Starch Broth Research Articles

Amylolytic microorganisms from diverse tropical environments: Isolation, identification, and amylase production

AbstractThis study aimed to identify microbes producing amylases from diverse unexplored environments in Kenya. Screening based on microorganisms' ability to grow on minimal media (M9) supplemented with starch resulted in the selection of 70 amylolytic isolates. The isolates were then subjected to primary screening based on starch hydrolysis ratio (SHR) obtained by halo formation on starch agar plates after flooding plates with Lugol's iodine solution. SHR narrowed down the isolates' number to 17 (SHR > 1.5), which were used for subsequent experiments. The 17 isolates were grown on M9 starch broth for the production of amylases. Crude amylases and commercial enzyme (control) were assayed for amylase activity using the 3,5‐dinitrosalicylic acid method. Specific enzyme activities of crude enzymes ranged between 0.0079 and 0.0629 U g−1, which were lower than 1.7188 U g−1 of the control. Five enzymes with the highest specific enzyme activity were used for cassava hydrolysis. All crude enzymes exhibited higher specific enzyme activity (0.0707–0.1853 U g−1) than the control (0.0052 U g−1) in hydrolyzing cassava. Alkaline lakes had the highest number (4) of isolates whose enzymes had the highest specific enzyme activity, while isolates from potato processing plant waste, termite gut, and black soldier fly gut had a representative each. Isolates with enzymes having the highest specific enzyme activity were identified by DNA sequencing and belonged to Lysinibacillus sp. (four isolates), Alternaria sp. (one isolate), and Bacillus sp. (two isolates). The study demonstrated the presence of microorganisms in tropical environments with amylase activity that can be scaled and optimized for practical applications.

Probiotic potential and amylolytic properties of lactic acid bacteria isolated from Chinese fermented cereal foods

This study investigated the probiotic potential and amylase properties of amylolytic lactic acid bacteria (ALAB) isolated from fermented cereal-based foods in China. Of 132 LAB isolates screened for amylase production, three (430, 445, and 472) were found to have high amylase activities (8.15, 9.23, and 8.06 U/mL in MRS-1% starch broth, respectively). These three strains were identified as Lactobacillus plantarum by 16 S rRNA sequencing. Optimization of the starch content in the culture broth, pH, and temperature during fermentation revealed that the highest amylase activity (15.89 ± 0.51 U/mL) was found in L. plantarum 445. Moreover, the three ALAB isolates were assessed for their probiotic properties, tolerance to low pH, bile salt resistance, and antimicrobial and aggregation activities. All three ALAB isolates showed good survival at pH 2.0 and 3.0 and were resistant to 0.3% and 0.6% bile salts after 48 h of incubation. They also had different antimicrobial activities against five food-borne pathogens and similar intrinsic and non-transmissible antibiotic susceptibilities. The isolates had higher auto-aggregation ability but weaker co-aggregation ability than the reference strain, Lactobacillus rhamnosus ATCC 53103. These data demonstrate that the three ALAB isolates possess potential probiotic characteristics, suggesting that they are suitable candidates for cereal-based probiotic products and starter cultures for improving the cereal fermentation process.

Screening and identification of amylase producing strains of Bacillus

An attempt was made to isolate and screen efficient amylolytic strains of Bacillus sp. Initial screening based on Starch hydrolysis ratio (SHR) resulted in selection of 72 amylolytic bacterial strains. Among these, 18 strains were selected for further studies. Secondary screening based on amylase production in starch broth medium led to selection of six amylolytic strains of Bacillus sp. The selected strains were grown in four different fermentation media (FMI-FMIV) in order to screen for three most efficient amyloytic strains for optimization and characterization. FMIV was the best basal medium for amylase production as it had the shortest incubation time (24 hours) and highest amylase production. Molecular identification based on 16S rDNA sequence revealed that three most efficient strains (BCM36 (KR1), BCM33 (KR2) & BCM25 (KR3)) belonged to Bacillus sp.

Endophytic Bacteria Isolated from Citrus Plants for Biological Control of Citrus Canker in Lime Plants.

