Functional characterization of the riboflavin-overproducing and dextran-producing Weissella cibaria BAL3C-5 C120T strain for the development of biofortified plant-based beverages.

Riboflavin (vitamin B2) is essential for human beings and it has to be provided by healthy nutrition. The use of fermentation with riboflavin-overproducing lactic acid bacteria (LAB) represents an ideal strategy to generate, by in situ biofortification, functional drinks. These beverages can positively contribute to consumer health and address nutritional deficiencies. In the present work, the functional capabilities of Weissella cibaria BAL3C-5 C120T for riboflavin-overproduction and dextran-production during fermentation of oat-, rice-, soybean- and almond-based drinks have been evaluated. It was confirmed that the strain was capable of producing riboflavin and dextran in the analysed drinks. This property was especially pronounced in the oat-based drink, where after 24 h of fermentation the strain was able to increase riboflavin and dextran levels up to 3.4 mg/L and 3.2 g/L, respectively. Moreover, under optimized conditions the strain was able to enrich the fermented oat-based drinks with the prebiotic oligosaccharide panose (up to 6.6 g/L). In addition, in the oat-based drinks BAL3C-5 C120T showed a good pH-lowering ability (from 7.0 to 3.8) as well as a high 80 % cell viability after one month of storage. Rheological analysis of the resulting fermented oat-based beverages revealed a thixotropic structure related to a gel-like behaviour which was not observed in the non-fermented control drinks. In summary, these results confirmed the unique characteristics of W. cibaria BAL3C-5 C120T strain for the development of biofortified and functional plant-based beverages with improved nutritional and rheological properties. Analysis of the BAL3C-5 C120T strain survival under gastrointestinal conditions and its autoaggregation properties, also indicated its potential use as a probiotic delivered in an oat-based fermented beverage. In this context, this study also promotes the utilization of W. cibaria species in health and food industries where it has not yet been used as a starter or adjunct culture.

Lactobacilli Cell-Free Supernatants Modulate Inflammation and Oxidative Stress in Human Microglia via NRF2-SOD1 Signaling.

Microglia are macrophage cells residing in the brain, where they exert a key role in neuronal protection. Through the gut-brain axis, metabolites produced by gut commensal microbes can influence brain functions, including microglial activity. The nuclear factor erythroid 2-related factor 2 (NRF2) is a key regulator of the oxidative stress response in microglia, controlling the expression of cytoprotective genes. Lactobacilli-derived cell-free supernatants (CFSs) are postbiotics that have shown antioxidant and immunomodulatory effects in several in vitro and in vivo studies. This study aimed to explore the effects of lactobacilli CFSs on modulating microglial responses against oxidative stress and inflammation. HMC3 microglia were exposed to lipopolysaccaride (LPS), as an inflammatory trigger, before and after administration of CFSs from three human gut probiotic species. The NRF2 nuclear protein activation and the expression of NRF2-controlled antioxidant genes were investigated by immunoassay and quantitative RT-PCR, respectively. Furthermore, the level of pro- and anti-inflammatory cytokines was evaluated by immunoassay. All CFSs induced a significant increase of NRF2 nuclear activity in basal conditions and upon inflammation. The transcription of antioxidant genes, namely heme oxygenase 1, superoxide dismutase (SOD), glutathione-S transferase, glutathione peroxidase, and catalase also increased, especially after inflammatory stimulus. Besides, higher SOD1 activity was detected relative to inflamed microglia. In addition, CFSs pre-treatment of microglia attenuated pro-inflammatory TNF-α levels while increasing anti-inflammatory IL-10 levels. These findings confirmed that gut microorganisms' metabolites can play a relevant role in adjuvating the microglia cellular response against neuroinflammation and oxidative stress, which are known to cause neurodegenerative diseases.

Substitution of Asp29 with Asn29 in the metallochaperone UreE of Streptococcus thermophilus DSM 20617T increases the urease activity and anticipates urea hydrolysis during milk fermentation.

