Production of kojic acid by Aspergillus flavus OL314748 using box-Behnken statistical design and its antibacterial and anticancer applications using molecular docking technique.

Kojic acid is a wonderful fungal secondary metabolite that has several applications in the food, medical, and agriculture sectors. Many human diseases become resistant to normal antibiotics and normal treatments. We need to search for alternative treatment sources and understand their mode of action. Aspergillus flavus ASU45 (OL314748) was isolated from the caraway rhizosphere as a non-aflatoxin producer and identified genetically using 18S rRNA gene sequencing. After applying the Box-Behnken statistical design to maximize KA production, the production raised from 39.96 to 81.59 g/l utilizing (g/l) glucose 150, yeast extract 5, KH2PO4 1, MgSO4.7H2O 2, and medium pH 3 with a coefficient (R2) of 98.45%. Extracted KA was characterized using FTIR, XRD, and a scanning electron microscope. Crystalized KA was an effective antibacterial agent against six human pathogenic bacteria (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Serratia marcescens, and Serratia plymuthica). KA achieves high inhibition activity against Bacillus cereus, K. pneumonia, and S. plymuthica at 100 µg/ml concentration by 2.75, 2.85, and 2.85 compared with chloramphenicol which gives inhibition zones 1, 1.1, and 1.6, respectively. Crystalized KA had anticancer activity versus three types of cancer cell lines (Mcf-7, HepG2, and Huh7) and demonstrated high cytotoxic capabilities on HepG-2 cells that propose strong antitumor potent of KA versus hepatocellular carcinoma. The antibacterial and anticancer modes of action were illustrated using the molecular docking technique. Crystalized kojic acid from a biological source represented a promising microbial metabolite that could be utilized as an alternative antibacterial and anticancer agent effectively.

New strategies for sterilization and preservation of fresh fish skin grafts.

The introduction of fish skin as a biological dressing for treating burns and wounds holds great promise, offering an alternative to existing management strategies. However, the risk of disease transmission is a significant concern. Therefore, this study aimed to examine how established sterilization and preservation procedures affected fish skin grafts' microbiological and histological properties for long-term usage. Lyophilization of the fish skin graft followed by rehydration in normal saline for 15 min did not change the collagen content. Furthermore, gamma irradiation of the lyophilized fish skin graft at different lengths 5, 10, and 25 KGy showed a significant reduction in microbial growth (aerobic bacteria, aerobic yeasts, and fungi) at 15- and 30 days after the irradiation. However, exposure to 10 KGy was found to be the most effective intensity among the different gamma irradiation lengths since it preserved the collagen fiber content and intensity in the lyophilized fish skin grafts at 15- and 30 days after the irradiation. These findings provide efficient preservation and sterilization methods for long-term usage of the fresh Tilapia skin grafts used for biological dressings.

Responses of Pea (Pisum sativum L.) to Single and Consortium Bio-Fertilizers in Clay and Newly Reclaimed Soils.

The huge development of climatic change highly affects our crop production and soil fertility. Also, the rise in the uncontrolled, excessive use of chemical fertilizers diminishes the soil prosperity and generates pollutants, threatening all environmental life forms, including us. Replacement of these chemical fertilizers with natural ones is becoming an inevitable environmental strategy. In our study, we evaluated the responses of Pisum sativum L. to the action of single species and consortiums of plant growth-promoting bacteria (Azotobacter chroococcum, Bacillus megaterium, and Bacillus cerkularice) in clay and new reclaimed soil types in terms of phenotype, yield components, and physiological and biochemical responses. Data analysis showed single or consortium microbial inoculation significantly increased the measured traits under clay and calcareous sandy soils compared to the control. Shoot physiological and biochemical activities, and seed biochemical activities were significantly enhanced with the inoculation of pea seeds with three types of bacteria in both soil types. The bud numbers, fresh weight, and seeds' dry weight increased in seeds treated with A. chroococcum and B. megaterium in the sandy soil. Taken together, these findings suggested that the inoculation of plants with PGP bacteria could be used to diminish the implementation of chemical fertilizer and improve the goodness of agricultural products. These findings expand the understanding of the responsive mechanism of microbial inoculation under different soil types, especially at physiological and biochemical levels.

Biodiesel Production from the Marine Alga Nannochloropsis oceanica Grown on Yeast Wastewater and the Effect on Its Biochemical Composition and Gene Expression.

