WO2003049547A2

WO2003049547A2 - Dairy process and product

Info

Publication number: WO2003049547A2
Application number: PCT/NZ2002/000276
Authority: WO
Inventors: Paul Mcjarrow; Jean Garman; Stephanie Harvey; Adrianna Van Amelsfort
Original assignee: New Zealand Dairy Board
Priority date: 2001-12-13
Filing date: 2002-12-13
Publication date: 2003-06-19
Also published as: AU2002356472A8; AU2002356472A1; TW200306161A; WO2003049547A3

Abstract

The invention provides a method for the combined production of a sialyl-oligosaccharide and desialylated glycomacropeptide (desialylated GMP). A sugar and glycomacropeptide (GMP) are reacted in the presence of a sialidase capable of desialylating GMP to produce desialylated GMP and sialyl-oligosaccharide and the sialyl-oligosaccharide produced is recovered.

Description

DAIRY PROCESS AND PRODUCT

TECHNICAL FIELD The present invention relates to a process for the combined production of sialyl-oligosaccharide and desialylated glycomacropeptide (GMP) and products produced by such a process and in particular to the production of sialyl-lactose and desialylated GMP.

The invention has been developed primarily for use in the combined production of the sialyl- lactose and desialylated GMP and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use. Other sialyl-oligosaccharides may also be produced by the method.

BACKGROUND ART

Human milk sialyl-oligosaccharides have been shown to provide a source of sialic acid for infants. Infants have a high demand for sialic acid, which is an essential component in the development of brain structures. Infant formulas contain 28% of the level of human milk oligosaccharide linked sialic acid and 50% of the total sialic acid found in human breast milk (Sanchez-Diaz et al. (1997) J Pediatr Gastroenterol Nutr 24, 405-410). Sialic acid is an essential sugar in gangliosides found in brain and central nervous tissue. These gangliosides are concentrated at the synaptic ends of nerves and have been postulated to aid in the binding of neurotransmitters. Epidemiological studies have shown that breast fed babies have a slight but significant advantage over formula fed babies in IQ tests, Peabody Picture Vocabulary Test Revised (PPVT-R), psychomotor development and neurological nonnormality. The IQ, PPNT-R and neurological nonnormality advantage persists well into childhood with the IQ studies at 7.5-8 years (Lucas et al. (1992) Lancet 339, 261-264), PPVT-R scores taken at 5 years (Quinn et al. (2001) JPaediatr Child Health 37, 465-469) and neurological nonnormality measurements at 9 years (Lanting et al. (1994) Lancet 344, 1319-1322). Sialyl-oligosaccharides are suggested to provide sialic acid for incorporation into gangliosides (Tram et al. (1997) Arch Dis Child 11, 315-318). The inclusion of a preparation containing sialyl-oligosaccharide into selected food products will therefore provide a source of sialic acid for infants or children to aid in brain development.

Previous patents have disclosed the use of sialic acid (JP 3251593), sialic acid methylglycoside (JP5244976), sialic acid dimer (JP 6038784) and colominic acid (JP 11196891) as the donor molecule for the reaction in association with a variety of sialidases/neuraminidases. Ajisaka et al (1994, Carbohydrate Research 259, 103-115) and Tanaka et al. (1995, Bioscience, Biotechnology and Biochemistry 59, 638-643) have used sialidases from Clostridium perfringens, Arthrobacter ureafaciens, Vibrio cholerae, Bacteroides fragilis and Newcastle disease sialidases with colominic acid, paranitrophenol-sialic acid, sialic acid dimer and methyl sialic acid as substrates. Patent WO 9908511 uses an alpha (2-3) trans-sialidase derived from Trypanosoma cruzi in association with an unfractionated cheese processing waste stream.

US 4992420 discloses the use of GMP (including desialylated GMP) for the treatment of dental caries.

Significant costs can be incurred in the preparation of sialyl-oligosaccharides. The provision of a process in which the by-products of sialyl-oligosaccharide production have a commercial value is therefore desirable.

