MAKERERE UNIVERSITY

COLLEGE OF NATURAL SCIENCES

SCHOOL OF BIOSCIENCES

DEPARTMENT OF ZOOLOGY, ENTOMOLOGY AND FISHERIES SCIENCES

THE FAECAL CONTAMINATION OF SILVER CYPRINID (Rastrineobola argentea)

SUNDRIED ON NETS LAID ON GRASS AT KATOSI LANDING SITE, MUKONO

DISTRICT

BY

BARBRA ASIIMWE (BSC. FISHERIES AND AQUACULTURE)

Reg. No.16/U/3692/PS; Student No. 216007213

SUPERVISOR: DR. ROBINSON ODONG

A RESEARCH DISSERTATION SUBMITTED TO THE DEPARTMENT OF

ZOOLOGY, ENTOMOLOGY AND FISHERIES SCIENCES IN PARTIAL

FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF BACHELORS

OF SCIENCE IN FISHERIES AND AQUACULTURE

AUGUST, 2019 DECLARATION

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APPROVAL

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TABLE OF CONTENTS DECLARATION ...... i

APPROVAL ...... ii

TABLE OF CONTENTS ...... iii

LIST OF FIGURES...... v

LIST OF TABLES ...... vi

LIST OF ABBREVIATIONS ...... vii

ABSTRACT ...... viii

CHAPTER ONE: INTRODUCTION ...... 1

1.1 BACKGROUND ...... 1

1.2 STATEMENT OF THE PROBLEM ...... 3

1.3 OBJECTIVES ...... 4

1.3.1 MAIN OBJECTIVE ...... 4

1.3.2 SPECIFIC OBJECTIVES ...... 4

1.4 NULL HYPOTHESIS ...... 4

CHAPTER TWO: LITERATURE REVIEW ...... 5

2.1 FISH AS A NURTITIONAL SOURCE...... 5

2.2 FISHERIES AND AQUACULTURE...... 5

2.3 SILVER CYPRINID IN UGANDA ...... 6

2.4 FISH SPOILAGE ...... 7

2.5 FISH PRESERVATION APPROACHES...... 9

2.6 SUN DRYING AS A FOOD PRESERVATION METHOD ...... 15

2.7 QUALITY ASPECTS OF SUN-DRIED SILVER CYPRINID ...... 16

CHAPTER THREE: MATERIALS AND METHODS ...... 17

3.1 DESCRIPTION OF STUDY AREA...... 17

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3.2 EXPERIMENTAL STRATEGY ...... 18

3.3 DETERMINATION OF THE FECAL CONTAMINATION OF THE SILVER CYPRINID ...... 19

3.3.1 MEDIA PREPARATION AND CASTING...... 19

3.3.2 SPREADING OF INOCULUM ON THE CAST PLATES AND INCUBATION OF THE SPREAD PLATES ...... 21

3.3.3 COLONY IDENTIFICATION AND COUNTING ...... 22

3.3.4 STATISITICAL ANALYSIS ...... 23

CHAPTER FOUR: RESULTS ...... 25

4.1 OBJECTIVE 1: LEVEL OF E. COLI ON SILVER CYPRINID SUN DRIED ON NETS LAID ON GRASS IN RELATION TO THE DIFFERENT NET LOCATIONS .... 25

4.2 OBJECTIVE 2: LEVEL OF ENTEROCOCCI ON SILVER CYPRINID SUN DRIED ON NETS LAID ON GRASS IN RELATION TO THE DIFFERENT NET LOCATIONS ...... 26

CHAPTER FIVE: DISCUSSION ...... 28

5.1 LEVEL OF E. COLI ON THE SUN DRIED SILVER CYPRINID AT THREE DIFFERENT NET LOCATIONS ...... 29

5.2 LEVEL OF ENTEROCOCCI ON THE SUN DRIED SILVER CYPRINID AT THREE DIFFERENT NET LOCATIONS ...... 30

CHAPTER SIX: CONCLUSIONS AND RECOMMENDATIONS...... 32

6.1 CONCLUSION ...... 32

6.2 RECOMMENDATIONS...... 33

REFERENCES ...... 34

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LIST OF FIGURES Figure 1 Uganda districts (Left) and section of map showing Mukono district (Right)...... 17 Figure 2 The demarcations used to distinguish between the three net locations ...... 19 Figure 3 Plates cast with MacConkey agar (A) and BEA agar (B) ...... 20 Figure 4 Labelled Borosilicate bottles containing 10g of the samples to be mixed in 90ml of peptone water ...... 22 Figure 5 A contaminated plate of MacConkey agar (Left) and BEA agar (Right) ...... 23 Figure 6 A water logged site at the shores of Katosi LS ...... 30

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LIST OF TABLES Table 1: Mean and Standard deviation of Total coliform and E.coli loads for the wet and dry samples from three net on grass locations. (T.C WET= Total Coliforms on wet samples. T.C DRY= Total Coliforms on dry samples, E.C WET= E.coli on wet samples and E.C DRY= E.coli on dry samples) ...... 25 Table 2: Mean and Standard Deviation of the Enterococci loads on wet and dry Silver cyprinid at three different net on grass locations ...... 26 Table 3 Mean and Standard deviation of Enterococci loads on Sundried Silver Cyprinid at each different net locations ...... 27

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LIST OF ABBREVIATIONS

BEA Bile Esculin Azide cfu Colony forming Unity FAO Food and Agricultural Organization FTI Fisheries Training Institute OAG the Office of the Auditor General

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ABSTRACT

Rastrineobola argentea, a small pelagic fish known as the Silver cyprinid and commonly referred to as ‘Mukene’ ranks among the top three species landed from Lake Victoria. It is majorly preserved by sun drying on surfaces such as net- on grass, net- on ground and ranks with the commonly used being net-on grass. Studies show that this particular surface has higher microbial loads thus poses a high risk of contaminating the sun dried fish. This study evaluated whether the location of the nets on grass had a significant effect on the level of fecal contamination on the sun dried fish. Three net locations were evaluated i.e. shore, mid-point and upland at Katosi LS. Wet and dry samples were collected on randomly selected nets at the three different net locations. Dry samples were collected after 8 hours on the same nets from which the wet samples were initially collected at the three different nets locations. The samples were analyzed for fecal contamination by examining for Escherichia coli and Enterococci fecal indicators. The findings showed that generally the location of nets laid on grass had a less significant effect on the level of fecal contamination. However this was greatly influenced by the kind of fecal indicator species examined because the level of fecal contamination with

Enterococci on sundried Silver cyprinid significantly varied at the three different locations.

