NZ717489B2 - Production of galacto-oligosaccharides. - Google Patents

Abstract

The invention relates to the enzymatic preparation of galacto-oligosaccharides (GOS). Provided is a method for preparing GOS from lactose, comprising (i) contacting a lactose feed with immobilized beta- galactosidase (EC 3.2.1.23) and (ii) allowing for GOS synthesis, wherein said lactose feed is an aqueous slurry of crystalline lactose. aqueous slurry of crystalline lactose.

Description

Title: Production of galacto-oligosaccharides.

The invention relates to methods for preparing galacto- oligosaccharides (G08). G08, also known as oligogalactosyllactose, oligogalactose, oligolactose or transgalactooligosaccharides (TOS), is an important food ingredient [1]. Because of its indigestible nature, GOS belongs to the group of prebiotics. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by ating the growth and/or activity of beneficial ia in the colon. The ability of GOS, when added to infant milk as, to replicate the genic effect of human milk, not only in bacterial numbers, but also with respect to the metabolic activity of the colonic microbiota, has significantly increased interest in their production and application in various food and pharmaceutical processes. For example, GOS occurs in commercial available products such as food for both infants and adult, ranging from infant formula to food for the critical ill.

As its name suggests, the G08 sis typically involves a number of galactosyl transfer processes catalyzed by B-galactosidase (8-D- galactohydrolase; EC 3.2.1.23), which uses lactose as galactosyl donor and lactose or the intermediate GOS species as osyl or.

The underlying mechanism is depicted in Scheme 1. In short, the enzyme and the galactosyl donor (lactose, B-D-Galp-(1,4)-B-D-Glcp) initially form a transient galactosyl-enzyme complex (En--Galp) and subsequently a true nt osyl-enzyme complex (En-Galp),which releases a glucose ule (B-D-Glcp) from the complex. Then, another galactosyl acceptor (lactose, B-D-Galp-(1,4)-B-D-Glcp or other GOS species) binds to the complex in the active center and performs a nucleophilic attack on this osyl- enzyme complex (En-Galp) generally with its non-reducing end (galactosyl moiety of lactose), thus resulting in the formation of a DP3 GOS species, which is expressed generally as lp-(lan)—B-D-Galp-(1,4)-B-D-Glcp products, where the n represents the position of the glycoside linkage, which can be 2,3,4, or 6, depending on the source of the enzyme and the applied reaction conditions.

Scheme 1 Schematic depiction of the GOS synthesis mechanism by B- galactosidase-catalyzed elongation of lactose by B-l,4 glycosidic linkages.

As the reaction progresses (see Scheme 1), more and more galactosyl moieties are erred to the existing GOS s, leading to the formation of GOS species with different degree of polymerization (DP).

Therefore, the GOS can be generally expressed as (Gal)m-Glc (where m varies usually n 2-9, depending on the source of the enzyme and the reaction conditions applied). Apart from the 2DP3 GOS species, new DP2 species such as allolactose are usually considered as the primary product of the alactosylation action of B-galactosidase. These new DP2 species formed by the transgalactosylation of B-galactosidase can r take part in the formation of higher DP GOS species, thus complicating the GOS product spectrum, as discussed below.

Although the enzyme B-galactosidase has us applications in the food and dairy industries, the moderate stability of enzyme is one of the limitations that hinder general implementation of biocatalysts at an industrial scale. Studies to explore their full potential as catalyst have resulted in various le strategies for enzyme ization. For example, the enzyme has been immobilized by s methods such as physical absorption, entrapment, and covalent binding method on different supports. Immobilization has also shown to have an advantageous effect on reducing production inhibition of B-galactosidase. Still further, enzyme immobilization facilitates enzyme reusability and continuous operation of the G08 synthesis process.

It is well known that ctosidase-catalyzed GOS synthesis is kinetically controlled [2]. This means that the actual amount of galacto- oligosaccharide formed at a certain point in time depends largely on the relative rates of the desired synthetic reactions versus and the undesired hydrolytic reactions of lactose and /or GOS. This, in turn, depends not only on the e tration but also on the G08 species that are formed, and moreover, on the interaction of the enzyme with the products resulting from the synthesis and the hydrolysis.

