Process for preparing high purity galactooligosaccharides

Abstract

The present invention relates to an enzymatic process that reduces the lactose content of galactooligosaccharide compositions and produces high purity galactooligosaccharides with high retention of disaccharides that support the growth of bifidobacteria.

Description

[Technical field]

[0001] The present invention relates to high purity galactooligosaccharide (GOS) compositions and processes for obtaining such compositions. [Background technology]

[0002] Galactooligosaccharides contain chains of multiple galactose units and one terminal glucose unit and are generated by sequential transgalactosylation reactions catalyzed by beta-galactosidase. A typical GOS composition contains mainly di- to hexasaccharides. Some GOS components occur naturally in human breast milk and bovine colostrum.

[0003] Various physiological functions of GOS have been reported, including their ability to stimulate the growth of bifidobacteria in the intestine, support normal intestinal transit, contribute to natural defenses, enhance mineral absorption, stimulate immune function, and reduce inflammation. GOS have attracted special attention due to their prebiotic effect, which promotes the growth of Bifidobacterium, Lactobacillus, and other intestinal bacteria. Therefore, GOS are used in infant formulas, Lactobacillus-fermented beverages, yogurt, juices, and drinks. Some of these GOS-containing foods have been approved as foods for specified health uses by the Consumer Affairs Agency of Japan, and GOS has been approved as a generally recognized as safe (GRAS) substance by the U.S. Food and Drug Administration (GRAS notices; GRNs 233, 236, 285, 286, 334, 484, 489, 495, 518, and 569).

[0004] Studies have shown that certain disaccharide GOS components (those with a degree of polymerization of 2; i.e., DP2 components), especially gal-β1,2-glc and gal-β1,3-glc, can stimulate the production of mucins, which are important for the colonization of bifidobacteria in the colon. Furthermore, in vitro fermentation studies have shown that these DP2 species are very rapidly digested, thereby suggesting that these DP2 GOS components are highly probiotic and have a high growth stimulatory effect on bifidobacteria (Lammerts van Bueren et al., (2017) Scientific Reports 7:40478).

[0005] GOS is traditionally produced by contacting lactose-containing raw materials with beta-galactosidase enzyme. The resulting GOS is a mixture of galactooligosaccharides and lactose with various degrees of polymerization (DP). A large portion of the world's population over the age of 3 suffers from lactose intolerance, which can result in abdominal pain, bloating, diarrhea, gas, and nausea upon ingestion of lactose-containing compositions. Conventional GOS compositions contain high amounts of lactose and thus can cause these symptoms. For example, commercially available GOS syrups, such as Vivinal® GOS, often contain about 60% by weight of oligosaccharides, 13-16% by weight of lactose, and 20-21% by weight of monosaccharides (e.g., 19-20% by weight of glucose and 1.5% by weight of galactose).

[0006] Therefore, it is necessary to produce GOS from which lactose has been largely removed.

[0007] There are various known methods to achieve this goal, such as fermentation with strains of yeast (Pazmandi et al., Yeast; 2020:37:515-530) or S. thermophilus (WO 2011 / 016008) or Kluyveromyces lactis (EP 3205727, CN 111334541 and CN 107523595).

[0008] The disadvantages of fermentation processes are: (i) the productivity of the fermentation process is relatively low due to the low substrate concentration required, (ii) the addition of nutrients is required for microbial growth, resulting in the presence and formation of contaminants and / or by-products such as ethanol or glycerol, and (iii) the potential formation of allergens.

[0009] Another approach to removing lactose from GOS is the enzymatic removal of lactose from pre-formed GOS compositions using the enzyme lactase.

[0010] This method is described in WO 2017 / 120678 and WO 2019 / 119102, which preferably use yeast lactase derived from Kluyveromyces lactis, at a concentration of 1-50 LU / g lactose and at a temperature of 30-45°C.

[0011] The GOS purified according to these disclosures was obtained by using beta-galactosidase from Aspergillus oryzae, and the GOS had a high content of lactose and a low content of other disaccharides. The content of disaccharides other than lactose in this GOS composition was largely retained upon lactose hydrolysis, which is not surprising since the initial lactose content was very high, the disaccharide / lactose ratio was low, and the lactose concentration remained above 8 wt% during the reaction. In other words, there was always enough lactose available for the enzyme, and the enzyme did not need to use other disaccharides as a substrate.