This showed a contradiction from the findings found in examining the dry samples for E.coli because the results indicated the level of contamination was significantly the same at the three net locations. Previous studies indicate that Enterococci is not purely found in human and animal fecal matter but can also be found on plants and in soil with evidences that Enterococci cells are capable of replicating in extra-enteric environments such as beach sands. Further studies to eliminate the possibility of human fecal contamination from the other environmental sources of the organisms which was not determined in this study is thus required.

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CHAPTER ONE: INTRODUCTION 1.1 BACKGROUND Fish is an important part of a healthy diet that contains high quality proteins, as well as a wide variety of vitamins and minerals (Immaculate et al., 2013). Fish is a versatile food commodity that has been widely accepted as a protein source (FAO, 2018). More so even in small quantities, fish addresses food and nutritional security challenges among the poor and vulnerable communities. Nevertheless, it is an extremely perishable commodity that spoils more rapidly than many other foods, resulting into higher post-harvest losses, globally estimated at 27% of the total fish production in 2016 (FAO, 2018).

Due to its high perishability in comparison to the many other foods, fish post-harvest handling practices such as preservation, processing and packaging require particular care to minimize post-harvest losses and also maintain nutritional attributes (FAO, 2018). Consequently, over

55% of fish traded in 2016 around the world was either under preservation or processed (FAO,

2018). Preservation techniques are applied to slow down the spoilage process, since rapid spoilage of fish is attributed to a combination of microbial, biochemical and enzymatic processes (Seema, 2015). These processes are driven by a number of factors, the major factor being moisture content (Abdu et al., 2018). The preservation methods employed such as drying and salting aim at reducing the moisture content (Rahman, 2006) in the fish in order to inhibit the stated processes from taking place.

Rastrineobola argentea, commonly known as ‘Mukene’ is a traditional commercially important fish species for food security and socio-economic development of rural areas around

Lake Victoria and beyond (Tieli et al., 2017). It is a relatively cheap source of animal protein,

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hence its demand is substantially increasing (Sifuna et al., 2008). Rastrineobola argentea fishing takes place at night, using canoes that do not have any forms of cold storage facilities

(Sifuna et al., 2008). Its preservation is often done by drying on different surfaces, majorly the ground at the landing beaches (Egesa et al., 2018). Besides sun-drying, other preservation methods include deep frying, smoking, freezing and curing (Egesa et al., 2018). However the adoption of these preservation methods is low due to higher investment and operational costs

(Egesa et al., 2018).

Sun drying of Silver cyprinid involves laying fish in the open air, mainly on bare ground, old fishing nets or mats laid on bare ground or grass and pebbles (LVFO, 2012; Egesa et al., 2018) that results into contamination with sand, microorganisms, as well as fecal matter carried by birds and flies. Food products that show evidence of fecal contamination are generally regarded as a greater risk to human health, as they are more likely to contain human-specific enteric pathogens (Sifuna et al., 2008). Indicator microorganisms in food microbiology have been used to predict the presence of potential risks associated with pathogenic microbes (Buchanan and

Oni, 2012). Escherichia coli (E.coli) are considered to be of fecal origin among the enteric bacteria, and exists only momentarily in other environments (Sifuna et al., 2008). E. coli is also found in abundance in almost any moist environment such as in soil, drainage water, shallow groundwater and the domestic environment (Sifuna et al., 2008).

In order to improve the quality of sun-dried Mukene within Lake Victoria basin, raised –rack sun-drying surfaces have been rapidly promoted as a faster and hygienic surface through projects such as “Increasing supply of Mukene (Rastrineobola argentea) for human consumption” carried out by the Department of Fisheries Resources with support from FAO

(Egesa et al., 2018). Previous studies have reported a higher coliform load on fish sun-dried on

2 bare-ground and net-on-grass that is attributed to potential contamination from the drying surfaces (Egesa et al., 2018). With an increasing population currently equivalent to 0.59% of the total world population and deteriorating sanitation at the various landing sites, research particularly to evaluate the levels of fecal contamination on the sun dried fish in relation to the surfaces is required to assess the effect of these increasing population. This will in turn enable management to sensitize the fishing communities on the issues of sanitation, eradicate the use of surfaces that permit contamination with unacceptable amounts of fecal matter and more so introduce a more controlled environment at the landing sites where drying of R.argentea can be done.

1.2 STATEMENT OF THE PROBLEM Preservation of fish and farmed products is one of the factors that has contributed to storage of food to cater for long term needs, since fish is more perishable than other proteinaceous animal foods (Ariyawansa et al., 2015). Open sun-drying is the simplest and cheapest preservation option utilized in developing countries by fish folk and small-scale farmers who cannot afford artificial dryers (Chiwaula, 2017).Open sun-drying involves the laying of the product on a desired surface such as on bare ground, net-on grass, peddle and net-on bare ground (Onyango et al., 2015)

These surfaces expose the product to contamination by bacteria, infestation by insects, birds, animals and rodents(Maathai, 2015).Net-on grass drying surfaces are known to have significantly higher total bacterial loads that are above the acceptability limit of 1×106 cfu /g for fish and fish products (Nunoo and Noono, 2013). Further studies are required to examine the factors that may influence this level of high level of fecal contamination that is observed on sun dried fish dried on nets on grass to best mitigate the problem.

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1.3 OBJECTIVES

1.3.1 MAIN OBJECTIVE

To determine the level of fecal contamination of Silver cyprinid sun-dried on nets laid on grass.

1.3.2 SPECIFIC OBJECTIVES

1. To determine the level of E. coli contamination on Silver cyprinid sun-dried on nets

laid on grass.

