Search

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.

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.

Circular Bioeconomy – the symbiosis of bio- and circular economies is widely accepted as a solution for ensuring wise and frugal use of bioresources, and provide means to mitigate climate change. In this chapter, we apply circularity concept to scrutinize the use of bioresources and discuss their upcycling circularity potential during and at the end of life of the products and materials. Furthermore, the relationship, including potential synergies and conflicts, between circular bioeconomy and climate change mitigation is studied. The covered sectors include food, packaging, buildings and infrastructure and energy use of regenerated and produced bioresources.

Feather degrading bacteria from birds' nests were characterized to find safe bacterial strains that could be utilized to convert feathers into soluble form and bacterial biomass for feed purposes. Of all tested 571 isolates 122 were keratinolytic. Partial sequencing of the 16S rRNA gene revealed that the keratinolytic isolates represented Proteobacteria (genera Pseudomonas and Stenotrophomonas) and Firmicutes (genera Bacillus, Exiguobacterium, Paenibacillus, Rummeliibacillus, and Sporosarcina). Bacillus cereus group and gram-negative bacterial isolates were not further characterized due to safety concerns related to potential pathogenicity or potential exposure of animals or workers to endotoxins (lipopolysaccharide part of the gram-negative cell wall). Keratinolytic strains showed clear differences in their ability to solubilize feathers with feather weight losses up to 30%. Amino acid composition of feather hydrolysates shifted from feather composition towards more bacterial-like composition. Especially the levels of lysine could be increased in the feather hydrolysate with bacterial fermentation. Feathers can be effectively hydrolyzed with non-pathogenic bacteria without any additional nutrients. The resulting feather hydrolysate is easier to digest than feather material and it has an improved amino acid composition regarding some limiting amino acids. The amino acid content of the final product can be tailored by varying the fermentation time and thus the ratio of bacterial cells to the feather hydrolysate.

Aqueous dispersion properties of a protein concentrate obtained from turnip rapeseed (Brassica rapa) press cake were investigated. Protein content of the press cake was enriched from 35.9% to 45.8% by sequential supercritical-CO2 extraction, milling and air classification. The protein denaturation degree and the solubility in the prepared protein concentrate were compared with a commercial canola protein isolate. Partial loss of the protein native structure was observed in the protein concentrate. The ζ-potential in the soluble fraction of the protein concentrate dispersion was ca. 20 mV lower (more negative) compared with the commercial isolate at pHs 4–10, which was attributed to the presence of charged carbohydrates such as pectin in the protein concentrate. To improve the dispersion stability of the protein concentrate, enzymatic crosslinking with transglutaminase and microfluidization at 1850 bars were applied. Microfluidization resulted in the formation of rod-like aggregates formed mainly of the insoluble cell wall polysaccharides glued together with proteins as imaged by confocal laser scanning microscopy. Improvement in colloidal stability was observed in all samples upon microfluidization. Pre-treatment with transglutaminase resulted in different distributions of the components in the rods and further improved dispersion stability against sedimentation. Heat treatment caused compaction of the structures.

The aims were to assess how particle size reduction and carbohydrate-hydrolyzing enzyme treatment influence protein recovery from rapeseed cold-pressed cake and to determine the effect of these pretreatments in protein extraction procedures varying in ionic strength, pH, and total solid content. Defatted press cake (median particle size 600 μm) was milled to 21–164 μm and 7 μm median particle sizes by pin disc milling and air-flow milling, respectively. The milled press cake samples were treated with a carbohydrate-hydrolyzing enzyme preparation, after which proteins were extracted in saline (pH 6) or alkaline (pH 12) buffer at 5 % solid content, or in water at 20 % solid content. Particle size reduction of the press cake did not influence enzyme action or protein yield, suggesting that protein release from the press cake is not physically limited by cell walls or internal cell structures. As an exception, protein release from the aleuronic cells appeared to be hindered by intact cell walls. Enzyme treatment improved protein recovery, more substantially when the extraction was carried out in water at 20 % solid content than in saline or alkaline conditions at 5 % solid content. The enzyme mediated its positive effect most probably by reducing the water holding capacity of the press cake, thereby facilitating solid-liquid separation, and releasing anionic compounds which improved protein solubility through electrostatic stabilization. The results suggest that carbohydrate-hydrolyzing enzymes are beneficial for rapeseed protein extraction at reduced water content or when no salt or alkali is added to increase protein solubility.