GMP, a complex mixture of macropeptides found in sweet wheys (rennet and cheese wheys), is rich in sialic acid. The applicant has recently discovered that desialylated GMP has a number of processing advantages. Desialylated GMP is both more heat and acid stable than sialylated GMP. GMP is used, among other things, in the formulation of diets for patients with phenylketonuria and for the prevention of plaque and caries. GMP contains 4-7% w/w sialic acid.

It is an object of the present invention to provide a method for the production of sialyl- oligosaccharide that includes a useful by-product or at least to provide the public with a useful choice. DISCLOSURE OF THE INVENTION

In a first aspect, the present invention provides a method for the combined production of a sialyl- oligosaccharide and desialylated GMP. The method comprises reacting a sugar and GMP in the presence of a sialidase capable of desialylating GMP to produce desialylated GMP and sialyl- oligosaccharide. The sialyl-oligosaccharide is then recovered.

Preferably the desialylated GMP is also recovered.

The term "sialyl-oligosaccharide" in this specification refers to an oligosaccharide having 2-6 saccharide units including one or more sialyl units.

The term "sugar" refers to a monosaccharide, disaccharide, trisaccharide, tetrasaccharide or a pentasaccharide.

Preferably the sugar and GMP are provided from a dairy stream or dairy streams.

Preferably the dairy stream is either cheese whey or rennet whey. More preferably the starting material comprises purified GMP and sugar.

Preferably the sugar is a galactosyl-sugar.

Most preferably the sugar is lactose.

Preferably the sialidase is an α-sialidase. One preferred sialidase is the Arthrobacter ureafaciens sialidase.

More preferred are sialidases from food-acceptable organisms, including from lactic acid bacteria. Suitable organisms may be selected from bacteria and yeast strains. For many species it is necessary to select a strain having a high sialidase activity because some strains will have low or undetectable sialidase activity. The organism may be selected from the group consisting of Bifidobacterium, Candida, Debromyces, Lactobacillus, Lactococcus, Leuconostoc, Pichia, Rhodotorula and Streptococcus species. Bifidobacterium species are particularly preferred.

Preferred organisms are strains of Bifidobacterium bifidum, Bifidobacterium infantis, Lactococcus lactis, Leuconostoc lactis, Leuconostoc mesenteroides, Pichia holstii and Streptococcus thermophilus selected for high sialidase activity. Suitable strains can be selected by conducting a sialidase activity assay using 2'-(4-methylumbelliferyl)-N-acetylneuraminic acid as a substrate (Potier et al. (1979) Analytical Biochemistry 94, 287-296). Bifidobacterium infantis is a particularly preferred organism for producing a sialidase for use in the invention. Particularly suitable strains can be identified after selection for high sialyl-transferase activity.

A particularly preferred strain is the Bifidobacterium infantis strain B5509 (Fonterra Research Centre culture collection) commercially available as for example ATCC 15697, DSM 20088 and NCTC 11817.

The reaction is preferably continued until the maximal amount of sialyl-oligosaccharides is produced. Preferably the GMP is totally desialylated.

All sialidases have the potential to catalyse the transfer of a sialyl residue from a substrate to an acceptor other than water given the correct reaction conditions. As literature shows, a wide range of substrates and acceptors are suitable but individual sialidases work best with different substrates and acceptors to produce individual product profiles. The sialidases from Arthrobacter ureafaciens and Bifidobacterium infantis are particularly effective in producing sialyl-lactose from GMP and lactose, and the preferred embodiment of the invention is a process using either the Arthrobacter ureafaciens or the Bifidobacterium infantis sialidase.

Using the Arthrobacter ureafaciens sialidase, sialyl-lactose is produced over the pH range 4 to 8. Better production is obtained when the pH is between 5 and 7. The desired temperature range for sialyl-lactose production is between 30°C and 55°C but preferably the temperature is between 37°C and 50°C. At lower temperatures the production is slow and at higher temperatures the reaction goes too swiftly to control accurately. Using the Bifidobacterium infantis cytoplasmic sialidase, sialyl-lactose is produced over the pH range 4 to 8. Better production is obtained when the pH is between 5 and 7. The desired temperature range for sialyl-lactose production is between 30°C and 80°C but preferably between 37°C and 60°C.

