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Phellinus Quél is one of the largest genera of Hymenochaetaceae; it comprises about 220 species widely distributed on Earth. Most Phellinus species are lignicolous mushrooms that accumulate bioactive compounds. This research studied the phenolic composition of Phellinus spp. and their relationship with antibacterial and antiviral capacity. Phenolics were extracted from Phellinus badius, P. fastuosus, and P. grenadensis; their antiviral and antibacterial activities were evaluated against Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157: H7; and the bacteriophages MS2 and Φ- × 174. Gallic acid, chlorogenic acid, caffeic acid, epicatechin, ferulic acid, catechin, 1,3-dicaffeoylquinic acid, p-coumaric acid, and rutin were found in different proportions among Phellinus spp. Total phenolic content ranged from 96 to 209 mg GAE/g, and total flavonoids from 10 to 27 QE/g. The minimum inhibitory concentrations of P. badius, P. grenadensis, and P. fastuosus against E. coli O157: H7 were 13, 20, and 27 mg/mL, against S. enterica were 20, 30, and 15 mg/mL, and against L. monocytogenes were 10, 15, and 25 mg/mL, respectively. The phenolic content was better correlated with the antibacterial effect against E. coli O157: H7 and L. monocytogenes (r = 0.8–0.9), but not against S. enterica (r = 0.05). The antiviral activity of the extracts (0.9 mg/mL) was 29 to 41% against MS2 and 27 to 38% for Φ-X174 virus (r = 0.8–0.9). In silico analysis showed binding energy values of − 7.9 and − 4.8 kcal/mol between the identified phenolic compounds and the M and G proteins of each virus. The antibacterial and antiviral properties of Phellinus species were correlated with the phenolic content.

High proteolytic activity and several biological functions (antimicrobial, antioxidant, antihypertensive, among others) have been attributed to lactic-acid bacteria (LAB) isolated from fish and peptides obtained from proteolysis. Therefore, the objective of this research was isolating, characterizing, and identifying LAB with proteolytic activity by spontaneous fermentation from common carp (Cyprinus carpio) reared in ponds and wild ones obtained from Lago de Chapala, Jalisco, Mexico. Spontaneous fermentation from complete carp specimens was observed, considering two sampling points (skin and intestines) at 15 °C at 5 and 10 days. Isolated LAB—from both reared and wild specimens—were identified and morphologically characterized; identification was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Proteolytic activity was assessed by the presence of the proteolytic halo. A total of five genera and eight different LAB proteolytic species were isolated from all the carp samples. At 10 days, greater proteolytic LAB diversity was obtained from the intestine (Tukey’s, p

Cordyceps acid is an active component of Cordyceps cicadae and has a variety of medicinal uses, including anti-tumor effects, the prevention of cerebral hemorrhaging and myocardial infarction, and the inhibition of a wide range of bacteria. The objectives of this study were to identify C. cicadae fungi and optimize the culture conditions to obtain a high yield of cordycepic acid. First, a wild C. cicadae was identified by morphological observation and rDNA sequence analysis. Secondly, the optimal fermentation conditions were determined using a single-factor method, a Plackett–Burman design, and a Box–Behnken response surface. Finally, using the yield of fruit bodies and the content of cordyceps acid as indices, combined with a single-factor experiment and a response surface design, the best combination of conditions for cultivation was determined. The results showed that the best combination was as follows: sucrose 2%, tryptone 2%, KH(2)PO(4) 0.4%, MgSO(4)·7H(2)O 0.4%, an initial pH of the fermentation liquid of 7.0, 5% inoculum, fermentation for 4.5 d, a ratio of medium to liquid of 1:1.7, illumination intensity 150 Lux, illumination time 15 h per day, and 70% humidity. The content of cordycepic acid in the fruiting bodies developed in cultivation was 2.07-fold higher than that in the wild C. cicadae. This study provides a theoretical basis for the large-scale cultivation of C. cicadae with a high concentration of cordycepic acid. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-022-00687-4.

