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.

Effect of Bioprocessing on Techno-Functional Properties of Climate-Resilient African Crops, Sorghum and Cowpea.

Sorghum and cowpea are very compatible for intercropping in hot and dry environments, and they also have complementary nutritional compositions. Thus, the crops have the potential to improve food security in regions threatened by climate change. The aim of this study was to investigate different enzymes (carbohydrate-degrading, proteases and phytases) and lactic acid bacteria (LAB) fermentation to improve the techno-functional properties of sorghum and cowpea flours. Results show that sorghum carbohydrates were very resistant to hydrolysis induced by bioprocessing treatments. Most of the protease treatments resulted in low or moderate protein solubilization (from ca. 6.5% to 10%) in sorghum, while the pH adjustment to 8 followed by alkaline protease increased solubility to 40%. With cowpea, protease treatment combined with carbohydrate-degrading enzymes increased the solubility of proteins from 37% up to 61%. With regard to the techno-functional properties, LAB and amylase treatment decreased the sorghum peak paste viscosities (from 504 to 370 and 325 cPa, respectively), while LAB and chemical acidification increased cowpea viscosity (from 282 to 366 and 468 cPa, respectively). When the bioprocessed sorghum and cowpea were tested in breadmaking, only moderate effects were observed, suggesting that the modifications by enzymes and fermentation were not strong enough to improve breadmaking.

Effect of pH and temperature on fibrous structure formation of plant proteins during high-moisture extrusion processing.

This study investigated the effect of pH on fibrous structure formation (protein alignment) during high-moisture extrusion processing of gluten, rice protein, as well as pea protein concentrate, and isolate. The pH of the raw material was shifted to 5 and 7 in water suspension with an acid or base and freeze-dried, after which, conductivity, solubility, water-holding capacity, particle size, and pH were measured. The pH-shifted raw materials were extruded at various temperatures (95-160 °C) and the extrudates were analysed for protein alignment (macro and microstructure), tensile strength, free thiol groups, and cooking properties. In general, all raw materials generated fibrous structure at lower temperatures (115-140 °C) at pH 7 than at pH 5 (135-160 °C). Higher pH and temperature values led to an increased tensile strength and pronounced protein alignment. No such unambiguous link could be observed between the raw material properties and enhanced structure formation. This study showed that the structure formation of the extrudate can be positively influenced by increasing the pH of the raw material, which facilitates the plant protein structuring into appealing meat analogue products.

Impact of Enzymatic Hydrolysis and Microfluidization on the Techno-Functionality of Oat Bran in Suspension and Acid Milk Gel Models.

Oat bran is a nutritionally rich ingredient, but it is underutilized in semi-moist and liquid foods due to technological issues such as high viscosity and sliminess. The aim of this work was to improve the technological properties of oat bran concentrate (OBC) in high-moisture food applications by enzymatic and mechanical treatments. OBC was hydrolyzed with ß-glucanase (OBC-Hyd) and the water-soluble fraction (OBC-Sol) was separated. OBC, OBC-Hyd and OBC-Sol were further microfluidized at 5% dry matter content. Enzymatic treatment and microfluidization of OBC reduced the molecular weight (Mw) of ß-glucan from 2748 kDa to 893 and 350 kDa, respectively, as well as the average particle size of OBC (3.4 and 35 times, respectively). Both treatments increased the extractability of the soluble compounds from the OBC samples (up to 80%) and affected their water retention capacity. OBC in suspension had very high viscosity (969 mPa·s) when heated, which decreased after both enzyme and microfluidization treatments. The colloidal stability of the OBC in suspension was improved, especially after microfluidization. The addition of OBC samples to acid milk gels decreased syneresis, improved the water holding capacity and softened the texture. The changes in the suspension and gel characteristics were linked with reduced ß-glucan Mw and OBC particle size.

Grains - a major source of sustainable protein for health.

Cereal grains are the main dietary source of energy, carbohydrates, and plant proteins world-wide. Currently, only 41% of grains are used for human consumption, and up to 35% are used for animal feed. Cereals have been overlooked as a source of environmentally sustainable and healthy plant proteins and could play a major role in transitioning towards a more sustainable food system for healthy diets. Cereal plant proteins are of good nutritional quality, but lysine is often the limiting amino acid. When consumed as whole grains, cereals provide health-protecting components such as dietary fiber and phytochemicals. Shifting grain use from feed to traditional foods and conceptually new foods and ingredients could improve protein security and alleviate climate change. Rapid development of new grain-based food ingredients and use of grains in new food contexts, such as dairy replacements and meat analogues, could accelerate the transition. This review discusses recent developments and outlines future perspectives for cereal grain use.

