Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates / Produção significativa de vanilina e amplificação in vitro do gene ech em isolados bacterianos locais

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.

Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.

Assessment of Acute Kidney Injury in Patients Undergoing Elective Coronary Angiography and Percutaneous Coronary Intervention.

Background: CIN has gained increased attention in the clinical setting, particularly during cardiac intervention but also in many other radiological procedures in which iodinated contrast media are used. There is at present good clinical evidence from well-controlled randomized studies that CIN is a common cause of acute renal dysfunction. Methodology: This was a prospective study conducted among the patients who underwent coronary angiography and percutaneous coronary intervention in the Department of Cardiology, Dhaka Medical College Hospital during January 2010 to December 2010. A total of 111 patients age range from 25 to 75 years were included in the study. Serum creatinine level at baseline and at the end of 48 hours was done in all these patients. Study population was divided into two groups according to development of acute kidney injury (AKI). Group-I = AKI, Group II = Not developed AKI. Results: AKI developed 11.7% of the study patient. DM and Preexisting renal insufficiency were significantly higher in group I patients. HTN was (61.5% Vs 44.9%) higher in group I but not significantly. History of ACE inhibitor/ARB, NSAID intake and LVEF <40% were significantly higher in group I patients. The mean±SD volume of CM (Contrast Media) were 156.9±44.8 ml and 115.4±30.0 ml in group I and group II respectively, which was significant. The mean±SD of serum creatinine after 48-72 hours of CAG/PCI was 1.4±0.37 mg/dl and 1.1±0.2 mg/dl in group I and group II respectively. The serum creatinine level increased significantly (p<0.05) after 48-72 hours of CAG/PCI in group I. In group II, S. creatinine level increased but not significant (p>0.05). Impaired renal function was found 76.9% and 2.0% in group I and group II respectively. DM, HTN, preexisting renal insufficiency, ACE inhibitor/ARB, NSAIDs, contrast volume (>150 ml), eGFR (<60 ml/min/ 1.73m2) and LVEF (<40%) are significantly (p0.05) associated for CIN development, Conclusion: CIN is an iatrogenic but preventable disorder results from the administration of contract media. Although rare in the general population, CIN occurs frequently in patients with underlying renal dysfunction and diabetes. In patients with pre angiographic normal renal function, the prevalence is low but in pre-existing renal impairment it may pose a serious threat. Thus risk factors are synergistic in their ability to predispose to the development of CIN. A careful risk-benefit analysis must always be performed prior to the administration of contrast media to patients at risk for CIN.

Study of Serum Insulin Level in Hypertensive Patients.

Background and aims: Hypertension is a frequent and almost ubiquitous health disorder, prevalent both in developed and developing countries. Hyperinsulinemia and insulin resistance have been suggested to be closely associated with the pathogenesis of essential hypertension. However there is considerable controversy in this regards. The present study was designed to explore the relationship between serum insulin and serum ionized calcium in non diabetic and diabetic hypertensive subjects. Subjects and Methods: A total of 57 hypertensive and diabetic hypertensive patients attending out patients department of the BIRDEM and NICVD were included in the study. Fasting serum glucose was measured by glucose oxidase method, lipid profile was measured by enzymatic method. Serum insulin was measured by Enzyme Linked Immunosorbent assay (ELISA) method and serum ionized calcium by the Ion Sensitive Electrode (ISE) technique. Results: Glucose-insulin ratios were calculated as the index for insulin. Serum insulin (pmol/ml), Mean ± SD, 147 ± 48 in DC and 170 ± 80 in DH groups vs 118 ± 21 in NC and 120 ± 41 in EH groups, p= 0.023 and p= 0.031 respectively. Although, from the serum insulin results, the diabetic groups seemed to have insulin resistance, the glucose-insulin ratios in the two groups were significantly lower compared to nondiabetic groups (Glucose-insulin ratio, mmol/pmol, 0.066 ± 0.025 in DC, 0.074 ± 0.025 in DH vs 0.044 ± 0.11 in NC, 0.043 ± 0.012 in EH, p= 0.005 - 0.0001). The serum ionized calcium in the healthy subject, first time reported in the country by an up to date method (1.17 ± 0.05 M ± SD), were within the range found in healthy subjects of the other populations. No significant difference in the serum Ca2+ could be found between any of the study groups. Also, serum Ca2+ did not correlate with blood pressure, glucose or insulin in any of the study groups or with all the patients as a whole. Serum total cholesterol, triglyceride, HDLc and LDLc levels in the DC, EH and DH group did not show any significant difference compared to NC group and among the groups. The lipid abnormality as reflected by the mean LDL-HDL cholesterol ratios was the highest in the DH group but the differences were not statistically significant compared to the NC, DC and EH group. Conclusions: The data suggest the following conclusions: a) Serum ionized calcium level in our population is similar to that reported for other population. b) Serum glucose and insulin by themselves do not have any direct influence on serum ionized calcium. c) Non obese diabetes mellitus subjects in our population do not show insulin resistance as the primary defect. Rather, there is significant decompensation of the insulin secretory capacity in the subjects. d) Insulin resistance should be measured directly in relation to blood pressure and Ca2+ in appropriate groups of subjects to explore the relationship between insulin resistance, hyperinsulinemia and serum ionized calcium.

Assessment of Colour Blindness and Erythrocyte G6PD Enzyme Status among the School Children of Dhaka City.

