Microbial Type Culture Collection Research Articles

Exploring prebiotic properties and its probiotic potential of new formulations of soy milk-derived beverages

IntroductionThe food and beverage industry has shown a growing interest in plant-based beverages as alternatives to traditional milk consumption. Soy milk is derived from soy beans and contains proteins, isoflavones, soy bean oligosaccharides, and saponins, among other ingredients. Because of its high nutritive value and versatility, soy milk has gained a lot of attention as a functional food.MethodsThe present work aims to explore the prebiotic properties and gastrointestinal tolerance potential of new formulations of soy milk-derived drinks to be fermented with riboflavin-producing probiotic Lactiplantibacillus plantarum MTCC (Microbial Type Culture Collection and Gene Bank) 25432, Lactiplantibacillus plantarum MTCC 25433, and Lactobacillus acidophilus NCIM (National Collection of Industrial Microorganisms) 2902 strains.Results and discussionThe soy milk co-fermented beverage showed highest PAS (1.24 ± 0.02) followed by soy milk beverages fermented with L. plantarum MTCC 25433 (0.753 ± 0.0) when compared to the commercial prebiotic raffinose (1.29 ± 0.01). The findings of this study suggested that the soy milk beverages exhibited potent prebiotic activity, having the ability to support the growth of probiotics, and the potential to raise the content of several bioactive substances. The higher prebiotics activity score showed that the higher the growth rate of probiotics microorganism, the lower the growth of pathogen. For acidic tolerance, all fermented soy milk managed to meet the minimal requirement of 106 viable probiotic cells per milliliter at pH 2 (8.13, 8.26, 8.30, and 8.45 logs CFU/mL, respectively) and pH 3.5 (8.11, 8.07, 8.39, and 9.01 log CFU/mL, respectively). The survival rate of soy milk LAB isolates on bile for 3 h ranged from 84.64 to 89.60%. The study concluded that lactobacilli could thrive in gastrointestinal tract. The sensory evaluation scores for body and texture, color, flavor, and overall acceptability showed a significant difference (p < 0.05) between the fermented probiotic soy milk and control samples. Soy milk fermented with a combination of L. plantarum MTCC 25432 & MTCC 25433 demonstrated the highest acceptability with the least amount of beany flavor. The findings of the study suggest soy milk’s potential in plant-based beverage market.

Comparative expression profile of selected genes in Venturia inaequalis Cooke (Wint.) infecting apple fruits and leaves

Scab is a noxious fungal disease of apples (Malus domestica Borkh) in all apple producing regions caused by a host-specific phytopathogen Venturia inaequalis Cooke (Wint.). Its symptoms include black or brown lesions on leaves as well as on fruits, and causes fruit deformation. In this study, V. inaequalis was isolated from infected fruit samples and expression level of selected pathogenesis related genes in scab infected fruits and leaves was studied. The isolated fungus formed a brown coloured mat-like colony having septate mycelium and produced acute ovoid shaped conidia. Molecular characterization of the fungus was done by amplification and sequencing of internal transcribed spacer (ITS) and Cytochrome (CYP51A1) regions of the isolate. The Sanger sequencing of amplicons and sequence analysis using BLAST tool of the NCBI against GenBank database confirmed that the obtained sequence of ITS and CYP51A1 regions had 99.6 % and 98 % sequence identity with the reference V. inaequalis, respectively. The isolated strain of V. inaequalis was submitted in the fungal repository of Microbial Type Culture Collection (MTCC), India. The phylogenetic analysis of ITS region of the isolate revealed that the isolated strain of V. inaequalis was more closely related to Fusicladium eriobotryae compared to other Venturia species. The expression levels of pathogenesis related genes viz. CIN1, CE5 and VICE16 were higher in infected leaves as compared to infected fruits. Whereas expression of VICE12 and GH28 were higher in infected fruits compared to infected leaves. VICE4 gene showed equal level of expression during infection in both fruits and leaves, which was also maximum as compared to the control treatment. The transcript analysis of aforementioned genes of V. inaequalis gives the insights of the pathogen's expression profile during pathogenesis in fruits and leaves. This expression analysis can form a basis selecting pathogenesis related genes as targets for developing dsRNA expressing constructs with an aim to reduce RNAi mediated silencing of specific genes, in a futuristic biotechnological based solution to deal with apple scab disease.

