Amylolytic And Beta-Glucanase Activities Of Yam Rot Fungi

Project and Seminar Material for Microbiology

Abstract

Yam is in the class of roots and tubers that is a staple of the Nigerian and West African diet but fungi organisms can degrade it causing yam rot by producing amylolytic and beta-glucanase enzymes which breakdown starch and cellulose, respectively into sugars. They were produced using yam rot fungi isolated on SDA medium. The isolates obtained were all Aspergillus sp, they were identified using their morphological characteristics, lactophenol cotton blue stain and slide culture test. Pathogenicity test was carried out to check if the isolates could cause rot. Agar plate screening was carried out to check for the enzyme activites of the isolates, this was done on starch agar and carboxymethyl cellulose agar. It was detected by the disappearance of the blue colour and red colour, respectively around the colony producing a clear zone. The Aspergillus isolates were used to produce amylase and β-glucanase enzyme in spectrophotometric enzyme assay in which the enzyme activity was determined at OD540nm. Amylase and beta-glucanase production was carried out using the 4 isolates in solid state fermentation. The amount of beta-glucanase produced was 0.0097U/ml for Aspergillus spC followed by Aspergillus spD which gave 0.0020U/ml, Aspergillus spA gave 0.0015U/ml and Aspergillus spB being the least producer measuring 0.0005U/ml. All isolates showed good results for the production of amylase, in other words, they are all good producers of amylase with Aspergillus spB being the least producer with 0.0335U/ml, Aspergillus spA is the best producer with 0.0800U/ml, followed by Aspergillus spC (0.0601U/ml) and Aspergillus spD (0.0500U/ml).

Chapter One

Introduction

Yam popularly called “Ji” in Igbo Language is a tropical crop belonging to the Family Dioscoreaceae in the genus Dioscorea. It has as many as 600 species out of which six are economically important staple species. These are: Dioscorea rotundata (white guinea yam), Dioscorea alata (water yam), Dioscorea bulbifera (aerial yam), Dioscorea esculant (Chinese yam) and Dioscorea dumetorum (trifoliate yam). Out of these, Dioscorea rotundata (white yam) and Dioscorea alata (water yam) are the most common species in Nigeria (Izekor and Olumese, 2010). Yams are grown in the coastal region in rain forests, wood savanna and southern savanna habitats. Yams are perennial herbaceous vines cultivated for the consumption of their starchy tuber in Asia, Africa, Central and South America, and Oceania. Nigeria is by far the world’s largest producer of yams, accounting for over 70–76 percent of the world production.

According to the Food and Agricultural Organization report, in 1985, Nigeria produced 18.3 million tones of yam from 1.5 million hectares, representing 73.8 percent of total yam production in Africa. The tubers themselves are also called “yams”. There are many different cultivars of yams example: Dioscorea rotundata, Dioscorea bulbifera, Dioscorea esculenta etc.

Yam is in the class of roots and tubers that is a staple of the Nigerian and West African diet, which provides some 200 calories of energy per capita daily. In Nigeria, in many yam-producing areas, it is said that “yam is food and food is yam”. Yam is one of the major sources of carbohydrate, minerals, vitamins B6 and C and also dietary fibre.

However, the production of yam in Nigeria is substantially short and cannot meet the growing demand at its present level of use. It also has an important social status in gatherings and religious functions, which is assessed by the size of yam holdings one possesses. Yams are prone to different diseases. These diseases are caused by some pathogenic agents that reduce the quantity of yam produced and also the quality. Yam is prone to infection right from the seedling stage through harvesting and even after harvesting in storage. Storage losses for yams are very high in Africa the major period of spoilage is during storage (Amusa et al., 2003).

At storage, excessive exposure to sunlight, poor ventilation, storage at insect and rat infested area contributes to yam rooting and spoilage. Yam are properly stored in a well ventilated area to remove the heat generated by respiration of the tubers, regular inspection during storage and removal of rotting tubers and then protection from direct sunlight (Linus , 2003).

Enzymes such as amylase and beta-glucanase are produced by these fungi which are both significant in the degradation of starch and cellulose, respectively. These enzymes (amylase and Beta-glucanase) are the centre of interest in this work. Starchy substances constitute the major part of the human diet for most people in the world, as well as many animals. They are synthesized naturally in plants. Similar to cellulose, starch molecules are glucose polymers that are joined together by the alpha-1,4 and alpha-1,6 glycosidic bonds as opposed to beta-1,4 glycosidic bond for cellulose. Because of the existence of two linkages, different structures are possible for starch molecules.

