Production of bovine beta-lactoglobulin and hen egg ovalbumin by Trichoderma reesei using precision fermentation technology and testing of their techno-functional properties.

The food protein ingredient market is dominated by dairy and egg proteins. Both milk whey and egg proteins are challenging proteins to replace, e.g. with plant proteins, due to the unique structural features of the animal proteins that render them highly functional. Thus, to provide a non-animal source of these important proteins the fungal host Trichoderma reesei was utilized for the biotechnical production of recombinant hen ovalbumin (TrOVA) and bovine beta lactoglobulin (TrBLG). These food proteins were investigated using two different promoter systems to test the concept of effectively expressing them in a fungal host. Both proteins were successfully produced in 24 well plate and bioreactor scale. The production level of TrBLG and TrOVA were 1 g/L and 2 g/L, respectively. Both proteins were further purified and characterized, and their functional properties were tested. TrBLG and TrOVA secondary structures determined by circular dichroism corresponded to the proteins of bovine and hen. The T. reesei produced proteins were found to be N-glycosylated, mostly with Man 5. TrBLG had emulsification properties matching to corresponding bovine protein. TrOVA showed excellent foaming characteristics and heat-induced gelation, although the strength of the gel was somewhat lower than with hen ovalbumin, possibly due to the partial degradation of TrOVA or presence of other host proteins. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming. Industrial relevance: The food protein ingredient market is dominated by dairy (largely whey proteins) and egg proteins. Whey proteins are valuable and versatile food ingredients due to their functional and nutritional quality. They are largely used in meat and milk products, low fat products, bakery, confectionary, infant formulas and sports nutrition. Similarly, egg white protein ovalbumin is a highly functional protein ingredient that facilitates structure formation and high nutritional quality in most food products. Together they comprise 40-70% of the revenue in the animal protein ingredients market. Both whey and egg proteins are extremely challenging proteins to replace, e.g., by plant proteins due to their unique structural features that render them with high functionality. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming.

Impact of ultra-fine milling and air classification on biochemical and techno-functional characteristics of wheat and rye bran.

Dry milling and air classification were applied to produce three different ingredients from wheat and rye brans. Dried and pin disc-milled brans having particle size medians of 89-131 µm were air classified to produce protein- and soluble dietary fibre-enriched hybrid ingredients (median particle size 7-9 µm) and additionally brans were ultra-finely milled (median particle size 17-19 µm). The samples were characterised in regard to their composition and techno-functional properties. In air classification, protein content increased from 16.4 and 14.7% to 30.9 and 30.7% for wheat and rye brans, which corresponded to protein separation efficiencies of 18.0 and 26.9%, respectively. Concurrently, the ratio between soluble and insoluble dietary fibre increased from 0.22 to 0.85 for wheat and from 0.56 to 1.75 for rye bran. The protein- and soluble dietary fibre-enriched wheat bran fraction showed improved protein solubility at alkaline pH when compared to pin disc- and ultra-finely-milled wheat bran, whereas less difference between the wheat ingredients was observed at native and acidic pH. The protein- and soluble dietary fibre-enriched rye bran fraction exhibited lower solubility than the pin disc- or ultra-finely-milled rye brans at all the studied pH-values. Ultra-fine milling alone decreased protein solubility and increased damaged starch content when compared to the pin disc-milled brans. Both protein enrichment and ultra-fine milling improved colloidal stability in comparison to the pin disc-milled raw materials. The lowest water and oil binding capacities were obtained for the protein-enriched fractions. Ultrasound-assisted emulsification of the protein- and soluble dietary fibre-enriched fractions and the ultra-finely-milled brans revealed no major differences in the visual quality or stability of the emulsions. The results suggest that modification of the techno-functional properties of cereal brans may be acquired via both air classification and ultra-fine milling.

Ovalbumin production using Trichoderma reesei culture and low-carbon energy could mitigate the environmental impacts of chicken-egg-derived ovalbumin.