Microalgae-based biodiesel synthesis is currently not commercially viable due to the high costs of culture realizations and low lipid yields. The main objective of the current study was to determine the possibility of growing Nannochloropsis oceanica on Saccharomyces cerevisiae yeast wastewater for biodiesel generation at an economical rate. N. oceanica was grown in Guillard F/2 synthetic medium and three dilutions of yeast wastewater (1, 1.25, and 1.5%). Biodiesel properties, in addition to carbohydrate, protein, lipid, dry weight, biomass, lipid productivity, amino acids, and fatty acid methyl ester (FAMEs) content, were analyzed and the quality of the produced biodiesel is assessed. The data revealed the response of N. oceanica to nitrogen-deficiency in the three dilutions of yeast wastewater. N. oceanica in Y2 (1.25%) yeast wastewater dilution exhibited the highest total carbohydrate and lipid percentages (21.19% and 41.97%, respectively), and the highest lipid productivity (52.46 mg L-1 day -1) under nitrogen deficiency in yeast wastewater. The fatty acids profile shows that N. oceanica cultivated in Y2 (1.25%) wastewater dilution provides a significant level of TSFA (47.42%) and can be used as a feedstock for biodiesel synthesis. In addition, N. oceanica responded to nitrogen shortage in wastewater dilutions by upregulating the gene encoding delta-9 fatty acid desaturase (Δ9FAD). As a result, the oleic and palmitoleic acid levels increased in the fatty acid profile of Y2 yeast wastewater dilution, highlighting the increased activity of Δ9FAD enzyme in transforming stearic acid and palmitic acid into oleic acid and palmitoleic acid. This study proved that the Y2 (1.25%) yeast wastewater dilution can be utilized as a growth medium for improving the quantity of specific fatty acids and lipid productivity in N. oceanica that affect biodiesel quality to satisfy global biodiesel requirements.

The optimal concentration of silver nanoparticles in sterilizing fish skin grafts.

Collagen integrity should be considered on using a sterilizing agent for fish skin grafts. This study defined the optimal concentration of silver nanoparticles (Ag NPs) for sterilization of fish skin grafts without disrupting collagen content based on microbiological and histological evaluation. Strips of tilapia skin (n = 5) were randomly allocated to be immersed in Ag NPs solution at different concentrations of 25, 50, 100, and 250 µg/mL, respectively, for 5 min. The treated skin strips underwent bacteriological and histological evaluation. Yeast and fungi were more sensitive to Ag NPs than bacteria. On increasing the nanoparticles concentration, the total counts of aerobic bacteria decrease giving 933.3 ± 28.67, 601 ± 27.66, 288 ± 16.8, 15 ± 4.08 (CFU/cm2 ± S.D) at 25, 50, 100, and 250 µg/mL, respectively, comparing with untreated sample (1453.3 ± 57.92). Yeasts and filamentous fungi also exhibited a similar response, achieving a complete inhibition at 100 and 250 µg/mL. Bacillus cereus and Escherichia coli were the dominant aerobic bacteria, Candida albicans and Rhodotorula glutinis were the dominant aerobic yeasts, whereas Aspergillus niger, Aspergillus fumigatus, and Rhizopus stolonifer were the dominant aerobic fungi. The collagen fibers were loose with a wavey pattern at 25 µg/mL, wavey and slightly disorganized at 50 µg/mL, highly disorganized at 100 µg/mL, and compactly arranged and slightly loose at 250 µg/mL. Ag NPs at a concentration of 250 µg/mL could be considered a reliable and feasible method for the sterilization of fish skin grafts before application on human skin with an effective antimicrobial effect and less disrupting impact on collagen content.

Induction of resilience strategies against biochemical deteriorations prompted by severe cadmium stress in sunflower plant when Trichoderma and bacterial inoculation were used as biofertilizers.