Thus, the s GOS species can be used not only as acceptors for further GOS synthesis but also as ates for hydrolysis. The hydrolytic reaction will dominate generally at the later stages of the reaction when the peak in GOS tration has passed and the main substrate lactose concentration in the reaction mixture is decreased to a low value. On the other hand, production inhibition may also occur, when more and more products accumulate, resulting in either the inhibition of GOS sis or GOS hydrolysis.

Various factors are know in the art to increase the yield of GOS synthesis from lactose using B-galactosidase. See Torres et al. for a review on the production, the properties and the applications of GOS. The reaction conditions for transgalactosylation should be high lactose concentration, elevated temperature, and low water activity in the reaction medium. The temperature, concentration of substrate, and enzyme origin play an ant role in the enzymatic sis of oligosaccharides. However, the influence of the initial lactose concentration can be much larger. Regarding the use of highly concentrated starting lactose solution, it has been shown that maximum GOS yield is largely influenced by initial lactose concentration mainly, until the concentration range is 30% to 40% (w/v). In general, more and larger galacto-oligosaccharides (GOSs) can be produced with higher initial lactose concentrations. Since lactose solubility is relatively low at room temperature but manifestly increases with increasing temperature, high temperatures are generally desired.

The higher temperatures can be beneficial in higher oligosaccharide yields. The higher yield at higher temperatures is an additional advantage when operating at high initial lactose concentrations and, consequently, elevated atures. On the other hand, the potentials of immobilized beta-galactosidase for the synthesis of GOS, e.g. reducing the cost bution of the enzyme by efficient recycling, easy g and process control and ng a continuous process have been widely recognized.

However, the industrial use of immobilized beta-galactosidase for the production of GOS has been not reported yet. In the example of the study by a et al., it was shown that immobilized B. circulans B-galactosidase was recyclable, when performing the on at 60°C in a 50% (w/w) lactose solution using a high enzyme dosage (~18% of the lactose (w/w)) under repeated batch operation.

Consequently, GOS synthesis is generally preferred to be med using immobilized enzyme under conditions with a high l lactose concentration in order to achieve a high GOS yield and to reduce the hydrolytic side reaction [3,4,5].

However, the present inventors found that the results ed from optimization studies performed in a research setting do not always provide a good indicator for GOS synthesis at an industrial scale, e.g. in food industries, wherein relatively low enzyme concentrations (and/or a smaller volume of immobilized enzyme) and long (>20 hours) incubation periods that are typically used for economical reasons, in order to achieve a high productivity and a high space-time yield. In particular, they observed that the use of a highly concentrated (55% w/w) lactose solution as lactose feed for GOS synthesis at 60°C in a repeated batch operation resulted in a loss of about 95% of the initial activity LU of the lized enzyme after the second cycle of operation.

Therefore, they set out to develop improved reaction conditions for GOS synthesis at an industrial scale which allows for repeated re-usage of immobilized enzyme while retaining substantial enzyme activity and without sacrificing a high GOS yield and a cost-effective ion.

It was surprisingly found that the above goals could be met by using a slurry of crystalline lactose d of a highly concentrated solution of dissolved lactose as a lactose feed. More specifically, 20% of the initial LU enzyme activity (measured as LU as defined below) was retained after the third reaction cycle when a lactose slurry of 55% w/w was incubated with immobilized enzyme at 58°C for 24 hours. The final GOS content was above 60% (dm) during the initial six batches. Without wishing to be bound by any theory, it is speculated that a conventional highly concentrated e solution prepared by complete dissolution at high ature, is to be considered as a "meta stable" solution,) that will undergo recrystallization at a lower reaction ature during ged incubation s., This causes lactose crystal formation on the surface and in the pores of the enzyme carrier, presumably leading not only to a reduced accessibility but also to inactivation (denaturation) of the immobilized . Surprisingly, this can be avoided by using a sion of "pre-crystallized" lactose n the lactose crystals function as a substrate pool and the dissolved lactose can freely access the pores of the enzyme carrier. Thus, the enzyme denaturation can be retarded by the use of ‘’pre-crystallized’’ lactose, without the need to dissolve lactose completely by ating the crystalized lactose to a higher temperature, thus resulting in a sustainable industrial process.

