Paper Disc Assay Research Articles

Transcriptomic analysis of the antimicrobial activity of prodigiosin against Cutibacterium acnes

Prodigiosin, a red pigment produced by Hahella chejuensis, a marine-derived microorganism, has several biological functions, including antimicrobial activity and inflammatory relief. In this study, the antibacterial activity of prodigiosin against skin microorganisms was explored. Paper disc assay on skin bacterial cells revealed that Cutibacterium acnes related to acne vulgaris highly susceptible to prodigiosin. MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) were determined on Cutibacterium species. The RNA-seq analysis of prodigiosin-treated C. acnes cells was performed to understand the antibacterial mechanism of prodigiosin. Among changes in the expression of hundreds of genes, the expression of a stress-responsive sigma factor encoded by sigB increased. Conversely, the gene expression of cell wall biosynthesis and energy metabolism was inhibited by prodigiosin. Specifically, the expression of genes related to the metabolism of porphyrin, a pro-inflammatory metabolite, was significantly reduced. Therefore, prodigiosin could be used to control C. acnes. Our study provided new insights into the antimicrobial mechanism of prodigiosin against C. acnes strains.

Biological control of cabbage head rot (Sclerotinia sclerotiorum) by Coniothyrium minitans

BackgroundSclerotinia sclerotiorum is the causal agent of white mould, stem and head rot diseases on a wide variety, including cabbage in the field and storage. Control of this pathogen by using commercial disease management methods is extremely difficult. Therefore, this study was performed to identify the potential biological control agent.ResultsThe antagonist fungal isolates, a mycoparasite of S. sclerotiorum, were isolated and described as KET Cm1, PAL Cm 2 and TDK Cm 3. They were subjected to morphological and microscopic observation. The growth of antagonist fungal isolates on different media revealed that PDA and half-strength Czapek dox agar medium showed the maximum mycelial growth (28.67 and 28.33 mm) and pycnidial production in the PDA medium. The studies on pH and temperature found that growth and sporulation of the antagonist fungal isolates were optimum at pH 5.5 (34 mm) and 20 °C. The molecular confirmation of antagonist fungal isolates was carried out using ITS 1 and ITS 4 primers, which target 18S-28S rRNA gene fragment. The isolates were partially sequenced and showed that more than 99% identity with Coniothyrium minitans already exist in the NCBI database. The accession numbers (MW093134, MW111282 and MW136938) were obtained for all the three isolates and also confirmed using specific primers Cm sp 1F and Cm sp 1R. Among the tested isolates, the antagonistic activity of C. minitans TDK Cm 3 showed the maximum mycelial growth inhibition of 48.8% S. sclerotiorum and was used for further studies. Penetration and formation of appressoria on the hyphae of S. sclerotiorum by C. minitans isolate TDK Cm 3 were observed through the scanning electron microscope. The ethyl acetate fractions of TDK CM 3 isolate were evaluated against S. sclerotiorum by agar well diffusion assay, paper disc assay and poison food technique in vitro. Through GC–MS analysis, the effective antifungal compounds were identified.ConclusionThe present study focused on the identification and characterization of effective Coniothyrium isolates as eco-friendly, integrated disease management strategies against cabbage head rot disease.

Isolation, Screening, and Active Metabolites Identification of Anti-Vibrio Fungal Strains Derived From the Beibu Gulf Coral.

The Beibu Gulf harbors abundant underexplored marine microbial resources, which are rich in diversified secondary metabolites. The genera Vibrio is a well-known pathogenic bacterium of aquatic animals. In this study, 22 fungal strains were isolated and identified from the Beibu Gulf coral via the serial dilution method and internal transcribed spacer (ITS) sequence analysis, which were further divided into three branches by phylogenetic tree analysis. The crude extracts of them via small-scale fermentation were selected for the screening of inhibitory activity against Vibrio alginalyticus, Vibrio coralliilyticus, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio owensii, and Vibrio shilonii. The results showed that eight fungal extracts displayed anti-Vibrio activity via the filter paper disk assay. Several of them showed strong inhibitory effects. Then, two tetramic acid alkaloids, equisetin (1) and 5′-epiequisetin (2), were identified from Fusarium equiseti BBG10 by bioassay-guided isolation, both of which inhibited the growth of Vibrio spp. with the MIC values of 86–132 μg/ml. The scanning electron microscope results showed that cell membranes of Vibrio became corrugated, distorted or ruptured after treatment with 1 and 2. Taken together, this study provided eight fungal isolates with anti-Vibrio potentials, and two alkaloid-type antibiotics were found with anti-Vibrio effects from the bioactive strain F. equiseti BBG10. Our findings highlight the importance of exploring promising microbes from the Beibu Gulf for the identification of anti-Vibrio for future antibiotic development.

