METHOD FOR PREPARING GOS-PREPARATION WITH BETA-GALACTOSIDASE FROM CRYPTOCOCCUS TERRESTRIS, GOS PREPARATIONS OBTAINABLE THEREBY AND USES THEREOF

Abstract

The invention relates to the field of nutritional ingredients, in particular to economically attractive methods for producing hypoallergenic galactooligosaccharides (HA-GOS) and the use thereof in food and feed items. Provided is a method for the production of a HA-GOS preparation, comprising contacting a lactose feed with a beta-galactosidase (EC 3.2.1.23) comprising 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 thereto, wherein the lactose feed is a cheese whey permeate (CWP) or a CWP that is enriched in sialyllactose (SL-CWP).

Claims

1: A method of producing a hypoallergenic galacto-oligosaccharide (HA-GOS) preparation, the method comprising contacting a lactose feed with a beta-galactosidase (EC 3.2.1.23) comprising 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 thereto, wherein the lactose feed comprises a cheese whey permeate (CWP) or a CWP that is enriched in sialyllactose (SL-CWP).

2: The method according to claim 1, wherein the lactose feed comprises a demineralized CWP, preferably CWP that has been demineralized, more preferably by electrodialysis (ED), to an ash content of up to 4 wt. % and/or a conductivity of up to 4 mS.

3: The method according to claim 1, wherein the lactose feed is contacted with beta-galactosidase at a temperature of about 65-70° C., at a lactose content in the range of 47-55 wt. % lactose, and/or an enzyme dosage of 0.75-1.0 LU/gram.

4: The method according to claim 1, wherein the lactose feed comprises a SL-CWP, preferably having sialyllactose content of at least 0.25 wt. %, more preferably at least 0.3 wt. % based on dry solids.

5: The method according to claim 4, wherein the SL-CWP is a CWP that has been subjected to a lactose-removal step, preferably followed by a step to remove multivalent anions.

6: The method according to claim 4, wherein the lactose-content of the SL-CWP is about 52-60 wt. %.

7: The method according to claim 4, wherein the lactose feed is contacted with beta-galactosidase at a temperature of about 55-60° C., at pH 6.0-7.0 and/or at an enzyme dosage of 0.75-1.0 LU/gram.

8: The method according to claim 1, wherein the HA-GOS thus obtained is subjected to at least one further purification step, preferably wherein said purification step comprises one or more of demineralization, de-proteinization, removal of mono-sugar components, enriching for sialyllactose and removal of coloured impurities.

9: The method according to claim 1, wherein the beta-galactosidase comprises an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95%, yet even more preferably at least 97%, and most preferably at least 98% identical to any of SEQ ID NO: 1, 2, 3 or 4.

10: The method according to claim 1, wherein the beta-galactosidase (EC 3.2.1.23) is derived from Cryptococcus terrestris strain MM13-F2171 (Accession Number: NITE BP-02177) or APC-6431 (Accession Number: NITE BP-02178).

11: A hypoallergenic GOS preparation obtained by the method according to claim 1.

12: The hypoallergenic GOS preparation according to claim 11, showing a decreased score in a Basophil Activation Test performed on a blood sample isolated from a subject when compared to a GOS preparation obtained by transgalactosylation of lactose using Bacillus circulans beta-galactosidase.

13: A method of at least partially preventing an (IgE-mediated) allergic response in a subject known to suffer or has an increased chance to suffer from hypersensitivity to a GOS preparation obtained by transgalactosylation of lactose using a beta-galactosidase derived from Bacillus circulans, the method comprising administering to the subject an effective amount of the hypoallergenic GOS preparation of claim 11.

14: A nutritional composition comprising the hypoallergenic GOS preparation according to claim 11.

15: The nutritional composition according to claim 14, wherein the nutritional composition is a MUM composition for pregnant women, a growing up milk (GUM), a follow-up formula, or an infant formula.

Description

LEGENDS TO THE FIGURES

[0089] FIG. 1: Illustration of OPL composition (in dry matter).

[0090] FIG. 2: Schematic illustration of enzymatically converting OPL into BOS.

[0091] FIG. 3: Illustration of “OPL to SL-enriched GOS” process.

[0092] FIG. 4A: Results of the Basophil activation in test subject #1 as measured by expression of the basophil activation marker CD203c (MFl=Mean Fluorescence).