Klebsiella variicola is generally known as endophyte as well as lignocellulose-degrading strain. However, their roles in goat omasum along with lignocellulolytic genetic repertoire are not yet explored. In this study, five different pectin-degrading bacteria were isolated from a healthy goat omasum. Among them, a new Klebsiella variicola strain HSTU-AAM51 was identified to degrade lignocellulose. The genome of the HSTU-AAM51 strain comprised 5,564,045 bp with a GC content of 57.2% and 5312 coding sequences. The comparison of housekeeping genes (16S rRNA, TonB, gyrase B, RecA) and whole-genome sequence (ANI, pangenome, synteny, DNA-DNA hybridization) revealed that the strain HSTU-AAM51 was clustered with Klebsiella variicola strains, but the HSTU-AAM51 strain was markedly deviated. It consisted of seventeen cellulases (GH1, GH3, GH4, GH5, GH13), fourteen beta-glucosidase (2GH3, 7GH4, 4GH1), two glucosidase, and one pullulanase genes. The strain secreted cellulase, pectinase, and xylanase, lignin peroxidase approximately 76–78 U/mL and 57–60 U/mL, respectively, when it was cultured on banana pseudostem for 96 h. The catalytically important residues of extracellular cellulase, xylanase, mannanase, pectinase, chitinase, and tannase proteins (validated 3D model) were bound to their specific ligands. Besides, genes involved in the benzoate and phenylacetate catabolic pathways as well as laccase and DiP-type peroxidase were annotated, which indicated the strain lignin-degrading potentiality. This study revealed a new K. variicola bacterium from goat omasum which harbored lignin and cellulolytic enzymes that could be utilized for the production of bioethanol from lignocelluloses. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00660-7.

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.

Paenibacillus elgii AC13 produces antimicrobial lipopeptides of agricultural and pharmaceutical importance. It secretes four cyclic lipopeptides named pelgipeptins, previously characterized in P. elgii B69. These lipopeptides result from the expression of a nonribosomal peptide gene cluster. P. elgii AC13 also produced two linear lipopeptides with ratios of [M + H] + 1105 and 1119 m/z. These compounds were previously observed in Paenibacillus sp. strain OSY-N, but due to purification difficulties, their characterization was executed using synthetically produced linear pelgipeptins. In the present study, purification was achieved from the supernatants of cultures from three complex media by high-performance liquid chromatography. The partial characterization of linear pelgipeptins revealed the similar antimicrobial activity and cytotoxicity of their synthetically produced counterparts, known as paenipeptins. Cyclic forms were highly stable to changes in pH, temperature, and organic extraction with n-butanol as shown by mass spectrometry (MALDI-TOF); therefore, these steps did not cause the hydrolysis of pelgipeptins. A low-activity thioesterase could also generate the linear isoforms observed; this enzyme catalyzes the cyclization process and is coded in the same gene cluster. Alternatively, the cyclic forms were hydrolyzed by an unknown protease produced during growth in the complex medium used in the present study. Although culture conditions are known to produce pelgipeptins with different yields and amino acid compositions, the occurrence of linear and cyclic forms simultaneously has not yet been reported. A mixture of cyclic and linear pelgipeptins presents a potential advantage of the higher antimicrobial activity of cyclic forms combined with the lower cytotoxicity of linear isoforms. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00597-x.

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.

Recombinant granulocyte colony–stimulating factor (G-CSF) protein produced in Escherichia coli has been widely used for the treatment of neutropenia induced by chemotherapy for decades. In E. coli cells, G-CSF is usually expressed as inactive inclusion bodies, which requires costly and inefficient denaturation and refolding steps to obtain the protein in its active form. However, following the findings of previous studies, we here successfully produced G-CSF in E. coli as non-classical inclusion bodies (ncIBs), which contained likely correctly folded protein. The ncIBs were easily dissolved in 0.2% N-lauroylsarcosine solution and then directly applied to a Ni-NTA affinity chromatography column to get G-CSF with high purity (> 90%). The obtained G-CSF was demonstrated to have a similar bioactivity with the well-known G-CSF containing product Neupogen (Amgen, Switzerland). Our finding clearly verified that the G-CSF production from ncIBs is a feasible approach to improve the yield and lower the cost of G-CSF manufacturing process.

