α-Amylase are enzymes that are used in the degradation of starch. This study focuses on optimizing the production of α-Amylase using locally isolated Aspergillus japonicus in defined and undefined medium.
Five grams of cocoyam waste, sweet potato waste, wheat waste and plantain peel were utilized as substrates during fermentation for the production of α-Amylase using 1 ml spore suspension as inoculum. The fermentation media contained in g/l (0.8 NaCl, 0.8 KCl, 0.1 CaCl2, 2.0 Na2HPO4, 0.1 FeSO4, 8.0 Fructose, 2.0 NH4Cl). Temperature, pH, sugar content and amylase activity of the culture filtrates were monitored after every 48 hours. For optimization, mycelia was used as inoculum and different inoculum sizes (5%, 10% and 15%) were used for fermentation in a defined medium containing starch as substrate. The effect of urea as nitrogen source on α -Amylase activity was monitored. Different concentrations (2g and 5g) of cocoyam waste and sweet potato waste were also utilized for optimization using 10% inoculum. The effect of temperature and pH on amylase was determined.
Two grams (2g) of sweet potato waste with NH4Cl as nitrogen source, using 10% inoculum gave the highest yield of α-Amylase after 96 hours. The optimum pH and temperature for α -amylase production were pH 5 and 50 oC respectively.
The ability of the amylase to act within an acidic pH suggests that it is stable within a wide range of acidic pH (2 – 6) and its ability to withstand relatively high temperature (40 o C – 60 o C) above the optimum growth temperature of A. japonicus suggests that it is thermo stable. The crude α-Amylase produced from A. japonicus retained 67% of its activity at 60oC.α-Amylase from this fungus has the potential to be utilized for various biotechnological processes. Agro industrial wastes which are often carelessly discarded into the environment causing health hazards can be utilized as cheap and readily available substrate for the production of alpha amylase hence, it is practicable to rid our environment of these hazardous wastes.
Keywords:α-Amylase, Aspergillus japonicus, Optimization, Agro industrial wastes, Environment
Word Count: 332
TABLE OF CONTENTS
Title page i
Table of Contents vi
List of Tables x
List of Figures xi
List of Plates xii
CHAPTER ONE: INTRODUCTION
1.1 Background to the Study 1
1.2 Statement of the Problem 3
1.3 Objective of the Study 4
1.4 Research Questions 4
1.5Significance of the Study 5
1.6Justification for the Study 5
CHAPTER TWO: REVIEW OF LITERATURE
2.1Agricultural Wastes 6
2.2 Plantain 6
2.3 Constituents of Plantain 6
2.3.1 Plantain peel 7
2.4 Cocoyam 7
2.4.1 Constituents of cocoyam 7
2.5 Sweet Potato 7
2.5.1 Constituents of Sweet Potato 8
2.6 Wheat 8
2.6.1 Constituents of wheat 9
2.7 Enzymes 9
2.7.1 Amylases 9
2.8 Microbial amylases 10
2.8.1 Fungal amylase 10
2.8.2 Bacterial amylase 11
2.8.3 Microbial amylase and degradable substrate 12
2.9 Starch 13
2.10 Amylase assays 14
2.11 Effect of temperature on microbial amylase 16
2.12 Effect of pH on microbial amylase 16
2.13Amylase and Industrial applications 16
CHAPTER THREE: METHODOLOGY
3.1 Research Design 19
3.2 Population 19
3.3 Sample size and sampling Technique 19
3.3.1Processing of plantain peel substrate (PPW) 19
3.3.2Submerged fermentation of PPW 19
3.3.3 Processing of wheat waste (WW) 20
3.3.4 Processing of cocoyam waste (CW) 20
3.3.5 Processing of sweet potato waste (SPW) 20
3.3.6 Submerged fermentation of CW, SPW and WW 20
3.3.7 Sample size 20
3.4Preparation of reagentsand buffer 21
3.5Isolation of Aspergillusjaponicus from cocoa 21
3.5.1Molecular analysis and identification 22
3.5.2Inoculum preparation (spore) 22
3.5.3Inoculum preparation (mycelia) 23
3.6 Determination of physicochemical parameters 23
Determination of sugar content 23
3.7 Extraction of crude enzyme 23
3.8 Amylase assay in crude enzyme 23
3.9 Production of α-Amylase using a chemically defined medium 24
3.10 Optimization 24
