Selection and microencapsulation of an “NADH-oxidizing” bacterium and its use for NAD regeneration

An alternative approach to the regeneration of coenzymes is described here using immobilized microorganisms possessing “NADH-oxidase” function. Bacteria containing NADH-oxidase activity are immobilized by microencapsulation within artificial cells. In this form, the microencapsulated bacteria can recycle NADH back to NAD in the presence of molecular oxygen as an electron acceptor. The only byproduct of the recycling reaction is water. In order to perform the biological regeneration of NAD, the activity of NADH-oxidase was investigated in 13 strains of aerobic bacteria and yeast. The NADH-oxidizing bacteriaLeuconostoc mesenteroides exhibited the highest activity among the microorganisms tested. The permeabilized bacteria showed 10% of their initial activity after microencapsulation. Light and electron microscopy studies of bacteria loaded microcapsules have been done. Enzymatic properties of microcapsule-immobilized bacteria were investigated in comparison with those of the free enzyme complex.Leuconostoc mesenteroides, containing NADH-oxidase, has been microencapsulated together with 3α-hydroxysteroid dehydrogenase (3α-HSDH) for stereospecific steroid oxidation. In a batch reactor, 2 mg of NAD, with recycling, allowed the same substrate consumption as 4.4 mg of NAD without recycling. The microencapsulated system can be used repeatedly. The system is functional for 10 h, during which time each molecule of NAD has been used 7.6 times.     

Application of isotherm calorimetry in the development of foods containing probiotic live flora and enriched with bioavailable Ca<Superscript>2+</Superscript>

The development of functional foods of probiotic effect based on the slime-producing strains isolated in the 1980s, and that of enriched with Ca on the utilization of the high Ca-containing whey of the quarg production in the Carpathian basin using fermentation. The probiotic properties of the slime-producing microbe strains isolated have been proved by in vitro and in vivo examinations. We have used an isotherm DSC method to identify the probiotic microbes. The percentile ratio of probiotic and other microbes was determined in the product by this technique. By utilization of quarg whey a special additive food for Ca-enrichment has been developed which is suitable to complete or enrich different foods (dairy, meat and bakery products). The products developed are: probiotic kefir (Synbiofir), probiotic sour cream, probiotic butter cream, poultry meat products completed with Ca, bakery products completed with Ca.     

Feasibility of biofilm production capacity by Levilactobacillus brevis isolated from motal cheese and evaluation of biofilm resistance produced in vitro and in yogurt

Traditional dairy products are a unique source which have been considered for the extraction of indigenous probiotic strains in recent years. In this study, biofilm formation power of Levilactobacillus brevis that isolated from Mutal traditional cheese were investigated. Survival was assessed during 21 days of storage time and at the presence of residues antibiotics as well as gastrointestinal conditions. The results showed after 120 min of treatment in high acidic conditions (pH 2.0), the survival rate decreased only 0.75 log CFU/mL in biofilm formed. The antibiotic susceptibility evaluated of probiotic to enrofloxacin, sulfadimidine, tetracycline, and oxytetracycline showed reducing the bacterial population in the biofilm form only 2.6 log. probiotic strain that isolated from indigenous dairy sources showed excellent resistance in the biofilm state. Therefore, extracting strong probiotic strains from indigenous resources, it can significantly improve functional products and fermentory engineering.     

The Development of the Antibacterial Microcapsules of Citrus Essential Oil for the Cosmetotextile Application: A Review

Essential oils (EOs) obtained from the Citrus genus were reported to exhibit good antimicrobial activity. Therefore, they can potentially be applied in daily necessities such as textile sectors as antibacterial functional fabric products. However, a packaging technique to retain such volatile and labile active substances is compulsory. In particular, microencapsulation was found to be a common coating technique employed to protect EOs from the effects of light, heat, humidity, stability, and controlled release of active substances. Various microencapsulation techniques have been introduced, but the most widely used method is complex coacervation, as it is simple, inexpensive, and capable of snaring high essential oils. Hence, this review focused on the microencapsulation of the most consumable citrus EOs with complex coacervation methods and their immobilization on commonly carried-out fabrics. In addition, it also discusses the isolation methods of the EOs, their chemical composition, and the mechanism of antibacterial action.     