Background: Cadmium (Cd) is a highly toxic heavy metal. Its emission is suspected to be further increased due to the dramatic application of ash to agricultural soils and newly reclaimed ones. Thereby, Cd stress encountered by plants will exacerbate. Acute and chronic exposure to Cd can upset plant growth and development and ultimately causes plant death. Microorganisms as agriculturally important biofertilizers have constantly been arising as eco-friendly practices owing to their ability to built-in durability and adaptability mechanisms of plants. However, applying microbes as a biofertilizer agent necessitates the elucidation of the different mechanisms of microbe protection and stabilization of plants against toxic elements in the soil. A greenhouse experiment was performed using Trichoderma harzianum and plant growth-promoting (PGP) bacteria (Azotobacter chroococcum and Bacillus subtilis) individually and integrally to differentiate their potentiality in underpinning various resilience mechanisms versus various Cd levels (0, 50, 100, and 150 mg/kg of soil). Microorganisms were analyzed for Cd tolerance and biosorption capacity, indoleacetic acid production, and phosphate and potassium solubilization in vitro. Plant growth parameters, water relations, physiological and biochemical analysis, stress markers and membrane damage traits, and nutritional composition were estimated. Results: Unequivocal inversion from a state of downregulation to upregulation was distinct under microbial inoculations. Inoculating soil with T. harzianum and PGPB markedly enhanced the plant parameters under Cd stress (150 mg/kg) compared with control plants by 4.9% and 13.9%, 5.6% and 11.1%, 55.6% and 5.7%, and 9.1% and 4.6% for plant fresh weight, dry weight, net assimilation rate, and transpiration rate, respectively; by 2.3% and 34.9%, 26.3% and 69.0%, 26.3% and 232.4%, 135.3% and 446.2%, 500% and 95.6%, and 60% and 300% for some metabolites such as starch, amino acids, phenolics, flavonoids, anthocyanin, and proline, respectively; by 134.0% and 604.6% for antioxidants including reduced glutathione; and by 64.8% and 91.2%, 21.9% and 72.7%, and 76.7% and 166.7% for enzymes activity including ascorbate peroxidase, glutathione peroxidase, and phenylalanine ammonia-lyase, respectively. Whereas a hampering effect mediated by PGP bacterial inoculation was registered on levels of superoxide anion, hydroxyl radical, electrolyte leakage, and polyphenol oxidase activity, with a decrease of 0.53%, 14.12%, 2.70%, and 5.70%, respectively, under a highest Cd level (150 mg/kg) compared with control plants. The available soil and plant Cd concentrations were decreased by 11.5% and 47.5%, and 3.8% and 45.0% with T. harzianum and PGP bacterial inoculation, respectively, compared with non-inoculated Cd-stressed plants. Whereas, non-significant alternation in antioxidant capacity of sunflower mediated by T. harzianum action even with elevated soil Cd concentrations indicates stable oxidative status. The uptake of nutrients, viz., K, Ca, Mg, Fe, nitrate, and phosphorus, was interestingly increased (34.0, 4.4, 3.3, 9.2, 30.0, and 1.0 mg/g dry weight, respectively) owing to the synergic inoculation in the presence of 150 mg of Cd/kg. Conclusions: However, strategies of microbe-induced resilience are largely exclusive and divergent. Biofertilizing potential of T. harzianum showed that, owing to its Cd biosorption capability, a resilience strategy was induced via reducing Cd bioavailability to be in the range that turned its effect from toxicity to essentiality posing well-known low-dose stimulation phenomena (hormetic effect), whereas using Azotobacter chroococcum and Bacillus subtilis, owing to their PGP traits, manifested a resilience strategy by neutralizing the potential side effects of Cd toxicity. The synergistic use of fungi and bacteria proved the highest efficiency in imparting sunflower adaptability under Cd stress.

Using fermentation waste of ethanol-producing yeast for bacterial riboflavin production and recycling of spent bacterial mass for enhancing the growth of oily plants.

AIM: This study aims to use fermentation waste of ethanol production (solid and liquid) for riboflavin and recycling of bacterial biomass as biofertilizers to enhance the growth of some oily crop plants. METHODS AND RESULTS: Out of 10 yeast isolates from fresh milk, Clavispora lusitaniae ASU 33 (MN583181) was able to ferment different concentrations of glucose (2.5%, 5%, 7.5%, 10%, 15% and 20%) into ethanol with high efficiency at 10%. Among seven non-Lactobacillus bacterial isolates recovered from cheese samples, two bacterial isolates Bacillus subtlis-SR2 (MT002768) and Novosphingobium panipatense-SR3 (MT002778) were selected for their high riboflavin production. Different media (control medium, fermentation waste medium and a mixture of the fermentation waste medium and control medium [1:1]) were used for riboflavin production. These media were inoculated by a single or mixture of B. subtlis-SR2, N. panipatense-SR3. The addition of the waste medium of ethanol production to the control medium (1:1) had a stimulatory effect on riboflavin production whether inoculated with either a single strain or a mixture of B. subtlis-SR2 and N. panipatense-SR3. A mixture of fermentation waste and control media inoculated with N. panipatense produced a high riboflavin yield in comparison with other media. Inoculation of Zea mays and Ocimum basilicum plants with either the bacterial biomass waste of riboflavin production (B. subtlis or N. panipatense) or a mixture of B. subtlis and N. panipatense) shows a stimulatory effect on the plant growth in comparison with control (uninoculated plants). CONCLUSIONS: These results demonstrate the possibility of minimizing the cost of riboflavin and biofertilizer manufacturing via interlinking ethanol and riboflavin with the biofertilizer production technology. SIGNIFICANCE AND IMPACT OF STUDY: This study outlines the methods of evaluating the strength of spent media by applying procedures developed in the vitamin production industries. Furthermore, bacterial biomass waste can act as an environmentally friendly alternative for agrochemicals.

