Supernatant Extract Research Articles

GC–MS-based metabolite fingerprinting reveals the presence of novel anticancer compounds in the microcolonial fungus Aureobasidium sp. TD-062 from the under-explored Thar Desert

Aureobasidium sp., strain TD-062, a micro colonial black yeast, was obtained as part of a screening program from the Thar Desert of India, which has been inadequately explored for novel microorganisms/bioactive metabolites. The anticancer activity of aqueous and organic solvent extracts of culture supernatant of TD-062 was investigated against A-375 myeloma and MCF-7 breast cancer cell lines. Following column chromatography, bioactive fractions were subjected to GC–MS analysis in two cycles. The GC–MS metabolite fingerprinting revealed 20 compounds belonging to various chemical groups. Further, based on observed and calculated mass ion spectra, retention time, and retention indices, many compounds could be considered novel. The second purification cycle resulted in three compounds- ATD-1, ATD-2 and ATD-3 with different calculated retention indices from that of the nearest matching compounds, squalene and tocopherol. In silico prediction study of their ADMET profiles, suggests that these new compounds have a suitable contour for use as safe anticancer drugs, without the toxicity normally associated with anticancer compounds.

Biosynthesis of antimicrobial nanoparticle from Swertia spp. (Chirayita) against bacterial pathogens of poultry- A way forward to green nanotechnology and nano-medicines

The increasing prevalence of multidrug-resistant microorganisms in poultry has led to a rise in bacterial infections, causing significant economic loss. Green nanotechnology, such as silver nanoparticles (AgNPs), has the potential to address this issue by providing potent antifungal, antiviral, and antibacterial properties. This study explored the combined potential of AgNPs and the local herb Swertia chirayita against established poultry pathogens, employing a non-factorial Central Composite Design (CCD) to evaluate the factors affecting the production of nanoparticles induced by silver nitrate from the selected herb. The optimal values for temperature, wavelength, silver nitrate concentration, incubation duration, and pH were found to produce the highest nanoparticles. The functional groups in Swertia chirayita stimulated nanoparticles were confirmed using FTIR spectroscopy, and the stability of ScNPs was elucidated using zeta potential. The crystalline structure of ScNPs was confirmed using diffraction intensity patterns. Silver nanoparticles demonstrated antibacterial activity against Salmonella spp. and Escherichia coli (E.coli), both known as significant poultry pathogens, using the agar well diffusion method, with inhibition zones of 25.0 mm and 35.0 mm, respectively.This study explored the green manufacturing of silver nanoparticles by using plants and microorganisms, focusing on their antibacterial properties. The exact mechanism of synthesis and action in AgNPs is still poorly understood. Researchers should prioritize the use of accessible, easy-to-extract plants or bacteria, especially non-pathogenic and fast-growing microorganisms for safe handling. Analyzing biomolecules in plant extract, microbial biomass, or culture supernatants, including probiotic bacteria, is crucial for creating and stabilizing AgNPs, which could be effective synthetic agents. It is crucial to optimize conditions for rapid, stable, and large-scale synthesis. Based on this research, Sc-NPs may be proposed as nanomedicine for treating infections in poultry caused by E. coli and Salmonella spp.

Trichoderma reesei JCM 2267, fungi-associated sponge Stylissa flabelliformis: Effects of applied different fermentation methods on their antibacterial activity

