Tracing the origin and authenticity of Spanish PDO honey using metagenomics and machine learning.

The Protected Designation of Origin (PDO) indication for foods intends to guarantee the conditions of production and the geographical origin of regional products within the European Union. Honey products are widely consumed due to their health-promoting properties and there is a general interest in tracing their authenticity. In this regard, metagenomics sequencing and machine learning (ML) have been proposed as complementary technologies to improve the traceability methods of foods. Therefore, the aim of this study was to analyze the metagenomic profiles of Spanish honeys from three different PDOs (Granada, Tenerife and Villuercas-Ibores), and compare them with non-PDO honeys using ML models (PLS, RF, LOGITBOOST, and NNET). According to the results obtained, non-PDO honeys and Granada PDO showed higher beta diversity values than Tenerife and Villuercas-Ibores PDOs. ML classification of honey products allowed the identification of different microbial biomarkers of the geographical origin of honeys: Lactobacillus kunkeei, Parasaccharibacter apium and Lactobacillus helsingborgensis for PDO honeys and Paenibacillus larvae, Lactobacillus apinorum and Klebsiella pneumoniae for non-PDO honeys. In addition, potential microbial biomarkers of some honey varieties including L. kunkeei for Albaida and Retama del Teide varieties, and P. apium for Tajinaste variety, were identified. ML models were validated on an independent set of samples leading to high accuracy rates (above 90 %). This work demonstrates the potential of ML to differentiate different types of honey using metagenome-based methods, leading to high performance metrics. In addition, ML models discriminate both the geographical origin and variety of products corresponding to different PDOs and non-PDO products. Results here presented may contribute to develop enhanced traceability and authenticity methods that could be applied to a wide range of foods.

Melipona stingless bees and honey microbiota reveal the diversity, composition, and modes of symbionts transmission.

The Melipona gut microbiota differs from other social bees, being characterized by the absence of crucial corbiculate core gut symbionts and a high occurrence of environmental strains. We studied the microbial diversity and composition of three Melipona species and their honey to understand which strains are obtained by horizontal transmission (HT) from the pollination environment, represent symbionts with HT from the hive/food stores or social transmission (ST) between nestmates. Bees harbored higher microbial alpha diversity and a different and more species-specific bacterial composition than honey. The fungal communities of bee and honey samples are also different but less dissimilar. As expected, the eusocial corbiculate core symbionts Snodgrassella and Gilliamella were absent in bees that had a prevalence of Lactobacillaceae - including Lactobacillus (formerly known as Firm-5), Bifidobacteriaceae, Acetobacteraceae, and Streptococcaceae - mainly strains close to Floricoccus, a putative novel symbiont acquired from flowers. They might have co-evolved with these bees via ST, and along with environmental Lactobacillaceae and Pectinatus (Veillonellaceae) strains obtained by HT, and Metschnikowia and Saccharomycetales yeasts acquired by HT from honey or the pollination environment, including plants/flowers, possibly compose the Melipona core microbiota. This work contributes to the understanding of Melipona symbionts and their modes of transmission.

Novel chromosomes and genomes provide new insights into evolution and adaptation of the whole genome duplicated yeast-like fungus TN3-1 isolated from natural honey.

Aureobasidium melanogenum TN3-1 strain and A. melanogenum P16 strain were isolated from the natural honey and the mangrove ecosystem, respectively. The former can produce much higher pullulan from high concentration of glucose than the latter. In order to know what happened to their genomes, the PacBio sequencing and Hi-C technologies were used to create the first high-quality chromosome-level reference genome assembly of A. melanogenum TN3-1 (51.61 Mb) and A. melanogenum P16 (25.82 Mb) with the contig N50 of 2.19 Mb and 2.26 Mb, respectively. Based on the Hi-C results, a total of 93.33% contigs in the TN3-1 strain and 92.31% contigs in the P16 strain were anchored onto 24 and 12 haploid chromosomes, respectively. The genomes of the TN3-1 strain had two subgenomes A and B. Synteny analysis showed that the genomic contents of the two subgenomes were asymmetric with many structural variations. Intriguingly, the TN3-1 strain was revealed as a recent hybrid/fusion between the ancestor of A. melanogenum CBS105.22/CBS110374 and the ancestor of another unidentified strain of A. melanogenum similar to P16 strain. We estimated that the two ancient progenitors diverged around 18.38 Mya and merged around 10.66-9.98 Mya. It was found that in the TN3-1 strain, telomeres of each chromosome contained high level of long interspersed nuclear elements (LINEs), but had low level of the telomerase encoding gene. Meanwhile, there were high level of transposable elements (TEs) inserted in the chromosomes of the TN3-1 strain. In addition, the positively selected genes of the TN3-1 strain were mainly enriched in the metabolic processes related to harsh environmental adaptability. Most of the stress-related genes were found to be related to the adjacent LTRs, and the glucose derepression was caused by the mutation of the Glc7-2 in the Snf-Mig1 system. All of these could contribute to its genetic instability, genome evolution, high stress resistance, and high pullulan production from glucose.

