فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 39 (پاییز 1400)

Alpha-amylase is one of the most widely-used amylase types in the industry. Bacterial amylase production is less expensive and easier. Also, solid-state fermentation is an efficient method for the production of industrial amylase due to its advantages such as significant cost reduction and recycling of nutrient-rich wastes. Agricultural residues containing nutrients such as wheat bran are more economical substrates for solid-state fermentation. The aim of the present study was to optimize the production of alpha-amylase from Bacillus subtilis using wheat bran under solid-fermentation and partial purification of this enzyme.

In this study, alpha-amylase production by Bacillus subtilis PTCC 1720 in solid-state fermentation (SSF) was investigated and optimized. The produced alpha-amylase in solid-state fermentation was precipitated, separated, concentrated, and dialyzed with 75% ammonium sulfate. Alpha-amylase activity was measured by the DNS method. The protein content after dialysis was estimated by the Bradford method and the purity of the enzyme was calculated. The molecular weight and partial purity of the enzyme were determined by polyacrylamide-sodium dodecyl sulfate gel electrophoresis (SDS-PAGE).

The results of the study showed optimal enzyme production was achieved when 10 gr of wheat bran mixed with 10 ml tap water and inoculated with 3 ml of bacterial inoculum with OD=1.0 (109 CFU/mL) and incubated at 37 °C for 72 h. The alpha-amylase activity after precipitation with 75% ammonium sulfate solutions was 3.563 U/mL.  As a result, the purity of alpha-amylase precipitated with 75% saturated ammonium sulfate was estimated to be 26.8% and its molecular weight was 63 KDa

Based on the results of the study, solid-state fermentation is a new and valuable way for amylase production using Bacillus subtilis.

Monascus purpureus is a filamentous ascomycetes fungus that has the ability to produce natural pigments. The aim of the present study was to investigate the effects of mutagenic chemical agents of Sodium Azide and Nitro Acid on the morphology of Monascus purpureus and stable pigment production by this fungus.

Initial sporulation of Monascus purpuoreus was affected by chemical mutagens (Sodium Azide and Nitrous Acid). Then, the morphological features (appearance and microscopy) of the suspected colonies were examined. Wild and selected colonies were cultured under the submerge fermentation for up to two generations to measure biomass and pigment under immersion conditions.

The production of biomass and yellow, orange, and red pigments in the first and second generation of NA2 (nitrous acid, 0.4 M, 45 min), NA3 (nitrous acid, 0.4 M, 30 min), and NA6 (nitrous acid, 0.2 M, 30 min) samples did not change significantly (P <0.05). NA2 sample significantly produced the maximum amount of pigment (P <0.05). The concentrations of yellow, orange, and red pigments produced by this sample were 0.64, 0.42, and 0.45 units per milliliter, respectively.

In the present study, the morphology of all selected samples showed changes compared to the wild sample. Most of the selected samples produced significantly more pigments than the wild strain which were stable for up to two generations (P <0.05). The treatment method of Monascos purpureus with nitrous acid can successfully lead to the production of samples with higher and more stable pigment production.

Nowadays, the widespread use of antimicrobial peptides as a natural preservative due to the side effects of synthetic preservatives (cancers and liver damages in medicines and foods) has received much attention.

In the present study, five antibacterial peptides including Nisin A of Lactococcus lactis, melitin of Apis mellifera, copsin from Coprinopsis cinerea, Terpene of Erythrolobus australicus, and thionin from Arabidopsis thaliana were studied using bioinformatics analysis.

The results showed that these peptides (peptides studied in eukaryotics and prokaryotics) had cytoplasmic targeting and the studied peptides were not protected in different organisms. There was a variation in the number of α helixes and β sheets among the studied peptides. The results of the phylogenetic tree by Mega5 showed that besides Apis mellifera, four other species were located in the same cluster. The domains were different in the five studied species, but all domains had antibacterial properties. These peptides showed a wide range of physicochemical properties. The substitution template, substitution model, and D-Tajima sequence of the studied peptides showed that Apis mellifera was isolated from other species during evolution. Three-dimensional (3-D) modeling of peptides by the homology modeling method and Swiss Model database showed that the three-dimensional structure of terpene and thionin had high quality. Using peptidecutter and allermatch websites, it was found that nisin A, melitin, copsine, terpene, and thionin were not allergenic. To determine the minimum lethal concentration and minimum inhibitory concentration, the disk diffusion method was used. The highest inhibition of nisin of Lactococcus Lactis was obtained in Staphylococcus aureus and Escherichia coli by disk diffusion. Blank discs were 19, 23 mm, 5, and 1 mm, respectively.

The results showed that nisin can be used as a natural preservative to delay food spoilage against gram-positive bacteria.

This research was conducted with the aim of isolating and identifying cowpea root and crown pathogens in Khuzestan province. In addition, the antifungal effectiveness of nine selected strains of five Trichoderma species was surveyed on four root and crown pathogens of cowpea.