Predicting the Properties of Industrially Produced Oat Flours by the Characteristics of Native Oat Grains or Non-Heat-Treated Groats.

The aim of this study was to determine whether the properties of the native oat grain or non-heat-treated groats (laboratory-scale dehulling) can be used to predict the quality of the industrially produced oat flour produced from heat-treated groats. Quality properties such as the color, hectoliter weight, thousand seed weight and hull content of Finnish native grains (n = 30) were determined. Furthermore, the relationship between the properties of the native grains and the chemical composition of the raw oat materials before and after the milling process were studied. A significant relationship (p < 0.01) was observed between the thousand seed weight of the native oat groats and the chemical composition of the industrially produced oat flour. Furthermore, the protein content of the native grains measured by NIT correlated with the chemical composition of the oat flours. These results suggest that the properties of oat flour produced on an industrial scale, including heat treatment, could be predicted based on the properties of native oat grains.

Role of ß-glucan content, molecular weight and phytate in the bile acid binding of oat ß-glucan.

There is controversy about the role of viscosity and co-migrating molecules on the bile acid binding of beta-glucan. Thus, this study aimed to investigate the impact of ß-glucan molecular weight and the content of both ß-glucan and phytate on the mobility of bile acids by modelling intestinal conditions in vitro. Two approaches were used to evaluate factors underlying this binding effect. The first studied bile acid binding capacity of soluble ß-glucan using purified compounds. Viscosity of the ß-glucan solution governed mainly the mobility of bile acid since both a decrease in ß-glucan concentration and degradation of ß-glucan by enzyme hydrolysis resulted in decreased binding. The second approach investigated the trapping of bile acids in the oat bran matrix. Results suggested trapping of bile acids by the ß-glucan gel network. Additionally, hydrolysis of phytate was shown to increase bile acid binding, probably due to better extractability of ß-glucan in this sample.

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.

Dietary Fiber from Oat and Rye Brans Ameliorate Western Diet-Induced Body Weight Gain and Hepatic Inflammation by the Modulation of Short-Chain Fatty Acids, Bile Acids, and Tryptophan Metabolism.

SCOPE: Dietary fiber (DF) induces changes in gut microbiota function and thus modulates the gut environment. How this modulation is associated with metabolic pathways related to the gut is largely unclear. This study aims to investigate differences in metabolites produced by the gut microbiota and their interactions with host metabolism in response to supplementation with two bran fibers. METHODS AND RESULTS: Male C57BL/6N mice are fed a western diet (WD) for 17 weeks. Two groups of mice received a diet enriched with 10% w/w of either oat or rye bran, with each bran containing 50% DF. Microbial metabolites are assessed by measuring cecal short-chain fatty acids (SCFAs), ileal and fecal bile acids (BAs), and the expression of genes related to tryptophan (TRP) metabolism. Both brans lowered body weight gain and ameliorated WD-induced impaired glucose responses, hepatic inflammation, liver enzymes, and gut integrity markers associated with SCFA production, altered BA metabolism, and TRP diversion from the serotonin synthesis pathway to microbial indole production. CONCLUSIONS: Both brans develop a favorable environment in the gut by altering the composition of microbes and modulating produced metabolites. Changes induced in the gut environment by a fiber-enriched diet may explain the amelioration of metabolic disturbances related to WD.

Effect of oat ß-glucan of different molecular weights on fecal bile acids, urine metabolites and pressure in the digestive tract - A human cross over trial.

While the development of oat products often requires altered molecular weight (MW) of ß-glucan, the resulting health implications are currently unclear. This 3-leg crossover trial (n = 14) investigated the effects of the consumption of oat bran with High, Medium and Low MW ß-glucan (average > 1000, 524 and 82 kDa respectively) with 3 consequent meals on oat-derived phenolic compounds in urine (UHPLC-MS/MS), bile acids in feces (UHPLC-QTOF), gastrointestinal conditions (ingestible capsule), and perceived gut well-being. Urine excretion of ferulic acid was higher (p < 0.001, p < 0.001), and the fecal excretion of deoxycholic (p < 0.03, p < 0.02) and chenodeoxycholic (p < 0.06, p < 0.02) acids lower after consumption of Low MW ß-glucan compared with both Medium and High MW ß-glucan. Duodenal pressure was higher after consumption of High MW ß-glucan compared to Medium (p < 0.041) and Low (p < 0.022) MW ß-glucan. The MW of ß-glucan did not affect gut well-being, but the perceptions between females and males differed.

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.

The role of rye bran acidification and in situ dextran formation on structure and texture of high fibre extrudates.

