ISOLATION AND IDENTIFICATION OF MICROORGANISMS INVOLVED IN COCOA PULP FERMENTATION

ABSTRACT

Fruit juices especially unpasteurized ones are well recognized by consumers for their fresh flavor, vitamin content and nutritive values. This study investigated a five-day microbial fermentation of the pulp of Theobroma cacao. The extracted juice samples were analyzed for total viable bacterial count (TVBC), total lactic acid bacterial count and total fungal (yeast and mould) count using the spread plating method with appropriate media. All isolates were characterized following standard methods. There was a microbial succession of a wide range of microorganisms including yeasts, lactic-acid, and acetic-acid bacteria during the fermentation period. Over-fermentation led to a rise in Bacilli and fungi that caused off-flavors in the samples. Results show that the mean viable bacterial count and lactic acid bacterial count of the juice samples ranged from 1.29 x 108cfu/ml to 2.41 x 108cfu/ml and 7.74 x 107cfu/ml to 1.04 x 108cfu/ml respectively. The microbial load decreased gradually during the study period. The pH recorded during the fermentation period ranged from 3.2 to 3.7. The pH decreased as the fermentation proceeded affirming the production of acidic products. The bacterial isolates were identified as Staphylococcus aureus, Bacillus spp., Acetobacter spp., and Lactobacillus spp. while the identities of the fungal isolates were Saccharomyces cerevisiae.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                              iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

Lists of Tables                                                                                                                         vii

List of Figures                                                                                                                         viii

Abstract                                                                                                                                  ix

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Cocoa Pulp                                                                                                                  1

1.2       Aim and Objectives                                                                                                    5

CHAPTER TWO: LITERATURE REVIEW

2.1       Background of Cocoa Pulp                                                                                        6

2.2       Fermentation                                                                                                               6

2.2.1    Phases of cocoa fermentation                                                                                                 7

2.3       Microorganisms involved in Fermentation of Cocoa Pulp                                         9

2.3.1    Yeasts                                                                                                                         9

2.3.2    Lactic acid bacteria                                                                                                     11       

2.3.3    Acetic acid bacteria                                                                                                    12

CHAPTER THREE

3.0       Materials and Methods                                                                                               15

3.2       Media Preparation                                                                                                       15

3.3       Microbiological Analysis                                                                                            15

3.3.1    Sample preparation                                                                                                     15

3.3.2    Inoculation                                                                                                                  15

3.3.3    Isolation and Characterization                                                                                    16

3.4       Gram Staining                                                                                                             16

3.5       Biochemical tests                                                                                                        17

3.6       Determination of pH Value                                                                                        19

3.7       Identification of Microbial Isolates                                                                            19

3.7.1    Bacterial Isolates                                                                                                         19

3.7.2    Yeast Isolates                                                                                                              19

CHAPTER FOUR:

Results                                                                                                                                    20

CHAPTER FIVE

5.0       Discussion                                                                                                                   25

5.1       Conclusion                                                                                                                  26

5.2       Recommendation                                                                                                        26

References                                                                                                                  28

LIST OF TABLES

Table                                          Title                                                                       Page

1          Yeasts isolated from Cocoa Fermentation in Four Countries                         11

2          Lactic-acid Bacteria isolated from Cocoa Fermentation in Four Countries   12

3          Acetic-acid Bacteria isolated from Cocoa Fermentation in Four Countries   13

4          Total viable count of Bacterial and fungi load obtained                                21

5          Biochemical characteristics of the isolates                                                      22

6          pH of the samples during the period of fermentation                                    23

7          Microorganisms isolated from the studied samples                                        24

LIST OF FIGURES

Figure                                     Title                                                                            Page

1          Extraction and fermentation of cocoa pulp juice                                            4

2          Microbial activities during cocoa fermentation                                              14

 CHAPTER ONE

1.0       Introduction

Theobroma cacao belongs to the family Sterculiaceae and is economically important due to its valuable seeds. The seeds, commonly known as cocoa beans are the principal raw material for chocolate production. Cocoa plants are cultivated on plantations in tropical regions throughout the world; Ivory Coast, Ghana, Nigeria, Cameroon, Indonesia and Brazil are the major producers. For many years, cocoa production and commercialization have been the basis of the economy in some Brazilian states and particularly in Bahia. Brazil is the world’s sixth largest cocoa producer while Nigeria is the world’s fourth largest cocoa producer according to the United National Food and Agriculture Organization (Freire, et al., 1999).

A specific type of bacteria Streptomyces camerooniansis was found to be beneficial for Theobroma cacao by helping plant growth by accelerating seed germination of T. cacao, inhibiting growth of various types of microorganisms (Such as; different Oomycetes, fungi and bacteria) and preventing rotting by Phytophthora megakarya.

Extraction of pulp does not interface with subsequent seed fermentation and reduction of pulp before fermentation may be beneficial to cocoa quality (Camu, et al., 2008).