Out of twenty strainsobtained, four of them were selected for further investigation including pathogenicity test and siderophore assay. The relative amount of the siderophore produced by nine selected strains from five Trichoderma speciesand pathogens was assayed by the chrome Azurol S method (CAS). The radial growth of the pathogen’s colonies in front of the Trichodermastrains was analyzed using a completely randomized design, with three replicates. The pathogenic strains of ITS region were amplified and sequenced. The obtained sequences were compared with the reference strains using BLASTn search and the maximum likelihood (ML) phylogenetic analysis.

In the present study, the following pathogens were identified: Rhizoctonia solani, Fusarium chlamydosporum, F. falciforme, and Macrophomina phaseolina. In CAS assay, all of the Trichoderma strains and pathogens produced the siderophore into growth media under iron starvation.Strains of T. koningiopsis SCUA-Arak-96 and T. pleuroticola SCUA-Isf-15 produced the minimum and maximum siderophore with 31% and 85%, respectively. In the dual culture test, most Trichoderma strains significantly reduced the growth of pathogenic fungi. The most antifungal effect was observed in T. virens SCUA-Ham-65 strain on F. chlamydosporum and F. falciforme pathogens, with 30% and 29% growth inhibitory, respectively.

The results of the present study showed thatR. solani, F. chlamydosporum, F. falciforme, and M. phaseolina were the most important pathogens of the cowpea causing root and crown rot in Khuzestan province. Trichoderma strains produced the siderophore which chelates minimal iron of environment. In addition, they inhibit the growth of the plant pathogens in vitro.

Investigating the vegetation of protected areas and its harmful agents such as Erysipelas fungi is an important issue for evaluating such areas. The aim of the present study was to investigate the biodiversity of Erysipelas fungi and their hosts in the central and peripheral zones of Oshtorankouh Mountain. The analysis could the basis for further studies and provide basic biometric information of plants in terms of infection with pathogens in these areas.

In order to study the biodiversity of these fungi and their host plants, 10 plots in the central zone and the same plot number of peripheral areas in Oshtorankouh Mountain were selected and infected plants and fungal species were identified using their morphological characteristics. Then, the biodiversity indices of Simpson and Shannon, uniformity indices of Eviness, and richness indices of Margalef and Mannheicks were estimated for these species by PAST software. After that, the relative abundance percentages and total abundance of these species were estimated. The correlation between these indices was analyzed using the Leven test.

Eighteen pathogenic fungi species belonging to 6 genera on 40 plant species from 15 genera were identified. The results of the study showed that the highest relative abundance of fungi species belonged to Leveillula the Astreaceae family for plants. There was no significant difference between biodiversity, richness, and uniformity indices of the studied areas. But, there was a difference between the elevation classes in terms of the relative abundance of pathogenic fungi. The correlations between diversity and richness indices of infected plants and between these two indices in pathogenic fungi were also positive and significant (p <0.01).

Most plant species hosts are new records for Iran or even in the world. Damages by such fungi along with other harmful factors and the critical situation of the area occupied by these species threaten the plants of the region.

So far, studies have shown that an important reason for Escherichia coli resistance to beta-lactam antibiotics is the presence of broad-spectrum beta-lactamases (ESBLs) which are the product of SHV and TEM gene expression. Some studies also show that the use of nanoparticles can be effective in eliminating bacterial resistance. Therefore, the present study aimed to investigate the effects of silver nanoparticles on the expression of the blaTEM beta-lactamase resistance gene and determin the pattern of antibiotic resistance in existing Escherichia coli samples.

In this cross-sectional descriptive study, 64 Escherichia coli were collected from 11 medical diagnostic laboratories. These samples were identified using standard laboratory methods and specific cultures. The PCR method was used to evaluate the frequency of the blaTEM gene. To evaluate the antibiotic susceptibility pattern of the strains, the disk diffusion method was performed based on the CLSI protocol. Silver nanoparticles were synthesized from the ginger extract and real-time PCR was used to investigate the effect of silver nanoparticles on blaTEMgene expression.

From 64 Escherichia coli resistant samples, 61 samples were beta-lactamase resistant. Phenotypic evaluation of the antibiotic resistance pattern of beta-lactamase-resistant Escherichia coli strains showed that 90% was resistant to penicillin, 66% to carbonicillin, 87% to isolates to erythromycin, 85% to cefotaxime 84% to ceftriaxone, 49% to gentamicin, 37% to spirofloxacin, 22% to imipenem, and 12% to linezolid. The highest antibiotic resistance belonged to penicillin (90%) and erythromycin (87%) and the lowest to imipetmo (22%) and linezolid (12%), respectively. Molecular analysis showed the presence of the blaTEMgene in all samples. The results of the real-time method showed that the effect of silver nanoparticles on blaTEMgene expression was significant.

The presence of 61 resistant samples out of 64 samples in the present study indicates an increase in the resistance of Escherichia coli to various antibiotics, which could be a serious concern for the treatment of infections caused by Escherichia coli. The effectiveness of silver nanoparticles on blaTEMgene expression suggests that it could be a good alternative to existing antibiotics.