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 ?-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 b 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.

SynopsisObjective Despite the great number of substances produced by the skincare industry, very few of them seem to truly have an effect on the skin. Therefore, given the social implications surrounding physical appearance, the search for new bioactive compounds to prevent or attenuate skin ageing and enhance self-image is a priority of current research. In this context, being rich in valuable compounds, such as proteins, phenolics, lipids and vitamins, this study is focused on the potential activity of rapeseed press cake hydrolysates to be used as raw materials for skincare applications. Methods In this study, the protein-rich press residue from the rapeseed oil industry was converted enzymatically into short-chain biologically active peptides using four protease products with varying substrate specificity – Alcalase 2.4L FG, Protex 6L, Protamex and Corolase 7089. The antioxidant, anti-wrinkle and anti-inflammatory activities of the obtained hydrolysates were evaluated in vitro while their biocompatibility with human skin fibroblasts was tested. Results All hydrolysates were biocompatible with skin fibroblasts after 24 h of exposure, while the non-hydrolysed extract induced cell toxicity. Alcalase 2,4L FG and Protex 6L-obtained hydrolysates were the most promising extracts showing improved bioactivities suitable for skin anti-ageing formulations, namely antioxidant activity, inhibiting approximately 80% cellular reactive oxidative species, anti-inflammatory and anti-wrinkle properties, inhibiting around 36% of myeloperoxidase activity and over 83% of elastase activity. Conclusion The enzymatic technology applied to the rapeseed oil industry costream results in the release of bioactive compounds suitable for skincare applications.

Milk fat was treated with the sn- 2 specific PAN-2 lipase from Pseudozyma antarctica with the aim of achieving selective removal of saturated fatty acids with chain lengths between C12 and C16, which are abundant at the sn- 2 position. Hydrolysis was also carried out using the non-specific CRU-NS from Candida rugosa and the 1,3-specific TLA-1,3 from Thermomyces lanuginosus . PAN-2 treatment decreased the proportion of the target fatty acids considerably (content of 28%, w/w) and increased the proportion of unsaturated ones (content of 52%, w/w) at the degree of hydrolysis of 48% (mol/mol) in relation to the unhydrolysed substrate (47%, w/w, C12:0-C14:0 and 32%, w/w, unsaturated). The fatty acid distributions of the TLA-1,3 and CRU-NS treated samples were similar to untreated milk fat. The proportion of crystalline fat was smaller and the firmness lower in the PAN-2 treated milk fat samples than in the untreated milk fat at 5 °C.

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 ?- 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.

Grass silage is a promising biorefinery feedstock with surplus production potential, providing a source of readily soluble protein and lignocellulosic fibre. This study presents a concept combining protein extraction with production of single cell protein from enzymatically saccharified grass silage fibre by fermentation of the filamentous fungus Paecilomyces variotii. Steam explosion and ammonia soaking were compared as pretreatments, leading to 81.2% and 88.1% carbohydrate hydrolysability, respectively. Microbial biomass yields of 51% from hydrolysate sugars were reached with a protein content of 51% of cell dry weight. A single-reactor ammonia pretreatment and hydrolysis process was demonstrated, including ammonia recovery of up to 66%, while the residual ammonia was synergistically utilized as a nitrogen source for protein production. The effect of cellulase dosage, hydrolysis time and solids concentration was empirically modelled and the model was applied for cost optimization of enzymatic hydrolysis as a part of a techno-economic assessment of the process.