Accordingly, the invention provides a method for preparing galactooligosaccharides (GOS) from lactose, comprising contacting a lactose feed with β-galactosidase which is immobilized, preferably on a solid r, and allowing for GOS synthesis, wherein said lactose feed is an aqueous slurry of crystalline lactose.

SUMMARY OF THE INVENTION In a first aspect of the invention there is provided a method for ing galacto-oligosaccharides (GOS) from lactose, comprising (i) dissolving e crystals in an aqueous phase at room temperature to provide an aqueous slurry of crystallized lactose that ns at least 53% (w/w) lactose (ii) g said aqueous slurry of crystallized lactose from room temperature to a temperature of 20-60°C (iii) contacting said heated aqueous slurry of crystallized lactose with Bacillus circulans beta-galactosidase immobilized on a porous carrier; and (iv) allowing for GOS synthesis, thereby preparing GOS from lactose.

As used herein, the term ‘’aqueous slurry’’ refers to a watery e (suspension) of insoluble e crystals i.e. a composition wherein not all lactose is dissolved and wherein the soluble lactose concentration is equal to its solubility at a given reaction ature. As indicated above, lactose solubility is strongly dependent on the temperature. Figure 1 shows the solubility-temperature on for lactose in water at neutral pH.

Typically, the aqueous lactose slurry for use in the present invention contains 17%-75% (w/w) lactose. In practice, a high lactose content in the slurry may result in a concentrated product in the reactor that requires less energy to further concentrate to obtain the finished GOS syrup of 75% (w/w). ore, both for economical reason and very good GOS yields, the lactose slurry is referred to contain 50-70 % (w/w) lactose in total. For example, the aqueous lactose slurry contains at least 53% (w/w), preferably at least 55% (w/w) lactose. By increasing the total dry matter content of the reaction e without sacrificing enzyme ity, the method of the invention provides a very high productivity and GOS output per reactor volume.

[Followed by page 7] A slurry for use in the present ion is easily prepared by adding crystalline e (monohydrate) to an aqueous liquid. The skilled person will be able to calculate the lactose concentration needed to obtain a slurry at a given temperature based on the lactose solubility curve. For example, the following empiric equation can be used to calculated soluble lactose at a given reaction temperature (T): solubility of lactose =0.003*TA2+0.2713*T+9.778 .

Accordingly, the lactose solubility at 58°C,was calculated to be 35.6% (w/w).

Therefore, the percentage insoluble lactose crystals will be the e concentration subtracted by the lactose solubility at 58°C. In this way, the percentage of the insoluble lactose crystals present in reaction system is calculated to be 19.4% (55% minus 35.6%) (w/w) Typically, a suitable amount of lactose is added directly to a buffer solution at room temperature and heated to a desired reaction temperature.

It is also possible to add lactose to water, followed by adjusting the pH of the lactose slurry to a working pH range of about pH3.5 - 7.5 that is within the enzyme activity range for example by adding caustic (NaOH) or a buffer solution.

The d people will realize that the optimum pH of individual immobilized beta-galactosidase depends on its source and the type of immobilization methods and that it might decrease during the enzymatic conversion. uently, the pH may need to be adjusted during the enzymatic conversion, e.g. by stepwise adding a caustic solution or via a pH stat mode.

As mentioned above, in st to conventional methods employing highly concentrated lactose solutions, a method of the ion does not require extensive heating of lactose to completely dissolve the lactose. This not only s the energy consumption and thus operational costs that are associated with the heating step, but also avoids the undesirable color change of lactose solution to a more yellowish ance.

