Solid-state cultivation of Aspergillus niger NCIM 548 for glucoamylase production on groundnut shell
culture condition, flask culture, glucoamylase, groundnut shell, solid-state fermentation
C Paulchamy. Solid-state cultivation of Aspergillus niger NCIM 548 for glucoamylase production on groundnut shell. The Internet Journal of Microbiology. 2007 Volume 5 Number 1.
Glucoamylase is a well recognized amylolytic enzyme used in food industry, which has been produced by Aspergillus genus under solid-state and submerged cultivation. In this study, the strain Aspergillus niger NCIM 548 was examined for glucoamylase production on the solid surface of groundnut shell (0.5 mm particle size) supplemented with different nutrient factors in Aspergillus minimal medium. With 50% initial moisture content, a maximum glucoamylase activity (726 U/gdm) was achieved by this strain on groundnut shell in presence of sucrose (1%, w/v) and yeast extract (0.5%, w/v) at 84 hrs. However, when groundnut shell was combined with other starchy wastes this strain did not produce a considerable enzyme yield. This strain was produced more glucoamylase yield on groundnut shell alone. Since groundnut shell is a very cheap, easily available source from oil industry and more suited for solid-state cultivation of A. niger for glucoamylase production, a detailed study on the large-scale fermentation processes using groundnut shell and potential uses are suggested.
Amylases are well known starch degrading enzymes, which are comprised in to α-, β-, and γ-amylase. Among these, glucoamylase (γ-amylase) is being a commercial interest in food industry. It has been exploited for the production of starch syrups, chocolate syrup and dextrose, the preparation of starch derived adhesives, grain alcohol and fruit cakes, improving utilization of enzymatically treated barely in poultry calf raising and the clarification of fruit juice for jelly manufacture in food processing industry (Joshi et al., 1999). Glucoamylase (amyloglycosidase, EC.126.96.36.199) is an exo-acting enzyme that yields β-D-glucose by hydrolyzing β-1-4 glycoside linkages from the non-reducing ends of amylose, amylopectin and glycogen (Forgarty and Kelley, 1980). The exclusive production of this enzyme is achieved by
Material and Methods
Solid-state cultivation in flask
Groundnut shell was collected from a local vegetable oil industry and grinded to obtain 0.5 mm particle size using a standard sieve, and preserved at room temperature. The chemical constituents of groundnut shell were analyzed according to the standard methods described by Ranade et al (1980). 10 g (w/w) of this substrate was distributed in a 250 mL wide-mouth flask and then moisturized with 40 mL of
Effect of media supplements on enzyme production
The effect of different media supplements including carbon, nitrogen and other starchy materials along with groundnut shell was studied in this work by using
Solid-state cultivation in tray
One mL (v/v) spore suspension (3 x 10 8 spores/mL) was transferred to a 500 ml wide-mouth flask containing 25 g (w/w) ground nut shell moisturized with 100 mL
Extraction of enzyme
After 4 days incubation, 50 mM citrate buffer (pH-4.8) (1:10 ratio) was added to fermented dough and homogenized with a constant stirring. This suspension was filtered through Whatman filter paper number 1 and the filtrate was again centrifuged at 6000 rpm for 15 min. This solid-free supernatant was used as enzyme source for assaying glucoamylase activity.
The enzyme activity was determined by a reactive mixture contains one mL of 50 mM citrate buffer (pH-4.8) and one mL starch solution (1 %, w/v) and 0.1 mL of crude enzyme. This reactive mixture was incubated at 50 o C for 20 min and then released reducing sugars were measured with 3, 5-dinitrosalicyclic acid (DNSA) reagent (Miller, 1959) using glucose as a standard. The protein concentration in crude enzyme was estimated by Folin-phenol method (Lowry et al., 1956) using BSA as standard. Glucoamylase activity was expressed as the amount of enzyme required to liberate one mole of reducing sugars from starch by multiplying the total amount of enzyme extracted from initial gram dry mass of substrate (gdm).
Results and Discussion
The chemical constituents of groundnut shell were analyzed and are listed in Table 1. The high concentration of starch suggested the possible use of groundnut shell for amylase induction in solid-state conditions.
Fig.1 shows the effect of incubation time on glucoamylase production by
The optimum moisture content reported for maximum synthesis of extracellular amylases at 30 o C was 50-55 % (Raimbault and Alazard, 1980; Wang et al., 2006). In this work, the similar moisture content was found to be favorable for glucoamylase synthesis. With moisture content below 30% was significantly lowering the formation of glucoamylase, and the biomass yield of
A maximum glucoamylase activity (726 U/gdm) was yielded by
Groundnut shell alone, without addition of minimal medium, served as a potential carbon source for the solid-state cultivation of
Additional carbon supplements, exclusively galactose, mannose and sucrose, were found to induce maximum glucoamylase activity where as its activity was lowered by adding xylose and fructose to minimal medium (data not shown). Similarly, media supplementation with sucrose in wheat bran increased enzyme production (271 U/gdm) as reported by Anto
Organic nitrogen source, corn steep liquor stimulated the growth of
Among different starchy raw materials checked as shown Table 4, the maximum enzyme activity (704 U/gdm) with specific activity (15.08 U/mg proteins) was observed on groundnut shell followed on potato starch (687 U/gdm). In contrast, many of the raw materials used including cereal flours and straws when in combination failed to boost enzyme yield in presence of sucrose and yeast extract. These results revealed that on comparison to other expensive raw substrates groundnut shell exhibited more suitability for solid-state cultivation of
Considering the results of solid-state cultivation of
Table 4: Showing the influence of production media supplemented with different starchy raw materials (10 g each) for glucoamylase production by
We are thankful to Vice-Chancellor and Registrar of Gujarat Vidyapith, Ahmedabad, India for permitting us to carry out this investigation. This work was financially supported by University Grants Commission, New Delhi, India.
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