Potential Assessment of Rumex spp. as a Source of Bioactive Compounds and Biological Activity

Secondary metabolites from the shoots and roots of three Rumex species collected from three different habitats were investigated (Rumex dentatus collected from cultivated land, R. pictus collected from the coastal desert and R. vesicarius collected from the inland desert) and tested for antioxidant activity as well as for anti-microbial activity against some human pathogenic bacteria. The present study indicated that the quantitative analysis of shoot and root extracts of three Rumex spp. were found to be rich in tannins and phenolics composition. The aerial parts of the three plants exhibited the highest significant values compared to the root parts. The MeOH extracts of Rumex species showed adequate antioxidant activity, wherein the IC50 values of the MeOH from the cultivated sample was 41.61 and 31.31 mg mL-1, coastal samples were 34.99 and 23.99 mg mL-1, while the sample of inland showed IC50 value of 41.59 and 31.67 mg mL-1, for root and shoot, respectively. Furthermore, using a filter paper disc assay, the MeOH extracts of the three Rumex species showed a substantial anti-microbial inhibitory effect on the growth of 10 pathogenic bacteria. According to sensitivity, the tested organisms could be sequenced as following: E. coli < K. pneumoniae ˂ S. typhi < P. aeruginosa for Gram-negative bacteria and B. subtilis < S. pneumoniae ˂ L. monocytoyenes < S. epidermis < S. aureus < B. cereus for Gram-positive bacteria. In addition, the antibacterial performance of R. dentatus root and R. vesicarius shoot MeOH extract is 100% broad spectrum against Gram-negative bacteria. A shoot of R. dentatus and R. pictus MeOH extract against Gram-positive bacteria is 83.3% broad spectrum. A further study is recommended for more characterization of the major compounds and assesses their efficiency and biosafety.

Antibacterial activity of Calotropis procera against some common human pathogens

Calotropis procera (Oshaar, family Asclepiadaceae) is a species of flowering plant used as a folk medicine for the treatment of various ailments. This study aimed to evaluate antibacterial and antioxidant activities of C. procera aerial parts collected from Eastern Desert of Egypt (Wadi Hagul). The different extracts of C. procera aerial parts showed a significant antimicrobial inhibitory effect (P < 0.05) on growth of nine pathogenic bacterial using filter paper disc assay. Qualitative and quantitative phytochemical screening was carried out on water and different extracts. The present study indicated that this plant has a good inhibitory activity against Klebsiella pneumonia (21.3 mm), Escherichia coli (22.5 mm), and Staphylococcus aureus (21.6 mm). Moreover, methanol extract of C. procera exhibited wide broad spectrum (100%) followed by petroleum ether and water extracts (60%, each). Activity index (AI) illustrated variable antibacterial activities, that support the folkloric usage of medicinal plants for treatment of infectious pathogenic microorganism. The crude extract of C. procera showed adequate antioxidant activity, wherein methanol, ethyl acetate and petroleum ether extracts have an IC50 value of 0.81, 0.88 and 1.09 mg mL-1, respectively. Therefore, phytochemicals from C. procera could be used as a raw material for producing cheaper pharmaceuticals, pesticides in addition to many more important commercialized products of public use.