Urease operon is highly conserved within the species Streptococcus thermophilus and urease-negative strains are rare in nature. S. thermophilus MIMO1, isolated from commercial yogurt, was previously characterized as urease-positive Ni-dependent strain. Beside a mutation in ureQ, coding for a nickel ABC transporter permease, the strain MIMO1 showed a mutation in ureE gene which code for a metallochaperone involved in Ni delivery to the urease catalytic site. The single base mutation in ureE determined a substitution of Asp29 with Asn29 in the metallochaperone in a conserved protein region not involved in the catalytic activity. With the aim to investigate the role Asp29vs Asn29 substitution in UreE on the urease activity of S. thermophilus, ureE gene of the reference strain DSM 20617T (ureEDSM20617) was replaced by ureE gene of strain MIMO1 (ureEMIMO1) to obtain the recombinant ES3. In-gel detection of urease activity revealed that the substitution of Asp29 with Asn29 in UreE resulted in a higher stability of the enzyme complexes. Moreover, the recombinant ES3 showed higher level of urease activity compared to the wildtype without any detectable increase in the expression level of ureC gene, thus highlighting the role of UreE not only in Ni assembly but also on the level of urease activity. During the growth in milk, the recombinant ES3 showed an anticipated urease activity compared to the wildtype, and analogous milk fermentation performance. The overall data obtained by comparing urease-positive and urease-negative strains/mutants confirmed that urease activity strongly impacts on the milk fermentation process and specifically on the yield of the homolactic fermentation.

Immunomodulatory Activity on Human Macrophages by Cell-Free Supernatants to Explore the Probiotic and Postbiotic Potential of Lactiplantibacillus plantarum Strains of Plant Origin.

Upon dietary administration, probiotic microorganisms can reach as live cells the human gut, where they interact with the microbiota and host cells, thereby exerting a beneficial impact on host functions, mainly through immune-modulatory activities. Recently, attention has been drawn by postbiotics, i.e. non-viable probiotic microbes, including their metabolic products, which possess biological activities that benefit the host. Lactiplantibacillus plantarum is a bacterial species that comprises recognised probiotic strains. In this study, we investigated in vitro the probiotic (and postbiotic) potential of seven L. plantarum strains, including five newly isolated from plant-related niches. The strains were shown to possess some basic probiotic attributes, including tolerance to the gastrointestinal environment, adhesion to the intestinal epithelium and safety. Besides, their cell-free culture supernatants modulated cytokine patterns in human macrophages in vitro, promoting TNF-α gene transcription and secretion, while attenuating the transcriptional activation and secretion of both TNF-α and IL-8 in response to a pro-inflammatory signal, and enhancing the production of IL-10. Some strains induced a high IL-10/IL-12 ratio that may correlate to an anti-inflammatory capacity in vivo. Overall, the investigated strains are good probiotic candidates, whose postbiotic fraction exhibits immunomodulatory properties that need further in vivo studies. The main novelty of this work consists in the polyphasic characterisation of candidate beneficial L. plantarum strains obtained from relatively atypical plant-associated niches, by an approach that explores both probiotic and postbiotic potentials, in particular studying the effect of microbial culture-conditioned media on cytokine pattern, analysed at both transcriptional and secretion level in human macrophages.

Molecular chaperone function of three small heat-shock proteins from a model probiotic species.

Small heat-shock proteins (sHSP) are ubiquitous ATP-independent chaperones that prevent irreversible aggregation of heat-damaged denaturing proteins. Lactiplantibacillus plantarum is a widespread Gram-positive bacterium with probiotic claims and vast potential for agro-food, biotechnological and biomedical applications. L. plantarum possesses a family of three sHSP, which were previously demonstrated to be involved in its stress tolerance mechanisms. Here, the three L. plantarum sHSP were heterologously expressed, purified and shown to have a chaperone activity in vitro, measuring their capacity to suppress protein aggregation, as assayed spectrophotometrically by light scattering. Their anti-aggregative capacity was found to be differently influenced by pH. Differences were also found relative to their holdase function and their capacity to modulate liposome membrane fluidity, suggesting interplays between them and indicating diversified activities. This is the first study assessing the chaperone action of sHSP from a probiotic model. The different roles of the three sHSP can increase L. plantarum's capabilities to survive the various types of stress characterising the diverse habitats of this highly adaptable species. Reported evidence supports the interest in L. plantarum as one of the model species for bacteria that have three different sHSP-encoding genes in their genomes.