Interrogating Salmonella Typhi biofilm formation and dynamics to understand antimicrobial resistance

AimsSalmonella Typhi biofilm-mediated infections are globally rising. Due to the emergence of drug resistance antibiotics did not show effective results against S. Typhi biofilm. Therefore, there is an urgent need for an in-depth interrogation of S. Typhi biofilm to understand its formation kinetics, compositions, and surface charge value. MethodsThis study utilized the S. Typhi MTCC-733 strain from a microbial-type culture collection in India. The S. Typhi biofilm was formed on a glass slide in a biofilm development apparatus. Typhoidal biofilm analysis was done with the help of various assays such as a crystal violet assay, SEM analysis, FTIR analysis, Raman analysis, and zeta potential analysis. Key findingThis article contained a comprehensive assessment of the typhoid biofilm formation kinetics, biofilm compositions, and surface charge which revealed that cellulose was a major molecule in the typhoidal biofilm which can be used as a major biofilm drug target against typhoidal biofilm. SignificanceThis study provided interrogations about typhoidal biofilm kinetics which provided ideas about the biofilm composition. The cellulose molecule showed a major component of S. Typhi biofilm and it could potentially involved in drug resistance, and offer a promising avenue for developing a new antibiofilm therapeutic target to conquer the big obstacle of drug resistance. The obtained information can be instrumental in designing novel therapeutic molecules in the future to combat typhoidal biofilm conditions effectively for overcoming antibiotic resistance against bacterial infection Salmonella.

Statistical optimization of process variables for anti-hypercholesterolemic metabolites production from Monascus purpureus MTCC 369 fermented finger millet

Monascus purpureus fermented rice has been used for centuries to treat hypercholesterolemia, indigestion, and diarrhea in East Asian countries. Monacolin K, also known as lovastatin, a secondary metabolite synthesized by Monascus sp. in fermentation, is mainly responsible for reducing the level of blood lipids. Besides statins, sterols produced in this process have also been reported to have hypolipidemic effects. In the current study, statin and total sterol production by M. purpureus microbial type culture collection and Gene Bank MTCC 369 on finger millet (FM) substrate were investigated. The central composite design of the response surface methodology (RSM) was used for the optimization of different fermentation process independent variables such as fermentation time period, temperature, inoculum volume, and pH for maximum production of statins and total sterols by M. purpureus MTCC 369. The maximum statin (9.78 mg/g) and sterol (0.46 mg/g) production was achieved at a temperature of 29.44°C, pH 5.0, inoculum size 5.37 ml, and fermentation time of 13.8 days. Fermentation process factors such as fermentation time period and temperature were significant positive factors, while pH and inoculum volume were found to be insignificant factors. The interactive effects of the positively influencing variables have been demonstrated. Compared with the traditional production of these metabolites, using FM as a substrate is promising for the high production of anti-hypercholesterolemic metabolites. The current study was aimed at optimization of the solid-state fermentation process of statin and total sterol using RSM for nutritional FM so as to improve M. purpureus-fermented FM (like red yeast rice) with health-promoting properties. This is the first report on the optimization of statin and sterols from M. purpureus MTCC 369 fermented FM with a significant yield that is on par with that reported so far.

Acrylonitrile adducts: design, synthesis and biological evaluation as antimicrobial, haemolytic and thrombolytic agent

In this work, five acrylonitrile adducts were screened for antibacterial activity against Gram-positive Bacillus subtilis, Microbial Type Culture Collection and Gene Bank (MTCC 1305) and Gram-negative Escherichia coli (MTCC 443). Synthesis was followed by aza-Michael addition reaction, where the acrylonitrile accepts an electron pair from the respective amines and results in the formation of n-alkyliminobis-propionitrile and n-alkyliminopropionitrile under microwave irradiation. Characterization of the compounds were performed using Fourier Transform Infrared (FTIR), Proton Nuclear Magnetic Resonance (1H NMR) and Electrospray Ionisation Mass Spectrometry (ESI–MS). The particle size characterization was done by Dynamic Light Scattering (DLS) technique. The antibacterial study showed higher inhibition rate for both Gram-positive and Gram-negative bacteria. The antibacterial ability was found to be dose dependent. The minimum inhibitory concentration against both bacteria were found to be 1, 3, 0.4, 1, 3 µl/ml for E. coli and 6, 6, 0.9, 0.5, 5 µl/ml for B. subtilis. Time-kill kinetics evaluation showed that the adducts possess bacteriostatic action. Further it was evaluated for high-throughput in vitro assays to determine the compatibility of the adducts for drug delivery. The haemolytic and thrombolytic activity was analysed against normal mouse erythrocytes. The haemolytic activity showed prominent results, and thereby projecting this acrylonitrile adducts as potent antimicrobial and haemolytic agent.

First Report of Neoscytalidium dimidiatum Causing Stem Canker of Dragon fruit (Hylocereus spp.) in India.