3.11 Effect of temperature on amylase activity 24
3.12 Effect of pH on amylase activity 24
3.13 Statistical analysis 24
CHAPTER FOUR: DATA ANALYSIS, RESULTS AND DISCUSSION OF FINDINGS
4.1 Results 25
4.1.1 Growth of A. japonicus on PDA 25
4.1.2 Optimization of temperature for fermentation 25
4.1.3 pH in the culture filtrates containing SPW, CW, PW and WW 28
4.1.4 Sugar content in the culture filtrates containing SPW, CW, PW and WW 28
4.1.5 Amylase activity in crude enzyme produced by A. japonicus in undefined medium 31
4.1.6 pH of culture filtrates in defined medium 31
4.1.7 Amylase activity of crude enzyme by A. japonicus in defined medium 34
4.1.8 Effect of urea on α-Amylase production by A. japonicus in defined medium 34
4.1.9 Optimizing α-Amylase production using 2 g of SPW and CW 37
4.1.10 Optimizing α-Amylase production using 5 g of SPW and CW 37
4.1.11 Effect of pH on amylase activity 40
4.1.12 Effect of temperature on amylase activity 40
4.2 Discussion 43
4.2.2 pH in the culture filtrates containing SPW, CW, PW and WW 43
4.2.3 Sugar content in the culture filtrates containing SPW, CW, PW and WW 43
4.2.4 Amylase activity in crude enzyme produced by A. japonicusin undefined medium 44
4.2.5 Amylase activity of crude enzyme in defined medium using different mycelia suspensions of A. japonicus as inoculum 44
4.2.6 Optimization of crude α-Amylase by A. japonicusin a defined medium using
urea as nitrogen source 45
4.2.7 Optimization of α-Amylase production using varying concentrations of CW
and SWP 45
4.2.8 Effect of pH on crude α-Amylase produced by A. japonicus 45
4.2.9Effect of temperature on crude α-Amylase produced by A. japonicus 46
CHAPTER FIVE: SUMMARY, CONCLUSION AND RECOMMENDATIONS 5.1 Summary 47
5.2 Conclusion 47
5.3 Recommendations 47
5.4 Limitation of the Study 48
LIST OF TABLES
1 Effect of temperature on fermentation 27
2 pH of the culture filtrates using 1 ml spore suspension of A. japonicus29
3 Sugar content in the culture filtrates using 1 ml spore suspension of A. japonicus 30
4 Amylase activity of the crude enzyme using 1 ml spore suspension of A. japonicus 32
5 pH of culture filtrates in a defined medium using different inoculum mycelia
suspensions of A. japonicus as inoculum 33
6 Amylase activity of crude in defined medium using different mycelia suspensions
of A. japonicus as inoculum 35
7 Optimization of crude α-Amylase in adefined medium using urea as nitrogen
LIST OF FIGURES
1 Optimization of α-Amylase production by A. japonicus using 2 g CW
and 2 g SWP 38
2 Optimization of α-Amylase production by A. japonicus using 5 g CW
and 5 g SWP 39
3 Effect of pH on crude α-Amylase produced by A. japonicus 41
4 Effect of temperature on crude α-Amylase produced by A. japonicus 42
LIST OF PLATE
1Aspergillus japonicus on PDA after 5 days of incubation 26
1.1 Background to the Study
Amylases are enzymes that are well known for their applications in starch, food, brewing, distilling, textile, paper and pharmaceutical industries (Gupta et al., 2003; Krishna et al., 2011; Pandey et al., 2000). They are currently utilized in various fields e.g. brewing industries, medicinal, analytical chemistry and food processing (Anto et al., 2006; Chimata et al., 2010; Nimkar et al., 2010). This wide range of applications is the reason for the industrial production of amylase (Khan & Yadav, 2011). Amylases are one of the most important and well-known enzymes that can hydrolyse starch or glycogen (Krishna et al., 2011). They hydrolyse α 1-4 glycosidic bonds of glycogen, amylopectin and other related compounds (Lehninger, 1982). It can be produced by submerged fermentation or solid state fermentation (Egas et al., 1998; Khan & Yadav, 2011; Krishna, 2011). The enzyme is one of the mostly sought after, as it has huge importance in biotechnology; comprising a group of industrial enzymes that controls about 25% of the total enzyme market of the world (Rajagopalam & Krishnan, 2008; Reddy et al., 2003).
