GOS with high lactulose content

Abstract

Presented herein is a galactooligosaccharide characterized by a high raffinose content and a method for its production.

Description

[0001] Invention Field

[0002] This invention relates to the field of food technology, and specifically to galactooligosaccharides characterized by high lactulose content and their production process. Technical Background

[0004] Galacto-oligosaccharides (GOS)

[0005]

[0006] Also known as galacto-lacto-oligosaccharides, galacto-oligosaccharides, or transgalacto-oligosaccharides (TOS), GOS belong to the class of prebiotics. GOS are widely found in commercially available products, such as infant formula and adult food.

[0007] Due to their glycosidic bond configuration, galactooligosaccharides (GOS) are highly resistant to hydrolysis by saliva and intestinal digestive enzymes. Therefore, GOS are classified as prebiotics, defined as indigestible food components that exert beneficial effects on the host by stimulating the growth and / or activity of beneficial bacteria in the colon. This enhanced activity of beneficial bacteria produces a variety of effects, both directly through the bacteria themselves and indirectly through the organic acids produced during their fermentation. Examples of these effects include stimulating immune function, absorbing essential nutrients, and synthesizing certain vitamins.

[0008] Galacto-oligosaccharides are substrates for bacteria such as Bifidobacteria and Lactobacilli. Studies on infants and adults have shown that foods or beverages rich in galacto-oligosaccharides can significantly increase the number of Bifidobacteria. These sugars are naturally found in human milk and are called human milk oligosaccharides. Examples include lactose-N-tetrasaccharide, lactose-N-neotetrasaccharide, and lactose-N-fucopentaose.

[0009] The human gut microbiota plays a crucial role in the intestinal immune system. Galacto-oligosaccharides (GOS) indirectly support the body's natural defense mechanisms by increasing the abundance of gut microbiota and inhibiting the binding or survival of Escherichia coli, Salmonella typhimurium, and Clostridium. GOS can positively impact the immune system by indirectly promoting the production of antimicrobial substances through reducing the proliferation of pathogenic bacteria. Constipation is a potential problem, especially in infants, the elderly, and pregnant women. For infants, formula feeding can lead to constipation and hard stools. GOS can improve bowel movement frequency and alleviate constipation-related symptoms.

[0010] Related existing technologies

[0011] EP 2620506 B1 (DUPONT) relates to the production of GOS from lactitol.

[0012] EP 3598901 B1 (HOCHSCHULE ANHALT) relates to a method for producing GOS, wherein a substance derived from Lactobacillus bulgaricus ( L. delbrueckii spp. bulgaricus β-galactosidase of lactose is incubated with a lactose-containing composition, such as milk, buffer, or whey, such as sweet whey, acid whey, whey concentrate, or whey osmotic solution, at 37°C or lower.

[0013] EP 3041945 B1 (FRIESLAND) provides a method for producing GOS from lactose, comprising (i) contacting a lactose feedstock with an immobilized β-galactosidase (EC 3.2.1.23); and (ii) performing GOS synthesis, wherein the lactose feedstock is an aqueous slurry of crystalline lactose.

[0014] WO 2008 037839 A1 (VALIO) relates to a method for producing dairy products containing GOS by treatment with β-galactosidase.

[0015] WO 2018 048305 A1 (UNIV GRONINGEN) describes the use of a GOS composition comprising branched and linear GOS species with a degree of polymerization (DP) of 3, wherein the branched DP3 GOS species are present in excess relative to the linear DP3 GOS species, for inducing the mucopolysaccharide utilization pathway of beneficial gut bacteria in animals.

[0016] WO 2018 210820 A1 (NOVOZYMES) claims a method in which a milk substrate having a lactose content of at least 20% by weight is treated with an enzyme having transgalactosylation activity. The transgalactosylation activity of the enzyme can be improved by incubating the enzyme with a high concentration of glucose at high temperature to glycosylate lysine and / or arginine residues.

[0017] WO 2020 049016 A1 (FRIESLAND) relates to the field of hypoallergenic oligosaccharides for use in food compositions, particularly oligosaccharides with prebiotic properties. This invention provides a hypoallergenic oligosaccharide composition comprising galactooligosaccharides (GOS), wherein (i) the content of galactooligosaccharides (GOS) is at least 40% of the total dry matter weight of the composition; (ii) the content of isolarose is at least 10% of the total dry matter weight of the composition; (iii) the content of 6'-GL is at least 30% of the total GOS in the composition by weight; and (iv) at least 0.5% of the total GOS has a degree of polymerization (DP) of 6 or higher.