[0093] FIG. 4B: Results of the Basophil activation in test subject #2 as measured by expression of the basophil activation marker CD203c (MFl=Mean Fluorescence).

[0094] FIG. 4C: Results of the Basophil activation in test subject #3 as measured by expression of the basophil activation marker CD203c (MFl=Mean Fluorescence).

EXPERIMENTAL SECTION

[0095] Beta-Galactosidase Derived from C. terrestris

[0096] The beta-galactosidase enzymes according to SEQ ID NO: 1, 2, 3 and 4 derived from C. terrestris (also referred to as “Tetris enzyme”) for use in the present invention were obtained from Amano Enzyme Inc. (Nagoya, Japan). These beta-galactosidase enzymes and methods for their preparation are disclosed in WO 2017/115826 by Amano. Methods for the preparation of (mutant) beta-galactosidase enzymes derived from C. terrestris, as well as the enzyme properties, are disclosed in WO 2017/115826.

Example 1: Preparation of Hypoallergenic GOS (HA-GOS)

[0097] This example describes the process of determining the optimal process conditions for the Tetris enzyme to prepare HA-GOS from Cheese Whey Permeate (CWP) as lactose feed.

[0098] Electrodialysis (ED) was used to demineralize the Cheese Whey Permeate from about 12 mS to 4 mS or below. After concentration, the demineralized CWP was used as lactose feed in the preparation of HA-GOS.

[0099] The composition of the cheese whey permeate before and after demineralization is shown in Table 3.

TABLE-US-00002 TABLE 3 Composition of cheese whey permeate (CWP). CWP (12.7 mS) Demineralized CWP (4 mS) Dry matter % 28.0 26.8 Total Protein % 0.77 0.73 NPN % 0.70 0.64 Lactose % 24.3 24.7 Ash % 2.52 0.93 Potassium % 0.78 0.188 Sodium % 0.193 0.101 Magnesium mg/kg 372 270 Calcium mg/mg 1500 1200 Phosphorous % 0.167 0.125 Chloride % 0.66 0.075 Nitrate mg/kg 334 116 Nitrite mg/kg <0.1 <0.1 Ash on DM % 9.00 3.5 Lactose on DM % 86.8 92.2 Protein on DM % 2.75 2.72

Experimental Set-Up

Temperature: 65° C., 67.5° C. or 70° C.

[0100] Lactose content (DM): 45, 50 or 55%
Enzyme dosage: 50%, 75% or 100%, wherein 100%=0.94 LU/gram lactose

[0101] Cheese whey permeate (CWP) was used as lactose feed. The CWP was first demineralized to 4% mineral content. This demineralized CWP was then concentrated (via evaporation) to a lactose content of either 45%, 50% or 55%. Then the GOS reaction was initiated by adding different dosages (0.5, 0.75 or 1.0) of the Tetris enzyme (batch BGP1150931SDR). The GOS reaction took place in a water bath using magnetic stirring, in the dark. No pH adjustments before or during the process were performed. The reaction temperature was either 65, 67.5 or 70° C.

[0102] The experimental details of the various experiments are summarized in Table 4.

TABLE-US-00003 TABLE 4 Details of various experiments to prepare HA-GOS from demineralized CWP. Lactose concentration Enzyme Temp. Exp. Nr. (% DM) dosage 65° C.  1 45 0.5   2 * 50 0.75  3 55 1 10 45 0.75 67.5° C. 13 45 0.75 14 50 1 15 55 0.5 22 45 0.75 70° C. 25 45 1 26 50 0.5 27 55 0.75 34 45 0.75 * this sample was used in the BAT test to determine hypoallergenicity.

GOS Yield

[0103] Tables 5, 6 and 7 show the amount of GOS, based on lactose, in several experiments at 65° C. (Table 5), 67.5° C. (Table 6) and 70° C. (Table 7). The results indicate how effective the enzyme is in converting lactose to GOS at various reactions conditions. Also it shows the amount of GOS based on dry matter (DM) and based on lactose. A third column is added with the amount of GOS based on total product. The highest yield is possible with the highest DM. It also shows that 0.5 of the normal dosage of enzyme is sufficient to make more than 45% GOS on DM even after 24 hours.

