wherein, the column temperature of the chromatographic column is 55℃; the purity of 2′-fucosylated lactose in the eluent is 97%;

[0084] The eluent contains 20% 2′-fucosylated lactose by mass, and after filtration, it is subjected to UHT.

[0085] (5) The eluent obtained in step (4) is concentrated in a rotary evaporator at 65°C under negative pressure until the mass content of 2′-fucosylated lactose is 75%;

[0086] (6) Cool the concentrated liquid to 0°C within 2 hours. Specifically, first, the liquid temperature is rapidly reduced to room temperature through a heat exchanger, and then cooled to 0°C through a jacketed cooling element (coolant temperature is -15°C). At the same time, the stirring speed is 100 rpm during the cooling process.

[0087] (7) After cooling to 0℃, continue stirring for 1 hour. During the stirring process, use an ultrasonic probe to perform ultrasonication 5 times, each time for 5 minutes. Stop stirring during ultrasonication, that is, alternating between ultrasonication and stirring, with an interval of 12 minutes each time, to obtain 2′-fucosylated lactose concentrate. The crystal particle size D90 was measured to be 81 micrometers.

[0088] Comparative Example 1

[0089] This comparative example provides a 2′-fucosylated lactose concentrate (concentration of 70%) prepared by a conventional method. That is, compared with Example 1, after obtaining the concentrate in step (5), there are no other process operations, and it is naturally cooled to room temperature.

[0090] Comparative Example 2

[0091] This comparative example provides a 2′-fucosylated lactose concentrate (concentration of 70%) prepared by a conventional method. That is, compared with Example 1, after obtaining the concentrate in step (5), it is allowed to cool naturally to room temperature and then placed in a non-rapidly cooled environment of 4°C for storage.

[0092] Effect Comparison

[0093] Take 1L of each of the 2′-fucosylated lactose concentrates obtained in each example and comparative example, place them in a glass jar and store at 0°C for two months.

[0094] 1. Observation revealed that there were no clumps of crystals at the bottom of the concentrates in Examples 1, 2, and 3. Examples 1 and 3 had no precipitate but were only cloudy in appearance. The small crystals at the bottom of Example 2 could be easily dispersed by shaking.

[0095] In contrast, the concentrated solutions of Comparative Examples 1 and 2 showed that the crystals at the bottom were clumped together and could not be dispersed.

[0096] 2. Solubility

[0097] The concentrates obtained in Examples 1 and 3 were heated to 30°C in a water bath and kept for 10 minutes with stirring at 50 rpm until completely dissolved. In Example 2, the concentrates were heated to 30°C in a water bath and kept for 15 minutes with stirring at 50 rpm until completely dissolved.

[0098] The concentrates of Comparative Examples 1 and 2 could not be completely dissolved when heated in a water bath to 30°C for 20 minutes and stirred at 50 rpm. They were completely dissolved after stirring for 45 minutes.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An HMO concentrate solution, characterized in that, The mass concentration of the HMO is above 60%, the HMO concentrate contains no nucleic acid molecules of genetically engineered microorganisms, genetically engineered microorganisms and proteins, and no organic solvents, the purity of the HMO is at least 95%, and 90% of the crystal particle size of the HMO concentrate is not greater than 150 microns.

2. The HMO concentrate of claim 1, wherein The HMO concentrate is placed at 0°C for more than one month, and can be completely redissolved within 20 minutes when the temperature is raised to 30°C, and the redissolution process includes stirring.

3. The HMO concentrate of claim 1 or 2, characterized in that, The HMO is fucosyl oligosaccharide, sialyl oligosaccharide, or oligosaccharide formed by a core sugar chain structure without fucosyl or sialyl; Preferably, the HMO is a neutral HMO; More preferably, one or more of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lactosyl-N-dulcosyl, lactosyl-N-neodulcosyl, lactosyl-N-fucosylpentose I, lactosyl-N-neofucosylpentose, lactosyl-N-fucosylpentose II, lactosyl-N-fucosylpentose III, lactosyl-N-fucosylpentose V, lactosyl-N-neofucosylpentose V, lactose-N-difucosylhexose I, lactose-N-difucosylhexose II, 6'-galactosyllactose, 3'-galactosyllactose, lactosyl-N-hexose, and lactosyl-N-neohexose.

4. A process for the preparation of an HMO concentrate according to any one of claims 1 to 3, characterized in that, The HMO concentrate of any one of claims 1-3. The HMO concentrate of any one of claims 1-3.

5. The method of preparing an HMO concentrate solution according to claim 4, characterized by, Preferably, the food is infant formula.

6. The method of preparing an HMO concentrate solution according to claim 5, characterized by, ​ 7. The method of preparing an HMO concentrate solution according to claim 4, characterized by, ​ 8. The method of preparing an HMO concentrate solution according to claim 4, characterized by, ​ ​ 9. A nutritional composition, characterized in that, ​ 10. A food product, characterized by, ​ ​