LACTOSE

BACKGROUND

1. Field

This disclosure relates to the enzymatic preparation of galactooligosacchande (GOS) from lactose. More particularly, this disclosure relates to the sequential use of two different microbial lactase enzymes to maximize the degree of transgalactosylation during the digestion of lactose.

2. Description of Related Art

Galactooligosaccharides (GOS) are non-digestible carbohydrates that serve as the building block of oligosaccharides in human milk. GOS modulate the growth and activity of gastrointestinal microorganisms, and are therefore believed to promote a healthy balance of microorganisms in the gut. Among other things, GOS are believed to reduce levels of blood serum cholesterol, improve mineral absorption, and prevent colon cancer development. The properties of GOS depend significantly on the chemical composition, structure, and degree of polymerization (DP).

GOS can be formed by the digestion of lactose with β-D-galactoside galactohydrolases. β-D-galactoside galactohydrolases catalyze the hydrolysis of the galactosyl moiety from the non-reducing end of lactose. In addition, β-D- galactoside galactohydrolases can catalyze transgalactosylation in which a galactosyl moiety is transferred to a nucleophilic acceptor other than water, i.e. potentially any sugar present in a reaction medium. Transgalactosylation is a kinetically controlled reaction, and represents competition between the reactions of hydrolysis and synthesis. The ability to favor synthesis over hydrolysis depends on several factors, including the origin of the β-D- galactoside galactohydrolase and the initial composition of acceptor sugars in the medium (e.g. lactose and galactose) with which the enzymes are presented. If lactose is the initial substrate, transgalactosylation results in the production of GOS comprising a mixture of di- (DP2), tri- (DP3), and even higher oligosaccharides (DP4 ⁺ ) with or without a terminal glucose. The chemical structure and composition of a GOS (e.g. the number of hexose moieties and the types of linkages) affects its properties, such as the fermentation pattern by probiotic bacteria in the gut. The chemical compositions, structure, degree of polymerization, and yield of GOS also depends on the origin of the β-D-galactoside galactohydrolases utilized.

Many adults are lactose intolerant, and thus it is desirable to hydrolyze as much lactose as possible during the preparation of GOS from lactose. However, reaction conditions that favor the enzymatic digestion of lactose to, for example, less than 20% of the initial lactose concentration tend to also favor the digestion of GOS that is synthesized. Accordingly, reducing lactose concentration may result in reduced yield of GOS. SUMMARY

This disclosure relates to a method of producing galactooligosacchande (GOS) from lactose. The method includes incubating an initial aqueous solution comprising lactose at an initial concentration with an acid fungal lactase to produce an intermediate aqueous solution comprising lactose and GOS in which the concentration of lactose is about 30% to about 70% of the initial concentration of the initial aqueous solution; adding a yeast lactase to the intermediate aqueous solution; and incubating the intermediate aqueous solution comprising the yeast lactase to produce a final aqueous solution in which the concentration of lactose is between 0% and 20% of the initial concentration of the initial aqueous solution. Incubating the initial aqueous solution to produce the intermediate aqueous may involve incubating the initial aqueous solution to produce the intermediate aqueous having about 40% of the initial concentration of lactose the initial aqueous solution. Incubating the initial aqueous solution to produce the intermediate aqueous may involve incubating the initial aqueous solution to produce the intermediate aqueous comprising 49% to 52% DP2 sugar (w/w) of total sugar in the intermediate aqueous solution. Incubating the intermediate aqueous solution with the yeast lactase to produce the final aqueous solution, may involve incubating the intermediate aqueous solution to produce the final aqueous solution comprising 23.5 % to 25% DP2 sugar (w/w) of total sugar in the final aqueous solution. The method may further include adjusting the pH of the intermediate aqueous solution to between 5.5 and 9.0 with KOH, MgCI ₂ , and citric acid prior to adding the yeast lactase. Adjusting the pH of the intermediate aqueous solution to between 5.5 and 9.0 with KOH, MgCI ₂ , and citric acid may include adjusting the pH to between 6.0 and 7.5. Adjusting the pH of the intermediate aqueous solution to between 5.5 and 9.0 with KOH, MgCI ₂ , and citric acid may include adjusting the pH to about 6.8. Adjusting the pH of the intermediate aqueous solution with KOH, MgCI ₂ , and citric acid may include sequentially adding KOH, MgCI ₂ , and citric acid to the intermediate aqueous solution. Sequentially adding KOH, MgCI ₂ , and citric acid to the intermediate aqueous solution comprises, in sequential order: adjusting the pH of the intermediate aqueous solution to about 9.2 with KOH; adding about 0.16 g of MgCI ₂ per 1 00 g of aqueous solution to the intermediate aqueous solution; and adjusting the pH of the intermediate aqueous solution from about 9.1 to about 6.8. The acid fungal lactase may be a fungal β-D-galactoside galactohydrolase.