[0012] The problem associated with this type of enzymatic reaction is that when the lactose concentration becomes very low, for example 1-2% by weight, the other disaccharide components, especially gal-β1,2-glc and gal-β1,3-glc, become suitable substrates for the lactase enzyme, resulting in hydrolysis of these species and a significant loss of efficacy in supporting the growth of bifidobacteria. Summary of the Invention [Means for solving the problem]

[0013] It has now been found that by selecting appropriate conditions, it is possible to obtain a GOS composition having a lactose content of less than 6% by weight based on the total carbohydrate weight and a relatively high content of gal-β1,2-glc and gal-β1,3-glc.

[0014] The present invention therefore relates to a process for reducing the lactose content of a galactooligosaccharide composition, the process comprising: a) providing an aqueous solution of a galactooligosaccharide composition having a lactose content of 10-30% by weight based on total carbohydrate weight and a weight ratio of (gal-β1,2-glc+gal-β1,3-glc):lactose in the range of 1.5:1 to 3.0:1, said solution having a dry matter content of up to 54% by weight; b) adding lactase enzyme to the solution; c) carrying out an enzymatic reaction at 30-50° C. until the lactose content of the solution is reduced to a maximum of 6% by weight of lactose based on the total carbohydrate content, in order to allow hydrolysis of lactose into monosaccharides; and d) removing monosaccharides from the solution obtained in step c). Includes.

[0015] The resulting GOS composition has a lactose content of 6.0% or less by weight based on the total carbohydrate weight.

[0016] The present invention also relates to a GOS composition comprising at least 90% by weight of oligosaccharides, 10-40% by weight of the disaccharides gal-β1,2-glc and gal-β1,3-glc and less than 6% by weight of lactose, based on the total carbohydrate weight. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The oligosaccharide content, lactose content, disaccharide content and total carbohydrate content are determined by HPAEC-PAD as disclosed by S. van Leeuwen et al., Carbohydrate Research 425 (2016) 48-58 and in the Examples below.

[0018] Starting GOS compositions with the required lactose content and (gal-β1,2-glc+gal-β1,3-glc):lactose ratio could be prepared by various beta-galactosidase enzymes, such as those produced in the microorganisms Bacillus circulans, Kluyveromyces fragilis, Sporobolomyces singularis, Lactobacillus fermentum, Papiliotrema terrestris, Bifidobacterium bifidum, and Lactobacillus bulgaris. In contrast to beta-galactosidase from, for example, Aspergillus oryzae, these enzymes allow the formation of adequate lactose and disaccharide levels to grow bifidobacteria and allow the retention of said disaccharides in the process of the invention.

[0019] Preferred enzymes are those produced by Bacillus circulans or Papiliotrema terrestris.

[0020] A highly preferred enzyme is beta-galactosidase produced by Papiliotrema terrestris, which not only provides adequate lactose and disaccharide levels for the growth of bifidobacteria and allows for the retention of said disaccharides in the process of the present invention, but the resulting GOS is also hypoallergenic in that the resulting GOS composition does not cause an elevated response in the basophil activation test.

[0021] GOS production produces GOS syrup. The lactose content of such GOS should be in the range of 10-30% by weight based on carbohydrate content. The weight ratio (gal-β1,2-glc+gal-β1,3-glc):lactose in said GOS syrup should be in the range of 1.5:1-3.0:1.

[0022] The dry matter content of such syrups usually ranges from 40 to 75% by weight.

[0023] According to the process of the present invention, an aqueous solution of the GOS composition to be purified is contacted with the lactase enzyme.

[0024] This GOS solution may be the (diluted) syrup resulting from the GOS preparation process, or it may be dissolved GOS powder. Any enzymes used in the GOS preparation process may be inactivated, optionally removed, or may still be in an active form at the start of the process of the invention. This optional inactivation may be performed, for example, by adjusting the pH to below 2 by adding HCl, or by heating, for example, to 95°C.