[0018] WO 2020 117548 A1 (DUPONT) relates to a method for providing a low-lactose dairy product containing GOS fiber, wherein a lactose-containing milk substrate is treated with transgalactosylase to provide GOS fiber and residual lactose; the transgalactosylase is inactivated; the GOS fiber-containing milk substrate is contacted with lactase to break down the residual lactose to provide a low-lactose dairy product containing GOS fiber; and the lactase is inactivated.

[0019] WO 2020 141032 A1 (FRIESLAND) relates to the field of food ingredients, and more particularly to an economically attractive method for producing hypoallergenic galacto-oligosaccharides (HA-GOS) and their application in food and feed. This invention provides a method for producing an HA-GOS formulation, comprising contacting a lactose feedstock with a specific β-galactosidase (EC 3.2.1.23), wherein the lactose feedstock is cheese whey permeate (CWP) or cheese whey permeate rich in sialic acid lactose (SL-CWP).

[0020] Purpose of the invention

[0021] Lactulose is a known growth factor for Bifidobacteria. Its efficacy in maintaining human health has been well-established. Furthermore, the benefits of lactulose are not limited to humans; it has also been shown to be used in animal feed, meaning its applications have been studied across a wide range of fields.

[0022] The effects of lactulose on the human body are reported, for example, in "The Many Faces of Lactulose: Recent Research Trends in Development and Physiological Effects" (Milk Science, Vol. 50, No. 2 (2001), pp. 39-47), which provides information on the growth activity of Bifidobacteria. Through this activity, lactulose exerts a range of effects, including improving the intestinal environment, promoting excretion, and accelerating intestinal motility, meaning its positive effects on human health are widely known. In recent years, consumer awareness of healthy eating has steadily increased. Taking probiotics is an important step for people who wish to improve their overall health and well-being. Therefore, it is necessary to provide consumers with new probiotic products. The basic idea of ​​this invention is to provide a new probiotic composition comprising two substances known for their probiotic effects.

[0023] Therefore, the object of this invention is to provide galactooligosaccharides with a high lactulose content. In addition to the high lactulose content, the galactooligosaccharides of this invention should also have a low content of lactose, monosaccharides, and oligosaccharides. Summary of the Invention

[0024] The first subject of this invention relates to galactooligosaccharides with a high lactulose content, which can be obtained by means of: (a) Provides an aqueous composition comprising lactose and fructose; (b) Sterilize the aqueous composition from step (a); (c) At an optimal temperature and pH range, lactose and fructose in the sterilized aqueous composition of step (b) are subjected to transgalactosylation for at least 30 minutes by adding at least one β-galactosidase to obtain a reaction mixture; (d) Inhibit the amount of enzyme contained in the reaction mixture in step (c); (e) Filter the intermediate product in step (d) to obtain a retentate R1 containing the inhibited enzyme and a permeate P1 containing GOS, lactulose, lactose, monosaccharides and oligosaccharides. (f) Optionally, the permeate P1 in step (e) is treated with lactose hydrolase and / or yeast capable of metabolizing lactose into CO2 and ethanol, and then subjected to ultrafiltration to obtain retentate R2 and permeate P2. (g) Filter the permeate P1 from step (e) or the permeate P2 from step (f) to obtain retentate R3 and permeate P3; (h) Retention material R3 in packaging step (g).

[0025] Another subject of the present invention relates to a method for producing galactooligosaccharides, particularly galactooligosaccharides with a high lactulose content, comprising or consisting of the following steps: (a) Provides an aqueous composition comprising lactose and fructose; (b) Sterilize the aqueous composition from step (a); (c) At an optimal temperature and pH range, lactose and fructose in the sterilized aqueous composition of step (b) are subjected to transgalactosylation for at least 30 minutes by adding at least one β-galactosidase to obtain a reaction mixture; (d) Inhibit the amount of enzyme contained in the reaction mixture in step (c); (e) Filter the intermediate product in step (d) to obtain a retentate R1 containing the inhibited enzyme and a permeate P1 containing GOS, lactulose, lactose, monosaccharides and oligosaccharides. (f) Optionally, the permeate P1 in step (e) is treated with lactose hydrolase and / or yeast capable of metabolizing lactose into CO2 and ethanol, and then subjected to ultrafiltration to obtain retentate R2 and permeate P2. (g) Filter the permeate P1 from step (e) or the permeate P2 from step (f) to obtain retentate R3 and permeate P3; (h) Retention material R3 in packaging step (g).

