Fructan oligosaccharide priming alters apoplastic sugar dynamics and improves resistance against Botrytis cinerea in chicory.

Carbohydrates such as fructans can be involved in priming or defence stimulation, and hence potentially provide new strategies for crop protection against biotic stress. Chicory (Cichorium intybus) is a model plant for fructan research and is a crop with many known health benefits. Using the chicory-Botrytis cinerea pathosystem, we tested the effectiveness of fructan-induced immunity, focussing on different plant and microbial fructans. Sugar dynamics were followed after priming and subsequent pathogen infection. Our results indicated that many higher plants might detect extracellular levan oligosaccharides (LOS) of microbial origin, while chicory also detects extracellular small inulin-type fructooligosaccharides (FOS) of endogenous origin, thus differing from the findings of previous fructan priming studies. No clear positive effects were observed for inulin or mixed-type fructans. An elicitor-specific burst of reactive oxygen species was observed for sulfated LOS, while FOS and LOS both behaved as genuine priming agents. In addition, a direct antifungal effect was observed for sulfated LOS. Intriguingly, LOS priming led to a temporary increase in apoplastic sugar concentrations, mainly glucose, which could trigger downstream responses. Total sugar and starch contents in total extracts of LOS-primed leaves were higher after leaf detachment, indicating they could maintain their metabolic activity. Our results indicate the importance of balancing intra- and extracellular sugar levels (osmotic balance) in the context of 'sweet immunity' pathways.

Dynamic co-culture metabolic models reveal the fermentation dynamics, metabolic capacities and interplays of cheese starter cultures.

In this study, we have investigated the cheese starter culture as a microbial community through a question: can the metabolic behaviour of a co-culture be explained by the characterized individual organism that constituted the co-culture? To address this question, the dairy-origin lactic acid bacteria Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Streptococcus thermophilus and Leuconostoc mesenteroides, commonly used in cheese starter cultures, were grown in pure and four different co-cultures. We used a dynamic metabolic modelling approach based on the integration of the genome-scale metabolic networks of the involved organisms to simulate the co-cultures. The strain-specific kinetic parameters of dynamic models were estimated using the pure culture experiments and they were subsequently applied to co-culture models. Biomass, carbon source, lactic acid and most of the amino acid concentration profiles simulated by the co-culture models fit closely to the experimental results and the co-culture models explained the mechanisms behind the dynamic microbial abundance. We then applied the co-culture models to estimate further information on the co-cultures that could not be obtained by the experimental method used. This includes estimation of the profile of various metabolites in the co-culture medium such as flavour compounds produced and the individual organism level metabolic exchange flux profiles, which revealed the potential metabolic interactions between organisms in the co-cultures.

Developments in effective use of volatile organic compound analysis to assess flavour formation during cheese ripening.

In the burgeoning demand for optimization of cheese production, ascertaining cheese flavour formation during the cheese making process has been the focal point of determining cheese quality. In this research reflection, we have highlighted how valuable volatile organic compound (VOC) analysis has been in assessing contingent cheese flavour compounds arising from non-starter lactic acid bacteria (NSLAB) along with starter lactic acid bacteria (SLAB), and whether VOC analysis associated with other high-throughput data might help provide a better understanding the cheese flavour formation during cheese process. It is widely known that there is a keen interest to merge all omics data to find specific biomarkers and/or to assess aroma formation of cheese. Towards that end, results of VOC analysis have provided valuable insights into the cheese flavour profile. In this review, we are pinpointing the effective use of flavour compound analysis to perceive flavour-forming ability of microbial strains that are convenient for dairy production, intertwining microbiome and metabolome to unveil potential biomarkers that occur during cheese ripening. In doing so, we summarised the functionality and integration of aromatic compound analysis in cheese making and gave reflections on reconsidering what the role of flavour-based analysis might have in the future.

A genome-scale metabolic network of the aroma bacterium Leuconostoc mesenteroides subsp. cremoris.