Antimicrobial Properties, Functional Characterisation and Application of Fructobacillus fructosus and Lactiplantibacillus plantarum Isolated from Artisanal Honey.

Honey is a valuable reservoir of lactic acid bacteria (LAB) and, particularly, of fructophilic LAB (FLAB), a relatively novel subgroup of LAB whose functional potential for human and food application has yet to be explored. In this study, FLAB and LAB strains have been isolated from honeys of different floral origins and selected for their broad antimicrobial activity against typical foodborne pathogenic bacteria and spoilage filamentous fungi. The best candidates, two strains belonging to the species Lactiplantibacillus plantarum and Fructobacillus fructosus, were submitted to partial characterisation of their cell free supernatants (CFS) in order to identify the secreted metabolites with antimicrobial activity. Besides, these strains were examined to assess some major functional features, including in vitro tolerance to the oro-gastrointestinal conditions, potential cytotoxicity against HT-29 cells, adhesion to human enterocyte-like cells and capability to stimulate macrophages. Moreover, when the tested strains were applied on table grapes artificially contaminated with pathogenic bacteria or filamentous fungi, they showed a good ability to antagonise the growth of undesired microbes, as well as to survive on the fruit surface at a concentration that is recommended to develop a probiotic effect. In conclusion, both LAB and FLAB honey-isolated strains characterised in this work exhibit functional properties that validate their potential use as biocontrol agents and for the design of novel functional foods. We reported antimicrobial activity, cytotoxic evaluation, probiotic properties and direct food application of a F. fructosus strain, improving the knowledge of this species, in particular, and on FLAB, more generally.

Biotechnological Potential and Safety Evaluation of Dextran- and Riboflavin-Producing Weisella cibaria Strains for Gluten-Free Baking.

Gluten consumption causes several immunological and non-immunological intolerances in susceptible individuals. In this study, the dextran-producing Weissella cibaria BAL3C-5 and its derivative, the riboflavin-overproducing strain BAL3C-5 C120T, together with a commercial bakery yeast, were used to ferment gluten-free (GF)-doughs obtained from corn and rice flours at two different concentrations and supplemented with either quinoa, buckwheat, or chickpea to obtain laboratory-scale GF bread. The levels of dextran, riboflavin, and total flavins were determined in the fermented and breads. Both strains grew in fermented doughs and contributed dextran, especially to those made with corn plus quinoa (~1 g/100 g). The highest riboflavin (350-150 µg/100 g) and total flavin (2.3-1.75 mg/100 g) levels were observed with BAL3C-5 C120T, though some differences were detected between the various doughs or breads, suggesting an impact of the type of flour used. The safety assessment confirmed the lack of pathogenic factors in the bacterial strains, such as hemolysin and gelatinase activity, as well as the genetic determinants for biogenic amine production. Some intrinsic resistance to antibiotics, including vancomycin and kanamycin, was found. These results indicated the microbiological safety of both W. cibaria strains and indicated their potential application in baking to produce GF bread.

Weissella cibaria riboflavin-overproducing and dextran-producing strains useful for the development of functional bread.

This work describes a method for deriving riboflavin overproducing strains of Weissella cibaria by exposing three strains (BAL3C-5, BAL3C-7, and BAL3C-22) isolated from dough to increasing concentrations of roseoflavin. By this procedure, we selected one mutant overproducing strain from each parental strain (BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2, respectively). Quantification of dextran and riboflavin produced by the parental and mutant strains in a defined medium lacking riboflavin and polysaccharides confirmed that riboflavin was only overproduced by the mutant strains, whereas dextran production was similar in both mutant and parental strains. The molecular basis of the riboflavin overproduction by the mutants was determined by nucleotide sequencing of their rib operons, which encode the enzymes of the riboflavin biosynthetic pathway. We detected a unique mutation in each of the overproducing strains. These mutations, which map in the sensor domain (aptamer) of a regulatory element (the so-called FMN riboswitch) present in the 5' untranslated region of the rib operon mRNA, appear to be responsible for the riboflavin-overproducing phenotype of the BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2 mutant strains. Furthermore, the molecular basis of dextran production by the six W. cibaria strains has been characterized by (i) the sequencing of their dsr genes encoding dextransucrases, which synthesize dextran using sucrose as substrate, and (ii) the detection of active Dsr proteins by zymograms. Finally, the parental and mutant strains were analyzed for in situ production of riboflavin and dextran during experimental bread making. The results indicate that the mutant strains were able to produce experimental wheat breads biofortified with both riboflavin and dextran and, therefore, may be useful for the manufacture of functional commercial breads.