Water retention in transglutaminase (TG)-treated acid milk gels was studied and linked with the gel formation dynamics. Heat-treated skim milk with and without pre-treatment by TG was acidified at 20 degrees C, 30 degrees C and 40 degrees C at constant glucono-delta-lactone (GDL) level to obtain different acidification rates. Formation dynamics and structural properties of acid-induced gels were followed by rheological and near-infrared light backscattering measurements as well as microscopy. TG-treated gels showed decreased tan delta values all through the acidification, which was pronounced around the gelation point. Backscattered light intensity was lowered in TG-treated gels compared to the controls indicating that TG-treated gels were comprised of smaller aggregates. Water holding capacity (WHC) was measured by using centrifugation at selected pH points (pH 5.2, 5.0, 4.8 and 4.6) during acidification. Both acidification temperature and TG treatment had significant effects on the water retention properties of the gels. Spontaneous syneresis observed at high acidification temperatures (>= 30 degrees C) was prevented upon TG-treatment. WHC of TG-treated gels was significantly higher compared to the control gels at all pH points. TG-treated milk gels showed a homogeneous network formed of smaller aggregate and pore sizes at the gelation point and did not show any large-scale re-organisation thereafter. Transglutaminase is likely to act as a fixative of the protein network at an early stage of gelation and thereby limiting network rearrangements that take place in acid milk gels formed at high acidification temperatures leading to contraction and subsequent wheying off. (C) 2012 Elsevier Ltd. All rights reserved.

Effective and controlled use of cross-linking enzymes in structure engineering of food systems depends on characterization of the favorable conditions for enzyme-substrate complex and the limiting factors for the desired modification. In this respect, we analyzed the susceptibility of bovine β-lactoglobulin (BLG) to enzymatic cross-linking by Trichoderma reesei tyrosinase (TrTyr) and transglutaminase (TG). Changes in BLG molecular structure were determined at pH 6.8, 7.5 and 9.0 before and after high-temperature heat treatment. The conformational change was linked to efficiency of protein cross-linking. BLG was not susceptible to TrTyr without heat treatment. TG, however, induced inter-molecular cross-links at pH 7.5 and 9.0. After the heat treatments, BLG molecules adopted a molten-globule-like conformation. Both of the enzymes were able to form inter-molecular cross-links between heat-denatured BLG molecules. Electrophoretic mobility and broadness of the oligomer bands created by both enzymes on SDS-PAGE gels showed differences which were linked to the availability and number of target amino acid residues. Evidence for intra-molecular cross-linking was obtained. Once adsorbed to air/water interface, BLG formed a viscoelastic surface film which was characterized by surface shear rheology. Application of cross-linking enzymes under a dense layer of BLG molecules at the interface led to decreasing G′ with time. Intra-molecular links were most probably favored against inter-molecular on packed BLG layer leading to constrained molecules. Results in general emphasize the importance of structural and colloidal aspects of protein molecules in controlling inter/intra-molecular bond formation by cross-linking enzymes.

Wool fibres are subject to a variety of industrial chemical treatments prior to finishing processes. The influence of such treatments on subsequent enzymatic treatments is still poorly characterised, but its importance is paramount to achieve maximal efficiency of enzymatic action on the fibres. The effect of such treatments on wool fibre structure and their significance on the accessibility of the enzymes transglutaminase and tyrosinase to their target amino acids within wool proteins was investigated. These enzymes are of great interest due to their potential as protein cross‐linkers and their capability to covalently graft beneficial compounds onto proteins. The accessibility of target amino acids was assessed using specific peptides tagged with fluorescent probes, followed by epifluorescence or confocal microscopy. Differences in the action of the different chemical pretreatments were found, with a treatment with permonosulphuric acid resulting in the greatest level accessibility to both enzymes, followed by chlorination and plasma treatments. This was confirmed by the increase in tensile strength of treated wool observed in the enzyme‐treated yarns. This work illustrates that the accessibility of these enzymes to their target aminoacids in solid substrates such as wool is key for their action and to achieve the desired property modifications. The techniques used have the advantage of permitting the visual monitoring of the diffusion of the active enzymes through the wool fibre structure and to relate this to the extent of enzymatic reaction. Crucially, this will allow an informed choice of the best wool processing stage at which to introduce such enzyme treatments.

