Production and characterization of Aspergillus niger GH29 family α-fucosidase and production of a novel non-reducing 1-fucosyllactose.

Fucosylated oligosaccharides are interesting molecules due to their bioactive properties. In particular, their application as active ingredient in milk powders is attractive for dairy industries. The objective of this study was to characterize the glycosyl hydrolase family 29 α-fucosidase produced by Aspergillus niger and test its ability to transfucosylate lactose with a view towards potential industrial applications such as the valorization of the lactose side stream produced by dairy industry. In order to reduce costs and toxicity the use of free fucose instead of environmentally questionable fucose derivatives was studied. In contrast to earlier studies, a recombinantly produced A. niger α-fucosidase was utilized. Using pNP-fucose as substrate, the optimal pH for hydrolytic activity was determined to be 3.8. The optimal temperature for a 30-min reaction was 60 °C, and considering temperature stability, the optimal temperature for a 24-h reaction was defined as 45 °C For the same hydrolysis reaction, the kinetic values were calculated to be 0.385 mM for the KM and 2.8 mmol/(mg*h) for the Vmax. Transfucosylation of lactose occurred at high substrate concentrations when reaction time was elongated to several days. The structure of the product trisaccharide was defined as 1-fucosyllactose, where fucose is α-linked to the anomeric carbon of the ß-glucose moiety of lactose. Furthermore, the enzyme was able to hydrolyze its own transfucosylation product and 2'-fucosyllactose but only poorly 3-fucosyllactose. As a conclusion, α-fucosidase from A. niger can transfucosylate lactose using free fucose as substrate producing a novel non-reducing 1-fucosyllactose.

Enzymatic synthesis of fucose-containing galacto-oligosaccharides using ß-galactosidase and identification of novel disaccharide structures.

Fucosylated oligosaccharides have an important role in maintaining a healthy immune system and homeostatic gut microflora. This study employed a commercial ß-galactosidase in the production of fucose-containing galacto-oligosaccharides (fGOS) from lactose and fucose. The production was optimized using experiment design and optimal conditions for a batch production in 3-liter scale. The reaction product was analyzed and the produced galactose-fucose disaccharides were purified. The structures of these disaccharides were determined using NMR and it was verified that one major product with the structure Galß1-3Fuc and two minor products with the structures Galß1-4Fuc and Galß1-2Fuc were formed. Additionally, the product composition was defined in more detail using several different analytical methods. It was concluded that the final product contained 42% total monosaccharides, 40% disaccharides and 18% of larger oligosaccharides. 290 µmol of fGOS was produced per gram of reaction mixture and 37% of the added fucose was bound to fGOS. The fraction of fGOS from total oligosaccharides was determined as 44%. This fGOS product could be used as a new putative route to deliver fucose to the intestine.

Mild protein hydrolysation of lactose-free milk further reduces milk-related gastrointestinal symptoms.

Gastrointestinal symptoms associated with milk are common. Besides lactose, milk proteins may cause symptoms in sensitive individuals. We have developed a method for mild enzymatic hydrolysation of milk proteins and studied the effects of hydrolysed milk on gastrointestinal symptoms in adults with a self-diagnosed sensitive stomach. In a double blind, randomised placebo-controlled study, 97 subjects consumed protein-hydrolysed lactose-free milk or commercially available lactose-free milk for 10 d. Frequency of gastrointestinal symptoms during the study period was reported and a symptom score was calculated. Rumbling and flatulence decreased significantly in the hydrolysed milk group (P < 0·05). Also, the total symptom score was lower in subjects who consumed hydrolysed milk (P < 0·05). No difference between groups was seen in abdominal pain (P = 0·47) or bloating (P = 0·076). The results suggest that mild enzymatic protein hydrolysation may decrease gastrointestinal symptoms in adults with a sensitive stomach.

Removal of Bovine Insulin From Cow's Milk Formula and Early Initiation of Beta-Cell Autoimmunity in the FINDIA Pilot Study.

OBJECTIVE To test whether weaning to a bovine insulin-free cow's milk formula (CMF) reduces type 1 diabetes mellitus-associated autoantibodies in children at genetic risk. DESIGN Randomized, double-blind pilot trial (Finnish Dietary Intervention Trial for the Prevention of Type 1 Diabetes [FINDIA]). SETTING Three pediatric hospitals in Finland from May 15, 2002, to November 22, 2005. PARTICIPANTS A total of 1113 infants with HLA-conferred susceptibility to type 1 diabetes were randomly assigned to receive study infant formulas; 908 children provided at least 1 follow-up blood sample (last follow-up, June 2009). INTERVENTION The CMF (n = 389), whey-based hydrolyzed formula (WHF) (n = 350), or whey-based FINDIA formula essentially free of bovine insulin (n = 365) during the first 6 months of life whenever breast milk was not available. MAIN OUTCOME MEASURES Primary outcome was beta-cell autoimmunity monitored at ages 3, 6, and 12 months and then annually until age 3 years. Autoantibodies to insulin, the 65-kDa isoform of glutamic acid decarboxylase, and the tyrosine phosphatase-related IA-2 molecule were screened, and islet cell autoantibodies and autoantibodies to zinc transporter 8 were analyzed in infants whose primary screening test results were positive. RESULTS In the intention-to-treat analysis, 6.3% of children in the CMF group, 4.9% of those in the WHF group, and 2.6% of children in the FINDIA group were positive for at least 1 autoantibody by age 3 years. The odds ratios were 0.75 (95% CI, 0.37-1.54) in the WHF group and 0.39 (0.17-0.91) in the FINDIA group when compared with the CMF group. In the treatment-received analysis, the corresponding odds ratios were 0.81 (95% CI, 0.37-1.76) and 0.23 (0.08-0.69). CONCLUSION In comparison with ordinary CMF, weaning to an insulin-free CMF reduced the cumulative incidence of autoantibodies by age 3 years in children at genetic risk of type 1 diabetes mellitus. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01055080.