Metal corrosion induced by microbial activity – Mechanism and control options

Microbe-influenced material damage is the result of contributions from different types of microbes and their physiological activity. This fact makes the understanding of microbial corrosion very difficult. Another interesting fact is that the biofilms formed by the bacterial action inhibit or promote the biocorrosion of metals depending on the local environmental conditions. A slight change in the living conditions such as nutrient composition, oxygen concentration, light, and temperature can alter the behavior of the microbial population from non-corrosive to aggressively corroding. The metabolic activity of certain bacterial strains and the biofilm produced by them helps to control biocorrosion. The use of bioengineered bacterial strains has also been found to offer promising results in biocorrosion control. The most widely practiced biocorrosion mitigation practices include the application of protective coatings and the use of biocides. Recently, incorporating the corrosion protective functional layers on the metal surface via polymerization reactions has gained importance. This review provides information on the type of microorganisms causing the biocorrosion, their mechanism of action, and the factors that influence the corrosion rate. The development and involvement of the biofilm in corrosion have also been discussed in detail. The techniques available for the control of biocorrosion have also been explored.     

Toward a new generation of therapeutics: artificial cell targeted delivery of live cells for therapy

Scientific evidence in the prevention and treatment of various disorders is accumulating regarding probiotics. The health benefits supported by adequate clinical data include increased resistance to infectious disease, decreased duration of diarrhea, management of inflammatory bowel disease, reduction of serum cholesterol, prevention of allergy, modulation of cytokine gene expression, and suppression of carcinogen production. Recent ventures in metabolic engineering and heterologous protein expression have enhanced the enzymatic and immunomodulatory effects of probiotics and, with time, may allow more active intervention among critical care patients. In addition, a number of approaches are currently being explored, including the physical and chemical protection of cells, to increase probiotic viability and its health benefits. Traditional immobilization of probiotics in gel matrices, most notably calcium alginate and kappa-carrageenan, has frequently been employed, with noted improvements in viability during freezing and storage. Conflicting reports exist, however, on the protection offered by immobilization from harsh physiologic environments. An alternative approach, microencapsulation in "artificial cells," builds on immobilization technologies by combining enhanced mechanical stability of the capsule membrane with improved mass transport, increased cell loading, and greater control of parameters. This review summarizes the current clinical status of probiotics, examines the promises and challenges of current immobilization technologies, and presents the concept of artificial cells for effective delivery of therapeutic bacterial cells.     

Indication of proliferation of lactic acid bacteria in mixed cultures

The supply and consumption of probiotic foods, and particularly probiotic dairy products, has grown steadily in recent years. In the production of dairy products of this type other microbes must also be used in addition to the microbes which provide the probiotic effect and which generally have a proliferation optimum at 37°C. The probiotic microbes have a neutral taste in dairy products consequently the taste of fermented dairy products is supplied by other microbes. These microbes are likewise lactic acid bacteria, and their proliferation optima are either below (mesophilic) or above (thermophilic) that of the probiotic microbes. It is imperative to have an indication of whether the probiotic bacteria have multiplied at the fermentation temperature used during the technology, since they provide the beneficial physiological effect of the product. Isothermic calorimetry appeared a suitable method for the indication of this process, because the amount of heat released during lactic acid bacterial proliferation differs from the probiotic one. In order to analyze the heat flow curves a deconvolutional program was developed which decomposed them into Gaussian curves, because the proliferation of individual microbes follows a lognormal distribution. The Gaussian curve characteristic for the culture was determined, and from the area under the curve the heat liberated during the creation of one microbe was calculated.     

Toward a new generation of therapeutics

Scientific evidence in the prevention and treatment of various disorders is accumulating regarding probiotics. The health benefits supported by adequate clinical data include increased resistance to infectious disease, decreased duration of diarrhea, management of inflammatory bowel disease, reduction of serum cholesterol, prevention of allergy, modulation of cytokine gene expression, and suppression of carcinogen production. Recent ventures in metabolic engineering and heterologous protein expression have enhanced the enzymatic and immunomodulatory effects of probiotics and, with time, may allow more active intervention among critical care patients. In addition, a number of approaches are currently being explored, including the physical and chemical protection of cells, to increase probiotic viability and its health benefits. Traditional immobilization of probiotics in gel matrices, most notably calcium alginate and κ-carrageenan, has frequently been employed, with noted improvements in viability during freezing and storage. Conflicting reports exist, however, on the protection offered by immobilization from harsh physiologic environments. An alternative approach, microencapsulation in “artificial cells,” builds on immobilization technologies by combining enhanced mechanical stability of the capsule membrane with improved mass transport, increased cell loading, and greater control of parameters. This review summarizes the current clinical status of probiotics, examines the promises and challenges of current immobilization technologies, and presents the concept of artificial cells for effective delivery of therapeutic bacterial cells.     

