Starch Hydrolysis Test Research Articles

Isolation and Molecular Identification of Novel Chicken Feather Degrading Bacteria Bacillus tropicus KRKJVR 5

The isolation and molecular identification of a novel chicken feather-degrading bacterium, Bacillus tropicus KRKJVR 5, were conducted to explore its potential in biotechnological applications. Soil samples collected from various sites in Suryapet district, Telangana, were screened using the enrichment culture method to identify potential keratinolytic producers. A total of ten bacterial isolates were initially identified, out of which five isolates demonstrated significant feather degradation on feather-containing agar media. Among these, Isolate 5 exhibited the highest keratinase activity, completely degrading feathers within 48 hours and producing 8.66 U/ml of keratinase, as determined by the Azocasein assay. Morphological and biochemical tests characterized Isolate 5 as a rod-shaped, Gram-positive Bacillus species with positive results for spore staining, Voges-Proskauer, catalase, starch hydrolysis, and carbohydrate fermentation tests, and negative results for indole, methyl red, citrate, H2S, and urease tests. Definitive genetic identification through 16S rRNA sequencing and comparison with the NCBI database confirmed Isolate 5 as Bacillus tropicus KRKJVR 5, with a 92% similarity to other Bacillus tropicus strains. The sequence data was submitted to NCBI and assigned the accession number PP859287. These findings highlight the potential of Bacillus tropicus KRKJVR 5 for efficient keratin degradation, making it a promising candidate for various industrial and environmental applications.

Morphogenetic characterization of Stenotrophomonas maltophilia infecting white stripe disease of rice (Oryza sativa L.)

Rice is a major cereal crop which ensure food security to more than half of the global population. Several biotic factors impact rice grain quality and its final production. White stripe disease, caused by pathogen Stenotrophomonas maltophilia is considered among the major limiting factor for reducing rice yields and quality. Present study was performed to understand the white stripe disease, which has been frequently misdiagnosed as bacterial leaf blight (BLB) due to similar symptoms. A survey was carried out based on accessibility and farmer participation to monitor incidence and sample collection. The survey was conducted in districts Faisalabad, Gujranwala, Sialkot, Sheikhupura, and Hafizabad, these districts were selected for their importance for rice cultivation in Pakistan. The total sample size was around 500 leaves distributed evenly throughout each study area. The results of study indicated presence of new pathogen of rice. These isolates were biochemically identified and confirmed by gram staining negative, 3% KOH positive, 5% salt tolerance positive, oxidase test negative, catalase test positive, starch hydrolysis test negative, nitrate reductase test positive, indole test negative, lactose test positive, maltose test positive, methyl red test negative, Voges-prokauer test negative and urea hydrolysis test negative. The pathogenicity test was confirmed that pathogen and the Sialkot isolate were the most aggressive isolate among the five isolates collected form the studied areas. The molecular characterization was accomplished by PCR and sequencing. The results of the phylogenetic study indicate that this pathogen belongs to a distinct group, as it is distantly related to Xanthomonas oryzae pv. oryzae (Xoo). This study provides important findings into a novel clade of pathogen causing white stripe disease in rice.

Isolation and Identification of Soil-Derived Amylase-Producing Bacteria

Soil is a vital ecosystem for various life forms, including bacteria. Bacteria are the main group of soil microbes, mostly because of their ability to produce various extracellular enzymes. Amylases are the enzymes that break down starch, which are essential in biotechnology industries such as food, fermentation, textiles, and paper manufacturing. The demand for amylase is often high since many enterprises do not have access to local supplies of the enzyme. The objective of the current investigation is to isolate and identify bacterial isolates that can produce the enzyme amylase from soil samples. Soil samples were collected from the unexplored Biligiriranga hills (11.9988° N, 77.1398° E) in south-western Karnataka Bacterial isolates were isolated from soil samples and were primarily screened on starch agar medium to determine the amylase production. Three out of fifteen bacterial isolates that produced amylase were selected based on the highest clear zone around the bacterial colony. These three bacterial isolates were identified both microscopically and biochemically. Gram staining demonstrated that the isolates are rod-shaped and gram-positive organisms. Biochemical studies indicated that the isolates were positive for the catalase and starch hydrolysis tests, whereas negative for the potassium hydroxide and methyl red tests. Besides, 16S rRNA gene amplification and sequencing confirmed the isolate as Bacillus subtilis. The 16S rRNA sequence of B. subtilis strain B has been deposited in the NCBI GenBank. The optimal growth conditions observed in B. Subtilis. The optimal growth conditions observed in B. Subtilis were 37° C temperature at pH 7 and incubation period for 37h. Thus, the isolation and identification of soil-derived amylase-producing bacteria are crucial for understanding their potential applications in various industrial processes, such as bioremediation, waste management, and the production of enzymes for food, pharmaceuticals, and biofuel industries.

