Intensification of extraction process through IVDV pretreatment from Eryngium creticum leaves and stems: Maximizing yields and assessing biological activities.

"Intensification of Vaporization by Decompression to the Vacuum" (IVDV) has initially emerged as a technology primarily employed for expanding and enhancing the texture of biological products. However, its recent applications have showcased significant promise in the realm of extracting bioactive molecules from various plant materials. In this context, optimization using response surface methodology was conducted to investigate the impact of IVDV pretreatment on the extractability of phenolic compounds from Eryngium creticum leaves and stems, as well as their biological activities. Using IVDV preceding the extraction led to higher total phenolic content (TPC) and enhanced antiradical activities in treated materials compared to untreated ones. The optimal processing conditions in terms of water content, steam pressure and treatment time were determined in order to maximize TPC (89.07 and 20.06 mg GAE/g DM in leaves and stems, respectively) and antiradical (DPPH) inhibition percentage (93.51% and 27.54% in leaves and stems, respectively). IVDV-treated extracts showed superior antioxidant, antibacterial and antibiofilm capacities compared to raw extracts. Using RP-UHPLC-PDA-MS, caffeic acid and rosmarinic acid were identified in IVDV-treated leaves. IVDV can be implemented as an innovative treatment applied prior to extraction to boost the recovery of biomolecules from plant matrices.

Chemical Composition and Biological Activities of Centranthus longiflorus Stems Extracts Recovered Using Ired-Irrad®, an Innovative Infrared Technology, Compared to Water Bath and Ultrasound.

Extraction of polyphenols from Centranthus longiflorus stems was conducted using ultrasound and infrared Ired-Irrad® techniques, and compared to the conventional water bath method. Response surface methodology was used to analyse the effect of time, temperature, and ethanol percentage, as well as to optimize the three extraction methods. The highest phenolic content (81 mg GAE/g DM) and antioxidant activity (76% DPPH inhibition) were recorded with the Ired-Irrad® extract obtained under the optimal conditions: 55 °C, 127 min, 48% (v/v) ethanol. Biological activities (antioxidant, antibacterial and antibiofilm) of the three extracts were assessed. All C. longiflorus stems extracts showed limited antibacterial effects regardless of the extraction method (MIC = 50 mg/mL), whereas Ired-Irrad® extract exhibited the highest biofilm eradication and prevention capacities (93% against Escherichia coli and 97% against Staphylococcus epidermidis, respectively). This bioactivity is likely related to abundant caffeoylquinic acid and quercetin rutinoside, as identified by RP-UHPLC-PDA-MS analysis. The results obtained further promote the effectiveness of Ired-Irrad® as a highly flexible and cost-efficient extraction technique.

Potential Protein Production from Lignocellulosic Materials Using Edible Mushroom Forming Fungi.

There is a need for new protein sources to feed the world in a sustainable way. Converting non-food-grade "woody" side streams into food containing proteins will contribute to this mission. Mushroom forming fungi are unique in their capability to convert lignocellulosic substances into edible biomass containing protein. Especially if substrate mycelium can be used instead of mushrooms, this technology could be a serious contribution to addressing the protein challenge. In this Perspective, we discuss challenges toward production, purification, and market introduction of mushroom mycelium based foods.

Multi-Step Biomass Fractionation of Grape Seeds from Pomace, a Zero-Waste Approach.