Detection of macrolide resistance genes, ermC and ermB, in Japanese honey using real-time PCR assays.

American foulbrood (AFB) is a honeybee disease caused by Paenibacillus larvae, and tylosin is used as the prophylactic in Japan. Honey contains macrolide-resistant bacteria that are a potential source of genes that may confer tylosin resistance to P. larvae. To investigate the potential risk of such genes in Japanese honey, we developed real-time PCR assays for the detection of important macrolide resistance genes, ermC and ermB, and analyzed 116 Japanese honey samples with known contamination status of P. larvae. Consequently, 91.38% of samples contained ermC and/or ermB, and 71.55% of samples contained both ermC and P. larvae, suggesting the possible emergence of tylosin-resistant P. larvae in Japan. Therefore, judicious use of the prophylactic is essential in maintaining its effectiveness.

Evaluation of different bacterial honey isolates as probiotics and their efficient roles in cholesterol reduction.

Continue to hypothesize that honey is a storehouse of beneficial bacteria, and the majority of these isolates are levansucrase producers. Accordingly, ten bacterial strains were isolated from different honey sources. Four honey isolates that had the highest levansucrase production and levan yield were identified by the partial sequencing of the 16S rRNA gene as Achromobacter sp. (10A), Bacillus paralicheniformis (2M), Bacillus subtilis (9A), and Bacillus paranthracis (13M). The cytotoxicity of the selected isolates showed negative blood hemolysis. Also, they are sensitive to the tested antibiotics (Amoxicillin + Flucloxacillin, Ampicillin, Gentamicin, Benzathine benzylpenicillin, Epicephin, Vancomycin, Amikacin, and Zinol). The isolates had strong alkaline stability (pHs 9, 11) and were resistant to severe acidic conditions (29-100 percent). The tested isolates recorded complete tolerance to both H2O2 and the bile salt (0.3% Oxgall powder) after 24 h incubation. The cell-free supernatant of the examined strains had antifungal activities against C. Albicans with varying degrees. Also, isolates 2M and 13M showed strong activities against S. aureus. The isolates showed strong adhesion and auto-aggregation capacity. Isolate 10A showed the highest antioxidant activity (91.45%) followed by 2M (47.37%). The isolates recorded different catalase and protease activity. All isolates produced cholesterol oxidase and lipase with different levels. Besides, the four isolates reduced LDL (low-density lipoprotein) to different significant values. The cholesterol-reducing ability varied not only for strains but also for the time of incubation. The previous results recommended these isolates be used safely in solving the LDL problem.

Purification and characterization of antifungal lipopeptide produced by Bacillus velezensis isolated from raw honey.

Raw honey contains a diverse microbiota originating from honeybees, plants, and soil. Some gram-positive bacteria isolated from raw honey are known for their ability to produce secondary metabolites that have the potential to be exploited as antimicrobial agents. Currently, there is a high demand for natural, broad-spectrum, and eco-friendly bio-fungicides in the food industry. Naturally occurring antifungal products from food-isolated bacteria are ideal candidates for agricultural applications. To obtain novel antifungals from natural sources, we isolated bacteria from raw clover and orange blossom honey to evaluate their antifungal-producing potential. Two Bacillus velezensis isolates showed strong antifungal activity against food-isolated fungal strains. Antifungal compound production was optimized by adjusting the growth conditions of these bacterial isolates. Extracellular proteinaceous compounds were purified via ammonium sulfate precipitation, solid phase extraction, and RP-HPLC. Antifungal activity of purified products was confirmed by deferred overlay inhibition assay. Mass spectrometry (MS) was performed to determine the molecular weight of the isolated compounds. Whole genome sequencing (WGS) was conducted to predict secondary metabolite gene clusters encoded by the two antifungal-producing strains. Using MS and WGS data, we determined that the main antifungal compound produced by these two Bacillus velezensis isolates was iturin A, a lipopeptide exhibiting broad spectrum antifungal activity.

