The impact of wounding and postharvest storage conditions on retention of soluble protein in sugar beet leaves.

Sugar beet leaves can be a viable and economically interesting source of high-quality protein for the food industry. We investigated how storage conditions and leaf wounding at harvest affect the content and quality of the soluble protein. After collection, leaves were either stored intact or shredded to mimic wounding induced by commercial leaf harvesters. Leaf material was stored in small volumes at different temperatures to assess leaf physiology or in larger volumes to assess temperature development at different locations in the bins. Protein degradation was more pronounced at higher storage temperatures. Wounding accelerated the degradation of soluble protein at all temperatures. Both wounding and storage at higher temperatures greatly stimulated respiration activity and heat production. At temperatures below 5°C, ribulose-1,5-biphosphate carboxylase oxygenase (RuBisCO) in intact leaves was preserved for up to 3 weeks. At temperatures of 30-40°C, RuBisCO degradation occurred within 48 h. Degradation was more pronounced in shredded leaves. In 0.8-m3 storage bins at ambient temperature, core temperatures rapidly increased, up to 25°C in intact leaves and up to 45°C in shredded leaves within 2-3 days. Immediate storage at 5°C greatly suppressed the temperature increase in intact but not in shredded leaves. The indirect effect of excessive wounding, that is, heat production, is discussed as the pivotal factor responsible for increased degradation of protein. For optimal retention of soluble protein levels and quality in harvested sugar beet leaves, it is advised to minimize wounding and to store the material at temperatures around -5°C. PRACTICAL APPLICATION: To preserve the soluble protein content and quality for at least 3 weeks, sugar beet leaves should be harvested with minimal wounding and stored at temperatures between 1 and 5°C. When aiming to store minimally wounded leaves in larger volumes, it must be ensured that the product temperature in the core of the biomass meets the temperature criterium or the cooling strategy must be adjusted. The principles of minimal wounding and low temperature storage are transferable to other leafy crops that are harvested for food protein.

Fibrous Structures from Starch and Gluten.

Starch is added to meat analogues for binding and water holding. In this study, we investigate whether starch can have an additional role as a structuring agent. Therefore, different types of starch were combined with wheat gluten at various amounts and sheared in a High Temperature Shear Cell to determine how starch influences the structuring behavior of gluten-starch blends. The starches were chosen based on their diverse amylose contents, leading to different technological properties. Remarkable differences were found between the starches investigated. The addition of Amioca starch (containing 1% amylose) had a strong negative influence on the ability of gluten to form fibers. Maize starch (25% amylose) and Hylon VII (68% amylose) formed fibrous materials up to high starch additions. The pre-gelatinizing of maize starch further increased the ability of gluten-starch mixtures to form fibrous structures. The influence of the different types of starch on the hardness, deformability, and stiffness of the sheared samples was also assessed, revealing a spectrum of achievable properties through the addition of starch. Most remarkable was the formation of a material with anisotropy in Young's modules. This anisotropy is also found in chicken meat, but not in protein-based fibrous materials. Furthermore, it was observed that the pre-gelatinization of starch facilitated fiber formation. A similar effect of pre-gelatinizing the starch was found when using faba bean meal with added wheat gluten, where fibrous structures could even be formed in a recipe that previously failed to produce such structures without pre-treatment.

Quantifying water distribution between starch and protein in doughs and gels from mildly refined faba bean fractions.

The development of novel and sustainable food products, such as cheese- and meat analogues, requires a better understanding of the use of less refined ingredients. We investigated the distribution of water between the protein and starch phase of doughs and heat-induced gels made from air-classified faba bean fractions by developing a method suited for investigation of such multi-component ingredients. The moisture contents of the protein and starch phases in the dough were determined using a method based on partial sorption isotherms of mixed doughs of protein- and starch-rich fractions at high water activity. Water content of the protein phase is higher than that of the starch phase in dough, showing that protein takes up more water than starch at room temperature. Also, the moisture content of the protein phase in the gels was calculated using a model based on the denaturation temperature of legumin. From the experiments and the modelling, it became evident that the moisture content of the protein phase in the gel is lower than the moisture content of the protein phase in the dough, showing the importance of considering moisture migration from the protein to the starch during heating.

