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Optimization of Conditions for Extracellular Amylase Production from Listeria Denitrificans.

M. N. MAHMUD
Department of Microbiology, University of Chittagong, Chittagong -4331, Bangladesh

M. N. ANWAR
Department of Microbiology, University of Chittagong, Chittagong -4331, Bangladesh

Abstract/ Executive Summary:

The foodborne Listeria denitrificans was isolated from degraded rice and studied as an amylase producer. This microorganism showed maximum amylase production at temperature 27 ºC and medium pH 7.0, and on 3rd day during incubation period. Maximum amylase production by the isolate was found in medium containing 3.0 % starch as carbon source and 0.50 % yeast extract as nitrogen source.

Keywords: Listeria denitrificans, Amylase activity, Factors influence

Location: Department of Microbiology, University of Chittagong, Chittagong

Start Date:

End Date:

Program Area: Quality and Nutrition

Commodity: Fortified food

Objectives:

1. This paper reports on the screening and identification of a bacterial isolate obtained from degraded rice and also on the determination of its optimum culture conditions involved in maximum production of amylase.

Methodology:

Microorganism - The isolate Listeria denitrificans (NB₂) was isolated from degraded rice by enrichment technique and purified by repeated pour plate and streak plate methods using nutrient agar (NA) medium. Screening of the isolates for amylolytic activity - For primary selection of the isolates, hydrolysis of starch agar plate method was followed. After primary selection, secondary screening of the isolates was made by measuring amylase activity in liquid medium by quantitative method. For in vitro production of amylase by the isolates, three different broth media, namely - Starch yeast extract medium, Defined salt medium and Standard production medium were used. After secondary Screening, on the basis of their better enzyme activity in broth medium the bacterial isolate (NB₂) was finally selected for detailed study. Measurement of enzyme activity - One ml of culture filtrate was added to 5 ml of 1% soluble starch prepared in 0.2 M citrate phosphate buffer pH 5.8 and 1 ml of 0.2 M citrate phosphate buffer was taken in a test tube and incubated at 40⁰C for 1 hour. The amount of reducing sugars released during incubation was measured by Nelson's modification of Somogyi method. Enzyme activity was expressed by amount of Glucose released/ml extracted enzyme /unit time (U/mL). Biomass yield - Bacterial biomass was determined by measuring the absorbance at 600 nm. Identification - The bacterial isolate NB₂ was identified on the basis of its morphological, cultural and physiological characteristics. These characteristics were compared with standard description of Bergey’s Manual of Determinative Bacteriology. Optimization of culture conditions for maximum production of amylase Effect of incubation period - The effect of incubation periods on the maximum production of amylase by selected isolate was studied. For this, 50 ml of selected medium was taken in each 100 ml conical flask and autoclaved. After cooling the flasks were inoculated and incubated for 24, 48, 72, 96 and 120 hours. The culture was centrifuged at 8000 rpm for 15 min at 4⁰C. Effect of medium pH - To observe the effect of medium pH on enzyme production, the medium was prepared at different pH (4.5, 6.0, 6.5, 7.0 and 8.5), inoculated and incubated at 37ºC. The effect of medium pH on biomass characteristics, biomass yield and amylase activity was recorded. Effect of temperature - After inoculation the culture medium was incubated at different temperature values such as 10⁰C, 27 ± 2⁰C, 37 ± 2⁰C, and 45 ± 2⁰C for maximum enzyme production. The effect of temperature on biomass characteristic, biomass yield and amylase production was recorded. Effect of carbon and nitrogen sources - Four carbon (starch, rice powder, potato, wheat powder) and five nitrogen sources (malt, yeast extract, ammonium di-hydrogen phosphate, ammonium sulphate and potassium nitrate) were added separately to the medium (Standard Production Medium) and the effect of this carbon and nitrogen sources on the production of amylase, extracellular protein and biomass yield was recorded. To ascertain optimum proportion of suitable carbon and nitrogen sources, the study was carried out with 0.5 to 4% carbon and 0.025 to 2% nitrogen sources keeping other experimental conditions at optimum level.

Source of Information: The Chittagong Univ. J. B. Sci.,Vol. 4(1 &2):73-81 , 2009

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Highest biomass yield was recorded with starch and malt when used as carbon and nitrogen sources, respectively. The bacterial isolate exhibited highest amylase production in starch and yeast extract containing medium and also showed that the optimum concentration of starch and yeast extract for maximum enzyme production were 3.0 % and 0.50 %, respectively. Maximum amylase production by Aspergillus fumigatus was reported when 0.25% (NH₄)₂HPO₄ and 4% starch were added in the growth medium.

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