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Baculoviruses have been applied for biocontrol of agricultural pests, such as velvetbean caterpillar (Anticarsia gemmatalis) and fall armyworm (Spodoptera frugiperda). Cell culture is an interesting approach for large-scale production of these viruses. Co-infection of a host cell with two distinct viruses can contribute to reduce costs due to saving cell culture media, bioreactor space and the resulting co-occluded polyhedra may help to reduce final biopesticide costs. The baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) and Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) were chosen to test a model for in vitro co-infection in SF21 cells. Different proportions of SfMNPV/AgMNPV were evaluated along three in vitro passages by optical microscopy analysis of cells and real-time PCR (qPCR) of DNA obtained from budded viruses (BVs) and occlusion bodies (OBs). The kinetics of viral protein synthesis was carried out for analysis of the co-infection in first passage and bioassays with the resulting OBs were performed against A. gemmatalis and S. frugiperda larvae. The results demonstrated successful co-infection in these cells. The quantity of SfMNPV and AgMNPV in supernatants and sediments tends to be maintained stable during the three passages, although the amount of AgMNPV was higher than SfMPNV in most of the experiments. Analysis of the kinetics of radiolabed proteins showed that the cell protein synthesis was shut off and two distinct bands of about 30 kDa, regarded to be the polyhedrin of each virus, were strongly detected at 48 and 72 hp.i. Although the pathogenicity of the produced viruses was not completely satisfactory, the bioassays confirmed occurrence of co-infected larvae with disproportional amount of each virus.

Microalgae cultivation for exopolysaccharide production has getting more attention as a result of their high hydrocarbon biosynthesis skill. The aim of this study is to examine the exopolysaccharide production potential of different species of microalgae. In this context, exopolysaccharides were produced from Chlorella minutissima, Chlorella sorokiniana and Botryococcus braunii microalgae and the effects of carbon and nitrogen content in the growth medium and illumination time on exopolysaccharide production were analyzed statistically using Box-Behnken experimental design. In addition, techno-economic assessment of exopolysaccharide production were also performed by using the most productive microalgae and optimum conditions determined in this study. As a result of the experiments, it was seen that C. minutissima, C. sorokiniana and B. braunii produced 0.245 ± 0.0025 g/L, 0.163 ± 0.0016 g/L and 0.117 ± 0.0007 g/L exopolysaccharide, respectively. Statistically, it was observed that there was an inverse relationship between the exopolysaccharide production and investigated parameters such as illumination period and carbon and nitrogen amounts of culture mediums. The techno-economic assessment comprising microalgal exopolysaccharide (EPS) bioprocess was carried out, and it showed that the system can be considered economically viable, yet can be improved with biorefinery approach.

Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A3 (4093 μg mL−1), Jujubogenin isomer of Bacopasaponin C (65,339 μg mL−1), and Bacopasaponin C (1325 μg mL−1) while Bacopaside II (13,030 μg mL−1) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides—Withaferrin A, Withanolide A (480 μg mL−1), and Withanolide B (1024 μg mL−1) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions. Supplementary Information The online version contains supplementary material available at 10.1007/s42770-021-00586-0.

Toxic heavy metals pollution posed severe health hazards to the environment and biodiversity. Therefore, the development of rapid and non-invasive bioassays is in the race to monitor toxic chemicals using novel approaches. This study isolated and characterized an intense blue luminescence-producing marine bacteria, Vibrio campbellii STF1, for biosensing applications. Species-level identification of this strain was confirmed based on various phenotypic tests and multilocus sequence approach using 16s rRNA, toxR, and luxA gene sequence analysis. Fatty acid methyl ester analysis revealed the presence of three predominant fatty acids C(15:0 anteiso) (21.73%), C(17:0 anteiso) (11.27%), and C(19:0 anteiso) (9.08%) in STF1. Luciferase enzyme from V. campbellii STF1 was extracted, partially purified, and molecular masses (alpha subunit 40 kDa and beta subunit 37 kDa) were determined by SDS-PAGE gel for in vivo assays. MALDI-TOF-MS analysis of V. campbellii cells’ protein extracts showed distinct mass spectral peaks at m/z of 2615, 3948, and 4232 da. V. campbellii STF1 is resistant to heavy metal lead, while other metals such as cadmium, copper, and mercury inhibited its growth and luminescence. Crude ethyl acetate extraction of V. campbellii demonstrated antibacterial activity against Shigella dysenteriae type 5 with a maximum inhibition zone of 27.0±1.0 mm. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00471-w.

Saline environments are extreme habitats with a high diversity of microorganisms source of a myriad of biomolecules. These microorganisms are assigned as extremophiles recognized to be producers of new natural compounds, which can be synthesized by helping to survive under harshness and extreme conditions. In Brazil, in the saline and semi-arid region of Areia Branca (Caatinga biome), halotolerant bacteria (able to growth at high NaCl concentrations) were isolated from rhizosphere of native plants Blutaparon portulacoides and Spergularia sp. and their biopolymer production was studied. A total of 25 bacterial isolates were identified at genus level based on 16S rRNA gene sequence analysis. Isolates were mainly Gram-positive bacteria from Bacillaceae, Staphylococcaceae, Microbacteriaceae, and Bacillales XII incertae sedis families, affiliates to Bacillus, Staphylococcus, Curtobacterium, and Exiguobacterium genera, respectively. One of the Gram-negative isolates was identified as member of the Pseudomonadaceae family, genus Pseudomonas. All the identified strains were halotolerant bacteria with optimum growth at 0.6–2.0 M salt concentrations. Assays for biopolymer production showed that the halotolerant strains are a rich source of compounds as polyhydroxyalkanoates (PHA), biodegradable biopolymer, such as poly(3-hydroxybutyrate) (PHB) produced from low-cost substrates, and exopolysaccharides (EPS), such as hyaluronic acid (HA), metabolite of great interest to the cosmetic and pharmaceutical industry. Also, eight bacterial EPS extracts showed immunostimulatory activity, promising results that can be used in biomedical applications. Overall, our findings demonstrate that these biomolecules can be produced in culture medium with 0.6–2.0 M NaCl concentrations, relevant feature to avoid costly production processes. This is the first report of biopolymer-producing bacteria from a saline region of Caatinga biome that showed important biological activities. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s42770-021-00426-1) contains supplementary material, which is available to authorized users.