Ovalbumin (OVA) produced using the fungus Trichoderma reesei (Tr-OVA) could become a sustainable replacement for chicken egg white protein powder-a widely used ingredient in the food industry. Although the approach can generate OVA at pilot scale, the environmental impacts of industrial-scale production have not been explored. Here, we conducted an anticipatory life cycle assessment using data from a pilot study to compare the impacts of Tr-OVA production with an equivalent functional unit of dried chicken egg white protein produced in Finland, Germany and Poland. Tr-OVA production reduced most agriculture-associated impacts, such as global warming and land use. Increased impacts were mostly related to industrial inputs, such as electricity production, but were also associated with glucose consumption. Switching to low-carbon energy sources could further reduce environmental impact, demonstrating the potential benefits of cellular agriculture over livestock agriculture for OVA production.

Limited hydrolysis of rice endosperm protein for improved techno-functional properties.

Limited hydrolysis of rice endosperm protein isolate was carried out with acid and neutral endoproteases to evaluate the relationship between degree of hydrolysis and techno-functional properties. The highest studied degree of hydrolysis was 5.4% corresponding to 55.2% protein solubility. Solubility increased as a function of degree of hydrolysis with higher efficiency by acid endoprotease. Colloidal stability of the protein suspensions steadily increased with increasing degree of hydrolysis. Higher colloidal stability values were achieved by neutral endoprotease (31-89%) compared to that by acid endoprotease (20-75%). On the other hand, the absolute values of zeta potential and surface hydrophobicity decreased as a function of degree of hydrolysis leading to higher values by neutral endoprotease (-21.4 mV and 21.7 mV) than by acid endoprotease (-813.4 mV and 11.7 mV). Foaming, gel formation and water holding properties improved only until degree of hydrolysis values of 1.5% (neural endoprotease) and 1.9% (acid endoprotease).

Structuring colloidal oat and faba bean protein particles via enzymatic modification.

Oat and faba bean protein isolates were treated with transglutaminase from Streptomyces mobaraensis and tyrosinase from Trichoderma reesei to modify the colloidal properties of protein particles in order to improve their colloidal stability and foaming properties. Transglutaminase crosslinked faba bean protein extensively already with 10nkat/g enzyme dosage. Oat protein was crosslinked to some extent with transglutaminase with higher dosages (100 and 1000nkat/g). Transglutaminase increased the absolute zeta-potential values and reduced the particle size of oat protein particles. As a result, the colloidal stability and foaming properties were improved. Tyrosinase had limited crosslinking ability on both plant protein materials. Tyrosinase greatly reduced the solubility of oat protein despite limited crosslinking. Tyrosinase did not have effect on zeta-potential or colloidal stability of either protein, but it impaired foaming properties of both. Thus, the crosslinking enzymes studied caused significantly different end product functionality, presumably due to the different mechanism of action.

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.

One-step method for isolation and purification of native ß-lactoglobulin from bovine whey.

BACKGROUND: The major whey protein ß-lactoglobulin (BLG) has been widely studied for its functional properties. The aim of this study was to develop an efficient, inexpensive and rapid one-step method for the isolation and purification of BLG while preserving its native structure. RESULTS: BLG was purified from defatted whey obtained from raw cow's milk by anion exchange chromatography. Protein purity and identity were determined using reverse phase high-performance liquid chromatography and mass spectrometry. Total BLG yield was 80% with protein purity from 97 to 99%. BLG isoforms A and B were separated into fractions of 91 and 99% purity respectively. The structure and native conformation of the isolated BLG were compared with those of standard commercial BLG by circular dichroism spectrometry, susceptibility to various crosslinking enzymes and enzyme-linked immunosorbent assay inhibition. CONCLUSION: The proposed method is very useful for the rapid preparation of BLG suitable for studying antigenic and molecular characteristics of this protein, as well as the effect of food processing on these properties. The procedure requires only 1 day for the purification of about 300 mg of BLG from a single run using a small column (2.5 cm × 20 cm) of diethylaminoethyl Sephadex and has potential for scaling up.

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.