Citrus canker caused by Xanthomonas citri subsp. citri is a disease affecting the yield and fruit quality of lime (Citrus aurantiifolia). This research investigated endophytic bacteria obtained from six healthy Citrus spp. to inhibit the pathogen and to control citrus canker on lime plants. Numbers of the endophytic bacteria isolated from C. aurantifolia, C. hystrix, C. maxima, C. nobilis, C. reticulata and C. sinensis were 28, 25, 29, 42, 12 and 34 isolates, respectively. The selected endophytic bacteria that were effective against X. citri subsp. citri were Bacillus amyloliquefaciens LE109, B. subtilis LE24 and B. tequilensis PO80. The optimum culture medium for an antagonistic effect on the pathogen in B. amyloliquefaciens LE109 and B. tequilensis PO80 was yeast extract peptone dextrose broth, and in B. subtilis LE24 was modified soluble starch broth. To control citrus canker in lime, young expanded leaves of lime plants were aseptically punctured and inoculated with 30 μl of bacterial suspension of the pathogen (108 CFU/ml in 0.85% NaCl) per punctured location. After the pathogenic inoculation for 24 h, the leaves were then inoculated with 30 μl of the selected endophytic bacteria (108 CFU/ml in 0.85% NaCl), and treated with 30 μl of the culture media containing bioactive compounds produced by the selected endophytic bacteria. The leaves inoculated with cell suspensions of B. amyloliquefaciens LE109 or B. subtilis LE24 could completely control citrus canker. However, the leaves inoculated with B. tequilensis PO80 displayed 10% disease incidence. Additionally, the leaves treated with the crude bioactive compounds of B. amyloliquefaciens LE109 or B. subtilis LE24 could completely control citrus canker. Notably, the leaves treated with the crude bioactive compounds of B. tequilensis PO80 displayed 5% disease incidence. The results of this study showed that the Bacillus strains play important roles in the biocontrol of citrus canker in lime.

Farm level survey of spore-forming bacteria on four dairy farms in the Waikato region of New Zealand.

The aim of our study was to determine the occurrence and diversity of economically important spore‐forming bacteria in New Zealand dairy farm systems. Farm dairy effluent (FDE) collected from Waikato dairy farms were tested for the presence of spore‐forming bacteria, using a new culture‐based methodology followed by genomic analysis. An enrichment step in which samples were inoculated in cooked meat glucose starch broth under anaerobic conditions, aided in the differential isolation of Bacillus and Clostridium species. Furthermore, the use of molecular methods such as ERIC genotyping, 16S rRNA gene sequence analysis identified different spore‐forming bacteria present in FDE. C. sporogenes signature PCR gave further information on the phylogenetic relationship of the different Clostridium spp. isolated in this study. In total 19 Bacillus spp., 5 Paenibacillus spp. and 17 Clostridium spp. were isolated from farm dairy effluent. Sequence types similar to economically important food spoilage bacteria viz: C. butyricum, C. sporogenes and members of the Paenibacillus Genus were isolated from all four farms, whereas, sequence types similar to potential toxigenic, B. cereus, C. perfringens, C. butyricum, and C. botulinum were found on at least three of the farms. Sampling of farm dairy effluent provides a good indicator of farm level prevalence of bacterial load as it is used to irrigate dairy pasture in New Zealand. This study highlights the presence of various spore‐forming bacteria in dairy waste water and indicates the implementation of good hygienic farm practices and dairy waste effluent management.

English

The use of fungi for the production of enzymes has been widely investigated, but there are few reports concerning the use of these organisms isolated from the Amazon Region for the production of hydrolytic enzymes. Considering the importance of hydrolases for different industrial applications, this work aimed to select Amazon fungi for the production of cellulase, protease, pectinase, amylase and xylanase. In order to verify the potential of the Amazon isolates for the production of these five hydrolytic enzymes, growth on solid media containing specific substrates was performed. The qualitative assay revealed that among 40 Amazon fungi, 16 produce cellulase; 13 present proteolytic activity; 13 have pectinase activity, 27 produce amylase and 16 present xylanolyitc activity. Two amylase producers were grown in liquid media in order to access their amylolytic activity. Fungi isolated from aloe vera presented higher amylase production within 48 h of cultivation on potato starch broth. Key words: Hydrolytic enzymes, Amazon fungi, solid media assays, amylase activity.