Grape seeds are the wineries' main by-products, and their disposal causes ecological and environmental problems. In this study seeds from the pomace waste of autochthonous grape varieties from Lebanon, Obeidi (white variety) and Asswad Karech (red variety) were used for a multi-step biomass fractionation. For the first step, a lipid extraction was performed, and the obtained yield was 12.33% (w/w) for Obeidi and 13.04% (w/w) for Asswad Karech. For the second step, polyphenols' recovery from the defatted seeds was carried out, resulting in 12.0% (w/w) for Obeidi and 6.6% (w/w) for Asswad Karech, with Obeidi's extract having the highest total phenolic content (333.1 ± 1.6 mg GAE/g dry matter) and antioxidant activity (662.17 ± 0.01 µg/mL of Trolox equivalent). In the third step, the defatted and dephenolized seeds were subsequently extracted under alkaline conditions and the proteins were isoelectric precipitated. The recovered protein extract was 3.90% (w/w) for Obeidi and 4.11% (w/w) for Asswad Karech seeds, with Asswad Karech's extract having the highest protein content (64 ± 0.2 mg protein/g dry matter). The remaining exhausted residue can be valorized in cosmetic scrubs formulations as a replacement for plastic microbeads. The designed zero-waste approach multi-step biomass fractionation has the potential to improve the valorization of the side products (grape seeds) of these two Lebanese autochthonous grape varieties.

The effect of me-substituents of 1,4-butanediol analogues on the thermal properties of biobased polyesters.

Biobased 1,4-butanediol analogues are used to tune the glass transition temperature and crystallization in a series of polyesters and allow for the formation of stereocomplexes.

ß-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin.

Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable ß-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50 °C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55 °C (144 h, 75% activity), 60 °C (48 h, 85% activity; half-life 82 h), and 70 °C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc)2-5, p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc)1-3-pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc)2. MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MthNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.

Water-soluble chitosan derivatives and pH-responsive hydrogels by selective C-6 oxidation mediated by TEMPO-laccase redox system.

Chitosan is a polysaccharide with recognized antioxidant, antimicrobial and wound healing activities. However, this polymer is soluble only in dilute acidic solutions, which restricts much of its applications. A usual strategy for improving the functionality of polysaccharides is the selective oxidation mediated by 2,2,6,6-tetra-methyl-1-piperidinidyloxy (TEMPO) using laccase as a co-oxidant. In this work, the TEMPO-laccase redox system was used for the first time to selectively oxidize chitosan in order to produce tailored derivatives. The reaction was performed at pH 4.5 under continuous air supply and the oxidized products were characterized structurally and functionally. The TEMPO-laccase oxidation successfully added aldehyde and carboxylate groups to chitosan structure resulting in derivatives with oxidation between 4 and 7%. These derivatives showed increased solubility and decreased viscosity in solution. If chitosan is dissolved in diluted hydrochloric acid prior to TEMPO-laccase oxidation, a crosslinked chitosan derivative was produced, which was able to form a pH-responsive hydrogel.

Chitinase Chi1 from Myceliophthora thermophila C1, a Thermostable Enzyme for Chitin and Chitosan Depolymerization.

A thermostable Chitinase Chi1 from Myceliophthora thermophila C1 was homologously produced and characterized. Chitinase Chi1 shows high thermostability at 40 °C (>140 h 90% activity), 50 °C (>168 h 90% activity), and 55 °C (half-life 48 h). Chitinase Chi1 has broad substrate specificity and converts chitin, chitosan, modified chitosan, and chitin oligosaccharides. The activity of Chitinase Chi1 is strongly affected by the degree of deacetylation (DDA), molecular weight (Mw), and side chain modification of chitosan. Chitinase Chi1 releases mainly (GlcNAc)2 from insoluble chitin and chito-oligosaccharides with a polymerization degree (DP) ranging from 2 to 12 from chitosan, in a processive way. Chitinase Chi1 shows higher activity toward chitin oligosaccharides (GlcNAc)4-6 than toward (GlcNAc)3 and is inactive for (GlcNAc)2. During hydrolysis, oligosaccharides bind at subsites -2 to +2 in the enzyme's active site. Chitinase Chi1 can be used for chitin valorisation and for production of chitin- and chito-oligosaccharides at industrial scale.

Nitrogen-to-Protein Conversion Factors for Three Edible Insects: Tenebrio molitor, Alphitobius diaperinus, and Hermetia illucens.

