Donkey Milk Fermentation by Lactococcus lactis subsp. cremoris and Lactobacillus rhamnosus Affects the Antiviral and Antibacterial Milk Properties.

BACKGROUND: Milk is considered an important source of bioactive peptides, which can be produced by endogenous or starter bacteria, such as lactic acid bacteria, that are considered effective and safe producers of food-grade bioactive peptides. Among the various types of milk, donkey milk has been gaining more and more attention for its nutraceutical properties. METHODS: Lactobacillus rhamnosus 17D10 and Lactococcus lactis subsp. cremoris 40FEL3 were selected for their ability to produce peptides from donkey milk. The endogenous peptides and those obtained after bacterial fermentation were assayed for their antioxidant, antibacterial, and antiviral activities. The peptide mixtures were characterized by means of LC-MS/MS and then analyzed in silico using the Milk Bioactive Peptide DataBase. RESULTS: The peptides produced by the two selected bacteria enhanced the antioxidant activity and reduced E. coli growth. Only the peptides produced by L. rhamnosus 17D10 were able to reduce S. aureus growth. All the peptide mixtures were able to inhibit the replication of HSV-1 by more than 50%. Seventeen peptides were found to have 60% sequence similarity with already known bioactive peptides. CONCLUSIONS: A lactic acid bacterium fermentation process is able to enhance the value of donkey milk through bioactivities that are important for human health.

Back to the past: "find the guilty bug-microorganisms involved in the biodeterioration of archeological and historical artifacts".

Microbial deterioration accounts for a significant percentage of the degradation processes that occur on archeological/historical objects and artworks, and identifying the causative agents of such a phenomenon should therefore be a priority, in consideration of the need to conserve these important cultural heritage items. Diverse microbiological approaches, such as microscopic evaluations, cultural methods, metabolic- and DNA-based techniques, as well as a combination of the aforementioned methods, have been employed to characterize the bacterial, archaeal, and fungal communities that colonize art objects. The purpose of the present review article is to report the interactions occurring between the microorganisms and nutrients that are present in stones, bones, wood, paper, films, paintings, and modern art specimens (namely, collagen, cellulose, gelatin, albumin, lipids, and hydrocarbons). Some examples, which underline that a good knowledge of these interactions is essential to obtain an in depth understanding of the factors that favor colonization, are reported. These data can be exploited both to prevent damage and to obtain information on historical aspects that can be decrypted through the study of microbial population successions.

Back to the past-forever young: cutting-edge biochemical and microbiological tools for cultural heritage conservation.

Ancient documents and milestones of human history such as manuscripts and textiles are fragile and during aging undergo chemical, physical, and biological deterioration. Among the different causes of damage, also human intervention plays a role since some restoration strategies proved to be transient and/or they generated further damage. Outdoor monuments undergo deterioration since they are exposed to pollution, weathering, microbial attack (giving rise to undesired pigmentation, discoloration or true dissolution, corrosion, and overall decay), as well as man-made damage (i.e., graffiti). This review article reports the best-fitting strategies used to restore wall paintings, outdoor monuments, textiles, and paper documents to their ancient beauty by employing "soft" biobased approaches such as viable bacteria or suitable enzymes.

Back to the past: deciphering cultural heritage secrets by protein identification.

The present review article reports the most innovative methods to detect proteins in historical and archeological samples as well as to characterize proteins used as binders in artworks. Difficulties to ascribe proteins to a certain animal species are often due to post-translational modifications originated by chemical or microbial deterioration during aging. Combining different techniques such as peptide mass fingerprinting and tandem mass spectrometry can solve some of these problems and also allow discrimination between taxonomically related species like sheep and goat. The most studied proteins in bones and textile samples are osteocalcin, collagen and keratin, whereas egg yolk and white proteins, casein and collagen are the most relevant for binders used in old paintings. With the suitable approaches (immune-based methods, DOT-blot, etc…) it is also possible to obtain in situ characterization or analyze the samples directly in the museum laboratories, with the advantage of avoiding artwork damage and expensive external commitments. Recent cutting-edge strategies allowed detection of proteinaceous infection markers that, for instance, were used to establish the cause of death of old Inca mummies and also proved the presence of Yersinia pestis in old documents dating from the period in 17th century in which the plague ravaged Europe.