The reaction time is important. If the reaction is stopped too soon the yield of sialyl-lactose is low. If the reaction goes too long the yield of sialyl-lactose decreases as the sialidase uses the sialyl-lactose as substrate hydrolysing it to produce sialic acid and lactose. Using the Arthrobacter ureafaciens sialidase under maximal conditions the reaction is best stopped between 0.5 and 2 hours.

The GMP concentration used is preferably between 5% w/v and 20% w/v. The lactose concentration is preferably at or near saturation (more preferably over 80% saturation). The optimum temperature is higher for the Bifidobacterium infantis sialidase than the Arthrobacter ureafaciens sialidase and thus allows the use of higher lactose concentrations.

It is anticipated that the Arthrobacter ureafaciens sialidase and the Bifidobacterium infantis sialidase will also work efficiently under similar conditions when the GMP and lactose are part of a dairy stream containing other dairy components.

The applicant has demonstrated sialidases from different organisms can be used in sialyl-transfer reactions.

Once the sialyl-transfer reaction has taken place, the sialyl-oligosaccharide can then be recovered from the mixture. This may be carried out in a number of ways known to those skilled in the art, for example by anion exchange chromatography or ultrafiltration. The sialyl-oligosaccharide may be recovered in substantially pure form or as a fraction enriched in sialyl-oligosaccharide relative to the reaction mixture. Also once the sialyl-transfer reaction has taken place, the GMP can then be recovered from the mixture by ways known to those skilled in the art, for example by ultrafiltration. In another aspect the invention provides a food-acceptable sialidase preparation wherein the sialidase is from a food-acceptable organism having a high sialidase activity. Preferably the sialidase is from a preferred organism described above.

Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a graph of the amount of sialyl-lactose produced per gram of GMP by the Arthrobacter ureafaciens sialidase against time.

Figure 2 shows a graph of the amount of sialyl-lactose produced per gram of GMP by the Bifidobacterium infantis sialidase against time.

EXAMPLES

EXAMPLE 1

Glycomacropeptide (15% w/v GMP) and lactose (40% w/v) were dissolved in 0.1 M sodium acetate buffer, pH 5.0. To 500 μL of this mixture 10 μL (50mUnits, one unit hydrolyses 1 μmol N-acetyl-neuraminyl-D-lactose/ min at 25°C) of Arthrobacter ureafaciens sialidase (Roche, Cat. No. 269 611) was added. The reaction was maintained at 45 ° C for 4 hours. Samples were taken at 0, 0.5, 1, 2, and 4 hours and the reaction stopped by heating to 100 ° C for 5 minutes.

The production of sialyl-lactose was followed by High Performance Anion Exchange Chromatography (HPAEC) as described by Kunz et al. (1996, J Chromatogr B Biomed Appl 685, 211-221).

The production of sialyl-lactose is shown in Figure 1. The highest levels of sialyl-lactose were obtained between 0.5 and 1 hour after which the levels dropped. The maximal yield of sialyl- lactose was 5.5 mg per gram of GMP. EXAMPLE 2

A survey of bacteria and fungi in the applicant's culture collection was conducted to look at the levels of sialidase activity secreted to the growth medium and intracellular levels of the same enzyme.

Sialidase assay

Enzyme levels were determined using a fluorescent assay of released 4-methylumbelliferone from 2'-(4-methylumbelliferyl)-N-acetymeuraminic acid (sialic acid MU, sodium salt, Sigma M- 8639) as the substrate. The assay conditions were adapted from those described in Potier et al. (1979).

