Nanostructured Antimicrobials for Quality and Safety Improvement in Dairy Products.

In the food sector, one of the most important economic activities is the dairy industry, which has been facing many challenges in order to meet the increasing demand by consumers for natural and minimally processed products with high quality. In this sense, the application of innovative and emerging technologies can be an interesting alternative, for example, the use of nanotechnology in packaging and as delivery systems. This technology has the potential to improve the quality and safety of dairy products, representing an interesting approach for delivering food preservatives and improving the mechanical, barrier and functional properties of packaging. Several applications and promising results of nanostructures for dairy product preservation can be found throughout this review, including the use of metallic and polymeric nanoparticles, lipid-based nanostructures, nanofibers, nanofilms and nanocoatings. In addition, some relevant examples of the direct application of nanostructured natural antimicrobials in milk and cheese are presented and discussed, as well as the use of milk agar as a model for a preliminary test. Despite their high cost and the difficulties for scale-up, interesting results of these technologies in dairy foods and packaging materials have promoted a growing interest of the dairy industry.

Proteomic dataset of Listeria monocytogenes exposed to sublethal concentrations of free and nanoencapsulated nisin.

The cellular proteins of L. monocytogenes exposed to free and liposome-encapsulated nisin at sublethal concentration were hydrolyzed by trypsin and examined by tandem mass spectrometry (MS/MS) to obtain proteomic data. In the present study, we use the STRING v11.05 database analyze the interactions among the 78 upregulated proteins from L. monocytogenes obtained after treatment with sublethal concentrations of free and nanoliposome-encapsulated nisin. As result, from the upregulated proteins by free nisin was determined a network with 140 edges with two relevant nodes, containing ribosomal proteins and transmembrane transport proteins (SecD and ABC transport system). These two sets of proteins present biological connection as a group, with strong interactions and are related to detoxification and other Listeria response mechanisms. In addition, a high amount of membrane proteins was identified in the free nisin treatment. On the other hand, in the interaction analysis of upregulated proteins by liposome-loaded nisin, was found 156 edges with a single protein network, the same observed in free nisin, related to ribosomal proteins. Therefore, according with this analysis, the encapsulation of nisin into liposomes cause upregulation of ribosomal and decrease of L. monocytogenes response proteins as compared with free nisin.

Ginger: a systematic review of clinical trials and recent advances in encapsulation of its bioactive compounds.

Recently, the numbers of studies on natural products have considerably increased owing to their exceptional biological activities and health benefits. Their pharmacological attributes have played an immense role in detecting natural and safe alternative therapeutics, consequently extending their industrial applications. In this line, ginger (Zingiber officinale) has been gaining wide attention owing to its bioactive compounds, such as phenolic and terpene compounds. Ginger has a great pharmacological and biological potential in the prevention and treatment of various diseases, namely colds, nausea, arthritis, migraines and hypertension. However, these bioactive compounds are unstable and susceptible to degradation, volatilization and oxidation during extraction and processing, mainly owing to their exposure to environments with adverse conditions, such as high temperature, the presence of O2 and light. In this sense, this current review covers a wide range of topics, starting from the chemical profile and biological properties of ginger bioactive compounds (GBCs), their clinical effectiveness for the treatment of diseases and the application of different encapsulation methods (molecular inclusion, spray drying, complex coacervation, ionic strength and nanoemulsions) to protect and improve their application in food products. This work summarizes the fundamental principles of, recent progress in and effectiveness of different methods regarding the physicochemical, structural and functional properties of encapsulated GBCs. The potential use of encapsulated GBCs as a promising active ingredient to be applied in different food products is discussed in detail.

Genomic characterization and production of antimicrobial lipopeptides by Bacillus velezensis P45 growing on feather by-products.

