Method for preparing GOS having reduced allergenicity

Abstract

The invention relates to the field of nutritional ingredients, in particular to methods for producing hypoallergenic galacto-oligosaccharides (GOS) and the use thereof in food and drink items. Provided is the use of a beta-galactosidase (EC 3.2.1.23) derived from Cryptococcus terrestris (recently renamed Papiliotrema terrestris) in the production of a hypoallergenic GOS preparation having a reduced capacity to induce an allergic response in a subject.

Claims

1. A method of at least partially preventing an allergic response to a galacto-oligosaccharide (GOS) preparation in a subject, the method comprising administering to the subject a hypoallergenic GOS preparation obtained by transgalactosylation of lactose using a Cryptococcus terrestris beta-galactosidase having EC 3.2.1.23, wherein the subject is known to suffer from or has hypersensitivity to a GOS preparation obtained by transgalactosylation of lactose using a Bacillus circulans beta-galactosidase.

2. The method according to claim 1, wherein the Cryptococcus terrestris beta-galactosidase is from Cryptococcus terrestris strain MM13-F2171 having National Institute of Technology and Evaluation (NITE) Accession Number BP-02177 or Cryptococcus terrestris strain APC-6431 having NITE Accession Number BP-02178.

3. The method according to claim 1, wherein the Cryptococcus terrestris beta-galactosidase comprises the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4.

4. The method according to claim 1, wherein the hypoallergenic GOS preparation has a decreased score in a Skin Prick Test in the subject and/or in a Basophil Activation Test performed on a blood sample isolated from the subject when compared to a GOS preparation obtained by transgalactosylation of lactose using a Bacillus circulans beta-galactosidase.

5. The method according to claim 1, wherein the subject is of South East Asian origin.

6. The method according to claim 1, wherein the subject is an adult, an adolescent, or a child.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1a-1i: amino acid sequences of exemplary beta-galactosidase (EC 3.2.1.23) enzymes derived from Cryptococcus terrestris. A) SEQ ID NO: 1: wild-type enzyme; B) SEQ ID NO: 2: mutant strain enzyme 1; C) SEQ ID NO: 3: mutant strain enzyme 2; D) SEQ ID NO: 4: mutant strain enzyme 3.

(2) FIGS. 2a and 2b: Basophil activation in test subject #1 as measured by expression of the basophil activation marker CD203c (FIG. 2a, MFl=Mean Fluorescence) and percentage of cells expressing the basophil activation marker CD63 (FIG. 2b). Squares represent HA-GOS (solid line in the graph). Diamonds represent REF-GOS (dotted line in the graph). For details see Example 2.

(3) FIGS. 3a and 3b: Basophil activation in test subject #2 as measured by expression of the basophil activation marker CD203c (FIG. 3a, MFl=Mean Fluorescence) and percentage of cells expressing the basophil activation marker CD63 (FIG. 3b). Squares represent HA-GOS (solid line in the graph). Diamonds represent REF-GOS (dotted line in the graph). For details see Example 2.

(4) FIGS. 4a and 4b: Basophil activation in test subject #3 as measured by expression of the basophil activation marker CD203c (FIG. 4a, MFl=Mean Fluorescence) and percentage of cells expressing the basophil activation marker CD63 (FIG. 4b). Squares represent HA-GOS (solid line in the graph). Diamonds represent REF-GOS (dotted line in the graph). For details see Example 2.

(5) FIGS. 5a and 5b: Basophil activation in test subject #4 as measured by expression of the basophil activation marker CD203c (FIG. 5a, MFl=Mean Fluorescence) and percentage of cells expressing the basophil activation marker CD63 (FIG. 5b). Squares represent HA-GOS (solid line in the graph). Diamonds represent REF-GOS (dotted line in the graph). For details see Example 2.

(6) FIGS. 6a and 6b: Basophil activation in test subject #5 as measured by expression of the basophil activation marker CD203c (FIG. 6a, MFl=Mean Fluorescence) and percentage of cells expressing the basophil activation marker CD63 (FIG. 6b). Squares represent HA-GOS (solid line in the graph). Diamonds represent REF-GOS (dotted line in the graph). For details see Example 2.

EXPERIMENTAL SECTION

(7) β-Galactosidase Derived from C. terrestris

(8) The beta-galactosidase enzymes derived from C. terrestris as used in the present invention were obtained from Amano Enzyme Inc. (Nagoya, Japan). These beta-galactosidase enzymes and methods for their preparation are disclosed in PCT/JP2016/089001 by Amano. The method for the preparation of beta-galactosidase enzymes derived from C. terrestris, as well as the enzyme properties, as disclosed in PCT/JP2016/089001 are described below in paragraphs 1 to 8.