Trichoderma is one of the fungi producing a wide range of metabolites with potent biological activities, including antibacterial. Selecting appropriate fermentation methods can improve the production of metabolite compounds and provide the best biological activity. This study investigates Trichoderma reesei JCM 2267’s antibacterial activity, which is fermented using submerged fermentation (SmF) and solid-state fermentation (SSF). The effect of fermentation time on antibacterial activities and yield weight was also determined. Antibacterial activity was tested against Staphylococcus aureus (S.a) ATCC 25923, Escherichia coli (E.c) ATCC 25922, and Pseudomonas aeruginosa (P.a) ATCC 27853 using the Agar well diffusion method. This research successfully demonstrated that the SmF and SSF methods provided extracts with antibacterial activity ranging from weak to strong, against bacteria tested. The extract fermented by SSF showed the best activity on day 25 of fermentation with a diameter clear zone 12.2 ± 0.20; 5.0 ± 0.17, and 3.3 ± 0.1 (mm) against S.a ATCC 25923, E.c ATCC 25922, and P.a ATCC 27853. The extract fermented by SmF, especially the supernatant extract, reached the higher inhibition on day 8 with diameter inhibition 6.90 ± 0.05 and 8.20 ± 0.04 (mm) against E.c ATCC 25922 and P.a ATCC 27853, whereas 15.3 ± 0.10 (mm) against S.a ATCC 25923 on day 14 of fermentation. The various times of fermentation affect the antibacterial activities. The maximum growth of TR JCM 2267 was reached on day 14 of fermentation. The SSF methods showed a higher yield of extract than SmF, increasing over time of fermentation. This research provides a possible alternative fermentation method suitable for fungal growth related to its antibacterial activity and extract yield.

Antibacterial Efficacy of AgNPs synthesized from Aloe vera extract and Staphylococcus aureus Culture Supernatant

Biological synthesis of silver nanoparticles (AgNPs) is an emerging method that avoids the need for costly equipment and hazardous chemicals. Therefore, in this study, Aloe vera (AV) extract and culture supernatant of Staphylococcus aureus (CS-S. aureus) were used to reduce and stabilise silver nanoparticles. The NPs were characterized using UV–vis spectrophotometry at 445 nm for AV-AgNP and 444.5 nm for CS-S.aureus AgNP, and the presence of silver was confirmed by energy dispersive X-ray (EDX). Furthermore, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that the AV-CS of S. aureus-AgNPs had irregular and spherical shapes, with average sizes of 18.5 nm for AvAgNPs and 7.03 nm for CS-S. aureus-AgNPs. Additionally, the minimum inhibitory concentration (MIC) of both AgNPs showed a similar reaction. K. pneumoniae, P. aeruginosa, and S. epidermidis were more sensitive to the AgNPs compared to methicillin-resistant Staphylococcus aureus (MRSA). In conclusion, our study used a green method to synthesise silver nanoparticles using AV extract and CS of S. aureus. The NPs showed a remarkable antibacterial effect, especially CS-S. aureus-AgNP, which could have application as a treatment against both Gram-positive and Gram-negative bacteria.

The influence of a composition based on exometabolites of Saccharomyces boulardii in relation to the pathogenic and persistent properties of fungi of the genus Candida

Clinical manifestations of mycoses vary widely from mild mucosal damage to severe invasive forms. Currently, the pharmaceutical market offers a wide range of antimycotic drugs, including oral and injectable drugs for systemic and external and local use. Varying degrees of effectiveness are due to the chemical structure and pharmacokinetics. A significant problem is the recurrence of mucosal candidiasis. One of the possible solutions to this problem can be considered the use of exometabolites of bacterial or fungal origin, affecting the morphofunctional structures of fungi of the genus Candida spp. The purpose of the study was to obtain cell-free supernatant of Saccharomyces boulardii (S. boulardii) and herbal extracts, which are the basis of a composition with a preventive effect against fungi of the genus Candida. The authors proposed a composition based on the exometabolite of S. boulardii and herbal extracts for the prevention of candidiasis. Propylene glycol served as a control. The effectiveness of the composition was assessed on clinical isolates of C. albicans, C. glabrata and C. krusei isolated from the vaginal biotope at a dilution of 103-106 CFU/mL. At the first stage, we studied the effect of S. boulardii metabolites on the biological properties of fungi. Next, the fungicidal activity of metabolites and extracts was determined by serial dilution against micromycete isolates. At the final stage, the effect of the composition with the most effective extracts was studied. Under the influence of the supernatant of S. boulardii, micromycetes lost the ability to form growth tubes, and a decrease in the catalase activity of Candida spp. was observed both during carriage and disease. Exometabolites significantly reduced the ability to form film in cultures isolated from patients with vaginal candidiasis. The most effective among the extracts were the extracts of St. John’s wort and coriander. The fungicidal properties of the composition were maintained for 12 hours after co-incubation with all isolates of Candida spp. Thus, the composition is effective against both C. albicans and C. non-albicans carriage and disease.

