Comparison of enzymatic and pH shift methods to extract protein from whole Baltic herring (Clupea harengus membras) and roach (Rutilus rutilus).

This study aimed to establish the differences between enzymatically extracted hydrolysates and pH shifted protein isolates from whole Baltic herring and roach in terms of polypeptide patterns, functionality, sensory properties, microbial quality, yield, and composition. Alkaline extraction resulted in the highest yields, whereas the hydrolysates showed the highest protein contents. The hydrolysates showed higher protein solubility (86.0-88.5%) than the protein isolates (5.1-14.5%) as well as the higher foam capacity for Baltic herring. However, for roach, alkaline extracted protein isolates exhibited the highest foam capacity. All hydrolysates showed poor foam stability (0-13%) while the protein isolates showed notably higher stability (30-55%). The hydrolysates showed relatively low bitterness, whereas alkaline extracted roach proteins were perceived as bitter. This study demonstrated that it was possible to produce protein isolates and hydrolysates from whole fish with good microbial quality. However, both processes need to be optimised according to the food application and fish species.

Ductile keratin films from deep eutectic solvent-fractionated feathers.

Feathers, an industrial by-product, are a valuable source of keratin that could be used, for example, in the preparation of films for biomedical and packaging applications. However, the utilisation of feather keratin requires scalable processes to convert feathers into a feasible keratin stream. This paper shows how deep eutectic solvent (DES) fractionated feathers could be converted into strong films. In the DES fractionation process, two keratin fractions with different molecular weights were obtained. The films made of the high molecular weight keratin fraction had better mechanical properties and stability against moisture than the films made of the low molecular weight keratin fraction. The strength properties were further improved by cross-linking the keratin with diglycidyl ether enabling the formation of a uniform keratin network, whereas glutaraldehyde did not show a clear cross-linking effect. These keratin films could be used, for example, in food packaging or medical applications such as wound care.

Green process to regenerate keratin from feathers with an aqueous deep eutectic solvent.

Poultry feathers, a source of keratin, are a significant side stream from the food industry, for which valorization is essential considering the circular economy aspects. For this, ecofriendly processes are the tools that allow the easy and feasible transformation of the feathers. Deep eutectic solvents (DESs) are generally considered as inexpensive, relatively simple, mild and environmentally friendly solvents which can dissolve proteins from protein-rich biomasses. In this work, feathers were processed with an aqueous DES to produce a uniform keratin feedstock. The proposed DES is composed of non-toxic sodium acetate and urea, with a small amount of water. After the DES treatment, water was used to dilute the DES components and regenerate the dissolved keratin. The processing conditions were optimized in terms of keratin yield and properties by varying the dissolution time from 2 h to 24 h and temperature from 80 °C to 100 °C. The yield of regenerated keratin was followed at different sodium acetate-urea molar ratios, and compared to the treatment performed with choline chloride-urea or 8 M urea as reference solvents. Sodium acetate-urea in the molar ratio of 1 : 2 at 100 °C and with 6 h dissolution time dissolved 86% of the feathers with a regenerated keratin yield of 45%. In the characterization of regenerated keratin, it was found that when the dissolution temperature was higher and the dissolution time longer, the disulfide and total sulfur content of feather keratin decreased, the range of molecular weights became wider, and some of the ordered secondary structure and crystallinity were lost.

The impact of fermentation with exopolysaccharide producing lactic acid bacteria on rheological, chemical and sensory properties of pureed carrots (Daucus carota L.).

Fermentation with lactic acid bacteria (LAB) offers a natural means to modify technological and nutritional properties of foods and food ingredients. This study explored the impact of fermentation with different exopolysaccharide (EPS) producing LAB on rheological, chemical and sensory properties of puréed carrots in water, as a vegetable model, with the focus on texture formation. The screening of 37 LAB strains for starter selection revealed 16 Lactobacillus, Leuconostoc and Weissella strains capable of EPS (dextran, levan, and/or ß-glucan) production in the carrot raw material. Fermentations with five out of six selected EPS producers modified perceived texture of the liquid carrot model (p<0.05). The formation of low-branched dextran correlated with perceived thickness, whereas the production of ß-glucan correlated with perceived elasticity. Low-branched dextran producing Weissella confusa and Leuconostoc lactis strains produced thick texture accompanied by pleasant odour and flavour. The fermentation with the selected EPS-producing LAB strains is a promising clean label approach to replace hydrocolloid additives as texturizers in vegetable containing products, not only carrot.