[0026] In one specific implementation scheme, the method according to the present invention is characterized by: (i) By setting the pH value outside the optimal range for activity, the amount of enzyme is wholly or partially inhibited; and (ii) The amount of inhibited enzyme is separated into a retentate (R1) and returned to step (c).

[0027] Surprisingly, it has been found that a galactooligosaccharide solution with a lactulose content of at least about 5% by weight can be produced by the method according to the invention, wherein the content of residual lactose, monosaccharides and oligosaccharides is less than 10% by weight.

[0028] According to the present invention, the lactulose content of the galactooligosaccharide mixture of the present invention or the galactooligosaccharide produced by the method according to the present invention is about 5% to 20% by weight, particularly about 10% to 15% by weight.

[0029] Starting materials

[0030] For the purposes of this invention, an aqueous composition containing sufficient amounts of lactose, particularly glycosidic galactose, and fructose is suitable as a raw material.

[0031] In a preferred embodiment, the aqueous composition is selected from fructose-rich lactose solution, fructose-rich acid whey, fructose-rich milk permeate, or syrup composed of a mixture of glucose, galactose, and fructose.

[0032] In another preferred embodiment, the fructose content of the above-described aqueous composition is about 10 to 25% by weight, particularly about 15 to 25% by weight.

[0033] In another preferred embodiment, the lactose content of the above-described aqueous composition is about 20 to 50% by weight, particularly about 25 to 35% by weight.

[0034] In another preferred embodiment, the above-mentioned aqueous composition has a lactose content of about 20 to 50% by weight, particularly about 25 to 35% by weight, and a fructose content of about 10 to 25% by weight, particularly about 15 to 25% by weight.

[0035] When using this syrup, it is preferable to prepare it in advance with lactose. Preferably, pure lactose (pharmaceutical grade) is used. If lower quality lactose is used, it can be purified first, for example, by removing calcium through cation exchange. Lactose is hydrolyzed using β-galactosidase to obtain glucose-galactose syrup. Subsequently, glucose isomerization is performed to obtain a syrup consisting of glucose (25 wt%), galactose (50 wt%), and fructose (25 wt%). This process is well known to those skilled in the art and therefore does not need to be described in detail (see Luzzi, G., Steffens, M., Clawin-Rädecker, I., Hoffmann, W., Franz, CMAP, Fritsche, J., and Lorenzen, PC (2020), En-hancing the sweetening power of lactose by enzymatic modification in the reformulation of dairy products. Int J Dairy Technol, 73: 502-512). Of course, fructose can be separated from syrup by chromatography, and the separated fructose can be used to enrich fructose in lactose aqueous solution, acid whey, or milk permeate.

[0036] To enable the method of the present invention to be carried out with economically feasible conversion and yield, it is recommended to use an aqueous composition with a sufficiently high dry matter content in step (a). A solution with a dry matter content of about 25 to about 50% by weight, preferably about 30 to about 35% by weight, is suitable for this purpose. If necessary, the aqueous composition can be concentrated accordingly, for example by reverse osmosis (“RO”).

[0037] sterilization

[0038] The aqueous composition in step (a) is sterilized. Sterilization refers to any method that reduces the colony count of the starting product to below the food approval threshold specified by the relevant national testing agency. Typically, the aqueous composition should be sterilized to below 1,000 colonies / mL, preferably below 500 colonies / mL, and particularly preferably from about 10 to about 50 colonies / mL. A preferred sterilization method is high-temperature treatment, wherein the aqueous composition is exposed to a temperature range of about 70 to about 150 °C, preferably from about 90 to about 120 °C, for about 3 to about 300 seconds, preferably from about 50 to about 200 seconds.

[0039] Enzymatic transgalactosylation

[0040] In step (c), the sterilized intermediate is subjected to an enzymatic transgalactosylation reaction. This refers to the conversion of galactose units into oligosaccharides in the presence of a suitable enzyme, in this case β-galactosidase, wherein the enzyme may be derived from Aspergillus oryzae (…). Aspergillus oryzae ), Bacillus circularis ( Bacillus circulans Enzymes that are either 1 or a mixture of the two.