Culture-dependent analysis of seed bacterial endophyte, Pseudomonas spp. EGN 1 against the stem rot disease (Sclerotium rolfsii Sacc.) in groundnut

BackgroundGroundnut, Arachis hypogaea L., crop is an important oil seed crop in India and is prone to attack by numerous fungal and viral diseases, among the soil-borne diseases, stem rot caused by Sclerotium rolfsii Sacc. is economically important.Main body of abstractIncidence (86.6%) of the stem rot disease was recorded in Coimbatore district, Tamil Nadu, India, during the roving survey. For its management, an attempt was made to isolate bacterial endophyte from peanut seeds of different groundnut cultivars by culture-dependent analysis. Totally, 16 bacterial endophytes (endophytes groundnut (EGN) 1 to EGN 16 along with standards TNAU-Pf1 and EPC 5) were obtained and characterized through morphological, biochemical and molecular studies and also phytostimulation activities were performed. Among the isolates, EGN 1 and EGN 4 showed positive results for indole acetic acid (IAA), siderophore, phosphate solubilization and protease tests in vitro. The dual culture analysis showed inhibition rates of 60.1% (dual plate assay), 68.23% (filter paper disc assay) and 100% (triangle method of streaking and culture filtrate assay) for EGN 1 against S. rolfsii. Further, the crude metabolite assay showed 97.7% inhibition in EGN 1, followed by 87.7% in EGN 4. The roll towel study showed a high vigour index of 4286.7 in EGN 1; hence, this isolate was chosen. Further, thin-layer chromatography (TLC) analysis showed various bands at 0.72 Rf, whereas GC-MS analysis indicated the prominent peaks of hexadecanoic acid and cis-vaccenic acid that may responsible for antifungal activity. In a molecular approach, the genomic DNA of EGN 1 strain was used to amplify a 1200 bp PCR-fragment and sequenced.Short conclusionThe overall outcome of this study showed that Pseudomonas spp. EGN 1 had a great potential as a bio-stimulant and biocontrol agent to manage effectively the stem rot in peanut.

In Vitro Study of Action Mode of Rhodotorula minuta Dmg 16 BEP as Biocontrol Agents on Alternaria solani

Rhodotorula sp. is widely known as a biocontrol agent and is reported effective in controlling several diseases on crops. Rhodotorula minuta Dmg 16 BEP is an antagonist yeast from Indonesia which was effective in controlling various plant diseases. The objective of this research was to study the in vitro mode of action of R. minuta Dmg 16 BEP against Alternaria solani. The antibiosis ability of R. minuta Dmg 16 BEP to A. solani might be based on the activity of volatile compounds and not from its metabolites products. The growth of A. solani in paper disk assay and dual culture tests were not inhibited nor inhibitory zones developed, whereas the inverse petridish test showed the growth inhibition of A. solani. The results of the chitinolytic activity test on chitin agar and proteolytic on skim milk agar showed that no clear zone was formed. R. minuta has a strong hyperparasitic ability according to the slide culture test as the yeast cells surrounded A. solani hyphae and caused damage to some parts of hyphae.

Comparative antibacterial and antifungal activities of sulfur nanoparticles capped with chitosan

The antimicrobial activity of sulfur nanoparticles (SNPs) was compared with elemental sulfur and sulfur-containing salts (sodium thiosulfate and sodium metabisulfite) against bacteria (Escherichia coli, Staphylococcus aureus) and fungi (Aspergillus flavus, Candida albicans) using the paper disc, broth microdilution, and time-kill assay methods. The results of the paper disc and MIC tests showed stronger antimicrobial activity of SNPs compared to the elemental sulfur and sulfur-containing salts. SNPs showed more potent activity against bacteria than fungi. Among the test microorganisms, E. coli (Gram-negative) was the most susceptible to SNPs, followed by S. aureus (Gram-positive), C. albicans (yeast), and A. flavus (mold). Scanning electron micrographs of microorganisms treated with SNPs showed different cell disruption patterns depending on the type of microorganisms.