Controlling Alternaria cerealis MT808477 Tomato Phytopathogen by Trichoderma harzianum and Tracking the Plant Physiological Changes.

Plant responses during the pathogen infection and the pathogen control reflect its strategies to protect its cells. This work represents the Alternaria cerealis MT808477 as a phytopathogen causing leaf spot disease in tomatoes. A. cerealis was identified morphologically and genetically by 18SrRNA, and its pathogenicity was confirmed by light and scanning electron microscopy. Trichoderma harzianum has the ability to control A. cerealis MT808477 by stimulating various cell responses during the controlling process. The cell behavior during the biological control process was observed by analyses of total phenol, flavonoids, terpenoids, antioxidant, malondialdehyde and antioxidant enzymes (catalase and peroxidase). The extracts of infected tomato leaves were tested against plant and human pathogenic microorganisms. Results showed that the biological control process activates the defense cell strategies by increasing the plant tolerance, and activation of plant defense systems. The total phenol, flavonoids, terpenoids, antioxidant and malondialdehyde were increased after 48 h. Catalase and peroxidase were increased in infected tomato plants and decreased during the biological control process, reflecting the decrease of cell stress. Leaves extract inhibited the growth of nine plant and human pathogenic microorganisms. Biological control represents a safe and effective solution to phytopathogens that decreases plant cell stress by stimulating various defensive agents.

Safe natural bio-pigment production by Monascus purpureus using mixed carbon sources with cytotoxicity evaluation on root tips of Allium cepa L.

By increasing the undesirable side effects of synthetic food pigments on human health, using safe natural food pigment become an urgent issue. Incorporate corn starch with oils conducted a high impact on red pigment production by Monascus purpureus. Fortification the medium with sesame oil raised the pigment production by 80% and the dry mass by 63% compared with free oil medium. Response surface methodology maximizes the production with 114.6% (12.8 A500) using medium constituents (g/l); Sesame oil 5; Corn starch 30; Yeast extract 1.5; KH2PO4 2.5 and MgSO4.7H2O 0.1. After evaluating red pigment stability in three common food components, citric acid showed a great effect on residual stability percentage compared with ascorbic and salicylic acid which decrease slightly the residual stability percentage at light and dark conditions. The mitotic index of red pigment was lower than the negative control at all tested concentrations. Different types of mitotic chromosomal abnormalities e.g. lagging chromosome, chromosomal bridge, chromosome and chromatin fragments, outside chromosome, chromosomal stickiness and micro nuclei were recorded. Insignificant increase in total mitotic aberrations percentage in all tested root tips treated with all concentrations of red pigment (1.23, 1.58, 1.63, 2.32 and 2.40%) compared with negative control (0.91%). There was a significant increase in total aberrations percentage after treatment with all concentrations (10, 15, 20 and 25%) of positive control (2.93, 3.00, 3.55 and 6.53 respectively) except (5%) which was insignificant (2.71%). From the previous data, this red pigment can be used as an alternative safe pigment in the food industry.

Nitrogen, Amino Acids, and Carbon as Control Factors of Riboflavin Production by Novosphingobium panipatense-SR3 (MT002778).

By increasing the environmental pollution, crop losses, and side effects of chemically synthesized vitamins; new vitamin sources should be included. Through this study, we introduce novel riboflavin bacterial producer Novosphingobium panipatense-SR3 (MT002778) and tested various nutritional factors with interactions effects on the production abilities. Yeast extract, maltose, and glycine were the best nitrogen, carbon, and amino acid sources for enhancing the production, respectively. The interaction between the previous factors with three concentrations of each (+, 0, -) studied statistically using Box-Behnken statistical quadric design 13- run. The perfect interaction increases the production to 497.12 mg/l (predicted 489.45 mg/l) using 30 g/l maltose, 10 g/l yeast extract, and 1 g/l glycine. The F and P- values of the tested model of riboflavin and OD600 indicating significant results with probability ≤ 0.05. Also, the evaluating statistical parameter coefficient (R2) was 0.994 of riboflavin and 0.992 of OD600 with adjusted R2 value 0.976, and 0.967, respectively, which indicated that the whole variations were explained highly by the statistical model. The novel producer proved its high riboflavin production ability especially under the optimized conditions comparing with previous producers and represents a new high-speed riboflavin producer that could utilize in the industrial process.

Correction: A cerium-based MOFzyme with multi-enzyme-like activity for the disruption and inhibition of fungal recolonization.

