Flavor Characterization of Animal Hydrolysates and Potential of Glucosamine in Flavor Modulation

Bovine (meat and heart) and porcine (hemoglobin and plasma) raw materials were hydrolyzed by Protease A (both endo- and exopeptidase activity), with or without glucosamine added during the enzyme inactivation step. Hydrolysates were characterized via peptide analysis (yield, UV- and fluorescence scanning spectroscopy, and peptide size distribution via size exclusion chromatography), sensory evaluation, and volatile compound analysis via gas chromatography mass-spectrometry (GC-MS) to determine if glucosamine-induced Maillard reaction improved taste and flavor. Porcine hemoglobin produced the most flavor-neutral hydrolysate, and could expectedly have the broadest application in food products. Both bovine meat and -heart hydrolysates were high in umami, and thereby good candidates for savory applications. Porcine plasma hydrolysate was high in liver flavor and would be suitable for addition to certain meat products where liver flavor is desirable. All hydrolysates had low perceived bitterness. Glucosamine-induced Maillard reaction had just a minor influence on the sensory profile via an increased perception of sweet taste (p = 0.038), umami taste (p = 0.042), and yolk flavor (p = 0.038) in the hydrolysates, irrespective of raw material. Glucosamine addition had a statistically significant effect on 13 of 69 volatiles detected in the hydrolysates, but the effect was minor and raw material-specific.

Biophysical characterization of the inactivation of E. coli transketolase by aqueous co-solvents

Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic, and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo- and holo-TK activity and structure to a range of polar organic co-solvents: acetonitrile (AcCN), n-butanol (nBuOH), ethyl acetate (EtOAc), isopropanol (iPrOH), and tetrahydrofuran (THF). The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not simply correlated to global conformational stability. The co-solvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts. Small amounts of aggregate formed in some cases, but this was not sufficient to explain the enzyme inactivation. TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) AcCN, or 6% (v/v) nBuOH over 3 h. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops.

Unravelling the regulation pathway of photosynthetic AB-GAPDH

Oxygenic phototrophs perform carbon fixation through the Calvin–Benson cycle. Different mechanisms adjust the cycle and the light–harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde–3–phosphate dehydrogenase (GAPDH) is a key enzyme of the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant formed by hetero-tetramers of A and B–subunits, and the homotetramer A4. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and for this reason is strictly regulated. While A4–GAPDH is regulated by CP12, AB–GAPDH is autonomously regulated through the C-terminal extension (CTE) of B–subunits. Reversible inactivation of AB–GAPDH occurs via oxidation of a cysteine pair located in the CTE, and substitution of NADP(H) with NAD(H) in the cofactor binding domain. These combined conditions lead to a change in the oligomerization state and enzyme inactivation. SEC–SAXS and single–particle cryoEM analysis disclosed the structural basis of this regulatory mechanism. Both approaches revealed that (A2B2)n–GAPDH oligomers with n=1, 2, 4 and 5 co–exist in a dynamic system. B–subunits mediate the contacts between adjacent A2B2 tetramers in A4B4 and A8B8 oligomers. The CTE of each B–subunit penetrates into the active site of a B–subunit of the adjacent tetramer, while the CTE of this subunit moves in the opposite direction, effectively preventing the binding of the substrate 1,3–bisphosphoglycerate in the B–subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H) by removing NADP(H) from A–subunits allows the entrance of the CTE in B–subunits active sites and hence inactive oligomer stabilization.

Experimental and Computational Study of Hyaluronidase Interactions with Glycosaminoglycans and their Ligands

: Covalent conjugation of hyaluronidase with copolymeric glycosaminoglycans (GAG, heparin and dermatan sulfate) considerably inactivates the enzyme, while conjugation with polymeric GAG (chondroitin sulfate and hyaluronan) improves its stability. These effects are associated with structural differences of these GAG caused by С-5 epimerization of glucuronic and iduronic acid residues and different effects of (α[1 – 4] and α[1 – 3] relative to β[1 – 4] and β[1 – 3]) glycosidic bonds. Pronounced effects of galactose C-4 epimers (in comparison with glucose) and disaccharide mixture (lactose, cellobiose, maltose) on endoglycosidase activity of hyaluronidase emphasize the importance of its diversified multi-contact microenvironment. For a better understanding of the mechanisms regulating hyaluronidase activity, molecular docking and molecular dynamics were chosen. Stabilization effect of chondroitin ligands on heat inactivation of hyaluronidase was demonstrated. An increase in denaturation temperature by 10-15oC hampers blocking of the active site entrance and prevents the enzyme inactivation. Enzyme-GAG interactions were examined by molecular docking with molecular dynamic elaboration. Gradual chemical modification of hyaluronidase was based on the calculated sequence of preferential binding of GAG. Theoretically, covalent binding of chondroitin sulfate trimers at cs7 or cs7, cs1 and cs5 on the enzyme surface provides complete protection against heparin inhibition. Computational investigation of hyaluronidase microenvironment and interactions which limit the enzyme activity allows identification of the best GAG regulators of hyaluronidase endoglycosidase activity and their experimental verification.

Thermosonication Combined with Natural Antimicrobial Nisin: A Potential Technique Ensuring Microbiological Safety and Improving the Quality Parameters of Orange Juice

Currently, thermal pasteurisation (TP) remains the most widely applied technique for commercial orange juice preservation; however, a high temperature causes adverse effects on the quality attributes of orange juice. In order to explore a novel non-thermal sterilization method for orange juice, the impacts of thermosonication combined with nisin (TSN) and TP treatments on the quality attributes including microbial and enzyme inactivation and the physicochemical, nutritional, functional, and sensory qualities of orange juice were studied. Both TP and TSN treatments achieved desirable bactericidal and enzyme inactivation effects, and nisin had a significant synergistic lethal effect on aerobic bacteria in orange juice (p < 0.05). Additionally, TSN treatment significantly improved the color attributes of orange juice and well maintained its physicochemical properties and sensory quality. More importantly, TSN treatment significantly increased the total polyphenols content (TPC) and total carotenoids (TC) by 10.03% and 20.10%, increased the ORAC and DPPH by 51.10% and 10.58%, and the contents of total flavonoids and ascorbic acid were largely retained. Correlation analysis of antioxidant activity showed that the ORAC and scavenging ability of DPPH radicals of orange juice are mainly attributed to TC and TPC. These findings indicate that TSN shows great potential application value, which could guarantee the microbiological safety and improve the quality attributes of orange juice.