(9) 1. Obtaining a Wild-Type Beta-Galactosidase from C. terrestris

(10) In order to obtain a β-galactosidase enzyme suitable for the production of galacto-oligosaccharides, various kinds of microorganisms were screened. As a result, it turned out that a microorganism of Cryptococcus terrestris contained in a soil sample that had been collected near Heho Airport in Myanmar in October 2013 was a promising producer strain for β-galactosidase. An attempt was made to purify β-galactosidase from this microbial strain (Cryptococcus terrestris strain MM13-F2171). Cryptococcus terrestris strain MM13-F2171 was deposited on Dec. 10, 2015 at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, under the name of Cryptococcus terrestris MM13-F2171, to which the Accession Number NITE BP-02177 was assigned.

(11) Cryptococcus terrestris strain MM13-F2171 was cultured in a liquid medium (2.0% lactose, 2.0% Yeast Extract, 0.1% KH.sub.2PO.sub.4, 0.05% MgSO.sub.4.Math.7H.sub.2O, pH 5.0) at 30° C. for 4 days with shaking (at 200 revolutions per minute). After the culturing was completed, about 3 L supernatant was collected by centrifugation, and then subjected to concentration and desalting treatment with an ultra-filtration membrane (AIP-1013D with a membrane inner size of 0.8 mm; Asahi Kasei Chemicals Corp.). In the desalting treatment, 20 mM acetate buffer (pH 6.0) was used.

(12) The concentrated solution was loaded onto an anion-exchange column HiTrap DEAE FF (GE Healthcare Biosciences), which had been equilibrated with 20 mM acetate buffer (pH 6.0). Absorbed fractions were eluted with a gradient using 20 mM acetate buffer (pH 6.0) containing 1 M NaCl, and measured for enzyme activity. Fractions with enzyme activity were pooled, and then subjected to dialysis against 20 mM acetate buffer (pH 6.0) containing 1.8 M ammonium sulfate. The enzyme-active fraction obtained after the dialysis was loaded onto a hydrophobic column HiTrap Phenyl HP (GE Healthcare Biosciences), which had been equilibrated with 20 mM acetate buffer (pH 6.0) containing 1.8 M ammonium sulfate. Absorbed fractions were eluted with a gradient using 20 mM acetate buffer (pH 6.0), and measured for enzyme activity. Fractions with enzyme activity were pooled, and then subjected to dialysis against 20 mM acetate buffer (pH 6.0) containing 0.2 M NaCl.

(13) The enzyme-active fraction obtained after the dialysis was loaded onto a gel filtration column HiLoad Superdex 200 prep grade (GE Healthcare Biosciences), which had been equilibrated with 20 mM acetate buffer (pH 6.0) containing 0.2 M NaCl, and then fractions with enzyme activity were collected. The enzyme had a molecular weight of about 266 kDa when determined by a gel filtration method using this HiLoad Superdex 200 prep grade column.

(14) When this result is considered in combination with the results of SDS-PAGE analysis (see below), it is supposed that the enzyme is in the form of a dimer.

(15) Subsequently, the molecular weight of the purified wild-type strain enzyme was determined by SDS-PAGE. First, samples of the purified wild-type strain enzyme were subjected to denaturation (in a denaturing buffer in a boiling water bath for 10 minutes), followed by treatments for removal of O-linked sugar chains (using both O-glycosidase and neuraminidase; O-Glycosidase & Neuraminidase Bundle, New England Biolabs) and/or N-linked sugar chains (using PNGase F; New England Biolabs). The conditions for these enzyme treatments followed the protocols provided with the respective enzymes. After the treatments, the molecular weights of the resulting products were determined by SDS-PAGE.

(16) The wild-type strain enzyme was found to have a molecular weight of 120 kDa after no treatment, 106 kDa after removal of O-linked sugar chains, 104 kDa after removal of N-linked sugar chains, and 104 kDa after removal of both O-linked and N-linked sugar chains.

(17) 2. Internal Amino Acid Sequences of the Purified Enzyme

(18) Analysis of the internal amino acid sequence of the purified enzyme revealed that the enzyme comprises the following internal amino acid sequences:

(19) TABLE-US-00001 (SEQ ID NO: 9) GVQYVDYNSPT (SEQ ID NO: 10) FLFGWATAAQQ (SEQ ID NO: 11) QAYQIGIFAEPIYNT (SEQ ID NO: 12) PSIWDWAS, and (SEQ ID NO: 13) EEPPFAYVPE.
3. Determination of the Gene Sequence of the Wild-Type Strain Enzyme