Functionalization of soy residue (okara) by enzymatic hydrolysis and LAB fermentation for B2 bio-enrichment and improved in vitro digestion.

The utilization of major edible soy-waste (okara) remains a challenge due to its poor digestion, nutritional imbalance (lack of B-vitamins), and undesirable off-flavors. Herein, fresh okara was enzymatically hydrolyzed and then fermented using the B2-overproducing Lactiplantibacillus plantarum UFG169 strain. SEM micrographs showed the microporous and honeycombed structures on the surface of okara. The off-flavors were reduced, and the essential amino acids content was significantly increased in fermented okara. The higher ß-glucosidase activity, increased aglycone isoflavones, and in situ riboflavin (B2) were associated with the enhanced antioxidant potential of the fermented okara. The in vitro digestion of okara resulted in reduced particle size, higher protein digestibility, improved aggregation, lower protein molecular chains, and increased polyphenols. Overall, our study indicated the improved nutrition and digestibility of B2 bio-enriched okara.

A single change in the aptamer of the Lactiplantibacillus plantarum rib operon riboswitch severely impairs its regulatory activity and leads to a vitamin B2 - overproducing phenotype.

Manufacturing of probiotics and functional foods using lactic acid bacteria (LAB) that overproduce vitamin B2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analysed for production and intra- and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for LAB, exceeding by ~ 25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~ 8-fold higher in B2. Mutation B2 could decrease the stability of the aptamer's regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavin, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods.

Dairy Lactic Acid Bacteria and Their Potential Function in Dietetics: The Food-Gut-Health Axis.

Fermented dairy products are the good source of different species of live lactic acid bacteria (LAB), which are beneficial microbes well characterized for their health-promoting potential. Traditionally, dietary intake of fermented dairy foods has been related to different health-promoting benefits including antimicrobial activity and modulation of the immune system, among others. In recent years, emerging evidence suggests a contribution of dairy LAB in the prophylaxis and therapy of non-communicable diseases. Live bacterial cells or their metabolites can directly impact physiological responses and/or act as signalling molecules mediating more complex communications. This review provides up-to-date knowledge on the interactions between LAB isolated from dairy products (dairy LAB) and human health by discussing the concept of the food-gut-health axis. In particular, some bioactivities and probiotic potentials of dairy LAB have been provided on their involvement in the gut-brain axis and non-communicable diseases mainly focusing on their potential in the treatment of obesity, cardiovascular diseases, diabetes mellitus, inflammatory bowel diseases, and cancer.

Bioprospecting Antimicrobials from Lactiplantibacillus plantarum: Key Factors Underlying Its Probiotic Action.

Lactiplantibacillus plantarum (L. plantarum) is a well-studied and versatile species of lactobacilli. It is found in several niches, including human mucosal surfaces, and it is largely employed in the food industry and boasts a millenary tradition of safe use, sharing a long-lasting relationship with humans. L. plantarum is generally recognised as safe and exhibits a strong probiotic character, so that several strains are commercialised as health-promoting supplements and functional food products. For these reasons, L. plantarum represents a valuable model to gain insight into the nature and mechanisms of antimicrobials as key factors underlying the probiotic action of health-promoting microbes. Probiotic antimicrobials can inhibit the growth of pathogens in the gut ensuring the intestinal homeostasis and contributing to the host health. Furthermore, they may be attractive alternatives to conventional antibiotics, holding potential in several biomedical applications. The aim of this review is to investigate the most relevant papers published in the last ten years, bioprospecting the antimicrobial activity of characterised probiotic L. plantarum strains. Specifically, it focuses on the different chemical nature, the action spectra and the mechanisms underlying the bioactivity of their antibacterial and antiviral agents. Emerging trends in postbiotics, some in vivo applications of L. plantarum antimicrobials, including strengths and limitations of their therapeutic potential, are addressed and discussed.