Leuconostoc mesenteroides subsp. cremoris is an obligate heterolactic fermentative lactic acid bacterium that is mostly used in industrial dairy fermentations. The phosphoketolase pathway (PKP) is a unique feature of the obligate heterolactic fermentation, which leads to the production of lactate, ethanol, and/or acetate, and the final product profile of PKP highly depends on the energetics and redox state of the organism. Another characteristic of the L. mesenteroides subsp. cremoris is the production of aroma compounds in dairy fermentation, such as in cheese production, through the utilization of citrate. Considering its importance in dairy fermentation, a detailed metabolic characterization of the organism is necessary for its more efficient use in the industry. To this aim, a genome-scale metabolic model of dairy-origin L. mesenteroides subsp. cremoris ATCC 19254 (iLM.c559) was reconstructed to explain the energetics and redox state mechanisms of the organism in full detail. The model includes 559 genes governing 1088 reactions between 1129 metabolites, and the reactions cover citrate utilization and citrate-related flavor metabolism. The model was validated by simulating co-metabolism of glucose and citrate and comparing the in silico results to our experimental results. Model simulations further showed that, in co-metabolism of citrate and glucose, no flavor compounds were produced when citrate could stimulate the formation of biomass. Significant amounts of flavor metabolites (e.g., diacetyl and acetoin) were only produced when citrate could not enhance growth, which suggests that flavor formation only occurs under carbon and ATP excess. The effects of aerobic conditions and different carbon sources on product profiles and growth were also investigated using the reconstructed model. The analyses provided further insights for the growth stimulation and flavor formation mechanisms of the organism.

The fructan syndrome: Evolutionary aspects and common themes among plants and microbes.

Fructans are multifunctional fructose-based water soluble carbohydrates found in all biological kingdoms but not in animals. Most research has focused on plant and microbial fructans and has received a growing interest because of their practical applications. Nevertheless, the origin of fructan production, the so-called "fructan syndrome," is still unknown. Why fructans only occur in a limited number of plant and microbial species remains unclear. In this review, we provide an overview of plant and microbial fructan research with a focus on fructans as an adaptation to the environment and their role in (a)biotic stress tolerance. The taxonomical and biogeographical distribution of fructans in both kingdoms is discussed and linked (where possible) to environmental factors. Overall, the fructan syndrome may be related to water scarcity and differences in physicochemical properties, for instance, water retaining characteristics, at least partially explain why different fructan types with different branching levels are found in different species. Although a close correlation between environmental stresses and fructan production is quite clear in plants, this link seems to be missing in microbes. We hypothesize that this can be at least partially explained by differential evolutionary timeframes for plants and microbes, combined with potential redundancy effects.

Levansucrase from Halomonas smyrnensis AAD6T: first halophilic GH-J clan enzyme recombinantly expressed, purified, and characterized.

Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies.

Development of a cost-effective production process for Halomonas levan.

Levan polysaccharide is an industrially important natural polymer with unique properties and diverse high-value applications. However, current bottlenecks associated with its large-scale production need to be overcome by innovative approaches leading to economically viable processes. Besides many mesophilic levan producers, halophilic Halomonas smyrnensis cultures hold distinctive industrial potential and, for the first time with this study, the advantage of halophilicity is used and conditions for non-sterile levan production were optimized. Levan productivity of Halomonas cultures in medium containing industrial sucrose from sugar beet and food industry by-product syrup, a total of ten sea, lake and rock salt samples from four natural salterns, as well as three different industrial-grade boron compounds were compared and the most suitable low-cost substitutes for sucrose, salt and boron were specified. Then, the effects of pH control, non-sterile conditions and different bioreactor modes (batch and fed-batch) were investigated. The development of a cost-effective production process was achieved with the highest yield (18.06 g/L) reported so far on this microbial system, as well as the highest theoretical bioconversion efficiency ever reported for levan-producing suspension cultures. Structural integrity and biocompatibility of the final product were also verified in vitro.

Genomic analysis of Brevibacillus thermoruber 423 reveals its biotechnological and industrial potential.