Polyhydroxyalkanoates (PHAs) are polymers with biodegradable and biocompatible properties accumulated in a wide variety of bacterial strains. In the present study, active sludge, wheat starch wastewater (WSW), and oil wastewater were used for the isolation and screening of PHA-accumulating bacteria. WSW was then implemented as a cheap and economical culture medium for the production of PHAs by the selected isolate. The extracted PHA was characterized, and the capability of produced biopolymer for preparing nanoparticles was evaluated. Based on the results, 96 different bacterial isolates were obtained, of which the strains isolated from WSW demonstrated the highest PHA-accumulation capability. The maximum PHA content of 3.07 g/l (59.50% of dry cell weight) was obtained by strain N6 in 21 h. The selected strain was identified by molecular approaches as Bacillus cereus. Afterward, the physicochemical characterization of an accumulated biopolymer was specified as a PHBV copolymer. Finally, spherical homogenous PHBV nanoparticles with a size of 137 nm were achieved. The PHBV nanoparticles showed a suitable small size and good zeta potential for medical applications. Hence, it can be concluded that isolated wild strain (B. cereus) has the potential exploitation capability for cost-effective PHBV production using the WSW. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00430-5.

n-Butanol is a renewable resource with a wide range of applications. Its physicochemical properties make it a potential substitute for gasoline. Saccharomyces cerevisiae can produce n-butanol via amino acid catabolic pathways, but the use of pure amino acids is economically unfeasible for large-scale production. The aim of this study was to optimize the production of n-butanol by S. cerevisiae from protein-rich agro-industrial by-products (sunflower and poultry offal meals). By-products were characterized according to their total protein and free amino acid contents and subjected to enzymatic hydrolysis. Protein hydrolysates were used as nitrogen sources for the production of n-butanol by S. cerevisiae, but only poultry offal meal hydrolysate (POMH) afforded detectable levels of n-butanol. Under optimized conditions (carbon/nitrogen ratio of 2 and working volume of 60%), 59.94 mg/L of n-butanol was produced using POMH and glucose as substrates. The low-cost agro-industrial by-product showed great potential to be used in the production of n-butanol by S. cerevisiae. Other protein-rich residues may also find application in biofuel production by yeasts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-020-00370-6) contains supplementary material, which is available to authorized users.

The development of an efficient transformation system is essential to enrich the genetic understanding of Trichoderma atroviride. To acquire an additional homologous selectable marker, uracil auxotrophic mutants were generated. First, the pyr4 gene encoding OMP decarboxylase was replaced by the hph marker gene, encoding a hygromycin phosphotransferase. Then, uracil auxotrophs were employed to determine that 5 mM uracil restores their growth and conidia production, and 1 mg ml(−1) is the lethal dose of 5-fluoroorotic acid in T. atroviride. Subsequently, uracil auxotrophic strains, free of a drug-selectable marker, were selected by 5-fluoroorotic acid resistance. Two different deletions in pyr4 were mapped in four auxotrophs, encoding a protein with frameshifts at the 310 and 335 amino acids in their COOH-terminal. Six auxotrophs did not have changes in the pyr4 ORF even though a specific cassette to delete the pyr4 was used, suggesting that 5-FOA could have mutagenic activity. The Ura(−)1 strain was selected as a genetic background to knock out the MAPKK Pbs2, MAPK Tmk3, and the blue light receptors Blr1/Blr2, using a short version of pyr4 as a homologous marker. The ∆tmk3 and ∆pbs2 mutants selected with pyr4 or hph marker were phenotypically identical, highly sensitive to different stressors, and affected in photoconidiation. The ∆blr1 and ∆blr2 mutants were not responsive to light, and complementation of uracil biosynthesis did not interfere in the expression of blu1, grg2, phr1, and env1 genes upregulated by blue light. Overall, uracil metabolism can be used as a tool for genetic manipulation in T. atroviride. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-020-00329-7) contains supplementary material, which is available to authorized users.

Bacterial biofilms play a key role in the pathogenesis of major oral diseases. Nanoparticles open new paths for drug delivery in complex structures such as biofilms. This study evaluated the antimicrobial effect of zein nanoparticles containing anacardic acid (AA) extracted from cashew shells of Anacardium occidentale on in vitro Streptococcus mutans biofilm formation and mature biofilms. The minimum inhibitory concentration (MIC), minimum bacterial concentration (MBC), and antibiofilm assays were performed. Streptococcus mutans UA159 biofilms were formed on saliva-coated hydroxyapatite disk for 5 days. To evaluate the preventive effect on biofilm formation, before contact with the inoculum, the disks were immersed once for 2 min in (1) hydroethanolic solution; (2) blank zein nanoparticles; (3) zein nanoparticles containing AA; and (4) 0.12% chlorhexidine gluconate. To determine the effect against mature biofilms, the disks containing 5-day preformed biofilms were further treated using the same procedure. The bacterial viability and dry weight were determined for both assays and used to compare the groups using ANOVA followed by Tukey’s test (p

In this study, we have investigated the effect of an antioxidant probiotic pretreatment toward an overdose of diclofenac in rats (100 mg/kg bw). Rats were treated daily with the probiotic Streptococcus salivarius St.sa (10(9) CFU) during seven successive days and then received a single treatment with diclofenac overdose in distilled water. Liver transaminases (alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase), histology, glutathione (GSH) and malondialdehyde (MDA) level were investigated. In addition, both antioxidant enzyme activity and its mRNA gene expression were studied to evaluate diclofenac hepatotoxicity. The results indicated that probiotic pretreatment reduced diclofenac-induced hepatotoxicity through enhancement of the studied hepatic markers and regulation of antioxidant enzyme expression and activity. These findings indicate that the probiotic pretreatment protects rat liver against the oxidative stress induced by diclofenac overdose.

Made available in DSpace on 2020-12-11T15:32:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-14 Currently, poultry farming is one of the sectors that have a significant impact on the global economy. In recent years, there has been an increase in the production of broilers, inflicting this segment of the industry to generate tons of keratin due to huge disposal of chicken feathers. This points to the need to degrade these chicken feathers, as they have emerged as a major threat to the environment. Thus, in this study we aimed to identify keratinases that are produced by the bacterium Citrobacter diversus and further investigate the biochemical characteristics of these keratin-degrading enzymes. In a submerged medium, the bacterium was capable of degrading chicken feathers almost completely after 36 h of fermentation. We found a maximum caseinolytic activity at pH 9-10.5 and 50-55 degrees C, and keratinolytic activity at pH 8.5-9.5 and 50 degrees C. Thus, given its stability at higher temperatures, upon incubation of this enzyme extract for 1 h at 50 degrees C, it showed approximately 50% of the keratinolytic and 100% of the caseinolytic activity. Further, under pH stability for 48 h at 4 degrees C, the enzyme extract maintained greater residual activity in the pH range 6-8. Caseinolytic activity was inhibited by EDTA and PMSF, whereas the keratinolytic activity was inhibited only by EDTA. Additionally, due to its alkaline activity and detergent compatibility, this enzyme extract could improve washing performance when added to a commercial detergent formulation. Using application tests, we could demonstrate a potential use of this bacterial enzyme extract as an additive in detergents to remove egg stains from cloth. Univ Estadual Paulista, Inst Biociencias Letras & Ciencias Exatas, Ibilce Unesp, R Cristovao Colombo 2265 Jd Nazareth, Sao Jose Do Rio Preto, SP, Brazil Univ Estadual Paulista, Inst Biociencias Letras & Ciencias Exatas, Ibilce Unesp, R Cristovao Colombo 2265 Jd Nazareth, Sao Jose Do Rio Preto, SP, Brazil