Identification and Characterization of Antagonism Band of Secondary Metabolite from T. asperellum MK045610 against F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici based on HPTLC and GC-MS

The present investigation was carried out on identification and characterization of an antagonism band of extract secondary metabolites from Trichoderma asperellum MK045610 against Fusarium oxysporum f. sp. ciceri and Fusarium oxysporum f. sp. lycopersici. In this experiment, analysis was performed by High Performance Thin Layer Chromatography (HPTLC), paper disc assay, gas chromatography-Mass spectrometry (GC-MS). Firstly, extract crude secondary metabolites were used for partial purification based on HPTLC. Secondly, the paper disc assay method was used for the determination of antifungal property on PDA plate from the partial purified compound. Thirdly, GC-MS was used for identification of partial purified compound based on peaks. Identified compounds are named as Phenol, 3, 5-bis (1,1-dimethylethyl), Pentadecanoic Acid, 14-methyl, methyl ester, Benzenepropanoic acid, 3,5,-bis (1,1-dimethyl ethyl)-4-hydroxy-methyl ester and represented to antifungal property . Conclusively, Secondary metabolite of Trichoderma asperellum MK045610 has a significant role in radial growth inhibition of Fusarium oxysporum f. sp. ciceri and Fusarium oxysporum f. sp. lycopersici.

Identification and Characterization of Antagonism Band of Secondary Metabolite from T. asperellum MK045610 against F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici based on HPTLC and GC-MS

The present investigation was carried out on identification and characterization of an antagonism band of extract secondary metabolites from Trichoderma asperellum MK045610 against Fusarium oxysporum f. sp. ciceri and Fusarium oxysporum f. sp. lycopersici. In this experiment, analysis was performed by High Performance Thin Layer Chromatography (HPTLC), paper disc assay, gas chromatography-Mass spectrometry (GC-MS). Firstly, extract crude secondary metabolites were used for partial purification based on HPTLC. Secondly, the paper disc assay method was used for the determination of antifungal property on PDA plate from the partial purified compound. Thirdly, GC-MS was used for identification of partial purified compound based on peaks. Identified compounds are named as Phenol, 3, 5-bis (1,1-dimethylethyl), Pentadecanoic Acid, 14-methyl, methyl ester, Benzenepropanoic acid, 3,5,-bis (1,1-dimethyl ethyl)-4-hydroxy-methyl ester and represented to antifungal property. Conclusively, Secondary metabolite of Trichoderma asperellum MK045610 has a significant role in radial growth inhibition of Fusarium oxysporum f. sp. ciceri and Fusarium oxysporum f. sp. lycopersici.

Chemical compositions and biological activities of marine invertebrates from the East Sea of South Korea

BackgroundMarine invertebrates are well known as pivotal bioresources with bioactive substances such as anti-inflammatory sterols, antitumor terpenes, and antimicrobial peptides. However, there are few scientific reports on chemical compositions and bioactivities of marine invertebrates from the East Sea of South Korea.MethodsIn this study, chemical compositions and biological activities were evaluated on both 70% EtOH and hot water extracts of 5 species of marine invertebrates (Crossaster papposus japonicus, Actinostola carlgreni, Stomphia coccinea, Actinostola sp., and Heliometra glacialis) collected from the East Sea of South Korea. The antioxidant activities were measured by ABTS radical scavenging assay. The cytotoxicity and anti-inflammatory activity were evaluated using MTT and Griess reagents. Moreover, the antibacterial effect was evaluated using paper disc assay and minimum inhibitory concentration (MIC) assay.ResultsIn the results of antioxidant activities, 70% EtOH extract of A. carlgreni showed the highest activity (IC50 0.19 ± 0.03 mg/ml) compared to other extracts. Moreover, 70% EtOH extract of A. carlgreni could significantly suppress the nitric oxide (NO) production in lipopolysaccharide-induced RAW 264.7. All extracts treated under 400 μg/ml have no cytotoxic effects on RAW 264.7 macrophages. In the antibacterial test, both 70% EtOH extracts of C. papposus japonicus and H. glacialis showed a significant antibacterial effect on Staphylococcus aureus. The MIC values were evaluated at 256 and 512 μg/ml, respectively.ConclusionsThese results suggested the bioactive potentials of marine invertebrates from the East Sea of South Korea in pharmaceutical and nutraceutical applications.