However, practically, it might be also desired to dissolve the lactose completely, in order to remove the insoluble protein aggregates or insoluble mineral salt such as calcium phosphate or calcium citrate that may be t in the lactose ls. Therefore, it is of course also possible to prepare a lactose slurry by heating a lactose feed to a high temperature (T1) to dissolve lactose and remove the insoluble particles as mentioned above and cooling down to the desired reaction temperature (T2) to let dissolved lactose crystalize out. As shown herein below, the heating of a completely solubilized lactose solution of >58% (w/w) followed by g to a ature of about 60°C resulted in the spontaneous crystallization of The formation of good crystals and crystal growth are critical to the extraction and purification of lactose. d-lactose crystallizes from super- saturated solutions at temperatures below 935°C to produce a variety of crystal shapes. The usual ones obtained le prism and tomahawk shapes, and are hard and only gly soluble. Above 935°C, B-lactose crystallises out, usually as an uneven-sided diamond. A molecule of water is associated with the crystalline (1- form of lactose and so it is referred to as the monohydrate. At temperatures over 120°C and under vacuum, however, this water is lost and the highly hygroscopic d-lactose ide is formed.

When lactose is dissolved in water, mutarotation occurs, ie. (1- and B- anomers interconvert to produce a solution of 62.7% B-lactose at 20°C. As (1- lactose is the far less soluble s, concentration of the solution results in d-lactose precipitating and further mutarotation takes place to maintain the same equilibrium on.

A method of the invention preferably comprises use of food grade or pharmaceutical grade lactose, or d lactose that is n the food and pharmaceutical grade lactose. Food grade lactose is produced by concentrating whey or permeate (a co-product of whey protein concentrate production) to supersaturated solution and lactose crystalizes out, then removing and drying the e crystals. Special processes of crystallization, as well as grinding and fractionated sifting, e types of lactose which differ in particle size distribution. Today, the ry offers several types of lactose ranging from superfine to extra coarse crystals for all applications. The lactose content is not less than 99%, with the sulfated ash content not more than 0.3%, both on a dry basis. The pH of a 10% solution is not less than 4.5 or more than 7.5.

To obtain a refined or a pharmaceutical grade of lactose, refining process is necessary. This involves redissolving the lactose crystals and treating the solution with virgin activated carbon, which absorbs a number of s including riboflavin and a variety of ns. Also absorbed are a group of polypeptides known as proteose peptones which are derived from B- casein. These peptones are produced by the action of plasmin, a protease enzyme which migrates from the bloodstream into the milk in the cow's udder. Further n may be absorbed onto the activated carbon by temporarily adjusting the liquor pH. The carbon is removed by flocculation and tion and then discarded. After crystallisation, uent tion of the crystals by centrifugation and cold water washing and drying, a high purity white pharmaceutical grade lactose is obtained. The crystals are milled or sifted to yield products with specific particle size distributions. ing contacting the lactose slurry with a ation comprising immobilized beta-galactosidase, the resulting reaction mixture is incubated under conditions favoring GOS synthesis. The skilled person will understand that the selected reaction temperature should both favor the G08 synthesis and the enzyme stability. ore, the optimum temperature is the temperature at which the immobilized enzyme can be recycled in economically optimal manner, for instance, disposing the immobilized enzyme when reaching the half-life of the immobilized enzyme at the individual ed immobilized beta-galactosidase. Typically, GOS synthesis is performed at a reaction temperature of between about 20 and about 600C. In one ment, the reaction is carried out at C, preferably 45-550C, like at about 500C.

The reaction mixture is incubated until the d amount of GOS has been obtained. Depending on the tion conditions and the amount of the immobilized enzyme used, GOS synthesis according to the invention is generally allowed to proceed from 0.5-100 hours. However, industrial production of GOS lly es a rational integration with other units of operation ed, such as purification, demineralization, decoloration and the like. Thus, GOS synthesis at an industrial scale is preferably performed during a reaction period of at least 6 or 10 hours, preferably 12- 36 hours for practical and economical reasons. For example, due to the high GOS yield and increased enzyme stability, a method of the invention is suitably practiced using an incubation time of about 18-24 hours. This allows for flexible accommodation in an employee working day while at the same time obviating the need for high amounts of enzyme, because a large amount of immobilized enzyme in the r will reduce the productivity and space-time yield For example, the dose of the immobilized beta-galactosidase can be used in an amount of up to 30 LU / gram initial lactose, preferably up to 25 LU/gram, more preferably in an amount of between about 10 and 20 LU/gram. As used herein, one lactase unit (LU) is defined as the quantity of enzyme that liberates 1 11mole of glucose per minute at the early stage of the reaction at 40°C, pH 6.0. When e is hydrolysed by lactase, it is converted into glucose and galactose. The lactase activity is determined by measuring the amount of liberated glucose.