(20) An attempt was made to determine the gene sequence encoding the β-galactosidase produced by Cryptococcus terrestris strain MM13-F2171. Cryptococcus terrestris strain MM13-F2171 was cultured in a liquid medium (2.0% lactose, 2.0% Yeast Extract, 0.1% KH.sub.2PO.sub.4, 0.05% MgSO.sub.4.Math.7H.sub.2O, pH 5.0) at 30° C. for 24 hours with shaking (at 200 revolutions per minute). After the culturing was completed, cells were harvested. Total RNA was prepared in accordance with the protocol of the RNeasy Mini Kit (QIAGEN) for RNA extract ion from yeast cells (mechanical disruption of cells). The synthesis of cDNAs from the resulting total RNA was performed using the SMARTer RACE 5′/3′ kit (TaKaRa), and then 5′ and 3′ RACE PCR react ions were carried out. The 5′RACE GSP primer used had the sequence GATTACGCCAAGCTTgcaaagatcccgatctggtacgcctg (SEQ ID NO: 14), and the 3′RACE GSP primer used had the sequence GATTACGCCAAGCTTttcctgtttggctgggcgaccgcc (SEQ ID NO: 15). The base sequences of the resulting RACE PCR products were analyzed to determine the full-length cDNA sequence (SEQ ID NO: 5). The putative amino acid sequence encoded by the full-length cDNA sequence is of SEQ ID NO: 1.

(21) By further investigation, the genomic DNA sequence encoding the β-galactosidase produced by Cryptococcus terrestris strain MM13-F2171 (SEQ ID NO: 16) was successfully determined.

(22) 4. Properties of the Purified Wild-Type Enzyme

(23) (1) Optimum pH and pH Stability

(24) The optimum pH and pH stability of the purified wild-type enzyme were examined using a lactose hydrolyzing activity as an indicator. Examinations for optimum pH were performed using 0.1 M glycine buffer in the pH range of pH 2 to 3, 0.1 M citrate buffer in the pH range of pH 3 to 6, 0.1 M acetate buffer in the pH range of pH 5 to 6, 0.1 M phosphate buffer in the pH range of pH 7 to 8, and 0.1 M sodium carbonate buffer in the pH range of pH 9 to 10. The results from enzyme activity measurements at different pHs showed that the purified enzyme has an optimum pH of 4 to 5.

(25) The pH stability of the purified enzyme was examined by heating it at 40° C. for 30 minutes in buffers of different pHs (using the above-described buffers) and then measuring the residual enzyme activity. The results from residual enzyme activity measurements at different pHs showed that the purified enzyme exhibited stable enzyme activity in the pH range of pH 2 to 8.

(26) (2) Optimum Temperature and Thermostability

(27) To examine the optimum temperature of the purified enzyme, acetate buffer (pH 6.0) was used and the lactose hydrolyzing activity was measured at different temperatures. The results from enzyme activity measurements at different temperatures showed that the purified enzyme was found to have an optimum temperature of 70° C. To examine the thermostability of the purified enzyme, the lactose hydrolyzing activity was measured after the enzyme was heated in acetate buffer (pH 6.0) for 30 minutes at different temperatures. The results from enzyme activity measurements at different temperatures showed that the purified enzyme was stable between 30° C. and 60° C. and the enzyme activity was retained at levels of 80% or more of the activity.

(28) 5. Examination of Oligosaccharide Production Ability of Wild-Type Enzyme

(29) (1) Methods

(30) The purified enzyme was examined for the ability to produce oligosaccharides. One unit (1 U) of the purified wild-type strain enzyme per 1 g of lactose was added to aliquots of a 53% lactose solution that had been preheated to specified reaction temperatures, which then were subjected to reaction at those temperatures for 24 hours. The reaction solutions after the reaction was completed were analyzed by HPLC (under the conditions described below) to determine the composition of sugars contained therein. The results from determination of the composition of sugars allow an evaluation of the transglycosylation activity.

(31) Examinations were made for the degrees of polymerization of galacto-oligosaccharides (GOSs) and of branching of trisaccharides when the production of galacto-oligosaccharides by the purified enzyme (wild-type strain enzyme) reached a yield of about 50%. Reactions were carried out in accordance with the above-described procedures, and at 65° C. for 24 hours as conditions where the production of GOSs by the purified enzyme (derived from Cryptococcus terrestris) reached a yield of about 50%.

(32) The degree of polymerization was determined by using an MCI™ GEL CK04S column (Mitsubishi Chemical Corporation); H.sub.2O as eluent; 0.4 ml/min flow rate; RI detector; 80° C. column temperature.

(33) The degree of branching was determined by using a Shodex column (registered trademark) Asahipak NH2P-40 3E (Showa Denko K.K.); MeCN:H.sub.2O=75:25 (vol:vol) as eluent; 0.35 ml/min flow rate; RI detector; 25° C. column temperature.