Amylases are a group of hydrolases that split the O-glycosidic bonds present in starch thereby breaking starch into simple units (Alva et al., 2007; Crabb & Mitchinson, 1997). They have been reported to be produced by microbial, plant and animal sources, although amylase produced by microorganims has been reported to be most effective (Khan & Yadav, 2011). A wide range of microorganisms, such as bacteria and fungi are utilized in the industrial production of amylases (Krishna et al., 2011). The use of microbes for the production of amylases is economical because microorganisms can be easily manipulated to produce metabolites e.g. enzymes (Aiyer, 2005). However, fungi are preferred over bacteria for enzyme production because of their filamentous nature, which helps in its penetration through solid substrate (Ramachandran et al., 2004).
The synthetic media utilized for the production of amylases are costly and this poses a major challenge to researchers especially in developing countries. Hence, researches are now focused on methods to reduce production cost (Khan & Yadav, 2011). Wastes from agro based industries have been reported to be good and readily available substrates for the cost effective production of α-Amylase (Kirankumar et al., 2011; Pandey et al., 2000). Agrarian nations possess inexhaustible supply of wastes annually generated from their breweries, rice mills, yam flour, plantain and banana chips producing outfits, processing units, and other small industries (Adeniran & Abiose, 2009).
The generation of waste materials (e.g. peels) emanating from the utilization of food and other food products pose potentially severe pollution problems and represent a loss of valuable biomass. Some of these wastes are usually carelessly dumped in the environment where they are left to decay. Soil and plant around the heaps of the waste are usually considered unproductive due to chemical and biological reactions that take place between the decomposing wastes, soil and the surrounding vegetation (Ajao et al., 2009; El-Shimi et al., 1987). Apart from their environmental pollution aspects, generally, these wastes may have the potential to be utilized as raw material for other industries or for their use as feed or food after biological treatment (Okolo et al., 1995). Agro industrial wastes include plantain peel, cocoyam waste, sweet potato waste, cocoyam waste, cassava waste, wheat bran, rice husk, banana peel, vegetable waste and citrus waste.
Plantain (Musa spp.) occupies a strategic position for rapid production of food in Nigeria. It is ranked third among starchy staples (IITA, 2014). The “total world production of plantain is estimated to be over 75 million metric tons (John & Marchal, 1995) out of which 12 million metric tons are produced annually in Africa (Fakayode et al., 2011)”. Nigeria is one of the leading producers of plantain worldwide, it is the largest producer in West Africa producing about 2.4 million metric tons yearly (FAO, 2006). However, Nigeria is not an exporter of plantain because production is more for local consumption (Fortaleza, 2012). About 15 million people depend on plantain as their major source of carbohydrate (Adeolu & Enesi, 2013). The high demand for plantain also generates wastes which are often discarded, and sometimes used as animal feeds (Olabanji et al., 2012).
In Nigeria, the ripe fruit are processed into different forms for consumption either by boiling, frying and roasting. Considerable interest has been generated, in the recent years, for value addition to plantain, such as the production of plantain chips, dodoikire (commonly sold along highway in the South Western part of Nigeria) and plantain powder because of improper storage facilities which usually lead to postharvest losses. During the course of producing some of these products, the plantain peel accumulates in bulk posing serious environmental problems.
