EP3402892A1 - Method for producing galactooligosaccharides from lactose - Google Patents
Method for producing galactooligosaccharides from lactoseInfo
- Publication number
- EP3402892A1 EP3402892A1 EP17738066.4A EP17738066A EP3402892A1 EP 3402892 A1 EP3402892 A1 EP 3402892A1 EP 17738066 A EP17738066 A EP 17738066A EP 3402892 A1 EP3402892 A1 EP 3402892A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- aqueous solution
- lactase
- lactose
- initial
- gos
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0036—Galactans; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01108—Lactase (3.2.1.108)
Definitions
- 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.
- GOS galactooligosacchande
- GOS Galactooligosaccharides
- DP degree of polymerization
- GOS can be formed by the digestion of lactose with ⁇ -D-galactoside galactohydrolases.
- ⁇ -D-galactoside galactohydrolases catalyze the hydrolysis of the galactosyl moiety from the non-reducing end of lactose.
- ⁇ -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 ⁇ -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 ⁇ -D-galactoside galactohydrolases utilized.
- 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 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.
- 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 ⁇ -D-galactoside galactohydrolase.
- the fungal ⁇ -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.
- 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 ⁇ -D-galactoside galactohydrolase.
- the yeast ⁇ -D-galactoside galactohydrolase may be derived from a
- Kluyveromyces species The Kluyveromyces species may be Kluyveromyces lactis.
- 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
- 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.
- the method includes deactivating the fungal acid lactase prior to adding the yeast neutral lactase.
- deactivating the fungal lactase comprises adjusting the pH of the intermediate aqueous solution to about 2 or less.
- 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.
- 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.
- 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.
- the fungal acid lactase and the yeast neutral lactase is removed from the final aqueous solution by ion exchange chromatography.
- 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.
- chromatographic separation comprises simulated moving bed chromatography.
- This disclosure further relates to a galactooligosaccharide (GOS) syrup produced according to a method as described above.
- the GOS syrup is at least 40% GOS w/w of the total carbohydrate in the GOS syrup.
- the GOS syrup is at least 65% GOS w/w of the total carbohydrate in the GOS syrup.
- 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 ⁇ -D-galactoside galactohydrolase derived from Aspergillus oryzae in combination with a ⁇ -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.
- GOS galactooligosaccharide
- the ⁇ -D-galactoside galactohydrolase derived from Aspergillus oryzae is for incubation with the aqueous solution prior to incubation of the aqueous solution with the ⁇ -D- galactoside galactohydrolase derived from Kluyveromyces lactis.
- 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 ⁇ -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 ⁇ -D-galactoside galactohydrolase derived from Aspergillus oryzae.
- GOS galactooligosaccharide
- the amount of GOS may be increased to at least 40% w/w of total carbohydrates in the solution.
- the diversity of GOS may be increased.
- This disclosure also relates generally to the use of a ⁇ -D-galactoside galactohydrolase derived from Aspergillus oryzae in combination with a ⁇ -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 ⁇ -D-galactoside galactohydrolase derived from Aspergillus oryzae is for incubation with the aqueous solution prior to incubation of the aqueous solution with the ⁇ -D-galactoside galactohydrolase derived from Kluyveromyces lactis.
- Figure 1 is a flow diagram of a method of producing GOS syrup as disclosed herein in Example 5.
- Figure 3 is a HPLC chromatogram following secondary transgalactosylation of lactose using yeast ⁇ -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.
- Figure 6 is a HPLC chromatogram following secondary transgalactosylation of lactose using yeast ⁇ -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.
- DP 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".
- 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 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 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.
- the acid fungal lactase is a fungal ⁇ -D-galactoside galactohydrolase.
- the ⁇ -D-galactoside galactohydrolase may be derived an Aspergillus species.
- the ⁇ -D-galactoside galactohydrolase is derived from Aspergillus oryzae, such as the ⁇ -D-galactoside galactohydrolase available from Enzyme Development Corporation (New York) as ENZECOTM Fungal Lactase Concentrate.
- ENZECOTM Fungal Lactase Concentrate ENZECOTM Fungal Lactase Concentrate.
- One LU may be defined as that quantity of enzyme which will liberate 1 .0 ⁇ " ⁇ / ⁇ " ⁇ 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.
- 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.
- the source of lactose may vary.
- the lactose can be provided in the form of milk permeate.
- 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.
- the initial concentration of lactose and in an initial aqueous solution will preferably be between about 30°Bx and about 60°Bx.
- the initial concentration of lactose and in the initial aqueous solution is about 45°Bx.
- 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 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,
- ENZECOTM 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 ENZECOTM Fungal Lactase Concentrate for example, has a pH optimum of between 4.5 and 5.0.
- 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.
- ENZECOTM Fungal Lactase Concentrate for example, has a temperature optimum of 55 °C at pH 4.5 and 6.5.
- the initial aqueous solution is incubated with the acid fungal lactase at a temperature between about 50 and about 56.5 °C.