(34) (2) Results

(35) Measurements results were used to calculate the content (%) of galacto-oligosaccharides (GOSs) in the total amount of sugars (total sugar), contained in the respective reaction solutions and the proportions (%) of respective GOSs with the indicated degrees of polymerization, at the indicated reaction temperatures. Results are shown in Table 1. The purified enzyme (wild-type strain enzyme) was found to have excellent GOS-producing ability. In addition, the wild-type strain enzyme was found to exhibit high levels of transglycosylation activity under high temperature conditions.

(36) TABLE-US-00002 TABLE 1 GOS production with wild-type β-galactosidase at varying temperatures. Reaction Ratio in GOS (%) Enzyme temperature Amount of GOS ≥ DP4 DP3 DP2* WT strain 65° C. 53.4 22.1 51.8 26.1 enzyme 70° C. 54.2 25.8 47.4 26.8 *Lactose not included

(37) Measurements results were used to calculate the proportions (%) of respective GOSs with the indicated degrees of polymerization. Typical results for the degrees of polymerization of GOSs when the purified enzyme (wild-type strain enzyme) was used are shown in Table 2. The wild-type strain enzyme (Cryptococcus terrestris derived enzyme) was found to have excellent GOS-producing ability and to efficiently produce oligosaccharides, particularly trisaccharides and higher saccharides.

(38) TABLE-US-00003 TABLE 2 Comparison of GOS production with various enzymes. Ratio in GOS (%) Strain (enzyme) ≥ DP4 DP3 DP2* Cryptococcus laurentii 18.0 55.8 26.1 Sporobolomyces singularis 13.5 54.5 32.0 C. terrestris MM13-F2171 (WT strain enzyme) 16.7 57.5 25.8 *Lactose not included

(39) Measurements results were used to calculate the proportions (%) of linear and branched oligosaccharide in the resultant trisaccharides and to compare the ratios of trisaccharides with branched chain (i.e. the degrees of branching of trisaccharides) between the enzymes derived from Cryptococcus terrestris and known other β-galactosidase-producing strains. The results for the degrees of branching of GOSs when the purified enzyme (wild-type strain enzyme) was used are shown in Table 3. The wild-type strain enzyme (Cryptococcus terrestris derived enzyme) was found to produce predominantly linear oligosaccharides. Thus, it was revealed that the wild-type strain enzyme has transglycosylation activity in which the sugar chain is specifically transferred via β-1,4-glycosidic linkage and in particular, is less capable of transglycosylating so as to form β-1,6-glycosidic linkage.

(40) TABLE-US-00004 TABLE 3 Comparison of Degree of branching of DP3 GOS produced with various enzymes. Ratio in DP3 (%) Strain (enzyme) β1-4 β1-6 β1-2, β1-3 Cryptococcus laurentii 71.9 12.0 16.1 Sporobolomyces singularis 70.1 5.7 24.3 C. terrestris MM13-F2171 (WT strain enzyme) 76.3 1.5 22.1
6. Obtaining β-Galactosidase Enzymes Produced by Mutant Strains, and Determination of Amino Acid Sequences and Molecular Weights Thereof

(41) Two mutant strains (M2 and M6) were obtained from Cryptococcus terrestris strain MM13-F2171 by means of mutagenesis with UV treatment. β-Galactosidase enzymes produced by these mutant strains were purified in procedures similar to those described above for the wild-type enzyme. Strains M2 and M6 each were found to have a high ability to produce mutant strain enzymes 1 to 3; strain M2 was observed to have a particularly high ability to produce mutant strain enzyme 1, and strain M6 to produce mutant strain enzymes 2 and 3. Cryptococcus terrestris strain M2 was deposited at Dec. 10, 2015 at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, under the name of Cryptococcus terrestris APC-6431, to which the Accession Number NITE BP-02178 was assigned.

(42) The amino acid sequences of the obtained purified enzymes, i.e., one enzyme derived from mutant strain M2 (mutant strain enzyme 1) and two enzymes derived from mutant strain M6 (mutant strain enzymes 2 and 3), were determined. First, N-terminal amino acid sequences of mutant strain enzymes 1 to 3 were determined using a protein sequencer (PPSQ-31A, SHIMADZU CORPORATION). Then, the cDNA sequence of the wild-type strain enzyme (SEQ ID NO: 5) was searched for the base sequence corresponding to the N-terminal amino acid sequence of each of the mutant strain enzymes, thereby to determine the cDNA sequence encoding each of the mutant strain enzymes. The amino acid sequence of mutant strain enzyme 1 (SEQ ID NO: 2) corresponds to one having a deletion of the N-terminal 130 amino acid residues of the full-length amino acid sequence of the wild-type strain enzyme (SEQ ID NO: 1), which is deduced from the cDNA sequence encoding the wild-type strain enzyme (SEQ ID NO: 5). Similarly, the amino acid sequence of mutant strain enzyme 2 (SEQ ID NO: 3) corresponds to one having a deletion of the N-terminal 136 amino acid residues of the full-length amino acid sequence of the wild-type strain enzyme (SEQ ID NO: 1), while the amino acid sequence of mutant strain enzyme 3 (SEQ ID NO: 4) corresponds to one having a deletion of the N-terminal 141 amino acid residues of the full-length amino acid sequence of the wild-type strain enzyme (SEQ ID NO: 1).