WO 34356 There are several manners to perform GOS synthesis using a lactose "slurry" reaction as disclosed in the present invention. In a first embodiment, the slurry is placed in a separate r and the insoluble lactose crystals are retained by a microfilter. Then, the soluble lactose is pumped to a packed bed reactor (PBR) where the immobilized beta- galactosidase is located. The outlet of the PBR is ed back to the reactor. In this way, after n time, the lactose will be completely dissolved and high GOS concentrations can be obtained.

In a second ment, lactose crystals are gradually added to the G08 solution. Lactose is dissolved and pumped to the PBR and the dissolved lactose will be converted to GOS.

In a third embodiment, not all the insoluble lactose crystals are added at once such that no difficulties with stirring the on mixture are encountered. For example, e crystals are directly added to an existing reaction mixture to replenish the lly present lactose crystals that have been dissolved and completely converted to GOS. Upon the visual detection of disappearance/dissolution of lactose crystals as the reaction proceeds, more lactose crystals can be added to continue the on until high, e.g. up to 75%, GOS solution can be obtained.

As will be understood, a method of the invention can be practiced using alactosidase from a wide variety of sources, such as microorganisms, plants and animals. Micro-organisms like bacteria, fungi and yeast are considered the preferred sources of beta-galactosidase for industrial applications. See ref Panesar 2010 for an overview of suitable microbial sources. Preferably, beta-galactosidase is isolated from a micro-organism selected from the group consisting of A. oryzae, A. niger, B. circulans, S. singularis, T. aquaticus, K. lactis, K. marxianus and E. coli. Some of the enzymes have high specificity to synthesize oligosaccharides of specific chain length and orientation of the linkage. For e, B-galactosidase WO 34356 sourced from B. circulans shows high specificity to B-l,4 es and in turn yields mainly B-l,4 linked osyl accharides (GOS) by transglycosylation, while B-galactosidase sourced from Aspergillus oryzae gives B-l,6 GOS mainly. Very good results were obtained with B. circulans beta-galactosidase, which is available from Daiwa Kasei, Amano, Japan in the form of the commercial enzyme preparation, Biolacta®N5.

A method of the invention is characterized by incubating a lactose slurry with immobilized beta-galactosidase. Various ways of enzyme immobilization are known in the art. They typically comprise a porous carrier onto which the beta-galactosidase is immobilized via covalent binding, via physical absorption (charge-charge or van der waals interaction), via gel encapsulation or a combination thereof. Table 2 of ref Panesar 2010 gives an overview of different sources of beta-galactosidase and methods of immobilization. Besides, the carrier-free immobilized enzymes such as CLEC (cross-linked enzyme crystals) or CLEAs - linked enzyme aggregates) might be also applied [6,7].

The invention is not limited to any type of enzyme immobilization.

However, carriers that can promote direct covalent binding of the enzyme are preferred, regarding the ease of operation and no leakage of the enzyme molecules into the reaction mixture.

As is shown herein below, good results were obtained with beta- galactosidase immobilized by nt binding to a solid r. Preferably, the solid carrier is an ted acrylic polymer, preferably a onalized polymethacrylate matrix. For example, a hexamethylenamino — functionalized polymethacrylate matrix eads) or a macroporous acrylic epoxy-activated resin, like Eupergit C 250L, can be used.

After the reaction has proceeded to a desired level, GOS synthesis can be terminated by methods known in the art. For instance, the immobilized beta-galactosidase is physically separated from the remainder of the reaction mixture by filtration or by retaining the les of the immobilized enzymes by a sieve installed on the bottom of the reactor.