Isolation and Characterization of Bioactive Compounds from Root of Rubus coreanus Miquel and their Antimicrobial Activity

Rubus coreanus Miquel (RCM), also known as Korean blackberry or bokbunja, is used as a South Korean traditional medicine to treat acne and inflammatory skin conditions. The antimicrobial activity of RCM root and its active compounds remain unclear. In this study, we prepared a 50% ethanol fraction, ethyl acetate fraction, and acid-treated ethyl acetate fraction (aglycone fraction) of RCM root, and evaluated antibacterial activities against the skin pathogens Staphylococcus aureus, Pseudomonas acnes, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. In a paper disc assay, all fractions of RCM root showed antimicrobial activities against the five skin pathogens. The ethyl acetate fraction displayed 6-, 12-, and 2-fold higher minimal inhibitory concentration (MIC) than the 50% ethanol fraction against S. aureus, E. coli, and P. acnes, respectively. The aglycone fraction displayed 2-fold higher MIC than methyl paraben against P. acnes, S. aureus, E. coli, and P. aeruginosa. The ethyl acetate fraction displayed a minimal bactericidal concentration (MBC) similar to that of methyl paraben, and the aglycone fraction showed 2- to 4-fold higher MBCs than those of methyl paraben. In particular, the ethyl acetate fraction was not cytotoxic and showed thermal stability after incubation at high temperatures (60-121℃). Finally, the ethyl acetate fraction was separated and four components were identified: procyanidin C, propelagonidin dimer, ellagic acid, and methyl ellagic acid acetyl pentose. The compounds showed high antibacterial activities. These results suggest that RCM root is potentially applicable as a natural preservative in cosmetics.

English

Rhizoctonia solani is the causative agent of rice sheath blight, which has become a major problem in rice production. Seaweed provides a rich source of structurally diverse and biologically active secondary metabolite and is proved to be better in decreasing foliar fungal diseases which ultimately increase its fertility and help in the growth of plants. The use of natural products is the ultimate way of combating this disease. In this context, five different seaweeds such as Sargassum wightii, Dictyota bartyrensiana, Ulva reticulate, Gelidiella acerosa and Odonus niger were used together with the fish powder extract in the control of sheath blight disease in rice were studied. Evaluation of marine products against R. solani was carried out by paper disc assay; agar well method and mycelial dry weight. Among the five marine extracts tested, extracts of S. wightii [brown seaweed algae] at a high concentration (20%) was found to be the best in the reduction of spore germination (19.60%). The leaf extracts of S. wightii [brown seaweed algae] at highest concentration of 20% showed a maximum reduction in both paper disc method and agar well method with 44.65 and 45.90% zone of inhibition, respectively. The antifungal compounds were identified through gas chromatography mass spectroscopy. The results revealed that, 18 compounds were present in S. wightii and among that n-hexadecanoic acid which was closely related to 9, 12-octadecadienoic acid may be responsible for the inhibition of the growth of R. solani. The present study revealed that the efficacy of seaweed extracts against fungal pathogens may be due to higher levels and early accumulation of phenolics and phytoalexins, and the pot study proved that R. solani can be controlled by the application of marine products which may be further used for field study. Key words: Seaweeds, Rhizoctonia solani, antifungal compounds, rice.

Statistical optimization of culture medium for improved production of antimicrobial compound by Streptomyces rimosus AG-P1441.

The nutritional requirements for antimicrobial activity of Streptomyces rimosus AG-P1441 were optimized using statistically-based experimental designs at a flask level. Based on a one-factor-at-a-time (OFAT) approach, glucose, corn starch and soybean meal were identified as the carbon and nitrogen sources having a significant effect on antimicrobial productivity. As a result of investigating the effect of glucose concentration, the highest antimicrobial activity was observed at 3% concentration. Response surface methodology (RSM) was then applied to optimize the growth medium components (corn starch, soybean meal, MgCl2 and glutamate). Antimicrobial productivity increased sharply when the medium consisted of 3% glucose, 3.5% corn starch, 2.5% soybean meal, 1.2mM MgCl2 and 5.9mM glutamate. The fermentation using optimized culture medium in a 5-L bioreactor allowed a significant increase in antimicrobial activity, evaluated by the paper disc assay, revealed a 29mm inhibition zone diameter against Phytophthora capsici.

