Crude Supernatant Research Articles

Antimicrobial effect of coconut oil on Staphylococcus aureus: an implication of Staphylococcus epidermidis induced fermentation

Staphylococcus epidermidis is a commensal bacterium of human skin. S. epidermidis possesses lipolytic activity to digest skin surface lipids into the smallest unit of fatty acids (FAs). Most FAs hold antimicrobial properties essential for protecting skin from invading microorganisms. In this study, we were interested in virgin coconut oil (VCO), the source of several medium-chain fatty acids (MCFAs) such as lauric acid and caprylic acid. Those MCFAs products demonstrated remarkable antibacterial activity. Our results showed that crude supernatant from the culture medium of S. epidermidis with VCO fermentation exhibited the growth inhibition effect on Staphylococcus aureus. This bacterium causes a wide range of skin diseases. A co-culture of S. epidermidis and S. aureus in a rich medium with 2.5% (v/v) VCO significantly reduced the growth of S. aureus compared to those without VCO (p-value <0.05). Moreover, time-kill assays indicated that the supernatant from the culture medium of S. epidermidis with VCO fermentation showed an efficient antimicrobial activity against S. aureus after 18 hours of incubation. Our results concluded that the culture of S. epidermidis with VCO plausibly induced fermentation of natural lipid sources aiming the production of MCFAs with antibacterial activity, particularly suppression of skin pathogen S. aureus growth. The skin commensal bacterium S. epidermidis might help produce MCFAs from skin products containing VCO and make more benefits for skin infection protection.

Aspergillus awamori endoglucanase-rich supernatant enhances lignocellulosic biomass liquefaction in high-solids enzymatic hydrolysis

Endoglucanases are particularly important in high-solids enzymatic hydrolysis of lignocellulosic biomass due to the operational need to decrease the medium viscosity via liquefaction, alleviating one of the processes' significant hindrances. This study evaluated an endoglucanase-rich supernatant produced by a particular strain of Aspergillus awamori to speed up biomass liquefaction in reactions with 30% solids. Supplementing commercial enzymes with the crude supernatant of A. awamori (Aa) comparatively decreased the media viscosity 10-fold and improved glucose release by 20% after 24 h hydrolysis. The A. awamori supernatant was fractionated by size-exclusion chromatography, and an endoglucanase-rich fraction was identified by liquid chromatography-mass spectrometry. The endoglucanase fraction and the crude Aa supplementations, under comparative conditions to a highly efficient commercial enzyme, resulted in equivalent improvements in medium viscosity and glucose release, indicating that A. awamori endoglucanases are particularly efficient for biomass liquefaction in conditions of high-solids hydrolysis.

Conversion of recombinant human ferritin light chain inclusion bodies into uniform nanoparticles in Escherichia coli for facile production.

Prokaryotic expression systems are widely used to produce many types of biologics because of their extreme conveniences and unmatchable cost. However, production of recombinant human ferritin light chain (rhFTL) protein is largely restrained because its expression in Escherichia coli tends to form inclusion bodies (IBs). In this study, a prokaryotic expression vector (FTL‐pBV220) harboring the rhFTL gene was constructed using a pBV220 plasmid. The tag‐free rhFTL was highly expressed and almost entirely converted to soluble form, and thus the rhFTL was successfully self‐assembled into uniform nanoparticles in E. coli. To establish a simplified downstream process, a precipitation procedure based on the optimized incubation temperature, pH condition, and ionic strength was developed to remove impurities from the crude lysate supernatant. The rhFTL retained in the clarified supernatant was subsequently purified in a single step using Capto Butyl column resulting in a considerable recovery and high purity. The purified rhFTL was characterized and verified by mass spectrometry and spectral and morphological analyses. The results revealed that rhFTL exhibited highly ordered and fairly compact structures and the spherical structures were preserved.

Isolation of Actinobacteria Strains From Environmental Samples and Assessment of Their Bioactivity