Technological screening and application of Saccharomyces cerevisiae strains isolated from fermented honey by-products for the sensory improvement of Spiritu re fascitrari, a typical Sicilian distilled beverage.

"Spiritu re fascitrari" is a Sicilian alcoholic beverage obtained through distillation of a decoction of spontaneously fermented honey by-products (FHP). The production process often leads to sensorial defects due to the unstable alcoholic fermentation. The objective of this work was to select Saccharomyces cerevisiae strains from FHP to be used as starter in decoction fermentation. Based on chemical, microbiological and technological data, from a total of 91 strains three S. cerevisae were selected for further testing to produce FHP at laboratory scale level. After FHP distillation, the analysis of volatile organic compounds showed a complex mixture of sensory active molecules, mainly alcohols and aldehydes. Among the alcohols, 3-methyl-1-butanol, 2-methyl-1-butanol, phenylethyl alcohol, hexadecanol and octadecanol were found at the highest concentrations. Among the carboxylic acids, acetic acid was mainly detected in the spontaneously fermented samples. FHP fermented with the three selected strains were not characterized by the presence of off-odors or off-flavours. The results obtained in this work demonstrate that the selected S. cerevisiae strains are promising starters to stabilize the production of distilled alcoholic beverages produced from honey by-products.

Comparison of individual hive and apiary-level sample types for spores of Paenibacillus larvae in Saskatchewan honey bee operations.

Three commercial honey bee operations in Saskatchewan, Canada, with outbreaks of American foulbrood (AFB) and recent or ongoing metaphylactic antibiotic use were intensively sampled to detect spores of Paenibacillus larvae during the summer of 2019. Here, we compared spore concentrations in different sample types within individual hives, assessed the surrogacy potential of honey collected from honey supers in place of brood chamber honey or adult bees within hives, and evaluated the ability of pooled, extracted honey to predict the degree of spore contamination identified through individual hive testing. Samples of honey and bees from hives within apiaries with a recent, confirmed case of AFB in a single hive (index apiaries) and apiaries without clinical evidence of AFB (unaffected apiaries), as well as pooled, apiary-level honey samples from end-of-season extraction, were collected and cultured to detect and enumerate spores. Only a few hives were heavily contaminated by spores in any given apiary. All operations were different from one another with regard to both the overall degree of spore contamination across apiaries and the distribution of spores between index apiaries and unaffected apiaries. Within operations, individual hive spore concentrations in unaffected apiaries were significantly different from index apiaries in the brood chamber (BC) honey, honey super (HS) honey, and BC bees of one of three operations. Across all operations, BC honey was best for discriminating index apiaries from unaffected apiaries (p = 0.001), followed by HS honey (p = 0.06), and BC bees (p = 0.398). HS honey positively correlated with both BC honey (rs = 0.76, p < 0.0001) and bees (rs = 0.50, p < 0.0001) and may be useful as a surrogate for either. Spore concentrations in pooled, extracted honey seem to have predictive potential for overall spore contamination within each operation and may have prognostic value in assessing the risk of future AFB outbreaks at the apiary (or operation) level.

Viable fructophilic lactic acid bacteria present in honeybee-based food products.

Fructophilic lactic acid bacteria (FLAB) are a group of lactic acid bacteria (LAB) with unique growth characteristics and are regarded as promising candidates for foods or food ingredients. The present study characterized levels of viable FLAB cells in honeybee-based food products, especially focusing on fresh honey. FLAB were recovered from 88% of fresh honey samples (harvested within a week) tested, and 29% of the fresh honey samples contained FLAB with levels of over 105 CFU/g. FLAB species found in the tested fresh honeys were mostly Apilactobacillus kunkeei and Fructobacillus fructosus. FLAB were not recovered from aged honey samples (aged over 2 weeks after harvest). Fresh comb honey and bee pollen samples occasionally contained low levels of FLAB. When FLAB were inoculated into honeys (linden honey and clover honey), their viability markedly reduced during a week. One of the reasons for this decrease was the presence of H2O2 in the honeys, and supplementation of catalase significantly improved their survivability in linden honey. The long history of human consumption of fresh honey suggests that viable FLAB cells do not present health concerns.