Partitioning of Proteins and Anti-Nutrients in Cassava (Manihot esculenta Crantz) Leaf Processing Fractions after Mechanical Extraction and Ultrafiltration.

Cassava plays a major role in improving food security and reducing malnutrition. The purpose of this study was to evaluate the influence of mechanical pressing coupled with ultrafiltration (UF) on the quality of different fractions of cassava leaves. Cassava leaves harvested from the greenhouse at the University of Hohenheim were passed through a mechanical screw press to extract the juice and separate the press cake. The juice was centrifuged and filtered to separate the sediment and clear supernatant. The clear supernatant was filtered using a 10 kDa UF system. The nutritional contents of the different fractions were analyzed at each processing step. The total phenolic content was significantly lower in the press cake that had a higher fiber and ash content. The juice and sediment fractions had higher crude protein and total phenolic content. Processing did not negatively affect the concentrations of essential amino acids except for tryptophan in the juice fraction. Non-protein nitrogen was mainly present in the UF permeate, illustrating the potential of UF for upgrading soluble protein fractions. The results indicated that the different fractions during processing could be a possible source of protein for food, feed (juice, sediment, and retentate), or fiber (press cake) for ruminant feed.

Insights in the Recalcitrance of Theasinensin A to Human Gut Microbial Degradation.

Due to low bioavailability of dietary phenolic compounds in small intestine, their metabolism by gut microbiota is gaining increasing attention. The microbial metabolism of theasinensin A (TSA), a bioactive catechin dimer found in black tea, has not been studied yet. Here, TSA was extracted and purified for in vitro fermentation by human fecal microbiota, and epigallocatechin gallate (EGCG) and procyanidin B2 (PCB2) were used for comparison. Despite the similarity in their flavan-3-ol skeletons, metabolic fate of TSA was distinctively different. After degalloylation, its core biphenyl-2,2',3,3',4,4'-hexaol structure remained intact during fermentation. Conversely, EGCG and PCB2 were promptly degraded into a series of hydroxylated phenylcarboxylic acids. Computational analyses comparing TSA and PCB2 revealed that TSA's stronger interflavanic bond and more compact stereo-configuration might underlie its lower fermentability. These insights in the recalcitrance of theasinensins to degradation by human gut microbiota are of key importance for a comprehensive understanding of its health benefits.

Microbial Metabolism of Theaflavin-3,3'-digallate and Its Gut Microbiota Composition Modulatory Effects.

Theaflavin-3,3'-digallate (TFDG), a bioactive black tea phenolic, is poorly absorbed in the small intestine, and it has been suggested that gut microbiota metabolism plays a crucial role in its bioactivities. However, information on its metabolic fate and impact on gut microbiota is limited. Here, TFDG was anaerobically fermented in vitro by human fecal microbiota, and epigallocatechin gallate (EGCG) was used for comparison. Despite the similar flavan-3-ol skeletons, TFDG was more slowly degraded and yielded a distinctively different metabolic profile. The formation of theanaphthoquinone as the main metabolites was unique to TFDG. Additionally, a number of hydroxylated phenylcarboxylic acids were formed with low concentrations, when comparing to EGCG metabolism. Microbiome profiling demonstrated several similarities in gut microbiota modulatory effects, including growth-promoting effects on Bacteroides, Faecalibacterium, Parabacteroides, and Bifidobacterium, and inhibitory effects on Prevotella and Fusobacterium. In conclusion, TFDG and EGCG underwent significantly different microbial metabolic fates, yet their gut microbiota modulatory effects were similar.

Reciprocal Interactions between Epigallocatechin-3-gallate (EGCG) and Human Gut Microbiota In Vitro.