2. To determine the level of Enterococci contamination on the Silver cyprinid that is

sun-dried on nets laid on grass.

1.4 NULL HYPOTHESIS 1. The location of the nets laid on grass has no effect on the level of E.coli on the sun-

dried Silver cyprinid.

2. The location of the nets laid on grass has no effect on the level of Enterococci on the

sun-dried Silver cyprinid.

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CHAPTER TWO: LITERATURE REVIEW 2.1 FISH AS A NURTITIONAL SOURCE Fish and fish products have a crucial role in nutrition and global food security (FAO, 2018;

Sifuna et al., 2008) as they represent a valuable source of nutrients of fundamental importance for diversified and healthy diets (FAO, 2018). Fish contains many of the vitamins and minerals required to address some of the most severe and widespread nutritional deficiencies such as nervous system development during the most crucial stages of an unborn or young child’s growth, mental health and prevention of cardiovascular diseases, stroke and age-related muscular degeneration (FAO, 2018). In low-income populations that depend heavily on a narrow range of calorie-dense staple foods, fish can represent much-needed means of nutritional diversification that is relatively cheap and locally available (FAO, 2018). Small quantities of fish can provide essential amino acids, fats and micronutrients, such as iron, iodine, vitamin D and calcium, which are often lacking in vegetable-based diets (FAO, 2018).

Globally, fish and fish products provide an average of only about 34 calories per capita per day

(FAO, 2018). In 2015, fish accounted for about 17% of animal protein, and 7% of all proteins, consumed by the global population (FAO, 2018). Since 1961, fish has provided about 3.2 billion people with almost 20% of their average per capita intake of animal protein (FAO,

2018).

2.2 FISHERIES AND AQUACULTURE There has been rapid growth in fisheries and aquaculture which has been observed by an increase in global fish production to about 171 million tonnes in 2016 (FAO, 2018). The total sale value of fisheries and aquaculture production in 2016 was estimated at USD 362 billion

(FAO, 2018). On average, the annual global fish consumption has increased by 3.2%. In per

5 capita terms, fish consumption grew from 9.0 kg in 1961 to 20.2 kg in 2015, at an average rate of about 1.5% per year globally (FAO, 2018).

Fish and fish products are among the most traded food items in the world today. In 2016, about

35% of global fish production was exported to the international market in various forms for human consumption or non-edible purposes (FAO, 2018). The 60 million tonnes of total fish and fish products exported in 2016 represent a 245% increase since 1976. More so the world trade in fish and fish products also grew significantly in value terms within the same period, with exports rising from USD 8 billion in 1976 to USD 143 billion in 2016 (FAO, 2018) . Of the 171 million tonnes of total fish production in 2016, about 88% i.e. over 151 million tonnes was utilized for direct human consumption, a share that has increased significantly in recent decades (FAO, 2018). The greatest part of the 12% used for non-food purposes i.e. about 20 million tonnes was reduced to fishmeal and fish oil (FAO, 2018).

2.3 SILVER CYPRINID IN UGANDA Rastrineobola argentea, locally known as Mukene is a small pelagic cyprinid, which occurs in

Lakes Victoria, Kyoga, and Nabugabo (Manyala and Ojuok, 2007). Prior to the 1960s, this species was of minimal economic importance, forming an insignificant proportion of fish landed from those lakes (Wandera, 1991). As a result of the decline in catches of the original commercially important species in Lakes Victoria and Kyoga, fishermen on Lake Victoria shifted to exploiting the Silver cyprinid in commercial quantities (Wandera, 1991). R. argentea ranks among the top three species landed from Lake Victoria, others being the Nile perch

(Lates niloticus) and Nile tilapia (Oreochromis niloticus) (Turyaheebwa, 2014).

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The fishery of R. argentea is currently based in the inshore areas and around the numerous islands of Lake Victoria where light fishing technology is possible (Mannini, 1992). On moon-less nights, kerosene pressure lamps are used to attract the fish which are then either scooped by lift nets, lampara nets or towed to the beach by a mosquito seine nets (Mannini,

1992). The catch is sun-dried for both direct human consumption and preparation of animal feeds (Wandera, 1991).

Other than its importance as a cheap source of protein to both humans and in the animal feeds manufacturing industries, R. argentea plays an important role in these lakes' ecosystem dynamics (Wandera, 1991). In the absence of the once abundant Haplochromine cichlids, R. argentea is the major food of the Nile perch which in turn accounts for over 50% of the total catches from these lakes (Wandera, 1991). It therefore serves as a bridging role in the transfer of energy from the lower to higher trophic levels. The Silver cyprinid provides a cheap source of fish proteins for both humans and domestic animals. This has partly been as a result of an increase in the export trade of processed table fish species i.e. the Nile perch and tilapia.

2.4 FISH SPOILAGE Food spoilage is considered as any change that renders the product unsafe for human consumption (Bourdichon et al., 2012). Spoilage of fish starts upon death due to autoxidation, autolytic and metabolic activities of microorganisms (Seema, 2015). It results when chemical, physical, and microbiological changes occur, rendering the food product unacceptable to the consumer (Petruzzi et al., 2017). Fish spoilage results from three basic mechanisms: enzymatic autolysis, oxidation and microbial growth (Ikape and Cheikyula, 2017). Among these three, fish spoilage due to bacteria gains greater concern as a health hazard (Ghaly et al., 2010).

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Composition of the micro flora on newly caught fish depends on the microbial contents of the water in which the fish live (Dhanya and Saleena, 2017). Bacteria are present on the surface slime, skin, gills and intestine of fish (Tawari et al., 2011). In dead fish, bacteria begin to invade the tissues causing spoilage and production of undesirable compounds leading to food safety risks (Tawari et al., 2011). The microbial spoilage of food can be as result of either growth of microbes in the food or release of their extracellular and intracellular enzymes (Tawari et al.,

2011). A number of organoleptical changes in color, odor and texture as well as slime formation, gas and fluid ccumulation are often utilized as indicators of food spoilage (Bibek and Bhunia, 2013).