Evaluating the antimicrobial activity of Nisin, Lysozyme and Ethylenediaminetetraacetate incorporated in starch based active food packaging film.

The pleothera of micro organisms obtained from contaminated food cultured in a starch broth was effectively tested against antibacterial agents, i.e. nisin, lysozyme and chelating agent EDTA. A variety of combination treatments of these antimicrobial agents and their incorporation in Starch based active packaging film according to their permissibility standards was done. 4 variables of Nisin concentration (ranging from 0 to 750IU/ml), 3 variables of lysozyme concentration (ranging from 0 to 500IU/ml) and 3 variables of EDTA concentration from (0 to 20μM) were chosen. Bacterial inhibition by combination of different levels of different factors without antimicrobial films was evaluated using a liquid incubation method. The samples were assayed for turbidity at interval of 2, 4 and 24h to check effectiveness of combined effects of antimicrobial agents which proved a transitory bactericidal effect for short incubation times. Zone of Inhibition was observed in the antimicrobial films prepared by agar diffusion method. Statistical analysis of experimental data for their antimicrobial spectrum was carried out by multi regression analysis and ANOVA using Design-Expert software to plot the final equation in terms of coded factors as antimicrobial agents. The experimental data indicated that the model was highly significant. Results were also evaluated graphically using response surface showing interactions between two factors, keeping other factor fixed at values at the center of domain. Synergy was also determined among antibacterial agents using the fractional inhibitory concentration (FIC) index which was observed to be 0.56 supporting the hypothesis that nisin and EDTA function as partial synergistically. The presented work aimed to screen in quick fashion the combinatorial effect of three antimicrobial agents and evaluating their efficacy in anti microbial film development.

Antagonistic Activity of Bacillus Bacteria against Food-Borne Pathogens

Bacillus bacteria have attracted the attention of scientists as promising probiotics because of their versatile antimicrobial activity and established health benefits on the host. In this study, seven Bacillus strains were identified and analyzed for antagonistic activity against broad spectrum of food borne pathogens. All strains were identified as B. subtilis, based on the results of morphological, biochemical characterization and 16S rDNA sequence analysis. B. subtilis strains demonstrated antagonistic activity against test-cultures of pathogens, including multiresistant strains. Reference Bacillus strains, derived from the commercial probiotics did not show antagonistic activity against tested strains of pathogens. Three the most active cultures were studied for production of biosurfactants. Crude biosurfactants were isolated and analyzed by oil spread test and inhibition activity against Salmonella, Shigella and Staphylococcus cultures. Biosurfactants from three tested B. subtilis strains gave positive oil spread test. Inhibition activity of biosurfactants was found only against Staphylococcus strains. Production of biosurfactants depended on the incubation conditions of Bacillus culture. Best results were obtained after cultivation of bacilli in starch broth at 30°C. The concentration of produced biosurfactant increased in time with growth of bacteria and reached the maximum at 30 hours of incubation.

Extracellular Amylase Activity of Amylolytic Bacteria Isolated from Quail’s (Coturnix japonica) Intestinal Tract in Corn Flour Medium

2 Abstract: Amylolitic microorganisms have capability of producing amylase which is an important enzyme in industries such as paper, textile and food industries. These microorganisms are found as a normal intestinal microflora in poultry's digestive tracts including quail (Coturnix japonica), especially with carbohydrates as a most nutrient in their ration. Corn becomes most important feed for quail because it has high energy related to its high amylum content. Amilolytic microorganisms in the digestive tract digest amylum by producing an extracellular enzyme (amylase) which breakdown amylum into simpler molecules facilitating for its absorbtion in the digestive tract. This research aimed to isolate amylolytic bacteria from quail's digestive tract, to determine their growth and amylolytic activity in corn flour medium. Isolation was done by using Starch Agar medium, then amylolytic activity was indicated based on intensity of clear zone formation in the media and by amylase assay using DNS method in Starch Broth medium. Growth curve and amylase assay was carried out in 2% Corn Flour Broth medium. Data were subjected to analysis of Pearson correlation. The results showed that six isolates called as BAP1, BAP2, BAP3, BAP4, BAP5 and BAP6 were found. BAP6 isolate showed the widest clear zone formation in Starch Agar medium, highest amylase activity and non pathogenic characters. Thus, it was selected and used in further analysis. BAP6 isolate grew well in 2% Corn Flour Broth medium with the highest cell number, 2.2 x 10 CFU/ml at 24 h incubation and the 8 highest amylase activity, 0.0201 Unit at 12 h incubation.