Insects are considered a nutritionally valuable source of alternative proteins, and their efficient protein extraction is a prerequisite for large-scale use. The protein content is usually calculated from total nitrogen using the nitrogen-to-protein conversion factor (Kp) of 6.25. This factor overestimates the protein content, due to the presence of nonprotein nitrogen in insects. In this paper, a specific Kp of 4.76 ± 0.09 was calculated for larvae from Tenebrio molitor, Alphitobius diaperinus, and Hermetia illucens, using amino acid analysis. After protein extraction and purification, a Kp factor of 5.60 ± 0.39 was found for the larvae of three insect species studied. We propose to adopt these Kp values for determining protein content of insects to avoid overestimation of the protein content.

Botryococcus braunii strains compared for biomass productivity, hydrocarbon and carbohydrate content.

Botryococcus braunii can produce both long-chain hydrocarbons as well as carbohydrates in large quantities, and is therefore a promising industrial organism for the production of biopolymer building blocks. Many studies describe the use of different strains of Botryococcus braunii but differences in handling and cultivation conditions make the comparison between strains difficult. In this study, 16 B. braunii strains obtained from six culture collections were compared for their biomass productivity and hydrocarbon and carbohydrate content. Biomass productivity was highest for AC768 strain with 1.8gL-1day-1, while hydrocarbon production ranged from none to up to 42% per gram biomass dry weight, with Showa showing the highest hydrocarbon content followed by AC761. The total carbohydrate content varied from 20% to 76% per gram of the biomass dry weight, with CCALA777 as the highest producer. Glucose and galactose are the main monosaccharides in most strains and fucose content reached 463mgL-1 in CCALA778.

Chitosan films and blends for packaging material.

An increased interest for hygiene in everyday life as well as in food, feed and medical issues lead to a strong interest in films and blends to prevent the growth and accumulation of harmful bacteria. A growing trend is to use synthetic and natural antimicrobial polymers, to provide non-migratory and non-depleting protection agents for application in films, coatings and packaging. In food packaging, antimicrobial effects add up to the barrier properties of the materials, to increase the shelf life and product quality. Chitosan is a natural bioactive polysaccharide with intrinsic antimicrobial activity and, due to its exceptional physicochemical properties imparted by the polysaccharide backbone, has been recognized as a natural alternative to chemically synthesized antimicrobial polymers. This, associated with the increasing preference for biofunctional materials from renewable resources, resulted in a significant interest on the potential for application of chitosan in packaging materials. In this review we describe the latest developments of chitosan films and blends as packaging material.

Production methods for heparosan, a precursor of heparin and heparan sulfate.

Heparin and heparan sulfate belong to the glycosaminoglycan family. Heparin which is known as a powerful anticoagulant has been also described to have potential in therapeutic applications such as in the treatment against cancer and prevention of virus infections. Heparan sulfate, an analog of heparin, which is not used for medical purposes yet, was reported to have the same pharmaceutical potential as heparin. Both heparin and heparan sulfate share a common precursor molecule known as heparosan. Heparosan determines the polymer chain length and the sugar unit backbone composition, which are determinant structural parameters for the biological activity of heparin and heparan sulfate. In this review we give an overview of the different methods used to synthesize heparosan, and we highlight the pro and cons of each method in respect to the synthesis of bioengineered heparin-like molecules. Advancements in the field of the synthesis of bioengineered heparin are also reported.

Enzymatic synthesis of oligo- and polysaccharide fatty acid esters.

Amphiphilic oligo- and polysaccharides (e.g. polysaccharide alkyl or alkyl-aryl esters) form a new class of polymers with exceptional properties. They function as polymeric surfactants, whilst maintaining most of the properties of the starting polymeric material such as emulsifying, gelling, and film forming properties combined with partial water solubility or permeability. At present carbohydrate fatty acid esters are generally obtained by chemical methods using toxic solvents and organic and inorganic catalysts that leave residual traces in the final products. Enzymatic reactions offer an attractive alternative route for the synthesis of polysaccharide esters. In this review the state of the art of enzymatic synthesis of oligo- and polysaccharides fatty esters has been described.