The fungal β-D-galactoside galactohydrolase may be derived from an Aspergillus species. The Aspergillus species may be Aspergillus oryzae. The concentration of the acid fungal lactase may be expressed in terms of lactase units (LU) per gram of lactose in the solution. The concentration of the acid fungal lactase in the initial aqueous solution may be between 1 and 300 LU per gram of lactose in the initial aqueous solution. The concentration of the acid fungal lactase may be between about 1 0 and about 20 LU per gram of lactose in the initial aqueous solution. The concentration of the acid fungal lactase may be between about 15 and about 1 7 LU per gram of lactose in the initial aqueous solution. The concentration of the acid fungal lactase may be about 16.7 LU per gram of lactose in the initial aqueous solution. Alternatively, the concentration of the acid fungal lactase may be about 5.6 LU per gram of lactose in the initial aqueous solution, or about 5.8 LU per gram of lactose in the initial aqueous solution.

The yeast neutral lactase may be a yeast β-D-galactoside galactohydrolase. The yeast β-D-galactoside galactohydrolase may be derived from a

Kluyveromyces species. The Kluyveromyces species may be Kluyveromyces lactis.

Adding the yeast neutral lactase to the intermediate aqueous solution may include adding the yeast lactase to a concentration of between 1 and 50 LU per gram of lactose in the intermediate aqueous solution. Adding the yeast neutral lactase to the intermediate aqueous solution may include adding the yeast lactase to a concentration of about 4 to about 5 LU per gram of lactose in the intermediate aqueous solution. Adding the yeast neutral lactase to the intermediate aqueous solution may include adding the yeast lactase to a concentration of about 4.7 LU per gram of lactose in the intermediate aqueous solution. Adding the yeast neutral lactase to the intermediate aqueous solution may include adding the yeast lactase to a concentration of about 4.4 LU per gram of lactose in the intermediate aqueous solution.

The initial concentration of lactose in the initial aqueous solution may be between 1 5 and 63 °Bx. The initial concentration of lactose in the initial aqueous solution may be between about 30°Bx and about 60 °Bx. The initial concentration of lactose in the initial aqueous solution may be about 45 °Bx. The initial concentration of lactose in the initial aqueous solution may be about

53 °Bx.

The initial aqueous solution may be incubated with the fungal acid lactase at a temperature between about 25 and 65 ^e C. The temperature may be between about 40 and about 55 ^e C. The initial aqueous solution may be incubated with the fungal lactase at a temperature of about 53.5 ^e C. The initial aqueous solution may be incubated with the fungal lactase at a pH between about 2.5 and about 8.0. The initial aqueous solution may be incubated with the fungal lactase at a pH between about 3.5 and about 6.5. In particular embodiments, the initial aqueous solution is incubated with the fungal lactase at a pH between about 4.5 and about 5.5.

In some embodiments, the method includes deactivating the fungal acid lactase prior to adding the yeast neutral lactase. In some embodiments, deactivating the fungal lactase comprises adjusting the pH of the intermediate aqueous solution to about 2 or less. In some embodiments, deactivating the fungal acid lactase includes adjusting the pH of the intermediate aqueous solution to about 2. The pH of the intermediate aqueous solution may be adjusted with hydrochloric acid (HCI) to deactivate the fungal lactase. In some embodiments, deactivating the fungal acid lactase includes heating to above 72 ^e C.

The intermediate aqueous solution may incubated with the yeast neutral lactase at a temperature between about 4 and about 50 ^e C. In some embodiments, the intermediate aqueous solution is incubated with the yeast lactase at a temperature between about 30 and about 45 ^e C. In some embodiments, the intermediate aqueous solution is incubated with the yeast lactase at a temperature of about 36.5 ^e C.

The method may further include deactivating the yeast lactase. In some embodiments, deactivating the yeast lactase includes adjusting the pH of the final aqueous solution to about pH 5.5. In some embodiments, the pH of the final aqueous solution is adjusted to about pH 5.5 with citric acid. In some embodiments, deactivating the yeast lactase includes incubating the final aqueous solution at 72 ^e C.

The method may further include partially removing glucose and galactose from the final aqueous solution by chromatography to produce a GOS- enriched solution. The method may further include removing the fungal acid lactase, the yeast neutral lactase, glucose and galactose from the final aqueous solution by chromatography.

In some embodiments, the fungal acid lactase and the yeast neutral lactase, is removed from the final aqueous solution by ion exchange chromatography.

In some embodiments, the glucose and/or galactose is at least partially removed from the final aqueous solution by ion exchange, filtration, chromatographic separation, or additional fermentation reactions. In some embodiments, chromatographic separation comprises simulated moving bed chromatography. This disclosure further relates to a galactooligosaccharide (GOS) syrup produced according to a method as described above. In some embodiments, the GOS syrup is at least 40% GOS w/w of the total carbohydrate in the GOS syrup. In some embodiments, the GOS syrup is at least 65% GOS w/w of the total carbohydrate in the GOS syrup.

In some embodiments, the wherein ratio of DP2:DP3:DP4 in the GOS syrup is about 2:3:1 .

This disclosure also relates generally to the use of a β-D-galactoside galactohydrolase derived from Aspergillus oryzae in combination with a β-D- galactoside galactohydrolase derived from Kluyveromyces lactis in the preparation of galactooligosaccharide (GOS) syrup from an aqueous solution comprising lactose, wherein the GOS syrup is at least about 40% GOS w/w of the total carbohydrate in the GOS syrup. The β-D-galactoside galactohydrolase derived from Aspergillus oryzae is for incubation with the aqueous solution prior to incubation of the aqueous solution with the β-D- galactoside galactohydrolase derived from Kluyveromyces lactis. ln some embodiments, the GOS syrup may be at least about 60% GOS w/w of the total carbohydrate in the GOS syrup. In some embodiments, the GOS syrup may be about 65% GOS w/w of the total carbohydrate in the GOS syrup.

