Medium Optimization for Production of A-Amylase by Bacillus Subtilis and Aspergillus Niger in Submerged Fermentation

Medium Optimization for Production of A-Amylase by Bacillus Subtilis and Aspergillus Niger in Submerged Fermentation

M. B. Khadse

Department of Agricultural Engineering Maharashtra Institute of Technology Aurangabad, Maharashtra, India

K. P. Babar

Department of Agricultural Engineering Maharashtra Institute of Technology Aurangabad, Maharashtra, India

Abstract The present study was carried out to optimize the fermentation medium for production of -amylase using Bacillus subtilis and Aspergillus niger strain in submerged fermentation. The effects of incubation period, temperature, pH, different carbon source, nitrogen source and metal salt of the medium were optimized. The maximum production of enzyme was found at 72 h (3.04 U/ml) in Bacillus subtilis and (4.35 U/ml) in Aspergillus niger. The optimal temperature and pH were found to be 350c at pH 7 in Bacillus subtilis and 450c at pH 7 in Aspergillus niger. Starch (3.04 U/ml) and peptone (3.92 U/ml) was observed to be best carbon and nitrogen source in Bacillus subtilis and in Aspergillus niger it was found to be starch (3.05 U/ml) and casein (1.36 U/ml) respectively. The effect of different metal salt were investigated and it as found that MgSO4 (3.14U/ml) and (2.96 U/ml) was observed to be best metal salt source in Bacillus subtilis and Aspergillus niger respectively.

Keywords Bacillus subtilis, Aspergillus niger, -amylase

1. INTRODUCTION

   In modern times most important products of biology origin are enzymes, because they have numerous applications in various industrial processes[1]. The world market for industrial enzymes is estimated to be 1.6 billion, split between food enzymes (29%), feed enzymes (15%) and general technical enzymes (56%)[2]. Food enzymes are the most widely used and still represent the major share in enzyme market.

   Amylase constitute a group of industrial enzymes, which alone covers approximately 30% of the enzyme market[3]. lpha amylase is a hydrolase enzyme that catalyses the hydrolysis of internal -1.4-glycosidic linkages in starch to yield products like glucose and maltose. It is a calcium metalloenzyme i.e. it depends on the presence of a metal co factor for its activity[4]. These uses have placed greater stress on search for novel -amylase for more efficient processes[5]. These enzymes are extensively employed in processed food industry such as baking, brewing, preparation of digestive aids, production of cakes, fruit juices and starch syrups[6].

   In recent year, the potential of using severa micro- organisms as biotechnological sources of industrially relevant enzymes has stimulated interest in the exploration of extracellular enzymatic activity[7]. Amylase can be produced by

   Fermentation is the technique of biological conversion of complex substrates into simple compounds by various micro- organisms such as bacteria and fungai.Fermentation has been classified into ssf and smf mainly based on the type of substrate used during fermentation. Smf utilizes free flowing liquid subtstrates, such as molasses and broths.The bioactive compounds are secreated into the fermentation broth.An additional advantage of the this technique is that purification of products is easier.Usually production of amylase has been carried out using well defined chemical media by submerged fermentation (smf).[10].

   The present study was carried out to optimize the fermentation medium for the production of -amylase using different micro-organism in submerged fermentation.

2. MATERIAL AND METHOD

   1. Procurement of raw material

      Bacillus subtilis (NCIM 2439) and Aspergillus niger (NCIM 1054) these two microbial strain were procured from National Laboratory (NCIM) Pune, Maharashtra, India.These strain was sub cultured on nutrient agar and potato dextrose agar slant respectively and maintained at 40c. Various chemicals and instruments used were obtained from Department of Agricultural Engineering Maharashtra Institute of Technology, Aurangabad.

   2. Methods

      1. Preparation of Inoculum and addition

         The inoculum was prepared by the addition of sterile distilled water in to the freshly grown agar slants, from this 0.5ml suspension was inoculated in to 100ml of sterilized fermentation medium and incubated in a shaking incubator at 120rpm[11].

