Combining the mechanical ball milling of the carbohydrate and the use of low solvent reaction media for the synthesis of fructose fatty acid esters by immobilized lipases.

Synthesis of carbohydrate fatty acid esters catalyzed by immobilized lipases is a pathway to obtain specific isomers from renewable feedstock, compared to unselective chemical esterification. While the use of low-solvent reaction media (≤ 10 %) offers advantages, the interactive effects of these media with biocatalysts and substrates should be modulated towards high catalytic efficiency and substrate availability. Among the investigated co-solvents, tert-butanol and DMSO in a mixture of lauric acid substrate/co-solvent (90/10; v/v) resulted in high bioconversion yields using either Novozym® 435 or Lipozyme® RM IM, as biocatalysts. Increased hydrophobicity of the Novozym® 435 immobilization support favored bioconversion, while polar substrate surface area enlargement by ball-milling improved productivity through enhancement of fructose availability. A compromise between bioconversion yield (19.7 %) and productivity (9.45 µmol/L min) was obtained in the reactions catalyzed by Novozym® 435 using ball-milled fructose at a concentration of 0.2 mol/L. Combining mechanical ball-milling of the substrates with low-solvent reaction media is expected to enhance and expand enzymatic synthesis of carbohydrate fatty acid esters.

Biotransformation of sucrose rich Maple syrups into fructooligosaccharides, oligolevans and levans using levansucrase biocatalyst: Bioprocess optimization and prebiotic activity assessment.

Maple syrup was investigated as a source to produce FOSs and ß-(2-6)-linked-oligolevans/levans. The modulation of this biotransformation was achieved through the control of Maple syrup °Bx and reaction conditions. Reaction time was identified as the most influential factor for the oligolevans/FOSs production in Maple syrup 30°Bx reaction system as well as for the oligolevans/levans synthesis in the 66°Bx one. In the predictive model of oligolevans/levans production in Maple syrup 60°Bx, the interactive effect between levansucrase unit and reaction time was significant (p-value of 0.0008). The optimal conditions for oligolevans/FOSs production (109.20 g/L) in Maple syrup 30°Bx were 3.73 U/mL, pH 6.60 and 23.12 h; while 5 U/mL, pH 6.04 and 29.92 h were identified as the optimal conditions for oligolevans/levans production (147.09 g/L) in Maple syrup 66°Bx. As compared to inulin-type commercial FOSs, the fermentation of oligolevans/FOSs from Maple syrup led to a higher count of Lactobacillus acidophilus and Bifidobacterium lactis and resulted in a higher production of lactic acid. This study lays the foundation for the biotransformation of Maple syrups into functional prebiotic ingredients.

A novel enzymatic approach based on the use of multi-enzymatic systems for the recovery of enriched protein extracts from potato pulp.

Ten commercial available multi-enzymatic systems have been explored for the efficient recovery of patatin and protease inhibitors from potato pulp. Their enzyme activity profile was characterized, where corresponding enzyme activity profile led to similar protein recovery yield. Of those assessed, Depol 670L (DEP) and Ceremix 2XL (CER) were efficient for the recovery of protein extract enriched with patatin (up to 60.0%) and protease inhibitors (up to 72.0%), respectively. The efficiency of DEP was significantly dependent on the interactive effect of enzymatic units and incubation time (p-value <0.015), while CER was found to be affected by the quadratric effect of both variables (p-value <0.022-0.052). The enriched patatin DEP-based protein extract possessed higher lipid acyl hydrolase activity emphasizing its preservation. The enriched protease inhibitors CER-based protein extract resulted in higher trypsin inhibiting activity, when compared to the industrial method. DEP and CER-based protein extracts were structurally characterized by peptide mass mapping.

Enzymatic generation of peptides from potato proteins by selected proteases and characterization of their structural properties.

The use of low grade starting material for the generation of peptides with bioactivity properties is of interest. The proteins from the potato starch industry byproduct is a promising source, as several health benefits may be associated with their hydrolysates. The efficiency of selected proteases (Novo Pro-D, Alcalase, Flavourzyme, and Papain), exhibiting different substrate specificities and cleavage action modes, to hydrolyze potato protein isolate (patatin and protease inhibitors) was investigated. Novo Pro-D resulted in the lowest degree of hydrolysis, whereas Alcalase, Flavourzyme, and Papain exhibited a high catalytic efficiency for the hydrolysis of potato proteins. The degree of hydrolysis behaved in a concentration dependent manner. However, the end-product profile (peptides and free amino acids) was dependent not only on the protease specificity, its cleavage action mode (endo/exo) and the availability of the intermediate substrates but also on the contribution of the protease inhibitors to the reaction kinetics through their inhibitory effects. Indeed, the dependence of the exo-activity on the catalytic efficiency of the endo-action of protease was shown to be significant. Papain generated more unique peptide sequences with homology assessment matching several potato proteins when compared with Flavourzyme. This can be attributed to the high exo-peptidase activity of Flavourzyme resulting in the generation of shorter peptides which were difficult to match. Flavourzyme produced more peptides originated from patatin fraction, whereas Papain resulted in the release of more peptides corresponding to the protease inhibitor fractions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:420-429, 2016.

Potato protein isolates: recovery and characterization of their properties.

An imitation of industrial potato fruit juice (PFJ) was prepared, using Canadian variety of potatoes, and was characterized of being composed of 22.9% patatin, 53.3% protease inhibitors, and 23.7% high MW proteins. To isolate potato proteins from PFJ, several extraction techniques were explored including thermal/acidic combination, acidic, FeCl3, MnCl2, ethanol and (NH4)2SO4 precipitations, and carboxymethyl cellulose complexation. (NH4)2SO4 precipitation led to the highest yield (98.6%) and to the recovery of protein isolates enriched in patatin with high resolubility. FeCl3 precipitation resulted in the highest purification factor (6.2) and isolates with the lowest relative proportion of high MW proteins (<4.6%); however, its optimal isolate showed a wide minimum solubility pH range of 3.0-6.0. FeCl3 and MnCl2 were identified as the best precipitating agents for the enrichment of isolates with >15kDa protease inhibitors. Trypsin inhibiting activities of protease inhibitors were highly preserved upon protein isolation than the chymotrypsin ones. Acidic-based protein isolate showed the highest specific lipid acyl hydrolase activity of patatin towards o-nitrophenyl butyrate, whereas FeCl3-based one exhibited the highest activity towards 4-nitrophenyl laurate.