This disclosure also relates generally to the use of a β-D-galactoside galacto hydrolase derived from Kluyveromyces lactis for increasing the amount of galactooligosaccharide (GOS) in an aqueous solution comprising lactose that has been previously treated with a β-D-galactoside galactohydrolase derived from Aspergillus oryzae. In some embodiments, the amount of GOS may be increased to at least 40% w/w of total carbohydrates in the solution. In some embodiments, the diversity of GOS may be increased.

This disclosure also relates generally to the use of a β-D-galactoside galactohydrolase derived from Aspergillus oryzae in combination with a β-D- galactoside galactohydrolase derived from Kluyveromyces lactis in reducing the concentration of lactose in an aqueous solution to less than 20% w/w of the initial concentration of lactose. The β-D-galactoside galactohydrolase derived from Aspergillus oryzae is for incubation with the aqueous solution prior to incubation of the aqueous solution with the β-D-galactoside galactohydrolase derived from Kluyveromyces lactis.

Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate embodiments of the invention,

Figure 1 is a flow diagram of a method of producing GOS syrup as disclosed herein in Example 5. is a HPLC chromatogram following primary transgalactosylation of lactose using fungal β-D-galactoside galactohydrolases from Aspergillus oryzae as disclosed herein in Example 5. Figure 3 is a HPLC chromatogram following secondary transgalactosylation of lactose using yeast β-D-galactoside galactohydrolases from Kluyveromyces as disclosed herein in Example 5. Figure 4 is a flow diagram of a method of producing GOS syrup as disclosed herein in Example 6. is a HPLC chromatogram following primary transgalactosylation of lactose using fungal β-D-galactoside galactohydrolases from Aspergillus oryzae as disclosed herein in Example 6.

Figure 6 is a HPLC chromatogram following secondary transgalactosylation of lactose using yeast β-D-galactoside galactohydrolases from Kluyveromyces as disclosed herein in Example 6.

Figure 7 is a HPLC chromatogram of final product after purification and enrichment as disclosed herein in Example 6.

DETAILED DESCRIPTION

Definitions

"DP" as used herein refers to the degree of polymerization of the GOS. A disaccharide GOS is characterized as a "DP2". A trisaccharide GOS is characterized as a "DP3". A tetrasaccharide GOS is characterized as a "DP4". The skilled person will understand that each grouping may include a plurality of species of GOS which differ in terms of the sequence of sugar moieties and the linkages between moieties.

"Initial aqueous solution" as used herein refers to the lactose solution that is prepared for and is digested by the acid fungal lactase as the primarily active lactase.

"Initial concentration of lactose" as used herein refers to the amount of lactose that is added to create the initial aqueous solution, including any lactose that may be added to the initial aqueous solution after incubation with the acid fungal lactase has commenced.

"Intermediate aqueous solution" as used herein refers to the resulting lactose solution upon the effective termination of the digestion of the initial aqueous solution by the acid fungal lactase that is then digested by the yeast neutral lactase.

"Final aqueous solution" as used herein refers to the resulting lactose solution upon the effective termination of the digestion of the intermediate aqueous solution by the yeast neutral lactase.

This disclosure relates to methods of producing galactooligosaccharide (GOS) from lactose using a combination of acidic lactases and neutral lactases. More particularly, the method comprises incubating an aqueous solution comprising lactose with an acid fungal lactase. The acidic fungal lactase hydrolyses the lactose in the solution to galactose and glucose. The lactase further catalyzes transgalactosylation reactions in which the galactosyl moiety is transferred to potentially any sugar moiety present in the solution (e.g. galactose, glucose, lactose, etc.) to produce GOS comprising a mixture of DP2, DP3, DP4, DP5, and even higher order oligosaccharides. Primarv Digestion with an Acid Fungal Lactase

In various embodiments of the methods disclosed herein, the acid fungal lactase is a fungal β-D-galactoside galactohydrolase. The β-D-galactoside galactohydrolase may be derived an Aspergillus species. In particular embodiments, the β-D-galactoside galactohydrolase is derived from Aspergillus oryzae, such as the β-D-galactoside galactohydrolase available from Enzyme Development Corporation (New York) as ENZECO™ Fungal Lactase Concentrate. The skilled person will understand that the determination of lactase units (LU) will be specified on the TDS for an enzyme. One LU may be defined as that quantity of enzyme which will liberate 1 .0 μη"ΐοΙ/η"ΐίη of o-nitrophenol under the conditions of the assay specified in the TDS. The concentration of the acid fungal lactase in the initial aqueous solution may be between 1 and 300 LU per gram of lactose in the initial aqueous solution. The concentration of the acid fungal lactase may be between about 10 and about 20 LU per gram of lactose in the initial aqueous solution. The concentration of the acid fungal lactase may be between about 1 5 and about 17 LU per gram of lactose in the initial aqueous solution. In particular embodiments, the concentration of the acid fungal lactase may be about 16.7 LU per gram of lactose in the initial aqueous solution. In particular embodiments, the concentration of the acid fungal lactase may be about 5.6 LU per gram of lactose in the initial aqueous solution. In particular embodiments, the concentration of the acid fungal lactase may be about 5.8 LU per gram of lactose in the initial aqueous solution. Nevertheless, the skilled person will understand that the methods disclosed herein may be performed with a wide range of acid lactase concentrations depending on a number of factors including the initial concentration of lactose in the aqueous solution, the length of time for which the reaction is allowed to proceed, the pH, and the reaction temperature.