[0041] Aspergillus oryzae ( Aspergillus oryzae More accurately, it is a variant of Aspergillus flavus rice ( Aspergillus flavus var. Oryzae This is a type of mold that plays an important role in Japanese cuisine (commonly known as "spray bottle mold"). It is *Kochia oryzae* (k. oryzae). The most important type of soybean (ji fungi). It is mainly used to ferment soybeans in solid-state bioreactors to produce miso and soy sauce.

[0042] Bacillus circularis ( Bacillus circulans Bacillus circulans is a bacterium that spreads in a ring on a culture medium, hence its name. It is an anaerobic, Gram-staining, rod-shaped, motile cell, 0.5 µm to 1 µm wide and 3.5 µm long. This bacterium ferments pentoses, hexoses, hexitols, and disaccharides. Bacillus circulans is found in the intestines of herbivorous fish, aiding digestion by secreting cellulase.

[0043] Like all enzymes, β-galactosidases also have a relatively narrow temperature and pH range in which they can perform at their best; these are well known to those skilled in the art.

[0044] Therefore, when using Aspergillus oryzae, the reaction is preferably carried out at a temperature of about 50°C to about 60°C and a pH of about 4 to about 5; while when adding enzymes from Bacillus circulans, the reaction is preferably carried out at a temperature of about 45°C to about 55°C and a pH of about 5.5 to about 6.5. Therefore, when using Aspergillus oryzae, the term "acidic process" is used; when using Bacillus circulans, the term "neutral process" is used.

[0045] A unique characteristic of the formation of galactooligosaccharides is that the chain structure does not remain stable indefinitely, but rather slows down after a period of time until the competing reaction, namely the cleavage of GOS, takes over.

[0046] Therefore, it has proven advantageous to consider the enzymatic reaction kinetics and to carry out the transgalactosylation reaction within approximately 30 to approximately 1200 minutes, particularly within approximately 60 to approximately 90 minutes. It should be noted that a high initial lactose concentration significantly reduces the tendency for hydrolysis, while a low enzyme concentration also delays hydrolysis.

[0047] Inhibitory effect: pH change

[0048] The transgalactosylation reaction is preferably carried out until the GOS concentration reaches its maximum value. This concentration depends on the enzyme and reaction conditions and can be monitored by sampling, thus easily determined by experts. Once the maximum GOS formation is reached, enzyme activity must be rapidly terminated to prevent reverse cleavage. This can be achieved, for example, by rapid high-temperature heating, but this will completely kill the enzyme. The present invention preferably employs a different method, namely, raising the pH to at least two units above the optimum value by adding a base, or lowering the pH to at least two units below the optimum value by adding an acid, thereby removing the enzyme from its optimal reaction conditions. Although this pH change does not immediately stop the reaction, it reduces enzyme activity by 80% to 90%, sufficient to prevent significant re-cleavage. To achieve this, it is sufficient to raise the pH to at least 7, preferably 8 to 12, particularly preferably 9 to 10, or lower it to 2 to 5, preferably 3 to 4.

[0049] When changing the pH value, it is essential to ensure that the enzyme does not undergo irreversible inactivation and that the corresponding pH value—especially in the acidic range—will not impede the subsequent use of the product. pH changes can be achieved by adding appropriate amounts of common inorganic bases, such as aqueous sodium hydroxide solution, mineral acids such as hydrochloric acid, or organic acids such as lactic acid. Increasing the pH value is preferable to decreasing it.

[0050] Enzyme isolation and reuse

[0051] The enzyme block is preferably separated by filtration, particularly by ultrafiltration—preferably continuously. This produces a residue R1 containing the inhibited enzyme and a permeate P1 containing GOS, lactulose, lactose, monosaccharides, and oligosaccharides.

[0052] In step (c), the isolated enzyme is returned to the reaction cycle. During this process, the enzyme is directly returned to its optimal temperature and pH range. If necessary, fresh enzyme can be added to maintain constant or at least approximately constant enzyme activity throughout the continuous process.

[0053] As previously mentioned, the enzyme is preferably separated by ultrafiltration. In a preferred embodiment, ultrafiltration is carried out at a temperature range of about 10 to about 55°C, preferably 10 to 20°C, and the pore size of the membrane used is preferably in the range of about 1 to about 50 kDa, preferably in the range of about 5 to about 25 kDa. Preferably, these membranes are made of polysulfone or polyethylene membranes and are so-called spiral wound membranes or plate and frame assemblies.