Autolytic enzymatic spoilage, the self-digestion action by enzymes, occurs shortly after capture (Ikape and Cheikyula, 2017). Enzymes have the ability to chemically combine with other organic compounds and work as catalysts for chemical reactions that finally end up in muscle self-deterioration (Mekonnen, 2015). These autolytic changes include proteolysis and fat hydrolysis which are prerequisites for microbial decomposition (Mekonnen, 2015).

Calpains, cathepsins and amino peptidases are the enzymes found to be responsible for the post mortem autolysis that occurs in fish body (Cheret et al., 2007). During the autolysis process, chemical and biological changes take place in the dead fish due to enzymatic breakdown of major molecules, reducing textural quality (Pushparajan et al., 2013). The digestive enzymes cause extensive autolysis which results into flesh softening, rupture of the belly wall and draining out of the body fluids which contains both protein and oils (Jeyosanta et al., 2018)

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Oxidative spoilage as a result of lipid oxidation is a major cause of deterioration and spoilage for fatty fish species (Ahmed et al., 2016), such as Nile perch. Lipid oxidation occurs on poly saturated fatty acids located on the cell membranes, hence fish rich in these fatty acids are highly susceptible to oxidative spoilage (Dhanya and Saleena, 2017). Oxidative spoilage proceed with radical chain reaction that takes place in three main stages i.e. initiation, propagation and termination (Ayala et al., 2014).

Initiation involves the formation of lipid free radicals, through catalysts such as heat, metal ions and irradiation, which react with oxygen to form peroxyl radicals (Ghaly et al., 2010).

During propagation, the peroxyl radicals react with other lipid molecules to form hydro peroxides and a new free radicals. Termination occurs when a buildup of these free radicals interacts to form non-radical products (Ghaly et al., 2010).

2.5 FISH PRESERVATION APPROACHES Between initial production and final consumption, different methods are used to preserve food products. Several preservation mechanisms have been and are being developed to tackle the quality and safety problems associated with fish and fishery products in order to assure best market quality, assure health safety of products, apply the most appropriate processing methods and reduce wastage to the least possible extent (Tawari et al., 2011).

Although every method can produce effective results, none ensures safety by entirely stopping the spoilage process. A number of preservation methods are utilized in the fish and fishery industry, these can be classified into three main methods; chemical, bio-preservation methods

9 and physical (Kamal et al., 2017). The methods have different modes of action but all aim at extending the shelf life of fish and fishery products (Abdu et al., 2018).

CHEMICAL METHODS OF FISH PRESERVATION

Curing: Curing is the addition of salt, sugar, nitrites, nitrates, seasonings or spices and phosphates to preserve food stuff (Sebranek, 2005). This particular processing also results in a characteristic color and flavor of the cured products (Gull et al., 2014). Salting is the main curing method (Islam et al., 2009). It exerts its preservative action by: causing high osmotic pressure resulting in the plasmolysis of microbial cells, dehydrating food and microorganisms by tying up the moisture, ionizing to yield the chloride ion which is harmful to microorganisms, and reducing the solubility of oxygen in water, sensitizing the cells against carbon dioxide (Seema, 2015). However, the use of salt as a preservative cannot arrest the growth of halophilic and halotolerant microorganisms whose growth often produce white patches on the fish fillets (Binici and Kaya, 2017).

Smoking: This is a simple method of preservation, for consumption either directly after curing or within twelve hours (Ugochukwu, 2017). Smoking has both antimicrobial and antioxidant effects, since the combined effect of phenolic compounds produced during the smoking process and high temperature condition results in reduced microbial growth and oxidation

(Abdu et al., 2018). More so, carbonyl compounds produced during the process contribute to the characteristic colour, texture and fishy odour of the product (Abdu et al., 2018).

The smoking process involves the treatment of food with given heat ranges. Fresh unsalted fish can be put over wood or husk fire (Ugochukwu, 2017). Smoking is majorly done in two methods; cold smoking and hot smoking which vary in the temperature ranges used to

10 preserve the fish (Luiza et al., 2010). The cold smoking process is in three stages, each of which is important to the product's potential shelf-life: salting, drying and smoking all at temperatures below 30°C (Sigurgisladottir et al., 2000). Hot smoking can either be wet or dry and both processes are carried out at temperatures above 80°C, which are high enough to cook the fish products not to require subsequent processing (Adeyeye and Oyewole, 2015).

Natural antimicrobial preservatives: Natural antimicrobials are secondary metabolites effective in reducing the activity of several pathogenic and spoilage microorganisms associated with foods that can be found in plants, animals, and microorganisms (Hayek et al.,

2013). These include substances such as essential oils (EOs) and extracts of various plants, spices, and herbs. These natural extracts are fused in packaging materials, as well as edible films and coatings to preserve and ensure the quality of minimally processed and delicate foods such as meat, fish and fruits (Abdu et al., 2018). The effectiveness of such antimicrobial substances depend greatly on temperature, since microorganisms tend to be more susceptible to antimicrobials at exposure temperatures close to their optimal due to their increased metabolic activity (Abdu et al., 2018).

Organic acids (OAs): OAs are compounds with acidic properties that occur naturally in a number of foods (Pereira and Adam, 2015). OAs are mainly present in fermented products as a result of hydrolysis, biochemical reactions and microbial activities (Anyasi et al., 2017). OAs consist of an inexpensive and effective means of reducing the prevalence and population size of pathogenic bacteria, thus are frequently used in decontamination applications in food commodities (Loretz et al., 2011)

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These weak acids are used as food additives, however not all of them have antimicrobial activities (Gao et al., 2012). The most effective antimicrobials are acetic, lactic, propionic, sorbic, and benzoic acids (Lucera et al., 2012). However microorganisms can adapt to the effect of the antimicrobials at pH of about 5.5, hence their use is generally limited to foods with a pH less than 5.5 (Davidson and Harrison, 2002).