Dragon fruit (Hylocereus spp.) is a tropical perennial plant (Family Cactaceae) that is popular for consumption in India. Originated from Central and South America and currently became popular in India. Hylocereus undatus and H. polyrhizus are the most commonly cultivated species in India. During August to December, 2021, stem cankers were observed in commercial orchards within the Satara, Pune and Solapur districts of Maharashtra. The disease incidence across four orchards was approx. 40% and severity ranged between 30 and 80%. Initial symptoms on infected cladodes were minute, circular, depressed chlorotic spots often with a brick red flecks followed by elevation of the centre of the lesion. Later the lesions turned necrotic and contained black, erumpent pycnidia, followed by chlorosis and stem rot. Twelve diseased cladodes from different orchards were collected and isolations were conducted. Edges of the lesions (5 to10 mm2) were excised and surface sterilized by exposure to 1% sodium hypochlorite (2 min) followed by triple rinsing with sterilized distilled water. Excess moisture was removed with sterilized blotter paper and pieces were plated on potato dextrose agar (PDA) amended with streptomycin sulphate (30 mg/L) for 3 days at 27 ± 2°C with a 12 h photoperiod. All twelve isolation attempts yielded uniform fungal colonies and out of these, purified colonies from each location viz., SLNeo, LNeo, MGNeo and KNeo were selected for etiology study. On PDA, initially white mycelial colonies turned to olive green to grayish with dark gray to black pigmentation. Colony growth was rapid (30 mm/day). Brown, branched septate hyphae fragmented to produce abundant arthroconidia in chains that were hyaline to dark brown, thick walled, 0 to 1 septate, ellipsoid to ovoid (10.1 ± 1.4 × 4.88 ± 1.1 μm), rod (9.0 ± 0.8 μm × 5.3 ± 0.8 μm), round (7.9 ± 1.6 μm) and capsule (14.2 ± 3.1 × 6.1 ± 0.8 μm) shape. Conidia from pycnidia developed on host tissues were aseptate, hyaline and ellipsoid-cylindrical (8.2 ± 0.8 × 2.2 ± 0.3 μm) in shape. Based on morphological characteristics, the isolates were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). Molecular characterization was done by amplifying the partial internal transcribed spacer (ITS) region, and translation elongation factor 1-α and β-tubulin genes of four isolates using ITS1/ITS4, EF1-728F/EF1-986R and BT2A/BT2B primers (White et al. 1990, Glass and Donaldson 1995, Carbone and Kohn 1999). Sequences were deposited in GenBank accesions (ITS: OM884028, OM884029, OM884030, OM899800; TUB2: OM927962, OM927963, ON099066; TEF1-α: OM927965, OM927966, OM927964, OM984744) showed 99 to 100% identity with the epitype CBS 499.66 accession numbers KF531820 for ITS and KF531798 for TEF1-α and KF531800 for TUB2. N. dimidiatum isolate designated as SLNeo has been deposited at Microbial Type Culture Collection (MTCC), Chandigarh, India with MTCC 13250. Phylogenetic analysis using maximum likelihood (ML) revealed these isolates were clustered with N. dimidiatum clade. To evaluate pathogenicity, stems of one year old dragon fruit plants were wounded with a sterilized needle and inoculated with 7 mm fresh mycelial discs. For each isolate three replications were kept. Non-inoculated controls were plants receiving only non-colonized PDA discs. Inoculated and control plants were kept separately at ambient temperature (35 ± 2°C during day and 22 ± 2°C at night). Symptom started to develop at 2 days after inoculation (DAI) and canker lesions with chlorotic halos and rotting were observed at 15 DAI. No symptoms were observed on the negative control. Pathogenicity testing was repeated twice and the pathogen was re-isolated from symptomatic tissues with 100% re-isolation frequency and found identical with N. dimidiatum. N. dimidiatum has been reported on H. undatus and H. polyrhizus in subtropical and tropical countries worldwide (Chuang et al. 2012; Ezra et al. 2013; Lan et al. 2012; Sanahuja et al. 2016; Serrato-Diaz and Goenaga 2021). To our knowledge, this is the first report of dragon fruit stem canker caused by N. dimidiatum from India. Since the disease poses a major threat to dragon fruit plantations, additional epidemiological studies may assist in developing management strategies.

Endophytic Streptomyces rochei BpR-2 GER (ON142045) from Bryophyllum pinnatum (Lam.) Oken with Antimicrobial and Antibiofilm Potential

Objectives: To explore the endophytic actinobacteria from the root tissue of Bryophyllum pinnatum (Lam.) Oken and its assessment for antimicrobial and antibiofilm activity against human pathogens. Methods: Surface sterilized root tissues of B. pinnatum have been seeded in different isolation media, to obtain colonies of endophytic actinobacteria. A polyphasic approach has been used to identify the isolate. Cross streak and disc diffusion methods have been used to perform the antimicrobial activity. However, the crystal violet assay assessed the antibiofilm activity, and its visualization was done under the light and fluorescent microscope. Findings: By polyphasic approach, Actinobacteria strain BpR-2 GER has been confirmed to be the member of genus Streptomyces, further through 16S rRNA gene sequence analysis, the strain has been identified as Streptomyces rochei that has 100% sequence similarity with Streptomyces rochei NRRL B-2410 (MUMD01000370). The isolate has exhibited extensive antagonistic interaction with Clinical and MTCC (Microbial Type Culture Collection) pathogens ranging from 9.750.71 to 19.381.30 mm. The highest zone of inhibition was recorded against Gram-positive bacteria B. subtilis (MTCC-441) of 19.381.30 mm and Gram-negative bacteria Proteus mirabilis (Clinical) of 16.250.71 mm. The strain has also exhibited significant antibiofilm potential against the MTCC-7443 (85.130.003 to 88.280.003%) and Clinical (85.130.003 to 88.280.003%) strain of Staphylococcus aureus. Novelty: This is the first report of S. rochei BpR-2 GER, a root endophyte of B. pinnatum with broad-spectrum antimicrobial activity and a significant antibiofilm property. Keywords: Antimicrobial activity; Antibiofilm activity; Bryophyllum pinnatum; Endophytic Actinobacteria; Streptomyces rochei