[0054] Optional post-processing

[0055] Optionally, the permeate P1 obtained in step (e) can be post-treated by adding lactose hydrolase and / or yeast capable of metabolizing lactose residues into CO2 and ethanol to the permeate P1.

[0056] Preferably, Kluyveromycin (lactic acid yeast) is used. Kluyvermomyces lactis The yeast was used as the pretreatment step. The post-treatment was also carried out within the optimal temperature and pH range for the hydrolytic enzyme and / or yeast—typically between 28 and 37°C—and lasted for at least 30 minutes. In this case, inhibition of the hydrolytic enzyme and / or yeast was not absolutely necessary, but may be beneficial depending on the intended subsequent use. A second ultrafiltration step was then performed to separate the enzyme or enzyme / yeast mixture. The resulting products were a retentate R2, which contained the enzyme or enzyme / yeast mixture; and a permeate P2, which contained components such as GOS and lactulose.

[0057] In a preferred embodiment, ultrafiltration is carried out at a temperature range of about 10 to about 55°C, preferably between 10 and 20°C, wherein the pore size of the membrane is preferably in the range of about 1 to about 50 kDa, preferably between about 5 and about 25 kDa. Preferably, these membranes are so-called spiral wound membranes or plate and frame assemblies made of polysulfone or polyethylene membranes.

[0058] Filtration and packaging (purification, concentration, and drying)

[0059] The permeate P1 from step (e) or the permeate P2 from step (f) is filtered to obtain a retentate R3 containing GOS and lactulose, and a permeate P3 containing undesirable byproducts such as monosaccharides, lactose residues and oligosaccharides.

[0060] In a preferred embodiment, the filtration in step (g) is performed by nanofiltration.

[0061] In another preferred embodiment, nanofiltration is carried out at a temperature range of about 6°C to about 60°C, preferably between 6°C and 20°C, wherein the pore size of the membrane is preferably between about 0.1 kDa and about 2 kDa, preferably between about 0.5 kDa and about 1 kDa. Preferably, these membranes are so-called spiral wound membranes made of polymer materials, or cartridge filters made of ceramic or alumina.

[0062] To obtain a packageable product, the retentate R3 is dried and, if necessary, purified and / or concentrated beforehand.

[0063] To increase the concentration of GOS, the retentate R3 can be further purified, for example by electrodialysis or membrane methods such as reverse osmosis. If necessary, the dry weight can be increased by evaporation.

[0064] Drying can be carried out by, for example, freeze drying, but spray drying is preferred, wherein the inlet temperature is typically from about 180 to about 260°C and the outlet temperature is from about 80 to about 105°C. The residual moisture content is at most 5% by weight, preferably from about 1% to about 2% by weight.

[0065] Referring to the following examples will help to better understand the present invention. However, these examples are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1

[0067] GOS is produced using a neutral process with a fructose-rich lactose solution as raw material.

[0068] 1000 kg of a lactose-fructose aqueous solution (30 wt% lactose, 18 wt% fructose) was heated to 98°C in a tubular heat exchanger for 120 seconds, and sterilized during heating. The sterilized solution was cooled to 55°C, transferred to a fermenter, and the pH was adjusted to 6.5 with lactic acid. It was then mixed with β-galactosidase from Bacillus circulatoryus at a substrate-enzyme ratio of 1:50 and stirred. The progress of the transgalactosylation reaction was monitored by sampling. The maximum GOS concentration was reached after approximately 90 minutes. Upon addition of 30 wt% sodium hydroxide solution, the pH rose to 10 within minutes, causing a sharp 80% drop in enzyme activity. The reaction mixture was then fed into an ultrafiltration unit equipped with a 10 kDa spiral wound membrane. The inactivated enzyme material was returned to the fermenter with retentate R1; to compensate for the loss, 5 wt% fresh enzyme was added based on the initial enzyme amount. Permeate P1 was stirred with lactose hydrolase at a 1:25 enzyme / yeast:substrate weight ratio at 35°C for approximately 10 hours. The reaction mixture was then fed into a second ultrafiltration unit, also equipped with a 10 kDa spiral wound membrane. The enzyme / yeast mixture was separated from the retentate R2, and permeate P2 was fed into a nanofiltration unit equipped with a 500-1000 Da ceramic membrane to obtain permeate P3 and retentate R3. Residual monosaccharides in the product were separated from permeate P3, while retentate R3 was fed into a reverse osmosis unit with a concentration ratio of 1:2. The resulting retentate P4 (i.e., concentrated water) was returned to the process flow, and retentate R4 (i.e., GOS concentrate containing lactulose) was heated to approximately 85°C in a plate heat exchanger for 30 seconds, and then sprayed onto the column. A white powder was obtained with a GOS content of more than 75% by weight, a lactulose content of 14% by weight, a residual moisture content of 1% by weight, and a monosaccharide content of 0.4% by weight.

