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PROJECT TOPIC ON PRODUCTION AND OPTIMIZATION OF GLUCOAMYLASE USING CANDIDA FAMATA ISOLATED FROM FERMENTED MASHED PINEAPPLE MUST UNDER SOLID STATE FERMENTATION

PRODUCTION AND OPTIMIZATION OF GLUCOAMYLASE USING CANDIDA FAMATA ISOLATED FROM FERMENTED MASHED PINEAPPLE MUST UNDER SOLID STATE FERMENTATION

ABSTRACT

The present study was an investigation on comparative glucoamylase production by Candida famata isolated from spontaneously fermented pineapple must, using wheat bran, maize bran and the combination of both substrates in equal ratios under solid state fermentation conditions. Various cultural parameters were monitored and subsequently optimized for the production of glucoamylase using different agro-industrial residues and processing mills. The pretreatment of substrates was milling to 2 – 3mm particle size. Mixed bran of maize and wheat gave the highest glucoamylase yield of 25.34 U/ml under predetermined optimum fermentation conditions (30°C, pH 5.0, 106 cells/ml inoculum concentration, 1:3 substrate to nutrient solution ratio and 3 days of fermentation). In contrast, lower activity of 17.93 and 13.28 U/ml were obtained in wheat bran and maize bran respectively. The mixed bran served as a better substrate in the production of glucoamylase. Highlighting the potential of this approach as an alternative strategy for waste management and sustainable production of enzymes could perhaps act as a springboard for applicable source in many biotechnological processes.

CHAPTER ONE

INTRODUCTION

1.1 Background

Glucoamylases also known as (α-1,4-glucan glucohydrolase, amyloglucosidase) are important enzymes that allow the hydrolysis of starch and related polymers to glucose, they can be obtained from microbial as well as other sources. The major application of glucoamylase is the saccharification of partially processed starch/dextrin to glucose, which is an essential substrate for numerous fermentation processes and a range of food and beverage industries. Glucoamylase consecutively hydrolyzes alpha 1,4-glycosidic bonds from the non-reducing ends of starch and alpha 1,6-glucosidic linkages in polysaccharides yielding glucose as the end-product, which in turn serves as a feedstock for biological fermentations (Norouzian et al., 2006; Siddhartha et al., 2012). A more technical name is (1,4) glucan glycohydrolase where glucan is the name for a series of glucose units attached and glycohydrolase describes the breaking of the bond between two glucose units. Glucoamylase has a wide range of use among industrial enzymes. This enzyme is used in the baking industry, the brewing process, and the most important application of glucoamylase is the production of high glucose syrups, it also has various applications in major areas of food processing, animal nutrition, fermentation biotechnology, paper making, fabric industries and in whole grain hydrolysis for the alcohol industry (Selvakumar et al., 1996; Zambare, 2010). Glucoamylases of microbial origin are divided into exo-acting, endo-acting, debranching and cyclodextrin producing enzymes. Glucoamylases hydrolyze α-1,4 and α-1,6 linkages and produce glucose as the sole end product from starch and related polymers (Svensson et al., 2000; Parbat and Singhal, 2011).

Glucoamylases are industrially important hydrolytic enzymes of biotechnological significance and are currently used for dextrose production, confectionary, baking and in pharmaceuticals. Glucoamylase is an economically important enzyme because of its capacity to hydrolyse starch and related polymers into β-D-glucose as the sole end product, the principal industrial use of glucoamylase is therefore, the production of glucose, which in turn serves as a feedstock for biological fermentations in the production of ethanol or high fructose syrups, it is a key enzyme in the production of saké and soy sauce. Glucoamylase is also used to improve barley mash for beer production. (Pandey et al., 2000; Pavezzi et al., 2008; Zambare, 2010).

Enzymes are produced by various microorganisms including bacteria, fungi and yeast and are considered as important products obtained for human needs through microbial sources. The advantage of using microorganisms for the production of enzymes is that bulk production is economical and microbes are easy to manipulate to obtain enzymes with desired characteristics. Fungal enzymes are preferred over other microbial sources owing to their widely accepted Generally Regarded As Safe (GRAS) status (Sindhu et al., 2009).

Solid State Fermentation (SSF) involves the growth of microorganisms on moist solid substrates in the absence of free water. The concept of using solid substrates is probably the oldest method used by man to make microorganisms work for him. In recent years, SSF has shown much promise in development of several bio-processes and products. Solid state offers greatest possibilities when fungi are used. Unlike other microorganisms, fungi typically grow in nature on solid substrates such as pieces of wood, seeds, stems, roots and dried parts of animals such as skin, bones and fecal matter that is low in moisture. In SSF, the moisture necessary for microbial growth exists in an absorbed state or in complex with solid matrix. However, SSF differs from solid substrate fermentation, since in solid substrate fermentation the substrate itself acts as a carbon source and occurs in the absence or near absence of free water by employing a natural substrate or inert substrate as solid support (Kumar et al., 2003).