The aim of this work was to study the potential of tyrosinase enzymes in structural engineering of acid-induced milk protein gels. Fat free raw milk, heated milk or a sodium caseinate solution were treated with tyrosinases from Trichoderma reesei (TrTyr) and Agaricus bisporus (AbTyr) and the reference enzyme transglutaminase (TG) prior to acid-induced gelation. TrTyr treatment increased the firmness of raw milk and sodium caseinate gels, but not that of heated milk gels, even though protein cross-linking was detected in heated milk. AbTyr did not cross-link proteins in any of the studied milk protein systems. TG was superior to TrTyr in gels prepared of heated milk. In acidified heated milk and sodium caseinate, TrTyr and TG treatment resulted in a decrease of the pore size. Scanning electron microscopy revealed more extensive particle interactions in the heated milk gels with TG than with TrTyr.

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.

The effect of transglutaminase (TG)-induced cross-linking on interfacial rheology and foam stability was investigated in sodium caseinate systems. Higher dilatational surface moduli values were found, when cross-linking was performed at an equilibrated interface. However, cross-linking prior to adsorption was not found to affect the moduli values, but increased the equilibrium surface tension value. Foaming ability of cross-linked sodium caseinate was little reduced, when short reaction times with the enzyme were used, but a considerable loss in foaming ability was observed with extensive cross-linking prior to foaming. Stability against drainage was increased with increasing reaction time. With 15 min reaction at 40 °C with 200 nkat/g TG, no liquid drainage was observed 2 h after foaming from 10% sodium caseinate foam. Much smaller bubbles were found in enzyme-treated foams as compared with the reference sodium caseinate foam at 30 min, 2 h and 24 h after foaming. TG-treatment retarded but did not inhibit bubble coarsening in foams. Most likely, the effects in stability were due to increase in continuous phase viscosity.

Trametes hirsuta laccase (EC 1.10.32) alone was not able to cross-link proteins effectively unless high dosages were used. Incorporation of ferulic acid enhanced the formation of intermolecular cross-links. Cross-linking was more effective when the reaction was carried out at 45 °C rather than at room temperature. Size exclusion chromatography showed that ferulic acid monomers disappeared from the solution in the presence of laccase. Force at rupture point of the caseinate gels treated with laccase without ferulic acid was not significantly different from the control, while stronger gels were achieved when ferulic acid was present. Rheological measurements showed that laccase treatment together with ferulic acid resulted in higher G′ values than the control. Gels were analyzed with confocal laser scanning microscope. A finer network was observed when the enzyme was used together with ferulic acid. Slight proteolytic activity resulted in a negative effect on the gel firmness and microstructure when laccase was used without ferulic acid.

Potato peel mass is a costream produced in large quantities by the food industry. Its availability and the presence of starch (46%), pectin, and cellulose make it a potential renewable raw material for polymer products. In this study, biopolymer films were produced from potato peel mass and glycerol. High-pressure homogenization (HPH) and HPH combined with heat treatment were investigated as pretreatment technologies before film casting. HPH-treated potato peel mass yielded biopolymer films with similar barrier and mechanical properties as films prepared from pure potato starch, including complete impermeability to grease. Additional heat treatment of the peel mass enhanced starch gelatinization, resulting in improved barrier properties and smoother surface topography of the films. Films with similar appearance and quality were also obtained from starch-free potato peel mass, indicating that potato fiber rich in pectin and cellulose is likewise a suitable material for biopolymer film preparation after HPH treatment. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 42862.