AIMS: To investigate the potential of novel Bacillus velezensis P45 as an eco-friendly alternative for bioprocessing poultry by-products into valuable antimicrobial products. METHODS AND RESULTS: The complete genome of B. velezensis P45 was sequenced using the Illumina MiSeq platform, showing 4455 protein and 98 RNA coding sequences according to the annotation on the RAST server. Moreover, the genome contains eight gene clusters for the production of antimicrobial secondary metabolites and 25 putative protease-related genes, which can be related to feather-degrading activity. Then, in vitro tests were performed to determine the production of antimicrobial compounds using feather, feather meal and brain-heart infusion (BHI) cultures. Antimicrobial activity was observed in feather meal and BHI media, reaching 800 and 3200 AU ml-1 against Listeria monocytogenes respectively. Mass spectrometry analysis indicates the production of antimicrobial lipopeptides surfactin, fengycin and iturin. CONCLUSIONS: The biotechnological potential of B. velezensis P45 was deciphered through genome analysis and in vitro studies. This strain produced antimicrobial lipopeptides growing on feather meal, a low-cost substrate. SIGNIFICANCE AND IMPACT OF STUDY: The production of antimicrobial peptides by this keratinolytic strain may represent a sustainable alternative for recycling by-products from poultry industry. Furthermore, whole B. velezensis P45 genome sequence was obtained and deposited.

Lipid-Based Nanostructures for the Delivery of Natural Antimicrobials.

Encapsulation can be a suitable strategy to protect natural antimicrobial substances against some harsh conditions of processing and storage and to provide efficient formulations for antimicrobial delivery. Lipid-based nanostructures, including liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid nanocarriers (NLCs), are valuable systems for the delivery and controlled release of natural antimicrobial substances. These nanostructures have been used as carriers for bacteriocins and other antimicrobial peptides, antimicrobial enzymes, essential oils, and antimicrobial phytochemicals. Most studies are conducted with liposomes, although the potential of SLNs and NLCs as antimicrobial nanocarriers is not yet fully established. Some studies reveal that lipid-based formulations can be used for co-encapsulation of natural antimicrobials, improving their potential to control microbial pathogens.

Antimicrobial activity of Baccharis dracunculifolia DC and its synergistic interaction with nisin against food-related bacteria.

The antimicrobial activities of Baccharis dracunculifolia DC essential oil (EO) and hydroalcoholic extract (HE) were evaluated. The EO showed broad antimicrobial activity and its synergistic combination with nisin was tested. Major components of EO were nerolidol, beta-pinene and D-limonene, while artepillin C, rutin and cafeic acid were major phenolics of HE. EO and HE were tested by agar diffusion assay against several strains of bacteria and yeasts, and mixed cultures of bacterial strains. The EO presented the largest spectrum of antimicrobial activity inhibiting all Gram-positive bacteria tested. Yeasts were not inhibited. The effect of EO against mixtures of sensitive and non-sensitive bacteria was tested on milk agar, being the inhibitory effect only observed on mixtures containing susceptible strains. The combination of EO and nisin at ½ MIC was evaluated on the growth curve of Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Salmonella Enteritidis during 24 h at 37 °C. The combination EO-nisin was effective and no viable counts of B. cereus, L. monocytogenes and S. Enteritidis was observed, while the individual antimicrobials caused no inhibition. The counts of S. aureus were about 4 log CFU/mL lower in comparison with EO or nisin alone. B. dracunculifolia DC may be a potential source of natural antimicrobials, and its synergistic effect with nisin would reduce the working concentration, minimizing the organoleptic effects associated with this plant antimicrobial.

Proteomic analysis reveals differential responses of Listeria monocytogenes to free and nanoencapsulated nisin.

The ability of Listeria monocytogenes grow on ready-to-eat food is a major concern in food safety. Natural antimicrobials, such as nisin, can be used to control this pathogen, but the increasing reports of nisin tolerance and resistance make necessary novel approaches to increase its effectiveness, such as encapsulation. The goal of this study was to investigate how L. monocytogenes ATCC7644 regulates and shapes its proteome in response to sublethal doses of nisin and nisin-loaded phosphatidylcholine liposomes (lipo-nisin), compared to untreated cells growing under optimal conditions. Total proteins were extracted from L. monocytogenes cells treated for 1 h with free and lipo-nisin. As result, of 803 proteins that were initially identified, 64 and 53 proteins were differentially upregulated and downregulated respectively, in the treatments with nisin and lipo-nisin. Changes of Listeria proteome in response to treatments containing nisin were mainly related to ATP-binding cassette (ABC) transporter systems, transmembrane proteins, RNA-binding proteins and diverse stress response proteins. Some of the proteins uniquely detected in samples treated with free nisin were the membrane proteins SecD, Lmo1539 and the YfhO enzyme, which are related to translocation of L. monocytogenes virulence factors, activation of the LiaR-mediated stress defense and glycosylation of wall teichoic acid, respectively. The L. monocytogenes treated with liposome encapsulated nisin showed no expression of some stress response factors as compared with the free nisin, suggesting a reduction of stress mediated response and production of nisin-resistance factors by exposure to encapsulated nisin.