Cocoa is very important ingredient in several kinds of foods, such as chocolate, cake, biscuit, child-food, ice creams and sweet consumed in developed countries (Guehi et al., 2007).

1.1       Cocoa Pulp

Raw cocoa beans are sheathed in an aromatic mucilaginous pulp, which constitutes about 40% of the seed fresh weight. This mucilaginous pulp is spongy and contains the sap (Pulp juice) known in the industry as “sweating”. It is made up of about 67% to 82% water, it is rich in sugars (10 to 15%), salts (1 to 2%, protein (2 to 3%), organic acids (1 to 2%) and proteins (0.6%) Cocoa sweating has relatively low pH (3.0 to 4.0) primarily due to high concentrations of citric acid (1 to 3%) (Schwan and Wheals, 2004).

Cocoa pulp is the raw material which serves as the basis for fermentation, as it is a rich medium for microbial growth which constitutes about 10% of the mass of the cocoa fruits. The relatively high content of pectin (2 to 3%) and other polysaccharides makes the pulp viscous limiting diffusion of air of the sugars present, about 60% to sucrose and 39% is a mixture of glucose and fructose, the concentration of sucrose, glucose and fructose is a function of cultivars and fruit age with unripe pods containing mainly fructose and glucose (Thompson et al., 2001).

Cocoa fermentation occur largely on the mucilaginous pulp, the reason is spontaneous (i. e. allowing naturally occurring microorganism to ferment the pulp sugars) and involves succession of microorganism.  The microbial activity in the cocoa pulp is a well defined temporal succession that is dominated  by yeast during the first few hours, followed by lactic acid  bacteria (LAB) which decline 48hrs of  fermentation and then  they are overcome by acetic acid bacteria (AAB) (Schwan and Wheals, 2004). The primary colonizers are yeasts, followed by lactic acid bacteria (LAB), acetic acid bacteria (AAB) and Bacillus species. During the early and mid-phase of the spontaneous fermentation of raw cocoa beans, yeasts produce ethanol from carbohydrates and assimilate citric acid under anaerobic and low pH conditions and cause depectinization of the pulp, consequently the pulp liquefies and the juice drain off allowing air to diffuse into the heap.

As air diffuses into the heap, microaerophilic LAB, citrate-fermenting, acid tolerant and ethanol tolerant Lactobacillus plantarum and Lactobacillus fermentum strains dominate this phase (Galvez, et al., 2007)

The great majority of lactic acid bacteria utilize glucose via the Embden-Meyerhof-Parnas pathway, yielding more than 85% lactic acid. While some species utilize glucose through the hexose monophosphate shunt, forming 50% lactic acid, as well as combinations of ethanol, acetic acid, glycerol, mannitol, and carbon dioxide causing a slight increase in pH of the pulp. Acetic acid bacteria are responsible for the oxidation of ethanol to acetic acid and oxidation of acetic acid to carbon dioxide and water.

Other pulp ingredients are presumably as nutritious but nutrient declarations are hard to come by at this early stage in the industry (Akinwale, 2000).

Cocoa pulp is the raw material on which the fermentation proceeds and a key determinant of both quality and financial viability of the process. Not only is the quantity of pulp crucial in affecting the efficiency and nature of the fermentation but excess pulp can also be sold as a high value commodity. Cocoa pulp can also be  used in preparation of soft drinks, fresh cocoa juice is collected, sterilized and bottled for the production of alcoholic drinks such as brandy (Camu, et al.,2007).

Cocoa pulp is a very interesting by-product of cocoa production and it forms the substrate for microbial growth during the fermentation process. Most of the pulp goes to waste during the processing of the cocoa beans and this has led to its utilization as a potential raw material for development of soft drink to enhance the importance of cocoa as a source of income for farmers.

A quick look at the historical use of cocoa pulp reveals that cocoa farmer and their immediate communities have long enjoyed the pulp as juice. This was discovered by Joseph Montgomery who owned cocoa farms in Ecuador and launched Suavva, the first commercial scale juice smoothie gotten from cocoa pulp.

Montgomery says that “Like most fruits, the pulp is where the sweetness (fruit sugar) is found in cocoa”. The pulp can be used to make jam, jelly, juice which can be used to do fermented beverage like coca wine and other products (Fowler, 2009).

The fermentation of cocoa pulp is one of the few remaining large scale spontaneous microbial processes in today’s food industry. The microbiota involved in cocoa pulp fermentations is complex and variable, which leads to inconsistent production efficiency and cocoa quality (Michael, 2001)

Pulp Extraction

The selection of cocoa pods is important for the production of quality juice. The most suitable pods will be those three quarters ripe and yellowish-green in colour (Adomako and Takrama, 1999).

Figure 1: Extraction and fermentation of cocoa pulp juice

1.2       Aim and Objectives

The aim of this work is to identify and isolate the microorganisms that are involved in the fermentation of the cocoa pulp.

1.      To evaluate the total heterotrophic bacterial and fungal load of the cocoa pulp samples.

2.      To characterize and identify the various organisms involved in the fermentation of the pulp.

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