Background: Visual impairment due to colour blindness is an unusual suffering of the school children. It may be associated with erythrocyte G6PD enzyme deficiency. Objective: To find out defective vision due to colour blindness in apparently healthy school children and to measure erythrocyte G6PD enzyme level among them. Methods: This cross sectional study was carried out in the Department of Physiology, SSMC from 1st July 2007 to 31st June 2008. Five hundred (500) apparently healthy school children of old Dhaka, age ranged from 6 to 12 years irrespective of gender and race was selected as study population. Colour vision test was done by Ishihara’s test. Erythrocyte G6PD enzyme was measured among the colour blind children. All the results were compared to that of children with normal colour vision. Results: Five male children were detected to have partial red-green type of colour blindness. The percentage of colour blindness was statistically not significant (p>0.05) when compared to that of children with normal colour vision. Mean erythrocyte G6PD enzyme level of colour blind children was significantly lower (p<0.05) compared to that of children with normal colour vision. Presence of G6PD enzyme deficiency among the colour blind children did not show any clinical abnormalities might be due to different non symptomatic G6PD variants. Conclusion: Visual defect due to colour which blindness particularly red-green type might be present in apparently healthy school children associated with erythrocyte G6PD enzyme deficiency.

Comparison between DOT EIA IgM and Widal Test as early Diagnosis of Typhoid Fever.

A recently developed DOT enzyme immunoassay known as "Typhidot" for detecting IgM antibody against 50 KDa OMP antigen of Salmonella typhi, was evaluated on 100 clinically suspected typhoid fever cases and 40 age-sex matched controls, in the Department of Microbiology, Mymensingh Medical College during, the period from June 2006 to July 2007. Blood culture, Widal test, and DOT EIA for IgM test were performed in all patients. Among 100 clinically suspected typhoid fever cases, 35 were subsequently confirmed on the basis of positive blood culture for S. typhi and/or significant rising titre of Widal test. The DOT EIA IgM test could produce results within 1 hour. The result of the DOT EIA IgM test showed a good diagnostic value for typhoid fever. The sensitivity, specificity, positive and negative predictive value of the test was found as 91.42%, 90.00%, 88.88% and 92.30% respectively. On the other hand corresponding values for Widal test were of 42.85%, 85.00%, 71.42% and 62.96% respectively. Thus, The DOT EIA IgM seems to be a practical alternative to Widal test for early diagnosis of typhoid fever.

A comparative study of body iron status and lipid profile in acute myocardial infarction patients.

This study was carried out to evaluate the relationship between body iron status and lipid profile in hospital admitted clinically diagnosed AMI patients considering the concept that there is a potential association between body iron status and coronary heart disease (CHD). Total 80 subjects were selected, of which 40 were healthy adults and 40 were AMI patients. Fasting blood samples were collected from healthy adults. Blood samples of AMI patients were collected within 24 hours of the attack of myocardial infarction. Body iron status was measured in term of 3 variables serum total iron concentration, TIBC and transferrin saturation. Lipid profile variables measure were total cholesterol, triglyceride, LDL-cholesterol and HDL-cholesterol. No correlation was found between serum iron and the variables of lipid profile. TIBC was found to maintain negative correlation with total cholesterol, triglyceride and LDL-cholesterol but positive correlation with HDL-cholesterol. Transferrin saturation was found to maintain strongly positive correlation with total cholesterol, triglyceride and LDL-cholesterol but strongly negative correlation with HDL-cholesterol. This correlation of TIBC and transferrin saturation with lipid profile supports the hypothesis that there is a potential association between body iron status and coronary heart disease.

Effect of graded head-up tilt on parasympathetic reactivity.

Thirteen healthy subjects were tested for parasympathetic reactivity during head-up tilt and reversal of the tilt. Head-up tilt (70 degrees) resulted in significant increase in baseline heart rate and diastolic blood pressure. Head-up tilt also led to increased parasympathetic reactivity as measured by Valsalva manoeuvre and hand grip test. Heart rate response to deep breathing test did not change. The reversal of the tilt led to returning of heart responses to original values. Responses indicate towards enhanced parasympathetic reactivity during head-up tilt position.

Prolonged stability of R plasmids KR61 and KR61-A in Salmonella host.

The R plasmids, KR61 and KR61-A, that were originally isolated from a clinical strain of Aerobacter aerogenes in 1971 and determined resistance to kanamycin (Km), neomycin (Nm), streptomycin (Sm), tetracycline (Tc); and ampicillin (Ap) respectively were found stable in Salmonella typhimurium LT2 even after 22 years of cultivation on antibiotic free media. KR61, carrying resistance to KmNmSmTc, not only maintained all its resistances but also maintained its conjugal transferability (RTF) as indicated by its subsequent transfer to Escherichia and Salmonella hosts. KR61-A that carried resistance to Ap and lacked an RTF could be mobilized by KR61 from S. typhimurium LT2, constructed to bear KR61-A and KR61, to E. coli recipients. S. typhimurium LT2 carrying KR61-A + KR61 (ApKmNmSmTc), showed the characteristic conjugal transfer of resistances in following three patterns: (i) Ap, (ii) KmNmSmTc and (iii) ApKmNmSmTc. The findings reported here are based on conjugal isolation of plasmids. Physical isolation of KR61 and KR61-A was never made.