[0025] If not already within this concentration range, the dry matter content of the GOS solution should be adjusted to a maximum of 54% by weight, preferably to a maximum of 50% by weight, more preferably in the range of 40-50% by weight, and most preferably in the range of 43-48% by weight.

[0026] The lactose content of the starting GOS solution is in the range of 10-30 wt.%, preferably 10-25 wt.%, more preferably 10-20 wt.%, based on total carbohydrate dry weight.

[0027] The weight ratio of (gal-β1,2-glc+gal-β1,3-glc):lactose in the GOS solution is in the range of 1.5:1 to 3.0:1, preferably 1.5:1 to 2.5:1, and more preferably 1.5:1 to 2.0:1.

[0028] The GOS solution preferably has a pH of 5.5 to 7.5, more preferably 6.0 to 7.0, and most preferably 6.3 to 6.8. The pH can be adjusted with a food grade buffer, such as a citrate buffer or a phosphate buffer, containing 10 mM KCl and 2.5 mM MgCl. 2 In addition, the concentration can be adjusted preferably to 5 mM to 20 mM.

[0029] The enzyme is preferably a lactase enzyme selected from the lactase enzymes produced by Kluyveromyces lactis (e.g. Maxilact® 5000, formerly DSM), Bifobacterium bifidum (e.g. Nola® Fit, formerly Chr. Hansen) and Lactobacillus bulgaris (e.g. Bonlacta™, formerly IFF). A particularly preferred enzyme is the lactase enzyme derived from Kluyveromyces lactis.

[0030] The dosage of lactase enzyme depends on the reaction temperature and reaction time: higher reaction temperatures and / or longer reaction times allow for lower enzyme concentrations.

[0031] The enzyme dosage, reaction time and temperature should be selected such that the lactose content is reduced to a maximum of 6 wt.% lactose based on the total carbohydrate weight, preferably a maximum of 5.5 wt.%, more preferably a maximum of 5.0 wt.%, even more preferably in the range of 3.0-5.0 wt.%, more preferably in the range of 3.5-5.0 wt.%, and most preferably in the range of 4.0-5.0 wt.%.

[0032] The enzyme may be used in powder form (e.g., freeze-dried, vacuum-dried or spray-dried) or in liquid form (e.g., dissolved in phosphate buffer, triethanolamine buffer, Tris-HCl buffer or Good's buffer).

[0033] In certain embodiments, the enzyme is used in immobilized form. Various techniques for immobilizing enzymes are known in the art. They typically include porous supports on which beta-galactosidase is immobilized via covalent bonding, physical adsorption (charge interactions or van der Waals interactions), gel entrapment or a combination thereof. In addition, immobilized enzymes without supports such as CLECs (crosslinked enzyme crystals) or CLEAs (crosslinked enzyme condensates) can also be applied.

[0034] Carriers that facilitate direct covalent attachment of the enzyme are preferred because they are easy to handle and do not leak into the reaction mixture. Examples of solid supports include activated acrylic polymers, preferably functionalized polymethacrylate matrices. For example, hexamethyleneamino-functionalized polymethacrylate matrices (Sepabeads) or microporous acrylic epoxy-activated resins such as Eupergit C 250L can be used.

[0035] The use of immobilized enzymes allows for a repeated batch operation system involving several successive batches ("cycles") of GOS purification. It also allows for recycling of the enzyme, which allows for semi-continuous operation and multiple reuse of the enzyme.

[0036] The enzymatic reaction is then carried out at 30-50° C., preferably 35-45° C., most preferably 38-42° C., to allow hydrolysis of lactose to the required extent. Reduction of the lactose content to the desired level usually takes about 1-8 hours, preferably 2-6 hours, most preferably 3-5 hours.

[0037] At the end of the reaction, the enzyme can be inactivated by conventional methods, such as adjusting the pH and / or increasing the temperature of the solution. For example, the pH can be adjusted to about 4.5 and / or the temperature can be increased to about 72° C.

[0038] Lactose is hydrolyzed to its monosaccharides glucose and galactose, which can be removed from the GOS solution by conventional methods such as nanofiltration or simulated moving bed chromatography (SMB), more preferably continuous simulated moving bed chromatography (SSMB). SSMB is the preferred method since it typically allows high product recovery (98-99.5%) and high purity (97-99.5%). Furthermore, SSMB requires less maintenance and lower water consumption than NF and consumes less solvent than discontinuous SMB.