Non-conventional yeasts from fermented honey by-products: Focus on Hanseniaspora uvarum strains for craft beer production.

The increasing interest in novel beer productions focused on non-Saccharomyces yeasts in order to pursue their potential in generating groundbreaking sensory profiles. Traditional fermented beverages represent an important source of yeast strains which could express interesting features during brewing. A total of 404 yeasts were isolated from fermented honey by-products and identified as Saccharomyces cerevisiae, Wickerhamomyces anomalus, Zygosaccharomyces bailii, Zygosaccharomyces rouxii and Hanseniaspora uvarum. Five H. uvarum strains were screened for their brewing capability. Interestingly, Hanseniaspora uvarum strains showed growth in presence of ethanol and hop and a more rapid growth than the control strain S. cerevisiae US-05. Even though all strains showed a very low fermentation power, their concentrations ranged between 7 and 8 Log cycles during fermentation. The statistical analyses showed significant differences among the strains and underlined the ability of YGA2 and YGA34 to grow rapidly in presence of ethanol and hop. The strain YGA34 showed the best technological properties and was selected for beer production. Its presence in mixed- and sequential-culture fermentations with US-05 did not influence attenuation and ethanol concentration but had a significant impact on glycerol and acetic acid concentrations, with a higher sensory complexity and intensity, representing promising co-starters during craft beer production.

Genome and Pangenome Analysis of Lactobacillus hilgardii FLUB-A New Strain Isolated from Mead.

The production of mead holds great value for the Polish liquor industry, which is why the bacterium that spoils mead has become an object of concern and scientific interest. This article describes, for the first time, Lactobacillus hilgardii FLUB newly isolated from mead, as a mead spoilage bacteria. Whole genome sequencing of L. hilgardii FLUB revealed a 3 Mbp chromosome and five plasmids, which is the largest reported genome of this species. An extensive phylogenetic analysis and digital DNA-DNA hybridization confirmed the membership of the strain in the L. hilgardii species. The genome of L. hilgardii FLUB encodes 3043 genes, 2871 of which are protein coding sequences, 79 code for RNA, and 93 are pseudogenes. L. hilgardii FLUB possesses three clustered regularly interspaced short palindromic repeats (CRISPR), eight genomic islands (44,155 bp to 6345 bp), and three (two intact and one incomplete) prophage regions. For the first time, the characteristics of the genome of this species were described and a pangenomic analysis was performed. The concept of the pangenome was used not only to establish the genetic repertoire of this species, but primarily to highlight the unique characteristics of L. hilgardii FLUB. The core of the genome of L. hilgardii is centered around genes related to the storage and processing of genetic information, as well as to carbohydrate and amino acid metabolism. Strains with such a genetic constitution can effectively adapt to environmental changes. L. hilgardii FLUB is distinguished by an extensive cluster of metabolic genes, arsenic detoxification genes, and unique surface layer proteins. Variants of MRS broth with ethanol (10-20%), glucose (2-25%), and fructose (2-24%) were prepared to test the strain's growth preferences using Bioscreen C and the PYTHON script. L. hilgardii FLUB was found to be more resistant than a reference strain to high concentrations of alcohol (18%) and sugars (25%). It exhibited greater preference for fructose than glucose, which suggests it has a fructophilic nature. Comparative genomic analysis supported by experimental research imitating the conditions of alcoholic beverages confirmed the niche specialization of L. hilgardii FLUB to the mead environment.

Yeast biodiversity in honey produced by stingless bees raised in the highlands of southern Brazil.

The physicochemical characteristics and yeasts diversity in honey samples from 17 species of stingless bees of the genera Nannotrigona, Melipona, Plebeia, Scraptotrigona, and Tetragonisca cultivated in Southern Brazil were determined. The sugar content, moisture, water activity, pH, reducing sugars/total sugar ratio, and total yeast population varied significantly among the honey from the different bee species. The highest yeast population was found in the Plebeia's honey samples and correlated with their high water-activity. Sixteen yeast species were identified based on the nuclear large subunit (26S) ribosomal RNA partial sequences. The genera Starmerella and Zygosaccharomyces were found predominant, with a high prevalence of Starmerella sp., S. etchellsii, and S. apicola. Some yeast species were only identified in honey samples from specific bee species indicating a close relationship between the yeasts and the insects. For the first time, Wickerhamomyces sydowiorum in honey is being reported. In general, the yeast species isolated from stingless bee honey samples demonstrated high osmotolerance and low sugar assimilation.