(43) Subsequently, the molecular weights of these mutant strain enzymes were determined by SDS-PAGE. Procedures and conditions for removing sugar chains were in accordance with those described above for the wild-type enzyme. The molecular weights of the respective mutant strain enzymes were determined when the enzymes were subjected to no treatment, and treatments for removal of N-linked sugar chains, O-linked sugar chains, and both N-linked and O-linked sugar chains. On the basis of the results of SDS-PAGE analysis, it was observed that strain M2 produces mutant strain enzymes 2 and 3, in addition to mutant strain enzyme 1.

(44) The mutant strain enzyme 1 was found to have a molecular weight of 71 kDa after no treatment, 65 kDa after removal of O-linked sugar chains, 64 kDa after removal of N-linked sugar chains, and 64 kDa after removal of both O-linked and N-linked sugar chains.

(45) The mutant strain enzyme 2 was found to have a molecular weight of 66 kDa after no treatment, 63 kDa after removal of O-linked sugar chains, 61 kDa after removal of N-linked sugar chains, and 61 kDa after removal of both O-linked and N-linked sugar chains.

(46) The mutant strain enzyme 3 was found to have a molecular weight of 66 kDa after no treatment, 62 kDa after removal of O-linked sugar chains, 61 kDa after removal of N-linked sugar chains, and 61 kDa after removal of both O-linked and N-linked sugar chains.

(47) 7. Examination of Oligosaccharide Production Ability of Mutant Strain Enzymes 1, 2 and 3

(48) (1) Methods

(49) To aliquots of a lactose solution was added the purified enzyme derived from strain M2 (mutant strain enzyme 1) or M6 (mutant strain enzyme 3), and the mixtures were subjected to reaction. Examinations were performed for the degrees of polymerization and branching of sugars contained in the reaction solutions after the reaction was completed. The reaction conditions and measurements of the degrees of polymerization and branching were in accordance with those described above under 5.

(50) (2) Results

(51) Measurements results were used to calculate the content (%) of GOSs in the total amount of the sugars (total sugar) contained in the respective reaction solutions and the proportions (%) of respective GOSs with the indicated degrees of polymerization, at the indicated reaction temperatures (Table 4). The purified enzyme (mutant strain enzyme) was found to have excellent GOS-producing ability. In addition, the mutant strain enzyme was found to exhibit high levels of sugar transfer activity under high temperature conditions. It can also be found that there were no differences in GOS producing ability between the wild-type stain enzyme and the mutant strain enzyme.

(52) TABLE-US-00005 TABLE 4 GOS production with mutant strain enzyme 3 at varying temperatures. Reaction Amount of Ratio in GOS (%) Enzyme temperature GOS ≥ DP4 DP3 DP2* C. terrestris M6 50° C. 46.9 6.4 68.9 24.7 (mutant strain 60° C. 52.1 16.1 58.2 25.8 enzyme 3) 65° C. 53.2 20.9 53.4 26.0 70° C. 53.9 24.5 49.2 26.3 *Lactose not included

(53) Measurements results were used to calculate the proportions (%) of respective GOSs with the indicated degrees of polymerization. Typical results for the degrees of polymerization of GO Ss when the purified mutated enzymes derived from strains M2 (mutant strain enzyme 1) and M6 (mutant strain enzyme 3) were used are shown in Table 5. The mutant strain enzymes were found to have excellent GOS-producing ability and to efficiently produce oligosaccharides, particularly trisaccharides and higher saccharides. It can also be found that there were no differences in GOS producing ability between the wild-type stain enzyme and the mutant strain enzymes.

(54) TABLE-US-00006 TABLE 5 Comparison of GOS production with various enzymes. Ratio in GOS (%) Strain (enzyme) ≥ DP4 DP3 DP2* Cryptococcus laurentii 18.0 55.8 26.1 Sporobolomyces singularis 13.5 54.5 32.0 C. terrestris M2 14.2 60.5 25.3 (mutant strain enzyme 1) C. terrestris M6 19.9 54.2 25.8 (mutant strain enzyme 3) *Lactose not included

(55) Measurements results were used to calculate the proportions (%) of linear and branched oligosaccharide in the resultant trisaccharides and to compare the ratios of trisaccharides with branched chain (i.e. the degrees of branching of trisaccharides) between the wild-type strain enzymes and two mutant strain enzymes Table 6.