Characterization and Evaluation of Native Rhizobium of Groundnut (Arachis hypogaea L.) and Soybean (Glycine max L.)

The present investigation was conducted to characterize and evaluate native Rhizobium isolates of groundnut and soybean to enhance the Legume-Rhizobium symbiosis as well as increase sustainable crop production. In this connection, all the native isolates under study were characterized biochemically and tested with N-free sand culture in the glasshouse to select effective native Rhizobium isolates through proper screening. 10 isolates of each crop were collected from the culture collection bank of the Department of Agricultural Microbiology, Indira Gandhi Krishi Vishwavidyalaya Raipur (Chhattisgarh). Out of 10 isolates, 3 isolates of each crop were selected on the basis of their growth performance and the purity of the culture. The biochemical studies of selected isolates include starch hydrolysis, catalase, urease, citrate utilization, indole production, gelatine iron, and methyl red tests, which were further evaluated for nodulation and biological nitrogen fixation in groundnut and soybean under controlled conditions with four treatments for each crop and five replications. The experiment result revealed that the evaluation of native Rhizobium isolate no. 1185 of groundnut and isolate no. 1076 of soybean was found to be superior among all the rhizobial isolates taken for study. These isolates accumulated 1.24, 1.44 mg/plant extra amount of atmospheric nitrogen over un-inoculated control plants, respectively. Further, it can be concluded that these native isolates may be the most effective nitrogen fixers for groundnut and soybean over the control.

Biochemical characterization of bacteria isolated from Rhizosphere soils of sugarcane grown in Uttar Pradesh

Rhizospheric soil samples of Sugarcane fields were collected from distinct regions of U.P. Further, these soil samples were used for the isolation and characterization of bacteria to identify their maximum PGPR traits and biochemical potential. A total of ten bacterial isolates were collected from the soil samples and sub-cultured in the nutrient agar plates and characterized through Biochemical test. One bacterial strain was found negative and the other 9 isolates were found positive for gram staining. All bacterial isolates were positive for citrate test. 3 isolates were found MR positive and 2 positives for VP test. 5 isolates were positive for gelatinase test and 9 isolates were positive for nitrate reductase test. 2 isolates were positive for starch hydrolysis test, while 7 isolates were found positive for phosphate solubilization test and 3 isolates were positive for siderophore production test. These results showed that the bacterial isolates from soil sample were Azotobacter Species, Azospirillum Species, Pseudomonas species, Bacillus Species, Rhizobium Species, Serratia Species and E. coli. These bacterial strains followed the maximum PGPR traits. After proper validation, these bacterial strains could be used for the bio-fertilizer formation for crop production enhancement by improving soil fertility.

Antimicrobial sensitivity pattern of aerobic bacteria isolated from hunting dogs in Maiduguri, Nigeria

Hunting dogs may be a potential source of transmission of pathogenic, zoonotic, or drug-resistant bacteria to people since they may carry the pathogenic bacteria in their oral cavities. As a result of clinic-based cross-sectional study conducted to evaluate the level of multidrug resistant bacteria, colonization of the bacteria in the oral cavities of hunting dogs that appeared apparently healthy were found in Maiduguri. Oral swab samples were taken from one hundred and ninety-three (193) apparently healthy hunting dogs. Using colony morphology and oxidase, catalase, starch hydrolysis, casein hydrolysis, indole, methyl-red and Voges-Proskauer test (MR-VP) the bacteria were isolated and identified. The isolates were tested for antibiotic susceptibility to ten (10) different antibiotics using the Kirby-Bauer disk diffusion method in accordance with standard procedure. Escherichia coli (E. coli) accounted for the greater proportion of 100 (51.8%) of the isolates. Others were Staphylococcus spp. 19 (18.8%), Klebsiella spp. 13 (6.7%), and Streptococcus spp. 7 (3.6%). Escherichia coli isolates were highly resistant to Penicillin 100 (100%), Ceftriaxone (100%) and Tetracycline (80%) while Klebsiella spp. was found resistant to Amikacin (100%), Gentamicin (100%) and Ceftriaxone (100 %). Similarly, Staphylococcus spp. obtained in the current study was found to be resistant to Ciprofloxacin (100%), Gentamicin (100%) and Amikacin (100%) while Streptococcus isolates were resistant to Tetracycline (100%), Penicillin (100%) and Chloramphenicol (100%). Susceptibility of the isolates were E. coli 100 (100%) for Chloramphenicol. Owners of dogs should refrain from using antibiotics indiscriminately. In conclusion, the study revealed the presence of dissimilar bacteria in the oral cavities of hunting dogs using cultural isolation, biochemical and antibiotics sensitivity tests in the study area.