TABLE-US-00004 TABLE 5 GOS concentrations of GOS, based on lactose, on DM and on total product for a temperature of 65° C., at various lactose concentrations, using CWP as the lactose feed. Reaction Enzyme Dosage GOS on GOS on GOS on time (h) lactose (LU/gram lactose) total DM Lactose DM product 24 45 0.50 49.09 54.20 49.86 24.48 48 45 0.50 49.09 58.53 53.85 26.44 24 50 0.75 54.55 57.71 53.09 28.96 48 50 0.75 54.55 61.42 56.51 30.82 24 55 1.00 59.34 59.21 54.47 32.32 48 55 1.00 59.34 62.07 57.10 33.89

TABLE-US-00005 TABLE 6 GOS concentrations of GOS, based on lactose, on DM and on total product for a temperature of 67.5° C., at various lactose concentrations, using CWP as the lactose feed. Reaction Enzyme Dosage GOS on GOS on GOS on time (h) lactose (LU/gram lactose) total DM Lactose DM product 24 45 0.75 49.09 52.35 48.16 23.64 48 45 0.75 49.09 60.95 56.07 27.53 24 50 1 54.55 57.19 52.61 28.70 48 50 1 54.55 62.06 57.10 31.14 24 55 0.5 59.67 53.32 49.05 29.27 48 55 0.5 59.67 58.59 53.90 32.16

TABLE-US-00006 TABLE 7 GOS concentrations of GOS, based on lactose, on DM and on total product for a temperature of 70° C., at various lactose concentrations, using CWP as the lactose feed. Reaction Enzyme Dosage GOS on GOS on GOS on time (h) lactose (LU/gram lactose) total DM Lactose DM product 24 45 1 49.09 60.1 55.29 27.14 48 45 1 49.09 60.62 55.77 27.38 24 50 0.5 54.55 55.51 51.07 27.86 48 50 0.5 54.55 58.74 54.04 29.48 24 55 0.75 60.00 59.08 54.35 32.61 48 55 0.75 60.00 61.34 56.43 33.86

[0104] Tables 8 and 9 show the average values for the HA-GOS yield for the different parameters that were varied in the series of experiments.

TABLE-US-00007 TABLE 8 Average values of GOS yield, expressed as GOS based on lactose content (meaning that x % of all lactose in sample is converted into GOS), after 24 h reaction time. Reaction time 24 h GOS, based on lactose (%) Temperature effect 65° C. 57.2 67.5° C.   55.7 70° C. 58.3 Enzyme dosage 0.5 54.5 0.75 57.2 1 59.5 Lactose content 45% 56.6 50% 57.3 55% 57.3

TABLE-US-00008 TABLE 9 Average values of GOS yield, expressed as GOS based on lactose content (meaning that x % of all lactose in sample is converted into GOS), after 24 h reaction time. Reaction time 42 h GOS, based on lactose (%) Temperature effect 65° C. 60.7 67.5° C.   58.4 70° C. 60.8 Enzyme dosage 0.5 56.7 0.75 61.4 1 61.7 Lactose content 45% 60.6 50% 60.6 55% 58.6

[0105] Further characteristics of a HA-GOS preparation according to the invention are shown in Tables 10 and 11. Table 10 shows the sugar profile of HA-GOS according to the invention compared to that of a reference GOS preparation obtained with purified lactose as lactose feed using the Tetris enzyme.

TABLE-US-00009 TABLE 10 Sugar profile of HA-GOS according to the invention and of a reference GOS. Ref. GOS .sup.1 HA-GOS .sup.2 DM % 51.67 Lactose % 46.50 Conc. on Brix % based on lactose galactose 1.25 0.93 Conc. on Brix % based on lactose glucose 19.57 17.97 Conc. on Brix % based on lactose allolactose 3.24 0.77 Conc. on Brix % based on lactose lactose 15.80 20.40 Conc. on Brix % based on lactose lactulose 0.57 0.59 % GOS % based on lactose 59.6 59.3 % GOS % based on DM 59.7 53.4 .sup.1 Ref. GOS is a reference GOS preparation obtained with purified lactose as lactose feed using the Tetris enzyme. .sup.2 HA-GOS preparation according to the invention, obtained from CWP using the Tetris enzyme.

[0106] Table 11 shows the distribution of di-to heptasaccharides (degree of polymerization (DP) 2 to 7) of HA-GOS according to the invention compared to that of a reference GOS obtained with purified lactose as lactose feed using the Tetris enzyme.

