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.

Kinetic studies on optimized extracellular laccase from Trichoderma harzianum PP389612 and its capabilities for azo dye removal.

BACKGROUND: Azo dyes represent a common textile dye preferred for its high stability on fabrics in various harsh conditions. Although these dyes pose high-risk levels for all biological forms, fungal laccase is known as a green catalyst for its ability to oxidize numerous dyes. METHODS: Trichoderma isolates were identified and tested for laccase production. Laccase production was optimized using Plackett-Burman Design. Laccase molecular weight and the kinetic properties of the enzyme, including Km and Vmax, pH, temperature, and ionic strength, were detected. Azo dye removal efficiency by laccase enzyme was detected for Congo red, methylene blue, and methyl orange. RESULTS: Eight out of nine Trichoderma isolates were laccase producers. Laccase production efficiency was optimized by the superior strain T. harzianum PP389612, increasing production from 1.6 to 2.89 U/ml. In SDS-PAGE, purified laccases appear as a single protein band with a molecular weight of 41.00 kDa. Km and Vmax values were 146.12 µmol guaiacol and 3.82 µmol guaiacol/min. Its activity was stable in the pH range of 5-7, with an optimum temperature range of 40 to 50 °C, optimum ionic strength of 50 mM NaCl, and thermostability properties up to 90 °C. The decolorization efficiency of laccase was increased by increasing the time and reached its maximum after 72 h. The highest efficiency was achieved in Congo red decolorization, which reached 99% after 72 h, followed by methylene blue at 72%, while methyl orange decolorization efficiency was 68.5%. CONCLUSION: Trichoderma laccase can be used as an effective natural bio-agent for dye removal because it is stable and removes colors very well.

Enhancement of ß-Glucan Biological Activity Using a Modified Acid-Base Extraction Method from Saccharomyces cerevisiae.

Beta glucan (ß-glucan) has promising bioactive properties. Consequently, the use of ß-glucan as a food additive is favored with the dual-purpose potential of increasing the fiber content of food products and enhancing their health properties. Our aim was to evaluate the biological activity of ß-glucan (antimicrobial, antitoxic, immunostimulatory, and anticancer) extracted from Saccharomyces cerevisiae using a modified acid-base extraction method. The results demonstrated that a modified acid-base extraction method gives a higher biological efficacy of ß-glucan than in the water extraction method. Using 0.5 mg dry weight of acid-base extracted ß-glucan (AB extracted) not only succeeded in removing 100% of aflatoxins, but also had a promising antimicrobial activity against multidrug-resistant bacteria, fungi, and yeast, with minimum inhibitory concentrations (MIC) of 0.39 and 0.19 mg/mL in the case of resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, respectively. In addition, AB extract exhibited a positive immunomodulatory effect, mediated through the high induction of TNFα, IL-6, IFN-γ, and IL-2. Moreover, AB extract showed a greater anticancer effect against A549, MDA-MB-232, and HepG-2 cells compared to WI-38 cells, at high concentrations. By studying the cell death mechanism using flow-cytometry, AB extract was shown to induce apoptotic cell death at higher concentrations, as in the case of MDA-MB-231 and HePG-2 cells. In conclusion, the use of a modified AB for ß-glucan from Saccharomyces cerevisiae exerted a promising antimicrobial, immunomodulatory efficacy, and anti-cancer potential. Future research should focus on evaluating ß-glucan in various biological systems and elucidating the underlying mechanism of action.

Exploring the antibacterial potential of plant extracts and essential oils against Bacillus thermophilus in beet sugar for enhanced sucrose retention: a comparative assessment and implications.