Characterizing the chemical and sensory profiles of traditional American meads.

The compositional and sensorial profiles of traditional American meads were determined using standard enological, volatile, and descriptive analyses. Forty-one commercial meads produced by 35 meaderies across 20 states were selected to encompass a broad product range. The meads were analyzed for ethanol content, residual sugar, pH, titratable acidity, acetic acid, and free and total sulfur dioxide. Forty-three volatile compounds (alcohols, esters, acids, terpenes, aldehydes, aromatic hydrocarbons, etc.) were tentatively identified using a nontargeted HS-SPME-GC-MS method. Ethyl octanoate, phenylethyl alcohol, ethyl decanoate, and ethyl acetate were the most relatively abundant volatile compounds across the sample set. A trained panel (n = 11) evaluated each mead using descriptive analysis and the chemical and sensory analyses were compared. Acidity, sweetness, and cloying and viscous mouthfeel sensations, and alcoholic heat were the most influential sensory attributes and were driven by titratable acidity, residual sugar, and ethanol content, respectively. Ethyl octanoate and ethyl decanoate were correlated with manure aroma, phenylethyl alcohol with yeast and green aromas, and ethyl acetate with citrus, solvent, and green olive aromas. This research further elucidates the empirical relationship between the chemical composition and sensory profiles of commercial meads. PRACTICAL APPLICATION: This work provides the mead industry with further understanding of the compositional drivers of the sensory profiles of commercial meads and demonstrates product categories (dry, semi-sweet, sweet) do not necessarily indicate compositional or sensory attributes.

Self-Assembled DNA Nanoflowers Triggered by a DNA Walker for Highly Sensitive Electrochemical Detection of Staphylococcus aureus.

With the development of DNA nanotechnology, DNA has been widely used to construct a variety of nanomachines. Among them, a DNA walker is a unique nanomachine that can move continuously along a specific orbit to fulfill diverse functions. In this paper, a dual signal amplification electrochemical biosensor based on a DNA walker and DNA nanoflowers is constructed for high sensitivity detection of Staphylococcus aureus (S. aureus). Two groups of double-stranded DNA are modified on the surface of a gold electrode. The binding of S. aureus with its aptamer induces the disintegration of the long double strands and releases the DNA walker. With the help of exonuclease III (Exo III), the DNA walker moves along the electrode surface and continuously hydrolyzes the anchored short double strands. The introduction of a specially customized circular DNA and phi29 DNA polymerase initiates the rolling circle amplification (RCA) reaction. DNA nanoflowers are formed at high local concentration of DNA in the solution, which provide binding sites for electroactive methylene blue (MB) and thus produce intense signal. Under the best conditions, the current response is linearly related to the logarithm of the concentration of S. aureus ranging from 60 to 6 × 107 CFU/mL, and the detection limit is 9 CFU/mL. In addition, the proposed biosensor has achieved satisfactory results in the detection of actual water samples and diluted honey samples, which confirm the practicability of the biosensor and its application potential in environmental monitoring and food safety.

Bombella favorum sp. nov. and Bombella mellum sp. nov., two novel species isolated from the honeycombs of Apis mellifera.