Background: Actinobacteria are widespread and live in a variety of habitats. Today, these bacteria are very important due to the production of various secondary metabolites with different biological activities. The present study aimed to isolate strains of Actinobacteria from different habitats (the Persian Gulf, Gandom Beryan area in the Lut Desert, and some plant roots). The anticancer and antimicrobial activities of secondary metabolites of these isolates were also investigated. Methods: Samples were taken from water of the Persian Gulf, soil of Gandom Beryan area in the Lut Desert, and plant roots. For isolation of Actinobacteria, samples were cultured in ISP2, ISP4, AIA, Gauze, M1, ISP3, and GYP media. Bacterial strains were identified based on the colony and bacterial morphology and confirmed using the specific primers for Actinobacteria. The anticancer and antimicrobial activities of crude metabolite extracts and supernatant of the isolates were evaluated on MCF-7 and Staphylococcus aureus PTCC 112 and Pseudomonas aeruginosa PTCC 1214 strains. Results: The results showed that the supernatants of 7 isolates (ga31, ez, sa, mar2, rz, ga33, and ga5) and the metabolite extracts of 4 strains (ga31, ga5, rz, and ez) had anticancer activity. Overall, ga31 was the best strain with anticancer activity of more than 75%. When evaluating the antimicrobial activity of bacterial secondary metabolites, we found that only two strains (ga31 and ga5) had antimicrobial activity against S. aureus PTCC 1112. Conclusions: In general, strain ga31, which has high anticancer and antimicrobial activities, could be a good candidate for new trials in the pharmaceutical industry.

Extracellular Secretion and Simple Purification of Bacterial Collagen from Escherichia coli

Because of structural similarities with type-I animal collagen, recombinant bacterial collagen-like proteins have been progressively used as a source of collagen for biomaterial applications. However, the intracellular expression combined with current costly and time-consuming chromatography methods for purification makes the large-scale production of recombinant bacterial collagen challenging. Here, we report the use of an adapted secretion pathway, used natively byEscherichia colito secrete curli fibers, for extracellular secretion of the bacterial collagen. We confirmed that a considerable fraction of expressed collagen (∼70%) is being secreted freely into the extracellular medium, with an initial purity of ∼50% in the crude culture supernatant. To simplify the purification of extracellular collagen, we avoided cell lysis and used cross-flow filtration or acid precipitation to concentrate the voluminous supernatant and separate the collagen from impurities. We confirmed that the secreted collagen forms triple helical structures, using Sirius Red staining and circular dichroism. We also detected collagen biomarkers via Raman spectroscopy, further supporting that the recombinant collagen forms a stable triple helical conformation. We further studied the effect of the isolation methods on the morphology and secondary structure, concluding that the final collagen structure is process-dependent. Overall, we show that the curli secretion system can be adapted for extracellular secretion of the bacterial collagen, eliminating the need for cell lysis, which simplifies the collagen isolation process and enables a simple cost-effective method with potential for scale-up.

Use of bacterial culture supernatants as anti-biofilm agents against Pseudomonas aeruginosa and Klebsiella pneumoniae.

Pseudomonas aeruginosa and Klebsiella pneumoniae are the most pervasive and challenging agents of bacterial nosocomial infections. Previous studies indicated that the microbial biofilms formed by these bacteria may play important roles in their pathogenesis and resistance to phagocytosis and antibiotics. The aim of this study was to explore the anti-biofilm activity of culture supernatants of Salmonella enterica subsp. enteric serovar Typhimurium SL1344 and P. aeruginosa PA01 against biofilms formed by P. aeruginosa PA01 and K. pneumoniae KR3167, respectively. Biofilm formation was quantified by crystal violet staining. A modified method was applied to separate planktonic and biofilm-forming cells. The viable cells in the planktonic and biofilm phases were quantitated by viable plate count. Dual-species interactions between P. aeruginosa PAO1 and Salmonella enterica subsp. enterica serovar Typhimurium SL1344 were investigated using different cell density ratios. Biofilm formation of P. aeruginosa PA01 was significantly inhibited by the heat resistant components from the culture supernatants of Salmonella enterica subsp. enterica serovar Typhimurium. Biofilm formed by K. pneumoniae KR3167 was also inhibited by the culture supernatants of P. aeruginosa PA01. The supernatants obtained from planktonic cell caused greater biofilm reduction than those extracted from biofilm-forming cells. This study is the first to report that sterile crude supernatants extracted from the cultures of Salmonella enterica and P. aeruginosa significantly inhibited biofilm formation of P. aeruginosa and K. pneumoniae, respectively. The active agents in the culture supernatants responsible for biofilm inhibition have not been determined yet. The culture supernatants of Salmonella enterica and P. aeruginosa should be further studied for their therapeutic potential to reduce biofilm formation produced by bacteria causing nosocomial infections.

Characterization and heterologous expression of a novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from Lysinibacillus sphaericus.