Interaction of tea phenolics with gut microbiota may play an integral role in the health benefits of these bioactive compounds, yet this interaction is not fully understood. Here, the metabolic fate of epigallocatechin-3-gallate (EGCG) and its impact on gut microbiota were integrally investigated via in vitro fermentation. As revealed by ultrahigh performance liquid chromatography hybrid quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS), EGCG was promptly degraded into a series of metabolites, including 4-phenylbutyric acid, 3-(3',4'-dihydroxyphenyl)propionic acid, and 3-(4'-hydroxyphenyl)propionic acid, through consecutive ester hydrolysis, C-ring opening, A-ring fission, dehydroxylation, and aliphatic chain shortening. Microbiome profiling indicated that, compared to the blank, EGCG treatment resulted in stimulation of the beneficial bacteria Bacteroides, Christensenellaceae, and Bifidobacterium. Additionally, the pathogenic bacteria Fusobacterium varium, Bilophila, and Enterobacteriaceae were inhibited. Furthermore, changes in concentrations of metabolites, including 4-phenylbutyric acid and phenylacetic acid, were strongly correlated with changes in the abundance of specific gut microbiota. These reciprocal interactions between EGCG and gut microbiota may collectively contribute to the health benefits of EGCG.

Modifying Faba Bean Protein Concentrate Using Dry Heat to Increase Water Holding Capacity.

We investigated the effect of dry-heat treatment on the properties of faba bean protein concentrate using soy protein concentrate as a benchmark. While soy protein-widely used as an ingredient in meat replacers-is recovered through a wet fractionation, protein recovery from starch bearing pulses like faba bean can be done via dry fractionation. This process does not require drying or heating steps and therefore, keeps the original protein functionality intact. This results in differences in properties such as water binding capacity of the protein fraction. Faba bean protein concentrate was dry-heated at temperatures from 75-175 °C, which resulted in higher water-holding capacity and less soluble protein, approaching values of soy protein concentrate. These changes were due to partial denaturation of protein, changing the structure of the protein, and exposing hydrophobic sites. This led to protein aggregation, as observed by light microscopy. Only noncovalent bonds caused the decrease of solubility of dry-heated faba bean protein concentrate. We conclude that dry-heating of dry fractionated faba bean protein can change the functional properties of the protein fraction to desired properties for certain applications. The effect is similar to that on soy, but the underlying mechanisms differ.

Pulsed Electric Field as an Alternative Pre-treatment for Drying to Enhance Polyphenol Extraction from Fresh Tea Leaves.

Drying is an essential pre-treatment prior to extraction of tea polyphenols from tea leaves, which is a time and energy-intensive process. In this study, pulsed electric field (PEF) was utilized to replace the conventional thermal dehydration procedure before the phenolic extraction. The influence of different PEF conditions on total polyphenol yield from fresh tea leaves combined with a solid-liquid extraction were compared. PEF treatment at 1.00 kV/cm electric field strength, 100 pulses of 100 µs pulse duration, and 5 s pulse repetition, which delivered 22 kJ/kg and induced 1.5 °C of temperature increase, was used for further study on the extraction kinetics of green tea catechins. The results indicated that compared to oven drying, PEF pre-treatment increased the extraction rate by approximately two times, without significantly altering the phenolic profiles, as revealed by using liquid chromatography combined with mass spectrometry. Scanning electron microscopy imaging revealed that PEF pre-treatment induced the formation of inhomogeneously distributed pores and protuberances on the surface of leaf tissues, which might facilitate the penetration of extraction solvent and the migration of phenolics. This study demonstrates that PEF as a time and energy efficient processing method is a promising alternative for the conventional drying process before further tea polyphenol extraction.

Effect of n-Caproate Concentration on Chain Elongation and Competing Processes.