Chemical Exposomics in Human Plasma by Lipid Removal and Large-Volume Injection Gas Chromatography-High-Resolution Mass Spectrometry.

For comprehensive chemical exposomics in blood, analytical workflows are evolving through advances in sample preparation and instrumental methods. We hypothesized that gas chromatography-high-resolution mass spectrometry (GC-HRMS) workflows could be enhanced by minimizing lipid coextractives, thereby enabling larger injection volumes and lower matrix interference for improved target sensitivity and nontarget molecular discovery. A simple protocol was developed for small plasma volumes (100-200 μL) by using isohexane (H) to extract supernatants of acetonitrile-plasma (A-P). The HA-P method was quantitative for a wide range of hydrophobic multiclass target analytes (i.e., log Kow > 3.0), and the extracts were free of major lipids, thereby enabling robust large-volume injections (LVIs; 25 μL) in long sequences (60-70 h, 70-80 injections) to a GC-Orbitrap HRMS. Without lipid removal, LVI was counterproductive because method sensitivity suffered from the abundant matrix signal, resulting in low ion injection times to the Orbitrap. The median method quantification limit was 0.09 ng/mL (range 0.005-4.83 ng/mL), and good accuracy was shown for a certified reference serum. Applying the method to plasma from a Swedish cohort (n = 32; 100 μL), 51 of 103 target analytes were detected. Simultaneous nontarget analysis resulted in 112 structural annotations (12.8% annotation rate), and Level 1 identification was achieved for 7 of 8 substances in follow-up confirmations. The HA-P method is potentially scalable for application in cohort studies and is also compatible with many liquid-chromatography-based exposomics workflows.

Pseudomonas protegens ML15 and Trichoderma koningiopsis Tr21 co-culture: A potent strategy for suppressing Fusarium cerealis infections in wheat through augmented antifungal metabolite production

Wheat (Triticum aestivum L.), a staple grain consumed worldwide, is heavily affected by Fusarium species, which cause harmful diseases that threaten its production. The present study was conducted to investigate the biocontrol activity of monocultures of Pseudomonas protegens ML15 and Trichoderma koningiopsis Tr21 as well as their co-culture, as a sustainable strategy to combat Fusarium cerealis. The cell-free supernatant and cell-free extract of co-culture demonstrated increased inhibitory effects against F. cerealis, compared to the monocultures. GC-MS analysis revealed that cell-free extract of P. protegens ML15, T. koningiopsis Tr21, and co-culture contained different bioactive metabolites. Pyrrolo[1,2-a]pyrazine-1,4-dione derivatives were major compounds in the cell-free extract of co-culture. Further analysis using NMR and HPLC, revealed that co-culture produced higher concentrations of pyoluteorin, 2,4-diacetylphloroglucinol, and 2,4-di-tert-butylphenol, compared to their respective monocultures. In vivo plant experiments indicated that co-culture treatment reduced F. cerealis infection and improved wheat development. Our findings highlight the exciting potential of co-culturing P. protegens ML15 and T. koningiopsis Tr21 as a formidable biocontrol duo, particularly effective against notorious fungal plant pathogens like Fusarium. This innovative approach holds promise for revolutionizing agricultural practices, offering sustainable solutions to combat crop diseases, and ensuring global food security.

2-heptanone and 2,3-butanediol from endophytic Bacillus subtilis GEB-1 against root-knot nematode, Meloidogyne enterolobii: a computational and experimental approach