This study aims to explore the potential aminopeptidases of Lysinibacillus sphaericus based on the unique metabolic characteristics of this species which cannot metabolize carbohydrates and may have a strong ability to metabolize amino acids. Fifteen peptidase-encoding genes predicted in L. sphaericus C3-41 have been heterologously expressed in Escherichia coli BL21, and of these genes, only Amp0279 shows a high ability to hydrolyze L-leucine-4-nitroanilide (Leu-pNA). Phylogenetic analysis, 3D-structure modeling, and enzyme assays indicated that Amp0279 should be a novel Co2+-dependent aminopeptidase belonging to the M29 family. The optimal conditions of Amp0279 were determined to be 50°C and pH 8.0 with the addition of 100μM Co2+, and under this condition, the specific activity of Amp0279 matched that of Flavourzyme® (3.54 × 104 vs. 3.37 × 104 U/mg for the protein ingredient of Flavourzyme®). Amp0279 is mainly expressed in the middle sporulation phase in wild-type L. sphaericus or in Bacillus subtilis under the control of the sporulation-dependent strong promoter pcry8E, which is carried by the recombinant vector pHT315-8E21b. Furthermore, the secretory expression systems based on B. subtilis and Corynebacterium glutamicum were used to enhance the soluble expression of Amp0279. Obvious expression and enzymatic activity were detected from the crude supernatant media of both host bacteria without further concentration and purification. Moreover, expression can occur in the vegetative phase in B. subtilis under the control of the Pgrac promoter. KEY POINTS: • A novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from L. sphaericus was characterized. • The activity of Amp0279 as a leucyl aminopeptidase matches that of Flavourzyme® under optimal conditions. • B. subtilis- and C. glutamicum-based expression systems are built to promote secretory (soluble) Amp0279 expression.

Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1.

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.

Antimicrobial activities of certain seaweeds from muddy shore places of kerala

A study was carried out to reveal the growth inhibitory effect of methanol crude extract (MCE) and methanol supernatant extract (MSE) of sea weeds: 1) Gracillaria corticata, 2) Hypnea musciforms, 3) Gelidium micropterum and 4) Hypnea valentiae against six bacterial pathogens, 1) Pseudomonas aeruginosa, 2) Bacillus licheniformis, 3) Serratia marcescens, 4) Aeromonas hydrophila, 5) Acinetobacter baumanii, 6) Escherichia coli and two fungal strains, 1) Aspergillus niger and 2) Candida albicans respectively. Well diffusion method using zone of inhibition as indicator for growth inhibition was adopted. The results showed that methanol extracts of seaweeds viz., Gracillaria corticata, Hypnea musciforms, and Hypnea valentiae prevented the growth of pathogenic bacteria and fungi. The effect on growth was observed as zone of inhibition, the diameter of which was indicated in the units of a millimeter. The growth of the bacterium, Serratia marcescens was affected by methanol supernatant extract of the three types of seaweeds, Gracillaria corticata, Hypnea musciforms, and Hypnea valentiae, and by the methanolic crude extract of Hypnea musciforms and Hypnea valentiae. However, the growths of other species of bacteria were not controlled by either of the extracts of the seaweeds except, Bacillus licheniformis which was controlled by only Gracillaria corticata. The growth of fungi: Aspergillus niger and Candida albicans were inhibited by the methanol extracts of Gracillaria corticata. Between the two forms of methanolic extracts i.e., supernatant and crude, the efficiency of the supernatant extract was greater than that of crude one. Further, among the three types of seaweeds which showed an effect on the growth of microbes, the level of the zone of inhibition caused by Gracillaria corticata was statistically higher than that of the other two, Hypnea musciforms, and Hypnea valentiae.

Optimization and Functional Properties of a Protease from a Fish Gut Isolate Pseudomonas sp. PD14 Grown on Fish Processing Wastes Substrate

Extracellular protease production from Pseudomonas sp. PD14 isolated from the gut of rabbitfish, Siganus guttatus, was investigated using fish processing wastes as nitrogen source. Three types of fish processing by-products – namely, heads, bones, and viscera – from yellowstripe scad (Selaroides sp.) and Japanese halfbeak (Hyporhampus sajori), as well as trimmings and viscera from squid (Loligo sp.) were used. Among the fish waste preparations tested, Japanese halfbeak viscera (HV) flour (1.0%) supported the maximum protease production of 365.0 U/mg protein. Effects of media components on protease production in the optimized concentration of HV flour revealed that the protease enzyme can be optimally produced using maltose (0.50%) as a carbon source, at pH 8.0, and incubated for 48 h at 40 °C. Partial characterization of the crude enzyme supernatant showed that the optimum pH and temperature were 8.0 and 50 °C, respectively. Furthermore, the crude protease exhibited thermostability retaining 50% activity at 60 °C and fairly stable up to pH 11.0. Considering the high costs of industrial enzyme production, the use of fish processing wastes for protease production offers a promising future for enzyme biotechnology.