OAs work on a principle of providing acidic environment that hinder the growth and multiplication of bacteria since they maintain their internal pH near neutral to prevent conformational changes to the cell (Kobayashi et al., 2000). The mechanism of action of OAs involve their penetration in the cell membrane bilayer in undissociated form (Anyasi et al.,

2017). Once inside the cell, the acid dissociates because the cell interior has higher pH than the exterior structural proteins, enzymes, nucleic acids and phospholipids (Davidson, Sofosi and Branen, 2005). Protons generated from intracellular dissociation of OA, acidify the cytoplasm and are extruded to the exterior (Yousef and Juneja, 2002).

MICROBIOLOGICAL METHODS OF FISH PRESERVATION

Lactic Acid Bacteria (LAB) are not indigenous microbes of the aquatic environment, but they are the most widely used in microbiological preservation of foods (Ghanbari and Mansooreh,

2013). They work on the principle of minimizing the growth of undesirable microorganisms by favoring the growth of competitive and harmless microorganisms in a food product either competing with the undesirable microbes for nutrients or producing the antimicrobial metabolites, or both (Abdu et al., 2018). LAB produce inhibitory metabolites which include hydrogen peroxide, benzoic acid, diacetyl, mevalonolactone, and reuterin (β-

12 hydroxypropionaldehyde (Flint and Garner, 2009). The protective cultures used in this method of preservation should not pose any adverse health effect and neither should their metabolites affect the sensory, chemical and physical properties of the end product in any way that confer the product with unattractive characteristics (Abdu et al., 2018).

PHYSICAL METHODS OF FISH PRESERVATION

The physical methods of fish preservation can be described as three distinct spoilage control measures, namely: moisture control, thermal control and non-thermal control technologies

(Abdu et al., 2018).

Thermal control: Fish handling after being caught or harvested should be immediately preserved, refrigerated or frozen. The use of cold treatments is mainly used during on-board handling (Gokoglu and Yerlikaya, 2015). These chilling treatments can be in various forms such as, wet ice, refrigerated seawater (RSW), chilled seawater (CSW), solid and liquefied forms of carbon dioxide, liquid nitrogen, and chilled air (Abdu et al., 2018). The cooling principle aims at quickly dropping the temperature of the fish as low as possible (Brown, 2014).

Cooling processes generally cannot prevent decay completely, but the colder the temperature the greater the reduction of bacteria and enzyme activity (Nasirin et al., 2016). Cooling can affect bacteriological and biochemical processes in the fish, but it is only delayed, not stopped

(Mashylom, 2011).

Non-thermal control: Non-thermal physical preservation approaches employed in the fisheries industry include: high pressure processing, packaging technologies, irradiation, ozonation, as well as pulsed electric fields and oscillatory magnetic fields treatments (Abdu et al., 2018). Approaches such as ozonation and ultraviolet (UV) irradiations involve oxidation processes that eliminate inorganic and organic contaminants and/or pollutants with

13 the help of reactive free radicals which are produced by ozone (O3), hydrogen peroxide

(H2O2), titanium oxide (TiO2), zinc oxide (ZnO), and UV radiations (Abdu et al., 2018).

Moisture control measures: Most foods have a water activity above 0.95 and that will provide sufficient moisture to support the growth of bacteria, yeast, and mold (Reed et al., 2011).

Water activity (aw) is a measure of how efficiently the water present can take part in a chemical (physical) reaction (Sandulachi, 2012). Preservation methods aim at reducing the amount of available moisture to a point which will inhibit the growth of the organisms. Since microorganisms are sensitive to the water status in their immediate environment and they can remain metabolically active only in a narrow range of high aw (Prokopov, 2007).

Fish and fishery products are known for their high moisture content in their fresh states, which makes them conducive environment for microbial growth (Abdu et al., 2018). Water activity can be reduced by partial removal of water by drying, reverse osmosis or by addition of substances such as glycerol, ethanol that increase the osmotic pressure of food or media

(Prokopov, 2007). The drying method works by removing water from the food matrix and thus, lower the aw of foods, which in turn minimizes microbial activity (Sunmola, 2014). The method has proved to be effective in extending the shelf life of fishery products. However rapid drying of the fish can result in fish layer hardening thus affecting the palatability features of the product, alternatively drying of the fish at a slower rate can result into survival and growth of undesirable microbes (Rahman, 2006).Various methods are utilized in the drying of food and meat products, these include sun-drying and, use of solar tent drying

(Ojutiku and Kolo, 2009).

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2.6 SUN DRYING AS A FOOD PRESERVATION METHOD Sun-drying is one of the traditional methods employed to preserve fish. The method varies from species to species as well as from region to region worldwide (Jain and Tiwari, 2000). It is the simplest and cheapest method of air drying used for foods. Effective open sun-drying depends mainly on the environmental temperature, relative humidity and wind speed (Jain and

Tiwari, 2000).

Traditional sun-drying is carried out in the open air using the energy of the sun to evaporate the water and air currents to carry away the vapor (Pravakar et al., 2013). Theoretically moisture content of the final product should be reduced to less than 15-16%, where most of the microbiological and enzymatic activities are slowed down or stopped (Pravakar et al.,

2013). A major problem associated with sun-drying of fish is the infestation of the products by fly and insect larvae during drying and storage, which deteriorate the quality of the products before consumption (Akhter et al., 2009).

The fish usually takes 5-7 days to dry during which it gets heavily contaminated (Huque et al.,

2013). Very small and thin fish such as the silver cyprinid can be dried straight away in the sun, starting early morning. If these conditions are not fulfilled the fish must be put for one night in brine, or dry salted. They can then be dried the next morning. However, it will be necessary to wash the salt off fish by soaking it in fresh or sea water for a couple of hours before drying, depending on the tastes required by consumers and on the purpose for which the fish is cured. Small fish are mostly sun-dried on nets laid on the ground, grass as well as, on racks. The fish laid on racks or other material to dry should be turned over every two hours so that they dry quickly.