Amylase is one of the most widely used enzymes in the industry. These enzymes are of great significance in biotechnological applications ranging from food, fermentation, detergent, pharmaceutical, brewing and textile to paper industries. Some factors affect this degradation which includes relative humidity, temperature, pH, substrate concentration and inhibitor concentration. (Okigbo and Ikediugwu, 2000).

1.1 Aim of Study

This work is aimed at studying the activities of these two enzymes amylase and beta-glucanase from fungi isolated from rotten yam.

Objectives

1. To collect sample from a yam rot and isolate the fungi associated with the rot.
2. Test and monitor the fungal isolates for their ability to produce amylase and beta-glucanase.
3. Production and characterization of the two enzymes.

Chapter Five

Discussion and Conclusion

5.1 Discussion

Studies on the fungi implicated in the spoilage of yam showed that it contained population of fungi. The presence of fungi isolated from the yam showed a great concern to post-harvest loss posed by the presence of those organisms. The prevalence of fungi that brings about spoilage is due to a wide range of factors which are encountered at each stage of handling from pre-harvest to consumption and is related to the physiological and physical conditions of the produce as well as the extrinsic parameters to which they are subjected (Akintobi et al., 2011). Damage inflicted on produce at the time of harvest is a major cause of infection since most of the spoilage microorganisms invade the produce through such damaged tissues. The incidence of infection is worsened by poor sanitary practice such as cross contamination and contact infection during the transportation (Singh et al., 2002).

Pathogenicity test was carried out to check the severity of spoilage and also the consistency of the isolates (Okigbo and Nmeka, 2005). It is as a result of the ability of the pathogens to utilize the nutrients of the tubers as substrate for growth and development, by producing enzymes that macerate the yam tissues.

The occurrence of amylolytic and cellulolytic organisms from yam rot agrees with the fact that they are good producers of these enzymes (Rehana, 1989). All the isolates selected from SDA produced detectable quantities of amylase and β-glucanase in the solid-state fermentation process. Amylase and β-glucanase are very important for the digestion of starch and cellulose. Amylase hydrolyses starch and β-glucanase hydrolyses cellulose. The isolates were identified macroscopically and microscopically and all the five isolates were found to be good producers of amylase and Beta-glucanase although Aspergillus niger was the most promising producer of β-glucanase.

The fungal isolates were screened primarily on starch agar and CMC agar for amylase and Beta-glucanase respectively and the quantitative amylase and Beta-glucanase activity was determined on the basis of clear zones formed around the colonies. All the isolates showed clear zones of amylase and Beta-glucanase activity although Aspergillus spC showed a better clear zone of the both. The enzyme assay was carried out using iodine reagent and congo red indicator for amylase and β-glucanase respectively.

Amylase and Beta-glucanase production was carried out using the 4 isolates in solid state fermentation. The amount of beta-glucanase it produced is 0.0097U/ml for Aspergillus spC followed by Aspergillus spD which gave 0.0020U/ml, Aspergillus spA gave 0.0015U/ml and Aspergillus spB being the least producer measuring 0.0005U/ml. All isolates showed good results for the production of amylase, in other words, they are all good producers of amylase with Aspergillus spB being the least producer measuring about 0.0335U/ml, Aspergillus spA is the best producer measuring 0.0800U/ml, Aspergillus spC is the next measuring 0.0601U/ml and Aspergillus spD measured about 0.0500U/ml.

Providing cost-effective enzymes is a big challenge for industrial application of enzymes. The use of waste materials as substrates and by using minimum steps in downstream recovery has reduced the cost. Many commercial enzyme manufacturers like Genencor, Danisco and Novozymes are striving to develop highly efficient and less expensive amylolytic and cellulytic enzymes (Chandel et al., 2012). Small scale production of these enzymes is possible but have some disadvantages which include time waste, but when produced in large scale using big pilot plants and fermenters, they are been produced in large quantity and marketable size. They have various uses in different fields such as the baking industry, paper and pulp industry, pharmaceutical industry, production of detergents etc.

5.2 Conclusion

The fungi causing rot isolated from yam rot produced amylase and glucanse enzymes which helped them degrade the starch and sugars present in the yam for their nutrition were identified as Aspergillus species by morphological and microscopical characteristics using lactophenol cotton blue stain. Other test like screening on starch agar and carboxymethyl cellulose agar for amylase and β-glucanase respectively. They were also assayed spectrophotometrically at OD540nm using a standard concentration of glucose to get the absorbance. Aspergillus spC showed a better result for the production of β-glucanase while all the isolates were good producers of amylase.

From this research, one could conclude that fungal isolates stand a better chance of producing these enzymes because they require cheap raw materials for its production and the microorganism could be genetically engineered to improve the production rate of these enzymes by them.

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