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.

Fungal secondary metabolites with antimicrobial properties are used for biological pest control. Their production is influenced by several factors as environment, host, and culture conditions. In the present work, the secondary metabolites from fermented extracts of Beauveria bassiana PQ2 were tested as antifungal agents against Gibberella moniliformis LIA. The L(18) (2(1) × 3(7)) orthogonal array from Taguchi methodology was used to assess 8 parameters (pH, agitation, sucrose, yeast extract, KH(2)PO(4), MgSO(4), NH(4)NO(3), and CaCl(2)) in B. bassiana PQ2 submerged fermentation. The ability of the fermented extracts to slow down the growth rate of G. moniliformis LIA was evaluated. The results from 18 trials were analyzed by Statistica 7 software by evaluating the signal-to-noise ratio (S/N) to find the lower-the-better condition. Optimal culture conditions were pH, 5; agitation, 250 rpm; sucrose, 37.5 g/L(−1); yeast extract, 10 g/L(−1); KH(2)PO(4), 0.8 g/L(−1); MgSO(4), 1.2 g/L(−1); NH(4)NO(3), 0.1 g/L(−1); and CaCl(2), 0.4 g/L(−1), being the agitation at the highest level the most significant factor. The optimal conditions were validated in a sparged bottle bioreactor resulting in a higher S/N value (12.48) compared to the estimate. The extract obtained has the capacity to inhibit the germination of G. moniliformis spores at 24 h. HPLC-ESI-MS(2) allowed to identify the water-soluble red pigment as oosporein (m/z 304.9). The secondary metabolites from B. bassiana PQ2 are a suitable alternative to control the growth and sporulation of G. moniliformis.

The use of lactic acid bacteria (LAB) and probiotic cultures in the breeding of animals such as poultry and swine are quite common. It is known that those strains can produce bacteriocins when grown in pure culture. However, the production of bacteriocin using co-culture of microorganisms has not been much studied so far. The present study contributes with innovation in this area by embracing the production of bacteriocin-like inhibitory substances (BLIS) by a newly isolated strain of Enterococcus faecium 135. Additionally, the co-cultivation of this strain with Ligilactobacillus salivarius and Limosilactobacillus reuteri was also investigated. The antimicrobial activity of the produced BLIS was evaluated against Listeria monocytogenes, Listeria innocua, Salmonella enterica, and Salmonella enterica serovar Typhimurium using two methods: turbidimetric and agar diffusion. In addition, the presence of enterocin genes was also evaluated. The BLIS produced showed a bacteriostatic effect against the bio-indicator strains, and the highest antimicrobial activities expressed by arbitrary units per mL (AU/mL) were obtained against L. monocytogenes in monoculture (12,800 AU/mL), followed by the co-culture of E. faecium with Limosilactobacillus reuteri (400 AU/mL). After concentration with ammonium sulfate, the antimicrobial activity raised to 25,600 AU/mL. Assays to determine the proteinaceous nature of the BLIS showed susceptibility to trypsin and antimicrobial activity until 90 °C. Finally, analysis of the presence of structural genes of enterocins revealed that four enterocin genes were present in E. faecium 135. These results suggest that BLIS produced by E. faecium 135 has potential to be a bacteriocin and, after purification, could potentially be used as an antimicrobial agent in animal breeding.

In a bioprospecting study of paramo soils cultivated with potato (Solanum tuberosum), 50 fungal isolates were obtained and evaluated for their nitrate reductase (NR) activity, given the role played by this enzyme in the biosynthesis of silver nanoparticles (AgNps). Five isolates strain with high NR activity belonging to Penicillium simplicissimum, Aspergillus niger, and Fusarium oxysporum species were selected, verifying the presence of the NR enzyme in their enzymatic extract. Later, these strains showed the ability to biosynthesize AgNps with distorted spherical shapes and sizes ranging from 15 to 45 nm. Subsequently, an antibiosis test was carried out by the agar diffusion method using glass fiber disks against the phytopathogenic agent Pectobacterium carotovorum, finding halos of inhibition of bacterial growth up to 15.3 mm using a 100 ppm solution of the AgNps obtained from F. oxysporum. These results contribute to generating the basis of a new alternative for the control of this phytopathogenic agent of potato, challenging to manage by traditional methods and of relevance at the post-harvest level.