Probiotic Attributes of Autochthonous Lactobacillus rhamnosus Strains of Human Origin

The study was aimed at evaluating the probiotic potential of indigenous autochthonous Lactobacillus rhamnosus strains isolated from infant feces and vaginal mucosa of healthy female. The survival of the selected strains and the two reference strains (L. rhamnosus GG and L. casei Actimel) was 67–81 % at pH 2 and 70–80 % after passage through the simulated gastrointestinal fluid. These strains are able to grow in the presence of 4 % bile salt, 10 % NaCl, and 0.6 % phenol. The cell surface of L. rhamnosus strains is hydrophilic in nature as revealed by bacterial adhesion to hydrocarbons (BATH) assay. Despite this, L. rhamnosus strains showed mucin adherence, autoaggregation and coaggregation properties that are strain-specific. In addition, they produce bile salt hydrolase (BSH) and β-galactosidase activities. L. rhamnosus strains exhibit antimicrobial activity against food spoilage organisms and gastrointestinal pathogens, as well as Candida and Aspergillus spp. L. rhamnosus strains have similar antibiotic susceptibility pattern, and resistance to certain antibiotics is intrinsic or innate. The strains are neither haemolytic nor producer of biogenic amines such as histamine, putrescine, cadaverine and tyramine. Lyophilized cells of L. rhamnosus Fb exhibited probiotic properties demonstrating potential of the strain for technological suitability and in the preparation of diverse probiotic food formulations.     

Synthesis of functional microcapsules containing suspensions responsive to electric fields

A sort of functional microcapsules, which contain a suspension responsive to electric fields, is prepared by in situ polymerization of urea and formaldehyde. The suspension is made up of pigment phthalocyanine green (PPG) and tetrachloroethylene. In order to solve the particles' separation from the suspension during the microencapsulation and to obtain microcapsules applying to electronic ink display, the dispersibility of the particles, the contact angles between the particles and the tetrachloroethylene, and the influences of different emulsifiers on the microencapsulation are investigated. It is found that the dispersion extent and lipophilicity of the PPG particles are improved due to their surface modification with octadecylamine. The contact angles between the modified PPG particles and the tetrachloroethylene increase, and the PPG particles modified with 2 wt% octadecylamine have the best affinity for tetrachloroethylene. The interfacial tension between C(2)Cl(4) and H(2)O with urea-formaldehyde prepolymer descends from 43 to 35 mN/m, which indicates that the polymer has certain surface activity. However, water-soluble emulsifiers have an important influence during the microencapsulation because they can absorb on the surfaces of internal phase and prevent the resin of urea-formaldehyde from depositing there. From the SEM images of shell surface and cross section, the microcapsules have relatively smooth surfaces and the average thickness is about 4.5 mum. When the microcapsules are prepared with agitation rates of 1000 and 600 rpm, the mean diameters of the obtained microcapsules are 11 and 155 mum, respectively. The particles in the capsules move toward positive electrode with a responsive time of several hundred milliseconds while providing an electric field.     

Using freezing and drying techniques of emulsions for the microencapsulation of fish oil to improve oxidation stability

Epidemiological investigations show that n-3-polyunsaturated fatty acids (PUFA) have a protective effect with respect to coronary heart disease. For a long time fish oil has been known as a source of PUFA. The successful incorporation of fish oil into the normal food components offers an opportunity to increase the intake of PUFA, but fish oil has to be protected against oxidation preferably by microencapsulation. The high temperatures during the traditional spray-drying microencapsulation process lead to an increased oxidation of the PUFA. The authors demonstrate the application of a freeze-drying technique with respect to the production of dried microencapsulated fish oil with high quality and oxidation stability. The influence of emulsion parameters, freezing parameters and formulation on the stability of a dried fish oil emulsion is presented and illustrated. The used procedure gave a product with a good oxidation stability which is available as pellets or powder. It was found that the stability was mainly influenced by the freezing rate and the used encapsulation carbohydrate, whereby the powder showed a better shelf life than the pellets. Concluding, it can be summarized that the production of microencapsulated fish oil by freezing and subsequent freeze-drying offers an opportunity to achieve a product with a good oxidation stability.     