Evidence that protein corona reduces the release of antimicrobial peptides from polymeric nanocapsules in milk.

The antimicrobial peptide produced by Bacillus velesensis P34 has a broad activity against Gram-positive bacteria, showing potential as natural food preservative. In this work, nanocapsules (NCs) containing the peptide P34 were produced using the polymers poly-ε-caprolactone (PCL) or Eudragit RS-100 (EUD), and their antimicrobial activities were assessed evaluating L. monocytogenes growth in synthetic media, milk and isolated milk proteins. As results, cationic and anionic nanocapsules were obtained, with zeta potential ranging from +15 to +28 mV for EUD and around -19 mV for PCL, and average diameter in the range of 104-130 nm and 224-245 nm, respectively. In the antimicrobial tests, only the P34-EUD NCs presented activity against L. monocytogenes in BHI broth, possibly due to the EUD high swelling and permeability properties, as compared with PCL. In whole and skimmed milk, the P34-EUD NCs caused no inhibition of L. monocytogenes growth, due to a possible interaction of casein proteins with the NCs surface resulting in protein corona formation, which interfered with the antimicrobial peptide release. Therefore, the application of polymeric NCs as antimicrobial delivery systems in foods could be limited by the polymer type, and the adhesion of specific matrix proteins that could form protein corona, reducing the bioactive compound release.

Structural features of myofibrillar fish protein interacting with phosphatidylcholine liposomes.

The interactions between liposomes and fish myofibrillar protein (surimi ground salted protein, SURP) were evaluated. Liposomes prepared with ultrapure phosphatidylcholine (UPC) or partially purified phosphatidylcholine (PPC) were dispersed at different weight ratio on SURP. Changes in protein stability and structure were evaluated using FTIR, intrinsic fluorescence and free sulfhydryl groups, and changes in liposome properties were studied by dynamic light scattering and electron microscopy. PPC promoted denaturation and aggregation of SURP, reflected in secondary structure loss, exposure of tyrosine residues and increment of free sulfhydryl. UPC produced partial unfolding and changes in the secondary structure of SURP from α-helical to ß-strand. Liposome size increased by about 40% and showed modified surface charge after SURP exposure, indicating the formation of protein corona. Surface charge and composition of liposomes influence SURP stability and could exert different effects on the myofibrillar protein network, which is important for liposome applications in surimi products.

Biochemical and functional properties of wheat middlings bioprocessed by lactic acid bacteria.

The present study aimed to investigate the bioprocessing of wheat middlings with different lactic acid bacteria (LAB) in order to improve biological activities of this by-product of wheat flour production. The concentration of lactic acid, reducing sugars, and total phenolics, as well as antioxidant, antibrowning, antibacterial and prebiotic activities of fermented samples were analyzed. All LAB strains were capable to growth on wheat middlings, and pH decreased in the medium associated with lactic acid production during cultivation. Samples inoculated with Lactobacillus plantarum DSM20174 presented the maximum growth, lactic acid concentration above 2 mg/ml, and pH values around 3.8. The amount or reducing sugars decreased after 24 hr growth, except for maltose. Bioprocessed wheat middlings exhibited antioxidant, antibrowning, antibacterial, and prebiotic properties, related with the increase of total phenolic content. Highest values for antioxidant activities were obtained for L. plantarum and Streptococcus thermophilus strains, reaching values around 400 and 640 µM Trolox equivalents (TE) ml-1 for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric ion reducing antioxidant power (FRAP) assays, respectively. Bioprocessing techniques using LAB can be an interesting approach to improve the availability of compounds with health-promoting properties from lignocellulosic waste material. PRACTICAL APPLICATIONS: The processing of secondary products from wheat milling can represent an important benefit to the industry. Wheat middlings bioprocessed with LAB showed improved biological activities and may represent an interesting ingredient to be incorporated in food and feed formulations.

Xylooligosaccharides production from wheat middlings bioprocessed with Bacillus subtilis.