Brevibacillus thermoruber 423 is a Gram-positive, motile, red-pigmented, spore-forming, aerobic, and thermophilic bacterium that is known to produce high levels of exopolysaccharide (EPS) with many potential uses in food, feed, cosmetics, and pharmaceutical and chemical industries. This bacterium not only is among the limited number of reported thermophilic EPS producers but also exceeds other thermophilic producers in light of the high level of polymer synthesis. By a systems-based approach, whole-genome analysis of this bacterium was performed to gain more insight about the biological mechanisms and whole-genome organization of thermophilic EPS producers and hence to develop rational strategies for the genetic and metabolic optimization of EPS production. Also with this study, the first genome analysis was performed on a thermophilic Brevibacillus species. Essential genes associated with EPS biosynthesis were detected by genome annotation, and together with experimental evidences, a hypothetical mechanism for EPS biosynthesis was generated. B. thermoruber 423 was found to have many potential applications in biotechnology and industry because of its capacity to utilize xylose and to produce EPS, isoprenoids, ethanol/butanol, lipases, proteases, cellulase, and glucoamylase enzymes as well as its resistance to arsenic.

Functional stability of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), and a major regulator of the fibrinolytic system. PAI-1 plays a pivotal role in acute thrombotic events such as deep vein thrombosis (DVT) and myocardial infarction (MI). The biological effects of PAI-1 extend far beyond thrombosis including its critical role in fibrotic disorders, atherosclerosis, renal and pulmonary fibrosis, type-2 diabetes, and cancer. The conversion of PAI-1 from the active to the latent conformation appears to be unique among serpins in that it occurs spontaneously at a relatively rapid rate. Latency transition is believed to represent a regulatory mechanism, reducing the risk of thrombosis from a prolonged antifibrinolytic action of PAI-1. Thus, relying solely on plasma concentrations of PAI-1 without assessing its function may be misleading in interpreting the role of PAI-1 in many complex diseases. Environmental conditions, interaction with other proteins, mutations, and glycosylation are the main factors that have a significant impact on the stability of the PAI-1 structure. This review provides an overview on the current knowledge on PAI-1 especially importance of PAI-1 level and stability and highlights the potential use of PAI-1 inhibitors for treating cardiovascular disease.

Investigating the cryoprotective efficacy of fructans in mammalian cell systems via a structure-functional perspective.

Fructans have long been known with their role in protecting organisms against various stress factors due to their ability to induce controlled dehydration and support membrane stability. Considering the vital importance of such features in cryo-technologies, this study aimed to explore the cryoprotective efficacy of fructans in mammalian cell systems where structurally different fructan polymers were examined on in vitro cell models derived from organs such as the liver, frequently used in transplantation, osteoblast, and cord cells, commonly employed in cell banking, as well as human seminal fluids that are of vital importance in assisted reproductive technology. To gain insights into the fructan/membrane interplay, structural differences were linked to rheological properties as well as to lipid membrane interactions where both fluorescein leakage from unilamellar liposomes and membrane integrity of osteoblast cells were monitored. High survival rates obtained with human endothelial, osteoblast and liver cells for up to two months clearly showed that fructans could be considered as effective non-permeating cryoprotectants, especially for extended periods of cryopreservation. In trials with human seminal fluid, short chained levan in combination with human serum albumin and glycerol proved very effective in preserving semen samples across multiple patients without any morphological abnormalities.

Co-production of levan with other high-value bioproducts: A review.

Levan is a homopolysaccharide of fructose that has both scientific and industrial importance, with various applications in health, pharmaceutical, cosmetic and food industries. Despite its broad spectrum of uses, there are only a limited number of commercial levan sources due to the high costs related to its production. To make production economically viable, efforts have been concentrated on the selection of levan-producing microorganisms, the genetic manipulation of new strains, and the use of inexpensive agro-industrial byproducts as substrates. Another efficient strategy involves the concomitant synthesis of other products with high market value and as such, the successful co-production of levan was demonstrated with fructooligosaccharides, ethanol, sorbitol, poly-ε-lysine, poly-γ-glutamic acid and polyhydroxyalkanoates. This paper offers a systematic review of important aspects regarding recent strategies involving the simultaneous synthesis of levan and other bioproducts of aggregate value reported to date and discusses the challenges and opportunities for its large-scale production and applications.