The source of lactose may vary. The lactose can be provided in the form of milk permeate. Alternatively, the lactose can be provided as edible crystalline lactose commonly available from commercial suppliers. The initial concentration of lactose in the initial aqueous solution should be in the range of 15 to 63 °Bx. Nevertheless, the skilled person will understand that, for commercial purposes, the initial concentration of lactose should be higher than 15°Bx, as lower concentration of lactose favors hydrolysis over the transgalactosylation, thereby leading to lower GOS yields. Moreover, lower initial concentrations of lactose necessitate larger volumes to be processed in order to obtain the same amount of products, and thus more resources for downstream separation such as chromatographic apparatuses and evaporators. Accordingly, the initial concentration of lactose and in an initial aqueous solution will preferably be between about 30°Bx and about 60°Bx. In particular embodiments, the initial concentration of lactose and in the initial aqueous solution is about 45°Bx. In particular embodiments, the initial concentration of lactose and in the initial aqueous solution is about 53 °Bx. The pH of the initial aqueous solution should be in the range of about 2.5 to about 8.0. The skilled person will understand, however, that the pH of the initial aqueous solution should be close to the optimal pH for the enzyme. Accordingly, in some embodiments, the pH of the initial aqueous solution will be between about 3.5 and about 6.5. In some embodiments, the pH of the initial aqueous solution will be between about 4.5 and about 5.5. For example,

ENZECO™ Fungal Lactase Concentrate has activity within a pH range of about 2.5 to about 2.8, although the activity may be slow outside a pH range of about 3.5 to about 6.5. The ENZECO™ Fungal Lactase Concentrate, for example, has a pH optimum of between 4.5 and 5.0.

The skilled person will understand that the pH of a solution comprising lactose may vary depending on the concentration of lactose and the source of lactose. Accordingly, it may be necessary to adjust the pH of the initial aqueous solution within the suitable pH range to bring the pH of the initial aqueous solution within the desired range.

The initial aqueous solution is incubated with the fungal lactase at a temperature between about 35 and about 65 °C. ENZECO™ Fungal Lactase Concentrate, for example, has a temperature optimum of 55 °C at pH 4.5 and 6.5. Thus, in some embodiments, the initial aqueous solution is incubated with the acid fungal lactase at a temperature between about 50 and about 56.5 °C. In some embodiments, the initial aqueous solution is incubated with the acid fungal lactase at a temperature between about 50 and about 55 °C. In particular embodiments, the initial aqueous solution is incubated with the acid fungal lactase at a temperature of about 53.5 ^< Ό.

The skilled person will understand that the methods disclosed herein are not limited by any specific reaction time for the incubation of the initial aqueous solution with the acid fungal lactase. Rather, the reaction is allowed to proceed until about 20% to about 70% of the lactose provided in the initial aqueous solution is hydrolyzed (i.e. until the concentration of lactose is between about 20% to about 70% of the initial concentration of lactose in the initial aqueous solution). In particular embodiments, the reaction is allowed to proceed until about 40% of the lactose provided in the initial aqueous solution is hydrolyzed (i.e. until the concentration of lactose is about 40% of the initial concentration of lactose in the initial aqueous solution) and/or until DP2 sugars comprise 49% to 52% (w/w) of total sugar in the intermediate aqueous solution. Accordingly, the concentration of lactose and other sugars in the initial aqueous solution may be monitored from time to time in order to identify an appropriate time to end the incubation with the acid fungal lactase. The skilled person will understand that incubation time depends on a combination of temperature, initial lactose concentration, pH, and lactase concentration. Reactions may be run quickly with a large concentration of enzyme if enzyme cost in not important. Alternatively, enzyme costs may be saved if a reaction is carried out more slowly. Parameters may also be adjusted depending on how the reaction time is to be logistically tied in downstream processes. Secondary Digestion with a Neutral Yeast Lactase

Once the desired concentration of lactose in the aqueous solution (and/or a DP2 sugar concentration of about 49% to 52% (w/w) of total sugar in the intermediate aqueous solution) is achieved, this intermediate aqueous solution is incubated with a yeast neutral lactase. Prior to adding the yeast neutral lactase, it may be preferable to deactivate the acid fungal lactase. Deactivating the acid fungal lactase may involve adjusting the pH of the intermediate aqueous solution to about 2 or less with, for example, HCI.

Deactivating the acid fungal lactase seeks to minimize hydrolysis of GOS by the acid fungal lactase, and thereby maximize GOS yield. However, the skilled person will understand that active steps to deactivate of the acid fungal lactase may not be completely necessary.