This work evaluated the effects of processing faba bean flour on textural, structural and sensory properties of gluten-free pasta. Pasta was prepared using faba bean flour, starch-rich fraction of faba bean flour or faba bean flour fermented with lactic acid bacteria. The impact of cross-linking enzyme transglutaminase (TG) on the quality of faba pasta was also studied. The structure, cooking quality, starch digestibility, textural and sensory characteristics of faba pasta samples were evaluated and compared to semolina pasta. Pasta prepared with faba bean and fermented faba bean flours had higher cooking loss (10.8–11.5%) and lower water absorption (130–160%) than semolina pasta (6 and 193%), but pasta prepared from fractionated faba flour had similar water absorption to semolina pasta. The texture of pasta made with faba bean flour was comparable to that of semolina pasta. Fermentation adversely affected the texture by increasing hardness, chewiness, sourness and flavour intensity. Starch hydrolysis index of pasta prepared with the three faba bean flours was similar to semolina pasta (46–50). TG reduced the in vitro starch hydrolysis index and increased some textural parameters of pasta made with faba bean flour, but no influence was observed on pasta made with fractionated or fermented faba.

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.

The effects of laccase and transglutaminase (TG) on the firmness and weight loss of cooked chicken meat homogenate gels were investigated at laboratory scale. The salt, trisodium pyrophosphate and meat contents were also used as variables. Laccase decreased firmness and increased weight loss of phosphate-free, low-meat (65%) and low-salt (1%) gels, although it modified myosin and troponin T and reacted with isolated myofibrils. By applying both low-salt (1%) and low-phosphate (0.17%) amounts, gel firmness decreased and weight loss increased (p

The effects of Trichoderma reesei tyrosinase-catalyzed cross-linking of isolated chicken breast myofibril proteins as a simplified model system were studied with special emphasis on the thermal stability and gel formation of myofibrillar proteins. In addition, tyrosinase-catalyzed cross-linking was utilized to modify the firmness, water-holding capacity (WHC), and microstructure of cooked chicken breast meat homogenate gels. According to SDS-PAGE, the myosin heavy chain (MHC) and troponin T were the most sensitive proteins to the action of tyrosinase, whereas actin was not affected to the same extent. Calorimetric enthalpy (DeltaH) of the major thermal transition associated with myosin denaturation was reduced and with actin denaturation increased in the presence of tyrosinase. Low-amplitude viscoelastic measurements at constant temperatures of 25 degrees C and 40 degrees C showed that tyrosinase substantially increased the storage modulus (G') of the 4% myofibrillar protein suspension in the 0.35 M NaCl concentration. The effect was the most pronounced with high-enzyme dosages and at 40 degrees C. Without tyrosinase, the G' increase was low. Tyrosinase increased the firmness of the cooked phosphate-free and low-meat chicken breast meat homogenate gels compared to the corresponding controls. Tyrosinase maintained gel firmness at the control level of the low-salt homogenate gel and weakened it when both salt and phosphate levels were low. Tyrosinase improved the WHC of the low-meat and low-salt homogenate gels and maintained it at the level of the corresponding controls of phosphate-free and low-salt/low-phosphate homogenate gels. Microstructural characterization showed that a collagen network was formed in the presence of tyrosinase.Chicken myofibrillar proteins; protein modification; cross-linking; tyrosinase; gelation; thermal stability; texture; water-holding capacity; microstructure.

Effects of microbial transglutaminase (mTG), mushroom tyrosinase and an apple powder containing transglutaminase (TG) and polyphenol oxidase (PPO) on the structure of industrial pork meat homogenate were studied. Apple powder and mTG both increased the value of storage modulus (G′). Mushroom tyrosinase was not able to affect gel forming with the used dosages and treatment conditions. All the enzymes studied were able to improve gel hardness of unheated meat homogenate at 4 °C to a certain extent. In gel hardness measurements added cysteine affected positively on the apple powder—treated pork meat and negatively on mTG—and tyrosinase-treated meat. Surprisingly cysteine addition nullified also the action of mTG. Hence powdered apple pulp, a recovered co-product of an industrial process, may contain suitable enzyme activities for food protein stabilization.

Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80°C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80°C.

