Production of galacto-oligosaccharides

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.

Claims

1. A method for preparing galacto-oligosaccharides (GOS) from lactose, comprising (i) dissolving lactose crystals 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 desired reaction temperature of between 20 C.-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.

2. The method according to claim 1, wherein said lactose is food grade or pharmaceutical grade lactose.

3. The method according to claim 1, wherein the pH of the aqueous slurry of crystallized lactose is pH 6.0-7.5.

4. The method according to claim 1, wherein GOS synthesis is performed at a temperature of between 40 C. and 60 C.

5. The method according to claim 1, wherein GOS synthesis is performed for at least 6 hours.

6. The method according to claim 1, wherein said immobilized beta-galactosidase is used in an amount of up to 30 LU/gram initial lactose.

7. The method according to claim 1, wherein said beta-galactosidase is immobilized on the porous carrier via covalent binding, via a charge-charge interaction or via gel encapsulation.

8. The method according to claim 7, wherein the porous carrier is an activated acrylic polymer carrier selected from the group consisting of a functionalized polymethacrylate matrix, a hexamethylenamino-functionalized polymethacrylate matrix or a macroporous acrylic epoxy-activated resin.

9. The method according to claim 1, further comprising, following a first cycle of GOS synthesis, the steps of: (a) washing the immobilized beta-galactosidase with water and/or buffer, (b) optionally storage of the washed immobilized beta-galactosidase until further use; and (c) at least one or more subsequent cycles of GOS synthesis using the washed immobilized beta-galactosidase of step (a) such that the immobilized enzyme is recycled.

10. The method according to claim 9, wherein the one or more subsequent cycles of GOS synthesis is at least 5 cycles of GOS synthesis.

11. The method according to claim 1, wherein the aqueous slurry of crystallized lactose contains at least 55% (w/w) lactose.

12. The method according to claim 5, wherein GOS synthesis is performed for 12-36 hours.

13. The method according to claim 6, wherein said immobilized beta-galactosidase is used in an amount of up to 25 LU/gram initial lactose.

14. The method according to claim 6, wherein said immobilized beta-galactosidase is used in an amount of between 10 and 20 LU/gram initial lactose.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Lactose solubility and temperature curve.

(2) FIG. 2: Comparison of activity retention of immobilized beta-galactosidase EC-HA catalyzed GOS synthesis using a lactose slurry (.square-solid.) or a lactose solution (.diamond-solid.) as lactose feed.

EXPERIMENTAL SECTION

(3) 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 -Galactosidase from Bacillus Circulans Using a Lactose Solution (55%)

(4) Experimental Conditions:

(5) 35 gram lactose was added to 25 gram 0.1M K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 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 reaction. The enzyme dosage is 10.2 LU/gram lactose.

(6) After 24 hours reaction time, the GOS was filtrated and the immobilized enzyme was washed with demineralized water and stored in 0.1M K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer, pH 6.0-7.0 at 4 C., prior to reuse.

(7) Carbohydrates (galactose, glucose, lactose and GOS) were analyzed as previously described by Coulier et al. (J. Agric. Food Chem. 2009, 57, 8488-8495) and Warmerdam et al. (Appl Biochem Biotechnol (2013) 170:340-358).

(8) The remaining enzyme activity and the GOS 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 initial LU activity, suggesting that the immobilized enzyme was quickly denatured.

(9) TABLE-US-00001 TABLE 1 A summary of GOS synthesis using immobilized beta-galactosidase in a completely solubilized lactose solution. Immo- Activity bilized retention enzyme/ GOS and sugar composition after each batch Time [% on dm] batch [%] No. [h] Galactose Glucose Lactose GOS 100 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 -Galactosidase from Bacillus Circulans in a Lactose Slurry (55% w/w) Reaction System of the Invention

(10) Experimental Conditions:

(11) 70 gram lactose was added directly to 51 gram 0.1M K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer, pH 6. and subsequently the reaction mixture was heated to the reaction temperature 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 specific activity of 141.8 LU/gram immobilized enzyme) was added to initialize the enzymatic reaction. The enzyme dosage is 15.2 LU/gram lactose.

(12) After 24 hours reaction time, the GOS was filtrated and the immobilized enzyme was washed with demineralized water and stored in 0.1M K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer, pH 6.0-7.0 solution at 4 C., prior to reuse.

(13) TABLE-US-00002 TABLE 2 A summary of synthesis of GOS with immobilized -galactosidase from Bacillus circulans in lactose slurry (55% w/w) in a consecutive mode Immo- LU Activity bilized retention enzyme/ Reaction GOS and sugar composition after batch batch time [% on dm] [%] No. [h] Galactose Glucose Lactose GOS 100 Batch 1 24 3.77 22.00 4.11 69.00 57.3 Batch 2 24 5.31 24.46 3.44 65.38 30.1 Batch 3 24 1.74 20.87 9.53 66.94 19.6 Batch 4 24 1.28 18.88 12.03 67.03 11.7 Batch 5 24 0.99 18.76 17.90 61.59 10.6 Batch 6 24 0.74 18.16 25.77 54.59 7.3 Batch 7 24 0.68 16.14 29.21 53.16 7.0 Batch 8 24 0.65 16.54 32.63 48.93 3.8

Example 3 (Comparative Example): Synthesis of GOS with Free -Galactosidase from Bacillus Circulans Using a Lactose Slurry (65%, w/w) Reaction System

(14) Experimental Conditions:

(15) 85 gram lactose was added directly to 65 gram 0.1M K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer, pH 6. and subsequently the reaction mixture was heated to the reaction temperature 58 C. (and maintained for at least 1 hour) and 85 mg free beta-galactosidase of Biolacta N5 (Amano) dissolved in 2 ml demi water was added to initialize the reaction. The enzyme dosage is 5 LU/gram lactose.

(16) The reaction mixture was incubated in a water batch with orbit shaker. After 24 hours reaction, the GOS 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 -Galactosidase from Bacillus Circulans (Carrier-Epoxy Eupergit C 250 L) in Lactose Slurry (65%, w/w) Reaction System

(17) 8 gram immobilized -galactosidase of Biolacta N5 on 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.

(18) After 24 hours, it was found that reaction mixture was completely clear and the GOS content was 57%.

(19) 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 product from the reactor is also close to the concentration of the final product (GOS syrup, 75% (w/w)).

REFERENCES

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