The aim of SSF is to bring cultivated fungi or bacteria in tight contact with the insoluble substrate and to achieve the highest nutrient concentration from the substrate for fermentation. This technology so far is run only on a small scale, but has an advantage over submerged fermentation. Two types of SSF systems have been distinguished depending on the type of solid phase used. The most commonly used system involves cultivation on a natural material and less frequently on an inert support impregnated with liquid medium (Ooijkaas et al., 2000). The use of agroindustrial wastes for enzymes production can become economically viable for the application of these biocatalysts in large scale, considering that one of the major problems in the enzymes utilization in industrial processes is the high cost of the microbial culture media, about 30 – 40% of the cost enzyme production (García-Martínez et al., 2010).

PRODUCTION AND OPTIMIZATION OF GLUCOAMYLASE USING CANDIDA FAMATA ISOLATED FROM FERMENTED MASHED PINEAPPLE MUST UNDER SOLID STATE FERMENTATION

1.2 Statement of Research Problem

A recent focus of research and development effort is the application of glucoamylases in the enzymatic degradation of carbohydrate-rich polysaccharides for the production of energy syrup. The development of microbial strains, media composition and process control all have contributed to the achievement of high levels of extracellular glucoamylases.

However, the cost of glucoamylase are still too high for the establishment of a cost effective production of energy syrup using it. One approach to overcome this obstacle is to employ Solid State Fermentation (SSF). This process (SSF) has the potential to significantly reduce the enzyme production costs because of lower energy requirements, increased productivity, smaller effluent volumes and simpler fermentation equipment. Agroindustrial residues are generally considered the best substrates for SSF processes and use of SSF for the production of enzymes is no exception to that (Ellaiah et al., 2002).

Commonly used carbon sources are dextrin, fructose, glucose, lactose, maltose and starch which are very expensive for commercial production of these enzymes. Various agricultural byproducts like wheat bran, rice husk, sugarcane bagasse, potato residue, rice bran, green gram bran, black gram bran, maize bran, can be abundantly used (Siddhartha et al., 2012).

The food, beverage and agro-industries produce large quantities of residues that pose serious problems of disposal, inspite of them being sources of biomass and nutrients. These substrates can be used for the production of valuable compounds such as enzymes and various secondary metabolites. There is an environmental concern that most of the agroindustrial wastes contain phenolic compounds and/or other compounds of toxic potential; which may cause deterioration of the environment when the waste is discharged to nature (Soccol et al., 2003; Solange et al., 2012).

1.3 Justification of the Study

The production of amylolytic enzymes, particularly glucoamylase on solid substrate is more advantageous for the fermentation industry. In the Solid State Fermentation process, the solid substrate not only supplies the nutrients to the culture, but also serves as a thriving environment for the microbial cells (Joshi et al., 1999).

The use of yeast to produce enzymes offer certain advantages, such as a moderate temperature for microbial growth, high metabolic diversity and rapid cell growth, which results in shorter fermentation cycles and easy adaptation to different cultivation conditions (Kato et al., 2007). The use of agroindustrial wastes as substrate in solid state fermentation reduces enzyme production cost and contributes to minimize environmental problems caused by the agroindustry. They are also inexpensive, readily available, renewable resource, has lower energy requirement and produce lesser waste water (Singhania et al., 2009).

Large amount of wastes is generated every year from the industrial processing of agricultural raw materials, most of these wastes are used as animal feed or burned as alternative for elimination. However, such wastes usually have a composition rich in sugars, minerals and proteins, and therefore, they should not be considered “wastes” but raw materials for other industrial processes. The economics of enzyme production using inexpensive raw materials can make an industrial enzyme process competitive (Solange et al., 2012; Shubhang and Vinod, 2014). Wheat bran, paddy husk, rice processing waste and other starch containing wastes have gained importance as supports for growth during enzyme production (Anto et al., 2006). Microbial enzymes are preferred to plant enzymes due to their short growth period, higher productivity and thermostability (Mishra and Behera, 2008). Yeasts from several genera, such as Ambrosiozyma, Arxula, Aureobasidium, Candida, Debaryomyces, Lipomyces, Saccharomyces, Saccharomycopsis (Endomycopsis) and Schwanniomyces, rank among the best producers of glucoamylases (Chi et al., 2009).

Recommended: SATISFIABILITY REASONING OVER VAGUE ONTOLOGIES USING FUZZY SOFT SET THEORY

1.4 Aim of the Study

The aim of this study was to produce and optimize glucoamylase using Candida famata under solid state fermentation conditions.

1.5 Specific Objectives

The specific objectives of this study were to:

  1. Isolate and characterize Candida famata from fermented pineapple must.
  2. Determine the proximate composition of the substrates (wheat bran and maize bran).
  3. Screen Candida famata for the production of glucoamylase and determine its glucoamylase-producing potential under solid state fermentation conditions.
  4. Optimize cultural parameters such as moisture, pH, incubation temperature, inoculum concentration and incubation time for the production of glucoamylase.

See also: ETERMINATION OF PROXIMATE COMPOSITION, MINERAL ELEMENTS, HEAVY METAL LEVELS AND MICROBIAL QUALITY OF KILISHI FROM SELECTED AREAS IN KADUNA STATE, NIGERIA

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