METHOD FOR PREPARING GALACTO-OLIGOSACHARIDES FROM LACTULOSE

Abstract

Process for the preparation of a galacto-oligosaccharide preparation, which process comprises the step of contacting a lactulose-containing feed with a beta-galactosidase derived from Papiliotrema terrestris. The resulting galacto-oligosaccharide is acceptable to subjects suffering from GOS-related allergy and subjects having lactose intolerance.

Claims

1. A process for the preparation of a galacto-oligosaccharide preparation, which process comprises the step of contacting a lactulose-containing feed with a beta-galactosidase (EC 3.2.1.23) derived from Papiliotrema terrestris.

2. The process according to claim 1 wherein the beta-galactosidase is derived from Papiliotrema terrestris strain MM13-F2171 (Accession Number: NITE BP-02177) or APC-6431 (Accession Number: NITE BP-02178).

3. The process according to claim 1 wherein the beta-galactosidase comprises an amino acid sequence according to any of SEQ ID NO: 1, 2, 3 or 4, or an amino acid sequence that is at least 80% identical to any of SEQ ID NO: 1, 2, 3 or 4.

4. The process according to claim 1 wherein the lactulose-containing feed comprises 40-58 wt. % lactulose.

5. The process according to claim 1 wherein the pH of the lactulose-containing feed is in the range 3.5-6.5.

6. The process according to claim 1 wherein the beta-galactosidase is used in a concentration of 1-20 LU/gram lactulose.

7. The process according to claim 1 wherein the lactulose is reacted with the beta-galactosidase at a temperature in the range 50-75° C.

8. A galacto-oligosaccharide preparation obtained by the process of claim 1.

9. A galacto-oligosaccharide preparation that is clinically free from lactose and which comprises, based on dry matter, 30-60 wt % of galacto-oligosaccharides other than lactulose.

10. The galacto-oligosaccharide preparation according to claim 9 comprising, based on dry matter, 40-70 wt % of a mixture of lactulose and monosugars.

11. The galacto-oligosaccharide preparation according to claim 8 for use in a nutritional composition for a subject known to suffer or having an increased chance to suffer from hypersensitivity to a GOS preparation obtained by transgalactosylation of lactose using a beta-galactosidase derived from Bacillus circulans or from Aspergillus oryzae.

12. The galacto-oligosaccharide preparation according to claim 8 for use in a nutritional composition for a subject known to suffer or having an increased chance to suffer from lactose intolerance.

13. A nutritional composition comprising the galacto-oligosaccharide preparation according to claim 8 and at least one further ingredient selected from the group consisting of a protein source, probiotics, LC-PUFA' s and carbohydrates.

14. The nutritional composition according to claim 13 wherein said composition is suitable for consumption by adults and children aged at least 18 months.

15. A method of at least partially preventing hypersensitivity to a galacto-oligosaccharide preparation in a subject, the method comprising administering to said subject the galacto-oligosaccharide preparation according to claim 8.

16. A method of at least partially preventing hypersensitivity to a galacto-oligosaccharide preparation in a subject, the method comprising administering to said subject the nutritional composition comprising the galacto-oligosaccharide preparation according to claim 13.

Description

EXAMPLES

[0053] Beta-galactosidases of the following sources were used in the experiments:

[0054] Aspergillus Oryzae (Lactase 14 DS, ex-Amano Enzyme),

[0055] Aspergillus Oryzae (Tolerase 100, ex-DSM)

[0056] Aspergillus Oryzae (Biolactase F, ex-Kerry Bioscience)

[0057] Papiliotrema terrestris (β-galactosidase PT, ex-Amano Enzyme)

[0058] The general reaction conditions were as follows: 35 gram lactulose was added to 35 gram 0.01 M sodium citrate buffer, pH 6.5. Subsequently, enzyme was dissolved in 10 ml water and added to initialize the reaction. Of most enzymes, 20 lactose units (LU)/gram lactulose were used. However, Lactase 14 DS and Tolerase 100 were used in a concentration of 200 lactose units (LU)/gram lactulose, due to their low activity at the general reaction conditions used. The reaction mixtures were placed in a water bath with an orbital shaker, thermostated at 50° C. After 48 hours reaction, the reaction was quenched by adding 1.5% 1M HCl and subsequently heated at 95° C. for 30 minutes.