Cladosporium cladosporioides causes leaf blight on garden pea in Telangana, India.

Garden pea (Pisum sativum L.) is an important vegetable and pulse crop grown worldwide. Leaf blight symptoms with chlorotic lesions were observed on the pea genotypes, 2006-2008A and Arkel in field conditions at Asian Vegetable Research and Development Centre (AVRDC), Hyderabad (17°23' N; 78°29' E) and greenhouse conditions at University of Hyderabad, Hyderabad, India (17° 27' N; 78° 19' E) since 2010. The blight symptoms showed black lesions coated by a fine velvety layer of olive-grey conidia. The disease incidence on field and greenhouse pea plants ranged from 3-6%. The Cladosporium isolates, Gp01 and Gp02 were isolated from pea genotypes, Arkel and 2006-2008A in greenhouse and field conditions respectively. One fungus was predominantly isolated from infected leaf tissue plated onto potato dextrose agar (PDA), and it was identified as Cladosporium sp. based on colony morphology and conidial appearance. Single-spored cultures grown at 26±2 °C on PDA showed olive green mycelia on the obverse-side and dark green on reverse-side (Fig. 1 a,b). The conidia were limoniform with dimensions ranging from 5.91-7.33 x 3.19-4.58 µm (Table 1) and were produced on solitary conidiophores containing 1-2 septa, with no constriction whereas the aerial hyphae were septate and unbranched or rarely branched (Fig.1 c-d). To verify the pathogenicity of the fungus, detached leaf assay was carried on 30-day old Arkel leaves by point inoculation with 20 µL (106 spores/ml) spore suspension using a needle-less syringe to infiltrate the spores into the abaxial surface and the inoculated leaves were incubated at 26±2 °C for 3 days in petri plates containing moistened autoclaved absorbent cotton. Pea leaves inoculated with water served as controls. The experiment was repeated with a minimum of three times with five leaves per replication and three replications per experiment for each of the isolates. The appressorial germ tube formation was observed by scanning electron microscopy (SEM) at 12 hours post inoculation (hpi) (Fig.1 e-f). Thirty-day old pea plants were spray-inoculated with a spore suspension of 1-2 ml per plant with a concentration of 106 spores/ ml while the control plants were mock-inoculated (sprayed) with water. 5 plants per replicate per isolate with three replications per experiment and a minimum of three experiments were carried. The inoculated plants were covered with polythene zip-lock bags for two days post inoculation (dpi) and maintained in greenhouse at 26±2 °C with natural photoperiod of 14 hours. Wilting and necrosis symptoms accompanied by chlorotic lesions were observed on pea plants at 7 days post inoculation (dpi) (Fig. 2). Complying with Koch's postulates, the pathogen, which was re-isolated from the first appearance at 3 dpi from infected pea showed similar morphological characteristics as those used for inoculation. Sequencing analysis of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers (White et al. 1990), D1-D2 region sequencing (Kwiatkowski et al. 2012), Translation elongation factor 1-alpha (TEF) and Actin (ACT) gene sequencing (Nam et al. 2015) of Gp01 and Gp02 showed significant similarity with C. cladosporioides (Table S1). Further, concatenated phylogenetic trees constructed by maximum likelihood method using all the four (ITS, D1-D2, TEF and ACTIN) gene sequences of these isolates along with other reported Cladosporium species (Fig. S1a) and specific isolates of C. cladosporiodes which are reported as "sensu stricto" (Bensch et al. 2010) collected from different substrata (Fig. S1b) confirmed them as C. cladosporioides. Gp01 and Gp02 were also confirmed by Microbial Type Culture Collection (MTCC, India) as C. cladosporioides with Acc. No. MTCC 9994 and MTCC 9995. C. cladosporioides has been previously reported on Dalbergia sp. and Eucalyptus sp in India and on garden pea in other countries (Bensch et al. 2010). However, owing to the impact of this pathogen on growth and productivity of pea crop, further in-depth study needs to be carried. To our understanding this is the first report of Cladosporium cladosporioides infecting garden pea in India.