Prebiotic compounds are substrates selectively metabolized by beneficial gut microbiota causing a health-promoting effect. Despite some prebiotic carbohydrates have been largely studied, xylooligosaccharides (XOS) are important prebiotics derived from arabinoxylans, which are polysaccharides found in cereals. This study aimed to investigate the production of xylanolytic enzymes and XOS during bioprocessing of wheat middlings, a product derived from wheat flour production, using a probiotic Bacillus subtilis. The composition of XOS and the enzymatic and prebiotic activities of resulting B. subtilis cultures were evaluated. The activity of xylanolytic enzymes continuously enhanced during the 72 h bacterial growth, where ß-xylosidase presented the highest value (70.31 U/mL). XOS profile and concentration varied considerably between control and bioprocessed samples and among these at different times. Maximum prebiotic activity score was found for the 24 h and 72 h bioprocessed samples (1.73 and 1.61, respectively) using the commercial probiotic Lactobacillus acidophilus LA-5. Wheat middlings showed to be a promising substrate for production of prebiotics like XOS and B. subtilis FTC01 appears to be a good source of xylanolytic enzymes.

Antifungal properties of phosphatidylcholine-oleic acid liposomes encapsulating garlic against environmental fungal in wheat bread.

Liposomes have gained great interest in the food and pharmaceutical industry as colloidal carriers of bioactive compounds. In this work, liposomes of phosphatidylcholine (PC) and oleic acid (OA) encapsulating garlic extract (GE) were developed to determine its aptitude as antifungal agent in wheat bread. The influence of GE on the properties of liposomes were followed by determination of size, Zeta potential, Fourier transform infrared patterns (FTIR), morphology, differential scanning calorimetry (DSC) and thermogravimetric (TGA) techniques. The produced PC-OA-GE liposomes showed spherical morphology with narrow size distribution, entrapment efficiency of 79.7% and zeta potential of -27.9 mV. In vitro antifungal test showed noticeable inhibitory activities for free and encapsulated GE against selected fungal strains. TGA analysis revealed that the presence of OA and GE in the formulation retards the liposomal thermal decomposition, as compared with the pure PC liposomes and the DSC enthalpy and main transition temperature variation in PC-OA-GE liposomes suggested a strong heat-induced rigidifying effect that could be attributed to the presence of garlic polysaccharides in the liposome surface, observed by FTIR. In the in situ test, the bread formulations with free or liposome-encapsulated GE (0.65 mL/100 g of dough) were microbiologically more stable as compared with the controls, showing mold inhibition for five days. Therefore, liposomes formulated with OA and GE showed potential as natural antifungal agent in bakery products.

Extraction and partial characterisation of antioxidant pigment produced by Chryseobacterium sp. kr6.

Pigments synthesised by Chryseobacterium sp. kr6 growing on feather waste were extracted and characterised. The pigment extract was characterised by KOH test, UV-vis, CIELAB colour system, HPLC-DAD-MS, FTIR and its antioxidant capacity was evaluated. A positive bathochromic shift was observed when kr6 colonies or pigment extracts were subjected to alkaline solution (20% KOH) and a λmax at 450 nm was detected for acetone extracts, although no typical fine structure of carotenoids was detected in the electomagnetic spectra. The HPLC profile of the extracted pigment showed that the compound has three different peaks with λmax near 450 nm. The FTIR analysis shows some principal functional groups from a flexirubin-like molecule. The pigmented compound also presents antioxidant activity evaluated by the scavenging of the ABTS radical.

Development and characterization of phosphatidylcholine nanovesicles, containing garlic extract, with antilisterial activity in milk.

Phospholipid nanovesicles were developed to improve the stability of garlic (Allium sativum L.) extract. Electron microscopy of liposomes revealed nanometric and spherical-shaped vesicles with a mean particle size of 174.6±17.3nm and polydispersity index of 0.26±0.02. The entrapment efficiency was 47.5±7.3% and the nanoliposomes had a zeta potential of -16.2±5.5mV. The antimicrobial activity of free and encapsulated garlic extract was evaluated against different strains of Listeria spp. in milk at 37°C for 24h. For free and encapsulated garlic extracts at 5% concentration, a decrease of 4log cycles in viable cell counts was observed at 10h, against four of the five strains of Listeria spp. tested. The results indicate that liposomes constitute a suitable system for encapsulation of unstable garlic active compounds and the encapsulation of garlic extract proves to be a promising technology for multiple applications, including antimicrobial agents.