Over the years, cocoyam has been a major crop in the system of farming in South Eastern and South Western part of Nigeria. It is one of the most important tuber crops grown in this region. The tubers contain starch that serves as dietary fibre, it can be boiled, roasted, fried and eaten with palm oil (Ezejiofor, 2012). Nigeria is the largest producer of cocoyam in the world (Okoye, et al., 2008; Onwueme, 1987). Over 20 million tonnes of cocoyam yields are annually wasted because of improper storage (IITA, 2009). It is consumed as vegetables by many rural inhabitats where they are available in large quantities (Okigbo, 1987). It is not expensive and usually available throughout the year (Braide & Nwaoguikpe, 2011). The high level of carbohydrate in cocoyam has not been completely harnessed in the industries (Nwufo & Fajola, 1998). Nigeria is one of the foremost producers of root crops such as cocoyam, which is one of the most under-utilized crops with huge economic potential (Eneh, 2013; Onwuka & Eneh, 1998).
Sweet potato is the seventh most important food crop in the world (Betiku et al., 2013). It is a vital food crop worldwide (Hasem et al., 2015). It is one the most important crops on fresh-weight basis in some developing nations after cassava, wheat, rice and maize (Ezeano, 2010). Ezeano (2010) reported that, there seems to be a rise in the growing and usage of sweet potato in Nigeria because of new development employed by farmers.
Wheat is cultivated worldwide; it has been cultivated in Nigeria for some time (Ohiagu et al., 1987; Olugbemi et al., 1979). Olabanji et al. (2007) reported that the cultivated varieties are relatively recent introduction. Wheat is used for the production of bread, semolina and for fermentation to make alcoholic beer (Poehlman, 1959), vodka (Palmer, 2001) or biofuel (Neil 2002). It is also used for the production of pasta and noodles (Li et al., 2014).
1.2 Statement of the Problem
The synthetic media used in the production of α-Amylase is expensive and this poses a major challenge for a developing country like Nigeria. There is a need to explore other cheaper and readily available substrates for the production of this enzyme. Nigeria is the largest producer of cocoyam and one of the leading producers of sweet potato and plantain in the world. However, cocoyam and sweet potato are under-exploited and a significant portion is often wasted. Most of the plantain produced in Nigeria is consumed locally; the epicarp of the plantain (plantain peel) is often considered as waste and is usually carelessly discarded. Additionally, Nigeria is not a major producer of wheat but this does not forestall the importation, processing and consumption of wheat and wheat products. The accumulation of wastes from these crops may contribute to environmental and health hazards in different parts of the country. This study aims at generating additional value to cocoyam, sweet potato, plantain and wheat by utilizing its waste as substrates for the production of α-Amylase.
1.3 Objective of the Study
The main objective of this study was to utilize different agro wastes for the production of α-Amylase enzyme using locally isolated Aspergillus sp. in submerged fermentation. The specific objectives are to:
- evaluate the production of α-Amylase by Aspergillus sp. in defined and undefined growth media;
- determine pH and sugar content in the culture filtrates;
- extract the crude alpha amylase produced during fermentation;
- determine the amylase activity;
- optimize α-Amylase production and
- determine the effect of pH of temperature on α-Amylase.
1.4 Research Questions
- Are there differences in amylase production using defined medium and/or undefined medium?
- Are there variations in the pH, temperature and sugar content in the different culture filtrates?
- How can the crude alpha amylase produced during fermentation be extracted?
- How can the α-Amylase activity be determined?
- How can α-Amylase production be optimized?
- How will varying temperature and pH affect the crude α-Amylase produced?
1.5 Significance of the Study
This study would provide baseline information about the ability of a locally isolated Aspergillus sp. to utilize pre-treated cocoyam and sweet potato waste for α-Amylase production. It would also add to the existing literature the possibility of utilizing wheat waste and plantain peel as a cheap and readily available substrate for α-Amylase production using locally isolated Aspergillus sp. from pulverise cocoa.
1.6 Justification for the Study
Wastes generated from plants poses serious environmental and health hazard to humans. This study would focus on utilizing agro wastes for α-Amylase production in a view to converting these agro industrial and potentially hazardous “wastes to wealth”.