Histological examination of horse chestnut infection by Pseudomonas syringae pv. aesculi and non-destructive heat treatment to stop disease progression.

Since its emergence in Northwest Europe as a pathogen that infects trunks and branches of Aesculus spp. (the horse chestnuts) approximately one decade ago, Pseudomonas syringae pv. aesculi has rapidly established itself as major threat to these trees. Infected trees exhibit extensive necrosis of phloem and cambium, which can ultimately lead to dieback. The events after host entry leading to extensive necrosis are not well documented. In this work, the histopathology of this interaction is investigated and heat-treatment is explored as method to eradicate bacteria associated with established infections. The early wound-repair responses of A. hippocastanum, both in absence and presence of P. s. pv. aesculi, included cell wall lignification by a distinct layer of phloem and cortex parenchyma cells. The same cells also deposited suberin lamellae later on, suggesting this layer functions in compartmentalizing healthy from disrupted tissues. However, monitoring bacterial ingress, its construction appeared inadequate to constrain pathogen spread. Microscopic evaluation of bacterial dispersal in situ using immunolabelling and GFP-tagging of P. s. pv. aesculi, revealed two discriminative types of bacterial colonization. The forefront of lesions was found to contain densely packed bacteria, while necrotic areas housed bacterial aggregates with scattered individuals embedded in an extracellular matrix of bacterial origin containing alginate. The endophytic localization and ability of P. s. pv aesculi to create a protective matrix render it poorly accessible for control agents. To circumvent this, a method based on selective bacterial lethality at 39 °C was conceived and successfully tested on A. hippocastanum saplings, providing proof of concept for controlling this disease by heat-treatment. This may be applicable for curing other tree cankers, caused by related phytopathogens.

Synthesis of heparosan oligosaccharides by Pasteurella multocida PmHS2 single-action transferases.

Pasteurella multocida heparosan synthase PmHS2 is a dual action glycosyltransferase that catalyzes the polymerization of heparosan polymers in a non-processive manner. The two PmHS2 single-action transferases, obtained previously by site-directed mutagenesis, have been immobilized on Ni(II)-nitrilotriacetic acid agarose during the purification step. A detailed study of the polymerization process in the presence of non-equal amounts of PmHS2 single-action transferases revealed that the glucuronyl transferase (PmHS2-GlcUA(+)) is the limiting catalyst in the polymerization process. Using experimental design, it was determined that the N-acetylglucosaminyl transferase (PmHS2-GlcNAc(+)) plays an important role in the control of heparosan chain elongation depending on the number of heparosan chains and the UDP-sugar concentrations present in the reaction mixture. Furthermore, for the first time, the synthesis of heparosan oligosaccharides alternately using PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) is reported. It was shown that the synthesis of heparosan oligosaccharides by PmHS2 single-action transferases do not require the presence of template molecules in the reaction mixture.

Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity.

Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUAß1-4GlcNAcα1-)(n) by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA(+)/PmHS2-GlcNAc(+) showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence, not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars, UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars.

Legumin allergens from peanuts and soybeans: effects of denaturation and aggregation on allergenicity.

Legumin proteins Ara h 3 from peanuts and glycinin from soybeans are increasingly described as important allergens. The stability of an allergen's IgE binding capacity towards heating and digestion is considered an important characteristic for food allergens. We investigated the effects of heating and digestion on the IgE binding of Ara h 3 and glycinin. Both proteins are relatively stable to denaturation, having denaturation temperatures ranging from 70 to 92 degrees C, depending on their quaternary structure and the ionic strength. Aggregates were formed upon heating, which were partly soluble for glycinin. Heating slightly decreased the pepsin digestion rate of both allergens. However, heating did not affect the IgE binding capacity of the hydrolyzates, as after only 10 min of hydrolysis no IgE binding could be detected any more in all samples. Peanut allergen Ara h 1, when digested under equal conditions, still showed IgE binding after 2 h of hydrolysis. Our results indicate that the IgE binding capacity of legumin allergens from peanuts and soybeans does not withstand peptic digestion. Consequently, these allergens are likely unable to sensitize via the gastro-intestinal tract and cause systemic food allergy symptoms. These proteins might thus be less important allergens than was previously assumed.