A standard curve for 4-methylumbelliferone (MU, Sigma M-1381) of 10 to 30 nM MU in glycine buffer pH 10.7 (0.133 M glycine, 0.06 M NaCl and 0.04 M Na₂CO₃) is prepared against a glycine buffer blank. The enzyme preparations (150 μL) were mixed with 1 mL of 1 μM sialic acid MU in 0.2 M sodium acetate buffer, pH 4.6, and were incubated at 37°C for up to 120 minutes. For cytoplasmic sialidase preparations the reaction was stopped by mixing 200 μL of the reaction solution with 2.5 mL of the glycine buffer. For secreted sialidase preparations 20 μL of the reaction mix was mixed with 2.5 mL glycine buffer to stop the reaction. Fluorescence of the stopped reaction was measured and enzyme levels calculated using the standard curve described above. One unit of activity is defined as releasing 1 nmole MU from sialic acid-MU per min. Fluorescence of sialic acid MU substrate in glycine buffer was used as a blank.

Assay for the production of sialyl-lactose

GMP (15% w/v) and lactose (40% w/v) are dissolved in 0.1 M sodium acetate buffer, pH 5.0. To 500 μL of this mixture 50 μL of sialidase is added, preferably containing >10 mU of sialidase as determined by the fluorescence assay, and incubated at 45°C until maximal sialyl-lactose production is achieved and the GMP desialylated.

Media used for growth of bacteria

Bifidobacterium strains were grown in PPC broth at 30°C for 24-48 hours with no shaking. Candida, Pichia and Rhodotorula strains were all grown in TSB broth at 30°C for 16-24 hours with shaking (200 rpm).

Streptococcus strains were all grown in J8 Broth at 37°C with no shaking for 16-24 hours.

Lactococcus strains were all grown in T5 broth at 30°C with no shaking for 16-24 hours.

Lactobacillus strains were all grown in PP broth with no shaking for 16-24 hours.

PPC Broth

(A) 10.0 g beef extract 10.0 gpolypeptone 5.0 g yeast extract 5.5 g Na₂HPO₄ 5.0 g KH₂PO₄

5.0 g sodium acetate

2.0 g tri ammonium citrate

1.0 g Tween 80

0.05 g cysteine 950 mL MilliQ (or equivalent) water

(B) Salts 11.5 g MgSO₄ 2.4 g MnSO₄ 250 mL MilliQ (or equivalent) water

(C) Salts/sugar solution 20.0 g glucose 10.0 mL of salts (B) MilliQ water (or equivalent) to 100 mL

Autoclave (A) and (C) separately then add 50 mL of (C) to 950 mL of (A). PP Broth

(A) 10.0 g beef extract lO.O gpolypeptone 5.0 g yeast extract

5.5 gNa₂HPO₄ 5.0 g KH₂PO₄ 5.0 g sodium acetate 2.0 g tri ammonium citrate 2.0 g Tween 80

950 mL MilliQ water (or equivalent)

(B) Salts 11.5 g MgSO₄ 2.4 g MnSO₄

250 mL MilliQ water

(C) Salts/sugar solution 20.0 g glucose 10.0 mL of salts (B)

MilliQ (or equivalent) to 100 mL

Autoclave (A) and (C) separately then add 50mL of (C) to 950 mL of (A).

T5 broth

(A) 2.0 g beef extract

5.0 g polypeptone

2.0 g phytone peptone

2.0 g yeast extract 8.5 gNa₂HPO₄

2.0 g KH₂PO₄

0.5 g ascorbic acid 950 mL MilliQ (or equivalent) water (B) salts/sugar solution 0.4 g MgCl₂ 20.0 g glucose MilliQ water (or equivalent) to 100 mL

Autoclave (A) and (B) separately and add 50 mL of (B) to 950 mL of (A)

J8 Broth (A) 2.0 g beef extract

5.0 g polypeptone

2.0 g phytone peptone

2.0 g yeast extract

5.5 gNa₂HPO₄ 5.0 g KH₂PO₄

0.5 g ascorbic acid

950 mL MilliQ water (or equivalent) (B) salts/sugar solution

0.4 g MgCl₂ 20.0 g glucose

MilliQ water (or equivalent) to 100 mL

Autoclave (A) and (B) separately and add 50 mL of (B) to 950 mL of (A)

MRS fde Man, Rugosa and Sharp) Broth

Made up as described by the manufacturers (Difco Laboratories)

TSB (Trvptic sov broth

Made up as described by the manufacturers (Difco Laboratories).