      2. Fermentation media

         Optimization of -amylase production by Bacillus subtilis and Aspergillus niger was carried out by using submerged fermentation. The fermentation medium for Bacilllus subtilis was (g/l) 6.0g bacteriological peptone, 0.5g

         MgSO .7H O, 0.5g KCl, 1.0g starch and fermentation medium

         different species of micro-organisms, but for commercial 4 2

         applications amylase is mainly derived from the genus Bacillus[8]. Several enzymes of industrial importance have been extracted from the fungai belonging to the genus Aspergillus[9].

         for Aspergillus niger was (g/l) 0.1g MgSO4, 1.0g (NH4)2SO4, 2.0gKH2PO4, 7.0g K2HPO4, 0.5g Na-citrate and the fermentation medium was maintained at pH 7[12][13].

      3. Enzyme production

         Erlenmeyer flasks (250 ml) containing 100ml of amylase producing fermentation medium were inoculated with

         0.5 ml suspension of culture and incubated at 120rpm at 350c[14].

      4. .Extraction of amylase enzyme from the fermentation medium

      Amylase after incubation, the fermentation medium was harvested by centrifuged at 5000 rpm for 20 minutes at 40c.The supernantant was collected and the amylase activity estimated[12].

      e . Assay of amylase

      The amylase activity was determined by incubating 1ml of soluble starch (1%) in 0.1M phosphate buffer (pH 6.9) are pre incubated at 370c for 10 minutes. The supernantant collected from the centrifugation of the production media was used as enzyme source, 0.1-0.5ml of this was added to the reaction mixture. The reaction was terminated by the addition of 1.0ml of 3,5-dinitrosalicylic acid reagent after incubation at 370c for 30 minutes. The contents were mixed well and kept in water bath for 10 minutes. Then they were cooled and diluted with 10 ml of distilled water. The colour developed was read at 540nm. One unit of enzyme activity was defined as the amount of enzyme that release 1µ mole of reducing sugar (maltose) per minute under the assay condition[15].

   3. Optimization studies for enzyme production

      In a sequential order, the various process parameter affecting the enzyme production were optimized for maximal enzyme production for which best amylase activity was observed. The different optimization process parameter are as follows.

      1. Effect of incubation time:-

   The effect of incubation period was optimized by incubating production medium for different incubation peroids(24hr, 48hr, 72hr)[16].

   1. Effect of temperature:-

      The effect of temperature on amylase production was investigated by incubating the fermentation medium at different temperatures (250c, 350c, 450c)[17].

   2. Effect of pH:-

      The effect of pH on amylase production was investigated by varying the pH values set to (6.0,7.0, 8.0)[13].

   3. Effect of carbon sources:-

      The effect of different carbon sources such as starch, maltose and glucose each at 1% concentration was investigated on amylase production[14].

   4. Effect of nitrogen sources:-

      The effect of different nitrogen sources such as peptone, casein, ammonium sulphate each at 1% concentration was investigated on amylase production[14].

   5. Effect of metal salts:-

      4

      The effect of metal sats on -amylase production was determined by adding different metal salts in the fermentation medium.The metal salts selected for present study are FeSO4, MgSO4, ZnSO [16].

3. EXPERIMENTS AND RESULTS

   The experiments were conducted for The optimization of culture conditions based on the stepwise modification of the governing parameter such as incubation time, incubation temperature, incubation pH, carbon source, nitrogen source and metal salt source. Data presented here show that Bacillus subtilis and Aspergillus niger produces – amylase. The optimal conditions for -amylase production have been determined under submerged fermentation conditions.

   The results obtained during the present investigation are presented and discussed under suitable heading. The results were discussed in the view of relevant scientific literature available in the country and elsewhere:

   1. Incubation time

      Effect of different incubation time was determined by incubating production medium for different incubation periods.