Novel pineapple leaf fibre composites coating with antimicrobial compound as a potential food packaging

AbstractBiodegradable packaging coated with antimicrobial compounds has emerged as a new type of food packaging with potential to enhance food safety, extend shelf life and minimize plastic pollution. We have developed a polylactic acid (PLA) composite pineapple leaf fibre (PALF) biopolymer to fulfil the high demand of biodegradable materials for a potential use in food packaging. Our formulation with 10% pineapple fibre PLA/PALF composites exhibited enhanced melting temperatures and heat resistance, with a 44% reduction in the tensile strength and 2.7‐fold reduction on the strain at the break when compared with neat PLA. To impart antimicrobial activity, the PLA/PALF was coated with crude supernatants isolated from Pediococcus pentosaceus PP04 containing 1.3% w/v of nisin. This material coated with the crude extract was shown to inhibit the growth of Bacillus cereus ATCC 11778, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311, and Escherichia coli ATCC 8739 in culture conditions, as well as minimizing the number of the tested pathogens on coated material surfaces.

Development of genus-specific universal primers for the detection of flaviviruses

BackgroundFlaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods.MethodsWe developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates.ResultsThe novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates.ConclusionThis PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology.

English

This study evaluated the preservative ability of protein-like cell free supernatants produced by lactic acid bacteria (LAB) isolates from samples of Ictalurus punctatus (Cat fish) and slurry of fermented Zea mays (Ogi). The LAB strains were separately isolated from understudied samples using De Man, Rogosa and Sharpe (MRS) media at 37°C for 48 h. The isolated strains were characterized with Gram staining, oxidase and catalase tests, microscopy study, carbohydrate fermentation, acid production and NaCl tolerance. Thereafter, the protein concentrations of crude bacteriocin supernatants from the Gram positive, rod shaped, oxidase and catalase negative strains were studied. Also, the growth inhibition of Bacillus subtilis, Staphyloccocus aureus and Escherichia coli, heat stability, pH tolerance, effect of proteolytic enzyme and biopreservation efficiency of protein-like cell free supernatants (crude bacteriocins) were determined.  Biopreservative efficiency of the crude bacteriocin samples was also determined in orange (Citrus sinenses) and Titus fish (Scomber scombrus). The isolates from intestine of I. punctatus and fermented Z. mays fermented carbohydrate, and grew optimally at 3% NaCl, and 10 and 37°C, respectively. They inhibited the multiplication of E. coli at various extents, but more effective on different strains. The bacteriocins from slurry of fermented Z. mays on the other hand, were more potent in E. coli (22.7 ± 0.8 mm) than S. aureus (7.9 ± 0.1 mm). The biopreservative efficiency of crude bacteriocin from I. punctatus was greater than that of Z. mays. The LAB obtained from the selected samples produced protein-like substances in form of bacteriocins with potent antibacterial and biopreservative proficiencies through the growth inhibition of tested pathogens and low colony counts on tested food samples, respectively. Bacterial isolates obtained from samples of I. punctatus and Z. mays can be successfully used in the preservation of food and vegetables.   Key words: Ictalurus punctatus, Zea mays, bacteriocin, protein-like substances, biopreservative ability.

A Potential Fungal Probiotic Aureobasidium melanogenum CK-CsC for the Western Honey Bee, Apis mellifera.

Aureobasidium melanogenum has been used as an animal feed additive for improving thehealth of pets, however, it has not yet been applied in honey bees. Here, a fungal strain CK-CsC isolated from bee bread pollen, was identified as A. melanogenum. Following characterizing CK-CsC fermentation broth, the 4-days fermentation broth (SYM medium or bee pollen) of the CK-CsC was used to feed newly emerged adult honey bees in cages under laboratory-controlled conditions for analysis of survival, gene expression of nutrient and antibacterial peptide, and gut microbiota of honey bees. It was found that the CK–CsC fermentation broth (SYM medium or bee pollen) is nontoxic to honey bees, and can regularly increase nutrient gene expression of honey bees. However, significant mortality of bees was observed after bees were fed on the supernatant liquid of the fermentation broth. Notably, this mortality can be lowered by the simultaneous consumption of bee pollen. The honey bees that were fed bee pollen exhibited more γ-Proteobacteria, Bacteriodetes, and Actinobacteria in their gut flora than did the honey bees fed only crude supernatant liquid extract. These findings indicate that A. melanogenum CK–CsC has high potential as a bee probiotic when it was fermented with bee pollen.