Claims

1. Galacto-oligosaccharides with high lactulose content can be obtained through the following methods: (a) Provides an aqueous composition comprising lactose and fructose; (b) Sterilize the aqueous composition from step (a); (c) At an optimal temperature and pH range, lactose and fructose in the sterilized aqueous composition of step (b) are subjected to transgalactosylation for at least 30 minutes by adding at least one β-galactosidase to obtain a reaction mixture; (d) Inhibit the amount of enzyme contained in the reaction mixture in step (c); (e) Filter the intermediate product in step (d) to obtain a retentate R1 containing the inhibited enzyme and a permeate P1 containing GOS, lactulose, lactose, monosaccharides and oligosaccharides. (f) Optionally, the permeate P1 in step (e) is treated with lactose hydrolase and / or yeast capable of metabolizing lactose into CO2 and ethanol, and then subjected to ultrafiltration to obtain retentate R2 and permeate P2. (g) Filter the permeate P1 from step (e) or the permeate P2 from step (f) to obtain retentate R3 and permeate P3; (h) Retention material R3 in packaging step (g).

2. A method for producing galactooligosaccharides, particularly galactooligosaccharides with a high lactulose content, comprising or consisting of the following steps: (a) Provides an aqueous composition comprising lactose and fructose; (b) Sterilize the aqueous composition from step (a); (c) At an optimal temperature and pH range, lactose and fructose in the sterilized aqueous composition of step (b) are subjected to transgalactosylation for at least 30 minutes by adding at least one β-galactosidase to obtain a reaction mixture; (d) Inhibit the amount of enzyme contained in the reaction mixture in step (c); (e) Filter the intermediate product in step (d) to obtain a retentate R1 containing the inhibited enzyme and a permeate P1 containing GOS, lactulose, lactose, monosaccharides and oligosaccharides. (f) Optionally, the permeate P1 in step (e) is treated with lactose hydrolase and / or yeast capable of metabolizing lactose into CO2 and ethanol, and then subjected to ultrafiltration to obtain retentate R2 and permeate P2. (g) Filter the permeate P1 from step (e) or the permeate P2 from step (f) to obtain retentate R3 and permeate P3; (h) Retention material R3 in packaging step (g).

3. The method according to claim 2, characterized in that: (i) By setting the pH value outside the optimal range for activity, the amount of enzyme is wholly or partially inhibited; and (ii) The amount of inhibited enzyme is separated into a retentate (R1) and returned to step (c).

4. The method according to claim 2, wherein the aqueous composition in step (a) is selected from a fructose-rich lactose solution, a fructose-rich acid whey, a fructose-rich milk permeate, or a syrup composed of a mixture of glucose, galactose, and fructose.

5. The method according to claim 2, wherein in step (a), an aqueous composition with a dry matter content of about 25 to about 50% by weight is used.

6. The method of claim 2, wherein sterilization is performed by high-temperature treatment.

7. The method of claim 2, wherein the fungus is derived from Aspergillus oryzae (… Aspergillus oryzae ) and / or Bacillus circularis ( Bacillus circulans The enzyme is used as β-galactosidase.

8. The method of claim 7, wherein transgalactosylation is carried out by adding β-galactosidase from Aspergillus oryzae at a temperature of about 50°C to about 60°C and a pH of about 4 to about 5.

9. The method of claim 7, wherein transgalactosylation is carried out by adding β-galactosidase from Bacillus circulatoryus at a temperature of about 45°C to about 55°C and a pH of about 5.5 to about 6.

5.

10. The method of claim 2, wherein the transgalactosylation in step (c) is carried out for about 30 to about 1200 minutes.

11. The method of claim 2, wherein the filtration in step (e) is ultrafiltration.

12. The method of claim 2, wherein the post-treatment is carried out for at least 30 minutes within the optimal temperature and pH range for the hydrolytic enzyme and / or yeast.

13. The method of claim 2, wherein the filtration in step (g) is nanofiltration.

14. The method of claim 2, wherein the retentate R3 is concentrated and dehydrated.

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