This study reports the optimization of milk-clotting protease production from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) in both one-variable-at-a-time and response surface methodology (RSM). The production and optimization of milk-clotting protease obtained from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) using different agro-industrial wastes as solid substrates were studied. The agro-industrial wastes used included wheat bran, rice bran, pea bran, and grass pea bran. The chemical composition of the best solid substrate was tested using standard methods. Others cultivation parameters were studied, and the results showed that the optimum fermentation medium composed of wheat bran, casein (1% w/w), and glucose (0.5% w/w) and the conditions for maximum milk-clotting protease production were at the moisture content of 55.0%, inoculum of 0.5*10(6) spores/mL, incubation temperature of 30 °C, pH of 6.0, and fermentation time of 5 days. The highest milk-clotting activity was obtained from the crude enzyme extracted using 0.1 M NaCl and partial purification of the crude enzyme using chilled acetone, and 80% (NH(4))(2)SO(4) increased the ratio of MCA/PA from 0.56 to 1.30 and 0.65, respectively. Moreover, the highest MCA (137.58 U/mL) was obtained at a casein concentration of 0.5%, pH 4.0, and 25 °C, using RSM. Thus, results from the present study showed that the optimization of milk-clotting protease production from A. oryzae DRDFS 13 under SSF by both one-variable-at-a-time and RSM significantly increased the milk-clotting activity. This is the first report from a fungus in the Ethiopian setting and a modest contribution to highlight the potential of harnessing microbial protease enzymes for industrial applications. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-020-00243-y) contains supplementary material, which is available to authorized users.

Plant growth-limiting factors, such as low nutrient availability and weak pathogen resistance, may hinder the production of several crops. Plant growth-promoting bacteria (PGPB) used in agriculture, which stimulate plant growth and development, can serve as a potential tool to mitigate or even circumvent these limitations. The present study evaluated the feasibility of using bacteria isolated from the maize rhizosphere as PGPB for the cultivation of this crop. A total of 282 isolates were collected and clustered into 57 groups based on their genetic similarity using BOX-PCR. A representative isolate from each group was selected and identified at the genus level with 16S rRNA sequencing. The identified genera included Bacillus (61.5% of the isolates), Lysinibacillus (30.52%), Pseudomonas (3.15%), Stenotrophomonas (2.91%), Paenibacillus (1.22%), Enterobacter (0.25%), Rhizobium (0.25%), and Atlantibacter (0.25%). Eleven isolates with the highest performance were selected for analyzing the possible pathways underlying plant growth promotion using biochemical and molecular techniques. Of the selected isolates, 90.9% were positive for indolepyruvate/phenylpyruvate decarboxylase, 54.4% for pyrroloquinoline quinine synthase, 36.4% for nitrogenase reductase, and 27.3% for nitrite reductase. Based on biochemical characterization, 9.1% isolates could fix nitrogen, 36.6% could solubilize phosphate, 54.5% could produce siderophores, and 90.9% could produce indole acetic acid. Enzymatic profiling revealed that the isolates could degrade starch (90.1%), cellulose (72.7%), pectin (81.8%), protein (90.9%), chitin (18.2%), urea (54.5%), and esters (45.4%). Based on the data obtained, we identified three Bacillus spp. (LGMB12, LGMB273, and LGMB426), one Stenotrophomonas sp. (LGMB417), and one Pseudomonas sp. (LGMB456) with the potential to serve as PGPB for maize. Further research is warranted to evaluate the biotechnological potential of these isolates as biofertilizers under field conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00596-y.

Bioemulsifiers form stable emulsions and lower surface tension between two phases with potent anti-microbial activities. Some applications of bioemulsifier are performed at high temperatures and hence production of bioemulsifiers that are stable at high temperature is required. This study aimed at the production of bioemulsifier by an unexplored bacterial strain isolated from a local hot spring. The parameters tested for bioemulsifier production (emulsification ability, surface tension measurement and product formation) showed that 24 h is the optimal time for the production of bioemulsifier by strain S3 with yield of 1.4 g/l. The logistic growth curve of bacterial strain was analysed and kinetic constants for substrate utilisation and product formation were determined by Luedeking-Piret kinetic models. The bacterial strain S3 was Gram-positive and was classified as a strain of Brevibacillus borstelensis. The specific growth rate of the organism was 0.0096 h(−1) with the kinetic rate constants as 11.246 (γ) and 10.626 (δ) for Luedeking-Piret substrate and 3.8423 (α) and − 1.9075 (β) for Luedeking-Piret product. Knowledge of these values will help in estimating the substrate utilisation or bioemulsifier formed at any time point. These studies will also help in understanding internal metabolic fluxes hence rigorous analysis of metabolic pathway of bioemulsan is also performed in this study. [Figure: see text]

Paracoccidioidomycosis (PCM) is a disease caused by fungi of the genus Paracoccidioides. The disease is responsible for high rates of premature deaths and socioeconomic repercussions. The limitations of antifungal agents against PCM have motivated the search for new compounds. In our ongoing exploration of Cerrado plants as potential sources of new antifungal agents, we selected Copaifera langsdorffii oil (Copaíba resin oil) in order to explore its bioactive potential and test a formulation to increase oil stability and solubilization employing Pluronic F-127 to obtain the nanoemulsion of the oil. We aim at testing both Copaíba resin oil and its nanoemulsion against four species of the Paracoccidioides genus. We performed cytotoxicity test in Balb/C3T3 cells, hemolytic activity and interaction of Copaíba resin oil and Copaíba resin oil nanoemulsion (CopaPlu) with the antifungal agents such as amphotericin B, co-trimoxazole, and itraconazole. Moreover, the Copaíba resin oil was analyzed by mass spectrometry to identify its chemical profile. Eventually, a new methodology to prepare the nanoemulsion is presented. The Copaíba resin oil and CopaPlu nanoemulsion inhibited Paracoccidioides sp. growth efficiently, and no cytotoxicity or hemolytic effect was observed at minimum inhibitory concentration (MIC). When combined with amphotericin B, Copaíba resin oil and its nanoemulsion showed an additive effect with reduction of MIC values. The Copaíba resin oil and CopaPlu nanoemulsion is a promising antifungal agent against Paracoccidioides.