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2.7 QUALITY ASPECTS OF SUN-DRIED SILVER CYPRINID

The landings of Silver cyprinid on Lake Victoria showed a steady increase from 418,590 tonnes to 674,616 tonnes between 2010 and 2015 (LVFO, 2016). However the value of the catch is often very low due to losses brought about by poor handling and storage that result into contamination of fish, hence decrease in quality (Kashindye, 2015). Less than 30% of

Silver cyprinid is utilized as human food, whereas the rest goes into industrial feed mills as raw material for production of feeds for poultry, fish and livestock (LVFO, 2016).

High post-harvest losses is a major concern in regards to the Mukene fishery with 43.3% being as result of rotting and 11.7% due to washing of by rain (LVFO, 2016). The sun-dried food is also prone to losing some of the more fragile vitamins because of being exposed directly to sunlight (Raquel et al., 2018). Thus most of the traditional sun-dried products available in the market are not satisfactory for human consumption (Maathai, 2015).

Preservation of these products involves laying the product on the ground, where it is exposed to contamination and attack by bacteria, infestation by insects, birds, animals and rodents

(Maathai, 2015). Contamination of food with fecal matter exposes the individual consuming the food to human-specific enteric pathogens that cause diseases such as gastrointestinal infections that include; Cholera, Salmonellosis, dysentery as well as infections from strains of

E. coli 0157:H7 which pose a threat to human health (Santamaria and Toranzos, 2003).

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CHAPTER THREE: MATERIALS AND METHODS 3.1 DESCRIPTION OF STUDY AREA Katosi landing site (LS) lies approximately 29 Km off Kampala-Jinja highway, on the shores of Lake Victoria, in Nsanja Parish, Ntenjeru Sub-county, Mukono District (Figure 1) lying at

0°9'9" North, 32°48'2" East (FTI, 2004). The site is a receptacle for commercial goods from nearby areas, and an outlet for Islanders from Ssese and Koome Islands in Lake Victoria (FTI,

2004). It is a designated landing site handling fish for the export market (OAG, 2012) thus the standards herein are therefore meant to meet international standards.

Figure 1 Uganda districts (Left) and section of map showing Mukono district (Right).

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3.2 EXPERIMENTAL STRATEGY Samples of Silver cyprinid sun dried on nets laid on grass were collected from Katosi LS in

Mukono district. The samples were collected from three different locations within the sun- drying area used at Katosi LS i.e. from nets laid on grass at the shore, mid-point and upland.

These demarcations were determined using the two murram paths dividing the sun-drying area

(Figure 2). The area between the shoreline and the first murram path was considered as the shore then the area between the two murram paths as the mid-point and finally the area after the second murram path as the upland (Figure 2). The already set nets from which the samples were to be collected were selected by simple random sampling. Wet and dry samples were collected from each selected net at the LS with the amount of wet samples being double that of the dry samples to account for the additional weight due to the presence of moisture. Two handfuls of wet samples were collected from each of the 3 nets laid on grass in each of the three locations early in the morning. The samples were then tightly wrapped in labeled sterile polythene bags then preserved under ice. After 8 hours, a handful of a dried mass of Silver cyprinid was sampled from each of the 3 initially sampled nets laid on grass at each of the three locations. The samples were then tightly wrapped in labeled sterile polythene bags and stored in a sterile larger polygene bag. All the collected samples were then transported to the

Microbiology Laboratory at the College of Veterinary Medicine for analysis.

18

Figure 2 The demarcations used to distinguish between the three net locations 3.3 DETERMINATION OF THE FECAL CONTAMINATION OF THE SILVER CYPRINID

3.3.1 MEDIA PREPARATION AND CASTING

MacConkey agar was used for culturing both Total coliforms and E.coli whereas Bile Esculin

Azide (BEA) agar for culturing Enterococcus. MacConkey agar is a selective media that contains crystal violet and bile salts that inhibit gram-positive organisms and allow gram- negative organisms to grow (Locatelli et al., 2019). It also contains lactose, a fermentable carbohydrate (Locatelli et al., 2019) that further differentiates between lactose fermenting

Gram- negative enteric bacilli from faeces and urine such as E.coli and those that are not. BEA agar is a selective and differential medium which is used to presumptively identify enterococci based on the ability of an organism to hydrolyze Esculin (Weiss et al., 2005). BEA agar also contains oxgall bile salts that inhibit the growth of gram positive organisms other than

19

Enterococci (Weiss et al., 2005). Prior to collection of samples from Katosi LS, the culture media required was prepared in the laboratory following the manufacturer’s guidelines indicated on each media container. The prepared media was then autoclaved at 121 oC for 25 minutes after which 20 ml of the prepared media was cast on each sterile plastic plates. The cast plates (Figure 3) were then incubated at 37oC overnight to check for any cases of contamination on the cast plates that may have occurred during the media casting process. The uncontaminated plates were then stored in a refrigerator calibrated within a range of 2- 8 oC until they were required for use.

Figure 3 Plates cast with MacConkey agar (A) and BEA agar (B)

20

3.3.2 SPREADING OF INOCULUM ON THE CAST PLATES AND INCUBATION OF THE SPREAD PLATES

In the laboratory, peptone water required for use in the bulk and serial dilution was prepared following the manufacturers guidelines indicated on the media container. 9 ml of peptone water to be used in serial dilution was measured using calibrated pipette and the media transferred into 15ml flacon tubes. The tips of the 15ml flacon tubes were then covered with cotton wool.

The bulk Peptone water and the15ml flacon tubes were then autoclaved at 121 oC for 25 minutes as well as all the instruments to be used in bulk dilution, serial dilution and introduction of the inoculum on the agar plates. These instruments include; 250-500ml borosilicate bottles to be used in mixing the 50ml of bulk peptone water and each sample,

0.1ml and 1ml pipette tips for introduction of the inoculum on the agar plates and serial dilution respectively then 50ml flacon tubes to be used in measuring the bulk peptone water for each sample. After autoclaving, 10g of each sample was measured using an analytical weighing balance, after which it was transferred into sterile 250-500ml borosilicate bottles (Figure 4).

90ml of the bulk peptone water was then measured using sterile 50ml flacon tube after which it was introduced in the 250-500ml borosilicate bottles containing the 10g of Silver cyprinid for each sample. The mouth of 1litre conical flask containing the bulk peptone water was flamed before every measurement to avoid contamination. The mixture in the borosilicate bottles was swirled about for several times in a circular movement until the peptone water had thoroughly washed the fish surface to form an inoculum.