(56) TABLE-US-00007 TABLE 6 Comparison of Degree of branching of DP3 GOS produced with various enzymes. Ratio in DP3 (%) Strain (enzyme) β1-4 β1-6 β1-2, β1-3 Cryptococcus laurentii 71.9 12.0 16.1 Sporobolomyces singularis 70.1 5.7 24.3 C. terrestris M2 79.0 0.4 20.7 (mutant strain enzyme 1) C. terrestris M6 75.2 2.7 22.1 (mutant strain enzyme 3)

(57) The mutant strain enzymes (mutated, Cryptococcus terrestris derived enzymes) were found to produce predominantly linear oligosaccharides. Thus, it was revealed that the mutant strain enzymes have transglycosylation activity in which the sugar chain is specifically transferred via β-1,4-glycosidic linkage and in particular, is less capable of transglycosylating so as to form β-1,6-glycosidic linkage. It was also observed that the wild-type strain enzyme and the mutant strain enzymes have a comparable ability to produce GOSs and do not have any substantial differences in terms of properties as β-galactosidase. Mutant strain enzyme 2 is a β-galactosidase enzyme of which the amino acid sequence is shorter by six amino acid residues at the N terminus than that of mutant strain enzyme 1 and longer by five amino acid residues at the N-terminus than that of mutant strain enzyme 3. Since it is apparent from the above results that an amino acid sequence in an N-terminal region does not affect enzymatic properties, it can be inferred that mutant strain enzyme 2 also have enzymatic properties equivalent to those of mutant strain enzymes 1 and 3.

(58) 8. Properties of Mutant Strain Enzymes 1 and 3

(59) The purified, mutated enzymes (see the above section described under 6) were used to examine properties of mutant strain enzymes 1 and 3. The experimental methods were similar to those described for the wild-type strain enzyme (see the above section described under 4).

(60) (1) Optimum pH and pH Stability

(61) Mutant strain enzymes 1 and 3 each were found to have an optimum pH of 4 to 5. Mutant strain enzymes 1 and 3 each were found to exhibit stable activity in the pH range of pH 2 to 9.

(62) (2) Optimum Temperature and Thermostability

(63) Mutant strain enzymes 1 and 3 each were found to have an optimum temperature of 70° C. Mutant strain enzymes 1 and 3 each were found to be stable between 30° C. and 65° C. and to retain enzyme activity at levels of 80% or more of the activity.

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

Synthesis of GOS Preparation

(64) Four batches of a GOS preparation (referred to as PT 731-741-631-431) were produced by the transgalactosylation of lactose by four batches of Cryptococcus terrestris β-galactosidase enzyme (obtained from Amano Enzyme Inc., batch numbers GFEO0450731SDR, GFEO0450741SDR, GFEN1052631SDR and GFEO0750431SDR). For each batch, a lactose slurry (˜50% (w/w) Lactopure, batch no. 502392; MFG:13-07-2014) was used as substrate. This lactose slurry was heated to 95° C. to dissolve the lactose. Subsequently the lactose solution was cooled down to the reaction temperature of 65° C. A very mild citrate buffer (2-3 mM) was added in order to adjust and stabilize the pH. Per gram lactose, 0.94 LU enzyme was used. During the reaction the pH was adjusted by citric acid and sodium hydroxide when needed. 42 Hours after the enzyme addition, the reaction was stopped by heating 30 min. at 90° C.

(65) Down Stream Processing

(66) After the enzyme reaction was terminated by heating, the solution was run on an Ion Exchange column (cation exchange followed by anion exchange)), followed by reduction of the pH to 3.2 using citric acid. After pasteurization at 75° C. the GOS was concentrated to approximately 75% dry matter.

(67) The analyses of the thus obtained batches of GOS preparation are indicated in Table 7. In this table it is shown that the specifications of the four batches produced by applying four batches of P. terrestris β-Galactosidase enzyme are highly consistent (low inter-batch variation). Differences with REF-GOS, which is prepared by transgalactosylation of lactose under the action of B. circulans, are very small.