Effects of temperature and pH on the growth of bacteria isolated from blown packs of vacuum-packaged beef.

Bacteria recovered from the microflora of blown packs of vacuum-packaged beef were identified as Leuconostoc mesenteroides, Lactococcus lactis, Carnobacterium maltaromaticum, and Clostridium estertheticum, with L. mesenteroides predominant. Isolates of these lactic acid bacteria all grew in peptone yeast extract glucose starch broth (PYGSB) at temperatures between -2 and 30 degrees C but generally grew more slowly and over a more restricted temperature range in meat juice medium (MJM). A C. estertheticum isolate and the type strain of C. estertheticum subsp. estertheticum (ATCC 51377) both grew in PYGSB and MJM at similar rates at temperatures between -2 and 17 degrees C and grew at 20 degrees C in MJM but not in PYGSB. Square root models of the variation of the growth rate with temperature indicated that the C. maltaromaticum isolate and the C. estertheticum strains grew at similar rates that were faster than those of the other isolates at temperatures between -2 and 0 degrees C. The L. mesenteroides and L. lactis isolates grew in PYGSB at pH 5.0, but the C. maltaromaticum isolate and both strains of C. estertheticum did not grow in PYGSB at pH <or= 5.3. C. estertheticum stopped growing in MJM buffered at pH 6.5 when glucose was exhausted, although these bacteria then utilized lactate. The findings suggest that, like carnobacteria, C. estertheticum may predominate during the early stages of development of the spoilage microflora of vacuum-packaged beef but that C. estertheticum will likely be inhibited by a falling pH and so may be only a minor part of the spoilage microflora when maximum numbers are attained.

The effect of 100% CO 2 on the growth of nonproteolytic Clostridium botulinum at chill temperatures

The growth of a cocktail of spores from six nonproteolytic Clostridium botulinum type B and E isolates at 5 and 10°C was used to assess the combined effect of NaCl (0.5–4.5% w/v), pH (5.5–6.5) and atmosphere (10% H 2:90% N 2, 5% CO 2:10% H 2:85% N 2, or 100% CO 2) in buffered peptone, yeast, glucose, starch broth with an E h of approximately −350 mV. Under all atmospheres growth tended to be slower as the concentration of NaCl increased and with NaCl combined with pH levels below 6.0. Of the atmospheres tested, growth occurred at a slower rate and over a narrower range of conditions when C. botulinum was exposed to 100% CO 2. This effect was enhanced when the incubation temperature was 5°C. The results indicate that while CO 2 decreased C. botulinum growth at chill temperatures, prevention of growth also depended on the NaCl concentration and the pH of the medium.

Clostridium gasigenes sp. nov., a psychrophile causing spoilage of vacuum-packed meat.

Two psychrophilic Clostridium strains, DB1AT and R26, were isolated from incidences of 'blown-pack' spoilage of vacuum-packed chilled lamb. Vacuum packs of meat inoculated with these strains developed gas bubbles and pack distension within 14 d storage at 2 degrees C. The two main gases responsible for pack distension were carbon dioxide and hydrogen. 1-Butanol, butyric and acetic acid and butyl esters were the major volatile compounds produced by the strains in the artificially inoculated packs. The unknown strains were Gram-positive motile rods producing elliptical subterminal spores during the late-stationary growth phase. At pH 7.0, they grew from -1.5 to 26 degrees C, and their optimum growth temperature was 20-22 degrees C. At 20 degrees C, the pH range for growth was 5.4-8.9 and the optimum pH for growth was 6.2-8.6. In peptone/yeast extract broth, the organisms grew little or not at all in the absence of fermentable carbohydrates. Both strains hydrolysed gelatin, aesculin and starch. The fermentation products formed in peptone yeast extract glucose starch broth were ethanol, acetate, butyrate, lactate, butanol, carbon dioxide and hydrogen. The G+C contents of the DNA of strains DB1AT and R26 were 29.4 and 28.3 mol%, respectively. Phylogenetic analyses indicated that the strains belong to cluster I of the genus Clostridium (sensu Collins et al. 1994). The new strains differed from the phylogenetically related clostridia in cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of rDNA analysis and phenotypic and phylogenetic characterization, the strains were assigned to a new species for which the name Clostridium gasigenes is proposed. Strain DB1AT (= DSM 12272T) is designated as the type strain.