ABSTRACT Meloidogyne enterolobii is regarded as one of the most severe and threatening pathogens worldwide. Guava production in India is quenched by the invasion of root-knot nematode, Meloidogyne enterolobii. Bacterial bio-control agents confer resistance against nematodes by producing various bioactive molecules. Exploiting these biomolecules will lead to the development of novel nematicidal formulations for managing plant parasitic nematodes. Considering this, our present investigation was focused on the isolation and identification of endophytic bacterial communities from wild guava Psidium eugeniaefolia. Amongst various endophytes, Bacillus subtilis GEB-1 exhibited a 90% reduction in the population of M. enterolobii. Cell-free supernatant extracts of B. subtilis GEB-1 revealed the presence of seventeen unique biomolecules. Attempts were made to dock these biomolecules against major protein targets of M. enterolobii such as E3 ubiquitin ligase, Neurotransmitter-gated ion-channel, G protein-coupled receptor kinase, Peptidase M12B domain-containing protein, Hyaluronidase through molecular docking techniques. The results of in-silico studies revealed that all seventeen biomolecules had a significant impact on protein targets of M. enterolobii. Among other bacterial metabolites, 2-heptanone and 2,3-butanediol were found to have a greater binding affinity with protein targets of M. enterolobii. Furthermore, the in-vitro evaluation of 2-heptanone and 2,3-butanediol at different concentrations confirmed their nematicidal activity against eggs and second-stage juveniles of M. enterolobii. Hence our present study revealed that bacterial metabolites, 2 heptanone, and 2,3-butanediol can be explored as novel nematicidal molecules against guava root-knot nematodes, M. enterolobii.

The antioxidant activity and metabolomic analysis of the supernatant of Streptococcus alactolyticus strain FGM

Strain-specific probiotics can present antioxidant activity and reduce damage caused by oxidation. Streptococcus alactolyticus strain FGM (S. alactolyticus strain FGM) isolated from the chicken cecum shows potential probiotic properties which have been previously demonstrated. However, the antioxidant properties of S. alactolyticus strain FGM remain unknown. In this view, cell-free supernatant (CFS), intact cells (IC) and intracellular extracts (CFE) of strain FGM and 3 strains of Lactobacillus (LAB) were prepared, and their scavenging capacities against DPPH, hydroxyl radicals and linoleic acid peroxidation inhibitory were compared in this study. The effects of strain FGM cell-free supernatant (FCFS) on NO production, activity of SOD and GSH-Px in RAW264.7 cells and LPS-induced RAW264.7 cells were analyzed. The metabolites in the supernatant were quantitated by N300 Quantitative Metabolome. It was shown that the physicochemical characteristics of CFS to scavenge DPPH, hydroxyl radicals, and linoleic acid peroxidation inhibitory were significantly stronger than that of IC and CFE in the strain FGM (P < 0.05), respectively 87.12% ± 1.62, 45.03% ± 1.27, 15.63% ± 1.34. FCFS had a promotional effect on RAW264.7 cells, and significantly elevated SOD and GSH-Px activities in RAW264.7 cells. 25 μL FCFS significantly promoted the proliferation of RAW264.7 cells induced by LPS, increased the activities of SOD and GSH-PX, and decreased the release of NO. Furthermore, among the differential metabolites of FCFS quantified by N300, 12 metabolites were significantly up-regulated, including lactic acid, indole lactic acid, linoleic acid, pyruvic acid etc., many of which are known with antioxidant properties. In conclusion, FCFS had good antioxidant properties and activity, which can be attributed to metabolites produced from strain FGM fermentation. It was further confirmed that S. alactolyticus strain FGM and its postbiotic have potential probiotic properties and bright application prospects in livestock and poultry breeding.

Automated sample preparation for electrospray ionization mass spectrometry based on CLOCK-controlled autonomous centrifugal microfluidics

We report a centrifugal microfluidic device that automatically performs sample preparation under steady-state rotation for clinical applications using mass spectrometry. The autonomous microfluidic device was designed for the control of liquid operation on centrifugal hydrokinetics (CLOCK) paradigm. The reported device was highly stable, with less than 7% variation with respect to the time of each unit operation (sample extraction, mixing, and supernatant extraction) in the preparation process. An agitation mechanism with bubbling was used to mix the sample and organic solvent in this device. We confirmed that the device effectively removed the protein aggregates from the sample, and the performance was comparable to those of conventional manual sample preparation procedures that use high-speed centrifugation. In addition, probe electrospray ionization mass spectrometry (PESI-MS) was performed to compare the device-treated and manually treated samples. The obtained PESI-MS spectra were analyzed by partial least squares discriminant analysis, and the preparation capability of the device was found to be equivalent to that of the conventional method.