Chain elongation is an open-culture fermentation process that facilitates conversion of organic residues with an additional electron donor, such as ethanol, into valuable n-caproate. Open-culture processes are catalyzed by an undefined consortium of microorganisms which typically also bring undesired (competing) processes. Inhibition of competing processes, such as syntrophic ethanol oxidation, will lead to a more selective n-caproate production process. In this study, we investigated the effect of n-caproate concentration on the specific activity of chain elongation and competing processes using batch inhibition assays. With "synthetic medium sludge" (originally operating at 3.4 g/L n-caproate), syntrophic ethanol oxidation was proportionally inhibited by n-caproate until 45% inhibition at 20 g/L n-caproate. Hydrogenotrophic methanogenesis was for 58% inhibited at 20 g/L n-caproate. Chain elongation of volatile fatty acids (volatile fatty acid upgrading; the desired process), was completely inhibited at 20 g/L n-caproate with all tested sludge types. "Adapted sludge" (operating at 23.2 g/L n-caproate) showed a 10 times higher volatile fatty acid upgrading activity at 15 g/L n-caproate compared to "nonadapted sludge" (operating at 7.1 g/L n-caproate). This shows that open cultures do adapt to perform chain elongation at high n-caproate concentrations which likely inhibits syntrophic ethanol oxidation through hydrogenotrophic methanogenesis. As such, we provide supporting evidence that the formation of n-caproate inhibits syntrophic ethanol oxidation which leads to a more selective medium chain fatty acid production process.

Development of an Effective Chain Elongation Process From Acidified Food Waste and Ethanol Into n-Caproate.

Introduction: Medium chain fatty acids (MCFAs), such as n-caproate, are potential valuable platform chemicals. MCFAs can be produced from low-grade organic residues by anaerobic reactor microbiomes through two subsequent biological processes: hydrolysis combined with acidogenesis and chain elongation. Continuous chain elongation with organic residues becomes effective when the targeted MCFA(s) are produced at high concentrations and rates, while excessive ethanol oxidation and base consumption are limited. The objective of this study was to develop an effective continuous chain elongation process with hydrolyzed and acidified food waste and additional ethanol. Results: We fed acidified food waste (AFW) and ethanol to an anaerobic reactor while operating the reactor at long (4 d) and at short (1 d) hydraulic retention time (HRT). At long HRT, n-caproate was continuously produced (5.5 g/L/d) at an average concentration of 23.4 g/L. The highest n-caproate concentration was 25.7 g/L which is the highest reported n-caproate concentration in a chain elongation process to date. Compared to short HRT (7.1 g/L n-caproate at 5.6 g/L/d), long HRT resulted in 6.2 times less excessive ethanol oxidation. This led to a two times lower ethanol consumption and a two times lower base consumption per produced MCFA at long HRT compared to short HRT. Conclusions: Chain elongation from AFW and ethanol is more effective at long HRT than at short HRT not only because it results in a higher concentration of MCFAs but also because it leads to a more efficient use of ethanol and base. The HRT did not influence the n-caproate production rate. The obtained n-caproate concentration is more than twice as high as the maximum solubility of n-caproic acid in water which is beneficial for its separation from the fermentation broth. This study does not only set the record on the highest n-caproate concentration observed in a chain elongation process to date, it notably demonstrates that such high concentrations can be obtained from AFW under practical circumstances in a continuous process.

Solubility of the Proteinogenic α-Amino Acids in Water, Ethanol, and Ethanol-Water Mixtures.

The addition of organic solvents to α-amino acids in aqueous solution could be an effective method in crystallization. We reviewed the available data on the solubility of α-amino acids in water, water-ethanol mixtures, and ethanol at 298.15 K and 0.1 MPa. The solubility of l-alanine, l-proline, l-arginine, l-cysteine, and l-lysine in water and ethanol mixtures and the solubility of l-alanine, l-proline, l-arginine, l-cysteine, l-lysine, l-asparagine, l-glutamine, l-histidine, and l-leucine in pure ethanol systems were measured and are published here for the first time. The impact on the solubility of amino acids that can convert in solution, l-glutamic acid and l-cysteine, was studied. At lower concentrations, only the ninhydrin method and the ultraperfomance liquid chromatography (UPLC) method yield reliable results. In the case of α-amino acids that convert in solution, only the UPLC method was able to discern between the different α-amino acids and yields reliable results. Our results demonstrate that α-amino acids with similar physical structures have similar changes in solubility in mixed water/ethanol mixtures. The solubility of l-tryptophan increased at moderate ethanol concentrations.