Phyto-extracts Mediated Biological Control of Citrus Canker

Citrus is second most important fruit crop and has significance economic importance in Pakistan. In Pakistan 2.17 million tonnes citrus was produced on area of 1.94 million acre. Citrus is mostly cultivated in Punjab and affected by different types of plant pathogens. Citrus canker is the most harmful disease among the major diseases of citrus which is caused by Xanthomonas axonopodis pv. citri. The copper based fungicides are mostly employed to manage the citrus canker in the field but excessive use of chemicals have negatively impacted the environment and warrant the need to find out organic solutions therefore the current study was aimed at identifying suitable plants extracts for the control of citrus canker bacterium. Isolation of the bacterium X.axonopodis was done through streaking on agar plate technique. Morphological and bio-chemical tests such as gram staining, KoH test, catalase test and starch hydrolysis test were performed to examine the bacteria X.axonopodispv. citri. Plant extracts were evaluated, in vitro, against the X.axonopodis pv. citri through paper disc method which showed the inhibition zone against the disease. The 100 percent extract of garlic, ginger, aloevera, moringa were used used along with the streptomycin as a positive control. The inhibition zone was 11.67mm and 10.83mm due to garlic and moringa extracts respectively while it was 16.67mm in case of positive control. The ginger and aloevera extracts proved ineffective against the citrus canker bacterium.The efficacy of garlic and moringa extracts were also checked against the X. axonopodis pv. citri under glasshouse conditions. The inoculum was sprayed on Kinnow plants, with three replicates, for each extract and data was recorded 15, 20, 25, 30 and 35 days post inoculation (dpi).The recorded disease severity percentage, after 35dpi, on garlic and moringa treated plants were 9.67% and 14.27% respectively while the 27% disease severity was recorded on control (plants sprayed with autoclaved distilled water). The garlic extract proved to be most effective followed by moringa extract which could be used as a potential organic solution to control citrus canker.

MORPHOLOGICAL CHARACTERIZATION OF ROOT INHABITING ENDOPHYTIC BACTERIA

Endophytic bacteria (EBs) are beneficial to stimulating plant growth. However, little information about the EBs associated with soybean plant roots is available. This study examined the diversity of ten EBs isolated from soybean root tissues. Morphological and biochemical characterization methods revealed significant variation among the isolates. Most isolates displayed smooth colony surfaces, regular shapes, and rod-shaped cells. However, Bacterial Strain-5 and 10 exhibited rough surfaces and irregular shapes, while Bacterial Strain-4 displayed round cell shapes instead of the typical rod morphology. Moreover, the isolates demonstrated diverse pigmentation, with strains showing various shades of white, creamy, light yellow, pinkish red, Creamish, yellow, and red. In KOH test, three strains (2, 6, and 9) showed positive reactions to KOH, while the remaining seven strains (1, 3, 4, 5, 7, 8, and 10) showed negative reactions. The catalase test confirmed that four strains (1, 2, 6, and 9) were gram-negative, and the six strains (3, 4, 5, 7, 8 and 10) were gram-positive. In the gram staining test, seven strains (1, 2, 3, 6, 8, 9, and 10) were gram-negative, while the remaining three strains (4, 5, and 7) were gram-positive. Finally, in the starch hydrolysis test, seven strains (1, 2, 3, 6, 8, 9, and 10) were gram-negative, whereas the three strains (4, 5 and 7) were gram-positive. This study will help us identify various EBs that could play a role in the nodule formation and adaptation of soybean plants in diverse soil conditions in Pakistan.