Autochthonous Biological Resources for the Production of Regional Craft Beers: Exploring Possible Contributions of Cereals, Hops, Microbes, and Other Ingredients.

Selected biological resources used as raw materials in beer production are important drivers of innovation and segmentation in the dynamic market of craft beers. Among these resources, local/regional ingredients have several benefits, such as strengthening the connection with territories, enhancing the added value of the final products, and reducing supply costs and environmental impacts. It is assumed that specific ingredients provide differences in flavours, aromas, and, more generally, sensory attributes of the final products. In particular, of interest are ingredients with features attributable and/or linked to a specific geographical origin. This review encompasses the potential contribution and exploitation of biodiversity in the main classes of beer inputs, such as cereals, hops, microbes, and adjuncts, with a specific emphasis on autochthonous biological resources, detailing the innovative paths already explored and documented in the scientific literature. This dissertation proposes an overview of the impact on beer quality for each raw material category, highlighting the benefits and limitations that influence its concrete applications and scale-up, from the field to the stain. The topics explored promote, in the sector of craft beers, trends already capitalised in the production of other alcoholic beverages, such as the preservation and revalorisation of minor and autochthonous varieties, the exploitation of yeast and bacteria strains isolated from specific sites/plant varieties, and the valorisation of the effects of peculiar terroirs on the quality of agricultural products. Finally, the examined tendencies contribute toward reducing the environmental impacts of craft beer manufacturing, and are in line with sustainable development of food systems, increasing the economic driver of biodiversity preservation.

Screening of Lactic Acid Bacteria for the Bio-Control of Botrytis cinerea and the Potential of Lactiplantibacillus plantarum for Eco-Friendly Preservation of Fresh-Cut Kiwifruit.

Botrytis cinerea, responsible for grey mold, represents the first biological cause of fruit and vegetable spoilage phenomena in post-harvest. Kiwifruit is a climacteric fruit particularly prone to this mold infestation during storage. Lactic acid bacteria (LAB) are food-grade bacteria that can synthesize several metabolites with antimicrobial activity and are, therefore, suggested as promising and eco-friendly resources for the bio-control of molds on fruits and vegetables. In this work, we propose the screening of a collection of 300 LAB previously isolated from traditional sourdoughs for their ability to counteract in vitro the growth of Botrytis cinerea CECT 20973. Only 2% of tested LAB strains belonging to Lactiplantibacillus plantarum species, exerted a strong antagonism against B. cinerea. The cell-free supernatants were partially characterized and results clearly indicated that high levels of lactic acid contributed to the antagonistic activity. PAN01 and UFG 121 cell-free supernatants were investigated as potential bio-control agents in a preliminary in vivo assay using freshly cut kiwifruits as a food model. The application of cell-free supernatants allowed to delay the growth of B. cinerea on artificially contaminated kiwifruits until two weeks. The antagonistic activity was greatly affected by the storage temperature (25 °C and 4 °C) selected for the processed fruits, suggesting the importance to include microbial-based solution in a broader framework of hurdle technologies.

Selection of Riboflavin Overproducing Strains of Lactic Acid Bacteria and Riboflavin Direct Quantification by Fluorescence.

Riboflavin (vitamin B2) is a vitamin of the B group involved in essential biological pathways, including redox reactions and the electron transport chain. Some lactic acid bacteria (LAB) can synthesize riboflavin and this capability is strain-dependent. In the last years, a growing interest has focused on the selection of riboflavin-overproducing food-grade LAB for the vitamin biofortification of fermented foods, as well as for the formulation of innovative functional products.In this chapter we report fast and inexpensive techniques in order to (1) screen LAB isolates able to produce riboflavin from different matrices, (2) select spontaneous roseoflavin-resistant riboflavin overproducing strains, and (3) quantify vitamin B2 in culture media by fluorescence detection.These protocols could be useful to select new overproducing strains and/or species from different ecological niches, as well as to optimize the conditions for vitamin bioproduction.