As explained herein above, a method of the invention is advantageously used in a repeated batch operating system involving l utive batches ("cycles") of GOS synthesis. Furthermore, a method of the invention allows for the recycling of immobilized enzyme during several batches since detrimental effects of lactose crystallization during the G08 synthesis reaction are avoided by the use of a lactose slurry as lactose feed.

This s semi-continuous operation and multiple reuse of the enzyme.

Accordingly, in one embodiment a method further comprises, ing a first cycle of GOS synthesis, the steps of : (a) washing the immobilized alactosidase, (b) optionally storage of the washed immobilized beta-galactosidase until further use; and (c) one or more uent cycles of GOS synthesis by contacting the washed immobilized beta-galactosidase of step (a) with a lactose slurry such that the enzyme is recycled.

Prior to enzyme washing, it is typically physically separated from the GOS-containing reaction mixture. For example, if the enzyme used for GOS synthesis is used in a ag"-like pouch, the tea-bag can simply be taken out of the reaction mixture. Alternatively, GOS can be removed from the reactor while the immobilized enzyme remains in the reactor during washing.

For example, the enzyme is washed several times with demineralized water and/or the same buffer used in the G08 sis on.

Immobilized enzyme can be sanitized e.g. to reduce the microbial count, by washing with an acetic acid solution of about pH 4.5. The enzyme may be stored in a buffer at a temperature below 10°C, preferably at around 4°C.

Suitable buffers include those in the range of pH 5.5-7 .5. For example, the enzyme is stored in 0.1M K2HPO4 / KH2PO4 buffer, pH 6.0 — 7.0 at 4°C, prior to reuse. In one embodiment, a method of the invention comprises at WO 34356 least 5, preferably at least 8, more preferably at least 10 cycles of GOS synthesis employing recycled immobilized beta-galactosidase.

In a further aspect, the invention provides a composition comprising an aqueous slurry of crystalline e and comprising beta-galactosidase which is immobilized on a solid carrier. Preferred lactose concentrations, enzyme sources and enzyme concentrations are disclosed herein above.

LEGEND TO THE FIGURES Figure 1: Lactose solubility and temperature curve.

Figure 2: ison of activity retention of immobilized beta-galactosidase EC-HA catalyzed GOS synthesis using a lactose slurry (I) or a e on (0) as lactose feed.

MENTAL N The examples herein below exemplify the advantageous effects of using a lactose slurry instead of a highly concentrated lactose solution in the manufacture of GOS using immobilized beta-galactosidase.

Example 1 (Comparative example): Synthesis of GOS with immobilized fi—galactosidase from Bacillus circulans using a lactose solution (55%) Experimental conditions: 35 gram lactose was added to 25 gram 0,1M K2HPO4 / KH2PO4 buffer, pH 6.3 and was completely dissolved at 95 °C then cooled down to 58°C.

Afterward, 2.7 gram carrier-bound enzyme immobilized on commercial carrier (Sepabeads EC-HA) via glutaraldehyde coupling with a specific activity of 131.16 LU/gram immobilized enzyme was added to initialize the enzymatic on. The enzyme dosage is 10.2 LU/gram lactose.

After 24 hours reaction time, the G08 was filtrated and the immobilized enzyme was washed with demineralized water and stored in 0.1M K2HPO4 / KH2PO4 buffer, pH 6.0 — 7.0 at 4 °C, prior to reuse.

Carbohydrates (galactose, glucose, lactose and GOS) were ed as previously described by Coulier et al. (J. Agric. Food Chem. 2009, 57, 8488— 8495) and Warmerdam et al. (Appl Biochem Biotechnol (2013) 0—358).

The remaining enzyme activity and the G08 yield after the first batch and second batch was summarized in Table 1. Notably, the remaining activity of the immobilized enzyme was only 5.4% of the l LU activity, suggesting that the immobilized enzyme was quickly denatured.

Table 1 A summary of GOS synthesis using immobilized beta-galactosidase in a completely lized lactose solution.