Isolation and Characterization of Nitrogen Fixing Bacteria from Rhizospher of Chickpea and Peanodules, Hossana, Ethiopia

One of the ways to increase the competitive survivability of rhizobial bio fertilizers and achieve better plant growth under such conditions is by modifying the rhizospheric environment or community by addition of non rhizobial nodule-associated bacteria (NAB) that cause better nodulation and plant growth when co-inoculated with rhizobia. To achieve this objective experimental design was carried out. Rhizobium is the root of legumes host nitrogen fixing bacteria which can invade root and get sugars from the plant. A study was performed to investigate the most commonly associated nodule-associated bacteria and the rhizospheric microorganisms associated with the legume plant. Present study on Isolation and characterization of Nitrogen Fixing Bacteria from agricultural rhizosphere was carried out with the aim of Isolation and characterization of Nitrogen Fixing Bacteria. The occurrence of Nitrogen Fixing Bacteria from root nodule of pea, white chickpea and red chickpea were investigated. Nitrogen fixing bacteria was isolated from root nodule of pea, white chickpea and red chickpea. The isolates were grown on yeast extract – mannitol agar (YEMA), and the morphologic characterization was done by Gram stain. A total of 21 isolates were isolated on Yeast Extract Mannitol (YEMA) agar medium. The isolates were circular, and irregular in form with smooth surface; entire, colony margins; with light pink coloured. The identification of isolated pure cultures through colony morphology analysis, cellular morphology and biochemical properties are including gram staining (negative), catalase test (negative), Methyl red test (negative), voges proskaeur test (positive), citrate utilization test (positive), starch hydrolysis test (positive) and mannitol salt agar (positive). Among the 21 bacterial isolates isolated, 18 were shown the characteristic of rhizobia. The effect of these 18 isolates those shows the characteristics of rhizobia were performed and all of them are significant, while compared with controls. These isolates will be useful to produce efficient bio fertilizers for Agriculture.

Determination of The Optimum pH and Enzyme Ratio for Starch Hydrolysis Test and Characterization of Steamed, Baked, and Fried Wheat Doughs

Digestibility is the most important nutritional functionality of starchy foods. Unfortunately, in Indonesia, this functionality has not been extensively studied due to the relatively challenging methods involving enzymes. This research aims to determine the optimal pH and enzyme ratio of α-amylase (AA) and glucoamylase (GA) for starch hydrolysis tests and apply them to characterize steamed, baked, and fried wheat doughs. For optimization, hydrolysis was carried out at 37 °C with an enzyme-to-substrate ratio (E/S) of 100 mL/g for 120 min. Samples of wheat dough obtained from three major dough producers in Banyumanik, Semarang, Indonesia was then tested for starch hydrolysis, texture, chemical analyses, sensory, and hedonic test. The collected data were analysed using principal component analysis (PCA). Under the determined conditions, the optimal pH and AA/GA ratio were found to be 6.6 and 0.5. Regarding quality of processed doughs, the steamed dough exhibited a cohesive texture, a soft sensory profile with a pleasant aroma, sweet taste, and was most preferred by the panellists. The baked dough was hard in texture, high in ash content, and brown in colour. The fried dough poses high starch hydrolysis, chewy texture, adhesion, solid particles, protein content, and relatively high lipid. The sensory evaluation indicated that the fried dough poses an oily, rancid, foreign aroma, and bitter and salty taste. It can be concluded that at a temperature of 37 °C, an enzyme-to-substrate ratio of 100 mL/g, and a reaction time of 120 min, the optimal starch hydrolysis of processed wheat dough can be achieved at a pH of 6.6 and an AA/GA ratio of 0.5. Furthermore, steaming resulted in a unique property of dough compared to the baked and fried, while the latter two yielded products with similar properties.

Microbiota from Intestinal Gut of Dawkinsia filamentosa (Valenciennes,1844) as Indicator of Environmental Condition