A new strain of Trichoderma reesei (teleomorph Hypocrea jecorina) with high cellulase production was obtained by exposing the spores from T. reesei QM9414 to an ultraviolet light followed by selecting fast-growing colonies on plates containing CMC (1% w/v) as the carbon source. The mutant T. reesei RP698 reduced cultivation period to 5 days and increased tolerance to the end-products of enzymatic cellulose digestion. Under submerged fermentation conditions, FPase, CMCase, and Avicelase production increased up to 2-fold as compared to the original QM9414 strain. The highest levels of cellulase activity were obtained at 27 °C after 72 h with Avicel®, cellobiose, and sugarcane bagasse as carbon sources. The temperature and pH activity optima of the FPase, CMCase, and Avicelase were approximately 60 °C and 5.0, respectively. The cellulase activity was unaffected by the addition of 140 mM glucose in the enzyme assay. When T. reesei RP698 crude extract was supplemented by the addition of β-glucosidase from Scytalidium thermophilum, a 2.3-fold increase in glucose release was observed, confirming the low inhibition by the end-product of cellulose hydrolysis. These features indicate the utility of this mutant strain in the production of enzymatic cocktails for biomass degradation.

The production of a highly concentrated probiotic preparation of Lactococcus lactis CECT 539 was studied in both batch and realkalized fed-batch fermentations in diluted whey (DW) media (DW25, DW50, DW75, DW100, and DW125) supplemented with MRS broth nutrients (except glucose and Tween 80) at 25, 50, 75, 100, and 125% of their standard concentrations in the complex medium. The fed-batch culture using DW100 medium provided the highest concentrations of probiotic biomass (5.98 g/L) and nisin (258.47 BU/mL), which were obtained at lower production costs than those estimated for the fed-batch culture in DW medium. The batch and fed-batch cultures reduced the initial chemical oxygen demand (COD) of the media by 29.1–41.7% and 31.2–54.2%, respectively. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00114-1) contains supplementary material, which is available to authorized users.

Recalcitrant characteristics and insolubility in water make the disposal of synthetic polymers a great environmental problem to be faced by modern society. Strategies towards the recycling of post-consumer polymers, like poly (ethylene terephthalate, PET) degradation/depolymerization have been studied but still need improvement. To contribute with this purpose, 100 fungal strains from hydrocarbon-associated environments were screened for lipase and esterase activities by plate assays and high-throughput screening (HTS), using short- and long-chain fluorogenic probes. Nine isolates were selected for their outstanding hydrolytic activity, comprising the genera Microsphaeropsis, Mucor, Trichoderma, Westerdykella, and Pycnidiophora. Two strains of Microsphaeropsis arundinis were able to convert 2–3% of PET nanoparticle into terephthalic acid, and when cultured with two kinds of commercial PET bottle fragments, they also promoted weight loss, surface and chemical changes, increased lipase and esterase activities, and led to PET depolymerization with release of terephthalic acid at concentrations above 20.0 ppm and other oligomers over 0.6 ppm. The results corroborate that hydrocarbon-associated areas are important source of microorganisms for application in environmental technologies, and the sources investigated revealed important strains with potential for PET depolymerization. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00093-3) contains supplementary material, which is available to authorized users.

To date, endophytic actinomycetes have been well-documented as great producers of novel antibiotics and important pharmaceutical leads. The present study aimed to evaluate potent bioactivities of metabolites synthesized by the strain LCP18 residing in the Vietnamese medicinal plant Litsea cubeba (Lour.) Pers towards human pathogenic bacteria and human cancer cell lines. Endophytic actinomycete strain LCP18 showed considerable inhibition against seven bacterial pathogens and three human tumor cell lines and was identified as species Streptomyces variabilis. Strain S. variabilis LCP18 was phenotypically resistant to fosfomycin, trimethoprim-sulfamethoxazole, dalacin, cefoxitin, rifampicin, and fusidic acid and harbored the two antibiotic biosynthetic genes such as PKS-II and NRPS. Further purification and structural elucidation of metabolites from the LCP18 extract revealed five plant-derived bioactive compounds including isopcrunetin, genistein, daidzein, syringic acid, and daucosterol. Among those, isoprunetin, genistein, and daidzein exhibited antibacterial activity against Salmonella typhimurium ATCC 14,028 and methicillin-resistant Staphylococcus epidermidis ATCC 35,984 with the MIC values ranging from 16 to 128 µg/ml. These plant-derived compounds also exhibited cytotoxic effects against human lung cancer cell line A549 with IC(50) values of less than 46 μM. These findings indicated that endophytic S. variabilis LCP18 can be an alternative producer of plant-derived compounds which significantly show potential applications in combating bacterial infections and inhibition against lung cancer cell lines.

Endophytes are microorganisms that form symbiotic relationships with their host. These microorganisms can produce a variety of secondary metabolites, some of which have inhibitory effects on pests and pathogens or even act to promote plant growth. Due to these characteristics, these microorganisms are used as sources of biologically active substances for a wide range of biotechnological applications. Based on that, the aim of this study was to evaluate the production of metabolites of the endophytic Aspergillus flavus CL7 isolated from Chromolaena laevigata, in four different cultivation conditions, and to determine the antimicrobial, cytotoxic, antiviral, and antioxidant potential of these extracts. The multiphasic approach used to identify this strain was based on morphology and ITS gene sequence analysis. The chemical investigation of A. flavus using potato dextrose and minimal medium, using both stationary and agitated methods, resulted in the isolation of kojic acid, α-cyclopiazonic acid, and 20,25-dihydroxyaflavinine. Another 18 compounds in these extracts were identified by UHPLC-HRMS/MS, of which dideacetyl parasiticolide A has been described for the first time from A. flavus. Aflatoxins, important chemomarkers of A. flavus, were not detected in any of the extracts, thus indicating that the CL7 strain is non-aflatoxigenic. The biological potential of all extracts was evaluated, and the best results were observed for the extract obtained using minimal medium against Trichophyton rubrum and Mycobacterium tuberculosis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00502-6.