Exploring the potential of Halomonas levan and its derivatives as active ingredients in cosmeceutical and skin regenerating formulations.

Demand on natural products that contain biological ingredients mimicking growth factors and cytokines made natural polysaccharides popular in pharmaceutical and cosmetic industries. Levan is the ß-(2-6) linked, nontoxic, biocompatible, water-soluble, film former fructan polymer that has diverse applications in pharmacy and cosmeceutical industries with its moisturizing, whitening, anti-irritant, anti-aging and slimming activities. Driven by the limited reports on few structurally similar levan polymers, this study presents the first systematic investigation on the effects of structurally different extremophilic Halomonas levan polysaccharides on human skin epidermis cells. In-vitro experiments with microbially produced linear Halomonas levan (HL), its hydrolyzed, (hHL) and sulfonated (ShHL) derivatives as well as enzymatically produced branched levan (EL) revealed increased keratinocyte and fibroblast proliferation (113-118 %), improved skin barrier function through induced expressions of involucrin (2.0 and 6.43 fold changes for HL and EL) and filaggrin (1.74 and 3.89 fold changes for hHL and ShHL) genes and increased type I collagen (2.63 for ShHL) and hyaluronan synthase 3 (1.41 for HL) gene expressions together with fast wound healing ability within 24 h (100 %, HL) on 2D wound models clearly showed that HL and its derivatives have high potential to be used as natural active ingredients in cosmeceutical and skin regenerating formulations.

First transcriptomic insight into the reprogramming of human macrophages by levan-type fructans.

Based on stimuli in the biological milieu, macrophages can undergo classical activation into the M1 pro-inflammatory (anti-cancer) phenotype or to the alternatively activated M2 anti-inflammatory one. Drug-free biomaterials have emerged as a new therapeutic strategy to modulate macrophage phenotype. Among them, polysaccharides polarize macrophages to M1 or M2 phenotypes based on the surface receptors they bind. Levan, a fructan, has been proposed as a novel biomaterial though its interaction with macrophages has been scarcely explored. In this study, we investigate the interaction of non-hydrolyzed and hydrolyzed Halomonas levan and its sulfated derivative with human macrophages in vitro. Viability studies show that these levans are cell compatible. In addition, RNA-sequencing analysis reveals the upregulation of pro-inflammatory pathways. These results are in good agreement with real time-quantitative polymerase chain reaction that indicates higher expression levels of C-X-C Motif Chemokine Ligand 8 and interleukin-6 genes and the M2-to-M1 reprogramming of these cells upon levan treatment. Finally, cytokine release studies confirm that hydrolyzed levans increase the secretion of pro-inflammatory cytokines and reprogram IL-4-polarized macrophages to the M1 state. Overall findings indicate that Halomonas levans trigger a classical macrophage activation and pave the way for their application in therapeutic interventions requiring a pro-inflammatory phenotype.

UV and chemically induced Halomonas smyrnensis mutants for enhanced levan productivity.

Halomonas smyrnensis AAD6T is a moderately halophilic bacterium proven to be a powerful biotechnological tool with its ability to accumulate valuable biopolymers such as levan and poly(3-hydroxybutyrate) (PHB). Levan is a fructose homopolymer with ß-2,6 fructofuranosidic linkages on the polymer backbone, and its distinctive applications in various industries such as food, pharmaceutical, medical, and chemical have been well-defined. On the other hand, PHB is a promising raw material to produce biodegradable plastics. Although it was shown in our previous studies that H. smyrnensis AAD6T exhibits one of the highest conversion yields of sucrose to levan reported to date, novel strategies are required to overcome high costs of levan production. In this study, we aimed at increasing levan productivity of H. smyrnensis AAD6T cultures using random mutagenesis techniques combined (i.e., ethyl methanesulfate treatment and/or ultraviolet irradiation). After several consecutive treatments, mutant strains BAE2, BAE5 and BAE6 were selected as efficient levan producers, as BAE2 standing out as the most efficient one not only in sucrose utilization and levan production rates, but also in final PHB concentrations. The mutants' whole genome sequences were analysed to determine the mutations occurred. Several mutations in genes related to central carbon metabolism and osmoregulation were found. Our results suggest that random mutagenesis can be a facile and efficient strategy to enhance the performance of extremophiles in adverse conditions.