TABLE-US-00010 TABLE 11 DP distribution of HA-GOS according to the invention compared to that of a reference GOS. Ref. GOS .sup.1 HA-GOS .sup.2 Galactose 1.7 0.7 Glucose 19.4 18.5 DP2 39.7 43.6 DP3 25.5 27.8 DP4 11.7 8.7 DP5 1.9 0.7 DP6 0.2 0.0 DP7 0.0 0.0 Total 100.0 100.0 .sup.1 Ref. GOS is a reference GOS preparation obtained with purified lactose as lactose feed using the Tetris enzyme. .sup.2 HA-GOS preparation according to the invention, obtained from CWP using the Tetris enzyme.

[0107] From the above it follows that when CWP is used as lactose feed in a method for the production of a HA-GOS preparation, excellent results are obtained when the minimal enzyme dosage is 0.65 LU/gram lactose, the minimal lactose concentration is >50% at a temperature of 70° C. for a 24 hour reaction time, when CWP is used as raw material. With a reaction time of 42 hours the optimal reaction conditions are an enzyme dosage of 0.75 LU/gram, a temperature of 65° C. and a lactose concentration of 50%.

[0108] When selecting the optimal conditions it is important to bear it in mind that the best conditions are in a certain range and the specific conditions can be defined when combining all 3 selected parameters in the optimal range. For instance, when selecting 0.75 LU/grams the enzyme dosage, the temperature is 65° C. and the lactose concentration is ≥50%.

Example 2: Preparation of HA-GOS from SL-CWP

[0109] This example describes how CWP enriched in SL (SL-CWP) can be obtained, and used as lactose feed for the cost-effective manufacture of HA-GOS.

[0110] CWP was first subjected to a lactose removal step by crystallization using procedures known in the art. To this end, CWP is concentrated typically to ˜48% (w/w) and the solution is cooled down to 15° C. and is stirred for 8-24 hour to allow for lactose crystallization. The so formed lactose crystals are separated by centrifugation. The mother liquor obtained is called OPL or DLP and typically contains about 24% (w/w) dry matter. OPL or DLP is concentrated to 44% (w/w) or higher for further application or treatment. The OPL or DLP is enriched in sialyllactose SL and can be subjected to various treatments mentioned above to obtain a HA-GOS enriched in SL with high purity (>75% GOS) after removal of salts and mono sugars.

[0111] The composition of the SL-CWP thus obtained is shown in Table 12.

TABLE-US-00011 TABLE 12 Typical chemical composition of SL-CWP (lactose-depleted whey; OPL) and CWP. SL-CWP CWP Dry matter content (%) 24 27 SA (sialic acid) (%) 0.11 0.03 3′-SL (%) 0.22 0.03 6′-SL (%) 0.08 0.06 SL total (%) 0.30 0.1 Lactose (%) 57 95 Protein 7.8 NPN (%) 9 1.5 Ash (%) 23.7
HA-GOS Preparation Using the Tetris Enzyme, with SL-CWP as Lactose Feed.

[0112] The reaction was performed at 65° C. using 65% (w/w) SL-CWP (prepared as described above) with an enzyme dosage of 2 LU/gram lactose. After 24 hour reaction time the formed HA-GOS was separated by centrifugation and the enzyme was denatured by heating at 100° C. for 15 minutes. The final product was analyzed by HPLC to determine the DP (degree of polymerization) distribution. It was found that the DP composition is highly similar to a reference GOS that was prepared with the Tetris beta-galactosidase using purified lactose as lactose feed (see Table 13). Following the removal of minerals, the GOS level is at least about 45 wt. % and the SL level is at least about 0.4 wt. %.

TABLE-US-00012 TABLE 13 Comparison between the DP composition of a conventional GOS preparation produced with Bacillus circulans beta- galactosidase (BC-GOS), a reference GOS obtained from purified lactose using “Tetris” enzyme derived from Cryptococcus terrestris (Ref-GOS) and HA-GOS obtained from SL-CWP using “Tetris” enzyme derived from Cryptococcus terrestris according to the invention. DP BC-GOS .sup.1 Ref-GOS .sup.2 HA-GOS .sup.3 DP7 0.53 0 0 DP6 1.8 0.19 0.38 DP5 4.79 1.98 2.08 DP4 10.74 12.06 11.15 DP3 22.02 25.47 24.28 DP2 37.83 39.06 39.06 Glucose 20.5 18.68 21.21 Galactose 1.8 2.55 1.85 .sup.1 BC-GOS is a conventional GOS preparation produced with purified lactose as lactose feed and with a Bacillus circulans beta-galactosidase. .sup.2 Ref. GOS is a reference GOS preparation obtained with purified lactose as lactose feed using the Tetris enzyme. .sup.3 HA-GOS preparation according to the invention, obtained from SL-CWP using the Tetris enzyme.