Gas Chromatography-Mass Spectrometry (GC-MS) Metabolite Profiling of Citrus limon (L.) Osbeck Juice Extract Evaluated for its Antimicrobial Activity Against Streptococcus mutans.

Purpose The present study aimed to determine the antimicrobial nature of Citrus limon juice extract against Streptococcus mutans and to identify its metabolic profile by gas chromatography-mass spectrometry (GC-MS) technique. Materials and methods The cariogenic bacteria S. mutans were procured from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India, and revived on brain heart infusion (BHI) agar. The C. limon (L.) Osbeck fruits were authenticated from the University of Trans-Disciplinary Health Sciences and Technology (TDU), Bengaluru, India. The antibacterial property of lyophilized lemon juice extract (LJE) dissolved in methanol was evaluated against S. mutans (MTCC 497) by the agar well diffusion assay. GC-MS technique was employed to identify the volatile metabolite profile of the methanolic LJE sample. The metabolite masses of the respective compounds were identified using the National Institute of Standards and Technology (NIST) library. Results The methanolic LJE sample concentration from 5 to 25 mg/ml did not demonstrate antimicrobial activity, while 30 to 100 mg/ml displayed antibacterial activity against S. mutans. Chlorohexidine (100 µg/ml) was used as the positive control, while methanol was used as the negative control. Significant antimicrobial metabolites were detected in the methanolic LJE sample by GC-MS analysis. Maleic anhydride, succinic anhydride, 6-Oxa-bicyclo[3.1.0] hexan-3-one, and 3-methyl-2,5-Furandione were the key metabolites identified in the methanolic LJE sample. Conclusion The present study reports that C. limon is a potential candidate for the pharmaceutical industry as it possesses bioactive compounds demonstrating therapeutic properties. Further investigations are warranted to determine the individual and synergetic effects of identified metabolites in the methanolic LJE sample for its antimicrobial property. Special attention needs to be focussed on translational research for the development of anti-caries products from C. limon.

Plant growth promoting, biochemical and antifungal properties of microbes isolated from cow dung

Cow dung is a rich source of microbial diversity and being used since long time in India for seed treatment, plastering cut ends of vegetative propagated plant parts and sprinkling of diluted solution on crops. In present study, microbes from fresh cow dung of indigenous cow were isolated and biochemically characterized. Cow dung enriched with diversity of microorganisms in different medium i.e 38 x 106cfu g-1 (Nutrient agar), 26.0 x 106 cfu g-1 (Actinomycetes isolation agar), 36 x 106 cfu g-1 (Kings B agar), 16 x 106 cfu g-1(CV agar),13 x 106 cfu g-1 (Methyl red), 15 x 106 cfu g-1 (Pikovskyas agar), 30 x 106 cfu g-1 (CRYEMA), Azospirillum 70 x 106 cfu g-1 (N-free maleate) and 30 x 106 cfu g-1(Jenson agar medium). Based on microbial enumeration, total 18 bacterial isolates were selected for biochemical, PGPR characterization and biocontrol activity. All isolates were tested positive for phosphate and Zn solubilisation. Isolate CD2, CD5, CD13, CD14 and CD19 were tested highly positive for zinc, phosphate, K-solubilisation, IAA and amylase production. Test isolates CD2, CD5, CD6, CD7, CD10 and CD13 were tested highly positive for β-galactosidase activity, ornithine utilization, nitrate reduction, citrate utilization, malonate utilization, esculin hydrolysis, trehalose, glucose and lactose utilization. All isolated microbes tested for biocontrol activity (antifungal property) against standard pathogen procured from microbial type culture collection (MTCC), i.e. C. gloeosporioides (MTCC 2190), F. oxysporum (MTCC 10247), C. fimbriota (MTCC 2281) and P. aphanidermatum (MTCC 284). CD2, CD34 and CD20 isolates significantly inhibited the growth of C. gloeosporioides and P. aphanidermatum. Result of the study advocates that cow dung and cow dung based formulations are cheap source for soil, plant health and management of Colletotrichum gloeosporioides, P. aphanidermatum and Fusarium oxysporum in various crops.

Mechanical Properties of Bacterial Concrete by Partial Replacement of Cement by Nano Silica in Different Grades of Concrete

Concrete with nano-silica also has a higher strength compared with normal concrete. In the current study, an examination of the mechanical characteristics of bacterial concrete containing nano-silica is conducted. The impact of concrete was examined in this study employing Bacillus subtilis for self-Healing Microbial Type Culture Collection (MTCC) Strain no.121, Among the bacteria concentrations of 104 , 105 , and 106 cells/ ml, a concentration of 105 cells/ml gives more strength, so further investigation is done using 105 cells/ml cell concentration for different percentages of nano-silica is examined. Firstly, Bacterial cell concentration is optimised. Each of the 40 sets of cubes (150 mm x 150 mm x 150 mm), cylinders (150 mm diameter and 300 mm height), and prisms (100 mm x 100 mm x 500 mm) are cast and tested at 28 days and 90 days as part of further research to determine the mechanical properties of bacterial concrete with different percentage of nano silica with and without bacteria. It has been noted that adding nano-silica to concrete increases its strength from 0% to 1.5% before being decreased to 2%. concrete splintering occurs due to a number of causes, including mechanical compression and low tensile strength, shrinkage, freeze-thaw reaction, and others. External loads can cause high tensile strains. For any combined nano-silica percentage when compared to regular concrete, bacterial concrete is stronger.