Production of Cheese and Ice Cream Enriched With Biomass and Supernatant of Spirulina platensis With Emphasis on Organoleptic and Nutritional Properties

Background: Spirulina platensis contains natural pigments along with phenolic and flavonoid compounds that may be excreted to the supernatant in addition to biomass during culture. Objectives: We prepared enriched cheese and ice cream using biomass and supernatant of S. platensis microalgae extract. We aimed to evaluate these algae’s organoleptic and nutritional properties. Methods: After preparing a 30-day culture of Spirulina, different concentrations of its biomass and supernatant extract were prepared to enrich cheese and ice cream. In the next step, the physicochemical and antioxidant properties of the products were measured by FRAP (ferric reducing antioxidant power) and DPPH (2,2-diphenyl 1-picryl hydroxyl) methods. Expert evaluators performed the sensory analysis according to the Iranian national standard method in terms of color, odor, taste, texture, and general acceptance on a scale of 0 to 5. Finally, statistical analysis of the data obtained from each experiment was performed with a one-way analysis of variance three times using SPSS software, version 24. Results: The results showed that the content of protein, fat, ash, and biomass was higher in enriched foods. However, in enriched ice creams, the content of protein, fat, sugar, and aeration rate increased significantly compared to the supernatant. In addition, cheese and ice cream fortified with supernatant had significantly greater antioxidant activity than biomass. The results of the hedonic test showed that with increasing concentrations of these agents in fortified cheeses and ice creams, the level of satisfaction in odor, taste, color, texture, and general acceptance factors was increased. Conclusion: The study results showed that adding biomass and supernatant of S. platensis to ice cream and cheese increased the nutritional value and improved the taste of these foods. Because these substances play an essential role in children’s diet, this microorganism can be used to combat malnutrition and a diet without micronutrients.

Transcriptomic and biochemical analyses reveal the antifungal mechanism of cell-free supernatant from Aspergillus luchuensis YZ-1 against Aspergillus flavus

Aspergillus flavus (A. flavus) and its metabolites, aflatoxins, are widespread in crops and foods, threatening public health. Using chemical fungicides to control A. flavus can pose safety hazards and cause environmental pollution. Therefore, there is an urgent need for safe and effective antifungal agents to control A. flavus. In this study, we isolated Aspergillus luchuensisYZ-1 cell-free supernatant (CFS), volatiles, and mycelial extracts to investigate their inhibitory effects on A. flavus. Results indicate that the CFS exhibited greater efficacy in impeding the growth and synthesis of aflatoxin B1 (AFB1) in A. flavus. Transcriptomic analysis revealed a significant downregulation of lipid metabolism, biosynthesis of other secondary metabolites, and glycan metabolism in A. flavus treated with CFS. Approximately 94% of the genes in the aflatoxin synthesis pathway were significantly downregulated. Furthermore, the CFS treatment significantly increased reactive oxygen species (ROS) production in A. flavus, affected its energy metabolism, and upregulated methane metabolism and the pentose phosphate pathway. We then investigated the inhibitory mechanism of YZ-1 against A. flavus by biochemical analysis. It was further confirmed that CFS had destructive effects in the cell wall and cell membrane of A. flavus, as indicated by leakage of nucleic acids, proteins, alkaline phosphatase (AKP), as well as decreased chitin and ergosterol content. In summary, this study provides novel insights into the mechanisms of CFS against A. flavus, suggesting its potential as a biocontrol agent in food and agriculture.

Restraining Staphylococcus aureus Virulence Factors and Quorum Sensing through Lactic Acid Bacteria Supernatant Extracts.