Lectin-Detectable Effects of Localized Pneumonia on Airway Mucous Cell Populations: Role of Cyclooxygenase Metabolites

We examined the airway secretory apparatus of adult sheep with experimental pneumonia to look for morphologic and lectin-binding correlates of increased mucus production. The animals were inoculated in the right caudal lobar bronchus either with starch broth containing Pasteurella haemolytica (INF, n = 6), starch broth alone (SHAM, n = 6), or with P. haemolytica and subsequently treated (INF/T, n = 5) with 2 mg/kg indomethacin, subcutaneously three times daily for 6 days. In the INF and INF/T groups, a localized pneumonic infiltrate containing P. haemolytica organisms was present. The bronchi (18-23rd generation) adjacent to the pneumonic lesion had an increased gland volume fraction (6.3 +/- 3.7% in INF, 11.3 +/- 2.4% in INF/T, and 3.1 +/- 1.9% in SHAM, p less than 0.05 among the three). The mean population densities of BSA-reactive (identifying alpha-D-gal) cells were 41.9 +/- 2.7% in the INF, 40.1 +/- 5.6% in the INF/T, versus 14.3 +/- 1.5% in the SHAM group (p less than 0.05), while the corresponding values for PNA-reactive [identifying beta-D-gal(1----3)-D-galNAc] cells were 28.8 +/- 5.1%, 0%, and 0%, respectively. Nor morphologic abnormalities were seen in the trachea, but BSA staining was shifted to morphologically different mucous cells in the INF and INF/T. We conclude that in localized P. haemolytica pneumonia in sheep (1) there are morphologic changes of the airway secretory apparatus adjacent to the lesion, (2) the glycoconjugate profile of secretory cells adjacent to and remote from the lesion is altered, and (3) cyclooxygenase products influence the chemical composition of secretory cells.

Further Investigations on the Nature of the Heat Resistance of Thermophilic Bacteria

AbstractWhen cells of Bacillus stearothermophilus, strain NCA 1503, were grown in tryptone starch broth and subsequently transferred to tris buffer, a fraction of the cells: rapidly died in ttie buffer. This fraction increased with increasing content of calcium chloride in the growth medium. The' addition of sodium, potassium or magnesium chloride to the growth medium had no such effect. The rapid dying of the cells in tris buffer was associated with a leakage of organic material and calcium ions from the cells. The results obtained are probably caused by a damage to the osmotic barrier of the cells during their contact with the buffer. Observations: made during the present investigation and a previous one (Ljunger 1970) indicate that the heat resistance of thermophilic bacteria depends on the maintenance of a high intracellular concentration of free calcium ions.

The Influence of Inorganic Ions on the Heat Stability of a Thermophilic Bacteriophage

AbstractA thermophilic bacteriophage was isolated from soil. Heat inactivation of this phage, suspended in tryptone starch broth at 65°C and 70°C, was found to be a monomolecular reaction. The phage was more heat stable in tryptone broth than in tris buffer. When the tris buffer was supplemented with calcium or magnesium ions, the survival percentage increased from 0.0 to 18.0 after two hours of heating at 65°C. The addition of sodium or potassium ions to the tris buffer had no significant effect. Equimolar solutions of calcium and magnesium chloride had the same effect on the heat stability of the phage. Maximum stability was attained in 2.5 mM solutions of these salts, and a further increase in the concentration up to 10.0 mM did not increase the percentage of surviving phages.