Eugenio Spadoni Andreani

Cocoa bean shells (CBS), a by-product of the cocoa industry, from two cacao varieties and obtained after selected processing conditions (fermentation, drying, roasting) were characterized in terms of their chemical composition, where they were found to be a great source of carbohydrates, specifically dietary fiber, protein, ash, and polyphenols, namely quercetin, epicatechin, and catechin. Cell wall polysaccharides were isolated by alkaline extraction (0.5 M or 4 M KOH) and were found to be enriched primarily in pectic polysaccharides (80.6–86%) namely rhamnogalacturonan and arabinogalactan as well as hemi- cellulosic polysaccharides (13.9–19.4%). Overall, 0.5 M KOH polysaccharides were favored having provided a diverse profile of neutral sugars and uronic acids. When tested for the promotion of the growth of selected probiotic strains, CBS cell wall polysaccharides performed similarly or more than inulin and rhamnogalacturonan based on the prebiotic activity scores. The short-chain fatty acid profiles were characterized by high amounts of lactic acid, followed by acetic and propionic acid.

The potential of poly- and oligosaccharides as functional ingredients depends on the type and glycosidic linkages of their monosaccharide residues, which determine their techno-functional properties, their digestibility and their fermentability. To isolate the pectic polysaccharides of cranberry, alcohol insoluble solids were first obtained from pomace. A sequential extraction with hot phosphate buffer, chelating agents (CH), diluted (DA) and concentrated sodium hydroxide was then carried out. Pectic polysaccharides present in CH and DA extracts were purified by anion exchange and gel filtration chromatography, then sequentially exposed to commercially available pectin-degrading enzymes (endo-polygalacturonase, pectin lyase and endo-arabinanase/endo-galactanase/both). The composition and linkages of the generated fragments revealed important characteristic features, including the presence of homogalacturonan with varied methyl esterification extent, branched type I arabinogalactan and pectic galactan. The presence of arabinan with galactose branches was suggested upon the analysis of the fragments by LC-MS.

Cranberry pomace obtained from industrial juice production was characterized by proximate composition analysis and monosaccharide profile of the dietary fiber. Extraction of carbohydrates from pomace was investigated using microwave‐assisted alkaline method and five commercial biocatalysts (pure endo‐galactanase and four multienzyme biocatalysts). The extracts obtained from microwave‐assisted approach had average total sugars yield of 21.3% and contained mostly oligosaccharides in the degree of polymerization range of 7 to 10. All multienzyme biocatalysts led to yields similar or higher than microwave‐assisted approach (23.4% to 42.0%), but mainly generated shorter oligosaccharides with a degree of polymerization of 2 to 5. Compared to cranberry pomace dietary fiber, microwave‐assisted extracts were enriched in pectic oligosaccharides, whereas the enzymatic extracts were enriched in glucans and had less rhamnose and galactose. Pomace ground for 5 min or more by ball mill assumed a powdery consistence. Longer milling did not affect particle size but increased their roughness. Such physical changes had no effect on the efficiency of multienzymatic treatment. Practical Application: The increased production of cranberries and cranberry products will continuously generate more pomace, a potentially valuable material for the generation of added‐value products. Up to 60% to 70% of cranberry pomace is composed of plant cell wall material. The properties of naturally occurring plant cell wall polysaccharides and their corresponding oligosaccharides have been of a great interest, and many of them find application as functional food ingredients. Despite the fact that the cranberry pomace is rich in plant cell polysaccharides, it has been mainly explored as a source of phenolic antioxidants. This study reveals the efficiency of cranberry pomace as a source of nondigestible oligosaccharides. The use of microwave‐assisted extraction and different biocatalysts for the enzymatic extraction led to oligosaccharides with well‐defined monosaccharide composition and molecular weight distribution. The study of the effects of these extraction techniques on the yield and the characteristics of generated oligosaccharides would allow the modulation of their properties. As an overall, the findings of this study would contribute to lay the scientific ground for the development of innovative process for the isolation of nondigestible oligosaccharides as functional ingredients from cranberry pomace by products.

Objectives To investigate the ability of the proteases, subtilisin and α-chymotrypsin (aCT), to inhibit the adhesion of Candida albicans biofilm to a polypropylene surface. Results The proteases were immobilized on plasma-treated polypropylene by covalently linking them with either glutaraldehyde (GA) or N′-diisopropylcarbodiimide (DIC) and N-hydroxysuccinimide (NHS). The immobilization did not negatively affect the enzyme activity and in the case of subtilisin, the activity was up to 640% higher than that of the free enzyme when using N-acetyl phenylalanine ethyl ester as the substrate. The efficacies against biofilm dispersal for the GA-linked SubC and aCT coatings were 41 and 55% higher than the control (polypropylene coated with only GA), respectively, whereas no effect was observed with enzymes immobilized with DIC and NHS. The higher dispersion efficacy observed for the proteases immobilized with GA could be both steric (proper orientation of the active site) and dynamic (higher protein mobility/flexibility). Conclusions Proteases immobilized on a polypropylene surface reduced the adhesion of C. albicans biofilms and therefore may be useful in developing anti-biofilm surfaces based on non-toxic molecules and sustainable strategies.

Polypropylene and polyethylene were coated with alpha‐Chymotrypsin (α‐CT) or subtilisin Carlsberg (SubC) or Burkholderia cepacia lipase (lipase BC) by different immobilization procedures, such as physical adsorption and covalent linking. This latter procedure was based on the chemical functionalization of the plastic surface by oxygen gas plasma treatment. Immobilization of the enzyme was carried out by using as cross‐linking agent i) glutaraldehyde (GA) or ii) N′‐diisopropylcarbodiimide (DIC) and N‐hydroxysuccinimide (NHS). The effects of duration of the plasma treatment and the type of the immobilization procedure on the transesterification activity of the enzyme were investigated. In general polypropylene resulted a better support than polyethylene. Moreover, α‐CT showed higher transesterification activity when immobilized with GA, while for SubC, DIC and NHS were better cross‐linking agents than GA. No activity was observed with these enzymes when immobilization was carried out by physical adsorption. On the contrary, lipase BC immobilized by physical adsorption was even more active than the free enzyme. Concerning thermal stability, immobilized SubC was less stable than the free enzyme. Overall, these results show that plastics endowed with biocatalytic properties could be obtained by simple immobilization protocols and that optimal immobilization conditions depend on the type of starting plastic, plasma treatment, cross‐linking method, and the nature of the enzyme.

Sensors are devices composed of an active sensing material with a signal transducer. Electrochemical sensors have more advantages over the others because the electrodes can sense the analyte that is present in the host without doing any damage to the host system. The immobilization of a protein on a metallic transducer can be a crucial step in the development of bionanodevices that find applications in the field of biomaterials, biocoatings, biofuel cells, etc. In the present study, we show the immobilization by electropolymerization of thioaniline functionalized glucose oxidase and Au nanoparticles on nanostructured gold films prepared by electrodeposition and compared to sputtered gold. This enzyme is employed in the preparation of biosensors of biomedical interest. The goodness of the Au film for enzyme binding is evaluated by comparison of the enzyme activity and of the interval of linearity for the determination of glucose concentration.