[0039] (Continuous) SMB chromatography is based on size exclusion. Suitable resins for such size exclusion chromatography are ion exchange resins and gel-type resins.

[0040] The GOS thus purified has a lactose content of not more than 6% by weight, preferably not more than 5.5% by weight, more preferably not more than a maximum of 5.0% by weight, even more preferably in the range of 3.0-5.0% by weight, more preferably in the range of 3.5-5.0% by weight and most preferably in the range of 4.0-5.0% by weight, based on the total carbohydrate content.

[0041] The monosaccharide content is less than 6% by weight, preferably less than 5.5% by weight, even more preferably less than 5% by weight, more preferably less than 4.5% by weight and most preferably less than 4% by weight.

[0042] The purified GOS has a content of the disaccharides gal-β1,2-glc and gal-β1,3-glc in the range of 10-40% by weight, preferably 15-30% by weight, based on the oligosaccharide content.

[0043] The process according to the invention is capable of retaining 60-80% of these disaccharides. The final purified GOS composition preferably has an oligosaccharide / lactose ratio of greater than 10, more preferably greater than 15, and most preferably greater than 20, which means that it is considered analytically lactose-free and does not cause any symptoms of lactose intolerance.

[0044] The resulting purified GOS can be added to or used as a nutritional composition or dietary supplement, it can be used as an aqueous solution / syrup, or it can be first dried, for example by spray drying, freeze drying or spray cooling, to form a powder.

[0045] The GOS composition can be administered to the subject in the form of a nutritional composition or a nutritional supplement.The subject is a mammal, particularly a human.The subject can be of any age, but the subject is preferably at least 18 months old, preferably at least 24 months old, even more preferably at least 3 years old (36 months old), most preferably at least 13 years old.In the most preferred embodiment, the subject is an adult.

[0046] Such nutritional compositions or dietary supplements may contain, in addition to the GOS composition, one or more additional ingredients, such as, for example, a protein source, probiotics, a lipid source, probiotics, human milk oligosaccharides and / or digestible carbohydrates.

[0047] The nutritional composition may have a liquid, semi-liquid or solid support.Examples of suitable forms are dairy products such as milk, milkshakes, chocolate milk, yogurt, cream, cheese, pudding and ice cream; bars such as nutritional bars, energy bars, snack bars, cereal bars and bars for diabetics; liquid products such as nutritional drinks, diet drinks, liquid complete meals, sports drinks and other nutritional drinks; savory snacks such as chips, tortillas, puffed baked snacks, crackers, pretzels and savory biscuits; bakery products such as muffins, cakes and biscuits; confectionery such as gummies and candies; and pasta such as spaghetti.

[0048] Dietary supplements may be in the form of pills, gummies, capsules, or dry powders. Dietary supplements may be ready to consume or may need to be dissolved in a liquid such as water. Products in dry powder form may come with a device such as a spoon for measuring the desired amount of powder (e.g., daily dose or unit dose).

[0049] The nutritional composition may be provided in a jar, bottle, sachet, carton, wrapping, or the like.

[0050] Examples of protein sources that may be present in the nutritional composition or dietary supplement include whey protein (e.g., whey protein concentrate or isolate), casein (e.g., micellar casein isolate), milk protein concentrate or isolate and / or vegetable protein such as soy protein. In a preferred embodiment, the protein source is a hypoallergenic or non-allergenic protein source. The protein source includes protein hydrolysates that can be administered to subjects intolerant to food proteins, more particularly milk proteins, without inducing an allergic reaction. Examples of such protein hydrolysates are hydrolyzed whey proteins containing hydrolyzed residues with molecular weights of less than 10,000 Da and casein hydrolysates with peptides of up to 3000 Da.

[0051] Examples of carbohydrate sources that may be present in the nutritional composition or dietary supplement are disaccharides such as sucrose, monosaccharides such as glucose, as well as maltodextrins, starches and carbohydrate sources with prebiotic effect. The presence of lactose is obviously undesirable.