[0113] Tables 14 and 15 summarize the composition of HA-GOS obtained using either purified lactose, CWP or SL-CWP as a lactose feed in a transgalactosylation method employing “Tetris” enzyme. Also included are BC-GOS, referring to a commercial GOS preparation obtained using B. circulans beta-galactosidase, and the composition of the crude starting materials CWP and SL-CWP.

TABLE-US-00013 TABLE 14 Composition of GOS obtained using purified lactose as a lactose feed in a transgalactosylation method employing “Tetris” enzyme (Ref-GOS) or B. circulans beta-galactosidase (BC-GOS). Ref-GOS.sup.2 Ref-GOS.sup.2 Ref-GOS.sup.2 Ref-GOS.sup.2 BC-GOS.sup.1 PT731 PT741 PT631 PT431 GOS % DM 58.14 62.2 63.14 63.8 64.82 % lactose glucose % on DM 20.36 18.4 18.16 18.39 17.24 lactose % on DM 20.04 18.3 17.6 16.62 17.07 galactose % lactose 1.46 1.03 1.1 1.19 0.88 sialic acid % on DM 3′SL % on DM 6′SL % on DM SL % on DM DM % 74.26 76.08 75.09 76.39 76.79 protein on DM 0.01 0.01 <0.01 <0.01 <0.01 NPN on DM 0.0016 0.0016 <0.0016 <0.0016 <0.0016 nitrite on DM 0.02 0.08 0.07 0.07 0.07 ash (525C) on DM 0.1 <0.01 0.02 0.02 0.02 calcium on DM magnesium on DM potassium on DM % phosphate sodium on DM phosphate inorganic % w/w pH 2.9 3.34 3.24 3.08 3.2 GOS DP DP2 (other than lactose) 37.4 41.15 41.21 40.59 41.25 DP3 % GOS cont. 22.02 26.02 25.99 25.63 26.43 DP4 % GOS cont. 10.76 11.3 11.26 11.67 11.67 DP5 % GOS cont. 4.88 1.56 1.53 1.71 1.6 DP6 % GOS cont. 1.9 0.14 0.14 0.16 0.15 DP7 % GOS cont. 0.6 — — — — .sup.1BC-GOS is a conventional GOS preparation produced with purified lactose as lactose feed and with a Bacillus circulans beta-galactosidase. .sup.2Ref. GOS is a reference GOS preparation obtained with purified lactose as lactose feed using the Tetris enzyme.

TABLE-US-00014 TABLE 15 Composition of HA-GOS obtained using either CWP or SL-CWP as a lactose feed in a transgalactosylation method employing “Tetris” enzyme, and the composition of the crude starting materials CWP and SL-CWP. HA-GOS SL-CWP HA-GOS CWP (CWP) 1 (OPL) (SL-CWP) 2 GOS % DM 53.4 ca. 55* % lactose 59.3 glucose % on DM 18.0 — 21.21 lactose % on DM 83 20.4 57.0 ca. 20* galactose % lactose 0.9 1.85 sialic acid % on DM 0.03 0.1 0.11 3′SL % on DM 0.03 0.2 0.22 6′SL % on DM 0.06 0.1 0.08 SL total % on DM 0.1 0.3 0.3 DM % 27.6 70.6 24.0 24 protein on DM 2.7 2.6 7.8 7.8 NPN on DM 0.4 0.4 9.0 9 nitrite on DM <0.1 ash (525C) on DM 9.02 3.3 23.7 23.7 calcium on DM 4166.7 magnesium on DM 1017.4 potassium on DM 7013.9 % phosphate sodium on DM 2951.4 phosphate inorganic % w/w 0.4 pH 5.1 GOS DP DP2 (other than lactose) 43.6 39.06 DP3 % GOS cont. 27.8 24.28 DP4 % GOS cont. 8.7 11.15 DP5 % GOS cont. 0.7 2.08 DP6 % GOS cont. — 0.38 DP7 % GOS cont. — — 1 HA-GOS (CWP) preparation according to the invention, obtained from CWP using the Tetris enzyme. 2 HA-GOS (SL-CWP) preparation according to the invention, obtained from SL-CWP using the Tetris enzyme.