Genomic Analysis Provides New Insights Into Biotechnological and Industrial Potential of Parageobacillus thermantarcticus M1.

Parageobacillus thermantarcticus strain M1 is a Gram-positive, motile, facultative anaerobic, spore forming, and thermophilic bacterium, isolated from geothermal soil of the crater of Mount Melbourne (74°22' S, 164°40' E) during the Italian Antarctic Expedition occurred in Austral summer 1986-1987. Strain M1 demonstrated great biotechnological and industrial potential owing to its ability to produce exopolysaccharides (EPSs), ethanol and thermostable extracellular enzymes, such as an xylanase and a ß-xylosidase, and intracellular ones, such as xylose/glucose isomerase and protease. Furthermore, recent studies revealed its high potential in green chemistry due to its use in residual biomass transformation/valorization and as an appropriate model for microbial astrobiology studies. In the present study, using a systems-based approach, genomic analysis of P. thermantarcticus M1 was carried out to enlighten its functional characteristics. The elucidation of whole-genome organization of this thermophilic cell factory increased our understanding of biological mechanisms and pathways, by providing valuable information on the essential genes related to the biosynthesis of nucleotide sugar precursors, monosaccharide unit assembly, as well as the production of EPSs and ethanol. In addition, gene prediction and genome annotation studies identified genes encoding xylanolytic enzymes that are required for the conversion of lignocellulosic materials to high-value added molecules. Our findings pointed out the significant potential of strain M1 in various biotechnological and industrial applications considering its capacity to produce EPSs, ethanol and thermostable enzymes via the utilization of lignocellulosic waste materials.

Resveratrol-Loaded Levan Nanoparticles Produced by Electrohydrodynamic Atomization Technique.

Considering the significant advances in nanostructured systems in various biomedical applications and the escalating need for levan-based nanoparticles as delivery systems, this study aimed to fabricate levan nanoparticles by the electrohydrodynamic atomization (EHDA) technique. The hydrolyzed derivative of levan polysaccharide from Halomonas smyrnensis halophilic bacteria, hydrolyzed Halomonas levan (hHL), was used. Nanoparticles were obtained by optimizing the EHDA parameters and then they were characterized in terms of morphology, molecular interactions, drug release and cell culture studies. The optimized hHL and resveratrol (RS)-loaded hHL nanoparticles were monodisperse and had smooth surfaces. The particle diameter size of hHL nanoparticles was 82.06 ± 15.33 nm. Additionally, release of RS from the fabricated hHL nanoparticles at different pH conditions were found to follow the first-order release model and hHL with higher RS loading showed a more gradual release. In vitro biocompatibility assay with human dermal fibroblast cell lines was performed and cell behavior on coated surfaces was observed. Nanoparticles were found to be safe for healthy cells. Consequently, the fabricated hHL-based nanoparticle system may have potential use in drug delivery systems for wound healing and tissue engineering applications and surfaces could be coated with these electrosprayed particles to improve cellular interaction.

Functional and Molecular Characterization of the Halomicrobium sp. IBSBa Inulosucrase.