The objective of this study was to evaluate the antibacterial effect of sophorolipid in combination with lactic acid against relevant bacteria isolated from the poultry industry. Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, and Escherichia coli were isolated from chicken meat and antibacterial tests with sophorolipid and lactic acid were performed. Checkerboard, time-kill, and scanning electron microscopy analyses were used to confirm the antibacterial action and the combined effects. Although no inhibitory effects were observed for E. coli and Salmonella, these compounds presented antibacterial activity against L. monocytogenes and S. aureus. Additionally, sophorolipid and lactic acid were not cytotoxic at the concentrations used in the tests. The combination of sophorolipid and lactic acid resulted in an additive interaction, reducing the concentration of the active compounds needed for effectiveness against S. aureus and L. monocytogenes, to 50% and 75%, respectively. These findings lead to the possibility of developing a new, sustainable, and natural antimicrobial solution that is considered noncytotoxic and has wide applicability in the poultry industry to reduce substantial losses in this sector.

Lignocellulosic hydrolysates will also contain compounds that inhibit microbial metabolism, such as organic acids, furaldehydes, and phenolic compounds. Understanding the response of yeasts toward such inhibitors is important to the development of different bioprocesses. In this work, the growth capacity of 7 industrial Saccharomyces cerevisiae and 7 non-Saccharomyces yeasts was compared in the presence of 3 different concentrations of furaldehydes (furfural and 5-hydroxymetil-furfural), organic acids (acetic and formic acids), and phenolic compounds (vanillin, syringaldehyde, ferulic, and coumaric acids). Then, Candida tropicalis JA2, Meyerozyma caribbica JA9, Wickerhamomyces anomalus 740, S. cerevisiae JP1, B1.1, and G06 were selected for fermentation in presence of acetic acid, HMF, and vanillin because they proved to be most tolerant to the tested compounds, while Spathaspora sp. JA1 because its xylose consumption rate. The results obtained showed a dose-dependent response of the yeasts toward the eight different inhibitors. Among the compared yeasts, S. cerevisiae strains presented higher tolerance than non-Saccharomyces, 3 of them with the highest tolerance among all. Regarding the non-Saccharomyces yeasts, C. tropicalis JA2 and W. anomalus 740 appeared as the most tolerant, whereas Spathaspora strains appeared very sensitive to the different compounds.

Made available in DSpace on 2020-12-12T02:04:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fungi in the genus Trichoderma are notorious producers of secondary metabolites with diverse applications, such as antibacterial, antifungal, and plant growth-promoting properties. Peptaibols are linear peptides produced by such fungi, with more than 440 compounds described to date, including tricholongins, longibrachins, trichobrachins, and trichovirins. Peptaibols are synthesized by non-ribosomal peptide synthetases and they have several biological activities. Our research group isolated four peptaibols (6DP2, 6DP3, 6DP4, and 6DP5) with antifungal activity against the plant pathogen Colletotrichum gloeosporioides and the proteasome (a cancer chemotherapy target) from Trichoderma sp. P8BDA1F1, an endophytic fungus from Begonia venosa. The ethyl acetate extract of this endophyte showed activity of 6.01% and 75% against C. gloeosporioides and the proteasome, respectively. The isolated compounds were identified by MS/MS and compared to literature data, suggesting the presence of trilongins BI, BII, BIII, and BIV, which are peptaibols containing 20 amino acid residues. The minimum inhibitory concentration against C. gloeosporioides was 40 μM for trilongin BI, 320 μM for trilongin BII, 160 μM for trilongin BIII, and 310 μM for trilongin BIV. BI–BIV trilongins inhibited proteasome ChTL activity, with IC50 values of 6.5 ± 2.7; 4.7 ± 1.8; 6.3 ± 2.2; and 2.7 ± 0.5 μM, respectively. The compounds were tested ex vivo against the intracellular amastigotes of Leishmania (L.) infantum but showed no selectivity. It is the first report of trilongins BI–BIV with antifungal activity against C. gloeosporioides and the proteasome target. Departamento de Ciências Exatas Universidade de São Paulo Escola Superior de Agricultura “Luiz de Queiroz”, CP 9 Centro Nacional de Pesquisa em Energia e Materiais Laboratório Nacional de Biociências, CP 6192 Instituto Adolfo Lutz Centro de Parasitologia e Micologia Departamento de Biologia Geral e Aplicada Universidade Estadual Paulista (UNESP) Centro de Estudos de Insetos Sociais Universidade Estadual Paulista (UNESP) Departamento de Biologia Geral e Aplicada Universidade Estadual Paulista (UNESP) Centro de Estudos de Insetos Sociais Universidade Estadual Paulista (UNESP) CNPq: 142079/2016-2 FAPESP: 2013/50228-8 FAPESP: 2014/10753-9 FAPESP: 2014/12021-5 FAPESP: 2014/15760-3 CAPES: 88881.133610/2016-01

Stenotrophomonas’ metabolic versatility plays important roles in the remediation of contaminated environment and plant growth promotion. We investigated two Stenotrophomonas strains isolated from textile polluted sewage for their ability to decolorize and degrade azo dyes. Two Stenotrophomonas strains (TepeL and TepeS) were isolated from textile effluents (Tepetitla, Mexico) using the selective agar Stenotrophomonas vancomycin, imipenem, amphotericin B agar (SVIA). Isolates’ identity was determined by the sequencing of their partial 16S rRNA fragments. Their abilities to decolorize dyes were tested in a Luria broth supplemented with varying concentrations (50 mg/L–1 g/L) of textile dyes (acidic red, methyl orange, reactive green, acidic yellow, and reactive black). Fourier-transform infrared (FTIR) spectroscopy and ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) metabolite analyses were used to determine the effect of the isolates’ growth on the dyes (acidic red, methyl orange). We also identified the enzymes that may be involved in the degradation process. Phylogenetic analysis based on the 16S rDNA sequences showed that the isolates belong to the genus Stenotrophomonas. Stenotrophomonas sp. TepeL and TepeS respectively decolorize all the azo dyes at the tested concentration except at 1 g/L and degraded the azo dyes. The degradation resulted in the formation of N, N-dimethyl p-phenylenediamine, and sodium 4-amino-1-naphthalenesulfonate from methyl orange and acid red. TepeL and TepeS rapidly decolorized and degraded the azo dyes tested. This result showed that the two isolates have a good potential for the decontamination of textile effluents. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00542-y.