      Table 1 Effect of different incubation time on amylase production from Bacillus subtilis and Aspergillus niger by submerged fermentation

      Sr. No.	Microbial strain used	Time in hours	O.D. at 540nm	Mg of maltose released	Enzyme activity (IU/ml)
      1.	Bacillus subtilis	24hr	0.623	1.020	2.82
      		48hr	0.679	1.075	2.97
      		72hr	0.690	1.100	3.04
      2.	Aspergillus niger	24hr	0.024	0.094	0.26
      		48hr	0.391	0.738	2.04
      		72hr	0.998	1.574	4.35

      *O.D.: Optical dentisity

      From the above table it is revealed that among the three timings activity tested for amylase production viz; 24, 48 and

      72 hours, the optimum incubation period for -amylase poduction was noted at 72hrs and production found decreased in the rest of the incubation period.

      Strain Bacillus subtilis showed the maximum activity of 3.04U/ml while starain Aspergillus niger showed the maximum activity of 4.35U/ml with the temperature of incubation period was kept at 350c and pH 7. Nearly similar observations were obtained by[12][13].

   2. Incubation temperature

      Effect of different incubation temperature was determined by incubating production medium for different incubation temperature.

      Table 2 Effect of Different Incubation Temperature On Amylase Production From Bacillus Subtilis And Aspergillus

      Niger By Submerged Fermentation

      Sr. No.	Microbial strain used	Temperature in (0c)	O.D. at 540nm	Mg of maltose released	Enzyme activity (IU/ml)
      1.	Bacillus subtilis	250c	0.291	0.550	1.52
      		350c	1.810	2.363	6.53
      		450c	0.865	1.321	3.65
      2.	Aspergillus niger	250c	0.491	0.839	2.32
      		350c	0.631	1.111	3.07
      		450c	0.923	1.495	4.13

      *O.D.: Optical dentisity

      Temparature is one of the important physical factor infuencing enzyme production. The production medium incubated at different temperature i.e. 250c, 350c and 450c.

      From the above table it is revealed that strain Bacillus subtilis when icubated at different temperature it was found that incubation temperature of 350c was found to be optimum for maximum amylase activity i.e. 6.53U/ml and for strain Aspergillus niger it was found to be 4.13U/ml at 450c with the production medium was kept for 72 hrs at 7 pH.

   3. Incubation pH

      Effect of different incubation pH was determined by incubating production medium for different incubation pH.

      Table 3 Effect of different incubation ph on amylase production from Bacillus subtilis and Aspergillus niger by submerged fermentation

      Sr. No.	Microbial strain used	PH	O.D. at 540nm	Mg of maltose released	Enzyme activity (IU/ml)
      1.	Bacillus subtilis	6	1.021	1.524	4.21
      		7	2.310	2.750	7.62
      		8	1.561	2.143	5.92
      2.	Aspergillus niger	6	0.381	0.684	1.89
      		7	0.601	0.973	2.69
      		8	0.176	0.405	1.12

      *O.D.: Optical dentisity

      Amylase production indicates greater influence by pH. The production medium incubated at different pH i.e. 6, 7 and 8. When pH is altered below or above the optimum activity appear to be decreased or becomes denatured. Different ogranism have different pH optima.

      From the above table it is revealed that strain Bacillus subtilis when icubated at different pH it was found that incubation PH of 7 was found to be optimum for maximum amylase activity i.e. 7.62 U/ml with temperature of production medium was kept at 350c for 72 hrs. and for strain Aspergillus niger maximum amylase activity was found to be 2.69 U/ml at optimum pH 7 with the production medium was kept for 72 hrs at 450c.

   4. Carbon source

      Effect of different carbon source (1% concentration) was determined by incubating production medium for different carbon sources.

      Table 4 Effect of Different Carbon Source on Amylase Production From Bacillus Subtilis And Aspergillus Niger By Submerged Fermentation

      Sr. No.	Microbial strain used	Carbon source	O.D. at 540nm	Mg of maltose released	Enzyme activity (IU/ml)
      1.	Bacillus subtilis	Glucose	0.443	0.756	2.09
      		Maltose	0.568	0.923	2.55
      		Starch	0.691	1.100	3.04
      2.	Aspergillus niger	Glucose	0.326	0.604	1.67
      		Maltoe	0.622	1.071	2.96
      		Starch	0.554	1.104	3.05

      *O.D.: Optical dentisity

      Different carbon sources like glucose, maltose and starch were used at 1% level in the current study. The result revealed that the different requirements of carbon source for different organisms.