Insights into the synthesis and mechanism of green synthesized antimicrobial nanoparticles,answer to the multidrug resistance

Emergence of the multidrug resistant human pathogenic strains is posing a serious health challenge. Resistant strains carry mutations which help them to resist conventional drugs. Therefore, it is required to produce more effective agents that are able to degrade the resistant pathogenic bacterial strains. The antimicrobial properties of nanoparticles (NPs) by eco-friendly green synthetic methods have pulled attention everywhere owing to their exceptional properties and small particle size of 100 nm. NPs are considered to belong to a group of antimicrobial agents which have ability to go inside microbial cells and kill them. In this comprehensive review, we are discussing the green synthetic methods used for the synthesis of NPs targeting the microbes. Additionally, several characterization techniques of antimicrobial NPs are also discussed. Subsequently, various methods used for the analysis of antimicrobial activities and their mechanisms are also examined.     

Chemical Approaches for the Preparation of Bacteria – Nano/Microparticle Hybrid Systems

Bacteria represent a class of living cells that are very attractive carriers for the transport and delivery of nano- and microsized particles. The use of cell-based carriers, such as for example bacteria, may allow to precisely direct nano- or microsized cargo to a desired site, which would greatly enhance the selectivity of drug delivery and allow to mitigate side effects. One key step towards the use of such nano-/microparticle – bacteria hybrids is the immobilization of the cargo on the bacterial cell surface. To fabricate bacteria – nano-/microparticle biohybrid microsystems, a wide range of chemical approaches are available that can be used to immobilize the particle payload on the bacterial cell surface. This article presents an overview of the various covalent and noncovalent chemistries that are available for the preparation of bacteria – nano-/microparticle hybrids. For each of the different chemical approaches, an overview will be presented that lists the bacterial strains that have been modified, the type and size of nanoparticles that have been immobilized, as well as the methods that have been used to characterize the nanoparticle-modified bacteria.     

Impact of a Purified Blueberry Extract on In Vitro Probiotic Mucin-Adhesion and Its Effect on Probiotic/Intestinal Pathogen Systems

Several arguments have been made to substantiate the need for natural antimicrobials for the food industry. With blueberry extracts, the most compelling are both their healthy connotation and the possibility of obtaining a multipurpose solution that can be an antioxidant, colorant, and antimicrobial. From an antimicrobial perspective, as blueberry/anthocyanin-rich extracts have been associated with a capacity to inhibit harmful bacteria while causing little to no inhibition on potential probiotic microorganisms, the study of potential benefits that come from synergies between the extract and probiotics may be of particular interest. Therefore, the present work aimed to evaluate the effect of an anthocyanin-rich extract on the adhesion of five different probiotics as well as their effect on the probiotics’ capacity to compete with or block pathogen adhesion to a mucin/BSA-treated surface. The results showed that, despite some loss of probiotic adhesion, the combined presence of extract and probiotic is more effective in reducing the overall amount of adhered viable pathogen cells than the PROBIOTIC alone, regardless of the probiotic/pathogen system considered. Furthermore, in some instances, the combination of the extract with Bifidobacterium animalis Bo allowed for almost complete inhibition of pathogen adhesion.     

Bimetallic Au–Ag Nanoparticles: Advanced Nanotechnology for Tackling Antimicrobial Resistance

To date, there are no antimicrobial agents available in the market that have absolute control over the growing threat of bacterial strains. The increase in the production capacity of antibiotics and the growing antibacterial resistance of bacteria have majorly affected a variety of businesses and public health. Bimetallic nanoparticles (NPs) with two separate metals have been found to have stronger antibacterial potential than their monometallic versions. This enhanced antibacterial efficiency of bimetallic nanoparticles is due to the synergistic effect of their participating monometallic counterparts. To distinguish between bacteria and mammals, the existence of diverse metal transport systems and metalloproteins is necessary for the use of bimetallic Au–Ag NPs, just like any other metal NPs. Due to their very low toxicity toward human cells, these bimetallic NPs, particularly gold–silver NPs, might prove to be an effective weapon in the arsenal to beat emerging drug-resistant bacteria. The cellular mechanism of bimetallic nanoparticles for antibacterial activity consists of cell membrane degradation, disturbance in homeostasis, oxidative stress, and the production of reactive oxygen species. The synthesis of bimetallic nanoparticles can be performed by a bottom-up and top-down strategy. The bottom-up technique generally includes sol-gel, chemical vapor deposition, green synthesis, and co-precipitation methods, whereas the top-down technique includes the laser ablation method. This review highlights the key prospects of the cellular mechanism, synthesis process, and antibacterial capabilities against a wide range of bacteria. Additionally, we also discussed the role of Au–Ag NPs in the treatment of multidrug-resistant bacterial infection and wound healing.     

