Method of production
a technology of oligosaccharides and production methods, applied in the field of enzymatic production of oligosaccharides, can solve the problems of adding time, complexity, energy and expense to the manufacturing process, and achieve the effects of reducing the number of calories, reducing the amount of sugar consumed, and achieving the desired sweetness and texture properties
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example 1
Manufacturing Oligosaccharides from Cellulose Using an LPMO
[0088]1. Phosphoric acid-swollen cellulose (PASO) was prepared by making a slurry of 1 g Avicel cellulose (Sigma-Aldrich) with 3 ml H2O before adding 30 ml ice-cold phosphoric acid and incubating at 0° C. for 1 h. The cellulose was then washed numerous times with water until the flowthrough had a neutral pH before use in reactions.
[0089]2. Apo-PaLPMO9E (SEQ ID NO:1) was pre-incubated for 0.5-1 h at 5° C. in 0.9 stoichiometric Cu(II)(NO3)2 immediately before enzyme reactions.
[0090]3. 25 μg PASO, 30 μg PaLPMO9E (pre-loaded with copper) and 500 nmol ascorbate were incubated in 100 μl 100 mM ammonium acetate pH 6 for 32 hours at 50° C. with intermittent shaking.
[0091]4. Samples were centrifuged and supernatants were dried in vacuo.
[0092]5. Supernatants were reductively labelled with ANTS and analysed by PACE (as per Goubet et al. 2002). FIG. 1 shows the resulting gel.
example 2
Manufacturing Oligosaccharides from Mixed-Linkage Glucan Using a Lichenase and Incorporation of Said Oligosaccharides into a Cake
[0093]1. 250 g ground porridge oat powder was boiled in 2 I water for 30 min.
[0094]2. Once cooled, 2 I ice-cold 96% (v / v) ethanol was added and the suspension was allowed to sit overnight at 5° C. The suspension was filter through miracloth until dry, resuspended in 50% (v / v) ethanol and again filtered through miracloth.
[0095]3. The remaining mass was boiled in 1 I water and incubated for 16 h at 30° C. with 2000 U of lichenase from Bacillus subtilis (SEQ ID NO:2, Megazyme).
[0096]4. Once cooled, 2 I ice-cold 96% (v / v) ethanol was added and the suspension was allowed to sit overnight.
[0097]5. The supernatant was collected by centrifugation and dried in vacuo, yielding 5.2 g mixed-linkage glucan oligosaccharides. An aliquot was reductively labelled with ANTS and analysed by PACE. FIG. 2 shows the resulting gel.
[0098]6. One medium egg was beaten with 50 g but...
example 3
Manufacturing Oligosaccharides from Xylan Using a GH30 Xylanase
[0107]1. Spruce wood chips were blended in suspension in a food blender until they broke into small particles, and then ball-milled.
[0108]2. 100 μl reaction mixtures containing 3.3 mg ball-milled spruce wood chips and 100 mM ammonium acetate pH6 were incubated for 16 h at 30° C. with (or without) 5 μg Ruminiclostridium thermocellum GH30 (sourced from NZYTech).
[0109]3. Reaction products were reductively labelled with ANTS and analysed by PACE. FIG. 4 shows the resulting gel.
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