      From the above table it is revealed that strain Bacillus subtilis when incubated at different carbon sources it was found that starch was found to be optimum carbon source for maxmum amylase activity i.e. 3.04 U/ml with temperature of production medium was kept at 350c for 72 hr and pH 7 and for strain Aspergillus niger, optimum carbon source was found to be starch for maximum amylase activity i.e. 3.05 U/ml with the production medium was kept at 450c for 72 hrs at pH7.

   5. Nitrogen source

      Effect of different nitrogen source (1% concentration) was determined by incubating production medium for different nitrogen sources.

      Table 5 Effect of different nitrogen source on amylase production from Bacillus subtilis and Aspergillus niger by submerged fermentation

      Sr. No.	Microbial strain used	Nitrogen source	O.D. at 540nm	Mg of maltose released	Enzyme activity (IU/ml)
      1.	Bacillus subtilis	Peptone	0.931	1.419	3.92
      		Casein	0.691	1.107	3.06
      		Ammonium sulphate	0.584	0.933	2.58
      2.	Aspergillus niger	Peptone	0.010	0.054	0.15
      		Casein	0.241	0.492	1.36
      		Ammonium sulphate	0.125	0.333	0.92

      *O.D.: Optical dentisity

      Different nitrogen sources like peptone, casein and ammonium sulphate were used at 1% level in the current study.

      From the above table it is revealed that strain Bacillus subtilis when incubated at different nitrogen sources it was found that peptone was found to be optimum nitogen source for maximum amylase activity i.e. 3.92 U/ml with temperature of production medium was kept at 350c for 72 hr and pH 7 and for strain Aspergillus niger, optimum nitrogen source was found to be casein for maximum amylase activity i.e. 1.36 U/ml with the production medium was kept at 450c for 72 hrs at pH7.

   6. Metal salt

   Effect of different metal salt (0.1% concentration) was determined by incubating production medium for different mteal salt sources.

   Table 6 Effect of Different Metal Salt Source on Amylase Production From Bacillus Subtilis And Aspergillus Niger By Submerged Fermentation

   Sr. No.	Microbial strain used	Carbon source	O.D. at 540nm	Mg of maltose released	Enzyme activity (IU/ml)
   1.	Bacillus subtilis	MgSO4	0.721	1.136	3.14
   		ZnSO4	0.00	0.00	No growth found
   		FeSO4	0.00	0.00	No growth found
   2.	Aspergillus niger	MgSO4	0.621	1.071	2.96
   		ZnSO4	0.00	0.00	No growth found
   		FeSO4	0.001	0.00	No groth found

   *O.D.: Optical dentisity

   Different metal salt sources like MgSO4, ZnSO4 and FeSO4 were used at 0.1% level in the current study. Supplementation of salts certain ions provided good growth of bacteria and fungai and thereby better enzyme production.

   From the above table it is revealed that strain Bacillus subtilis when incubated at different metal salt source it was found that MgSO4 was found to be optimum metal salt source for maximum amylase activity i.e. 3.14 U/ml with temperature of production medium was kept at 350c for 72 hr and pH 7 and used starch and peptone as a best optimum carbon and nitrogen source respectively.

   For strain Aspergillus niger, optimum metal salt source was found to be MgSO4 for maximum amylase activity i.e.2.96 U/ml with the production medium was kept at 450c for 72 hrs at pH7 and used starch and casien as a best optimum carbon and nitrogen source respectively.

4. CONCLUSION

Thus in the light of scientific data of the present investigation it may be concluded that bacterial species and fungal species can be a good source for the production of a very important enzyme amylase being used industrially. The amylases were produced by optimizing the various parameter and it was revealed that in Bacillus subtilis, maximum amylase activity was found to be 3.14U/ml with optimum time, temperature, pH,carbon , nitrogen and metal source was found to be 72hr, 350c, pH 7 ,starch ,peptone and MgSO4 respectively. While in Aspergillus niger, maximum activity was found to be

1. U/ml with optimum time, temperature, pH, carbon, nitrogen and metal salt was 72hr, 450c, pH 7, starch casein and MgSO4 respectively.

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