Purified endoxylanase from Thermomyces lanuginosus PC7S1T was immobilized in calcium alginate, resulting in a yield of 78.5% and a reusability for 11 cycles. The stability of the immobilized enzyme was given for a pH range of 4 to 9 for 96 h. Endoxylanase immobilized in calcium alginate at 65 °C exhibited thermal stability equal to the soluble enzyme for 5 h, and at high temperatures of 75 °C and 85 °C showed half-lives of 4 and 3 h, respectively. Both soluble endoxylanase and immobilized forms were able to hydrolyze hemicellulose, obtained from low-lignin sorghum biomass pretreated with 5% H(2)O(2) and 2% NaOH, after 1 h of incubation at 65 °C, releasing a mixture of short-chain xylooligosaccharides (X2–X6). The highest amounts of XOS generated were those for X5 (24 to 40%), X4 (33 to 39%), and X3 (11 to 22%). These XOS acted as prebiotics, promoting the growth of the probiotic L. acidophilus, similar to glucose in the MRS broth. These results show the potential of low-lignin sorghum to generate XOS with prebiotic activity, suggesting the application of these compounds in the food industry.

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.

Bombyx mori gloverin A2 (BMGlvA2) is an induced antimicrobial insect protein isolated from Bombyx mori. This study was conducted to explore the effect and potential mechanisms of BMGlvA2 on inflammatory responses and cellular functions in intestinal epithelial cells (IPEC-J2) exposure to enterotoxigenic E. coli (ETEC). IPEC-J2 cells pretreated with or without BMGlvA2 (12.5 μg/mL) were challenged by ETEC K88 (1×10(6) CFU/well) or culture medium. We show that BMGlvA2 pretreatment increased the cell viability and improved the distribution and abundance of tight junction protein ZO-1 in IPEC-J2 cells exposure to ETEC (P

Made available in DSpace on 2021-06-25T11:12:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Derived compounds from lignin have been used as substrates for chemical and biological processes for obtainment bioproducts. The ferulic acid is a lignocellulosic biomass whose biotransformation in flavors compounds was described. The objective of this study was the bioconversion of ferulic acid to 4-vinylguaiacol by Klebsiella pneumoniae TD 4.7. The biotransformation of commercial ferulic acid into 4-vinylguaiacol in a semi synthetic liquid medium containing the ferulic acid at an initial concentration of 300 mg L−1 reached 32.4%. The ferulic acid obtained from alkaline hydrolysis of the sugar cane bagasse at 300 mg L−1 allowed the yield of 1.3 mmol L−1 of 4-vinylguaiacol, corresponding to 81.7% of the ferulic acid content. The data indicated that the bacterial strain decarboxylated the ferulic acid to 4-vinylguaiacol and the presence of an active cell associated ferulic acid decarboxylase. The enzyme showed maximum activity at pH 5.5 and 40 °C and was stable at pH range 4.5 to 9.0 and temperature up 20 to 45 °C. According to these biochemical properties and performance to bioconversion of ferulic acid to 4-vinylguaiacol, this enzyme could be viable for application in food industry. Department of Biology São Paulo State University-UNESP Institute of Biosciences Humanities and Exact Sciences Department of Chemistry and Environment Science São Paulo State University-UNESP Institute of Biosciences Humanities and Exact Sciences Department of Biology São Paulo State University-UNESP Institute of Biosciences Humanities and Exact Sciences Department of Chemistry and Environment Science São Paulo State University-UNESP Institute of Biosciences Humanities and Exact Sciences

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.

