Optimization of kojic acid production conditions from cane molasses using Plackett-Burman design

Fungal synthesis of kojic acid has gained more interest in these days as an alternative way to chemical synthetic. The aspect of the microbial fermentation process is to develop a suitable culture medium to obtain the maximum amount of kojic acid using statistical methods. In this study; different selected three isolates of Aspergillus flavus (No 1, 2 and 3) were screened for their ability to produced kojic acid and the isolate No 3 was the highest kojic acid producer one. The capability of A. flavus No 3 to produce kojic acid was improved using Plackett-Burman design. From ten different agro-industrial wastes cane molasses recorded the highest kojic acid productivity with 2.24 g/l-1 day-1 and was the most effective parameter plays a crucial role in Plackett-Burman design. Maximum kojic acid production (24.65 g/l) by A. flavus (No. 3) obtained under the fermentation conditions: incubation temperature at 25oC, incubation time 9 days, pH 3, inoculum size 0.5%, shaking rate at 150 rpm and medium constituents: Cane molasses 60 g/l, yeast extract 7 g/l, KH2PO4 2 g/l, ZnSO4·7H2O 100 µg/l and MgSO4·7H2O 1 g/l with regression analysis (R2) 99.45% and 2.33-fold increase in comparison to the production of the original level (10.6 g/l). DOI: http://dx.doi.org/10.5281/zenodo.121151

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.Peer Reviewe

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. Method name: AED in reducing sugar quantification, Keywords: Organic proton transfer salts, AED, DNS, Application in reducing sugar determination, Greener rout

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

Abstract 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 (R 2 ) 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

Phenotypic and Genotypic Characterization of Exopolysaccharide Producing Bacteria Isolated from Fermented Fruits, Vegetables and Dairy Products

Exopolysaccharides (EPS) particularly, from Lactic acid bacteria have received increasing attention in food, medical, and pharmaceutical applications. The present work aims to isolate, characterize and identify exopoly saccharide-producing bacteria from fermented fruits and vegetables and dairy products. A total of 55 isolates were isolated from fermented fruits, vegetables, and dairy products depending on the mucoid appearance of the colonies. Based on total EPS production, the most promising nine strains were selected, phenotypically and genotypically characterized. They were facultative anaerobe, arranged in pairs/chains (cocco bacillus), oxidase, and catalase-negative, non-spore forming and non-motile Gram-positive bacteria. All the strains were capable of growing at optimum pH between 5-7, tolerate to NaCl up to 7% (w/v), growing at 20-37°C with optimum growth at 30°C, no growth was observed at 45°C. In addition they could utilize small range of organic compounds, except isolate S1 was differ from the others by their ability to utilize a varied range of organic compounds. Construction of phylogenetic tree, on the basis of partial 16S rRNA gene sequences indicated that isolate S1 was similar to Leuconostoc citreum with similarity of 91.3%, while, isolates S2 and S3 were similar to Leu. fallax and Leu. mesenteroides with similarity of 99.40 % and 97.73%, respectively. Isolates S4, S5, S7, S8, and S9 were similar to Leu. holzaapfelii with similarity of 98.3, 98.7 and 99.8, 98.5 and 98.1, respectively, while isolate S6 was similar to Leu. lactis with similarity of 97.9%. None of sugars such as lactose, glucose, and fructose except sucrose were support EPS production from these strains. The highest yield of EPS was recorded for isolates S6, S1 and S7 which were 61.90, 61.80 and 60 gl-1, respectively, followed by isolates S4, S9, S5 and S8 which were 58.40, 53.06, 51.61 and 33.53 gl-1, respectively. Although, the lowest yield was observed for the isolates S3 and S2 which were 22.08 and 18.80 g l-1, respectively. Finally, it could be concluded that EPS production from these strains in the current study, considering them to be the alternative choice for enhancing production of EPS with increased yields, with promising realistic importance in food, pharmaceutical, as well as dairy industries

Table_1_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.pdf

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.</p