As part of a study investigating the microbiome of bee hives and honey, two novel strains (TMW 2.1880T and TMW 2.1889T) of acetic acid bacteria were isolated and subsequently taxonomically characterized by a polyphasic approach, which revealed that they cannot be assigned to known species. The isolates are Gram-stain-negative, aerobic, pellicle-forming, catalase-positive and oxidase-negative. Cells of TMW 2.1880T are non-motile, thin/short rods, and cells of TMW 2.1889T are motile and occur as rods and long filaments. Morphological, physiological and phylogenetic analyses revealed a distinct lineage within the genus Bombella. Strain TMW 2.1880T is most closely related to the type strain of Bombella intestini with a 16S rRNA gene sequence similarity of 99.5 %, and ANIb and in silico DDH values of 94.16 and 56.3 %, respectively. The genome of TMW 2.1880T has a size of 1.98 Mb and a G+C content of 55.3 mol%. Strain TMW 2.1889T is most closely related to the type strain of Bombella apis with a 16S rRNA gene sequence similarity of 99.5 %, and ANIb and in silico DDH values of 85.12 and 29.5 %, respectively. The genome of TMW 2.1889T has a size of 2.07 Mb and a G+C content of 60.4 mol%. Ubiquinone analysis revealed that both strains contained Q-10 as the main respiratory quinone. Major fatty acids for both strains were C16 : 0, C19 : 0 cyclo ω8c and summed feature 8, respectively, and additionally C14 : 0 2-OH only for TMW 2.1880T and C14 : 0 only for TMW 2.1889T. Based on polyphasic evidence, the two isolates from honeycombs of Apis mellifera represent two novel species of the genus Bombella, for which the names Bombella favorum sp. nov and Bombella mellum sp. nov. are proposed. The designated respective type strains are TMW 2.1880T (=LMG 31882T=CECT 30114T) and TMW 2.1889T (=LMG 31883T=CECT 30113T).

Postbiotic and Anti-aflatoxigenic Capabilities of Lactobacillus kunkeei as the Potential Probiotic LAB Isolated from the Natural Honey.

The use of probiotic, postbiotic, and anti-aflatoxigenic capabilities of the lactic acid bacteria (LAB) isolated from stressful niches is a major strategy to prepare functional cultures and bio-preservatives for food industries. In the present study, abundant LAB strains isolated from natural honey were screened based on their tolerance to continuous pH and bile salt treatments. Then, the pro-functional properties of the selected LAB were investigated. In accordance with the screening data, a bacilli isolate was selected for further characterization. Sequencing results led to the identification of Lactobacillus kunkeei as the selected LAB isolate. In vitro antibacterial and antifungal activities of the LAB and in situ antifungal activity of the isolate cell-free supernatant (CFS) were verified against food-borne indicators. Accordingly, in vitro antibacterial and antifungal effects of Lact. kunkeei ENH01 on respective Escherichia coli and Aspergillus niger were significantly (P < 0.05) higher than the other indicators. Furthermore, in situ inhibitory effect of Lact. kunkeei CFS on Candida albicans (as the highest in situ effect) was equal to 76.36%. The presence of three antibacterial peptides was also verified in the Lact. kunkeei CFS according to the results of liquid chromatography mass spectrometry (LC/MS) assay. Antibiotic susceptibility profile and auto-aggregation ability of the isolate were noticeable. Anti-mycotoxigenic capabilities of Lact. kunkeei ENH01 as viable and heat-killed cells were also revealed against total aflatoxins according to the HPLC-based analysis. In vivo safety of the isolate was also attested through the evaluation of blood biochemistry and hematological parameters in the Lact. kunkeei ENH01 fed-mice compared with the control. Based on the findings, probiotic properties of Lact. kunkeei ENH01 and postbiotic capabilities of the isolate CFS and its heat-killed cells were approved.

Antibacterial Activity of Honey/Chitosan Nanofibers Loaded with Capsaicin and Gold Nanoparticles for Wound Dressing.

This paper describes the preparation, characterization, and evaluation of honey/tripolyphosphate (TPP)/chitosan (HTCs) nanofibers loaded with capsaicin derived from the natural extract of hot pepper (Capsicum annuumL.) and loaded with gold nanoparticles (AuNPs) as biocompatible antimicrobial nanofibrous wound bandages in topical skin treatments. The capsaicin and AuNPs were packed within HTCs in HTCs-capsaicin, HTCs-AuNP, and HTCs-AuNPs/capsaicin nanofibrous mats. In vitro antibacterial testing against Pasteurella multocida, Klebsiella rhinoscleromatis,Staphylococcus pyogenes, and Vibrio vulnificus was conducted in comparison with difloxacin and chloramphenicol antibiotics. Cell viability and proliferation of the developed nanofibers were evaluated using an MTT assay. Finally, in vivo study of the wound-closure process was performed on New Zealand white rabbits. The results indicate that HTCs-capsaicin and HTCs-AuNPs are suitable in inhibiting bacterial growth compared with HTCs and HTCs-capsaicin/AuNP nanofibers and antibiotics (P < 0.01). The MTT assay demonstrates that the nanofibrous mats increased cell proliferation compared with the untreated control (P < 0.01). In vivo results show that the developed mats enhanced the wound-closure rate more effectively than the control samples. The novel nanofibrous wound dressings provide a relatively rapid and efficacious wound-healing ability, making the obtained nanofibers promising candidates for the development of improved bandage materials.