Strains of bacteria/yeast investigated for sialidase activity Table 1. Species FRC number Other collection numbers

Bifidobacterium animalis B5507 DSM 20104

Bifidobacterium bifidum B5502 NCDO1453

Bifidobacterium bifidum B5508 DSM 20456

Bifidobacterium bifidum B5505

Bifidobacterium brevis B5503 NCDO2257

Bifidobacterium brevis B5504

Bifidobacterium infantis B5509 ATCC 15697

Bifidobacterium lactis B5506

Bifidobacterium longum B5510 ATCC 15707

Bifidobacterium pseudocatenulatum B5512 ATCC 27919

Bifidobacterium sp B5500

Candida famata B9011

Candida kefir B9015

Candida kefir B9006 NCYC 143

Candida kefir B9007 NCYC 744

Candida kefir B9008 NCYC 1441

Candida lipolytica B9013

Candida lipolytica B9014

Candida paras, losis B9012

Candida tropicalis B9009 NCYC 997

Debromyces hansenii B9010

Lactobacillus acidophilus B3000

Lactobacillus brevis B4007

Lactobacillus casei B3010

Lactobacillus delbruekii subsp lactis B4012

Lactobacillus fermentum B4008

Lactobacillus helveticus B4001

Lactobacillus helveticus B3003 NCDO 257

Lactobacillus helveticus B3012 NCDO 261 Lactobacillus paracasei B3044

Lactobacillus paracasei B3015

Lactobacillus paracasei B3019

Lactobacillus plantarum B3017

Lactobacillus plantarum B3018

Lactobacillus plantarum B3022 NCDO 82

Lactobacillus rhamnosus B3127

Lactococcus lactis subsp cremoris B0026

Lactococcus lactis subsp cremoris B0039

Lactococcus lactis subsp cremoris B0098

Lactococcus lactis subsp cremoris B0107

Lactococcus lactis subsp cremoris B0051

Lactococcus lactis subsp cremoris B0076

Lactococcus lactis subsp cremoris B0126

Lactococcus lactis subsp lactis BO 100

Lactococcus lactis subsp lactis B1051

Lactococcus lactis subsp lactis B0097

Lactococcus lactis subsp lactis B1614

Lactococcus lactis subsp lactis B1000

Lactococcus lactis subsp lactis B0114

Lactococcus lactis subsp lactis var diacetylactis B0103

Leuconostoc lactis B5007 NCFB 533

Leuconostoc lactis B5038 NCFB 546

Leuconostoc mesenteroides subsp cremoris B5004

Leuconostoc mesenteroides subsp cremoris B5041 NCFB 828

Leuconostoc mesenteroides subsp dextranicum B5013 ATCC 8082

Leuconostoc mesenteroides subsp mesenteroides B5047 NCFB 519

Leuconostoc paramesenteroides B5000

Leuconostoc paramesenteroides B5050 NCFB 883

Pediococcus sp B7004

Pichia holstii B9005 NCYC 560 Rhodotorula glutinis B9002 NCYC 59 Rhodotorula glutinis B9003 NCYC 162 Rhodotorula glutinis B9004 NCYC 2439 Streptococcus dysgalactiae B2290 Streptococcus acidominimus B2254 Streptococcus acidominimus B9575 Streptococcus sp B2270 Streptococcus sp B2272 Streptococcus sp B2274 Streptococcus sp B2280 Streptococcus thermophilus B2507 Streptococcus thermophilus B2289 Streptococcus thermophilus B2505 Streptococcus thermophilus B2516 Streptococcus thermophilus B2502 Streptococcus thermophilus B2504 Streptococcus thermophilus B2510 Streptococcus thermophilus B2511 Streptococcus thermophilus B2520 Streptococcus thermophilus B2517 Streptococcus thermophilus B2512 Streptococcus thermophilus B2518

ATCC, American Type Culture Collection; DSM, Deutsche Sammlung von Mikroorganismen (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen); NCDO, National Collection of Dairy Organisms (c/o NCIMB Ltd., Aberdeen, Scotland, United Kingdom); NCFB, National Collection of Food Bacteria (c/o NCIMB Ltd., Aberdeen, Scotland, United Kingdom); NCYC, National Collection of Yeast Cultures.