High insoluble dietary fibre content causes challenges with structure and texture in extrusion. This paper focused on studying the structure of extrudate enriched with rye bran modified in different ways. Fermentation of rye bran with dextran-producing Weissella confusa (with 10 g/100 g, 5 g/100 g and 0 g/100 g added sucrose as substrate for dextran production), in situ enzymatic production of dextran in the bran and chemical acidification of bran with lactic acid were compared in extrusion trials. Endosperm rye flour was the base in extrusion, of which 32 g/100 g was substituted for rye bran. Fermentation with dextran production showed similar improvement in extrudate expansion as chemically acidified bran samples (489 and 493%), in comparison with native bran (420%). Similarly, these treatments decreased extrudate hardness and increased crispiness index (CI) (16 N, 0.06 and 14 N, 0.071 respectively) compared to the control (39 N, 0.008). Enzymatically produced dextran did not affect expansion, although it decreased hardness (26 N) and increased CI compared to the control (0.023). Chemical changes in the fermented and acidified rye bran included reduction in insoluble dietary fibre (DF) (19 g/100 g â†’ 17 g/100 g) and increase in soluble DF (5.17 g/100 g â†’ 5.51-7.19 g/100 g), as well as soluble protein (8 g/100 g â†’ 11 g/100 g) content. Lactic acid bacteria fermentation or acidification is therefore a promising method to increase the functionality of rye bran in extrusion.

Development and Consumer Perception of a Snack Machine Producing Customized Spoonable and Drinkable Products Enriched in Dietary Fiber and Protein.

The aim of the study was to evaluate consumer perceptions toward customized snacks produced with a Healthy Snack Machine (HSM) prototype, at-site of the purchase and consumption. The present study had a multi-disciplinary approach including both snack product and HSM development (hardware and user interface). Snack development included both instrumental (viscosity, colloidal stability) and sensory characterization (by trained sensory (N = 10) and consumer (N = 55) panels) of spoonable and drinkable, oat- and dairy-based snack products, fortified with protein and/or dietary fiber. The protein and fiber addition reduced viscosity in spoonable products but did not affect the consistency of drinkable samples. Oat-based samples differed from dairy-based in multiple attributes in sensory profiling. In consumer sample testing, sample odor and taste were the most and least preferred aspects, respectively. In the snack machine testing, a qualitative consumer study (N = 33) showed that the HSM was easy to use, the user interface was clear, the ordering process was quick, and the participants were interested in using the HSM in the future. The snack choices (spoonable/drinkable and dairy/oat base) made by the consumers were distributed equally, but the berry-flavor was preferred over cocoa and vanilla. The most common HSM usage scenarios were "between work/school and hobbies" and "in transit from one place to another".

Comparison of Whole and Gutted Baltic Herring as a Raw Material for Restructured Fish Product Produced by High-Moisture Extrusion Cooking.

Interest in using undervalued forage fish for human consumption has recently increased due to its environmental benefits. However, feasible strategies to process the undervalued fish species to food use are limited. Therefore, this study investigated the possibility to utilise whole (ungutted) Baltic herring as a raw material for hybrid plant-fish meat analogues produced by high-moisture extrusion cooking. The sample properties were compared with ungutted Baltic herring. Produced meat analogues showed sufficiently high microbial quality, with spoilage microbes showing growth levels of under 1.4 log CFU/g. Whole fish and gutted fish extrudates showed uniform flavour- and odour-related sensory profiles. Colour values of the whole fish (L* 57.8) extrudates were similar to the values of gutted fish extrudates (L* 62.0). The whole and gutted fish extrudates had tensile strength in a cross-cut direction of 25.5 and 46.3 kPa, respectively. This correlated with the tearing force of the extrudates analysed by a trained sensory panel. Furthermore, a more explicit protein network was microscopically observed in gutted fish than in whole fish extrudates. The present study showed that high-moisture extrusion cooking enables the use of whole small-sized fish for human consumption.

In vitro study for investigating the impact of decreasing the molecular weight of oat bran dietary fibre components on the behaviour in small and large intestine.