The neutral yeast lactase is added to the intermediate aqueous solution comprising GOS and about 20 to about 70% of the initial lactose to a concentration. In various embodiments of the methods disclosed herein, the neutral yeast lactase is a yeast β-D-galactoside galactohydrolase. The β-D- galactoside galactohydrolase may be derived from a Kluyveromyces species.

In particular embodiments, the β-D-galactoside galactohydrolase is derived from Kluyveromyces lactis, such as the β-D-galactoside galactohydrolase available from Enzyme Development Corporation (New York) as ENZECO™ Lactase NL 2.5X. The yeast neutral lactase may be added to the intermediate aqueous solution at a concentration of between 1 and 50 LU per gram of lactose in the intermediate aqueous solution. The yeast neutral lactase may be added to the intermediate aqueous solution at a concentration of about 4 to about 5 LU/g lactose in the intermediate aqueous solution. The yeast neutral lactase may be added to the intermediate aqueous solution at a concentration of about 4.7 LU per gram of lactose in the intermediate aqueous solution. The yeast neutral lactase may be added to the intermediate aqueous solution at a concentration of about 4.4 LU per gram of lactose in the intermediate aqueous solution. Nevertheless, the skilled person will understand that the methods disclosed herein may be performed with a wide range of yeast lactase concentrations depending on a number of factors including the initial concentration of lactose in the intermediate aqueous solution, the length of time for which the reaction is allowed to proceed, the pH, and the reaction temperature. ln certain embodiments, e.g. where the neutral yeast β-D-galactoside galacto hydrolase is derived from a Kluyveromyces species, it may be necessary to add potassium and magnesium for enzyme activity. In embodiments where the pH must be adjusted up to 5.5 or higher, e.g. where the pH of the intermediate aqueous solution has been adjusted to about 2.0 or zero or less to deactivate the acid fungal lactase, the pH and salt can be adjusted using potassium, magnesium chloride, and citric acid. ENZECO™ Lactase NL 2.5X has a pH optimum of about 6 to about 7.

Accordingly, the skilled person will understand that it may be necessary to adjust the pH of the intermediate aqueous solution between 6 and 7.5 to facilitate the activity of the neutral yeast lactase. In particular embodiments, adjusting pH of the intermediate solution with potassium hydroxide, magnesium chloride and citric acid involves adjusting the pH to about 6.8.

Such pH adjustments can lead to turbidity of the mixture, which can plug downstream separation equipment. However, this turbidity can largely be avoided by adding the salts in a specific sequence. More particularly, adjusting the pH of the intermediate aqueous solution to the desired pH and salt concentration by sequentially adding the potassium hydroxide, magnesium chloride and citric acid can avoid turbidity. More particularly, sequentially adding potassium hydroxide, magnesium chloride and citric acid to the intermediate aqueous solution in the following amount and order can largely avoid turbidity:

• adding potassium hydroxide to arrive at a pH of about 9.2;

• adding magnesium chloride to arrive at a pH of about 9.1 ; and

• adding citric acid to a pH of about 6.8.

The temperature of the intermediate aqueous solution is adjusted to between 30 and 45 °C prior to addition of the neutral yeast lactase. However, the skilled person will understand that while temperature may be adjusted for optimal enzyme activity, the yeast lactase may perform at a much slower rate outside this range, e.g. between about 4.0 and about 50.0 °C. In particular embodiments disclosed herein, the temperature of the intermediate aqueous solution is adjusted to about 36.5 °C for incubation with the neutral yeast lactase. As with the acid fungal lactase, the reaction time will depend on temperature, pH, lactase concentration, and initial concentration of lactose in the intermediate aqueous solution. Again, the reaction rate can be increased if enzyme cost is not a concern. Alternatively, the reactions may be run more slowly to save on the cost of enzyme.

The intermediate aqueous solution is incubated with the neutral yeast lactase to produce a final aqueous solution in which the concentration of lactose is between zero and about 20% of the initial concentration of lactose in the initial aqueous solution. In some embodiments, the intermediate aqueous solution is incubated with the neutral yeast lactase until a final aqueous solution comprising 23.5 % to 25% DP2 sugar (w/w) of total sugar in the final aqueous solution is achieved. Deactivation of the Yeast Lactase

Once a final concentration of between zero and 20% of the initial concentration of lactose and the initial aqueous solution has been achieved, the neutral yeast lactase may be deactivated. In some embodiments, deactivating the neutral yeast lactase involves adjusting the pH of the final aqueous solution to about pH 5.5, at or below which pH the enzyme effectively has no activity. In addition to adjusting the pH to 5.5, or as an alternative to adjusting pH to 5.5, deactivating the yeast lactase may involve incubating the final aqueous solution at 72°C. The necessity of the pH adjustment step may depend on how quickly the final aqueous solution can be heated, and how quickly the reaction is proceeding prior to such heat treatment. If heating can be accomplished quickly enough so that there is no change in sugar composition (e.g. hydrolysis of GOS) while the final aqueous solution is being heated, then a pH adjustment may be unnecessary. On the other hand, the skilled person will appreciate that it may be unnecessary to heat treat the final aqueous solution to deactivate the neutral yeast lactase if pH is used to deactivate the reaction, the reaction rate is very slow, or the enzyme a little to no activity remaining.