Correction for 'A cerium-based MOFzyme with multi-enzyme-like activity for the disruption and inhibition of fungal recolonization' by Hani Nasser Abdelhamid et al., J. Mater. Chem. B, 2020, DOI: 10.1039/d0tb00894j.

A cerium-based MOFzyme with multi-enzyme-like activity for the disruption and inhibition of fungal recolonization.

A cerium-based metal-organic framework (Ce-MOF, denoted as AU-1) was synthesized using a solvothermal method by employing 4,4',4''-nitrilotribenzoic acid (H3NTB) as the linker and cerium clusters as the metal center. The material was considered as a MOFzyme based on the peroxidase-like activity of Ce-MOF that could eliminate and kill fungi, such as Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Candida albicans, and Rhodotorula glutinis. Ce-MOF showed high antifungal activity against airborne opportunistic human pathogens isolated from the outside of a hospital. The antifungal activity of CeMOF was evaluated using the colony-forming units, dry mass method, soluble proteins, and microscopic imaging. It exhibited an inhibition efficiency of 93.3-99.3% based on the colony-forming unit method. The Ce-MOF caused extensive deformation of the conidiophores, vesicles, and phialides with growth inhibition between 7.55-77.41% (based on the dry mass method). Ce-MOF showed different efficiencies in inhibiting the different fungal species. The biological activity of Ce-MOF was due to its enzymatic activity, such as those of antioxidants, catalase, superoxide dismutase, and peroxidase. Ce-MOF exhibited excellent enzymatic activity towards the fungal cells. Our results may facilitate the design of a MOFzyme system and pave the way for more profound applications of nanozymes.

Zn(II) and Cd(II) thiosemicarbazones for stimulation/inhibition of kojic acid biosynthesis from Aspergillus flavus and the fungal defense behavior against the metal complexes' excesses.

The complexes {[ZnL1Cl] C1, [ZnL2Cl].0.5H2O C2, [CdL1Cl] C3, and [CdL2Cl] C4} were prepared from tridentate thiosemicarbazones {HL1 = 4-(3-nitrophenyl)-1-((pyridin-2-yl)methylene) thiosemicarbazide and HL2 = 4-(2,4-dimethoxyphenyl)-1-((pyridin-2-yl)methylene)thiosemicarbazide} and identified by elemental CHNS, spectroscopic {IR and UV-Vis.}, thermal and DMF solution electrical conductivity data. On another hand, kojic acid (KA) which represents important secondary metabolite with numerous hot spot applications was successfully biosynthesized from Aspergillus flavus and structurally analyzed by single crystal analysis. The Zn(II) complexes C1&C2 (0.3 mM) enhanced the KA biosynthesis by 70.87% and 42.26%, while 76.09% of C1 and 72.78% of C2 were absorbed by the fungal cells. The Cd(II) complexes C3&C4 at 0.3 mM inhibited KA production by 87.95% and 97.03% with Cd(II) consumption reaching to 40.09% & 37.3%, while 0.4 mM of C3&C4 resulted in 100% inhibition of kojic acid biosynthesis. Light microscopic analysis showed the fungal structural abnormalities and the cell antioxidant behavior was detected. These complexes could be highly applicable as new stimulators and inhibitors of kojic acid production.

Evaluation and Microanalysis of Parasitic and Bacterial Agents of Egyptian Fresh Sushi, Salmo salar.

The present study aimed to evaluate the quality of fresh sushi in Egypt. Fifty samples of sushi (Salmo salar) were collected from restaurants in Alexandria, Egypt. Paraffin, semi-thin and ultra-thin sections were used for parasitological analysis by light and transmission electron microscopy. Bacteria were isolated by the dilution plate and direct plate methods and identified by a Vitek system. Twenty (40%) of the total examined samples showed microsporidia and helminth metacercariae infections. Histochemical stains showed distinct pinkish-red pyriform microspores embedded in muscular tissue stained with Gram, periodic acid-Schiff (PAS), and Ziehl-Neelsen (ZN) stains. Semi-thin sections showed double membrane xenoma-inducing granulomas containing spores at different developmental stages. Empty sporophorous vesicles and free spores were observed in the electron microscopic images. A bacteriological assay showed forty samples (80%) contaminated with human pathogenic bacteria with the average total bacterial counts ranging from 32 to 526 CFU/g. Four species of human pathogenic bacteria were identified in the examined samples, namely Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Serratia plymuthica in 40, 38, 11, and 6 samples, respectively. These constitute the first record of fresh sushi product in Egypt and indicate the potential pathogenicity associated with raw seafood products.