Production of Fibrinolytic Protease from a Halobacterium Bacillus licheniformis VITLMS Isolated from Marine Sponges of Rameshwaram Coast, India

Background: Marine bacteria serve as excellent sources of therapeutic enzymes, metabolites and natural products, which possess novel therapeutic properties. Increasing death rates due to cardiovascular diseases urge for cost-effective production of the fibrinolytic enzyme. Methods: In this study, marine sponge samples were screened for potent fibrinolytic producing bacteria. The primary screening was done for protease production, and clot lysis activity. The secondary screening was done for casein plasminogen activity and fibrinolytic activity. The strain which had potent fibrinolytic activity among them was further subjected to morphological, biochemical and molecular characterization. Media optimization was carried out to enhance enzyme production. The enzyme produced was subjected to purification using ammonium sulfate precipitation, gel filtration and characterized using HPLC and FTIR analysis. Results: Sponge was identified to be Desmapsamma anchorata. Thirteen bacterial isolates were isolated from the sponge sample. The 16S rRNA sequencing revealed that the potential strain had 99% similarity with Bacillus licheniformis. Amongst the isolates, most were found to be morphologically identical to the Bacillus genus. Gram’s staining and SEM analysis of the potent isolate were performed to identify the spore formation and rod-shaped morphology of the bacteria. The optimal temperature and pH for the production of the enzyme were 37°C and 8, respectively. The carbon source maltose and nitrogen sources were malt extract and yeast extract that were found to be optimal. The optimum incubation time was found to be 4 to 5 days. The crude supernatant was purified with ammonium sulfate precipitation and gel filtration chromatography. The retention time of 11.3 min and the presence of functional groups show the purity of the enzyme. The partially purified enzyme showed 96.4% clot lysis in artificial clot lysis activity. Conclusion: Although the secretion of fibrinolytic enzymes from Bacillus species is not new, based on our investigation, there are no reports regarding Bacillus licheniformis being isolated from marine sponges. However, there are reports of Bacillus licheniformis secreting fibrinolytic enzymes isolated from fermented food samples. This study identifies the marine environment as a potential source of new exploration for drug discovery.

Postbiotics, Metabolic Signaling, and Cancer.

Postbiotics are health-promoting microbial metabolites delivered as a functional food or a food supplement. They either directly influence signaling pathways of the body or indirectly manipulate metabolism and the composition of intestinal microflora. Cancer is the second leading cause of death worldwide and even though the prognosis of patients is improving, it is still poor in the substantial part of the cases. The preventable nature of cancer and the importance of a complex multi-level approach in anticancer therapy motivate the search for novel avenues of establishing the anticancer environment in the human body. This review summarizes the principal findings demonstrating the usefulness of both natural and synthetic sources of postbotics in the prevention and therapy of cancer. Specifically, the effects of crude cell-free supernatants, the short-chain fatty acid butyrate, lactic acid, hydrogen sulfide, and β-glucans are described. Contradictory roles of postbiotics in healthy and tumor tissues are highlighted. In conclusion, the application of postbiotics is an efficient complementary strategy to combat cancer.

Antifungal Properties of Protein Extracts from Thai Medicinal Plants to Opportunistic Fungal Pathogens

Antifungal proteins or peptides (AFPs) are the natural products produced by several life forms including plants as the first-line defenses to infections. The AFPs from Thai plants can be used as new therapeutic agents in the world with growing resistance to conventional antifungals. This study determined the antifungal activities of protein extracts from Thai medicinal plants against important human opportunistic fungi, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Talaromyces marneffei. Total crude protein supernatants and their precipitated proteins from 10 Thai medicinal plants with the historical usage for treatment of fungal infection were prepared. Most of the protein extracts showed antifungal activities to the tested fungi. The most effective reactivity found in the extracts from Rhinacanthus nasutus, Andrographis paniculata, and Psidium guajava by showing highest activity to T. marneffei ATCC200051 (yeast phase), followed by C. neoformans ATCC90112, C. albicans ATCC90028, T. marneffei ATCC200051 (mold phase), and A. fumigatus NCPF7367. The precipitated proteins from R. nasutus and A. paniculata containing antifungal properties were selected for partial purification by size cut-off membrane centrifugation and tested for antifungal activities. A colorimetric broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) to anti-T. marneffei. The partially purified fractions from A. paniculata, and R. nasutus showed anti-T. marneffei activity with the MIC and MFC values ranged from 2 to 128 μg/mL and 16 to >128 μg/mL, respectively. Therefore, A. paniculata and R. nasutus can be further subjected to the study of the therapeutic antifungals.