(68) TABLE-US-00008 TABLE 7 Proximate analysis of the various batches of HA-GOS HA- REF- GOS HA-GOS HA-GOS HA-GOS GOS* Test Parameter PT731 PT741 PT631 PT431 (ref Dry matter (%) 76.08 75.09 76.39 76.79 74.26 Galacto- 62.2 63.14 63.8 64.82 58.14 oligosaccharides (% on dry matter) Nitrogen 0.0016 <0.0016 <0.0016 <0.0016 0.0016 (% on dry matter) Protein 0.01 <0.01 <0.01 <0.01 0.01 (% on dry matter) Sulphated ash <0.01 0.02 0.02 0.02 0.1 (% on dry matter) Lactose 18.3 17.6 16.62 17.07 20.04 (% on dry matter) Glucose 18.4 18.16 18.39 17.24 20.36 (% on dry matter) Galactose 1.03 1.1 1.19 0.88 1.46 (% on dry matter) Nitrite 0.08 0.07 0.07 0.07 0.02 pH 3.34 3.24 3.08 3.2 2.9 *Conventional GOS prepared by transgalactosylation of lactose under the action of B. circulans.

(69) GOS Degree of Polymerisation (DP) analysis was performed according to established methods. Table 8 shows that the results of the Degree of Polymerization analysis of hypoallergenic GOS (HA-GOS) of the present invention are highly similar to those found in the reference GOS preparation obtained using the enzyme from B. circulans (REF-GOS).

(70) TABLE-US-00009 TABLE 8 Analytical results for DP-analysis HA-GOS Galactose Glucose DP2 DP3 DP4 DP5 DP6 DP7 Total PT731 0.91 18.93 41.15 26.02 11.30 1.56 0.14 — 100 PT741 1.19 18.68 41.21 25.99 11.26 1.53 0.14 — 100 PT631 1.13 19.11 40.59 25.63 11.67 1.71 0.16 — 100 PT431 0.82 18.08 41.25 26.43 11.67 1.60 0.15 — 100 REF-GOS 1.31 21.12 37.42 22.02 10.76 4.88 1.90 0.60 100

Example 2: Oral Challenge Test

(71) This example describes a double-blind placebo-controlled oral challenge test to demonstrate the reduced allergenicity of HA-GOS in in multiple human subjects with known galacto-oligosaccharide allergy. In addition, a skin prick test and basophil activation test were performed with HA-GOS.

(72) Materials

(73) HA-GOS (batch PT731; see Example 1) and a commercial GOS preparation obtained using B. circulans enzyme (REF-GOS) were included in the tests. The materials were stored at room temperature in the dark until use.

(74) Subjects

(75) 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., 2015.sup.10. Seven eligible subjects were approached for participation in the study. Two subjects refused to participate. Therefore, five adult subjects with confirmed GOS-related allergy were recalled to the clinic for a skin prick test, blood sample drawing and twice for a double-blind placebo-controlled oral challenge test with HA-GOS (minimum time between challenges was 2 weeks). The study was approved by the hospital's institutional ethical review board (IRB protocol “Testing hypoallergenicity of a modified galacto-oligosaccharide in patients with known galacto-oligosaccharide allergy”. Written consent of all subjects was obtained prior to the start of the study.

(76) Skin Prick Test, Basophil Activation Test and Oral Challenge Test

(77) Skin prick testing to HA-GOS was carried out by the clinical research coordinator on the middle of the back or the forearm. Histamine and REF-GOS were used as positive controls. The wheal size for each sample was recorded and used as the degree of skin test reactivity.

(78) 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).sup.7.

(79) An Oral Food Challenge Test was performed by administration of escalating dosages of HA-GOS at 30 minute intervals to achieve a total cumulative dose of 4 grams (see Table 9 below). The total dose of 4 grams was chosen, as this was the maximum dose that triggered a clinical reaction in 5 patients who had anaphylaxis to REF-GOS, as reported by Chiang et al..sup.7. A solution of 0.8 g GOS/100 ml water was used in the study and prepared fresh on the day of the challenge.

(80) TABLE-US-00010 TABLE 9 Dose regimen of the oral food challenge test Proportion of GOS mixture Amount of Amount of Cumulative Time to meal size GOS in grams GOS in ml dose in grams  0′ 5% 0.10 12.5 0.10 30′ 25% 0.50 62.5 0.60 60′ 70% 1.4 175 2.00 90′ 100% 2.00 250 4.00

(81) As placebo, we used a mixture of glucose, lactose and citric acid to mimic the non-oligosaccharide composition and taste of HA-GOS. Preparation of the test solution for the oral challenges was done by laboratory personnel not involved in the actual oral challenge testing. Furthermore, the preparation of the solution was checked by a second person to avoid any mix up. The physician performing the oral challenge test was blinded for the test material (HA-GO S or Placebo solution).