Example 3: Basophil Activation Test

[0114] This example describes a basophil activation test to demonstrate the reduced allergenicity of HA-GOS in multiple human subjects with known galacto-oligosaccharide allergy. The test was performed with HA-GOS prepared with cheese whey permeate (CWP) as lactose feed and with HA-GOS prepared with cheese whey permeate enriched in SL (SL-CWP) as lactose feed. HA-GOS prepared with CWP as lactose feed is herein referred to as HA-GOS (CWP). HA-GOS prepared with SL-CWP as lactose feed is herein referred to as HA-GOS (SL-CWP).

Materials

[0115] HA-GOS (CWL) (batch PT731; see Example 1), HA-GOS (SL-CWP) (see Example 2) and a commercial GOS preparation obtained using B. circulans enzyme (BC-GOS) were included in the tests. The materials were stored at room temperature in the dark until use.

Subjects

[0116] Eligible subjects were selected from the cohort previously studied for the prevalence of GOS-allergy in a Singapore atopic population, as described in the paper by Soh et al. (Anaphylaxis to galacto-oligosaccharides—an evaluation in an atopic population in Singapore, Allergy, 2015, 70, 1020-1023). The study was approved by the National University Hospital Singapore institutional ethical review board. Written consent of all subjects was obtained prior to the start of the study.

Basophil Activation Test

[0117] A Basophil Activation Test was performed on patient blood samples. Heparinized peripheral blood aliquots (100 μL) were pre-incubated at 37° C. for 5 minutes and then incubated with 100 μL of PBS (negative control), anti-IgE antibody (positive control, G7-18; BD Biosciences, San Jose, Calif.) or diluted GOS samples for 15 minutes (37° C.). After incubation, cells were washed in PBS-EDTA (20 mmol/L) and then incubated with phycoerythrin-labeled anti-human IgE (Ige21; eBioscience, San Jose, Calif.), biotin-labeled anti-human CD203c (NP4D6; BioLegend, San Jose, Calif.), and fluorescein isothiocyanate-labeled anti-human CD63 (MEM-259, BioLegend) mAbs for 20 minutes at 48° C. Expression of CD203c and CD63 are both markers for basophil activation. After washing the cells with 1% BSA/PBS, allophycocyanin-conjugated streptavidin (BD Biosciences) was added and incubated for 15 minutes at 48° C. Thereafter, samples were subjected to erythrocyte lysis with 2 mL of FACS Lysing Solution (BD Biosciences). Cells were then washed, resuspended in 1% BSA/PBS, and analysed by means of FACSCalibur (BD Biosciences). Basophils were detected on the basis of side-scatter characteristics and expression of IgE (IgEhigh).

Results

[0118] FIGS. 4a-4c show the results of the basophil activation test performed on three subjects. FIG. 4a shows the results of the Basophil activation in test subject #1 as measured by expression of the basophil activation marker CD203c (MFl=Mean Fluorescence). Similarly, FIGS. 4b and 4c show the Basophil activation in test subjects #2, and #3, respectively,

[0119] The results show that all three subjects had a positive basophil activation response to BC-GOS, which indicates that the subjects are allergic to BC-GOS and thus confirms previous findings (Soh et al., 2015). However, incubation of basophils with HA-GOS (CWP) or HA-GOS (SL-CWP) showed no or hardly any basophil activation in any of the subjects. Only at the highest concentrations tested, HA-GOS (CWP) and HA-GOS (SL-CWP) showed very slight basophil activation, which was clearly decreased as compared to the reference BC-GOS response.

CONCLUSIONS

[0120] Basophil activation tests in BC-GOS allergic subjects showed that HA-GOS (CWP) and HA-GOS (SL-CWP) have a markedly reduced allergenicity as compared to BC-GOS. It can be concluded that HA-GOS (CWP) and HA-GOS (SL-CWP) according to the invention are clearly hypoallergenic.