Aims: To isolate and identify the gut associated micro-organisms present in the gastrointestinal tract of freshwater fish Dawkinsia filamentosa. 
 Place and Duration of Study: Sampling site: The fish samples were collected from the river Gadana, located at the foot hills of Western Ghats, Alwarkurichi, Tenkasi district, Tamil Nadu, India. Place: Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Azhwarkurichi, Tenkasi, Tamilnadu, India (Latitude 8o20’N; Longitutde 77o10’E). Duration: The research was done during December 2021 and April 2022.
 Methodology: The fish samples were anaesthetized and surface sterilized followed by aseptic dissectionby using sterile scalpel and needle. The gut contents were removed and homogenized well and isolation of Gut micro biome was done by means of serial dilution. The identification of gastrointestinal bacteria was done by performing various biochemical tests such as Gram staining, Motility, Indole test, Methyl red test, Voges Proskauer test, Citrate utilization test, Triple sugar Iron test, Catalase test, Oxidase test, Urease test, Nitrate test, Starch hydrolysis and Carbohydrate Fermentation Test. The fungi present in gastrointestinal tract is identified with lacto phenol cotton blue staining followed by microscopic observations.
 Results: The overall bacterial load of gastrointestinal material ranged from 47 x10-7 to 219 x10-5 cfu/g. A total of six bacterial strains and four fungi strains were isolated. Several biochemical tests were used to identify the bacterial isolates. The six bacterial isolates were tentatively identified as Pseudomonas sp., Aeromonas sp., Staphylococcus sp., Bacillus sp., Enterobacter sp., and Vibrio sp. and the four fungi species isolated from the gutwere Aspergillus flavus, Aspergillus niger, Beauveria basiana, and Penicillium sp.
 Conclusion: The current research revealed that, Dawkinsia filamentosa has a variety of microbiota in its gut. Microbial species from freshwater environments may be able to augment fish feed with these bacteria in commercial aquaculture operations. Additionally, these findings have aided in the development of possible remedies, enhanced knowledge of host-microbe interactions in other vertebrates, and enhanced aquaculture practices. The benefits of identifying gut microbiota in fishes is, it helps to study about the host nutrient catabolism and immune defence mechanism against the disease-causing pathogens.

Black rot disease incited by Indian race 1 of Xanthomonas campestris pv. campestris in Brassica juncea L. cv. Pusa Bold in India.

Mustard (Brassica juncea L.) is an important oil seed crop in the Brassicaceae family. It is widely cultivated in India for its edible leaves, oil and medicinal properties. In January 2022, we noticed necrotic symptoms typical black rot disease on Brassica juncea (L.) cv. Pusa Bold grown in Indian Agricultural Research Institute, India. Initially, chlorotic lesions emerged on the leaf margin, which progressed to angular V-shaped necrotic lesions and blackened veins. Disease progression became a necrotic appearance in the leaf results browning and papery leaf texture appeared. The suspected causal agent was isolated from three different diseased plants of Pusa Bold on nutrient sucrose agar medium that formed pale yellow, mucoid, and fluidal colonies. Three representative isolates originated from three different plants were sub-cultured on YGCA medium. These isolates are Gram-negative, oxidase negative, KOH positive, nonfluorescent on King's Medium B agar, and positive for starch hydrolysis test (Schaad and White 1974). The 16S ribosomal RNA gene and avirulence genes - AvrBs1 and AvrGf1 were amplified and sequenced in these three isolates with other Xanthomonas campestris pv. campestris (Xcc) isolates. The DNA sequence analysis revealed that these isolates are within the species of X. campestris. The race 1 specific marker namely xcc-b100_4389 was used to characterized the race by detection of 1090bp fragment respectively from gDNA of Xcc isolates (Rubel et al., 2017). The pathogenicity of these isolates was tested twice on youngest leaves of 30-day-old plants of Pusa Bold to convey Koch postulates. Inoculum of three isolates were prepared in nutrient broth at 28°C for 48-h. The pathogenicity test was conducted by small scissors dipped in a bacterial suspension (~ 108 cfu/ml) to cut leaf near margins at 10 points per leaf and the three youngest leaves per plant with three replications. The number of infected points per leaf and the severity of symptoms were assessed 15 and 30 days after inoculation (Singh et al., 2011; 2016). The chlorotic lesions with V-shaped symptoms were appeared on all inoculated plants after 15 and 30 dpi (days post-inoculation). The bacteria were reisolated from inoculated plants and has the same identity as original isolates by using 16S rRNA, avr genes and race 1 specific marker PCR, thereby confirming Koch's postulates. The bacterial inoculation was repeated and the same symptoms appear. Most of the crucifers are infected with black rot disease e.g., cauliflower, cabbage, Brussels, sprout etc. (Vicente et al., 2001). The nucleotide BLAST analysis of 16S rRNA, AvrBs1, AvrGf1 showed a 100% identity with different Xcc strains reported from Germany (B100; AM920689), Brazil (ATCC 33913; AE008922), India (Xcc-C7; CP077958), France (CFBP 5817; CM002673) and China (8004; CP000050) (Singh et al. 2022). Whilst, the nBLAST analysis of xcc-b100_4389 showed 100% nucleotide identity with Xcc race 1 (B100; AM920689), Germany. The sequences were deposited in GenBank (16S rRNA: OM839780; AvrBs1: OM994397; AvrGf1: OM994398; xcc-b100_4389: OM994399). The XccAK1 strain (ITCCBH_0014) was deposited in Indian Type Culture Collection, ICAR-IARI, New Delhi, India. Presently, it is a first report of necrotic black rot on B. juncea cv. Pusa Bold incited by Xcc race 1, India. Previous research reported the black rot disease on other species of the Brassica genus e.g., B. oleracea, and B. napus in Serbia (Popovic et al., 2013) and Argentina (Gaetan et al., 2005).