The objective of this work was to evaluate the role of ß-glucan molecular weight (Mw) and the presence of other carbohydrates on the physiological functionality of oat bran via an in vitro digestion study. A complete approach using three different in vitro digestion models (viscosity of the small intestine digest, reduction of bile acids and on-line measurement of gas evolution) was used to predict the physiological functionality of enzymatically modified oat bran concentrate (OBC). OBC was enzymatically treated with two ß-glucanase preparations at three different levels in order to specifically decrease ß-glucan Mw (Pure: purified ß-glucanase) or ß-glucan and other cell wall polysaccharides (Mix: commercial food-grade cell wall degrading enzyme preparation). The Mw of ß-glucan in OBC was tailored to high (1000 kDa), medium (200-500 kDa) and low (<100 kDa) values. The amount of arabinoxylan-oligosaccharides varied from 0.3 to 4.7 g per 100 g of OBC when OBC was treated with the Mix enzyme at the highest dosage. When the enzymatically treated OBCs were studied in an upper gut model, a decrease in the viscosity of the digest simultaneously with the reduction of ß-glucan Mw was observed. At a similar ß-glucan Mw range, OBC samples treated with the Pure enzyme had lower viscosity than the samples treated with the Mix one, which also contained arabinoxylan-oligosaccharides. After enzymatic hydrolysis, the capacity of OBC to reduce bile acid was decreased regardless of the enzyme treatment used, and a positive correlation was found between ß-glucan Mw and bile acid reduction (r = 0.99**). The production of colonic gases by the enzymatically treated OBC samples in an in vitro colon model showed an inverse correlation between ß-glucan Mw and initial rate of gas formation (r = -0.9**), but no impact of arabinoxylan-oligosaccharides was observed. This study emphasised the complexity of factors affecting the functionality of oat components under physiological conditions and demonstrated the possibility to produce Mw-tailored oat fibre ingredients that could contribute to gut mediated health benefits.

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).

Study into the effect of microfluidisation processing parameters on the physicochemical properties of wheat (Triticum aestivum L.) bran.

The physicochemical properties of wheat bran have an effect on its technofunctional and nutritional profile. The possibility to induce physicochemical modifications in wheat bran using microfluidisation was investigated. An I-optimal experimental design was used to investigate the effect of microfluidisation processing parameters (pressure, number of passes, bran concentration and initial particle size) on important properties of wheat bran (particle size, microstructure, chemical composition, water retention capacity (WRC), extractability, viscosity and sedimentation). With the parameters used in this study, microfluidisation reduced wheat bran median particle size to 14.8 µm and disintegrated starch granules from the attached endosperm. This coincided with an increased extractability of starch and arabinoxylan. While the initial particle size was of minor importance, a higher pressure, larger number of passes and lower bran concentration during microfluidisation resulted in a smaller particle size, higher WRC and extractability, and an increased viscosity and stability in a 2% wheat bran suspension.

Biochemical characterization and technofunctional properties of bioprocessed wheat bran protein isolates.

The effect of three combinations of bioprocessing methods by lactic acid fermentation, cell wall hydrolyzing enzymes and phytase on the biochemical (protein, fat, carbohydrate composition) and technofunctional properties (protein solubility, emulsifying and foaming properties) of wheat bran protein isolates were evaluated. The bioprocessing increased the protein (up to 80%) and fat content (up to 22.8%) in the isolates due to the degradation of starch and soluble pentosans. Additional proteins, globulin 3A and 3C, chitinase, ß-amylase and LMW glutenins, were identified from the electrophoretic pattern of the protein isolate bioprocessed with added enzymes. Generally, the bioprocessed protein isolate had lower protein solubility and stronger net charge in pH below 7, when compared to the protein isolate made without bioprocessing. The emulsifying properties of the protein isolates were not affected by bioprocessing. However, the foaming stability of the protein isolates was nearly doubled by bioprocessing with cell wall hydrolyzing enzymes and phytase.

A Small In Vitro Fermentation Model for Screening the Gut Microbiota Effects of Different Fiber Preparations.

The development of prebiotic fibers requires fast high-throughput screening of their effects on the gut microbiota. We demonstrated the applicability of a mictotiter plate in the in vitro fermentation models for the screening of potentially-prebiotic dietary fibers. The effects of seven rye bran-, oat- and linseed-derived fiber preparations on the human fecal microbiota composition and short-chain fatty acid production were studied. The model was also used to study whether fibers can alleviate the harmful effects of amoxicillin-clavulanate on the microbiota. The antibiotic induced a shift in the bacterial community in the absence of fibers by decreasing the relative amounts of Bifidobacteriaceae, Bacteroidaceae, Prevotellaceae, Lachnospiraceae and Ruminococcaceae, and increasing proteobacterial Sutterilaceae levels from 1% to 11% of the total microbiota. The fermentation of rye bran, enzymatically treated rye bran, its insoluble fraction, soluble oat fiber and a mixture of rye fiber:soluble oat fiber:linseed resulted in a significant increase in butyrate production and a bifidogenic effect in the absence of the antibiotic. These fibers were also able to counteract the negative effects of the antibiotic and prevent the decrease in the relative amount of bifidobacteria. Insoluble and soluble rye bran fractions and soluble oat fiber were the best for controlling the level of proteobacteria at the level below 2%.