Cannabinoid Delivery Systems for Pain and Inflammation Treatment.

There is a growing body of evidence to suggest that cannabinoids are beneficial for a range of clinical conditions, including pain, inflammation, epilepsy, sleep disorders, the symptoms of multiple sclerosis, anorexia, schizophrenia and other conditions. The transformation of cannabinoids from herbal preparations into highly regulated prescription drugs is therefore progressing rapidly. The development of such drugs requires well-controlled clinical trials to be carried out in order to objectively establish therapeutic efficacy, dose ranges and safety. The low oral bioavailability of cannabinoids has led to feasible methods of administration, such as the transdermal route, intranasal administration and transmucosal adsorption, being proposed. The highly lipophilic nature of cannabinoids means that they are seen as suitable candidates for advanced nanosized drug delivery systems, which can be applied via a range of routes. Nanotechnology-based drug delivery strategies have flourished in several therapeutic fields in recent years and numerous drugs have reached the market. This review explores the most recent developments, from preclinical to advanced clinical trials, in the cannabinoid delivery field, and focuses particularly on pain and inflammation treatment. Likely future directions are also considered and reported.

Comparative proteomics of oxidative stress response of Lactobacillus acidophilus NCFM reveals effects on DNA repair and cysteine de novo synthesis.

Probiotic cultures encounter oxidative conditions during manufacturing, yet protein abundance changes induced by such stress have not been characterized for some of the most common probiotics and starters. This comparative proteomics investigation focuses on the response by Lactobacillus acidophilus NCFM to H2 O2, simulating an oxidative environment. Bacterial growth was monitored by BioScreen and batch cultures were harvested at exponential phase for protein profiling of stress responses by 2D gel based comparative proteomics. Proteins identified in 19 of 21 spots changing in abundance due to H2 O2 were typically related to carbohydrate and energy metabolism, cysteine biosynthesis, and stress. In particular, increased cysteine synthase activity may accumulate a cysteine pool relevant for protein stability, enzyme catalysis, and the disulfide-reducing pathway. The stress response further included elevated abundance of biomolecules reducing damage such as enzymes from DNA repair pathways and metabolic enzymes with active site cysteine residues. By contrast, a protein-refolding chaperone showed reduced abundance, possibly reflecting severe oxidative protein destruction that was not overcome by refolding. The proteome analysis provides novel insight into resistance mechanisms in lactic acid bacteria against reactive oxygen species and constitutes a valuable starting point for improving industrial processes, food design, or strain engineering preserving microorganism viability.

Recombinant Lactococcus lactis for efficient conversion of cellodextrins into L-lactic acid.

Lactic acid bacteria (LAB) are among the most interesting organisms for industrial processes with a long history of application as food starters and biocontrol agents, and an underexploited potential for biorefineries converting biomass into high-value compounds. Lactic acid (LA), their main fermentation product, is among the most requested chemicals owing to its broad range of applications. Notably, LA polymers, that is, polylactides, have high potential as biodegradable substitutes of fossil-derived plastics. However, LA production by LAB fermentation is currently too expensive for polylactide to be cost-competitive with traditional plastics. LAB have complex nutritional requirements and cannot ferment inexpensive substrates such as cellulose. Metabolic engineering could help reduce such nutritional requirements and enable LAB to directly ferment low-cost polysaccharides. Here, we engineered a Lactococcus lactis strain which constitutively secretes a ß-glucosidase and an endoglucanase. The recombinant strain can grow on cellooligosaccharides up to at least cellooctaose and efficiently metabolizes them to L-LA in single-step fermentation. This is the first report of a LAB able to directly metabolize cellooligosaccharides longer that cellohexaose and a significant step toward cost-sustainable consolidated bioprocessing of cellulose into optically pure LA.