Synthesis of novel azole functionalized trifluoromethyl pyrido[2,3-d] pyrimidinone derivatives and their antimicrobial activity

Novel azole functionalized pyrido[2,3-d] pyrimidinone derivatives 6a-l were prepared starting from 2-Amino- 6-(thiophen-2-yl)-4-(trifluoromethyl)nicotinonitrile 1 reacting with trifluoroacetic acid in the presence of conc. H2SO4 and further reaction with bromoethyl acetate to obtain 3a and 3b. Compounds 3a and 3b further reacted with hydrazine hydrate to afford hydrazide compounds 4a and 4b. Compounds 4a and 4b on treating with phenylisothiocyanate give compound 5a and b. Compound 5a and 5b on reaction with different reagents (NaOH, H2SO4 and N2H4.H2O) produced different azole-substituted (triazolothione, thiadiazole, triazole) functionalized trifluoromethyl pyrido[2,3-d] pyrimidinone derivatives 6a-l respectively. All the products 6a–l were screened against gram +ve, gram –ve bacteria and fungal strains. Compounds 6ad showed promising activity against Bacillus subtilis microbial-type culture collection (MTCC) 121 at <25.6 micromolar concentration. Promising compounds were further screened for minimum bactericidal concentration against B. subtilis MTCC 121 using ciprofloxacin as standard reference and found to show very good activity. These compounds also screened for biofilm inhibition activity against B. subtilis MTCC 121 using erythromycin as standard confirming the high activity.

Influence of Trichoderma spp on Macrophoma theicola branch canker disease in South Indian tea gardens, India

Abstract Branch canker disease caused by the fungus Macrophoma theicola is a major stem disease that reduces the yield of south Indian tea plantations. Hence the present study aimed to assess the efficacy of the biocontrol agent Trichoderma spp against various isolates of Macrophoma spp. For this matter, different tea-growing regions of south India were surveyed for the isolation and characterization of Macrophoma spp. Then, fungal biocontrol strains (Trichoderma viride, Trichoderma atroviride, Trichoderma harzianum, and Gliocladium virens) were procured from microbial type culture collection Centre (MTCC) to screen their antagonistic potential on different isolates Macrophoma spp. The spores of Macrophoma spp were examined through a light microscope and identified by their peculiar morphological features such as non-septum pycnidiospores present in the sac and oval shape spore with stalk and confirmed using 18S rRNA gene sequence. The results revealed that the biocontrol G. virens followed by T. harzianum showed a higher inhibitory effect on different isolates of Macrophoma spp in the dual plate and culture filtrate studies. In the well diffusion method, the fungal biocontrol agents were found to be exhibit non-significant differences on different isolates of branch canker pathogen. The hyphal interactions studies showed that the pathogenic hyphal wall shrunk and penetrated by the interaction of G. virens.

Enrichment of dimerumic acid in Monascus‐fermented rice and its in vivo antioxidant activity

AbstractMonascus‐fermented rice (MFR) has been used in traditional Chinese and Japanese medicine for the revitalization of blood, digestion problems, and cardiovascular disorders. Through a biotechnological approach, MFR is produced by the solid‐state fermentation of rice by Monascus spp. MFR has remarkable therapeutic properties as it contains several bioactive compounds, and dimerumic acid is one of the important siderophore‐based antioxidant fermentative metabolites found in it. This study aimed to screen out the medium composition for the production of dimerumic acid (DMA) and evaluate its antioxidant potential. Different strains of Monascus purpureus Microbial Type Culture Collection & Gene Bank (MTCC) 410, 369, 1090 and Monascus ruber MTCC 1880, 2326 and Monascus pilosus CBS 286.34, CBS 290.34 with different solid substrate were screened for maximum yield of DMA. Solid‐state fermentation of long‐grain rice (Oryza sativa) by Monascus pilosus CBS 290.34 was the best for DMA production. The optimized medium parameters in rice for the highest yield of DMA were glucose (75.13 mg/g), soya extract (19.33 mg/g), malt extract (35.14 mg/g), and calcium carbonate (0.29 mg/g). The predicted values for medium parameters were in good agreement with the experimental data. MFR enriched with DMA showed potent antioxidant and lipid lowering capacity in both in vitro and in vivo. It can be explored further for the development of nutraceuticals as a natural antioxidant.