INFLUENCE OF FOLIAR SPRAY OF BIO-INOCULANT AND MICRONUTRIENT ON SOIL BIOLOGY AND YIELD IN BROCCOLI

The experiment was carried out in the years 2021-22, at the Department of Agricultural Microbiology, Indira Gandhi Krishi Vishwavidyalaya, Raipur (Chhattisgarh). The study involved application of foliar spray of bio-inoculants and micronutrient; Ammonium Molybdate. Bio-inoculants; Azotobacter and Azospirillum inoculation strains were collected from Microbiology repository and were revived for biochemical characteristics on Jensen’s and Okon’s media respectively. Both the isolates showed positive reaction for starch hydrolysis test, catalase test, urease test and oxidase test. Plant parameters evaluated include biomass accumulation and fruit weight per plant. The experimental results revealed that significantly maximum fruit weight per plant (359.0g), dry shoot biomass (16.6g/ plant), dry root biomass (8.6g/ plant) were recorded under T4 (IF + Foliar Application of Bio-inoculants and Ammonium molybdate 0.2%). Besides higher microbial population, Azotobacter (44.3 x 103 cfu/ g of soil), Azospirillum (28.5x 103 cfu/ g of soil) were also observed to be significant under T4. Dehydrogenase activity in rhizosphere soils was found maximum 27.3 µg TPF/g soil/h under T4 (IF + Foliar Application of Bio-inoculants and Ammonium molybdate 0.2%) at 60DAT. Thus application of Bio-inoculants along with Ammonium molybdate (0.2%) as foliar spray along with 50% inorganic fertilizer proved to be the most effective and may be adopted in Broccoli cultivation.

Characterization of Immobilized β-Amylase Enzyme Isolated from Sweet Potato and prepared by Entrapment Method

Aim: This study attempted to isolate β-amylase from sweet potato and enzyme immobilized by encapsulation method, and characterized with various parameters. 
 Methods: The enzyme β-amylase was isolated with phosphate-buffered saline and purified by centrifugation with ammonium sulfate. The purified enzyme was immobilized on chitosan (0.25 g) and sodium alginate (0.25 g) polymers by entrapment method in the presence of calcium chloride (0.5 M). The immobilized enzyme was characterized by a starch hydrolysis test, the optimal pH and temperature were studied and the stability of the immobilized enzyme was also determined. SEM analysis was performed and Vm and Km were also found. 
 Results: The starch hydrolysis test showed positive results on the starch agar plates for immobilized enzymes. The thermal inactivation showed a severe loss in the activity of the free enzymes (49.3 %) while the temperature profile of the immobilized enzymes was much broader (84.55 %) at higher temperatures (80° C). The optimal pH and stability indicated that the immobilized enzyme has higher stability in the pH range of 5-8. The Km and Vmax value of free and immobilized enzyme was 7.67 mmol, 21.15 µmol (R2 0.8880), and 4.72 mmol,16.79 µmol (R2 0.8446) respectively. The storage of free and immobilized enzymes for one month showed that 83.5 % and 40 % of free enzymes and 11.6 % and 8.6 % of immobilized enzymes lost activity at 25° C and 4° C, respectively. SEM analysis shows the smooth, porous surface. 
 Conclusion: Immobilized enzymes (natural polymers) exhibit higher thermal stability the optimal pH and stability indicate immobilized enzyme has higher stability in the pH range of 5-8, and achieves a relative activity of 69.7 %. After 6 uses, the reuse efficiency of the immobilized enzyme decreased from 99.8 % to 52.3 %. The storage of the immobilized enzyme showed much higher stability than the found-free enzyme.