Enzymatic laundry for old clothes: immobilized alpha-amylase from Bacillus sp. for the biocleaning of an ancient Coptic tunic.

The classification and conservation of ancient artworks (belonging to collections) is of important cultural, historical, and economic concern. However, ancient textiles often display structural damage that renders them fragile and unsuitable for exhibition. One of the most common types of damage is linked to erroneous restoration treatments, among which the application of glues to consolidate cuts. Harsh strategies, such as mechanical or chemical treatments, are not suitable since they can cause further impairment of the fabric, whereas mild approaches, like wet cleaning, are often ineffective, as also demonstrated by the present study. Here, we have explored the possibility of using gellan-immobilized enzymes of bacterial origin (Bacillus alpha-amylase) to obtain a satisfactory starch removal from a damaged archaeological tunic-shroud from the Turin Egyptian Museum (Italy), without altering the original yarns or textile fibers. This method, already applied to clean casein-damaged wall paintings, as well as cotton, silk, and linen fabrics, has proved to be optimal for the treatment of a wool burial shroud and to be able to definitively solve fragile textile restoration problems. Moreover, efforts have been made to obtain insights into the artwork: a multidisciplinary approach has allowed to obtain a correct chronological attribution (radiocarbon dating) and fabric fiber characterization (SEM-EDX) as well as shed light on the colored parts and dark stains (FORS+IRFC and XRF). Finally, the evaluation of the type of glue, by Fourier transform infrared spectroscopy, has suggested the best enzyme for glue removal. These results have demonstrated that a mild bio-based approach is a successful tool for the treatment of archaeological textiles in critical conditions.

Antimicrobial Activity of Lactoferrin-Related Peptides and Applications in Human and Veterinary Medicine.

Antimicrobial peptides (AMPs) represent a vast array of molecules produced by virtually all living organisms as natural barriers against infection. Among AMP sources, an interesting class regards the food-derived bioactive agents. The whey protein lactoferrin (Lf) is an iron-binding glycoprotein that plays a significant role in the innate immune system, and is considered as an important host defense molecule. In search for novel antimicrobial agents, Lf offers a new source with potential pharmaceutical applications. The Lf-derived peptides Lf(1-11), lactoferricin (Lfcin) and lactoferrampin exhibit interesting and more potent antimicrobial actions than intact protein. Particularly, Lfcin has demonstrated strong antibacterial, anti-fungal and antiparasitic activity with promising applications both in human and veterinary diseases (from ocular infections to osteo-articular, gastrointestinal and dermatological diseases).

Privileged incorporation of selenium as selenocysteine in Lactobacillus reuteri proteins demonstrated by selenium-specific imaging and proteomics.

An analytical approach was developed to study the incorporation of selenium (Se), an important trace element involved in the protection of cells from oxidative stress, into the well-known probiotic Lactobacillus reuteri Lb2 BM-DSM 16143. The analyses revealed that about half of the internalized Se was covalently incorporated into soluble proteins. Se-enriched proteins were detected in 2D gels by laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) and identified by capillary HPLC with the parallel ICP MS ((78)Se) and electrospray Orbitrap MS/MS detection. On the basis of the identification of 10 richest in selenium proteins, it was demonstrated that selenium was incorporated by the strain exclusively as selenocysteine. Also, the exact location of selenocysteine within the primary sequence was determined. This finding is in a striking contrast to another common nutraceutical, Se-enriched yeast, which incorporates Se principally as selenomethionine.

Short-term response of different Saccharomyces cerevisiae strains to hyperosmotic stress caused by inoculation in grape must: RT-qPCR study and metabolite analysis.