Determination of pepsin-susceptible and pepsin-resistant epitopes in native and heat-treated peanut allergen Ara h 1.

This study was aimed at the determination of the pepsin-susceptible and pepsin-resistant epitopes in native and heat-treated Ara h 1, a major allergen from peanuts. Both the oligomeric structure and the trimeric structure of the allergen were investigated. Under the in vitro conditions applied, oligomeric Ara h 1, either unheated or preheated, was hydrolyzed by pepsin at a lower rate than trimeric Ara h 1. Peptides with relatively high molecular masses were shown to be able to bind IgE, whereas peptides with lower molecular masses (<2 kDa) did not. In these latter fractions, fragments of 15 previously published epitopes of mature Ara h 1 were identified. As a result, these epitopes are not likely responsible for the induction of systemic food allergic reactions to peanuts. Using sequential chymotrypsin digestion, the pepsin-resistant IgE-binding peptides were deduced to contain the previously identified intact epitopes EDWRRPSHQQ (amino acids 50-59) and PRKIRPEG (amino acids 60-67). The presence of four additional earlier published intact epitopes (covering amino acids 6-13, 14-21, 24-31, and 40-47) on the pepsin-resistant peptides could be neither deduced nor ruled out. The two deduced and four possible pepsin-resistant epitopes are all situated in the N-terminal part of Ara h 1, which does not show homology with other vicilin proteins. Consequently, this unique N-terminal part of Ara h 1 is proposed to be responsible for the allergen's ability to induce systemic allergic reactions.

Bifidobacterium carbohydrases-their role in breakdown and synthesis of (potential) prebiotics.

There is an increasing interest to positively influence the human intestinal microbiota through the diet by the use of prebiotics and/or probiotics. It is anticipated that this will balance the microbial composition in the gastrointestinal tract in favor of health promoting genera such as Bifidobacterium and Lactobacillus. Carbohydrates like non-digestible oligosaccharides are potential prebiotics. To understand how these bacteria can grow on these carbon sources, knowledge of the carbohydrate-modifying enzymes is needed. Little is known about the carbohydrate-modifying enzymes of bifidobacteria. The genome sequence of Bifidobacterium adolescentis and Bifidobacterium longum biotype longum has been completed and it was observed that for B. longum biotype longum more than 8% of the annotated genes were involved in carbohydrate metabolism. In addition more sequence data of individual carbohydrases from other Bifidobacterium spp. became available. Besides the degradation of (potential) prebiotics by bifidobacterial glycoside hydrolases, we will focus in this review on the possibilities to produce new classes of non-digestible oligosaccharides by showing the presence and (transglycosylation) activity of the most important carbohydrate modifying enzymes in bifidobacteria. Approaches to use and improve carbohydrate-modifying enzymes in prebiotic design will be discussed.

Heat denaturation of Brazil nut allergen Ber e 1 in relation to food processing.

Ber e 1, a major allergen from Brazil nuts, is very stable to in vitro peptic digestion. As heat-induced denaturation may affect protein digestibility, the denaturation behaviour of Ber e 1 was investigated. The denaturation temperature of Ber e 1 varies from approximately 80-110°C, depending on the pH. Upon heating above its denaturation temperature at pH 7.0, the protein partly forms insoluble aggregates and partly dissociates into its polypeptides, whereas heating at pH 5.0 does neither induce aggregation, nor dissociation of the protein. The denaturation temperature of approximately 110°C at pH values corresponding to the general pH values of foods (pH 5-7) is very high and is expected to be even higher in Brazil nuts themselves. As a result, it is unlikely that heat processing causes the denaturation of all Ber e 1 present in food products. Consequently, the allergen is assumed to be consumed (mainly) in its native form, having a high stability towards pepsin digestion.