[0059] The fGOS content of the reaction mixtures was analyzed by HPLC (ThermoFisher Scientific Dionex type ICS 3000), based on the peak area percentage of individual sugars. The fGOS content was calculated by the following formula:

fGOS content (%,ds)=100%−galactose %−glucose %−lactose %−lactulose %−fructose %.

[0060] The fGOS content obtained with the different enzymes is summarized in Table 1. Table 1 shows that the enzyme obtained from Papiliotrema terrestris gave a far higher yield than the beta-galactosidase from Aspergillus oryzae; the best enzyme according to C. Guerrero et al.

[0061] The ratio between the two building blocks of lactulose, namely fructose and galactose, is used as an indicator of the enzyme performance. When the ratio is <1, it indicates that the fGOS content achieved cannot be increased anymore by prolonging the reaction time, because it can only hydrolyze the substrate or fGOS formed, as is the case with Aspergillus oryzae-derived beta-galactosidase. For the P. terrestris-derived beta-galactosidase, the ratio of fructose/galactose is above 2, suggesting that the fGOS yield can be further optimized with prolonged reaction time.

TABLE-US-00001 TABLE 1 fGOS composition formed by different beta-galactosidases Enzyme Enzyme origin Gal Glu Fruc lactulose fGOS* Fruc/Gal Lactase 14-DS Aspergillus Oryzae 23.68 2.51 24.54 11.86 37.41 1.04 Tolerase 100 Aspergillus Oryzae 28.61 2.85 26.74 7.55 34.25 0.93 Biolactase F Aspergillus Oryzae 26.34 2.78 25.88 9.2 35.8  0.98 Amano PT Papiliotrema terrestris 5.22 1.99 11.63 27.59 53.57 2.23 *Estimated by the peak percentage of the mono sugars and the substrate left.

[0062] The HPLC chromatogram showed much more peaks for the fGOS produced by the Papiliotrema terrestris-derived enzyme compared to the fGOS produced with the Aspergillus oryzae-derived enzymes. Structure diversity of oligosaccharides is very important to serve the nutritional needs of the different bifidobacteria in the gut and the decoy function for binding pathogens. Therefore, the fGOS derived from the process of the present invention seems of higher nutritional value.

[0063] The degree of polymerization (DP) of the fGOS formed by the different enzymes is summarized in Table 2. Since it is not possible to distinguish lactulose from other DP2 fGOS components formed by lactulose, the total fGOS+lactulose content for each enzyme is given. Since lactulose itself is non-digestible (prebiotic) sugar, there is no need to separate it from fGOS. In the experiment with the P. terrestris enzyme, the fGOS+lactulose content is around 85%. In contrast, the fGOS+lactulose obtained from the Aspergillus oryzae enzymes is below 35%, which is probably due to the their intrinsic high hydrolytic activity as shown by their high mono sugar contents.

TABLE-US-00002 TABLE 2 DP composition of fGOS formed by different beta-galactosidases Mono sugars fGOS + Gal + Fru Glc DP2 DP3 DP4 DP5 DP6 Gal + Glu + Fru lactulose Lactase 14-DS 68,1 0,12 20,72 6,1 1,68 0,54 0,19 68,22 31,78 Biolactase F 75,38 0 18,73 3,68 0,71 0,14 0,03 75,38 24,62 Tolerase too 81,59 0,2 13,64 1,99 0,25 0 0 81,79 18,21 Amano PT 13,77 0,89 59,54 19,74 2,76 0,59 0 14,66 85,34

[0064] The experiment with P. terrestris was repeated with lactose as the substrate. The results are displayed in Table 3. It shows that the DP2-content of the fGOS (obtained from lactulose) is significantly higher than that of GOS obtained from lactose.

[0065] This high DP2 fGOS component may constitute an advantage for the infant gut microbiota, because the growth of infant type of bifidobacteria such as B. Breve in the infant colon may be selectively promoted.

TABLE-US-00003 TABLE 3 Comparison of Lactose-GOS and Lactulose GOS formed by P. Terrestris beta- galactosidase Mono sugars Substrate Glc DP2 DP3 DP4 DP5 DP6 Gal + Glu + Fru GOS + lactulose Lactulose 0,89 59,54 19,74 2,76 0,59 0 14,66 85,34 Lactose 20,04 39,3 24,45 11,35 2,26 0,23 22,23 60