- the initial aqueous solution is incubated with the acid fungal lactase at a temperature between about 50 and about 55 °C.
- the initial aqueous solution is incubated with the acid fungal lactase at a temperature of about 53.5 ⁇ ⁇ .
- 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.
- 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.
- 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.
- this intermediate aqueous solution is incubated with a yeast neutral lactase.
- 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.
- the neutral yeast lactase is a yeast ⁇ -D-galactoside galactohydrolase.
- the ⁇ -D- galactoside galactohydrolase may be derived from a Kluyveromyces species.
- the ⁇ -D-galactoside galactohydrolase is derived from Kluyveromyces lactis, such as the ⁇ -D-galactoside galactohydrolase available from Enzyme Development Corporation (New York) as ENZECOTM 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.
- 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.
- the neutral yeast ⁇ -D-galactoside galacto hydrolase is derived from a Kluyveromyces species, it may be necessary to add potassium and magnesium for enzyme activity.
- the pH and salt can be adjusted using potassium, magnesium chloride, and citric acid.
- ENZECOTM Lactase NL 2.5X has a pH optimum of about 6 to about 7.
- adjusting pH of the intermediate aqueous solution involves adjusting the pH to about 6.8.
- Such pH adjustments can lead to turbidity of the mixture, which can plug downstream separation equipment.
- 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:
- the temperature of the intermediate aqueous solution is adjusted to between 30 and 45 °C prior to addition of the neutral yeast lactase.
- the yeast lactase may perform at a much slower rate outside this range, e.g. between about 4.0 and about 50.0 °C.
- the temperature of the intermediate aqueous solution is adjusted to about 36.5 °C for incubation with the neutral yeast 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.
- 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
- the neutral yeast lactase may be deactivated.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- this disclosure further relates to use of the combination of a first ⁇ -D-galactoside galactohydrolase derived from an Aspergillus oryzae with a second ⁇ -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 ⁇ -D-galactoside galactohydrolase followed by incubation with the second ⁇ -D-galactoside galactohydrolase.
- 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. ⁇ -D-galactoside galactohydrolase derived from Aspergillus oryzae (ENZECOTM Fungal Lactase Concentrate from Enzyme Development Company) was added to the initial aqueous solution to a concentration of 20 LAU/g lactose.
- ENZECOTM Fungal Lactase Concentrate from Enzyme Development Company
- the initial aqueous solution was incubated with the ⁇ -D-galactoside galactohydrolase derived from Aspergillus oryzae for 6 hours under constant agitation.
- the ⁇ -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%
- 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, ⁇ - D-galactoside galactohydrolase derived from Kluyveromyces lactis (ENZECOTM 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 ⁇ -D-galactoside galactohydrolase derived from Kluyveromyces lactis for 10 hours under constant agitation.
- the ⁇ -D-galactoside galactohydrolase derived from Kluyveromyces lactis was then deactivated by adjusting the pH to about 3.0 with HCI.
- 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 ⁇ -galactosidase from
- Demineralized, deproteinized, ultrafiltered milk permeate was evaporated to 35 e Bx, and incubated with ⁇ -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 ⁇ -galactosidase from Aspergillus Oryzae and Kluyveromyces Lactis.
- the initial aqueous solution was incubated with the ⁇ -D- galactoside galactohydrolase derived from Aspergillus oryzae for 1 5 minutes under constant agitation.
- the ⁇ -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.
- ⁇ -D-galactoside galacto hydrolase derived from Kluyveromyces lactis (ENZECOTM 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 ⁇ -D-galactoside galacto hydrolase derived from Kluyveromyces lactis for 100 minutes under constant agitation.
- the ⁇ -D-galactoside galactohydrolase derived from Kluyveromyces lactis was then deactivated by adjusting the pH to about 3.0 with HCI.
- 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 ⁇ -D-galactoside galactohydrolase derived from Aspergillus oryzae (ENZECOTM 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 ⁇ -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 .
- 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 ⁇ -D-galactoside galactohydrolase derived from Kluyveromyces lactis (ENZECOTM 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 ⁇ -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.
- 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 ⁇ -D-galactoside galactohydrolase derived from Aspergillus oryzae (ENZECOTM 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.
- increasing the initial lactose concentration and enzyme concentration reduced the required reaction time from 17 h to 1 1 h.
- the fungal ⁇ -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.
- 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.
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PCT/CA2017/050042 WO2017120678A1 (en) | 2016-01-12 | 2017-01-12 | Method for producing galactooligosaccharides from lactose |
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US8425930B2 (en) * | 2009-03-13 | 2013-04-23 | The Regents Of The University Of California | Prebiotic oligosaccharides |
WO2011093907A1 (en) * | 2010-02-01 | 2011-08-04 | Corn Products International, Inc. | High-purity galactooligosaccharides and uses thereof |
CN101831389B (en) * | 2010-04-19 | 2011-12-07 | 江南大学 | Strain for generating beta-D-galactosidase and method for producing galacto-oligosaccharides with same |
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