Culture supernatants from strains of Melanocarpus albomyces, Myceliophthora thermophila, Chaetomium thermophilum, and Sporotrichum thermophilum were tested for their ability to release dye in neutral pH conditions from indigo-dyed cotton-containing fabric in biostoning applications. The supernatants from M. albomyces worked well in biostoning, with low backstaining. Three cellulases were purified to homogeneity from the culture medium of this species. Two of the cellulases were endoglucanases with apparent molecular masses of 20 and 50 kDa. The 20 kDa endoglucanase was a relatively heat-stable cellulase with high pH optimum. The partially purified enzyme crystallized spontaneously at pH 4.0 and 7 °C. The 50 kDa endoglucanase also had activity against 4-methylumbelliferyl-β-d-lactoside (MUL) and was active over a wide range of pH values. The third purified cellulase was the 50 kDa cellobiohydrolase with low MUL activity at acidic pH and detectable activity towards filter paper and acid swollen Solca Floc-cellulose, but no endoglucanase activity. The purified 20 kDa endoglucanase performed well in biostoning of denim fabric at neutral pH. Addition of the purified 50 kDa endoglucanase or the 50 kDa cellobiohydrolase to the 20 kDa endoglucanase decreased backstaining in biostoning.

The effects of Myceliophthora thermophila laccase are studied alone and in combina tion with the mediators violuric acid (VA) or l-hydroxybenzotriazole (HBT) on wool fibers, cystine, and tyrosine. Without a mediator, laccase is unable to oxidize wool or the amino acids. In the presence of either VA or HBT, oxygen consumption is observed, indicating oxidation of the secondary substrates of the laccase, i.e., wool, cystine, or tyrosine. The oxidation levels are low. Laccase/HBT reacts more efficiently with wool and tyrosine and laccase/VA with cystine. With the chosen enzyme dosage and mediator concentration, about 2-4% of cystine and about 1 % of tyrosine are oxidized. The slight oxidation of wool fibers is not sufficient to affect the surface chemistry or alkaline solubility of the fibers, although staining of the fibers by the oxidized mediators is significant.

Salmon backbones, co-streams of salmon processing industry, were transformed into stable, odour-free ingredients for cosmetics. First, the backbones were hydrolysed using commercial proteases (Bromelain + Papain, Trypsin, Corolase® 7089 and Protamex®) in order to accomplish the release of fish protein hydrolysates (FPH), which showed antioxidant activity and aptitude to inhibit skin-degrading and inflammatory enzymes. However, due to the FPH instability in aqueous solution and propensity for microbial contamination, their bioactive properties were entirely lost only after 24 h. To overcome the low stability and prevent the effect loss, a sonochemical technology was then employed to transform the FPH into stable tea tree oil-filled bioactive peptide-shell nanospheres (NS). Such transformation boosted the FPH antioxidant potential, which was further reflected in protection of fibroblasts from UV damage. In the form of NSs, the FPH resisted microbial contamination for more than 6 months and presented antimicrobial activity against Escherichia coli and Staphylococcus aureus. In addition, the fish odour was eliminated after the NSs processing, thus addressing this important challenge for using fish raw materials in cosmetics. This work suggests an alternative high value use of the fishery co-streams and expands their application potential beyond their current use as fish or animal feed

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.

A stable strain of Saccharomyces cerevisiae secreting glucoamylase (EC 3.2.1.3) with high debranching activity was constructed using recombinant DNA technology. An expression cassette without bacterial sequences, containing Hormoconis resinae glucoamylase P cDNA and the dominant selection marker MEL1 was integrated into the yeast chromosome using ARS1 homology. The glucoamylase expression level of the integrant yeast strain was increased by chemical mutagenesis. The yeast strains secreting glucoamylase were able to grow on soluble starch (5%, w/v) and ferment it to ethanol.

Kuluttajat haluavat korkealuokkaisia, mutta kuitenkin kohtuuhintaisia lihatuotteita. näihin toiveisiin perustuen lihateollisuuden käyttöön on kehitetty entsymaattisia menetelmiä, joiden avulla heikompilaatuisista ruhonosista saadaan valmistettua hyvälaatuisia, mureita arvolihatuotteita.