Isolate Characterization and Amylolytic Properties of Lactic Acid Bacteria from Traditional Fermented Dadih

This research aimed to explore, isolate, select, and characterize lactic acid bacteria (LAB) potentially used as amylolytic bacteria from traditional fermented food “dadih”. Dadih was collected from Lareh Sago Halaban and Lintau, West Sumatera Province. The LAB was isolated by the streak method to obtain a uniform color and size colonies. The selected isolates were characterized by the reaction gram and catalase test. The result showed that isolates had the form of coccus, coccobacillus, and basil with gram-positive, catalase-negative, and endospore-negative reactions. The further characterization using the KIT API 50 CHL identified Lactobacillus plantarum (mH1), Lactobacillus paracasei ssp paracasei (mL3), Lactobacillus plantarum (mH4), Lactococcus lactis subsp lactis (mH6), Lactobacillus plantarum (mH7), Lactobacillus plantarum (mH8), Lactobacillus brevis (mH13), and Lactobacillus paracasei subsp paracasei (mL14). The amylolytic and starch hydrolysis test showed that the isolates with higher amylolytic index ≥2 were Lactobacillus paracasei ssp paracasei mL3, Lactobacillus plantarum mH4, and Lactobacillus paracasei ssp paracasei mL14. The Lactobacillus plantarum mH8 had a lower amylolytic index of 0.7. Therefore, the four isolates (mL3, mH4, mL14, and mH8) are potentially used as fermenting bacteria for high- carbohydrate food.

ISOLATION & FORMULATION OF POMEGRANATE JUICE BASED ON PROBIOTIC PROPERTIES

Probiotics are mainly living microorganisms that used to improve or restore gut flora when ingested. The consumption of probiotics is mainly regarded as safe.A strain of lactobacillus bacteria strain Streptococcus faecalis strains were isolated from bifilac probiotic medicine and identified by colony morphologies cultured on MRS (Man, Rogosa and Sharpe) media and biochemical testing were done in this study. Isolated bacteria were chosen for probiotic screening and further investigation was done based on biochemical properties and further screening. In this study, Gram staining, Catalase test, carbohydrate fermentation, protease test, starch hydrolysis test, motility test, urease, catalase, nitrate test, indole test, growth optimization (pH optimization, and temperature optimization) were all used . Based on the OD (Optical Density), isolated bacteria may thrive at pH 9.0 and at room temperature of 25 ℃, making them suitable for use as a probiotic.The shelf life of prepared health drink was also check by checking the growth of bacteria on to the EMB agar media. So, different test revealed that S. faecalis can used as a probiotic and also for the development of probiotic health drink. So Probiotics used in gut microbiota by eliciting an immune response that recognises them as beneficial and forms a protective barrier in the intestine against infections.

Isolation, Screening, Characterization and Maintenance of Amylase Producing Bacteria from Soil

Abstract: Amylases are amongst most widely used enzymes in industries such as food, fermentation, starch processing, textile, and paper. In the present investigation, bacteria were isolated from soil samples of different areas of Padmashri Vikhe Patil College, Pravaranagar, Maharashtra, India screened for the production of amylase. Soil samples were serially diluted in normal saline and plated on sterile nutrient agar plates. The colonies obtained from higher dilutions were subjected to Gram staining, biochemical reactions, and starch hydrolysis test. Bacterial colony yielding positive starch hydrolysis test were subjected to Amylase activity test by DNSA method. Characteristic feature of the strain indicates that it belongs to the genus Bacillus and will be later used for further characterization. Maximum yield of amylase was obtained after 48hrs of incubation. The isolated amylase producing organism was maintain by using agar slant & glycerol stock method.

Assessment of metal-reducing bacteria residing in industrial wastewater: a bacterial-based bioremediation approach

Background: Metals are required in minor amounts in the human body to perform vital functions, but beyond that level, they become toxic and cause many harmful effects. At present, polluted water is a large source of these heavy metals inside our body. Bioremediation is one of best treatments for the removal of these heavy metals from water. Objectives: The main objective of the current study was to isolate metal-reducing bacteria from soil and wastewater samples from different industries. Furthermore, the metal reducing potential of the bacteria was also evaluated under various environmental conditions (pH, temperature, incubation time, and UV exposure). Methodology: Different bacterial strains were isolated that were resistant to different concentrations of zinc (Zn) and chromium (Cr) from wastewater and soil samples from four different industries (Riaz textile mills, Sitara chemical industry, Mandiali paper mills and Siddique leather works). Morphological characterization was carried out with the help of Gram staining, spore staining and motility tests. Biochemical tests were performed, such as catalase, oxidase, and H2S production tests, starch hydrolysis tests and Simon citrate tests. Chromium reduction after UV exposure was calculated to check mutation effects on chromium reduction. Results: A total of 14 bacterial strains were isolated from the soil and wastewater samples. Six strains were Zn resistant, while eight strains were chromium resistant. A total of five strains were isolated from wastewater out of fourteen strains, while the remaining nine strains were isolated from soil samples of these industries. The bacterial strains were rod-shaped cocci and coccobacillus. The growth of bacterial strains under different environmental conditions, such as temperature, pH and incubation time, was observed, and the best growth was found at 37◦C, pH 7.0 and after 48 hrs. Maximum reduction at different concentrations was observed at pH 7.0 and 37◦C and after 48 hrs. Conclusion: In conclusion, the bacterial strains isolated from industrial wastes and soils showed significant resistivity against various concentrations of Zn and Cr and reduced it efficiently under different conditions (pH, incubation time, temperature and UV light). The use of microorganisms for bioremediation is an environmentally friendly and cost-effective approach to reduce heavy metals present in our water and soil.