[0052] Examples of lipid sources that may be present in the nutritional composition or dietary supplement are tri-, di- and monoglycerides, phospholipids, sphingolipids, fatty acids and their esters or salts. Lipids may be of animal, vegetable, microbial or synthetic origin. Of particular interest are polyunsaturated fatty acids (PUFAs) such as gamma linolenic acid (GLA), dihomogamma linolenic acid (DHGLA), arachidonic acid (AA), stearidonic acid (SA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA) and conjugated linoleic acid (CLA). CLA is important in protecting against eczema and respiratory diseases in children. This is particularly related to the cis-9, trans-11 and cis-12 isomers of CLA. Examples of suitable vegetable lipid sources include sunflower oil, high oleic sunflower oil, coconut oil, palm oil, palm kernel oil, soybean oil, etc. Examples of suitable lipid sources of animal origin include milk fat, e.g. anhydrous milk fat (AMF), cream, etc. In a preferred embodiment, a combination of milk fat and vegetable derived lipids is used.

[0053] Examples of probiotics that may be present in the nutritional composition or dietary supplement are (probiotic) bacteria such as Bifidobacteria and / or Lactobacillus.

[0054] Additionally, the nutritional composition or dietary supplement may contain one or more conventional trace ingredients such as vitamins, antioxidants, minerals, free amino acids, nucleotides, taurine, carnitine, and polyamines. Examples of suitable antioxidants are BHT, ascorbyl palmitate, vitamin E, alpha and beta carotene, lutein, zeaxanthin, lycopene, and phospholipids. EXAMPLES

[0055] Example 1 The GOS-containing solutions having the dry matter contents listed in Table 1 were obtained by diluting Biotis™ GOS-O syrup having a dry matter content of 75% by weight. The GOS syrup contained 13.2% by weight lactose based on the total carbohydrate content and 24% by weight gal-β1,2-glc and gal-β1,3-glc (represented as "DP2-GB" in Table 1) based on the total carbohydrate content.

[0056] In a 100 ml vial, the pH of the GOS solution was adjusted to 10 mM sodium phosphate buffer, 2.5 mM MgCl 2 and adjusted to 6.5 using 10 mM KCl.

[0057] Lactose hydrolysis was carried out by adding various amounts of Maxilact® 5000 (Kluyveromyces lactis, formerly DSM) to the solution (see Table 1). The reaction mixture was heated in a water bath at 40° C. while stirring with a magnetic stirrer. The reaction time was 4 hours.

[0058] After the 4-hour reaction time, a 1 ml sample was taken. 1.5% (w / v) of 1.5M HCl solution was added to the sample and the resulting sample was heated at 95°C for 20 minutes to denature the enzyme. After cooling to room temperature, the sample was analyzed by HPLC using a Dionex ICS-3000 workstation equipped with a CarboPac PA-1 column (250 x 4 mm, Dionex) and an ICS-3000 ED pulsed amperometric detector (PAD) with a complex gradient A: 100 mM NaOH, B: 600 mM NaOAc in 100 mM NaOH, C: Milli-Q water and D: 50 mM NaOAc as disclosed in S. van Leeuwen et al, Carbohydrate Research 425 (2016) 48-58. Fractionation was performed at 1.0 mL / min with a gradient from 10% A, 85% C and 5% D for 25 min to 40% A, 10% C and 50% D, followed by a 35 min gradient to 75% A, 25% B, immediately followed by a 5 min wash with 100% B and a 7 min regeneration with 10% A, 85% B and 5% D.

[0059] As shown in Table 1, an enzyme dosage of 2.5 U / gram dry matter failed to reduce the lactose content below 6 wt% lactose in 4 h, whereas an enzyme dosage of 7.5 U / gram and a dry matter content of 55 wt% resulted in lower retention of DP2 oligosaccharides and lower selectivity for lactose hydrolysis than the other experiments. [Table 1]

[0060] Example 2 Experiment 5 of Example 1 was repeated in a 10 L glass reactor with downward stirring mode. The resulting GOS composition was analyzed at various time points before monosaccharide removal and summarized in Table 2 (in wt % relative to total carbohydrates). [Table 2]

[0061] After inactivation, the gal-β1,2-glc and gal-β1,3-glc content was 16.8% by weight based on the total carbohydrate content.