Sugar beet is one of the greatest sources for producing sugar worldwide. However, a group of bacteria grows on beets during the storage process, leading to a reduction in sucrose yield. Our study focused on identifying common bacterial species that grow on beets during manufacturing and contribute to sucrose loss. The ultimate goal was to find a potential antibacterial agent from various plant extracts and oils to inhibit the growth of these harmful bacteria and reduce sucrose losses. The screening of bacterial species that grow on beet revealed that a large group of mesophilic bacteria, such as Bacillus subtilis, Leuconostoc mesenteroides, Pseudomonas fluorescens, Escherichia coli, Acinetobacter baumannii, Staphylococcus xylosus, Enterobacter amnigenus, and Aeromonas species, in addition to a dominant thermophilic species called Bacillus thermophilus, were found to be present during the manufacturing of beets. The application of 20 plant extracts and 13 different oils indicated that the extracts of Geranium gruinum, Datura stramonium, and Mentha spicata were the best antibacterials to reduce the growth of B. thermophilus with inhibition zones equal to 40, 39, and 35 mm, respectively. In contrast, the best active oils for inhibiting the growth of B. thermophilus were Mentha spicata and Ocimum bacilicum, with an inhibitory effect of 50 and 45 mm, respectively. RAPD-PCR with different primers indicated that treating sugar juice with the most effective oils against bacteria resulted in new recombinant microorganisms, confirming their roles as strong antibacterial products. The characterization of Mentha spicata and Ocimum bacilicum oils using GC/MS analysis identified cis-iso pulegone and hexadecanoic acid as the two main bioactive compounds with potential antibacterial activity. An analysis of five genes using DD-PCR that have been affected due to antibacterial activity from the highly effective oil from Mentha spicata concluded that all belonged to the family of protein defense. Our findings indicate that the application of these pure antibacterial plant extracts and oils would minimize the reduction of sucrose during sugar production.

2-Aminoethanaminium 2-(ethoxycarbonyl)-4,6-dinitrophenolate as a greener route in reducing sugar quantification.

3,5-dinitrosalicylic acid (DNS) reducing sugar assay is the most convenient method for quantification of total reducing sugar in biomass hydrolysate, fermentation samples, sugar industry and biotechnology laboratories. The dimeric proton transfer salt 2-aminoethanaminium 2-(ethoxycarbonyl)-4,6-dinitrophenolate (AED) is an intensely colored derivative of DNS and in turn its reduced form intense color showed a superior properties in reducing sugar quantification eliminating phenol and rochelle salt additives using the same practical methodology of DNS giving an overall methodology advantageous than using DNS assay as a greener route. The proton transfer salt has already been X-ray imaged and deposited in Cambridge Crystallographic Data Centre CCDC 1441586 and a comparison was done between DNS and this salt using a salt and sodium hydroxide concentrations as the same as DNS assay as well as the latter phenol-rochelle salt free environment giving correlation coefficient 0.999 and absorptivity nearly two thirds the obtained in case of use DNS assay with added phenol for enhancing absorptivity and rochelle salt for produced color stabilization for the same detection range 0.1-0.5 mg/ml according to Miller procedures. The sensitivity and the reduced form color stability of this proton transfer salt could be interpreted on both of its molecular structure has a double oxidizing groups as well as it has an intense color as compared with DNS and a good reduced form solubility respectively. •DNS in reducing sugar quantification, Miller procedures involving phenol and rochelle salt addition.•AED in reducing sugar quantification as the same detection range as DNS with the elimination of phenol and rochelle salt from the assay.•A greener convenient route in reducing sugar quantification.

Kojic Acid production from agro-industrial by-products using fungi.

A total of 278 different isolates of filamentous fungi were screened using synthetic medium for respective ability to produce kojic acid. Nineteen, six, and five isolates proved to be low, moderate, and high kojic acid producers, respectively. Levels of kojic acid produced were generally increased when shaking cultivation was used rather than those obtained using static cultivation. A trial for the utilization of 15 agro-industrial wastes or by-products for kojic acid production by the five selected higher kojic acid producer isolates was made. The best by-product medium recorded was molasses for kojic acid. A. flavus numbers 7 and 24 were able to grow and produce kojic acid on only 12 out of 15 wastes or by-products media. The best medium used for kojic acid production by A. flavus number 7 was rice fragments followed by molasses, while the best medium used for kojic acid production by A. flavus number 24 was the molasses followed by orange, pea, and rice fragments. An attempt for production of kojic acid using a 1.5 L laboratory fermentor has been made. Aspergillus flavus number 7 was used and grown on molasses medium; maximum level (53.5 g/L) of kojic acid was obtained after eight days of incubation.