The escalating prevalence of antibiotic-resistant bacteria poses a grave threat to human health, necessitating the exploration of novel alternatives to conventional antibiotics. This study investigated the impact of extracts derived from the supernatant of four lactic acid bacteria strains on factors contributing to the pathogenicity of three Staphylococcus aureus strains. The study evaluated the influence of lactic acid bacteria supernatant extracts on the growth, biofilm biomass formation, biofilm metabolic activity, and biofilm integrity of the S. aureus strains. Additionally, the impact on virulence factors (hemolysin and coagulase) was examined. Gas chromatography coupled with mass spectrometry was used to identify the bioactive compounds in the extracts, while molecular docking analyses explored potential interactions. Predominantly, the extracts contain eight 2,5-diketopiperazines, which are cyclic forms of peptides. The extracts demonstrated inhibitory effects on biofilm formation, the ability to disrupt mature biofilms, and reduce the biofilm cell metabolic activity of the S. aureus strains. Furthermore, they exhibited the ability to inhibit α-hemolysin production and reduce coagulase activity. An in silico docking analysis reveals promising interactions between 2,5-diketopiperazines and key proteins (SarA and AgrA) in S. aureus, confirming their antivirulence and antibiofilm activities. These findings suggest that 2,5-diketopiperazines could serve as a promising lead compound in the fight against antibiotic-resistant S. aureus.

Enhancing the nutritional value and functional properties of mango pulp via lactic acid bacteria fermentation

The probiotic properties of four lactic acid bacteria (LAB) strains, Lacticaseibacillus paracasei OR1 and PP1 and Lacticaseibacillus rhamnosus MA3 and PP3, were evaluated. OR1 showed the highest levels of self-aggregation (61.27%) and coaggregation (17.70%) with Staphylococcus aureus. Compared to the crude cell-free supernatant (CFS) and ethyl acetate extract fraction, the water extract fraction of the CFS showed greater antimicrobial activity against Escherichia coli and Bacillus cereus. All LAB strains exhibited the highest levels of total carotenoids, phenolics, flavonoids and antioxidant activity in the CFS, suggesting that they can release water-soluble bioactive compounds into the growth medium. The phytochemical profile of Mahachanok mango, which is rich in bioactive compounds, was improved through LAB fermentation. LC–MS/MS analysis revealed 60 compounds, including phenolic acids (1.11-fold), organic acids (1.21-fold), and amino acid derivatives (1.28-fold), that were more abundant in the fermented Mahachanok mango pulp (FMP) samples than in the fresh samples. Interestingly, the antioxidant proline level increased 377.28-fold, and novel dipeptides and tripeptides were present in the FMP fermented with L. paracasei OR1. Mango pulp is a potential source of nutrients for LAB growth and metabolism; thus, LAB fermentation could be used to produce a functional food product rich in antioxidants and bioactive peptides.

Panoptic elucidation of algicidal mechanism of Raoultella sp. S1 against the Microcystis aeruginosa by TMT quantitative proteomics

Harmful algal blooms (HABs) due to eutrophication are becoming a serious ecological disaster worldwide, threatening human health and the optimal balance of aquatic ecosystems. The traditional approaches to eradicate HABs yield several drawbacks in practical application, while microbial algicidal technology is garnering mounting recognition due to its high efficiency, eco-friendliness, and low cost. In our previous study, we isolated a bacterium strain Raoultella sp. S1 from eutrophic water with high efficiency of algicidal properties. This study further investigated the flocculation and inactivation efficiency of S1 on Microcystis aeruginosa at different eutrophic stages by customizing the algal cell densities. The supernatant extract of S1 strain exhibited remarkable flocculation and inactivation effects against low (1 × 106 cell/mL)and medium (2.7 × 106 cell/mL)concentrations of algal cells, but unexceptional for higher densities. The results further revealed that algal cells at low and medium counts manifested a more apparent antioxidant defense response, while the photosynthetic efficiency and relative electron transport rate were considerably reduced within 24 h. TEM observations confirmed the disruption of thylakoid membranes and cell structure of algal cells by algicidal substances. Moreover, TMT proteomics revealed alterations in protein metabolic pathways of algal cells during the flocculation and lysis stages at the molecular biological level. This signified that the disruption of the photosynthetic system is the core algicidal mechanism of S1 supernatant. In contrast, the photosynthetic metabolic pathways in the HABs were significantly upregulated, increasing the energy supply for the NADPH dehydrogenation process and the upregulation of ATPases in oxidative phosphorylation. Insufficient energy provided by NADPH resulted in a dwindled electron transport rate, stagnation of carbon fixation in dark reactions, and blockage of light energy conversion into chemical energy. Nonetheless, carbohydrate metabolism (gluconeogenesis and glycolysis) proteins were down-regulated and hampered DNA replication and repair. This study aided in unveiling the bacterial management of eutrophication by Raoultella sp. S1 and further arrayed the proteomic mechanism of algal apoptosis.