The role of quorum sensing in the pathogenicity of the cunning aggressor Pseudomonas aeruginosa

Recent decades have revealed that many bacterial species are capable of communicating with each other, and this observation has been largely responsible for a paradigm shift in microbiology. Whereas it was previously believed that bacteria lived as individual cells, it is now acknowledged that bacteria preferentially live in communities in the form of primitive organisms in which the behavior of individual cells is coordinated by cell–cell communication, known as quorum sensing (QS). Bacteria use QS for regulation of the processes involved in their interaction with each other, their environment, and, particularly, higher organisms We have focused on Pseudomonas aeruginosa, an opportunistic pathogen producing more than 30 QS-regulated virulence factors. P. aeruginosa causes several types of nosocomial infection, and lung infection in cystic fibrosis (CF) patients. We review the role of QS in the protective mechanisms of P. aeruginosa and show how disruption of the QS can be used as an approach to control this cunning aggressor.     

5-Aminolaevulinic acid (ALA) induced formation of different fluorescent porphyrins: a study of the biosynthesis of porphyrins by bacteria of the human digestive tract

Aminolaevulinic acid (ALA) induces porphyrin formation in almost all living cells. The fluorescence spectra of porphyrins produced from a variety of 31 bacterial strains from the human oral cavity and other parts of digestive tract have been examined. Many of the bacteria exposed to ALA were able to induce protoporphyrin IX (PpIX) fluorescence, but under aerobic condition some bacteria can also produced different fluorescent porphyrins, in particular water-soluble porphyrins that can arise from an oxidation of the corresponding porphyrinogen precursors. The formation of fluorescent porphyrins can be different from one bacterial strain to another, but also one specific bacterium can form different fluorescent porphyrins. Irradiation of the ALA incubated cultures led to a rapid formation of water-soluble porphyrins exhibiting fluorescence maxima at wavelengths of 618-620 nm. This light induced formation of water-soluble porphyrins could be attributed to a photooxidation of the non-fluorescent (Uro/Copro)-porphyrinogen precursors. Addition of detergents to some of the bacterial cultures led to a strong PpIX fluorescence increase, indicating that some of the PpIX originally produced can be present in a non-fluorescent, probably aggregated, form. The large abundance of bacteria in the oral cavity and other parts of digestive tract, with their capacity to easily produce fluorescent porphyrins, indicates that such bacterial fluorescence should be suppressed during the ALA-based diagnosis of tumours in order to eliminate false positive results.     

Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification

In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2 × 10⁶ mL⁻¹. Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.     

Deciphering the nanometer-scale organization and assembly of Lactobacillus rhamnosus GG pili using atomic force microscopy

In living cells, sophisticated functional interfaces are generated through the self-assembly of bioactive building blocks. Prominent examples of such biofunctional surfaces are bacterial nanostructures referred to as pili. Although these proteinaceous filaments exhibit remarkable structure and functions, their potential to design bioinspired self-assembled systems has been overlooked. Here, we used atomic force microscopy (AFM) to explore the supramolecular organization and self-assembly of pili from the Gram-positive probiotic bacterium Lactobacillus rhamnosus GG (LGG). High-resolution AFM imaging of cell preparations adsorbed on mica revealed pili not only all around the cells, but also in the form of remarkable star-like structures assembled on the mica surface. Next, we showed that two-step centrifugation is a simple procedure to separate large amounts of pili, even though through their synthesis they are covalently anchored to the cell wall. We also found that the centrifuged pili assemble as long bundles. We suggest that these bundles originate from a complex interplay of mechanical effects (centrifugal force) and biomolecular interactions involving the SpaC cell adhesion pilin subunit (lectin-glycan bonds, hydrophobic bonds). Supporting this view, we found that pili isolated from an LGG mutant lacking hydrophilic exopolysaccharides show an increased tendency to form tight bundles. These experiments demonstrate that AFM is a powerful platform for visualizing individual pili on bacterial surfaces and for unravelling their two-dimensional assembly on solid surfaces. Our data suggest that bacterial pili may provide a generic approach in nanobiotechnology for elaborating functional supramolecular interfaces assembled from bioactive building blocks.