During the winemaking process, glycerol synthesis represents the first adaption response of Saccharomyces cerevisiae to osmotic stress after inoculation in grape must. We have implemented an RT-qPCR (Reverse Transcription-quantitative PCR) methodology with a preventive evaluation of candidate reference genes, to study six target genes related to glycerol synthesis (GPD1, GPD2, GPP2 and GPP1) and flux (STL1 and FPS1), and three ALD genes coding for aldehyde dehydrogenase involved in redox equilibrium via acetate production. The mRNA level in three strains, characterized by different metabolite production, was monitored in the first 120 min from inoculation into natural grape must. Expression analysis shows a transient response of genes GPD1, GPD2, GPP2, GPP1 and STL1 with differences among strains in term of mRNA abundance, while FPS1 was expressed constitutively. The transient response and different expression intensity among strains, in relation to the intracellular glycerol accumulation pattern, prove the negative feedback control via the HOG (High Osmolarity Glycerol) signalling pathway in S. cerevisiae wine strains under winery conditions. Among the ALD genes, only ALD6 was moderately induced in the hyperosmotic environment but not in all strains tested, while ALD3 and ALD4 were drastically glucose repressed. The intensity of transcription of ALD6 and ALD3 seems to be related to different acetate production found among the strains.

Ormosil gels doped with engineered catechol 1,2 dioxygenases for chlorocatechol bioremediation.

Enzymes entrapped in wet, nanoporous silica gel have great potential as bioreactors for bioremediation because of their improved thermal, chemical, and mechanical stability with respect to enzymes in solution. The B isozyme of catechol 1,2 dioxygenase from Acinetobacter radioresistens and its mutants of Leu69 and Ala72, designed for an increased reactivity toward the environmental pollutant chlorocatechols, were encapsulated using alkoxysilanes and alkyl alkoxysilanes as precursors in varying proportions. Encapsulation of the mutants in a hydrophobic tetramethoxysilane/dimethoxydimethylsilane-based matrix yielded a remarkable 10- to 12-fold enhancement in reactivity toward chlorocatechols. These gels also showed a fivefold increase in relative reactivity toward chlorocatechols with respect to the natural substrate catechol, thus compensating for their relatively low activity for these substrates in solution. The encapsulated enzyme, unlike the enzyme in solution, proved resilient in assays carried out in urban wastewater and bacteria-contaminated solutions mimicking environmentally relevant conditions. Overall, the combination of a structure-based rational design of enzyme mutants, and the selection of a suitable encapsulation material, proved to be a powerful approach for the production and optimization of a potential bioremediation device, with increased activity and resistance toward bacterial degradation.

An EPR, thermostability and pH-dependence study of wild-type and mutant forms of catechol 1,2-dioxygenase from Acinetobacter radioresistens S13.

Intradiol dioxygenase are iron-containing enzymes involved in the bacterial degradation of natural and xenobiotic aromatic compounds. The wild-type and mutants forms of catechol 1,2-dioxygenase Iso B from Acinetobacter radioresistens LMG S13 have been investigated in order to get an insight on the structure-function relationships within this system. 4K CW-EPR spectroscopy highlighted different oxygen binding properties of some mutants with respect to the wild-type enzyme, suggesting that a fine tuning of the substrate-binding determinants in the active site pocket may indirectly result in variations of the iron reactivity. A thermostability investigation by optical spectroscopy, that reports on the state of the metal center, showed that the structural stability is more influenced by the type rather than by the position of the mutation. Finally, the influence of pH and temperature on the catalytic activity was monitored and discussed in terms of perturbations induced on the tertiary contact network of the enzyme.

Different protein expression profiles in cheese and clinical isolates of Enterococcus faecalis revealed by proteomic analysis.