Immobilized Time Activity eNILzyme/batch [h] GOS and sugar composition 2:332:31 [% on dm] batch Galactose Glucose Lactose GOS EC-HA— 24 2.86 23.71 16.02 57.41 26.1 Batch 1 EC-HA— 24 1.39 21.27 28.13 49.21 5.4 Batch 2 Example 2: Synthesis of GOS with immobilized fi—galactosidase from Bacillus circulans in a lactose slurry (55% w/w) reaction system of the invention.

Experimental conditions: 70 gram lactose was added directly to 51 gram 0,1M K2HPO4 / KH2PO4 buffer, pH 6. and subsequently the reaction mixture was heated to the reaction ature 58 °C (and maintained for at least 1 hour) and 12 gram carrier-bound enzyme immobilized on commercial carrier (Sepabeads EC-HA) Via glutaraldehyde coupling with a c activity of 141.8 LU/gram immobilized enzyme) was added to initialize the enzymatic reaction. The enzyme dosage is 15.2 m lactose.

After 24 hours reaction time, the G08 was filtrated and the lized enzyme was washed with demineralized water and stored in 0.1M K2HPO4 / KH2PO4 buffer, pH 6.0 — 7.0 solution at 4 °C, prior to reuse.

Table 2 A summary of synthesis of GOS with immobilized B-galactosidase from Bacillus circulans in lactose slurry (55 % w/w) in a consecutive mode Immobilized Reaction LU /batch time G08 and sugar c0mp0s1t10n_ _ Activity N0. retention after [% on dm] batch Batch 3 66.94 19.6 Batch 4 67.03 11.7 Batch 5 61.59 10.6 Batch 6 54.59 7.3 Batch 7 53.16 7.0 Batch 8 48.93 3.8 Example 3 (comparative example): Synthesis of GOS with free [5— galactosidase from Bacillus circulans using a lactose slurry (65%, w/w) reaction system.

Experimental conditions: 85 gram lactose was added directly to 65 gram 0,1M K2HPO4 / KH2PO4 buffer, pH 6. and subsequently the on mixture was heated to the reaction temperature 58 °C (and maintained for at least 1 hour) and 85 mg free alactosidase of Biolacta N5 )dissolved in 2 ml demi water was added to initialize the reaction. The enzyme dosage is 5 LU/gram lactose.

The on mixture was incubated in a water batch with orbit shaker.

After 24 hours reaction, the G08 content in the final reaction mixture was only 40% and there was still a lot of insoluble lactose left in the reaction mixture. This suggests that, in such a high lactose concentration slurry system, the free enzyme was less active than immobilized enzyme.

Example 4: Synthesis of GOS with immobilised fi—galactosidase from us circulans (carrier-epoxy Eupergit C 250 L) in lactose slurry (65%, w/w) reaction system 8 gram immobilized B-galactosidase 0f Biolacta N5 0n Eupergit C 250 L was added to 200 gram lactose slurry with 20 mM potassium citrate buffer, pH7.0 and incubated at 60°C. The reaction mixture was stirred with a magnetic stirrer. The enzyme dosage was 7 LU/gram lactose.

After 24 hours, it was found that reaction mixture was completely clear and the G08 t was 57%.

This result suggests that a combination of high slurry concentration and immobilized enzyme is ideal for GOS synthesis at an industrial process; (i) there is no need to dissolve lactose completely, and (ii) less energy consumption for the concentration of the final product, when the concentration of the t from the reactor is also close to the tration of the final product (GOS syrup, 75% (w/w)).

REFERENCES 1. Rivero-Urgell M and Santamaria-Orleans A (2001) Oligosaccharides: application in infant food. Early Human Development 65 Suppl. S43—S52 2. Torres D, Goncalves M, Teixeira J, Rodrigues L: Galacto- oligosaccharides: production, properties, applications, and significance as prebiotics. Compr Rev Food Sci, Food Safety 2010, 9:438—454. 3. Urrutiaa P, Mateob C, Guisan on, Illanes LA (2013)Immobilization of Bacillus circulans B-galactosidase and its application in the synthesis of o-oligosaccharides under repeated- batch operation. Biochemical Engineering Journal 77:41— 48 4. Panesar PS, Panesar R, Singh RS, Kennedy JF and Kumar H (2006) Microbial production, immobilization and applications of B-D-galactosidase.