Fructans are fructose-based (poly)saccharides with inulin and levan being the best-known ones. Thanks to their health-related benefits, inulin-type fructans have been under the focus of scientific and industrial communities, though mostly represented by plant-based inulins, and rarely by microbial ones. Recently, it was discovered that some extremely halophilic Archaea are also able to synthesize fructans. Here, we describe the first in-depth functional and molecular characterization of an Archaeal inulosucrase from Halomicrobium sp. IBSBa (HmcIsc). The HmcIsc enzyme was recombinantly expressed and purified in Escherichia coli and shown to synthesize inulin as proven by nuclear magnetic resonance (NMR) analysis. In accordance with the halophilic lifestyle of its native host, the enzyme showed maximum activity at very high NaCl concentrations (3.5 M), with specific adaptations for that purpose. Phylogenetic analyses suggested that Archaeal inulosucrases have been acquired from halophilic bacilli through horizontal gene transfer, with a HX(H/F)T motif evolving further into a HXHT motif, together with a unique D residue creating the onset of a specific alternative acceptor binding groove. This work uncovers a novel area in fructan research, highlighting unexplored aspects of life in hypersaline habitats, and raising questions about the general physiological relevance of inulosucrases and their products in nature.

Effect of temperature on the hydrolysis of levan treated with compressed hot water fluids.

The hydrolysis of levan using compressed hot water for the production of functional fructooligosaccharides (FOSs) was investigated. Levans from Erwinia herbicola (EH) and Halomonas smyrnensis (HS) were characterized using scanning electron microscopy and light scattering techniques, and hydrolyzed using compressed hot water at four temperatures (120, 140, 160, and 180°C). The hydrolysates were analyzed using high-performance liquid chromatography and electrospray ionization-mass spectrometry. Levan HS showed a crystalline morphology, whereas levan EH showed an aggregated structure. Both levans had molar masses on the order of 106 g/mol, but levan EH had a smaller radius of gyration, hydrodynamic radius, and intrinsic viscosity. Levan EH hydrolyzed into FOSs at approximately 120°C, whereas levan HS required a temperature of at least 160°C, possibly because of differences in the degree of branching of the two levans. Both samples were degraded to fructose when treated at 180°C.

Levan-based hydrogels for controlled release of Amphotericin B for dermal local antifungal therapy of Candidiasis.

Hydrogels from Halomonas levan polysaccharide were prepared at different crosslinking densities. Swelling results demonstrated pH dependent rather than temperature dependent swelling of the hydrogel and the highest swelling value was achieved at basic conditions with a swelling ratio of 9.1 ± 0.1 which is the highest reported for levan based hydrogels. SEM images show a porous network architecture, which indicates a large surface area of the hydrogels. Rheological analyses showed the viscoelastic behavior of the hydrogels. Biocompatibility of the hydrogels was confirmed by cell culture experiments. For drug release experiments Amphotericin B (AmB) was used. 51% of the loaded AmB was released into the PBS buffer and the released AmB had a significant antifungal activity against Candida albicans.

Fructans Prime ROS Dynamics and Botrytis cinerea Resistance in Arabidopsis.

Naturally derived molecules can be used as priming or defense stimulatory agents to protect against biotic stress. Fructans have gained strong interest due to their ability to induce resistance in a number of crop species. In this study, we set out to establish the role of fructan-induced immunity against the fungal pathogen Botrytis cinerea in Arabidopsis thaliana. We show that both inulin- and levan-type fructans from different sources can enhance Arabidopsis resistance against B. cinerea. We found that inulin from chicory roots and levan oligosaccharides from the exopolysaccharide-producing bacterium Halomonas smyrnensis primed the NADPH-oxidase-mediated reactive oxygen species (ROS) burst in response to the elicitors flg22, derived from the bacterial flagellum, and oligogalacturonides (OGs), derived from the host cell wall. Neither induced a direct ROS burst typical of elicitors. We also found a primed response after infection with B. cinerea for H2O2 accumulation and the activities of ascorbate peroxidase and catalase. Sucrose accumulated as a consequence of fructan priming, and glucose and sucrose levels increased in fructan-treated plants after infection with B. cinerea. This study shows that levan-type fructans, specifically from bacterial origin, can prime plant defenses and that both inulin and levan oligosaccharide-mediated priming is associated with changes in ROS dynamics and sugar metabolism. Establishing fructan-induced immunity in Arabidopsis is an important step to further study the underlying mechanisms since a broad range of biological resources are available for Arabidopsis.