Separation

Chromatography may then be used to remove the enzymes, stabilizing agents, glucose and galactose from the final aqueous solution to produce a GOS-enriched solution. Ion exchange chromatography may be initially carried out on the final aqueous solution to remove the lactase enzymes, cations, anions, and components contributing to color.

After ion exchange, the further separation may be conducted to partially remove glucose and galactose and enrich the GOS fraction. The skilled person will be aware of the standard methods that may be available, including ion exchange, filtration, chromatographic separation (SMB), or additional fermentation reactions.

For example, simulated moving bed chromatography may be used to enrich the GOS in the GOS syrup from about 40% w/w of total carbohydrate in the final aqueous solution to greater than 60% w/w of total carbohydrates after separation.

GOS Products

The composition of different GOS species in a GOS syrup is unpredictable and will depend on the specific lactase with which lactose solution is incubated, the concentration of lactose, and the concentration of lactose. Accordingly, the skilled person will appreciate that the GOS syrups disclosed herein have a unique balance of di- (DP2), tri- (DP3), tetra- (DP4), penta- (DP5) and higher GOS. Accordingly, this disclosure also relates to GOS syrups with novel GOS balances that are produced according to methods disclosed herein. Accordingly, this disclosure further relates to use of the combination of a first β-D-galactoside galactohydrolase derived from an Aspergillus oryzae with a second β-D-galactoside galactohydrolase derive from a Kluyveromyces lactis in the preparation of GOS syrup from an aqueous solution comprising lactose. The GOS syrup may comprise at least 40% GOS w/w of total carbohydrate in the GOS syrup. The use involves incubation of the aqueous solution with the first β-D-galactoside galactohydrolase followed by incubation with the second β-D-galactoside galactohydrolase.

EXAMPLE 1

An aqueous solution of edible lactose with a starting concentration of 45°Bx as adjusted to pH=5 using hydrochloric acid and equilibrated to 53.5°C. β-D- galactoside galactohydrolase derived from Aspergillus oryzae (ENZECO™ Fungal Lactase Concentrate from Enzyme Development Company) was added to the aqueous solution to a concentration of 280 LU per gram of lactose in the aqueous solution. The initial aqueous solution was incubated with the β-D-galactoside galactohydrolase derived from Aspergillus oryzae for

1 95 minutes under constant agitation. Samples of the aqueous solution were taken at 1 min, 2.5 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60, min, 75 min, 90 min, 1 20 min, and 1 95 min. The composition of the carbohydrate fractions of the aqueous solution at the different time points are indicated in Table 1 .

EXAMPLE 2 4.5 kg of edible lactose was suspended in 5.5 kg of water. The temperature of the suspension was brought to above 90°C under constant agitation until the lactose was completely dissolved to produce an initial aqueous solution. The pH of the initial aqueous solution was adjusted to about 4.5 using hydrochloric acid. The temperature of the initial aqueous solution was equilibrated to 55 °C. β-D-galactoside galactohydrolase derived from Aspergillus oryzae (ENZECO™ Fungal Lactase Concentrate from Enzyme Development Company) was added to the initial aqueous solution to a concentration of 20 LAU/g lactose. The initial aqueous solution was incubated with the β-D-galactoside galactohydrolase derived from Aspergillus oryzae for 6 hours under constant agitation. The β-D-galactoside galactohydrolase was then deactivated by adjusting the pH to about 2.0 with HCI. The resulting intermediate solution comprising of GOS, glucose, galactose, and unreacted lactose, was analyzed by HPLC to ensure that the lactose concentration was reduced to less than 60% of the initial concentration of lactose in the initial aqueous solution (see Figure 1 , Table 2). The pH of the intermediate solution was adjusted to about pH 8 with 50%

KOH. The pH of the intermediate solution was then adjusted to 6.75 with a salt solution comprising of 3.72% w/w citric acid, 6.01 % w/w magnesium chloride hexahydrate, and 15.55% w/w dipotassium hydrogen phosphate, β- D-galactoside galactohydrolase derived from Kluyveromyces lactis (ENZECO™ Lactase NL 2.5x from Enzyme Development Company) was then added at a dosage of 8.8 LAU/g lactose. The intermediate solution was incubated with β-D-galactoside galactohydrolase derived from Kluyveromyces lactis for 10 hours under constant agitation. The β-D-galactoside galactohydrolase derived from Kluyveromyces lactis was then deactivated by adjusting the pH to about 3.0 with HCI.

Table 1. GOS produced using lactase (280 LU/g lactose) from Aspergillus Oryzae (Enzeco Fungal lactase) at starting lactose of 45 BRIX, T=53.5C, pH =5.