We studied the expression of Bacillus amyloliquefaciens transglutaminase cloned in Escherichia coli BL21(DE3)pLysS harboring the plasmid pBAD/3C/bTGase, a bicistronic expression system, in bioreactor cultivation. Batch and fed-batch controlled as DO-stat strategies were employed for the production of the recombinant enzyme. In 30 h-batch cultivations using Terrific broth (TB), 6 g/L of biomass and 3.12 U/mg(protein) of transglutaminase activity were obtained. DO-stat fed-batch cultivations under the control of oxygen concentration (DO-stat) using TB as medium but fed with glucose allowed the increment in biomass formation (17.5 g/L) and enzyme activity (6.43 U/mg(protein)). DO-stat fed-batch using mineral medium (M9) and fed with glucose under the same conditions produced even higher enzymatic activity (9.14 U/mg(protein)). The pH effect was investigated, and the best enzymatic activity could be observed at pH 8. In all cultivations, the bicistronic system remained stable, with 100% of plasmid-bearing cells. These results show that E. coli bearing bicistronic plasmid constructs to express recombinant TGase could be cultivated in bioreactors under DO-stat fed-batch using mineral medium and it is a promising strategy in future optimizations to produce this important enzyme.

Pectinase is a general term for a class of enzymes that decompose pectin. To obtain a fungal strain with high-activity pectinase of potential commercial importance, we screened microorganisms from the soil of vineyards, performed mutation breeding by ultraviolet (UV) and nitrosoguanidine (NTG) mutagenesis, and performed comparisons to commercially available pectinases. We found that the derived pectinase-producing strain Rn14-88A had the highest pectinase activity of 8363.215 U/mL, and identified it using internal transcribed spacer sequence analysis as Aspergillus tubingensis. Rn14-88A was the original strain for UV mutagenesis, from which mutant strain R-7-2-4 had the highest pectinase enzyme activity (9198.68 U/mL), which was a 9.99% increase compared to that of Rn14-88A. Following NTG mutagenesis of R-7-2-4, mutant strain Y1-3-2-6 had a pectinase enzyme activity of 9843.34 U/mL, which reflects a 6.36% increase compared to the pectinase activity of R-7-2-4. Subsequently, another round of NTG mutagenesis was performed on Y1-3-2-6, and the mutagenic strain Y2-6-3-4 exhibited an improved enzyme activity of 21,864.34 U/mL, which was 161.44% higher than that of Rn14-88A. Through liquid fermentation experiments of A. tubingensis Y2-6-3-4, it was determined that pectinase activity was the highest at a fermentation time of 20 h. Therefore, we conclude that A. tubingensis Y2-6-3-4 has potential for use in commercial production.

Microbes from hypersaline environments are useful in biotechnology as sources of novel enzymes and proteins. The current study aimed to characterize halophilic bacteria from the rhizosphere of halophytes (Salsola stocksii and Atriplex amnicola), non-rhizospheric, and brine lake-bank soils collected from Khewra Salt Mine and screening of these bacterial strains for industrially important enzymes. A total of 45 bacterial isolates from the rhizosphere of Salsola, 38 isolates from Atriplex, 24 isolates from non-rhizospheric, and 25 isolates from lake-bank soils were identified by using 16S rRNA gene analysis. Phylogenetic analysis showed that bacterial strains belonging to Bacillus, Halobacillus, and Kocuria were dominant in the rhizosphere of halophytes (Salsola and Atriplex), and Halobacillus and Halomonas were dominating genera from non-rhizospheric and lake-bank soils. Mostly identified strains were moderately halophilic bacteria with optimum growth at 1.5–3.0 M salt concentrations. Most of the bacterial exhibited lipase, protease, cellulase, amylase, gelatinase, and catalase activities. Halophilic and halotolerant Bacilli (AT2RP4, HL1RS13, NRS4HaP9, and LK3HaP7) identified in this study showed optimum lipase, protease, cellulase, and amylase activities at 1.0–1.5 M NaCl concentration, pH 7–8, and temperature 37 °C. These results indicated that halophilic and halotolerant bacteria can be used for bioconversion of organic compounds to useful products under extreme conditions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00044-y) contains supplementary material, which is available to authorized users.

Sclareol is an important intermediate for ambroxide synthesis industries. Hyphozyma roseonigra ATCC 20624 was the only reported strain capable of degrading sclareol to the main product of sclareol glycol, which is the precursor of ambroxide. To date, knowledge is lacking about the effects of sclareol on cells and the proteins involved in sclareol metabolism. Comparative proteomic analyses were conducted on the strain H. roseonigra ATCC 20624 by using sclareol or glucose as the sole carbon source. A total of 79 upregulated protein spots with a > 2.0-fold difference in abundance on 2-D gels under sclareol stress conditions were collected for further identification. Seventy spots were successfully identified and finally integrated into 30 proteins. The upregulated proteins under sclareol stress are involved in carbon metabolism and nitrogen metabolism, and replication, transcription, and translation processes. Eighteen upregulated spots were identified as aldehyde dehydrogenases, which indicating that aldehyde dehydrogenases might play an important role in sclareol metabolism. Overall, this study may lay the fundamentals for further cell engineering to improve sclareol glycol production. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00040-2) contains supplementary material, which is available to authorized users.

This work covers soymilk fermentation by starter and probiotic cultures and explores the influence of cooling protocol on cell viability, organic acid production, sugar consumption, fatty acid profile, and cell survival to in vitro gastrointestinal stress. After fermentation at 37 °C by mono- or co-cultures of Streptococcus thermophilus (St), Lactobacillus bulgaricus (Lb), and Lactobacillus paracasei (Lp), fermented soymilk was cooled directly at 4 °C for 28 days or cooled in two phases (TPC), i.e., by preceding that step by another at 25 °C for 8 h. Soybean milk fermentation by Lb alone lasted longer (15 h) than by StLb or StLbLp (9 h). In ternary culture, TPC increased Lp viability, linoleic, and lactic acid concentrations by 3.8, 22.6, and 96.2%, respectively, whereas the cooling protocol did not influence Lp and St counts after in vitro gastrointestinal stress. Graphical abstract.