Plasma‐activated biaxially oriented polypropylene (BOPP) films and paper substrate have been coated with functional chitosan solutions. Plasma treatment increased the amount of surface peroxide groups and carboxyl groups on the BOPP films. As a result of plasma activation, the surface energy increased from 30 to 50 dynes/cm. The enzyme tyrosinase catalysed the grafting of octyl gallate and dodecyl gallate to amino groups of chitosan polysaccharide. Resulting coatings exhibited strong antimicrobial activity against Gram‐positive Staphylococcus aureus and Gram‐negative Listeria innocua. After 24 h of incubation, a total reduction in both bacteria cell numbers varied between >4.9 and 1.4 logarithmic units. Grafted dodecyl gallate and octyl gallate at pH 6 were found to have the lowest reduction values of S. aureus, while 1.4 logarithmic reduction value was obtained for grafted dodecyl gallate at pH 6 against L. innocua. Chitosan coatings were also effective barrier layers against oxygen transmission although the transmission rates clearly increased in high‐humidity conditions. In dry conditions, however, the transmission rate of 2 cm3/(m2 · 24 h) was obtained with chitosan‐coated BOPP. Coatings did not have any effects on water vapour transmission. Both gallates were successfully grafted at pH 6. As increased flocculation and colour formation indicated, the tyrosinase‐catalysed grafting was more powerful with octyl gallate. Dodecyl gallate containing chitosan coatings was more hydrophobic as compared to octyl gallate. Total migration of substances into 95% ethanol was ≥5 mg/dm2, thus materials may be exploitable in packaging purposes in direct contact with certain foodstuffs.

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 β-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 β-casein could be cross-linked by TrTyr already in three hours, but large and compact BSA was not cross-linked even in 24 h. 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.

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.

Publisher Summary This chapter explains that food texture plays a major role in food product quality. The rheological properties of a food are determined by the number of weak and strong physical interactions and the permanent covalent bonds, crosslinks, present in the food matrix. Covalent crosslinks make the major contribution to the firmness of food matrices. Crosslinks can be introduced to a food matrix by chemical, enzymatic, and physical means as reviewed by Munindar P. Singh (1991) and Scott N. Gerrard (2002) . Enzymatic crosslinking of food biopolymers is an attractive option owing to the specificity of enzymes and mild reaction conditions. Both food proteins and carbohydrates can be crosslinked by enzymes. Sensory perception of texture plays an important role in different types of foods. The significance of food texture has further been increased with the trend toward low fat products and “natural” additive-free products. Enzymes provide specific and natural means for tailoring food structure. By the use of enzymes, it will be possible to transform inherently available food components into functional ingredients during food processing and manufacturing. Enzymatic crosslinking in the food matrix can occur via proteins or certain carbohydrates. The type of enzyme used affects the chemistry of the crosslink formed and subsequently the structure of the biopolymer network of the food product.

The effect of laccase and transglutaminase (TG) on cross-linking, gelation, and thermal stability of salt-soluble chicken-breast myofibril proteins was investigated at pH 6. Both enzymes modified the protein pattern detected by SDS-PAGE. Identification of proteins by peptide mass mapping showed that myosin heavy chain (MHC) and troponin T were the most affected proteins. These proteins faded or disappeared as a function of the incubation time with both enzymes on SDS-PAGE. The molecular weight of actin was not, however, affected by either enzyme. The effects that the enzymes had on the gel formation of chicken-breast myofibrils were studied in 0.35 and 0.60 M NaCl solutions at 3% protein content and a constant temperature of 40 degrees C by using a small deformation viscoelastic measurement. TG substantially increased the storage modulus (G') of 3% protein in 0.35 M NaCl. Without the enzymes, gelation was insignificant in 0.35 M NaCl. The increased solubility of the proteins at 0.60 M NaCl intensified gelation with TG. G' increased 32 and 64% at dosages of 10 and 100 nkat of TG, respectively. Also, laccase increased G' of the gel in 0.60 M salt concentration. However, a high laccase dosage decreased the magnitude of G' below the control level. Differential scanning calorimetric (DSC) measurements indicated slightly reduced myosin heat stability after TG pretreatment and increased actin heat stability with both enzymes. Maximum transition temperatures did not alter with either enzyme.