Impact of total solid content and extraction pH on enzyme-aided recovery of protein from defatted rapeseed (Brassica rapa L.) press cake and physicochemical properties of the protein fractions.

Pectinase treatment was used to facilitate protein recovery from defatted rapeseed (Brassica rapa) cold-pressing residue in water-lean conditions and without pH adjustment. Effect of extraction pH on protein yield and physiochemical properties of the protein concentrates was assessed. Enzymatic hydrolysis of carbohydrates was feasible at high (40%) solid content and improved protein recovery at pH 6. Comparable protein yields (40-41% of total protein) from enzyme-aided water extraction (pH 6) and nonenzymatic alkaline extraction (pH10) at 10% solid content suggested that after enzymatic treatment, rapeseed protein could be extracted without exposure to alkali. However, water extraction required dilute conditions, whereas alkaline extraction was feasible also at 20% solid content. The water extracts possessed better protein solubility, higher ζ-potential, and smaller particle size than isoelectric precipitates from alkaline extraction, indicating higher dispersion stability. This is suggested to be mediated by electrostatic interactions between proteins and pectic carbohydrates in the water extracts.

Effect of enzyme-aided cell wall disintegration on protein extractability from intact and dehulled rapeseed (Brassica rapa L. and Brassica napus L.) press cakes.

Cell-wall- and pectin-degrading enzyme preparations were used to enhance extractability of proteins from rapeseed press cake. Rapeseed press cakes from cold pressing of intact Brassica rapa and partially dehulled Brassica napus seeds, containing 36-40% protein and 35% carbohydrates, were treated with pectinolytic (Pectinex Ultra SP-L), xylanolytic (Depol 740L), and cellulolytic (Celluclast 1.5L) enzyme preparations. Pectinex caused effective disintegration of embryonic cell walls through hydrolysis of pectic polysaccharides and glucans and increased protein extraction by up to 1.7-fold in comparison to treatment without enzyme addition. Accordingly, 56% and 74% of the total protein in the intact and dehulled press cakes was extracted. Light microscopy of the press cakes suggested the presence of pectins colocalized with proteins inside the embryo cells. Hydrolysis of these intracellular pectins and deconstruction of embryonic cell walls during Pectinex treatment were concluded to relate with enhanced protein release.

Structural changes and allergenic properties of ß-lactoglobulin upon exposure to high-intensity ultrasound.

SCOPE: The aim of this work was to investigate the effects of high-intensity ultrasound (sonication), on the structure and allergenicity of the major cow's milk allergen, beta-lactoglobulin (BLG). METHODS AND RESULTS: Structural changes upon sonication of BLG were monitored by circular dichroism spectroscopy, tryptophan emission fluorescence, hydrophobic dye and retinol binding, as well as digestibility and phenol-oxidase cross-linking capacity. Allergenicity was monitored in individual patients' sera, basophil activation test, and skin prick testing in 41 cow's milk allergy patients. Uncontrolled local temperature changes induced modifications in BLG secondary structure accompanied by formation of dimers, trimers, and oligomers of BLG that were more digestible by pepsin and had reduced retinol binding. Controlled temperature conditions induced changes in secondary structure of BLG without causing formation of oligomers, or changing protein's capacity to bind retinol. Both sonicated forms of BLG had more exposed hydrophobic surfaces than native BLG and underwent facilitated cross-linking reaction with phenol-oxidase. Sonication had a minor effect on IgE-binding properties of BLG. CONCLUSION: Sonication-induced structural changes in major whey allergen were not clinically significant in cow's milk allergy patients. Ultrasound can be a safe procedure for dairy processing as it maintains the nutritional value and does not increase allergenic potential of BLG.

Crosslinking food proteins for improved functionality.

Different possibilities for protein crosslinking are examined in this review, with special emphasis on enzymatic crosslinking and its impact on food structure. Among potential enzymes for protein crosslinking are transglutaminase (TG) and various oxidative enzymes. Crosslinking enzymes can be applied in cereal, dairy, meat, and fish processing to improve the texture of the product. Most of the current commercial applications are based on TG. The reaction mechanisms of the crosslinking enzymes differ, which in turn results in different technological properties.

Oxidation of peptides and proteins by Trichoderma reesei and Agaricus bisporus tyrosinases.