1ml of the inoculum was then serially diluted into 9ml of peptone water in the 15ml falcon tubes until the 10-5 concentration.0.1ml of the required concentrations was then transferred onto the agar plates and spread using a spread loop sterilized by flaming using 70% ethanol.

For instance 10-2, 10-3, 10-4 and 10-5 concentrations of the inoculum were introduced and spread 21 on four MacConkey agar plates respectively whereas 10-1, 10-3 and 10-5 concentrations on three BEA agar plates respectively.

The spread plates were then incubated at different temperatures to facilitate growth of coliforms. To culture E.coli cells, spread plates of inoculum concentrations of 10-3 and 10-5 were incubated overnight at 44 oC. To culture Enterococci cells, spread plates of inoculum concentrations of 10-1, 10-3 and 10-5 were incubated overnight at 37 oC. To culture Total coliforms, spread plates of inoculum concentration 10-2 and 10-4 were incubated overnight at

37 oC. The wet samples were analyzed first and later the dry samples to minimize increase in microorganisms on the samples due to spoilage.

Figure 4 Labelled Borosilicate bottles containing 10g of the samples to be mixed in 90ml of peptone water

3.3.3 COLONY IDENTIFICATION AND COUNTING For the MacConkey agar inoculated plates incubated at 37 oC, all the pink colonies were identified as total coliforms (Figure 5), counted and recorded. However for the MacConkey agar inoculated plates incubated at 44 oC, only the colonies that appeared pink, flat, and dry

22 with a surrounding darker pink area where identified as E.coli colonies (Figure 5), counted and recorded for each sample. For the colonies formed on BEA agar, only the colonies that had a blackening of the media around them were identified as Enterococci cells (Figure 5), counted and recorded.

Figure 5 A contaminated plate of MacConkey agar (Left) and BEA agar (Right)

3.3.4 STATISITICAL ANALYSIS The results were entered into Excel 2013. The means counts from the various concentrations for Total coliforms, E.coli and Enterococci were calculated for each sample. The results were then expressed as colony forming units per g of Mukene (CFU/g). The data was then imported into IBM SPSS Statistics 25 for analysis. A normality test was first carried out on the data to determine which test was appropriate for analysis. The one-way analysis for variance

(ANOVA) analysis was then carried out to determine whether the net location had a significant effect on the level of fecal contamination of the sun dried Silver cyprinid. A Generalized Linear

Model Multivariate analysis was carried out to obtain relationships and patterns between the

23 levels of fecal contamination for both the wet and dried Silver cyprinid in relation to net location. All statistical tests will be determined at significance level of p<0.05.

24

CHAPTER FOUR: RESULTS

4.1 OBJECTIVE 1: LEVEL OF E. COLI ON SILVER CYPRINID SUN DRIED ON

NETS LAID ON GRASS IN RELATION TO THE DIFFERENT NET LOCATIONS

The wet samples had significantly higher levels of contamination with total coliforms and E. coli than the dry samples in relation to the three net locations (Table 1). For the dried samples, negligible contamination was observed on the Silver cyprinid sun dried on nets laid on grass at the shores with no contamination was registered to those on nets laid on grass at the mid- point and upland.

Table 1: Mean and Standard deviation of Total coliform and E.coli loads for the wet and dry samples from three net on grass locations. (T.C WET= Total Coliforms on wet samples. T.C DRY= Total Coliforms on dry samples, E.C WET= E.coli on wet samples and E.C DRY= E.coli on dry samples)

VARIABLE MEAN±SD (cfu/g) NUMBER OF SAMPLES

(n)

T.C WET 35.4±7.6 9

T.C DRY 0 9

E.C WET 4.2±1 9

E.C DRY 0 9

25

The results from the one-way ANOVA run on the level of contamination with E.coli in relation to the three net locations indicated that there was no significant difference between levels of fecal contamination with E.coli at the three net locations at significance level of p<0.05 (one- way ANOVA,F(2,6)=1,p=0.422). Implying that the location of the nets laid on grass on which

Silver cyprinid is sun-dried has no significant effect on the level of contamination with E.coli on the sun- dried Silver cyprinid.

The same outcome of there being no significant difference between levels of contamination with total coliforms at significance level of p<0.05 was observed in regards to the three different net locations (one-way ANOVA, F (2, 6) =3.270, p=0.110).

4.2 OBJECTIVE 2: LEVEL OF ENTEROCOCCI ON SILVER CYPRINID SUN DRIED

ON NETS LAID ON GRASS IN RELATION TO THE DIFFERENT NET LOCATIONS

The dry samples had significantly higher levels of contamination with Enterococci than the wet samples in relation to the three net locations (Table 2).

Table 2: Mean and Standard Deviation of the Enterococci loads on wet and dry Silver cyprinid at three different net on grass locations

NUMBER OF VARIABLE MEAN± SD (cfu/g) SAMPLES (n) WET SAMPLES 2.4±0.9 9

DRY SAMPLES 2.6±0.6 9

For the dried samples, the silver cyprinid dried on nets laid on grass at the shores had the highest level of fecal contamination with Enterococci in comparison with the other net locations, followed by those laid on the upland (Table 3). The silver cyprinid sun dried on nets

26 laid on grass at the mid-point had the least level of fecal contamination with Enterococci in comparison with the other net locations (Table 3)

Table 3 Mean and Standard deviation of Enterococci loads on Sundried Silver Cyprinid at each different net locations

VARIABLE NET LOCATION MEAN±SD (cfu/g) NUMBER OF SAMPLES (n)

DRY SAMPLES SHORE 4.5±1.0 3

MIDPOINT 0 3

UPLAND 3.4±1.0 3

The results of one-way ANOVA run on the level of fecal contamination with Enterococci of the Silver cyprinid sun dried at three different locations indicated that there was a significant difference in the level of fecal contamination with Enterococci at the three different net locations on grass locations at significance level of p<0.05 (one-way ANOVA, F (2, 6) =5.625, p=0.042). Therefore, the location of the nets laid on grass had a significant effect on the level of fecal contamination with Enterococci on the sun dried silver cyprinid (one-way ANOVA, F

(2, 6) =5.625, p=0.042)

27

CHAPTER FIVE: DISCUSSION Rastrineobola argentea, locally known as Mukene is a small pelagic cyprinid, which occurs in

Lakes Victoria, Kyoga, and Nabugabo (Manyala and Ojuok, 2007). The Silver cyprinid provides a cheap source of proteins for both humans and domestic animals. Sun-drying is one of the traditional methods employed to preserve fish. This method works by removing water from the food matrix and thus, lower the aw of foods, which in turn minimizes microbial activity (Sunmola, 2014). The method has proved to be effective in extending the shelf life of fishery products.

