Keywords

Enzymes, Bacillus Megatarium

Introduction

Potato (Solanum tuberosum L.) is an important crop in India. It is a primary source of starch for vegetarian diet. In India, it was cultivated in an area of about 1.83 million hectares with production of 34.39 Million Tonne in 2008-09, whereas in Gujarat in the same year it was cultivated in 57,000 hectares with production of 4.21 Million Tonne 1 There are about 35 potato processing units in Gujarat 2 Balaji foods (P.) Ltd, Rajkot is one of the biggest units. This unit was visited by the staff members of scheme, to get information about the utilization of potato peel – a waste product of wafer making unit. The capacity of the plant is about 150 tonnes potato per day, and potato peels production is about 30 tonnes potato peel @ 200 g / kg potato. Presently the potato peels are mainly used as a composting material in farm as well as selling for the cattle feed (according to Balaji foods (P.) Ltd, Rajkot)

Table 1

Proximate composition of Potato Peel

Component (%) Potato waste (peel)
Starch 66.78
Dry matter 17.82
Alcohol-insoluble solids 62.70
Total soluble sugars 1.40
Reducing sugars 0.91
Cellulose 2.20
Crude protein 14.70
pH 5.99

* The data obtained from United States department of Agricultural national nutrient database (USDA 2009)

Most Bacillus and some other species of bacteria have a wide range of hydrolytic enzyme systems and are often capable of utilizing the organic matter consisting of complex mixtures; moreover, with the exception of the certain group of bacteria, they are harmless microbes which are included in the groups of organisms generally recognized as saprophytes. These organisms are easy to grow and require no expensive growth factors. They are able to produce extra cellular enzymes in high concentrations which can isolate. The Bacillus will also be tried for the extraction of extra cellular enzymes from potato peels. The extra cellular enzymes are used for a variety of different purposes, including starch modification in the paper & pulp industries, brewing, pharmaceutical industries, leather production industries etc. The wastes coming out from potato plants in the form of liquid and solid arise from peeling, trimming, slicing, cleaning, and rinsing operations which creates pollution problem. Looking to the above it was thought to utilize the byproduct such as potato peels of the potato industries for the extraction of extra cellular enzyme and also to solve the problem of pollution. There are some review are done for same case. 3-20

Materials and methods

For obtaining correct situation about potato peel as waste materials, AICRP on PHT staff made an industrial visit at Apricot foods industries, Rajkot and Rajen wafers, Rajkot. Huge amount of peels were produced and the main consumers of that was farmers used for the cattle feed and also used for bio-fertilizers. Bacterial cultures obtained from Microbial Type Culture Collection Centre (MTCC), Chandigarh. MTCC is a culture collection bank i.e. different cultures having different usefulness. Potato peel act as source of starch obtained from local market of Junagadh i.e. from Sagar Namkeen. For standardize the conditions for growth of microbes it is necessary to maintain Potato peel and nutrient broth (a chemical) ratio and a continual practice it was maintained at 1:10. For 1 gram of potato peel substrate it is necessary to take 10 ml of nutrient broth.

Process

Make growth of bacterial cultures on Nutrient agar media & after it on Peel nutrient agar media then Starch hydrolyzation was confirmed by iodine test and after the confirmation, the both bacterial cultures are inoculated in a sterile nutrient broths separately. After 24 hrs incubation, 0.1 ml bacterial cultures from nutrient broth inoculated in the treatments T1 to T8 (Bacillus megatarium culture add in T1 to T4 flasks) while T5 to T8 treatment flasks were without inoculated (T5 to T8 flasks act as control treatments) and put all the flasks at it for their respective temperature and time. After that centrifuge the flasks material at 2000 rpm for 10 minutes for obtaining crude enzymes. Make Enzyme assay of supernatant and measure the absorbance (Optical Density) at their respective wavelength. Starch consumption data are also carried out. Microbiological Growth Media used for the growth of bacteria like Nutrient agar media, starch agar media, peel agar media and peel nutrient broth.

Result and discussion

Table 2

Treatment Explanation

Sr. No. Treatments Bacteria Strains Incubation Temperature Incubation Hours
T1 B2T1H1 B. megatarium 370C 24
T2 B2T2H1 B. megatarium R.T. 24
T3 B2T1H2 B. megatarium 370C 48
T4 B2T2H2 B. megatarium R.T. 48
T5 (control) B0T1H1 No bacteria 370C 24
T6 (control) B0T2H1 No bacteria R.T. 24
T7 (control) B0T1H2 No bacteria 370C 48
T8 (control) B0T2H2 No bacteria R.T. 48

Growth Habit Of Bacteria (B. Megatarium) On Nutrient Agar Media, Starch Agar Media and amp; On Peel Agar Media

Table 3

Colony morphology and amp; Microscopic analysis of Bacillus group of bacteria on N-agar media

Colony Colony characteristics on Nutrient agar media Bacterial strain Microscopic analysis
Form Elevation Colour Margin
2 Circular Raised White Entire Bacillus megatarium Gram positive rods

Standardize The Conditions For Growth Of Bacteria Using Potato Peel.

Table 4

Growth of bacteria on peel agar media

Sr. no. pH Potato peel (in gram) Average growth of Bacillus megatarium (in CFU/ml)
1 7.5 5 0.78 x 107
2 7.5 10 1.23 x 107
3 7.5 20 – peel ratio is high, so agar can’t properly solidify and bacterial growth can’t observe properly –
4 7.7 5 0.93 x 107
5 7.7 10 1.45 x 107
6 7.7 20 – peel ratio is high, so agar can’t properly solidify and bacterial growth can’t observe properly –
7 7.9 5 1.29 x 107
8 7.9 10 2.08 x 107
9 7.9 20 – peel ratio is high, so agar can’t properly solidify and bacterial growth can’t observe properly –

As per the above results it is concluded that the pH of peel agar media was maintained at 7.9 and the ratio of potato peel substrate + nutrient agar / broth was maintained 1:10. i.e. 10 gram of potato peel substrate was necessary for 100 ml of nutrient agar / broth for better hydrolysis of starch by Bacillus type of bacteria

Measurement Of The Enzyme Efficacy

A general formula for glucose or enzyme production was carried out as follow

Standard Glucose Estimation By Anthrone Method

100 mg in 100 ml distilled water. Working standard – 10 ml of stock diluted to 100 ml with distilled water and estimation is carried out by Anthrone method. Graph factor obtained by this estimation is very useful for starch consumption.

Table 5

Optical Density and graf factor of standard glucose determined by Anthrone metho

ml concentration in μg O.D. O.D./ml graf factor
0.00 0.00 0.000 0.00 129.87
0.2 20 0.150 0.750
0.4 40 0.357 0.893
0.6 60 0.461 0.769
0.8 80 0.698 0.873
1 100 0.749 0.749

Figure 1: Graph of Concentration v/s O.D. of standard glucose determined by Anthrone method

Table 6

Starch consumption by bacteria in various treatments

Sr. No. Treatments Average Glucose produced (in mg/g) Starch consumed = glucose produced x 0.9 (in mg/g)
T1 B2T1H1 36.82 33.14
T2 B2T2H1 60.78 54.7
T3 B2T1H2 46.56 41.9
T4 B2T2H2 89.22 80.3
T5 (control) B0T1H1 0 0
T6 (control) B0T2H1 0 0
T7 (control) B0T1H2 0 0
T8 (control) B0T2H2 0 0

Table 6 reveals that starch consumption was found higher (96.27 mg/g) in treatment T4 i.e. bacterial strain B.megatarium consume maximum amount of starch at room temperature during 48 hours of time period.

Standard Glucose Estimation By Dnsa Method

100 mg glucose dissolved in 100 ml distilled water. Working standard – 10 ml of stock diluted to 100 ml with distilled water and estimation was carried out by Di Nitro Salicylicylic Acid (DNSA) method. Graph factor obtained by this estimation is very useful for estimation of amylases enzyme activity.

Table 7

Optical Density and graf factor of standard glucose determined by DNSA method

Ml concentration in μg O.D. O.D./ml graf factor
0.00 0.00 0.000 0.00 158.5624
0.2 20 0.136 0.682
0.4 40 0.265 0.663
0.5 50 0.338 0.676
0.6 60 0.473 0.788
0.8 80 0.550 0.688
1 100 0.625 0.625

Figure 2: Graph of Concentration v/s O.D. of standard glucose determined by DNSA method

Table 8

Amylase production by bacteria in various treatments

Sr. No. Treatments Average Amylase enzyme produced (in mg/g)
T1 B2T1H1 7
T2 B2T2H1 13.19
T3 B2T1H2 14.51
T4 B2T2H2 21.22
T5 (control) B0T1H1 0
T6 (control) B0T2H1 0
T7 (control) B0T1H2 0
T8 (control) B0T2H2 0

Table 8 reveals that α – Amylase production was found higer, 56.9 mg/g, in treatment T4 i.e. bacterial strain B. megatarium produce more amount of α – amylase enzyme at room temperature during 48 hours of time period.

Table 9

Protease production by bacteria in various treatments

Sr. No. Treatments Average O.D. at 440 nm Treatment average – control average Protease enzyme produced = (Treatment average – control average) x 0.2 (in mg/g)
T1 B2T1H1 0.195 0.123 0.02
(T5-T9)
T2 B2T2H1 0.225 0.181 0.04
(T6-T10)
T3 B2T1H2 0.307 0.242 0.05
(T7-T11)
T4 B2T2H2 0.49 0.445 0.09
(T8-T12)
T5 (control) B0T1H1 0.072 0
T6 (control) B0T2H1 0.044 0
T7 (control) B0T1H2 0.064 0
T8 (control) B0T2H2 0.045 0

Table 9 reveals that Protease production was found higher, 0.18 mg/g, in treatment T4, i.e. bacterial strain B. megatarium produce more amount of protease enzyme at room temperature during 48 hours of time period.

Table 10

Statistical Analysis of individual factors as well as interaction

Effect Starch Consumption Amylase production Protease production
Bacterial strain (B)
Bacillus megatarium (B2) 53.383 13.914 0.248
No bacteria (B0) 0 0 0
Incubation temperature (T)
37oC (T1) 37.136 14.045 0.164
Room Temperature (T2) 49.335 22.225 0.344
S.Em± 0.7973 0.5904 0.0038
CD at 5 % 2.3272 1.7233 0.011
Incubation hours (H)
24 (H1) 34.816 13.854 0.193
48 (H2) 51.656 22.416 0.315

Table 11

Brief results regarding various treatments

Sr. No. Treatments Starch consumption (in mg/g) Amylase enzyme activity (in mg/g) Protease enzyme activity (in mg/g)
T1 B2T1H1 33.14 7 0.02
T2 B2T2H1 54.7 13.19 0.04
T3 B2T1H2 41.9 14.51 0.05
T4 B2T2H2 80.3 21.22 0.09
T5 B0T1H1 0 0 0
T6 B0T2H1 0 0 0
T7 B0T1H2 0 0 0
T8 B0T2H2 0 0 0