(82) Results

(83) Results Skin Prick Test and Oral Challenge Test

(84) Seven subjects with prior proven REF-GOS-related allergy were eligible for an oral challenge with HA-GOS. These subjects had had reactions typical of an acute allergic reaction within 30 minutes of the threshold dose during challenge with REF-GOS. All had positive skin prick tests to REF-GOS.

(85) Eventually, four subjects were challenged with HA-GOS and placebo in the current study (subject #1, #2, #4 and #5). Prior to the HA-GOS challenge, all were skin prick test negative to HA-GOS.

(86) The detailed results of the oral challenge (OC) and skin prick test (SPT) with HA-GOS are presented in the table below. The response to a REF-GOS challenge (performed roughly two-and-a-half years before the current challenge with HA-GOS) is also described in table 10, for reference.

(87) TABLE-US-00011 TABLE 10 Results of the oral challenge (OC) and skin prick test (SPT) with HA-GOS Subject number Allergy REF-GOS OC REF-GOS OC SPT REF-GOS SPT HA-GOS HA-GOS OC HA-GOS OC (male/female) background (date) response (wheal) (wheal) (date) response #1 (F) AR 7 Mar. 2014 Sneeze, cough, 3 × 4 0 21 Nov. 2016 C none chest tightness 12 Dec. 2016 P failed* #2 (M) AS 13 Jun. 2014 Wheeze, sneeze, 5 × 5 0 9 Dec. 2016 P none chest tightness 19 Dec. 2016 C none #3 (F) AD, AR 15 Aug. 2014 Sneeze, cough, 3 × 3 0 14 Nov. 2016 C none itchy eyes, no chest tightness erythema #4 (F) AR, AD 28 Feb. 2014 Chest tightness, 5 × 5 0 7 Nov. 2016 P none cough, itchy throat 5 Dec. 2016 C none #5 (F) AR 4 Apr. 2014 Cough, sneeze, 5 × 5 0 31 Oct. 2016 P none rash 11 Nov. 16 C none AR = allergic rhinitis, AS = asthma, AD = atopic dermatitis, OC = oral challenge, SPT = skin prick test, C = HA-GOS, P = placebo *Subject #1 failed the placebo challenge test; she experienced an itch on the back, chest and tongue and felt giddy.

(88) Subject #1 experienced symptoms of an allergic response (itch on the back, chest and tongue; giddy feeling) during the challenge with placebo, whereas she passed the oral challenge test with HA-GOS without any complaints. It was confirmed by medical practitioners that the subject had received placebo prior to experiencing these allergy symptoms. This is a known phenomenon that sometimes occurs during placebo-controlled challenges, and is probably caused by anxiety of the subject.

(89) The data in Table 10 show that all four subjects which underwent a double-blind placebo-controlled challenge with HA-GOS showed no allergic symptoms upon consumption of HA-GOS.

(90) Results Basophil Activation Test

(91) FIG. 2-6 show the results of the basophil activation test performed on all five subjects prior to undergoing the oral challenge test.

(92) FIG. 2 shows the results of the Basophil activation in test subject #1 as measured by expression of the basophil activation marker CD203c (FIG. 2a, MFl=Mean Fluorescence) and percentage of cells expressing the basophil activation marker CD63 (FIG. 2b). Squares represent HA-GOS (solid line in the graph). Diamonds represent REF-GOS (dotted line in the graph).

(93) Similarly, FIGS. 3, 4, 5 and 6 show the Basophil activation in test subjects #2, #3, #4 and #5, respectively, as measured by expression of the basophil activation marker CD203c (FIGS. 3a, 4a, 5a and 6a; MFl=Mean Fluorescence) and percentage of cells expressing the basophil activation marker CD63 (FIGS. 3b, 4b, 5b and 6b). Squares represent HA-GOS (solid line in the graph). Diamonds represent REF-GOS (dotted line in the graph).

(94) The results show that three subjects (#1, #3 and #4) had a negative basophil activation response to REF-GOS. This was also previously seen during other basophil activation tests with REF-GOS. These subjects, however, are confirmed allergic to REF-GOS, since they have a positive skin prick test and oral challenge test result in response to REF-GOS. The basophil response to HA-GOS was also negative in these subjects.

(95) Subjects #2 and #5 showed a clear basophil activation response to REF-GOS, whereas HA-GOS did not result in basophil activation.

CONCLUSIONS

(96) HA-GOS consumption showed no allergic responses during a placebo-controlled oral challenge test in n=4 REF-GOS-allergic subjects. Skin prick tests with HA-GOS were negative in all subjects tested. Also in the basophil activation test in REF-GOS allergic subjects, clearly reduced allergenicity was found with HA-GOS as compared to REF-GOS. Taken together, it can be concluded that HA-GOS is clearly hypoallergenic in comparison to REF-GOS.

REFERENCES

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