Microbial-based Biocontrol Solutions for Fruits and Vegetables: Recent Insight, Patents, and Innovative Trends.

BACKGROUND: Fruits and vegetables are susceptible to colonisation by undesired microflora, which, in pre- and post-harvest conditions, negatively impact the quality of these products, leading to a reduction of yield, shelf-life, and marketability. In the few last years, the use of microbial Biological Control Agents (BCAs) has assumed international relevance in order to control harmful microorganisms, as a promising alternative to chemical interventions. OBJECTIVE: The purpose of this review is to discuss the microbial-based solutions applicable for the biocontrol of the main microbial spoilers, phytopathogens, and human food-borne pathogens affecting fruits and vegetables during their production and storage. RESULTS: A comprehensive overview of the scientific literature investigating the effectiveness of BCA-based products available on the market is provided, as well as of the most recent patents protecting biotechnological applications in this field. Innovative trends are discussed, with a particular focus on the integration of BCAs to minimise spoilage phenomena and microbiological risks adopting combined approaches. CONCLUSION: This study underlines the growing interest about biocontrol strategies to counteract the growth of spoilage and/or pathogenic microorganisms indicating that in the next years a considerable increase of commercial products and patents will be developed worldwide to exploit innovative biotechnological solutions in the sector.

Knock out of sHSP genes determines some modifications in the probiotic attitude of Lactiplantibacillus plantarum.

OBJECTIVE: We investigated whether the knock out of small heat shock protein (sHSP) genes (hsp1, hsp2 and hsp3) impact on probiotic features of Lactiplantibacillus plantarum WCFS1, aiming to find specific microbial effectors involved in microbe-host interplay. RESULTS: The probiotic properties of L. plantarum WCFS1 wild type, hsp1, hsp2 and hsp3 mutant clones were evaluated and compared through in vitro trials. Oro-gastro-intestinal assays pointed to significantly lower survival for hsp1 and hsp2 mutants under stomach-like conditions, and for hsp3 mutant under intestinal stress. Adhesion to human enterocyte-like cells was similar for all clones, though the hsp2 mutant exhibited higher adhesiveness. L. plantarum cells attenuated the transcriptional induction of pro-inflammatory cytokines on lipopolysaccharide-treated human macrophages, with some exception for the hsp1 mutant. Intriguingly, this clone also induced a higher IL10/IL12 ratio, which is assumed to indicate the anti-inflammatory potential of probiotics. CONCLUSIONS: sHSP genes deletion determined some differences in gut stress resistance, cellular adhesion and immuno-modulation, also implying effects on in vivo interaction with the host. HSP1 might contribute to immunomodulatory mechanisms, though additional experiments are necessary to test this feature.

Editorial: Microbiological Safety of Foods.

The management of food safety represents a global and transdisciplinary issue of great relevance for human health and crucial economic sectors [...].

Botrytis cinerea and Table Grapes: A Review of the Main Physical, Chemical, and Bio-Based Control Treatments in Post-Harvest.

Consumers highly appreciate table grapes for their pleasant sensory attributes and as good sources of nutritional and functional compounds. This explains the rising market and global interest in this product. Along with other fruits and vegetables, table grapes are considerably perishable post-harvest due to the growth of undesired microorganisms. Among the microbial spoilers, Botrytis cinerea represents a model organism because of its degrading potential and the huge economic losses caused by its infection. The present review provides an overview of the recent primary physical, chemical, and biological control treatments adopted against the development of B. cinerea in table grapes to extend shelf life. These treatments preserve product quality and safety. This article also focuses on the compliance of different approaches with organic and sustainable production processes. Tailored approaches include those that rely on controlled atmosphere and the application of edible coating and packaging, as well as microbial-based activities. These strategies, applied alone or in combination, are among the most promising solutions in order to prolong table grape quality during cold storage. In general, the innovative design of applications dealing with hurdle technologies holds great promise for future improvements.