Sialidase activity of the cultures

The cultures in Table 1 were grown in their preferred media as described above. OD_όoo_nm absorption was measured then the cells separated from the culture medium by centrifugation (6500 g, 4°C, 20 rnin) and both processed separately. The growth medium (10 mL of a 200 mL culture) was concentrated at least 15 fold using spin concentrators (2600 g, 18°C, 90 min) (Vivaspin, 15 mL concentrator, 30 kDa membrane, Vivascience Ltd, Binbrook Hill, Binbrook, Lincoln, UK). The concentrated growth medium was used directly in the sialidase assay as described above.

The cells (from a 200 mL culture) were washed twice with 50 mL pre-chilled (4°C) 0.2 M sodium acetate buffer, pH 6.5 with the cells recovered after each wash by centrifugation (6500 g, 4°C, 20 min). The washed cells were then resuspended in a small volume of 0.2 M sodium acetate buffer and the cells disrupted by 2 passes (18000 psi) through a French Press Homogeniser (Spectronic Unicam, 820 Linden Avenue, Rochester, NY 14625, USA). The disrupted cells were then centrifuged (13000 rpm, 10 min, microfuge) to pellet the cell debris. The supernatant (cell free extract) was used directly in sialidase assays as described above.

The level of activity was expressed as per OD unit of the original culture. The survey results are shown in Table 2.

Table 2.

Strain Cytoplasmic Sialidase Secreted Sialidase μU/OD mU/OD

B5507 12 ND

B5502 23 ND

B5508 266 ND

B5505 27 ND

B5503 2 ND

B5504 16 ND

B5509 2800-7100 170-300

B5506 1 ND

B5510 41 ND

B5512 3 ND

B5500 5 ND

B9011 13 0.4 B9015 11 ND

B9006 15 ND

B9007 25 ND

B9008 13 ND

B9013 13 0.2

B9014 17 ND

B9012 13 ND

B9009 39 ND

B9010 11 0.3

B3000 3 ND

B4007 3 ND

B3010 4 ND

B4012 3 ND

B4008 7 ND

B4001 19 ND

B3003 3 ND

B3012 34 ND

B3044 ND 0.6

B3015 4 ND

B3019 3 ND

B3017 4 0.8

B3018 3 ND

B3022 2 ND

B3127 ND ND

B0026 63 0.5

B0039 4 ND

B0098 126 ND

B0107 39 ND

B0051 56 ND

B0076 90 ND

B0126 82 ND B0100 5 0.3

B1051 ND ND

B0097 25 ND

B1614 47 ND

B1000 25 ND

B0114 63 ND

B0103 27 ND

B5007 1 ND

B5038 4 1.6

B5004 11 ND

B5041 ND 1.9

B5013 12 0.1

B5047 2 0.5

B5000 26 ND

B5050 3 0.9

B7004 ND ND

B9005 41 1.0

B9002 11 ND

B9003 12 ND

B9004 12 ND

B2290 14 ND

B2254 35 ND

B9575 54 ND

B2270 20 ND

B2272 19 ND

B2274 1 ND

B2280 ND ND

B2507 85 ND

B2289 21 ND

B2505 29 ND

B2516 27 ND B2502 31 ND

B2504 6-40 ND

B2510 14 ND

B2511 54 20.9

B2520 10 ND

B2517 70 ND

B2512 23 ND

B2518 30 ND ND, no activity detected

Ability to produce sialyl-lactose in the GMP/lactose system

From the above screening exercise eight enzymes were selected on the basis of high levels of sialidase activity. These were

• Bifidobacterium bifidum B5508 cytoplasmic sialidase

• Bifidobacterium infantis B5509 cytoplasmic and secreted sialidases

• Lactococcus lactis subsp cremoris B0098 cytoplasmic sialidase

• Leuconostoc lactis B5038 secreted sialidase • Leuconostoc mesenteroides subsp cremoris B5041 secreted sialidase

• Pichia holstii B9005 secreted sialidase

• Streptococcus thermophilus B2511 secreted sialidase

One of these, the Bifidobacterium infantis B5509 enzyme was selected for use in Example 3.