KURT KIZILDOGAN, ASLIHAN/0000-0002-9323-0993 WOS: 000530831200012 PubMed: 31833007 lysA gene encoding meso-diaminopimelic acid (DAP) decarboxylase enzyme that catalyzes l-lysine biosynthesis in the aspartate pathway in Streptomyces clavuligerus was overexpressed, and its effects on cephamycin C (CephC), clavulanic acid (CA), and tunicamycin productions were investigated. Multicopy expression of lysA gene under the control of glpF promoter (glpFp) in S. clavuligerus pCOlysA led to higher expression levels ranging from 2- to 6-fold increase at both lysA gene and CephC biosynthetic gene cluster at T-36 and T-48 of TSBG fermentation. These results accorded well with CephC production. Thus, 1.86- and 3.14-fold higher volumetric as well as 1.26- and 1.71-fold increased specific CephC yields were recorded in S. clavuligerus pCOlysA in comparison with the wild-type and its control strain, respectively, at 48th h. Increasing the expression of lysA provided 4.3 times more tunicamycin yields in the recombinant strain. These findings suggested that lysA overexpression in S. clavuligerus made the strain more productive for CephC and tunicamycin. The results also supported the presence of complex interactions among antibiotic biosynthesis pathways in S. clavuligerus. Ondokuz Mayis UniversitesiOndokuz Mayis University [PYO.ZRT.1902-A.15.001]

The growing demand of consumers for synthetic chemical-free foods has increased the search for natural preservatives such as bacteriocins and bacteriocin-like inhibitory substances (BLIS) to give them adequate microbiological safety, sensory characteristics, and shelf life. In this study, the antimicrobial activity of BLIS produced by Pediococcus pentosaceus ATCC 43200 was compared with that of nisin. Lactobacillus sakei ATCC 15521, Listeria seeligeri NCTC 11289, Enterococcus En2052 and En2865, and Listeria monocytogenes CECT 934 and NADC 2045 exhibited larger inhibition halos in BLIS-treated than in Nisaplin-treated samples, unlike Listeria innocua NCTC 11288. In artificially contaminated ready-to-eat pork ham, BLIS was effective in inhibiting the growth of L. seeligeri NCTC 11289 for 6 days (counts from 1.74 to 0.00 log CFU/g) and ensured lower weight loss (2.7%) and lipid peroxidation (0.63 mg MDA/kg) of samples compared with the control (3.0%; 1.25 mg MDA/kg). At the same time, coloration of ham samples in terms of luminosity, redness, and yellowness as well as discoloration throughout cold storage was not influenced by BLIS or Nisaplin taken as a control. These results suggest the potential use of P. pentosaceus BLIS as a biopreservative in meat and other food processing industries.

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily cellobiose, because it is the major soluble by-product of cellulose and acts as a strong inhibitor, especially for cellobiohydrolase, which plays a key role in cellulose hydrolysis. Commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose; accordingly, genetic engineering efforts have been made to transfer β-glucosidase genes enabling cellobiose utilization. Nonetheless, laboratory yeast strains have been employed for most of this research, and such strains may be difficult to use in industrial processes because of their generally weaker resistance to stressors and worse fermenting abilities. The purpose of this study was to engineer industrial yeast strains to ferment cellobiose after stable integration of tabgl1 gene that encodes a β-glucosidase from Thermoascus aurantiacus (TaBgl1). The recombinant S. cerevisiae strains obtained in this study secrete TaBgl1, which can hydrolyze cellobiose and produce ethanol. This study clearly indicates that the extent of glycosylation of secreted TaBgl1 depends from the yeast strains used and is greatly influenced by carbon sources (cellobiose or glucose). The recombinant yeast strains showed high osmotolerance and resistance to various concentrations of ethanol and furfural and to high temperatures. Therefore, these yeast strains are suitable for ethanol production processes with saccharified lignocellulose. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00192-1) contains supplementary material, which is available to authorized users.

Biological nitrogen fixation (BNF) with the soybean crop probably represents the major sustainable technology worldwide, saving billions of dollars in N fertilizers and decreasing water pollution and the emission of greenhouse gases. Accordingly, the identification of strains occupying nodules under field conditions represents a critical step in studies that are aimed at guaranteeing increased BNF contribution. Current methods of identification are mostly based on serology, or on DNA profiles. However, the production of antibodies is restricted to few laboratories, and to obtain DNA profiles of hundreds of isolates is costly and time-consuming. Conversely, the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique might represent a golden opportunity for replacing serological and DNA-based methods. However, MALDI-TOF databases of environmental microorganisms are still limited, and, most importantly, there are concerns about the discrimination of protein profiles at the strain level. In this study, we investigated four soybean rhizobial strains carried in commercial inoculants used in over 35 million hectares in Brazil and also in other countries of South America and Africa. A supplementary MALDI-TOF database with the protein profiles of these rhizobial strains was built and allowed the identification of unique profiles statistically supported by multivariate analysis and neural networks. To test this new database, the nodule occupancy by Bradyrhizobium strains in symbiosis with soybean was characterized in a field experiment and the results were compared with serotyping of bacteria by immuno-agglutination. The results obtained by both techniques were highly correlated and confirmed the viability of using the MALDI-TOF MS technique to effectively distinguish bacteria at the strain level.

The effects of different three carbon sources, that is, glucose, fructose, and sucrose, on production, molecular properties and antiproliferative activity of exopolysaccharide (EPS), were evaluated in the submerged culture of Scleroderma areolatum Ehrenb. Among carbon sources examined, the addition of sucrose maximizes the mycelia production, while fructose could maximize the EPS yield. Although the predominant carbohydrate compositions identified were gluconic acid and mannose, the monosaccharide composition of EPSs was also different significantly. FT-IR spectral analysis revealed there was no significant difference among the prominent characteristic groups in three EPSs. The molecular weight of EPSs was also affected by carbon source, being generally lower compared with that with glucose. However, all EPSs molecule existed as nearly globular shape form in aqueous solution. The variation of carbon sources also affected antiproliferative activity examined in vitro using cell proliferation assay. Fructose was optimal carbon source giving higher antiproliferative activity probably due to the relatively high contents of xylose in the EPS with low molecular weight.

The effect of gene dosage on the production of Candida antarctica lipase B (CalB) in the methylotrophic yeast Komagataella phaffii, at high densities in a simple medium containing crude glycerin as the sole carbon source, is described. The use of crude glycerin, the main by-product of biodiesel production from vegetable oils, will reduce the production cost of the bioprocess. Two K. phaffii strains were constructed with one or three copies of LipB, an optimized version of the gene encoding CalB under the control of the constitutive P(PGK1) promoter. These two constructs were tested and compared on batches using minimal-salts medium with crude glycerin. The strain with three copies achieved a higher enzyme yield (48,760 U/L, 2.3-fold higher than the one-copy strain), with 42 g/L biomass, with no effects on growth. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00056-8) contains supplementary material, which is available to authorized users.