Glycerol, trehalose and vacuoles had relations to pullulan synthesis and osmotic tolerance by the whole genome duplicated strain Aureobasidium melanogenum TN3-1 isolated from natural honey.

In our previous study, it was found that Aureobasidium melanogenum TN3-1 was a high pullulan producing and osmotic tolerant yeast-like fungal strain. In this study, the HOG1 signaling pathway controlling glycerol synthesis, glycerol, trehalose and vacuoles were found to be closely related to its pullulan biosynthesis and high osmotic tolerance. Therefore, deletion of the key genes for the HOG1 signaling pathway, glycerol and trehalose biosynthesis and vacuole formation made all the mutants reduce pullulan biosynthesis and increase sensitivity of the growth of the mutants to high glucose concentration. Especially, abolishment of both the VSP11 and VSP12 genes which controlled the fission/fusion balance of vacuoles could cause big reduction in pullulan production (less than 7.4 ± 0.4 g/L) by the double mutant ΔDV-5 and increased sensitivity to high concentration glucose, while expression of the VSP11 gene in the double mutant ΔDV-5 made the transformants EV-2 restore pullulan production and tolerance to high concentration glucose. But cell growth of them were the similar. The double mutant ΔDV-5 had much bigger vacuoles and less numbers of vacuoles than the transformant EV-2 and its wild type strain TN3-1 while it grew weakly on the plate with 40% (w/v) glucose while the transformant EV-2 and its wild type strain TN3-1 could grow normally on the plate even with 60% (w/v) glucose. The double mutant ΔDV-5 also had high level of pigment and its cells were swollen. This was the first time to give the evidence that glycerol, trehalose and vacuoles were closely related to pullulan biosynthesis and high osmotic tolerance by A. melanogenum.

Microbial metabarcoding highlights different bacterial and fungal populations in honey samples from local beekeepers and market in north-eastern Italy.

Due to its chemical properties, honey does not foster the growth of microorganisms, however it may contain a rich microbial community, including viable, stressed, and not viable microbes. In order to characterize honey microbiota focusing on the difference between products from beekeepers and large retail in the present study a culture-independent approach based on DNA metabarcoding was applied. Honey samples were collected from Local Beekeepers (LB) and Market sales (M) during four years with the aim to investigate the microbiological quality in the honey market. Extraction and amplification of DNA from honey samples showed reduced efficiency with increasing age of honey, with the loss of 50-80% of samples four years old (2014). For this reason, only samples of similar age were compared and the analysis of microbial communities focused on year 2017, for a total of 75 samples. Differences in alpha and beta-diversity were evidenced comparing microbial communities between LB and M samples. In particular, contaminant bacteria dominated the microbiota in M samples while LB samples were enriched in Lactic Acid Bacteria (LAB) that cannot be isolated with culture-dependent approaches.

Fe3O4@CuS-based immunochromatographic test strips and their application to label-free and dual-readout detection of Escherichia coli O157:H7 in food.

Herein, a label-free and dual-readout immunochromatographic test strip (ITS) for the sensitive detection of Escherichia coli (E. coli) O157:H7 by taking advantages of the strong capture ability of Fe3O4@CuS nanostructures (NSs) towards bacteria and their ultrahigh photothermal effects (PTEs) was reported. Especially, without the customarily antibody (Ab)-labeled probe, Fe3O4@CuS NSs could be adsorbed onto the surfaces of bacteria to form Fe3O4@CuS-bacteria conjugates and then trapped by immobilized Abs on the test line (T-line), forming a characteristic yellow band. After direct immunoreactions, the PTEs of Fe3O4@CuS NSs endowed T-line to be irradiated by an 808 nm infrared light, obtaining satisfactory sensitivity and accuracy. Under optimal conditions, E. coli O157:H7, as low as 103 and 102 CFU/mL, could be monitored in colorimetric and photothermal modes. Additionally, E. coli O157:H7 was successfully detected in beef, chicken, milk and honey samples by this proposed platform with a recovery of 80-120%.