Antifungal activity of pinosylvin fromPinus densifloraon turfgrass fungal diseases

The objective was to examine the antifungal activity of Pinus densiflora extract for the control of turfgrass fungal diseases. Antifungal activities of the various fractions of n-hexane, methylene chloride (Ch), ethyl acetate (EtOAc), and n-butanol from P. densiflora were evaluated against Rhizoctonia solani AG1-1B, R. solani AG2-2IV, Sclerotinia homoeocarpa, R. cerealis, Pythium spp., and Colletotrichum graminicola. The Ch and EtOAc fractions showed antifungal activity against Pythium sp. and C. graminicola in paper disc assay. The effective concentration to produce 50% mycelial inhibition (EC50) using five discriminatory concentrations of pinosylvin (1) from the Ch fraction of P. densiflora was evaluated on R. solani AG1-1B, R. solani AG2- 2IV, R. cerealis, and S. homoeocarpa. S. homoeocarpa showed the highest sensitivity with the lowest mean EC50 value (8.426 μg/ mL) among the four pathogens. Among the three Rhizoctonia pathogens, R. cerealis had the highest mean EC50 value (99.832 μg/mL) and R. solani AG2-2IV, with the lowest sensitivity, had the lowest EC50 value (39.696 μg/mL). These results suggested that pinosylvin (1) from P. densiflora could be a valuable lead compound in the improvement of a novel antifungal agent.

Antimicrobial activities of green tea extract on the retardation of kimchi fermentation

Kimchi fermentation is complex process which really depends on the composition of microfloa under the optimum ripening environment such as salt concentration, acidity, pH and temperature conditions. Most kinds of bacteria belonging to Lactobacillus group have been identified in kimchi, which continuously produce lactic acids after ripening and cause the change of kimchi tasty and physical texture. Green tea extract as a natural preservative was investigated on the antimicrobial activity and the change of pH, in terms of retardation of kimch fermentation. Minimal Inhibitory Concentration (MIC), paper disc assay, bacterial growth, and pH change were tested against four microorgasnisms, Leuconostoc mesenteroides, Lactobacillussake, Lactobacillus plantarum, and Weissellakoreensis. 2 mg/mL of green tea extract completely inhibited the growth of test organisms after 5–7 hours. Furthermore, it showed the maximized clear zone (24–34 mm) was observed at the 2mg/disc concentration. pH change resulted in a slow downward drift for fermentation period. Moreover, green tea extract showed long-term buffer effects for over 35 days (5 weeks) at 4°C.

한약재 및 해조류 추출물에 의한 고등어 육중의 Histamine 생성 억제 효과

This study was conducted in order to investigate the inhibitory effects of natural materials on histamine production in mackerel. Antimicrobial activities on Photobacterium phosphoreum of medicinal herbs and seaweeds were investigated using the paper disc assay and MIC (minimum inhibitory concentration) test. According to the results, Sargassum sagamianum and Ecklonia cava ethanol extracts exhibited antibacterial activity. In particular, Sargassum sagamianum ethanol extract showed excellent antibacterial activity at 0.015625 mg/mL by the MIC test. Anti-histamine release activities of natural materials were further investigated by examining their inhibitory effects on histidine decarboxylase (HDC) activity in the crude enzyme preparation from Photobacterium phosphoreum. The ethanol extracts of Ecklonia cava and Eisenia bicyclis exhibited the strongest HDC inhibitory activity, with 32% and 22%, at a concentration of 1 mg/mL, respectively. Therefore, natural materials may reduce histamine poisoning through decrease of histamine production in mackerel.