Uji Antimikroba Ekstrak Kapang Endofit RLC 5 Akar Tanaman Kayu Jawa (Lannea coromandelica (Houtt.) Merr.)

Java root (Lannea coromandelica (Houtt.) Merr.) contains many saponins, steroids and alkoloids which are known to have potential as antimicrobials. This study aims to examine the antimicrobial activity of isolates of endophytic molds from the roots of Javanese wood plants with RLC code 5 against pathogenic fungi and bacteria. The research was carried out by rejuvenating RLC 5 isolates for 7 days and then carrying out macroscopic and microscopic characteristics. Fermentation was carried out for 21 days to reach the stationary phase in producing secondary metabolites, then extraction was carried out from the fermented RLC 5 isolate using a vacuum rotary evaporator. The metabolite screening test was carried out by testing for flavonoids, saponins, tannins, alkaloids, and triterpenoids and steroids. Antimicrobial testing was carried out using the disc diffusion method to see the clear zones that formed. The results showed that the endophytic mold RLC 5 is a mold of the genus Penicillium which is capable of synthesizing secondary metabolites in the form of saponins, alkaloids and steroids. The antimicrobial properties of the supernatant extract of the RLC 5 mold isolate were stronger than the biomass extract, with inhibition zones respectively of 9.03 mm, 11.09 mm and 19.83 mm against Candida albicans, Malassezia furfur and Trichophyton mentagrophytes and 12.51 mm and 13.03 mm against Bacillus subtilis and Escherichia coli. Thus, RLC 5 mold has the potential to become a fungicide and biostatic antibiotic for pathogenic microbes. Keyword : Antimicrobial, Endophytic mold, Lannea coromandelica, Java Wood Plant, RLC 5 isolate

Fibrinolytic Protease Production: Impact of Initial pH and Temperature in Solid-State Fermentation by Rhizopus microsporus var. oligosporus FNCC 6010

Background: Fibrinolytic enzyme is one of the cardiovascular disease therapies. Rhizopus microsporus var. oligosporus is microorganism that has been evaluated to produce fibrinolytic protease by fermentation. This study conducted fermentation of helianthi annui semen (sunflower seed) by Rhizopus microsporus var. oligosporus to produce fibrinolytic enzyme. Objective: This study aims to determine the effect of Initial pH and incubation temperature and its optimization in the production of fibrinolytic protease by Rhizopus microsporus var. oligosporus FNCC 6010 in solid-state fermentation on helianthi annui semen (sunflower seed) substrate. Optimum condition was determined by maximum protease and fibrinolytic activity. Method: A crude enzyme of protease fibrinolytic was obtained from the supernatant extract of fermented sunflower seed. Protease activity was measured by the skimmed milk agar (SMA) plate method, and fibrinolytic activity was determined by the fibrin agar plate method. Result: It was found that the starting pH affects both the proteolytic and fibrinolytic activity of enzymes that are produced in fermentation. The starting pH of 5.0 showed higher fibrinolytic and proteolytic activity values compared to the starting pH of 7.0. The incubation temperature 33±1 °C had the higher activity compared to 28±1 °C or 37±1 °C. Conclusion: Initial pH and incubation temperature affect the proteolytic and fibrinolytic activity of crude enzyme extracted from fermented sunflower seed by Rhizopus microsporus var. oligosporus. The optimum condition for producing fibrinolytic protease in the state fermentation method was an initial pH of 5.0 and an incubation temperature of 33±1°C.

Investigating the antidiabetic efficacy of dairy-derived Lacticaseibacillus paracasei probiotic strains: modulating α-amylase and α-glucosidase enzyme functions.