Controlling Ethanol Use in Chain Elongation by CO2 Loading Rate.

Chain elongation is an open-culture biotechnological process which converts volatile fatty acids (VFAs) into medium chain fatty acids (MCFAs) using ethanol and other reduced substrates. The objective of this study was to investigate the quantitative effect of CO2 loading rate on ethanol usages in a chain elongation process. We supplied different rates of CO2 to a continuously stirred anaerobic reactor, fed with ethanol and propionate. Ethanol was used to upgrade ethanol itself into caproate and to upgrade the supplied VFA (propionate) into heptanoate. A high CO2 loading rate (2.5 LCO2·L-1·d-1) stimulated excessive ethanol oxidation (EEO; up to 29%) which resulted in a high caproate production (10.8 g·L-1·d-1). A low CO2 loading rate (0.5 LCO2·L-1·d-1) reduced EEO (16%) and caproate production (2.9 g·L-1·d-1). Heptanoate production by VFA upgrading remained constant (∼1.8 g·L-1·d-1) at CO2 loading rates higher than or equal to 1 LCO2·L-1·d-1. CO2 was likely essential for growth of chain elongating microorganisms while it also stimulated syntrophic ethanol oxidation. A high CO2 loading rate must be selected to upgrade ethanol (e.g., from lignocellulosic bioethanol) into MCFAs whereas lower CO2 loading rates must be selected to upgrade VFAs (e.g., from acidified organic residues) into MCFAs while minimizing use of costly ethanol.

A new formula to calculate activity of superoxide dismutase in indirect assays.

To calculate superoxide dismutase (SOD) activity rapidly and accurately by indirect SOD assays, a formula based on the ratio of the catalytic speed of SOD to the reaction speed of the indicator with superoxide anion was deduced. The accuracy of this formula was compared with the conventional formula based on inhibition in five indirect SOD assays. The new formula was validated in nearly the entire SOD activity range, whereas the conventional formula was validated only during inhibition of 40-60%. This formula might also be used for the assays of other enzymes.

Production of hydrophobic amino acids from biobased resources: wheat gluten and rubber seed proteins.

Protein hydrolysis enables production of peptides and free amino acids that are suitable for usage in food and feed or can be used as precursors for bulk chemicals. Several essential amino acids for food and feed have hydrophobic side chains; this property may also be exploited for subsequent separation. Here, we present methods for selective production of hydrophobic amino acids from proteins. Selectivity can be achieved by selection of starting material, selection of hydrolysis conditions, and separation of achieved hydrolysate. Several protease combinations were applied for hydrolysis of rubber seed protein concentrate, wheat gluten, and bovine serum albumin (BSA). High degree of hydrolysis (>50 %) could be achieved. Hydrophobic selectivity was influenced by the combination of proteases and by the extent of hydrolysis. Combination of Pronase and Peptidase R showed the highest selectivity towards hydrophobic amino acids, roughly doubling the content of hydrophobic amino acids in the products compared to the original substrates. Hydrophobic selectivity of 0.6 mol-hydrophobic/mol-total free amino acids was observed after 6 h hydrolysis of wheat gluten and 24 h hydrolysis of rubber seed proteins and BSA. The results of experiments with rubber seed proteins and wheat gluten suggest that this process can be applied to agro-industrial residues.

Synergy between bio-based industry and the feed industry through biorefinery.