The capability of a novel tyrosinase from Trichoderma reesei (TrTyr) to catalyse the oxidation and oxidative cross-linking of l-tyrosine (l-Y) and tyrosine side-chains in GYG and EGVYVHPV peptides, in bovine serum albumin (BSA) and beta-casein proteins as well as in proteinaceous wool fibres was studied by oxygen consumption measurement, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase high-performance liquid chromatography (RP-HPLC), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and fluorescence microscopy. TrTyr was compared to the well-characterised tyrosinase from Agaricus bisporus (AbTyr) in terms of oxidation and cross-linking. According to the results obtained TrTyr was capable of cross-linking peptides and proteins more efficiently than AbTyr. However, the size and three-dimensional structure of the proteinaceous substrates proved to be crucial for the success of the enzymatic catalysis. Random coil beta-casein could be cross-linked by TrTyr already in three hours, but large and compact BSA was not cross-linked even in 24h. TrTyr could also be used to incorporate a diphenolic compound, l-dihydroxyphenyl alanine (l-dopa), into protein fibres whereas incorporation of a monophenol, l-Y was less efficient. Thus TrTyr is a potential tool for protein cross-linking and/or modification.

Production in Trichoderma reesei of three xylanases from Chaetomium thermophilum: a recombinant thermoxylanase for biobleaching of kraft pulp.

Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5-7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.

Tyrosinase-catalyzed grafting of sericin peptides onto chitosan and production of protein-polysaccharide bioconjugates.

The capability of Agaricus bisporus tyrosinase to catalyze the oxidation of tyrosine residues of silk sericin was studied under homogeneous reaction conditions, by using sericin peptides purified from industrial wastewater as the substrate. Tyrosinase was able to oxidize about 57% of sericin-bound tyrosine residues. The reaction rate was higher than with silk fibroin, but lower than with other silk-derived model peptides, i.e. tryptic and chymotryptic soluble peptide fractions of silk fibroin, suggesting that the size and the molecular conformation of the substrate influenced the kinetics of the reaction. The concentration of tyrosine in oxidized sericin samples decreased gradually with increasing the enzyme-to-substrate ratio. The average molecular weight of sericin peptides significantly increased by oxidation, indicating that cross-linking occurred via self-condensation of o-quinones and/or coupling with the free amine groups of lysine and, probably, with sulfhydryl groups of cysteine. The high temperature shift of the main thermal transitions observed in the differential scanning calorimetry curves confirmed the formation of peptide species with higher molecular weight and higher thermal stability. Fourier transform-infrared spectra of oxidized sericin samples showed slight changes related to the loss of tyrosine and formation of oxidation products. Oxidized sericin peptides were able to undergo non-enzymatic coupling with chitosan. Infrared spectra provided clear evidence of the formation of sericin-chitosan bioconjugates under homogeneous reaction conditions. Spectral changes in the NH stretching region seem to support the formation of bioconjugates via the Michael addition mechanism.

Enhanced production of cellobiohydrolases in Trichoderma reesei and evaluation of the new preparations in biofinishing of cotton.

In the search for suitable cellulase combinations for industrial biofinishing of cotton, five different types of Trichoderma reesei strains were constructed for elevated cellobiohydrolase production: CBHI overproducers with and without endoglucanase I (EGI), CBHII overproducers with and without endoglucanase II (EGII) and strains overproducing both CBHI and CBHII without the major endoglucanases I and II. One additional copy of cbh1 gene increased production of CBHI protein 1.3-fold, and two copies 1.5-fold according to ELISA (enzyme-linked immunosorbent assay). The level of total secreted proteins was increased in CBHI transformants as compared to the host strain. One copy of the cbh2 expression cassette in which the cbh2 was expressed from the cbh1 promoter increased production of CBHII protein three- to four-fold when compared to the host strain. T. reesei strains producing elevated amounts of both CBHI and CBHII without EGI and EGII were constructed by replacing the egl1 locus with the coding region of the cbh1 gene and the egl2 locus with the coding region of cbh2. The cbh1 was expressed from its own promoter and the cbh2 gene using either the cbh1 or cbh2 promoter. Production of CBHI by the CBH-transformants was increased up to 1.6-fold and production of CBHII up to 3.4-fold as compared with the host strain. Approximately similar amounts of CBHII protein were produced by using cbh1 or cbh2 promoters. When the enzyme preparation with elevated CBHII content was used in biofinishing of cotton, better depilling and visual appearance were achieved than with the wild type preparation; however, the improvement was not as pronounced as with preparations with elevated levels of endoglucanases (EG).