Purification of an antifungal compound, cyclo(l-Pro-d-Leu) for cereals produced by Bacillus cereus subsp. thuringiensis associated with entomopathogenic nematode

Mold spoilage is the main cause of substantial economic loss in cereals and might also cause public health problems due to the production of mycotoxins. The aim of this study was to separate and purify and to identify antifungal compounds of bacterium associated with novel entomopathogenic nematode and check the antifungal property of identified compound in particular food model systems. The antifungal compound was purified using silica gel column chromatography, TLC and HPLC and its structure was elucidated using NMR (1H NMR, 13C NMR, 1H–1H COSY, 1H–13C HMBC), HRMS and Marfey's method. Based on the spectral data, the active compounds were identified as diketopiperazine [cyclo(l-Pro-d-Leu)]. The antifungal activity of cyclo(l-Pro-d-Leu) was studied by MIC and paper disk assay against Aspergillus flavus MTCC 277 and Aspergillus niger MTCC 282 and best MIC value of 8μg/ml was recorded against A. flavus. Cyclo(l-Pro-d-Leu) strongly inhibit mycelia growth of fungus and thereby affecting aflatoxin production. To investigate the potential application of the cyclo(l-Pro-d-Leu) and to eliminate fungal spoilage in food and feed, soybean and peanut were used as models. White mycelia and dark/pale green spores of A. flavus were observed in the control soybeans after 2-day incubation. However the fungal growth was not observed in soybeans treated with cyclo(l-Pro-d-Leu). Almost the same result was observed for peanuts treated with cyclo(l-Pro-d-Leu) for A. niger. The cyclo(l-Pro-d-Leu) was nontoxic to two normal human cell lines (FS normal fibroblast and L231 lung epithelial) up to 200μg/ml. Thus the diketopiperazine derivative identified in the study may be a promising alternative to chemical preservatives as a potential biopreservative which prevent fungal growth and mycotoxin formation in food and feed.

Hydroxamate-based colorimetric method for direct screening of transglutaminase-producing bacteria

Microbial transglutaminase (MTGase) is a commercial enzyme that has been applied to many protein containing foods to improve their textural property. The screening of MTGase-producing microorganisms from various sources might lead to the discovery of a new MTGase with different characteristics. This report demonstrates the use of a direct detection method for MTGase-producing bacteria grown on an agar plate by filter paper disc (FPD) assay. The principle of the assay is the formation of a red burgundy color by the hydroxamate-ferric complex. The color developed intensity was linearly correlated by the concentration of hydroxamic acid in the range of 0.1-0.8 μM and was visually scored at 4 levels: 0, 1, 2 and 3. Streptoverticillium mobaraense DSM 40847, a positive MTGase-producer, was chosen for the verification and improving of the proposed method. The colonies grown on the nutrient agar plate at 37°C for 24 h were covered with FPDs and 30 μl of substrates (CBZ-Gln-Gly and hydroxylamine). After incubation, 10 μl of the ferric-TCA-HCl solution was placed on the FPD. The optimal time taken to catalyze the formation of CBZ-Gln-Gly-hydroxamic acid by the MTGase and the time taken for the hydroxamate-ferric complex to form color were 180 and 60 min, respectively. Using this assay, 30 of 189 colonies isolated from wastewater and floating-floc samples showed MTGase-positive colonies which were well correlated to the quantitative screening of MTGase activity (R(2) = 0.9758). The results revealed that the FPD assay could be used for the qualitative screening of MTGase-producing bacteria.

In vitro evaluation of solvent extracted compounds from edible macromycetes against phytopathogenic fungi

Extraction of antimicrobial compounds from medicinal mushrooms, namely, Auricularia polytricha (Jew's ear), Lentinulla edodes (Shiitake) and Volvariella volvacea (paddy straw), has been done with different solvent systems (chloroform, ethyl acetate, ether and methanol) and tested against a wide range of phytopathogens by filter paper disk assay. All the three basidiomycetes inhibited the phytopathogens tested so far. This enumeration was based on the number of organisms inhibited and the diameter of inhibitory zones produced. From the results obtained, it could be observed that ethyl acetate was the best solvent for extracting antimicrobial substances from these medicinal mushrooms. Thin layer chromatography revealed the presence of several compounds with different Retention Factor (RF) values. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis profiling of V. volvacea showed the presence of several antimicrobial proteins with various molecular weights. Western blotting revealed the presence of thaumatin-like glycoproteins of molecular weight more than 45 kDa.