Laccases (EC 1.10.3.2) and tyrosinases (EC 1.14.18.1) are copper-containing redox enzymes. They oxidize typically various phenolic compounds and use molecular oxygen as an electron acceptor. They are common enzymes in nature and can be found in plants, mammals, and microbes. Laccases have been studied in numerous applications including pulp bleaching, fibre functionalization, baking, and textile dye decolorization (1). Laccases are currently used in large scale applications e.g. in denim bleaching. The enzyme is commercially available. Tyrosinases have been used in enzymatic synthesis or modification of high-value-added compounds like coumestrol, and L-DOPA. Reports on the use of tyrosinases for grafting of chitosan on proteinous fibres have also been published (2). Laccases contain four tightly coordinated copper atoms in their active site. They oxidize their substrates with a one-electron removal mechanism, which usually results in formation of free radicals. Radicals can start to react non-enzymatically e.g. to form polymers or they can be further oxidized enzymatically (3). The biological role of laccases is related to synthesis or degradation of lignin, pigment production, sporulation and pathogenesis. Most of the studies have been performed with fungal laccases, which usually have very broad substrate specificity and reasonably high redox potential. Tyrosinases have two copper atoms in their active site. Laccases and tyrosinases have been traditionally differentiated on the bases of their substrate specificity and sensitivity to inhibitors, although their substrate specificity do overlap. Tyrosinases are capable of oxidizing various mono- and diphenolic compounds, however, the reaction mechanism in tyrosinase-catalyzed reaction is different from the reaction mechanism of laccases (4). Tyrosinases catalyze ortho-hydroxylation of monophenols and subsequent oxidation of diphenols to quinones. Radicals are not formed in tyrosinase-catalyzed reactions. The physiological role of tyrosinases is related to pigment formation. Plant tyrosinases are involved in enzymatic browning reactions. References 1. Xu, F. (1999) Recent progress in laccase study: properties, enzymology, production, and applications. In The Encyclopedia of Bioprocessing Technology: Fermentation, Biocatalysis and Bioseparation (Flickinger, M.C. & Drew, S.W. eds), pp. 1545-1554. John Wiley & Sons, NY. 2. Sampaio, S., Taddei, P., Monti P., Buchert, J. and Freddi G. (2005) Enzymatic grafting of chitosan onto Bombyx mori silk fibroin: kinetic and IR vibrational studies. J. Biotechnology 116:21-33. 3. Thurston, C. (1994) The structure and function of fungal laccases. Microbiology 140:19-26. 4. Decker H., and Tuczek F. (2000) Tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism, TIBS 25: 392-397.

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).

The chemical properties of two different sugar beet pectin preparations and their gel formation due to laccase was studied. The pectins differed in their molecular weight, acetylation and methylation degree. No gelling was observed without laccase addition with the two pectins. The response of laccase to the gelling was also different and gels made of sugar beet pectin SPB with the aid of laccase with or without calcium were significantly harder than that of RU-L sugar beet pectins. In the pH range 3.5–5.0, the hardest texture was measured at pH 4.0. The rate of cross-linking increased with increasing enzyme activity. However, at the highest enzyme activity levels the magnitude of storage modulus after 5.5 h was lower than that of gels containing 100 and 150 nkat laccase/g pectin. The hardness of gels was also measured at large deformation after 1 day storage time. The gel with 67 nkat/g pectin laccase gave hardest gel. At higher laccase activity (100–500 nkat/g) the gel was softer suggesting that gels formed at slower rate had better properties. In the presence of Ca the hardness of gels was independent on laccase activity in the range 67–500 nkat/g. Viscoelastic measurements suggest that Ca retards the rate of covalent cross-linking and improves the texture at high enzyme activities.

The chemical properties of sugar pectin preparate and its gel formation due to laccase were studied. The sugar beet pectin contained significant amounts of galacturonic acid and also rhamnose, arabinose, galactose and ferulic acid groups. No gel formation occurred without laccase. The rate of cross-linking increased with increasing enzyme activity. The gels were softer at higher enzyme activities.