In the present study, we synthesis nanoparticles using biosynthesis methods because of the eco-friendly approach. Gold nanoparticles were synthesized using extracellular metabolites of marine bacteria (Rastrelliger kanagurta, Selachimorpha sp., and Panna microdon). After the synthesis gold nanoparticles checked their antibacterial and antimycobacterial activities. Here we have few techniques that have been used for characterizing the gold nanoparticles followed by ultraviolet (UV)-visible spectrophotometer analysis, Fourier transform-infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), and transmission electron microscopy (TEM). We observed the formation of gold nanoparticles using UV-Vis spectroscopy (UV-Vis). FT-IR spectroscopy results of the extracellular metabolites showed that different characteristic functional groups are responsible for the bioreduction of gold ions. In the recent years, we used zebrafish for an animal model to estimate nanoparticle toxicity and biocompatibility. We tested toxicity of the gold nanoparticle using the zebrafish larvae that are growing exponentially. Sample 1 showed a good antimicrobial activity, and sample 5 showed a good antimycobacterial activity. Based on the UV spectrophotometer, sample 1 is used for further studies. Color change and UV spectrum confirmed gold nanoparticles. Based on the TEM and SEM particles, size was measured and ranged between 80 and 45 nm, and most of the particles are spherical and are in rod shape. XRD result showed the gold nanoparticles with crystalline nature. Toxicity studies in the zebrafish larvae showed that 50 μg ml(−1) showed less toxicity. Based on the studies, gold nanoparticle has good antibacterial and antimycobacterial activities. The present was concluded that gold nanoparticles have potential biocompatibility and less toxicity. Gold nanoparticles will be used as a drug molecule in pharmaceutical company and biomedicine application.

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.

White-rot basidiomycetes such as Lentinus crinitus produce laccases with potential use in dye biodegradation. However, high productivity and enzymes with specific properties are required in order to make viable laccase production. We aimed to produce laccase from Lentinus crinitus grown in sugarcane bagasse for dye decolorization. Solid state cultivation medium had sugarcane bagasse added with a nutrient solution of 10 g/L glucose, 1 g/L KH(2)PO(4), 0.5 g/L MgSO(4), 0.001 g/L FeSO(4), 0.01 g/L ZnSO(4), and 0.01 g/L MnSO(4). The addition of different nitrogen sources (peptone, urea, or peptone plus urea) and different nitrogen concentrations (0, 0.4, 0.6, 0.8, 1.0, and 1.2 g/L) were evaluated. Enzymatic extract was used in the decolorization of azo dyes, reactive blue 220 (RB220) and reactive black 5 (RB5), and anthraquinone dye, Remazol brilliant blue R (RBBR). The greatest laccase activity (4800 U/g dry mass) occurred when the peptone and urea mixture was added to the solid state cultivation medium. When the nitrogen concentration was 1 g/L, the laccase activity increased to 6555 U/g dry mass. The laccase activity peak occurred on the 10th day, and the maximum decolorization within 24 h was observed with enzymatic extracts obtained on different cultivation days, i.e., 6th day for RB220, 10th day for RB5, and 9th day for RBBR. Manganese and lignin peroxidases were not produced when nitrogen was added to the cultivation medium. The crude enzymatic extract was more effective in the decolorization of azo dyes (RB220 and RB5), more than 90% of decolorization, than anthraquinone dye with 77% decolorization.

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.

l-asparaginase II (ASNase) is the biopharmaceutical of choice for the treatment of acute lymphoblastic leukaemia. In this study, E. coli BL21 (DE3) transformed with the pET15b + asnB vector which expresses recombinant ASNase was used as a source to obtain this enzyme. The ideal conditions to produce ASNase would be a high level of secretion into the extracellular medium, which depends not only on the application of molecular biology techniques but also on the development of a strategy to modify cell permeability such as the addition of substances to the culture medium that stimulate destabilisation of structural components of the cell. Thus, the growth of E. coli BL21 (DE3) in modified Luria–Bertani broth, supplemented with 0.8% (w/v) glycine and 6% (v/v) n-dodecane, increased the total yield of ASNase by about 50% (15,108 IU L(−1)) and resulted in a 16-fold increase in extracellular enzymatic productivity (484 IU L(−1) h(−1)), compared to production using the same medium without addition of these substances. Most of the enzyme (89%) was secreted into the culture medium 24 h after the induction step. This proposed approach presents a simple strategy to increase extracellular production of ASNase in E. coli.

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.