Comparing Sterilization of Endodontic Hand Files by Four Different Methods: An In Vitro Study.

The aim and objective of this study is to compare the efficacy of four various approaches of sterilizing endodontic hand files via Autoclave, Glass-bead sterilizer, Glutaraldehyde solution, and Diode laser. Fifty-two k-files of size #25 and length 21 mm were taken for the study. All the 52 files were presterilized in an endodontic instrument box via autoclave. Bacillus stearothermophilus spore suspension was prepared and all the presterilized files were contaminated with stearothermophilus spore suspension in a sterile Petri dish under vacuum hood safety. Later, the test files were randomly divided into four groups of 13 each and subjected to four different methods of sterilization- Autoclave, Glass-bead sterilizer, Glutaraldehyde solution, and Diode laser. Files were then be placed in thioglycollate media containing test tubes and incubated in an incubator at 55°C and checked for turbidity at 3 days and 21 days. The result revealed that there was no Turbidity present in test tubes on both the 3rd and 21st day for autoclave. In all the remaining sterilization procedures there was some amount of turbidity present. In terms of sterilization provided autoclave provides complete sterilization and glutaraldehyde solution is the least effective.The specificity of Bacillus stearothermophilus was then confirmed with a sugar test viz., starch hydrolysis test which gave a positive result confirming the presence of Bacillus stearothermophilus. We can conclude that autoclave is the perfect process of sterilization providing 100% sterility and although Glass-bead didn't provide 100% sterility, it can be used as an alternative if autoclave is not available. Ameer B, Khatib MS, Peerzade SM, et al. Comparing Sterilization of Endodontic Hand Files by Four Different Methods: An In Vitro Study. Int J Clin Pediatr Dent 2022;15(2):149-152.

Production of Bioethanol by Co-Culture of Aspergillus niger and Saccharomyces cerevisiae using Watermelon Peels as Substrate

Bioethanol production from lignocellulosic biomass as an alternative fuel source has received considerable attention from researchers worldwide. Bioethanol was produced from watermelon peels by fermentation in the current work, using Aspergillus niger and Saccharomyces cerevisiae co-cultures isolated from soil and local beverage ( kunun zaki ), respectively. The isolates were characterised both macroscopically and microscopically. While starch hydrolysis test was carried out for the mould isolates, thermotolerance, glucose and ethanol tolerance tests were used to identify the yeast isolates. According to standard procedures described by the Association of Official Analytical Chemists (AOAC), Proximate analyses were carried out on the watermelon peels. The ethanol yield was determined using the specific gravity method. Isolate BGD of the moulds had the highest (40 mm) zone of clearance on starch agar. The identified yeasts showed the best growth at a maximum of 35 °C while tolerating up to 50% wt/v and 18% v/v of glucose and ethanol, respectively. The yeasts also fermented all the different sugars tested but lactose. The proximate composition of the substrate revealed low protein (3.5%) and ash contents (7.38%) but a remarkably high carbohydrate content of 79.22%. At optimal fermentation conditions (30 °C, pH 6.0, 4% g/L substrate, 200rpm and five (5) days fermentation period), the maximum ethanol yield produced by the co-culture of Aspergillus niger and Saccharomyces cerevisiae was 57 g/L. However, only 13.5 g/L and eight g/L were produced by individual cultures of A. niger and S. cerevisae, respectively. The bioethanol produced has a specific gravity of 0.994, miscible in water with a pleasant smell. This colourless liquid boils at a temperature of 78 °C with a pH of 3.02 and ignites at room temperature. It was concluded that watermelon peels are a suitable substrate for bioethanol production and was used successfully to produce an ethanol yield of 57 g/L by co-culture of A. niger and S. cerevisiae .