Time

(min) 1 2.5 5 10 15 20 25 30 40 50 60 75 90 120 195

DP5+ 0.063 0.286 0.790 1.601 2.250 2.634 2.877 3.009 3.155 3.134 3.001 2.867 2.671 2.210 1 .320

DP4 1 .143 2.594 4.288 5.821 6.449 6.605 6.669 6.522 6.299 6.051 5.649 5.247 4.917 4.214 2.957

DP3 12.532 16.610 18.862 19.508 18.954 18.265 17.792 17.223 16.197 15.652 14.742 13.831 13.053 1 1 .815 9.206

Lactose 78.856 69.258 57.818 48.774 42.769 38.460 35.558 33.051 28.909 26.122 23.476 20.830 18.760 15.493 10.976

DP2 0.532 0.382 2.339 2.913 4.257 5.630 6.342 7.100 8.657 9.674 10.650 1 1.626 12.136 13.289 13.673

Glucose 5.881 9.039 12.695 16.687 19.316 21 .209 22.778 24.148 26.257 27.719 29.345 30.970 32.289 34.402 38.059

Galactose 0.993 1 .830 3.207 4.697 6.004 7.197 7.984 8.947 10.526 1 1 .648 13.138 14.629 16.174 18.578 23.810

TOTAL

GOS 14.271 19.872 26.280 29.843 31 .910 33.134 33.680 33.854 34.308 34.510 34.041 33.572 32.777 31 .528 27.155

Table 2. % Composition of sugars in GOS mixture following primary transgalactosylation of lactose using fungal β-galactosidase from Aspergillus oryzae

The resulting final aqueous solution comprising of GOS, glucose, galactose and unreacted lactose, was analyzed by HPLC to ensure that the lactose concentration was to 10% or less than the initial concentration of lactose in the initial solution (see Figure 2, Table 3). Table 3. % Composition of sugars in GOS mixture following secondary transgalactosylation of lactose using yeast β-galactosidase from

Kluyveromyces

EXAMPLE 3

Demineralized, deproteinized, ultrafiltered milk permeate was evaporated to 35 ^e Bx, and incubated with β-D-galactoside galactohydrolases derived from Aspergillus oryzae and Kluyveromyces lacti as described in Example 2. The composition of sugars in the GOS mixture following two-stage transgalactosylation of lactose from ultrafiltered milk permeate is shown in Table 4.

Table 4. % Composition of sugars in GOS mixture following two-stage transgalactosylation of lactose from ultrafiltered milk permeate using yeast β-galactosidase from Aspergillus Oryzae and Kluyveromyces Lactis.

EXAMPLE 4

27.4 kg of edible lactose was suspended in 20.8 kg of water to produce a solution of 54°Bx. The temperature of the suspension was brought to above 95°C under constant agitation until the lactose was completely dissolved to produce an initial aqueous solution. The pH of the initial aqueous solution was 5.4. The temperature of the initial aqueous solution was equilibrated to 58.5 °C. β-D-galactoside galactohydrolase derived from Aspergillus oryzae (ENZECO™ Fungal Lactase Concentrate from Enzyme Development Company) was added to the initial aqueous solution to a concentration of 277

LU/g lactose. The initial aqueous solution was incubated with the β-D- galactoside galactohydrolase derived from Aspergillus oryzae for 1 5 minutes under constant agitation. The β-D-galactoside galactohydrolase was then deactivated by adjusting the pH to about 2.0 with HCI. The resulting intermediate solution comprising of GOS, glucose, galactose, and unreacted lactose, was analyzed by HPLC to ensure that the lactose concentration was reduced to less than 60% of the initial concentration of lactose in the initial aqueous solution (see Table 5).

The intermediate solution was diluted to 50BRIX. The pH was adjusted to about pH 9.3 with 50% KOH. Magnesium chloride hexahydrate (25g) was added and the pH adjusted to 6.80 using 50% citric acid. β-D-galactoside galacto hydrolase derived from Kluyveromyces lactis (ENZECO™ Lactase NL 2.5x from Enzyme Development Company) was then added at a dosage of 40.4 LU/g lactose. The temperature of the solution was adjusted to 40 ^. The intermediate solution was incubated with β-D-galactoside galacto hydrolase derived from Kluyveromyces lactis for 100 minutes under constant agitation. The β-D-galactoside galactohydrolase derived from Kluyveromyces lactis was then deactivated by adjusting the pH to about 3.0 with HCI.

Table 5. % Composition of sugars in GOS mixture following primary transgalactosylation of lactose using fungal β-galactosidase from

Aspergillus oryzae

The resulting final aqueous solution comprising of GOS, glucose, galactose and unreacted lactose, was analyzed by HPLC (see Table 6). Table 6. % Composition of sugars in GOS mixture following secondary transgalactosylation of lactose using yeast β-galactosidase from