Endophytes are microorganisms that form symbiotic relationships with their own host. Included in this group are the species Phyllosticta capitalensis, a group of fungi that include saprobes that produce bioactive metabolites. The present study aimed to identify the cultivable endophytic fungal microbiota present in healthy leaves of Tibouchina granulosa (Desr.) Cogn. (Melastomataceae) and investigate secondary metabolites produced by a strain of P. capitalensis and their effects against both Leishmania species and Trypanossoma cruzi. Identification of the strains was accomplished through multilocus sequencing analysis (MLSA), followed by phylogenetic analysis. The frequency of colonization was 73.66% and identified fungi belonged to the genus Diaporthe, Colletotrichum, Phyllosticta, Xylaria, Hypoxylon, Fusarium, Nigrospora, and Cercospora. A total of 18 compounds were identified by high-resolution mass spectrum analysis (UHPLC-HRMS), including fatty acids based on linoleic acid and derivatives, from P. capitalensis. Crude extracts had activity against Leishmania amazonensis, L. infantum, and Trypanosoma cruzi, with inhibitory concentration (IC(50)) values of 17.2 μg/mL, 82.0 μg/mL, and 50.13 μg/mL, respectively. This is the first report of the production of these compounds by the endophytic P. capitalensis isolated from T. granulosa. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00221-z) contains supplementary material, which is available to authorized users.

Microlipid vesicles (MLV) have a broad spectrum of applications for the delivery of molecules, ranging from chemical compounds to proteins, in both in vitro and in vivo conditions. In the present study, we developed a new set of nanosize multilayer lipid vesicles (NMVs) containing a unique combination of lipids. The NMVs enable the adsorption of histidine-tagged proteins at the vesicle surface and were demonstrated to be suitable for the in vivo delivery of antigens. The NMVs contained a combination of neutral (DOPC) and anionic (DPPG) lipids in the inner membrane and an external layer composed of DOPC, cholesterol, and a nickel-containing lipid (DGS-NTA [Ni]). NMVs combined with a recombinant form of the B subunit of the Shiga toxin (rStx2B) produced by certain enterohemorragic Escherichia coli (EHEC) strains enhanced the immunogenicity of the antigen after parenteral administration to mice. Mice immunized with rStx2B-loaded NMVs elicited serum antibodies capable of neutralizing the toxic activities of the native toxin; this result was demonstrated both in vitro and in vivo. Taken together, these results demonstrated that the proposed NMVs represent an alternative for the delivery of antigens, including recombinant proteins, generated in different expression systems. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-018-0035-0) contains supplementary material, which is available to authorized users.

Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (β-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00060-y) contains supplementary material, which is available to authorized users.

Ergosterol, a unique component of fungal cells, is not only important for fungal growth and stress responses but also holds great economic value. Limited studies have been performed on ergosterol biosynthesis in Aspergillus oryzae, a safe filamentous fungus that has been used for the manufacture of oriental fermented foods. This study revealed that the ergosterol biosynthesis pathway is conserved between Saccharomyces cerevisiae and A. oryzae 3.042 by treatment with ergosterol biosynthesis inhibitors and bioinformatics analysis. However, the ergosterol biosynthesis pathway in A. oryzae 3.042 is more complicated than that in S. cerevisiae as there are multiple paralogs encoding the same biosynthetic enzymes. Using RNA-seq, this study identified 138 and 104 differentially expressed genes (DEG) in response to the ergosterol biosynthesis inhibitors tebuconazole and terbinafine, respectively. The results showed that the most common DEGs were transport- and metabolism-related genes. There were only 17 DEGs regulated by both tebuconazole and terbinafine treatments and there were 256 DEGs between tebuconazole and terbinafine treatments. These results provide new information on A. oryzae ergosterol biosynthesis and regulation mechanisms, which may lay the foundation for genetic modification of the ergosterol biosynthesis pathway in A. oryzae. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-018-0026-1) contains supplementary material, which is available to authorized users.

The Asparagaceae family is endemic from America, being the Agave genus the most important. The Agave species possess economic relevance and are use as raw material to produce several distilled alcoholic beverages, as bacanora, tequila, and mezcal. The fermentation process has been carry out either spontaneously or by adding a selected yeast strain. The latter is generally responsible for the production of ethanol and volatile compounds. This study comprised five Agave species (A. angustifolia, A. cupreata, A. durangensis, A. salmiana, and A. tequilana) and eight endogenous yeast strains: five of them were non-Saccharomyces (Torulaspora delbrueckii, Zygosaccharomyces bisporus, Candida ethanolica, and two Kluyveromyces marxianus) and three Saccharomyces cerevisiae strains. The results showed that the S. cerevisiae strains were not able to grow on A. durangensis and A. salmiana juices. The Kluyveromyces marxianus strains grew and fermented all the agave juices and displayed high ethanol production (48–52 g L(−1)) and volatile compounds. The ethanol production was higher on A. angustifolia juice (1.1–2.8-fold), whereas the volatile compound was dependent on both yeast strain and the Agave species. The use of endogenous non-Saccharomyces yeast strains is feasible, as they may outperform S. cerevisiae regarding the production of fermented beverages from agave plants with a high content of ethanol and aromatic compounds. [Figure: see text]

Antibacterial activity of cell-free supernatant from Escherichia coli E against selected pathogenic bacteria in food and aquaculture was the highest against Edwardsiella tarda 3, a significant aquaculture pathogen. Biochemical properties of the bacteriocins were studied and bacteriocin was found to be sensitive to proteinase K, demonstrating its proteinaceous nature. In addition, pH and temperature affected bacteriocin activity and stability. The bacteriocins were partially purified by ammonium sulfate precipitation. The antibacterial activity was only detected in 20% ammonium sulfate fraction and direct detection of its activity was performed by overlaying on the indicator strains. The inhibition zone associated with the antibacterial activity was detected in the sample overlaid by E. tarda 3 and Staphylococcus aureus DMST8840 with the relative molecular mass of about 27 kDa and 10 kDa, respectively. Bacteriocin showed no cytotoxic effect on NIH-3T3 cell line; however, two virulence genes, aer and sfa, were detected in the genome of E. coli E by PCR. The characteristics of bacteriocins produced by E. coli E exhibited the antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria and the safe use determined by cytotoxicity test which may have interesting biotechnological applications. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-018-0014-5) contains supplementary material, which is available to authorized users.

The present work refers to a process involving the use of dilute nitric acid pretreatment and enzymatic hydrolysis for the transformation of rice straw into simple sugars. Acid pre-treated rice straw was separated into the pulp and supernatant through centrifugation and filtration. The two fractions are then converted into simple sugars by combined action of microbes producing cellulase and laccase enzymes. These microbes were isolated from soil samples which were collected from different locations with varying altitudes, expected to harbour microbes with high-hydrolysing activity. The nitric acid pretreatment was carried out at 30 °C, 200 rpm for 72 h. After 72 h, the culture supernatants were analysed for the presence of glucose with the help of HPLC. The supernatant fraction separated after the acid pre-treated rice straw produced highest amount of glucose (205 mg/g of rice straw) upon subsequent hydrolysis with synergistic action of cellulase and laccase-producing microbes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-018-0013-6) contains supplementary material, which is available to authorized users.