BIOCATALYSIS FOR BIOMASS VALORIZATION: PROTEIN HYDROLYSATES AND SUGAR ESTERS FROM AGRI-FOOD WASTES
Creators: name: AVRAMIDOU, KALLIOPI
Publisher: name: Universit� degli Studi di Milano
Year: 2020

description: During this doctorate work, two research topics have been studied within the aim of valorization of waste and by-products derived from the agri-food industry using a biotechnological approach for the production of high-value chemicals. The first topic was the preparation and characterization of hydrolysates from rice bran protein. Rice bran (RB) is a waste derived from the milling process of the rice and is a rich source of highly nutritional proteins, lipids, carbohydrates, and a number of micronutrients (e.g. vitamins, minerals, antioxidants, and phytosterols). The sequential treatment of RB with carbohydrases and proteases was used to prepare mixtures of water-soluble peptides which were tested for their biological activity (ACE-inhibition) and as flavor enhancers. Carbohydrases, that catalyze the hydrolysis of the glycosidic linkages of rice bran polysaccharides, enhanced the extractability of the entrapped protein components. Then, proteases (Flavourzyme or/and Alcalase) allowed converting the protein fraction of rice bran into mixtures of more water-soluble peptides. The prepared samples were submitted to ultrafiltration by using membranes with molecular weight cut-off of 10, 5 and 1 kDa and characterized by SDS-PAGE (Sodium dodecyl sulphate-polyacrylamide gel electrophoresis), Gel Permeation Chromatography (GPC) and by sensory analysis. All samples with a molecular weight under 10 kDa exhibited ACE-inhibitory activity. The highest activity was found for the samples P4?? (68.70%) with a molecular weight under 1 kDa and P2? (60.19 %) with a molecular weight under 5 kDa and the lowest activity for the sample ?P5? (20.28 %) with a molecular weight under 5 kDa. It is noticeable that the choice of the enzyme for the first step treatment (carbohydrases) has a great effect on the ACE ? inhibitory activity of the final hydrolysate. Interestingly, the sensory analysis revealed that the resulting protein hydrolysates exert only sweet and umami taste. It should be mentioned that the bitter taste was completely eliminated, which could be considered very promising for the application and utilization of the rice bran protein hydrolysates as food enhancers. The second topic of this PhD work was the enzymatic synthesis of sugar-fatty acid esters that can be used as bio-surfactants. Surfactants constitute an important class of chemicals widely used in almost every sector of industry. Environmental and health concerns about the effects of the conventional surfactants have increased the demand for surfactants from natural raw materials that possess good biodegradability and low toxicity, along with the desired functional performance. Sugar fatty acid esters (SFAEs), usually called sugar esters, are fully biodegradable, non-ionic surfactants which are characterized by excellent emulsifying, stabilizing and detergency properties. Depending on carbon chain length and nature of the sugar head group, together with the many possibilities for linkage between the hydrophilic sugar and the hydrophobic alkyl chain, SFAEs cover a wide range of hydrophilic-lipophilic balance (HLB) values which result in tunable surfactant properties. Chemical synthesis of SFAEs requires harsh reaction conditions which result, in most cases, in complex mixtures of isomers and by-products. Enzyme-based synthesis is an alternative strategy that can overcome the above-mentioned drawbacks. Sugar fatty acid esters can be prepared, indeed, through an esterification reaction between a sugar and a fatty acid catalyzed by a lipase. SFAEs, including glucose monooleate (GluMO), monostearate (GluMS), monopalmitate (GluMP), monolaurate (GluML), and galactose monooleate (GalMO), monostearate (GalMS), monopalmitate (GalMP), monolaurate (GalML), were synthesized by enzymatic esterification of fatty acids and the corresponding sugar. After a screening of several lipases both in free and immobilized form, an immobilized lipase CALB (Candida antarctica lipase B) was selected as the biocatalyst to promote the ester bond formation. Reactions were carried out in organic solvent by using molecular sieves (4 �) to scavenge the water by-product and thus shift the reaction toward sugar ester formation. Reaction yields and product characterization were assessed by NMR. Rational design of enzymatic reactions was carried out by using the synthesis of GluMP as the model reaction. Sugar: fatty acid ratio, temperature, and reaction time were selected as variables (response: product yield).