Kluyveromyces

EXAMPLE 5

Edible crystalline lactose (Lynn Proteins, Inc., Granton, Wl) was dissolved in water at 90 °C to a final concentration of 45 ^e Bx to produce an initial aqueous solution of lactose. The temperature of the initial lactose solution was equilibrated to about 53.5 ^e C, and the pH was adjusted to between 4.5 and 5.0 using HCI. Fungal β-D-galactoside galactohydrolase derived from Aspergillus oryzae (ENZECO™ Fungal Lactase Concentrate from Enzyme Development Company) was then added to the initial aqueous solution to a concentration of 5.6 LU/g of lactose. The solution was incubated 17 hours under constant agitation to produce an intermediate aqueous solution comprising lactose at a concentration about 40% of the initial concentration of lactose in the initial aqueous solution. The fungal β-D-galactoside galactohydrolase was then deactivated by adjusting the pH to about 2.0 with HCI using a 15% w/w aqueous solution of HCI. After 60 minutes of steady agitation, a 50% w/w solution of KOH was slowly added to the intermediate aqueous solution, thereby adjusting the pH to about 9.30. A 25% w/v solution of magnesium chloride hexahydrate was then added to a concentration of 0.16% w/w of the intermediate aqueous solution, thereby adjusting the pH to about 9.21 . Then, a 50% solution of citric acid was slowly added to the intermediate aqueous solution until the pH reached about 6.8. The intermediate aqueous solution was then equilibrated to 36.5 ^e C. Yeast β-D-galactoside galactohydrolase derived from Kluyveromyces lactis (ENZECO™ Lactase NL 2.5x from Enzyme Development Company) was then added to a concentration of 4.7 LU/g of lactose to the intermediate aqueous solution. The intermediate aqueous solution was incubated for 17h under steady agitation to produce a final aqueous solution comprising lactose at a concentration less than 20% of the lactose concentration in the initial aqueous solution. The pH of the final aqueous solution was adjusted to pH 5.5 with citric acid to deactivate the β-D- galactoside galactohydrolase derived from Kluyveromyces lactis. The final aqueous solution was then heat treated at 72 ^e C for 15 seconds. The carbohydrate composition of the final aqueous solution from five trials is provided in Table 7. The final aqueous solution was subjected to ion exchange purification to remove the salts, lactase enzymes and color components. After ion exchange, the partially purified solution was subjected to a purification step to enrich the GOS fraction. The carbohydrate composition of the GOS syrup final aqueous solution from five trials is provided in Table 7.

Table 7. Carbohydrate composition of GOS syrup produced using a combination of lactases from Aspergillus oryzae and Kluyveromyces lactis prior to chromatographic separation.

% w/w of the total carbohydrate

Name Stage 1 (t=17h) Stage 2 (t=1 7h) Enrichment

DP6 0.450 0.396 0.683

DP5 1 .973 1 .743 2.946

DP4 6.743 6.638 1 1 .133

DP3 18.770 1 8.851 32.972

DP2 4.069 1 2.652 18.216

Lactose 40.792 8.830 14.891

DP2 + Lactose 44.861 21 .482 88.1 07

Glucose 20.581 33.526 17.346

Galactose 6.622 1 7.364 1 .814

TOTAL GOS 32.006 40.280 65.950 EXAMPLE 6

Edible crystalline lactose (Lynn Proteins, Inc., Granton, Wl) was dissolved in water at 95 °C to a final concentration of 53 ^e Bx to produce an initial aqueous solution of lactose. The temperature of the initial lactose solution was equilibrated to about 55-56.5 ^e C, and the pH was adjusted to between 4.5 and 5.5. Fungal β-D-galactoside galactohydrolase derived from Aspergillus oryzae (ENZECO™ Fungal Lactase Concentrate from Enzyme Development Company) was then added to the initial aqueous solution to a concentration of 5.8 LU/g of lactose. The solution was incubated 1 1 hours under constant agitation to produce an intermediate aqueous solution comprising lactose at a concentration about 40% of the initial concentration of lactose in the initial aqueous solution, and DP2 sugar at 49% to 52% of total sugar. Thus, increasing the initial lactose concentration and enzyme concentration reduced the required reaction time from 17 h to 1 1 h. The fungal β-D-galactoside galactohydrolase was then deactivated by adjusting the pH to about 2.0 with HCI using a 15% w/w aqueous solution of HCI. a 20% w/w solution of KOH was slowly added to the intermediate aqueous solution, thereby adjusting the pH to about 9.30. A 25% w/v solution of magnesium chloride hexahydrate was then added to a concentration of 0.1 6% w/w of the intermediate aqueous solution, thereby adjusting the pH to about 9.21 , and essential ions were added to the second enzymatic reaction. Then, a 20% solution of citric acid was slowly added to the intermediate aqueous solution until the pH reached about 6.8. The intermediate aqueous solution was then equilibrated to 36.5 ^e C. Yeast β-D-galactoside galactohydrolase derived from Kluyveromyces lactis

(ENZECO™ Lactase NL 2.5x from Enzyme Development Company) was then added to a concentration of 4.4 LU/g of lactose to the intermediate aqueous solution. The intermediate aqueous solution was incubated for 17h under steady agitation to produce a final aqueous solution comprising lactose at a concentration less than 20% of the lactose concentration in the initial aqueous solution, and DP2 sugar at 23.5% to 25% of total sugar). The final aqueous solution was then heat treated at 72 ^e C for 15 seconds to deactivate the β-D- galactoside galactohydrolase derived from Kluyveromyces lactis. The carbohydrate composition of the final aqueous solution from ten trials is provided in Table 8.

The final aqueous solution was subjected to ion exchange purification to remove the salts, lactase enzymes and color components. After ion exchange, the partially purified solution was subjected to a purification step to enrich the GOS fraction. The carbohydrate composition of the GOS syrup final aqueous solution from five trials is provided in Table 8.

Table 8 Carbohydrate composition of GOS syrup produced using a combination of lactases from Aspergillus oryzae and Kluyveromyces lactis prior to chromatographic separation.

While specific embodiments of the invention have been described and illustrated, such embodiments should be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims.