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Alpha-amylase Production by Bacillus licheniformis 1331 using
Jackfruit Seeds as Substrate
Rica Mae A. Palongpalong1, Percival G. Garcia2
1 Student, School of Chemical Engineering and Chemistry, Mapua Institute of Technology
2 Adviser, School of Chemical Engineering and Chemistry, Mapua Institute of Technology
AbstractAmylases have great significance in biotechnology and are commercially important enzymes in

various starch processing industries. The microbial amylases meet industrial demands and have almost completely replaced chemical hydrolysis of starch in starch processing industry. This study explored the use of jackfruit seed as carbon source in the production of alpha-amylase. A production medium composed of ground jackfruit seed and yeast extract powder was mixed with 10% inoculum of Bacillus licheniformis 1331 cultured in nutrient broth at 30 C for 18 hours. The effects of temperature and carbon to nitrogen

ratio were determined. Triplicate analysis was done at 10%, 15%, 20% carbon source with 20% nitrogen
source set at pH 7 and temperature of 30 C and 37 C. It was found out that alpha-amylase production
was optimal at 10% carbon, 20% nitrogen, pH 7, and 37 C. Amylase production was conducted in 1000

ml medium and incubated with shaking. Samples were taken at regular intervals at 0, 1, 2, 4, 6, 8, 18, 24, 27, and 30 hours of fermentation. Enzyme activity and protein concentration were determined using Alpha- amylase assay and Bradford assay, respectively. Biomass was determined by dry cell weight (DCW) analysis. Alpha-amylase production was highest during exponential growth at 4-8 hours of fermentation.

Keywords: alpha-amylase, Bacillus licheniformis, jackfruit seed, yeast extract
1. Introduction

In the environment abound possible sources of essential materials or substances


the development or production of certain products like enzymes, which will be useful to humans, plants, and other animals. These substances can be produced in significantly large amounts with the advent of biotechnology, but primarily the materials and the factors that influence its production source must be identified. Papain was prepared from high quality latex of Carica papaya (Baines, B. S. and Brocklehurst, K., 1978). Some wastes from fruits and vegetables can also be a good substrate for enzyme production. One study was done on banana fruit stalk, which was

used as a substrate for alpha-amylase production by Bacillus subtilis under solid-state fermentation (Krishna and Chandrasekaran, M., 1996). A similar study was also done using potato peelings (Shukla, J. and Kar, R., 2000). Trypsin/ chymotrypsin inhibitor was isolated from jackfruit by ammonium sulfate fractionation and chromatography (Sai Annapurna, S. and Siva Prasad, D., 1990). These studies showed that some enzymes could be produced using components of fruits and vegetables as substrate.

This particular study aims to produce an enzyme by a Bacillus sp. using the seeds of a locally grown fruit as substrate. This particular fruit is jackfruit

Artocarpus heterophyllus or locally

known as \u201cnangka\u201d or \u201clangka\u201d, which is a favorite dessert of Filipinos and is a widely grown fruit crop in the Philippines. It contains carbohydrates, protein,


sodium, potassium, B-complex, ascorbic acid, and small amounts of fats, ash, and iron. Analysis of jackfruit food composition per 100 gram edible portion showed that the carbohydrate content of the seed is 34.90 g (Agriculture and Fisheries Information Science, Department of Agriculture).

Based on the above composition, the jackfruit seed having a high content of carbohydrate can be used as a substrate for the production of the enzyme amylase. The production of amylase is of great significance in biotechnology. It is commercially important in some processing industries such as beverages, food, textile, detergents, and paper industries (Pandey et al., 2000).

This enzyme can be produced using a specific microorganism or fungi under


The amylases can be derived from several sources ranging from bacteria to plants to humans. Bacteria and fungi secrete amylases to the outside of their cells to carry out extracellular digestion. When they have broken down the insoluble starch, the soluble end products such as glucose or maltose are absorbed into their cells. This particular study will use

Bacillus licheniformis for amylase
This is an exploratory study to
determine alpha-amylase production by
Bacillus licheniformis using jackfruit
seed as substrate. This study is limited to

and environmental conditions such as temperature and carbon/nitrogen ratio.

characterization will be done.

The significance of this study is that another substrate will be added to the existing list of carbon sources for amylase production by microorganisms. Many industrial processes use waste materials from plants or animals. Part of solving the environmental problem of waste management can be addressed by the utilization of waste products. Jackfruit seeds as waste from fruit can be a potential material for enzyme production. Being a substrate it will become a source of income for fruit vendors and farmers. This study becomes important to the biotechnology industry and likewise contributes to environmental

economic stability.
2. Methods
2.1 Preparation of jackfruit seed
One hundred (100) g of jackfruit
seeds was autoclaved in 1L beaker at

121\u00b0C (15 lbs psi) for 15 minutes. The outer seed coating was removed. Seeds were grinded by using a coffee grinder. Grinded seeds were placed in zip locked plastic bag and stored in the freezer.

2.2 Bacterial strain
licheniformis 1331, was obtained from

the Philippine National Collection of Microorganisms (PNCM), BIOTECH, University of the Philippines, Los Ba\u00f1os, Laguna.

2.3 Medium Preparation
HIMEDIA\u00ae M001 was used for
composition of nutrient agar is shown in
Appendix A.

Twenty-eight (28) g of nutrient agar was mixed with 1L-distilled water in 1L cotton plugged Erlenmeyer flask. The medium was sterilized by

autoclaving at 121\u00b0C (15 lbs psi) for 15
From the pure culture, cells were
subcultured by streaking them on

nutrient agar plate and incubated at 30\u00b0C for 30 h. Several growing colonies were transferred to nutrient agar slants and

incubated at 30\u00b0C for 18 h. Nutrient agar slants were stored in refrigerator and subcultured every three (3) weeks.

2.4 Preparation of inoculum
Nutrient broth was used for
preparation of inoculum. The medium
contains (in g/L): HIMEDIA\u00ae RM 027
yeast extract, 3; peptone, 5; and NaCl, 8.
The medium was sterilized at 121\u00b0C (15
lbs psi) for 15 minutes.
Three (3) loopfuls ofB.
licheniformis 1331 from nutrient agar

slant was inoculated in 100 ml nutrient broth in 250 ml cotton plugged Erlenmeyer flask. It was

incubated at 30\u00b0C for 18 h.
2.5 Production medium
The production medium consists
of grinded jackfruit seeds as carbon
source and HIMEDIA\u00ae RM 027 yeast
extract powder as nitrogen source.
One hundred (100) ml of

ml Erlenmeyer flask contains jackfruit seed as carbon source at 10%, 15%, and 20%

and HIMEDIA\u00ae RM 027 yeast extract powder as nitrogen source at 20%. The pH of the production medium was 7. The inoculum size was 10% (v/v) of the production medium. The culture was

incubated at 30\u00b0C for 24 h.
2.6 Optimization of cultural conditions

Factors such as temperature and source of carbon and nitrogen affecting production of amylase were optimized by varying parameter one ats a time. The experiment was conducted in 250 ml cotton


flask containing 100 ml of production medium. The pH of 7, temperature at

30\u00b0C and 37\u00b0C, carbon source at 10%, 15%, and 20%, and nitrogen source at 20% were used. The optimum C/N ratio was determined by varying carbon and nitrogen source of production medium. A factorial design shown below was conducted.

Table 1. Factorial design at pH 7 and
Carbon source (w/v)
Nitrogen source (w/v)
Table 2. Factorial design at pH 7 and
(10%, 20%) (15%, 20%) (20%, 20%)

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