Effects of fermentation and enzymatic treatment on phenolic compounds and soluble proteins in oil press cakes of canola (Brassica napus).

To develop novel processes for valorizing agro-industry side-streams, canola (Brassica napus) oil press cakes (CPC) were treated with lactic acid bacteria, carbohydrase, and protease. Altogether 29 protein-rich liquid fractions were obtained, of which the composition was analyzed using chromatographic and mass spectrometric methods. A clear association was revealed between the treatments and phenolic profile. Applying certain lactic acid bacteria enhanced the release of sinapic acid, sinapine, glycosylated kaempferols, and other phenolic compounds from CPC. Co-treatment using protease and Lactiplantibacillus plantarum was effective in degrading these compounds. The fraction obtained after 16 h of hydrolysis (with Protamex® of 2% dosage) and 48 h of fermentation (using L. plantarum) contained the lowest phenolic content (0.2 g/100 g DM) and a medium level of soluble proteins (78 g/100 g) among all samples studied. The fractions rich in soluble proteins and low in phenolics are potential food ingredients with improved bioavailability and sensory properties.

Impact of enzymatic pre-treatment on composition of nutrients and phytochemicals of canola (Brassica napus) oil press residues.

The study aimed to develop a biorefining process to recover proteins and dietary fibres from a food industry side-stream, canola (Brassica napus) oil pressing residues. The materials were treated with commercial protease, carbohydrase, and phytase to obtain protein-rich supernatants and fibre-rich precipitates. The compositions of these fractions were analyzed using LC-MS (glucosinolates and phenolics) and GC-MS (sugars, acids, and amino acids). Compared to raw material, the supernatants were richer in proteins, sugars, acids, amino acids, phenolic acids, and flavonols; the precipitates had higher levels of minerals and dietary fibres. The enzymatic treatment decreased the contents of phytic acid, glucosinolates, and phenolic alkaloids in all fractions. The applied enzymes effectively enhanced solubility of proteins, despite the lower yield of crude proteins compared to the alkaline extraction (40-82 vs 91 g/100 g dry matters). The impact of enzymes on other chemical components was also revealed by using principal component analysis.

Impact of Fermentation and Phytase Treatment of Pea-Oat Protein Blend on Physicochemical, Sensory, and Nutritional Properties of Extruded Meat Analogs.

Plant materials that are used for the production of extruded meat analogs are often nutritionally incomplete and also contain antinutrients, thus there is a need to explore alternative plant proteins and pre-treatments. This study demonstrates application of phytase and fermentation to a pea-oat protein blend with a good essential amino acid profile and subsequent texturization using extrusion cooking. Enzymatic treatment reduced the content of antinutrient phytic acid by 32%. Extrusion also degraded phytic acid by up to 18%, but the effect depended on the material. Differences in physicochemical, sensorial, and textural properties between untreated and phytase-treated extruded meat analogs were small. In contrast, fermented material was more difficult to texturize due to degradation of macromolecules; physicochemical and textural properties of extrudates were markedly different; sensory analysis showed enhancement of flavor, but also detected an increase in some unwanted taste attributes (bitterness, cereal and off-taste). Phytic acid was not degraded by fermentation. Analysis of volatile compounds showed extrusion eliminated volatiles from the raw material but introduced Maillard reaction products. Overall, phytase treatment and fermentation demonstrated the potential for application in extruded meat analogs but also highlighted the necessity of optimization of process conditions.

Inhibition of CREB phosphorylation by conjugates of adenosine analogues and arginine-rich peptides, inhibitors of PKA catalytic subunit.

Bisubstrate inhibitors of protein kinases associate simultaneously with two substrate-binding sites of the kinase and thus potentially possess better inhibitory potency and selectivity than inhibitors binding to only the conserved ATP-site of the kinase. We have previously used conjugates of adenosine analogues and arginine-rich peptides (ARCs) to develop proteolytically stable cell plasma membrane-permeable bisubstrate inhibitors whose biochemical affinities towards several basophilic protein kinases of the AGC group are in the picomolar range. The potency of bisubstrate inhibitors to affect the phosphorylation of proteins in living cells has been described in a limited number of publications. In this study, the effect of ARCs on the protein kinase A (PKA)-catalysed cAMP response element-binding protein (CREB) phosphorylation pathway was studied in living mammalian cells. Our results demonstrate that at low micromolar extracellular concentration N-myristoylated ARCs are capable of reducing the activity of transcription factor CREB through inhibition of PKA.

Protein-induced long lifetime luminescence of nonmetal probes.

Time-resolved luminometry-based assays have great potential for measurements in complicated biological solutions and living cells as the measured signal can be easily distinguished from nanosecond lifetime background fluorescence of organic compounds and autofluorescence of cells. In the present study we discovered that binding of a thiophene- or a selenophene-containing heteroaromatic moiety (luminescence donor) to the purine-binding pocket of a protein kinase (PK) induces long lifetime photoluminescence signal that is largely intensified through efficient energy transfer to a fluorescent dye present in close proximity to the luminescence donor. The developed ARC-Lum probes possessing 19-266 µs luminescence lifetime when associated with the target kinase can be used for determination of activity of basophilic PKs, characterization of inhibitors of PKs, and as cAMP sensors. An ARC-Lum probe was also used for the determination of kinetic parameters of inhibitor binding to the catalytic subunit of protein kinase A (PKAc). Effective real-time monitoring of the activation of PKA by Forskolin and the displacement of an ARC-Lum probe from its complex with PKA by inhibitor H89 was performed in live cells. The discovered phenomenon, protein-induced long lifetime luminescence of aromatic probes is very likely to occur with all PKs and many other proteins.

Effect of the structure of adenosine mimic of bisubstrate-analog inhibitors on their activity towards basophilic protein kinases.

Previously reported structural fragments that associate with the ATP-binding pocket of basophilic protein kinases were conjugated with d-arginine-containing peptides. Inhibitory potency of the resulting bisubstrate-analog inhibitors towards PKA and ROCK-II extended to subnanomolar range. The conjugates incorporating 2-pyrimidyl-5-amidothiophene fragment had the highest activity and at 100 nM concentration exhibited over 80% inhibition of most of the tested basophilic kinases of the AGC group.