Effect of Bacteria Inoculums on Compressive Strength

The use of bio-concrete is increasing in the present day context and researchers are working on strength and durability characteristics of concrete using bacteria species which have shown calcite precipitation. Three different species of bacteria namely P. Fluorescence, B. Pumilis and B. Subliis that have calcite precipitation properties have been investigated in this study. The investigations were carried first on cement mortar (CM) cubes using these three bacteria species suspension of 20%; 40% and 60% having colony forming units P. Fluorescence (108 CFU/ml), B. Pumilis (106 CFU/ml) and B. Subtilis (108 CFU/ml) respectively. The 40% suspension in all the three cases has shown increased compressive strength as compared to 20% and 60%. The compressive strength measured showed increase (CS) of 18%; 12% for P. Fluorescence; B. Subtilis and decrease of 35% with B. Pumilis respectively. B. Subtilis with optimized 40% suspension having CFU 10x108/ml showed 4.32% ; 5.56%; and 3.81% increase in CS of CC cubes with 3 days; 7 days and 28 days respectively and 5.92% overall increase in CS of CC cubes as compared to the 3 days CS of control cube. ABBREVIATIONSSDW: Sterile Distilled Water; SHC: Self-Healing Concrete; PCR: Polymerase Chain Reaction; BC: Bacterial Concrete; CP: Calcite precipitation; CS: Compressive Strength; CC: Cement Concrete; CM: Cement Mortar; MTCC: Microbial Type Culture Collection; CFU: Colony Forming Unit/ml

Evaluation of Antioxidant and Antimicrobial Efficacy of Camellia Sinensis and Alstonia Scholaris Extracts on Streptococcus Mutans and Lactobacillus Acidophilus - An in Vitro Study.

Context Dental caries is showing an upward trend in India and there is a need to explore innovative strategies to prevent the disease. Literature evaluating antibacterial activity of Camellia sinensis and Alstonia scholaris plant extracts on Streptococcus mutans and Lactobacillus acidophilus is practically non-existent. Objectives - To assess the minimum inhibitory concentration (MIC) and antimicrobial efficacy of Camellia sinensis and Alstonia scholaris on S. mutans and L. acidophilus. Materials and method: This was an in vitro study carried over a period of three months. The leaves of Camellia sinensis and Alstonia scholaris were collected, and crushed to obtain coarse powder. Plant extraction was performed using Soxhelet appartus. Anti- oxidant assay was performed for both the plant extracts against DPPH radical using Spectrophotometer at 517nm. Inhibition percentage was calculated through absorbance value measured from spectrophotometer. Anti- microbial activity of both the plant extracts against Microbial Type Culture Collection strains of Streptococcus mutans and Lactobacillus acidophilus was assessed using Agar well diffusion method. 0.2% Chlorhexidine was used as positive control and ethanol as negative control. The experiment was performed in triplicates. Mean inhibition zone in each set of experiment was computed using three readings after accounting for well diameter. One Way Analysis of Variance (ANOVA), Tukey’s post hoc test and independent sample‘t’ test were performed to compare the mean inhibition zone. Results: The plant extracts were effective against Streptococcus mutans and Lactobacillus acidophilus. Camellia sinensis at 4% concentration produced a mean inhibition zone of 30.3± 3.9 mm against Streptococcus mutans and 23.8± 2.2 mm against Lactobacillus acidophilus. Alstonia scholaris at 10% concentration produced a mean inhibition zone of 21.6± 2.8 mm against Streptococcus mutans and 24.1± 1.6 mm against Lactobacillus acidophilus. Conclusion: Camellia sinensis and Alstonia scholaris have significant anti- oxidant and anti- microbial property against Streptococcus mutans and Lactobacillus acidophilus.

Development of slow melting dietary fiber‐enriched ice cream formulation using bacterial cellulose and inulin

Effect of addition of bacterial cellulose (BC) and inulin on properties such as melting rate, over run, and sensory properties of ice cream was investigated. BC-supplemented ice creams were characterized by a significant reduction in the melting rate, while inulin contributed to enhance the over run and sensory properties. Sensory perceptions varied for BC at 17% and 30% (wet weight) when compared to inulin addition at 0.7% and 1.4% (dry weight). Eight formulations of ice cream were prepared, varying inulin concentration (0.7 and 1.4 g/100 g), BC concentration (17 and 30 g/100 g), and additional water (13.6 ml/100 ml) and two control samples were prepared. An interaction effect among BC, inulin, and additional water content was found for tested parameters. According to the model obtained, the ice cream formulation with 17 g/100 g BC and 1.4 g/100 g inulin observed significant differences for reduction in melting rate and increased fiber content. Novelty impact statement Food applications of bacterial cellulose (BC), a pure form of cellulose obtained from Gluconacetobacter xylinus, are described in the present study. Ice cream formulation optimized by 2 × 3 factorial experiments, using BC and inulin had significant melting resistance and desirable sensory properties as well as rich in dietary fiber. BC obtained from G. xylinus (MTCC 25105) is of significance as a robust producer suitable for the scale-up application of dietary fiber-enriched BC ice cream, and deposited at Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh.