The use of Enterococcus faecalis in the food industry has come under dispute because of the pathogenic potential of some strains of this species. In this study, we have compared the secretome and whole-cell proteome of one food isolate (E. faecalis DISAV 1022) and one clinical isolate (E. faecalis H1) by 2-DE and iTRAQ analyses, respectively. Extracellular protein patterns differed significantly, with only seven proteins common to both strains. Notably, only the clinical isolate expressed various well-characterized virulence factors such as the gelatinase coccolysin (GelE) and the extracellular serine proteinase V8 (SprE). Moreover, various other putative virulence factors, e.g. superoxide dismutase, choline- and chitin-binding proteins and potential moonlighting proteins, have been detected exclusively in the secretome of the clinical isolate, but not in the food isolate. The iTRAQ analysis of whole-cell proteins of the two strains highlighted a stronger expression of pathogenic traits such as an endocarditis-specific antigen and an adhesion lipoprotein in the pathogenic strain E. faecalis H1. Subsequently, six food isolates (including E. faecalis DISAV 1022) and six clinical isolates (including E. faecalis H1) were tested for the presence of gelatinase and protease activity in the culture supernatants. Both enzymatic activities were found in the clinical as well as the food isolates which clearly indicates that protease expression is strain specific and not representative for pathogenic isolates. Genetic analyses revealed that not only the gelatinase and serine protease genes but also the regulatory fsr genes must be present to allow protease expression.

X-ray crystallography, mass spectrometry and single crystal microspectrophotometry: a multidisciplinary characterization of catechol 1,2 dioxygenase.

Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

Norepinephrine and Serotonin Can Modulate the Behavior of the Probiotic Enterococcus faecium NCIMB10415 towards the Host: Is a Putative Surface Sensor Involved?

The human gut microbiota has co-evolved with humans by exchanging bidirectional signals. This study aims at deepening the knowledge of this crucial relationship by analyzing phenotypic and interactive responses of the probiotic Enterococcus faecium NCIMB10415 (E. faecium SF68) to the top-down signals norepinephrine (NE) and serotonin (5HT), two neuroactive molecules abundant in the gut. We treated E. faecium NCIMB10415 with 100 µM NE and 50 µM 5HT and tested its ability to form static biofilm (Confocal Laser Scanning Microscopy), adhere to the Caco-2/TC7 monolayer, affect the epithelial barrier function (Transepithelial Electrical Resistance) and human dendritic cells (DC) maturation, differentiation, and cytokines production. Finally, we evaluated the presence of a putative hormone sensor through in silico (whole genome sequence and protein modelling) and in vitro (Micro-Scale Thermophoresis) analyses. The hormone treatments increase biofilm formation and adhesion on Caco-2/TC7, as well as the epithelial barrier function. No differences concerning DC differentiation and maturation between stimulated and control bacteria were detected, while an enhanced TNF-α production was observed in NE-treated bacteria. Investigations on the sensor support the hypothesis that a two-component system on the bacterial surface can sense 5HT and NE. Overall, the data demonstrate that E. faecium NCIMB10415 can sense both NE and 5HT and respond accordingly.

Proteomic characterization of a selenium-metabolizing probiotic Lactobacillus reuteri Lb26 BM for nutraceutical applications.

Selenium (Se), Se-cysteines and selenoproteins have received growing interest in the nutritional field as redox-balance modulating agents. The aim of this study was to establish the Se-concentrating and Se-metabolizing capabilities of the probiotic Lactobacillus reuteri Lb26 BM, for nutraceutical applications. A comparative proteomic approach was employed to study the bacteria grown in a control condition (MRS modified medium) and in a stimulated condition (4.38 mg/L of sodium selenite). The total protein extract was separated into two pI ranges: 4-7 and 6-11; the 25 identified proteins were divided into five functional classes: (i) Se metabolism; (ii) energy metabolism; (iii) stress/adhesion; (iv) cell shape and transport; (v) proteins involved in other functions. All the experimental results indicate that L. reuteri Lb26 BM is able to metabolize Se(IV), incorporating it into selenoproteins, through the action of a selenocysteine lyase, thus enhancing organic Se bioavailability. This involves endo-ergonic reactions balanced by an increase of substrate-level phosphorylation, chiefly through lactic fermentation. Nevertheless, when L. reuteri was grown on Se a certain degree of stress was observed, and this has to be taken into account for future applicative purposes. The proteomic approach has proven to be a powerful tool for the metabolic characterization of potential Se-concentrating probiotics.