The current study aims to evaluate and characterize the probiotic andantidiabetic properties of lactic acid bacteria (LAB) obtained from milk and other dairy-based products. The strains were tested physiologically, biochemically, and molecularly. Based on biochemical tests and 16S rRNA gene amplification and sequencing, all three isolates RAMULAB18, RAMULAB19, and RAMULAB53 were identified as Lacticaseibacillus paracasei with homology similarity of more than 98%. The inhibitory potential of each isolate against carbohydrate hydrolysis enzymes (α-amylase and α-glucosidase) was assessed using three different preparations of RAMULAB (RL) isolates: the supernatant (RL-CS), intact cells (RL-IC), and cell-free extraction (RL-CE). Additionally, the isolate was evaluated for its antioxidant activity against free radicals (DPPH and ABTS). The strain's RL-CS, RL-CE, and RL-IC inhibited α-amylase (17.25 to 55.42%), α-glucosidase (15.08-59.55%), DPPH (56.42-87.45%), and ABTS (46.35-78.45%) enzymes differently. With the highest survival rate (>98%) toward tolerance to gastrointestinal conditions, hydrophobicity (>42.18%), aggregation (>74.21%), as well as attachment to an individual's colorectal cancer cell line (HT-29) (>64.98%), human buccal and chicken crop epithelial cells, all three isolates exhibited extensive results. All three isolates exhibited high resistance toward antibiotics (methicillin, kanamycin, cefixime, and vancomycin), and other assays such as antibacterial, DNase, hemolytic, and gelatinase were performed for safety assessment. Results suggest that the LAB described are valuable candidates for their significant health benefits and that they can also be utilized as a beginning or bio-preservative tradition in the food, agriculture, and pharmaceutical sectors. The LAB isolates are excellent in vitro probiotic applicants and yet additional in vivo testing is required.

Optimization of the biosynthesis of silver nanoparticles using bacterial extracts and their antimicrobial potential

In the present study, silver nanoparticles (AgNPs) were biosynthesized using the supernatant and the intracellular extract of Cupriavidus necator, Bacillus megaterium, and Bacillus subtilis. The characterization of the AgNPs was carried out using UV–Vis spectroscopy, FTIR, DLS and TEM. Resazurin microtiter-plate assay was used to determine the antimicrobial action of AgNPs against Escherichia coli. UV–Visible spectra showed peaks between 414 and 460 nm. TEM analysis revealed that the synthesized AgNPs showed mostly spherical shapes. DLS results determined sizes from 20.8 to 118.4 nm. The highest antimicrobial activity was obtained with the AgNPs synthesized with supernatant rather than those using the intracellular extract. Therefore, it was determined that the bacterial species, temperature, pH, and type of extract (supernatant or intracellular) influence the biosynthesis. This synthesis thus offers a simple, environmentally friendly, and low-cost method for the production of AgNPs, which can be used as antibacterial agents.

Inhibition of IL-17 ameliorates keratinocyte-borne cytokine responses in an in vitro model for house-dust-mite triggered atopic dermatitis

A subgroup of patients suffering from atopic dermatitis (AD) does not respond to biologics therapy targeting the key players of type-2 inflammation, and it is an ongoing discussion whether skin-infiltrating Th17 cells may underlie this phenomenon. This study aimed to investigate the potential of allergen-induced, immune-cell derived IL-17 on the induction of inflammatory processes in keratinocytes. Peripheral blood mononuclear cells derived from respectively sensitized AD patients were stimulated with house dust mite (HDM) extract and cell culture supernatants were applied subsequently in absence or presence of secukinumab to primary human keratinocytes. Hereby we confirm that the immune response of sensitized AD patients to HDM contains aside from type-2 cytokines significant amounts of IL-17. Blocking IL-17 efficiently reduced the stimulation-induced changes in keratinocyte gene expression. IL-17-dependent transcriptional changes included increased expression of the cytokines IL-20 and IL-24 as well as Suppressor of Cytokine Siganling 3 (SOCS3), a negative feedback-regulator of the STAT3/IL-17/IL-24 immune response. We conclude that the immune response to HDM can induce pro-inflammatory cytokines from keratinocytes in AD, which in part is mediated via IL-17. Targeting IL-17 may turn out to be a reasonable alternative therapy in a subgroup of patients with moderate to severe AD and HDM sensitization.