Processing biomass into multi-functional components can contribute to the increasing demand for raw materials for feed and bio-based non-food products. This contribution aims to demonstrate synergy between the bio-based industry and the feed industry through biorefinery of currently used feed ingredients. Illustrating the biorefinery concept, rapeseed was selected as a low priced feed ingredient based on market prices versus crude protein, crude fat and apparent ileal digestible lysine content. In addition it is already used as an alternative protein source in diets and can be cultivated in European climate zones. Furthermore, inclusion level of rapeseed meal in pig diet is limited because of its nutritionally active factors. A conceptual process was developed to improve rapeseeds nutritional value and producing other bio-based building blocks simultaneously. Based on the correlation between market prices of feed ingredients and its protein and fat content, the value of refined products was estimated. Finally, a sensitivity analysis, under two profit scenario, shows that the process is economically feasible. This study demonstrates that using biorefinery processes on feed ingredients can improve feed quality. In conjunction, it produces building blocks for a bio-based industry and creates synergy between bio-based and feed industry for more efficient use of biomass. © 2015 Society of Chemical Industry.

Towards plant protein refinery: Review on protein extraction using alkali and potential enzymatic assistance.

The globally increasing protein demands require additional resources to those currently available. Furthermore, the optimal usage of protein fractions from both traditional and new protein resources, such as algae and leaves, is essential. Here, we present an overview on alkaline plant protein extraction including the potentials of enzyme addition in the form of proteases and/or carbohydrolases. Strategic biomass selection, combined with the appropriate process conditions can increase protein yields after extraction. Enzyme addition, especially of proteases, can be useful when alkaline protein extraction yields are low. These additions can also be used to enable processing at a pH closer to 7 to avoid the otherwise severe conditions that denature proteins. Finally, a protein biorefinery concept is presented that aims to upcycle residual biomass by separating essential amino acids to be used for food and feed, and non-essential amino acids for production of bulk chemicals.

How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves.

Leaf protein can be obtained cost-efficiently by alkaline extraction, but overuse of chemicals and low quality of (denatured) protein limits its application. The research objective was to investigate how alkali aids protein extraction of green tea leaf residue, and use these results for further improvements in alkaline protein biorefinery. Protein extraction yield was studied for correlation to morphology of leaf tissue structure, protein solubility and hydrolysis degree, and yields of non-protein components obtained at various conditions. Alkaline protein extraction was not facilitated by increased solubility or hydrolysis of protein, but positively correlated to leaf tissue disruption. HG pectin, RGII pectin, and organic acids were extracted before protein extraction, which was followed by the extraction of cellulose and hemi-cellulose. RGI pectin and lignin were both linear to protein yield. The yields of these two components were 80% and 25% respectively when 95% protein was extracted, which indicated that RGI pectin is more likely to be the key limitation to leaf protein extraction. An integrated biorefinery was designed based on these results.

Pressure-aided proteolysis of beta-casein.

Beta-casein, which is present in the form of micelles at atmospheric pressure, has been hydrolyzed during pressure treatment to improve the accessibility of the protein. Two proteolytic enzymes with different specificities were used. Trypsin was aimed at mainly hydrolyzing hydrophilic segments of beta-casein and chymotrypsin at hydrolyzing hydrophobic segments of beta-casein. Measurements on aggregation during hydrolysis at atmospheric pressure showed that probably not micelle disruption, but disruption of much larger aggregates, occurs in the process. Peptide profiles were measured via reversed-phase chromatography. Measurements on enzyme activity after pressure treatments showed that trypsin was inactivated by pressure, which could explain all differences in peptide profiles compared to atmospheric experiments. Pressure did not influence the reaction mechanism, probably because the hydrophilic part of beta-casein is sufficiently accessible. However, chymotryptic proteolysis under pressure yielded new peptides that could not be explained by a change in enzyme activity. Here, pressure altered the mechanism of hydrolysis, by changing either enzyme specificity or substrate accessibility, which led to different peptides that can have different properties.