Insecticidal activity of supernatant and crude extract of Bacillus thuringiensis-based bio-insecticide towards oil palm pests Oryctes rhinoceros (Coleoptera: ‘/Scarabaeidae)

Oryctes rhinoceros is an important pest in oil palm plants. Larvae and imago are stadia which attack plants. Control by using synthetic chemical insecticides often create a negative impact on non-target insects and the environment. B. thuringiensis can be used as an alternative. The aim of the study was to study the insecticidal activity of B. thuringiensis isolates from South Sumatra on the larvae of Oryctes. The study was conducted at Entomological Laboratory, Department of Plant Protection, Faculty of Agriculture, Sriwijaya University, from March to May 2019. The study was designed by a factorial complete randomized design with two factors in the form of Bt isolates (four isolates) and the presence of spores (crude extract and supernatant) with each repeated 3 times. The treatments were B. thuringiensis isolates propagated in Biourin media and applicated in the form of crude and supernatant. B. thuringiensis propagated in NB media was used as control. Tested insects were 1st instar larvae of Oryctes. The results showed application of crude solution of B. thuringiensis resulted in higher mortality compared to supernatant treatment. It was assumed in crude solution there was a mixtured of spores and proteins compared with the solution of the supernatant which only contains spores. KJ3P1 isolate was able to cause the highest mortality in crude treatment.

Interaction between the probiotic Bacillus subtilis and Salmonella Typhimurium in Caco-2 cell culture.

Background and Objectives:Bacillus probiotics have been recently considered in biotechnological researches, and food additives. The present study was aimed to investigate the effects of Bacillus subtilis probiotics (PY79 and ATCC 6633) and their metabolites on Salmonella Typhimurium in Caco-2 cells.Materials and Methods:Cytotoxicity of B. subtilis ATCC 6633 crude supernatant (CS) was evaluated by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. S. Typhimurium invasion assay was performed in the presence of the probiotics. Cell viability, apoptosis, and necrosis were evaluated in presence of S. Typhimurium, B. subtilis strains, and CS (4%, 8%) using flow cytometry.Results:Results showed a significant reduction in the invasive ability of S. Typhimurium to Caco-2 cells by employing B. subtilis probiotics, and CS (p < 0.05). The less invasion was indicated in B. subtilis PY79 and Salmonella co-cultural group. Furthermore, the cell survival rates, and apoptosis/necrosis were respectively increased and decreased in co-culture groups (p < 0.05).Conclusion:Hence, it seems that B. subtilis strains could be suggested as beneficial candidates to overcome the invasion and cytotoxicity of Salmonella on the intestinal cells. However, additional in vivo models are suggested to validate our results.

Investigating the anti-inflammatory effects of high molecular weight secretions from Limosilactobacillus reuteri PTCC 1655 on LPS-stimulated PMA-differentiated THP-1 cells.

This study was done to investigate the anti-inflammatory effects of high molecular weight secretions from Limosilactobacillus reuteri PTCC 1655 probiotic bacteria on lipopolysaccharide (LPS)-stimulated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. After culturing the bacterium, the crude cell-free supernatant was fractionated on the basis of molecular weights using ultrafiltration. Also, a heat-killed and sonicated fraction was obtained from the biomass of the bacterial culture. All fractions were used to measure their anti-inflammatory effects on PMA-differentiated THP-1 cells following LPS stimulation by quantifying various cellular markers of inflammation. The results demonstrated that various L. reuteri PTCC 1655-derived fractions, especially the >100kDa supernatant fraction decreased some of the inflammatory cytokines and mediators, including tumour necrosis factor-α, interleukin-1, nitric oxide, cyclooxygenase-2, matrix metalloproteinase-9 and interleukin-6, which are critical for the pathogenesis of some inflammatory diseases. It is concluded that the L. reuteri PTCC 1655-derived high molecular weight fractions significantly reduce inflammation and therefore could be appropriate candidates for future medical studies. Providing new insights about the significance of L. reuteri PTCC 1655-derived extracts and their potential to modulate inflammation.