Fish is mainly preserved by sun drying on bare ground, nets on grass, peddle and net-on bare ground; surfaces that can expose the Silver cyprinid to further contamination and infestation

(Onyango et al., 2015). These infestation by fly and insect larvae during drying and storage can deteriorate the quality of the products before consumption (Akhter et al., 2009).

However, the bare- ground and net-on grass drying surfaces are known to have significantly higher total bacterial loads that are above the acceptability limit of 1×106 CFU/g for fish and fish products (Nunoo and Noono, 2013). The net on grass drying surfaces are majorly used to sun dry fish for human consumption whereas the bare- grounds are for sun drying fish to be used as food for domestic animals as per the observation at Katosi LS. This study therefore examined whether the location of the nets on grass had an effect on level of fecal contamination on the sun dried Silver cyprinid.

28

5.1 LEVEL OF E. COLI ON THE SUN DRIED SILVER CYPRINID AT THREE DIFFERENT NET LOCATIONS From the study, the level of contamination with E. coli and Total coliforms on the wet samples was significantly higher than on the dry samples at all the three net on the grass locations, suggesting that there was reduced bacterial growth and/or contamination during the drying process, implying that there was minimal or no contamination from external sources of contamination.

This was further backed up by the observation that there was no significant difference in the level of fecal contamination by E. coli on the Silver cyprinid sun dried at the three different net locations at significance level of p<0.05 (one-way ANOVA,F(2,6)=1,p=0.422). Therefore, there was no or minimal external sources of contamination to influence a variation in the levels of fecal contamination by E. coli at the three different net locations.

The results were in agreement with the findings of Baniga et al., 2015 who found 6.7 log cfu/g for the fresh samples then 6.4 and 4.2 log cfu/g for the dried samples sundried on ground and raised ranks respectively. This observation poses a contradiction with the findings made by

Egesa et al., 2018 who found that the total bacterial loads in wet fish were significantly lower than in all dried fish samples implying there was continued contamination during the drying process.

More so according to the study, the Silver cyprinid sun dried on nets laid on grass at the shores had the highest level of contamination by total coliforms as compared to those on Silver cyprinid sun dried on nets laid on grass at the mid-point and upland. This could be as a result of the water logged nature of the grass at the shore on which the nets are laid (Figure 6) that may affect the effectiveness of the surface during the sun drying process.

29

Figure 6 A water logged site at the shores of Katosi LS 5.2 LEVEL OF ENTEROCOCCI ON THE SUN DRIED SILVER CYPRINID AT THREE DIFFERENT NET LOCATIONS

From the study, there was a significant difference in the level of fecal contamination with

Enterococci at the three different net-on the grass locations at significance level of p<0.05

(one-way ANOVA, F (2, 6) =5.625, p=0.042). Implying that the location of the nets laid on grass had a significant effect on the level of fecal contamination with Enterococci on the sun dried silver cyprinid. This could be as a result that Enterococci is not purely found in human and animal fecal matter (Layton, Walter, Lam and Boehm, 2010) but can also be found on plants and in soil (Goto and Yan, 2011).

There is evidence that Enterococci cells are capable of replicating in extra-enteric environments such as beach sands and in water containing plankton (Mote, Turner and Lipp,

2012) explaining why perhaps the Silver cyprinid dried on nets laid on grass at the shores had the highest level of fecal contamination with Enterococci in comparison with the other net

30 locations. Since the shores are rich in plankton materials that is carried by the lake water currents to the shoreline.

From the study, there is evidence that there were external sources of contamination considering that the fecal contamination levels with Enterococci for the dry samples was slightly higher than that of the wet samples (Table 2). However, there is need for further identification of human-specific enterococci species or genotypes to be able to eliminate the possibility of human fecal contamination from the other environmental sources of the organisms which was not determined in this study.

31

CHAPTER SIX: CONCLUSIONS AND RECOMMENDATIONS 6.1 CONCLUSION

The location of nets on grass used for sun drying had less significant effect on the level of fecal contamination on sun dried Silver cyprinid. This level of contamination at the different net locations was greatly influenced by the kind of fecal indicators examined. The level of fecal contamination by E. coli on sun dried Silver cyprinid indicated that there was no significant difference in the E. coli contamination on the sun dried fish at the three net locations i.e. shore, mid-point and upland.

However, this was not the case with the levels of fecal contamination with Enterococci on the sun dried Silver cyprinid that significantly varied at the three different location. This was owed to the fact there was external sources of contamination since the study also observed that the level of fecal contamination with Enterococci on the dry samples was slightly higher than that of the wet samples from the three net locations.

Enterococci is not purely found in human and animal fecal matter (Layton, Walter, Lam and

Boehm, 2010) but can also be found on plants and in soil (Goto and Yan, 2011). Thus there is need for further identification of human-specific enterococci species or genotypes to be able to eliminate the possibility of human fecal contamination from the other environmental sources which was not determined in this study.

32

6.2 RECOMMENDATIONS  Nets to be used in the sun drying of the Silver cyprinid should be laid on dry grass to ensure the effectiveness of the drying process  The landing site should take up the use of raised racks as surfaces for drying fish to be used for human consumption as this ensures effective and efficient sun drying of the Silver cyprinid.

33

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