[0062] Example 3 Example 2 was repeated on a 1,000 L scale. The reaction was carried out for 3 hours. The resulting crude HP-GOS was subjected to SSMB to remove monosaccharides.

[0063] The GOS composition (in wt % of total carbohydrate) at various stages in the process is presented in Table 3. HP GOS extract is a middle distillate of SSMB containing monosaccharides. Raffinate is the product resulting from SSMB.

[0064] Table 4 shows the oligosaccharide distribution in the HP GOS raffinate after normalization of oligosaccharides to 100%.

[0065] As shown in these tables, a highly pure GOS composition was prepared having an oligosaccharide content of 93% by weight, a lactose content of 5.2% and a DP2 content (other than lactose) of 27.6%, all based on total carbohydrate. The retention of DP2 species other than lactose was 83% after lactose hydrolysis and 78% after SSMB. [Table 3] [Table 4]

[0066] After inactivation, the content of gal-β1,2-glc and gal-β1,3-glc was 16.7% by weight based on the total carbohydrate content.

Claims

1. 1. A process for reducing the lactose content of a galactooligosaccharide composition, comprising: a) providing an aqueous solution of a galactooligosaccharide composition having a lactose content of 10-30% by weight based on total carbohydrate weight and a weight ratio of (gal-β1,2-glc+gal-β1,3-glc):lactose ranging from 1.5:1 to 3.0:1, said solution having a dry matter content of up to 54% by weight; b) adding lactase enzyme to said solution; c) carrying out an enzymatic reaction at 30-50°C to allow the hydrolysis of lactose into monosaccharides, said reaction being carried out until the lactose content of the solution is reduced to a maximum of 6% lactose by weight based on the total carbohydrate content; d) removing monosaccharides from the solution obtained in step c). A process involving:

2. 2. The process according to claim 1, wherein the enzymatic reaction of step c) is carried out until the lactose content of the solution is reduced to a maximum of 5.5 wt.-%, preferably a maximum of 5.0 wt.-%, more preferably 3.0-5.0 wt.-%, even more preferably 3.5-5.0 wt.-%, and most preferably 4.0-5.0 wt.-% based on the total carbohydrate content.

3. 2. The process of claim 1, wherein the monosaccharides are removed by nanofiltration, simulated moving bed chromatography (SMB) or continuous simulated moving bed chromatography (SSMB), preferably continuous simulated moving bed chromatography.

4. 2. The process of claim 1, resulting in a galactooligosaccharide composition having a lactose content of less than 6% by weight, preferably less than 5.5% by weight, more preferably less than 5.0% by weight, and most preferably between 3.0 and 5.0% by weight, based on carbohydrate content.

5. The aqueous solution of galactooligosaccharide composition used in step a) may be selected from the group consisting of Bacillus circulans, Kluyveromyces fragilis, Sporobolomyces singularis, Lactobacillus fermentum, Papiliotrema terrestris, Bifidobacterium bifidum, and Lactobacillus vulgaris.

2. The process according to claim 1, wherein the lactose is obtained by conversion of lactose using beta-galactosidase derived from Bacillus bulgaris, preferably Bacillus circulans or Papiliotrema terrestris.

6. 2. The process according to claim 1, wherein the lactase enzyme added in step b) is selected from lactase enzymes derived from Kluyveromyces lactis, Lactobacillus bugaricus or Bifidobacterium bifidum, preferably Kluyveromyces lactis.

7. A galactooligosaccharide composition obtainable by the process of any one of claims 1 to 6, comprising 10-40% by weight of the disaccharides gal-β1,2-glc and gal-β1,3-glc, less than 6% by weight of lactose and less than 6% by weight of monosaccharides, based on the carbohydrate content.

8. 8. A galactooligosaccharide composition according to claim 7, comprising less than 5.5% by weight, even more preferably less than 5% by weight, more preferably less than 4.5% by weight, and most preferably less than 4% by weight of monosaccharides based on the carbohydrate content.

9. A nutritional composition comprising the galactooligosaccharide composition described in claim 7.

10. A nutritional supplement comprising the galactooligosaccharide composition described in claim 7.