Production of Chitinase from Chromobacterium violaceum Using Agro Industrial Residues under Solid State Fermentation

Chitinases have huge potential applications and biological value in industries, they are used in generating single cell proteins, sweeteners, insecticides, antifungal drugs, anti-cancer agents, biopesticides, food processing agents, degrading agents for sea waste etc. Chitinases are used for the conversion of chitin a polysaccharide into monomers. Chitin is produced enormously in the biosphere annually from fungal cell walls, exoskeletons of insects and crustaceans. Chitinases producing microorganisms Bacillus licheniformis, Chromobacterium violaceum, Rhizobium radiobacter, Streptomyces griseous were obtained from microbial type culture collection. Solid state fermentation was done on Naturally available substrates like Fish scale waste, Prawn shell waste, Wheat bran, Potato peel, Grape leaf to check the enhanced production of Chitinase. Prawn shell waste was found to be the best natural substrate for high activity of Chitinase (464.98 U/g). The overall maximum yield of Chitinase, Chtinolytic activity (531.66 U/gds) was achieved with the optimized process parameters of incubation time(60th h), temperature(34°), inoculum age(3rd d), inoculums volume(20 % v/w), pH(7.0) moisture content 60 % v/w), 1.5 % w/w starch as Carbon source supplement and 1 % w/w urea as Nitrogen source supplement. Both physico-chemical and nutritional parameters had played a significant role in the production of the Chitinase enzyme.

In vitro Antibacterial, Antioxidant and Mechanism of Antifungal Activities of Selected Indian Traditional Plants against Human Oral Microbes

Morus alba, Asparagus racemosus and Murraya koenigii are commonly used in Indian traditional medicines for the various ailments. The plant extracts were analysed for antibacterial, antifungal and antioxidant activities and their mechanism of action towards human oral fungal pathogens. In the present study, methanol extracts were obtained using a soxhlet apparatus and microbes were obtained from microbial type culture collection and gene bank, Pune. The total phenol and flavonoid were found to be high in Murraya koenigii (2.77 mg gallic acid equivalents/g) and Morus alba (160 µg Quercetin equivalent/mg) respectively. The maximum radical scavenging activity was shown by Murraya koenigii in 2,2-diphenylpicrylhydrazyl (71 %) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (82.57 %) methods. The maximum antibacterial and antifungal activities were observed in Asparagus racemosus towards Lactobacillus acidophilus (23 mm) and Candida krusei (10 mm) respectively. The minimum inhibitory concentration was also determined. The target of antifungal activity of Asparagus racemosus towards Candida krusei was not cell wall whereas targets the cell membrane which is confirmed by sorbitol assay, transmission electron microscopy and flow cytometry. This research can be used as a base to find the active components responsible for antibacterial, antifungal and antioxidant activities.

Antifungal Efficacy of Wrightia tinctoria (Roxb.) R.Br on Candida Species Isolated from the Oral Cavity: an Invitro Study

Introduction: Nature is a valuable source of active ingredients that needs to be explored, especially its utilization in the medical field. Owing to the limited availability of antifungal drugs and also considering their side effects, there is always a constant need for a safe and competent alternative. Wrightia tinctoria, a medicinal tree, has been reported to possess potent antifungal activity against commercially available candida strains [American type culture collection (ATCC) and microbial type culture collection (MTCC)]. This could be beneficial clinically only if its antifungal activity could be proved against candida species isolated from clinical samples as the commercially available candida strains might have lost its pathophysiological characteristics on repeated subcultures. Hence, with this background, we performed this study to determine the antifungal efficacy of the extracts obtained from the leaves of W. tinctoria against the candida species isolated from the oral cavity. The aim of this study was to determine the antifungal efficacy of W. tinctoria on candida species isolated from the oral cavity. Materials and Methods: To determine the antifungal activity, acetone, ethyl acetate, and chloroform extracts of leaves of W. tinctoria were used. The study population consisted of five healthy volunteers above 18 years of age without any harmful habits and five patients at increased risk of candida infection. Saliva samples were collected by oral rinse technique using phosphate buffered saline. Fluconazole was used as a positive control and the antifungal efficacy was determined using disk diffusion method. Kruskal-Wallis test was used to determine the significant difference between the extracts. Results: The mean zone of inhibition of acetone, ethyl acetate, and chloroform extracts of leaves of W. tinctoria was 10.8667, 11.0000, 10.1333 mm, respectively. Conclusion: Acetone, ethyl acetate, and chloroform extracts of W. tinctoria possess antifungal activity against candida species isolated from oral cavity.