EXAMPLE 3

GMP (15% w/v) and lactose (40% w/v) were dissolved in 0.1 M sodium acetate buffer pH 5.0. To 500 μL of this mixture 50 μL of Bifidobacterium infantis cytoplasmic enzyme (1.99 mU) was added and incubated at 45°C for 3 hours.

The amount of sialyl-lactose produced was quantified by HPAEC chromatography. The column was a CarboPac PAl (Dionex, Dionex Corporation, Sunnyvale, California). The HPLC a Dionex DX 500 with GP50 pump and ED40 electrochemical detector. The flow rate was ImL/min and the column was held at 30°C. The method consisted of 10 minutes of 200 mM sodium hydroxide (Solvent A) followed by 10 minutes of a mixture of 55% solvent B (100 mM sodium acetate containing 20mM sodium hydroxide) and 45% solvent C (100 mM sodium acetate containing 200 mM sodium hydroxide) to equilibrate the column before injection. The injection (25 μl) was followed by a 10 minute elution with a mixture of 55% solvent B and 45% solvent C.

The yield of sialyl-lactose was 0.9 mg per gram of GMP. The time course is shown in Figure 2.

Although the invention has been described with specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms. For example the sugar starting material may differ and the reaction conditions and the source of the sialidase may be varied.

Claims

CLAIMS:

1. A method for the combined production of a sialyl-oligosaccharide and desialylated glycomacropeptide (desialylated GMP) comprising reacting a sugar and glycomacropeptide (GMP) in the presence of a sialidase capable of desialylating GMP to produce desialylated GMP and sialyl-oligosaccharide and recovering the sialyl-oligosaccharide produced.

2. A method as claimed in claim 1 wherein the desialylated GMP is also recovered.

3. A method as claimed in claim 1 or claim 2 wherein the sugar and GMP are provided from a dairy stream or dairy streams.

4. A method as claimed in claim 3 wherein the dairy stream is either cheese whey or rennet whey.

5. A method as claimed in claim 1 or claim 2 wherein the starting material comprises purified GMP and sugar.

6. A method as claimed in any one of claims 1-5 wherein the sugar is a galactosyl sugar.

7. A method as claimed in claim 6 wherein the sugar is lactose.

8. A method as claimed in any one of claims 1-7 wherein the sialidase is an α-sialidase.

9. A method as claimed in any one of claims 1-8 wherein the sialidase is Arthrobacter ureafaciens sialidase.

10. A method as claimed in any one of claims 1-8 wherein the sialidase is from a food- acceptable organism.

11. A method as claimed in claim 10 wherein the sialidase is from an organism selected from bacteria and yeast strains.

12. A method as claimed in claim 11 wherein the sialidase is from an organism selected from the group consisting of Bifidobacterium, Candida, Debromyces, Lactobacillus, Lactococcus, Leuconostoc, Pichia, Rhodotorula and Streptococcus species.

13. A method as claimed in claim 12 wherein the sialidase is from an organism selected from strains of Bifidobacterium bifidum, Bifidobacterium infantis, Lactococcus lactis, Leuconostoc lactis, Leuconostoc mesenteroides, Pichia holstii and Streptococcus thermophilus chosen for having high sialidase activity.

14. A method as claimed in claim 12 wherein the sialidase is from Bifidobacterium infantis and is chosen for having high sialyl-transferase activity.

15. A method as claimed in any one of claims 1-14 in which the reaction is continued until the maximal amount of sialyl-oligosaccharides is produced.

16. A sialyl-oligosaccharide produced by the process of any one of claims 1-15.

17. Sialyl-lactose produced by the process of any one of claims 1-15.

18. Desialylated GMP produced by the process in any one of the claims 1-15.