Journal of Chemical logy and hnology J Chem Technol Biotechnol 8 1530—543 5. Grosova Z., Rosenberg M., Rebros M. (2008) Perspectives and applications of immobilised B-galactosidase in food industry — a review.

Czech J. Food Sci., 26: 1—14. 6. Gaur R, Pant H, Jain R, Khare SK: Galacto-oligosaccharide sis by lized B-galactosidase. Food Chem. 2006, 97, 426—430. 7. Cao LzImmobilized s. In Comprehensive Biotechnology (Second Edition) 2011, 2:461-476.

Claims (19)

1.Claims

2. A method for preparing galacto-oligosaccharides (GOS) from e, comprising 5 (i) dissolving lactose ls in an aqueous phase at room temperature to provide an aqueous slurry of crystallized lactose that contains at least 53% (w/w) lactose (ii) heating said aqueous slurry of crystallized lactose from room temperature to a temperature of 20-60°C 10 (iii) contacting said heated aqueous slurry of crystallized lactose with Bacillus circulans beta-galactosidase immobilized on a porous carrier; and (iv) allowing for GOS synthesis, thereby preparing GOS from e. 15 2. Method according to claim 1, wherein the aqueous e slurry contains 50-70% (w/w) lactose.

3. Method ing to claim 2, wherein the lactose is food grade or pharmaceutical grade lactose.

4. Method according to claim 2, wherein the aqueous lactose slurry contains at least 55% (w/w) lactose.

5. Method according to any one of claims 1-3, wherein the pH of the 25 lactose slurry is pH 3.5 - 7.5.

6. Method according to any one of the preceding claims, wherein GOS synthesis is performed at a reaction temperature of between 40 and 60ºC.

7. Method according to any one of the preceding , wherein GOS synthesis is med during a reaction period of at least 6 hours.

8. Method according to claim 7, wherein the GOS synthesis is performed 5 during a reaction period of 12-36 hours.

9. Method according to any one of the preceding claims, wherein said immobilized beta-galactosidase is used in an amount of up to 30 lactase unit (LU)/gram initial lactose.

10. Method according to claim 9, wherein said immobilized betagalactosidase is used in an amount of up to 25 LU/gram.

11. Method according to claim 10, wherein said immobilized beta- 15 galactosidase is used in an amount of between about 10 and 20 LU/gram.

12. Method according to any one of the preceding claims, wherein betagalactosidase is immobilized on the porous carrier, via covalent 20 binding, via a charge-charge interaction or via gel encapsulation.

13. Method according to claim 8, wherein the beta-galactosidase is immobilized on an activated acrylic polymer r. 25

14. Method ing to claim 13, wherein the beta-galactosidase is immobilized on a functionalized polymethacrylate matrix.

15. Method ing to claim 14, wherein the alactosidase is immobilized on a hexamethylenamino-functionalized 30 polymethacrylate matrix or a macroporous acrylic epoxy-activated resin.

16. Method according to any one of the preceding claims, further sing, following a first cycle of GOS synthesis, the steps of : 5 (a) washing the immobilized beta-galactosidase with water and/or buffer, (b) optionally storage of the washed lized beta-galactosidase until further use; and (c) at least one or more subsequent cycles of GOS synthesis by 10 contacting the washed immobilized beta-galactosidase of step (a) with a lactose slurry such that the immobilized enzyme is recycled.

17. Method ing to claim 16, involving at least 5, preferably at least 8cycles of GOS synthesis employing recycled immobilized beta- 15 galactosidase.

18. Method according to claim 17, involving at least 8 cycles of GOS synthesis employing recycled immobilized alactosidase. 20

19. Method according to claim 18, involving at least 10 cycles of GOS synthesis employing recycled immobilized beta-galactosidase. FrieslandCampina Nederland B.V. By the Attorneys for the Applicant 25 SPRUSON & FERGUSON Per: