PROXIMATE AND MICROBIAL QUALITIES OF DIFFERENT FISH FEEDS

SOLD IN OWERRI METROPOLIS, IMO STATE, NIGERIA

BY

ANYANWU CHIBUNNA ULTIMATE

22BH/0435/ST-MB

A PROJECT SUBMITTED TO THE

DEPARTMENT OF SCIENCE LABORATORY TECHNOLOGY
(BIOLOGY/MICROBIOLOGY OPTION)
FEDERAL POLYTECHNIC NEKEDE, OWERRI
IMO STATE

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD

OF HIGHER NATIONAL DIPLOMA (HND) SCIENCE LABORATORY
TECHNOLOGY (BIOLOGY/MICROBIOLOGY OPTION)

NOVEMBER, 2024

1
 CERTIFICATION

This project “ Proximate and Microbial Qualities of Different Fish Feeds Sold in

Owerri Metropolis, Imo State, Nigeria” , was carried out by Anyanwu Chibunna

Ultimate, with Registration Number 22BH/0435/ST-MB, from the Department of

Science Laboratory Technology (Biology/Microbiology Option), School of Industrial

and Applied Sciences, Federal Polytechnic Nekede, Owerri.

…………………………. ………………………..
Dr. (Mrs.) I.J. Ibe Date
(Supervisor)

……………………………… ………………………….
Mrs. Osikwe, A. K. Date
HOD (Biology/Microbiology)

2
 DEDICATION

I dedicate this work to Almighty God for His protection and guidance to me and how

far He has helped me till this level of my Higher Diploma Programme.

3
 ACKNOWLEDGMENTS

My profound gratitude goes to God almighty for is infinite mercy and love towards

me throughout my Higher National Diploma till this very moment.

This acknowledgment won't be complete without recognizing my seminar supervisor,

Dr. (Mrs.) Ibe, I.J., for her efforts and encouragement, she always counsels me never

to give up, I am grateful Ma.

I am grateful to the Head of Department of Biology/Microbiology, Mrs. Osikwe, A.K

and other lecturers, technologists, academic and non-academic staff, for their efforts

in training the students with current knowledge in science and technology. May God

bless you all, Amen.

Notwithstanding, no achievement in life is without the help of many known and

unknown individuals who contributed to our lives.

On this note, I wish to express my immense appreciation to my parents for always

being there for me especially their prayers.

Finally, I appreciate my friends and family for their supports and encouragement, and

every other person who have contributed in one way or the other to make my HND a

success, please multiply my thanks by infinity, I really appreciate.

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 ABSTRACT

Fish feed is the significant primary step in the aquaculture generation chain. This
study evaluated the proximate and microbial qualities of different fish feeds sold in
Owerri Metropolis, Imo State. Seven (7) samples of fish feed sizes were randomly
purchased from different sellers around Relief market Owerri, Imo State. Standard
methods by Association of Official Analytical Chemists were adopted in the
determination of the proximate compositions of the fish feeds. Standard
microbiological methods were adopted in the determination of the microbial load,
isolation and identification of the microorganisms associated with the fish feeds. The
proximate compositions were; moisture (6.10% to 8.07%), ash (7.85% to 11.16%),
fats and oil (11.13% to 14.29%), protein (39.45% to 44.65%), fibre (3.01% to 4.99%)
and carbohydrate (20.26% to 27.70%). The microbial load of the fish feed samples
showed that total viable bacterial counts ranged from 6.0 x 103 cfu/g to 3.4 x 104 cfu/g
while total fungal counts ranged from 1.0 x 103 cfu/g to 4.4 x 104 cfu/g. The bacterial
isolates from the fish feed samples were; Staphylococcus, Micrococcus, Bacillus and
Corynebacterium species. The fungal isolates from the different fish feed samples
were; Aspergillus, Penicillium, Mucor and Kluyveromyce species. The presence of
these organisms in fish feeds may also arise from poor hygienic and sanitary practices
employed in the manufacturing, processing and packaging of fish feed. Therefore,
there should be proper storage conditions and hygienic handling during storage so as
to reduce microbial contamination of fish feeds.

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 TABLE OF CONTENTS

Title page i

Certification page ii

Dedication Page ii

Acknowledgment iv

Abstract v

Table of contents vi
CHAPTER ONE
1.0 Introduction 1
1.1 Background to the study 1
1.2 Statement of Problem 3
1.3 Aim and Specific Objectives of Study 4
1.3 Significance of the Study 4
CHAPTER TWO
2.0 Literature Review 5
2.1 Fish Feed Processing and Production Technology 6
2.2 Composition and Microbial Qualities of Some Fish Feeds 10
CHAPTER THREE
3.0 Materials and Methods 16
3.1 Collection of Samples 16

3.2 Sterilization of materials 16

3.3 Proximate compositions of the fish feed 16

3.4 Microbiological analysis 19

3.5 Microbial plate count 20

3.6 Colonial morphology identification 20

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3.7 Purification and preservation of isolates 20

3.8 Gram staining 21

3.9 Biochemical tests with the bacterial isolates 21

3.10 Identification of the fungal isolates 24

CHAPTER FOUR
4.0 Results and discussion 19
4.1 Results 19
4.2 Discussion 22
CHAPTER FIVE
5.0 Conclusion and recommendations 25
5.1 Conclusion 25

5.2 Recommendations 26
REFERENCES 25

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 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the study

Fish like other animals have a requirement for essential nutrients in order to grow

properly. In the wild, natural foods are available and as the fish forage for these,

they are able to meet their body needs. When fish is removed from its natural

environment to an artificial one, enough food must be supplied in order to enable

them grow. Artificial diets may be either complete or supplemental, Complete diet

supply with all the ingredients (protein, carbohydrates, fats, vitamins and minerals)

is necessary for the optimal growth and health of the fish, Supplemental diet do

not contain a full complement of nutrients needed but are used to help fortify the

naturally available diets (Nwabueze, 2018).

Riche and Garling (2018) reported that fish reared in intensive tank systems

require all nutrients in a complete pelleted diet since natural food is limited and

fish cannot forage freely for natural foods. This has the advantage of high quality

and consistency of diet. Good nutrition in fish production system is essential to

economically produce healthy, high quality fish products (Sanal et al., 2018).

Fish farming involves commercial breeding of fish, most often for food, in fish

tanks or artificial enclosures such as fish ponds. It is a particular type of aquaculture,

which is the controlled cultivation and harvesting of aquatic animals such as

fish, crustaceans, molluscs and so on, in natural or pseudo-natural environments (Food

and Agricultural Organization, 2014).

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Commercially available fish feeds are provided to the cultured farm fishes which can

meet the protein demand of the fishes. Feed types can be divided into three groups,

such as industrially compounded feeds, farm-made feeds and raw organisms. Artificial

diets may be either complete or supplemental (Uddin et al., 2019). Fish feeds are

prepared commercially mainly using the animal byproducts (extreta, bones, meat) as

well as plant originated components (cereal seeds, bran, rapeseed or soybean meal or

cake, legume seeds) which are better in delivering more nutrition. But the byproducts

can readily transmit the associated pathogenic and opportunistic pathogenic bacteria in

the feed and consequently they are responsible to cause illness by producing toxins in

the fish (Ahmad & Ibrahim, 2016).

Fish feeds are constantly in contact with environmental organisms and become readily

colonized by various microbial species. Environmental factors during storage cause

the microbial spoilage of the fish feeds. The presence of bacteria in feeds causes their

decomposition and subsequently, fish diseases. Bacteria such as Salmonella, E. coli

and other bacteria strains have been reported to contaminate fish feeds. Fungal

contamination of fish feed has been reported to result in aflatoxicosis. Aflatoxins are

chemical produced by fungi like Aspergillus flavus and A. parasiticus commonly

known as mold. Other fungal toxins include patulins and trichotecens which are

strongly carcinogenic and mutagenic. Fungal contamination occurs mainly during the

storage in poor environmental conditions (Ubeibi, 2017).

Any equipments, production stages, entire production plant can be responsible for

potential contamination as a source. The growth and proliferation in the feed depends

9
on numerous factors, such as moisture, temperature, type of feed, aerobic and

anaerobic conditions, chemical and physical properties of raw material, feed pH value,

presence of feed supplements, storage periods and conditions as well as feed

decomposition products (Ubeibi, 2017; Uddin et al., 2019). The microbiological

indices of feed quality include total plate count (TPC), Escherichia coli count,

coliforms, Enterobacteriaceae Count, yeast and mould count etc. Therefore, in the

present study the microbiological quality of different fish feeds will be analyzed to

understand the quality of fish feed available in Owerri metropolis.

1.2 Statement of problem

The quality of fish feeds and the hygienic levels of technological process employed

during feed formulation determine the level of risk of microbial contamination aided

by storage temperature. Storage conditions especially temperature and humidity are

important factors affecting microbial quality of fish feeds. Improper storage

temperature may prolong survival of the microorganisms in fish feeds by enhancing

their multiplication and production of toxic substances which may be injurious to fish.

Rearing of fish in concrete tanks using artificial diet has become a common practice in

Owerri. Several outlets for sale of artificial fish feeds now exist. However, the owners

of the fish ponds care less about the microbiological and proximate compositions of

the fish feeds used in rearing fish. There is a need to investigate the proximate and

microbiological qualities of fish feeds sold in Owerri and to ascertain their storage

conditions in order to ensure their safety as food for fish.

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1.3 Aim and objectives of the study

1.3.1 Aim of the study

The aim of this study was to investigate the proximate and microbiological qualities of

fish feeds sold in Owerri and to ascertain their storage conditions in order to ensure

their safety as food for fish.

1.3.2 Objectives of the study

1. To determine the proximate compositions of some fish feeds sold in Owerri

Metropolis.

2. To determine the microbial load of some fish feeds.

3. To isolate the microorganisms associated with some fish feeds.

4. To identify the isolated microorganisms from fish feeds.

1.4 Significance of the Study

The findings from this study will help to reveal the proximate compositions of the

different brands of fish feeds in order to help the users identify the one with most

important nutrient needed for rearing of fish in the fish ponds. The findings will also

reveal the microbiological qualities of the fish feeds in order to know if they are

within the acceptable microbial quality for animal feeds. This will help to forestall

contamination of fish feed and by implication prevent transmission of harmful toxic

effects on human consumers of fish.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

Fish and fish products are a popular source of protein and animal fat to the people and

with the overgrowing people, the demand for protein is increasing. To meet the

demand, fisheries sectors have been established and are increasing rapidly worldwide.

To grow properly and rapidly, the fishes need proper nutrients in the artificial

environment as they are removed from natural environment. Fish is cheaper than other

animal sources of protein such as poultry, pork, cow and accepted by the people from

all religions (Uddin et al., 2019).

Fish feed is the significant primary step in the aquaculture generation chain. In Nigeria,

the generation of fish feed is basically focused on at exceedingly savage species such

as the salmonids, cod, bass and bream. These carnivorous species require a large

protein bolster. The fish feed has customarily been based on marine fixings fish oil

and fish support. An inexpensive source of fish feeds will make fish farming attractive

due to its profitability (Ombugadu et al., 2021). To decrease feed cost and make

strides the fish cultivating supportability, the latest advancement is the fractional

substitution of marine components of animal sources (fish oil and meal) with

vegetable fixings. It may be a common perception that vegetable feed ingredients are

more vulnerable to development of parasites, due to the natural capacity and transport

related variables, compared to bolster fixings from an animal ancestry (Omola et al.,

2022).

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Feed quality is subordinate on a few components such as crude materials utilized,

preparing conditions and feed management practices amongst others (Tangendjaja,

2015). Fish feed formulators are mindful that the diet’ s composition can influence

fish’ s pigmentation due to the choice of crude materials and nourish fixings at their

disposal. Nutrition hence, has critical impact on flesh quality. Care is practiced in like

manner to permit for these inputs and hence define feeds to meet the dietary

prerequisites of the fish and accomplish best execution in terms of flesh coloration and

texture (Komolka et al., 2020). The supplement balance of feed moreover impacts

feed utilization and development of fish. It is fundamental to know the supplement

prerequisite especially for protein, lipid and vitality, for the ideal growth of a fish

species and to define a balanced diet (Ayuba & Iorkohol, 2013).

Dietary protein and vitality levels are known to influence the development and body

composition of fish species (Lovell, 2013). Inadequate vitality in diets causes a protein

to squander due to the increased amount of dietary protein utilized for vitality. The

produced ammonia can contaminate the water and make it unfit for fish farming (Isah

et al., 2021). Fish nutrition and feed quality specifically influence fish wellbeing and

efficiency (Wong et al., 2016).

2.1 Fish feed processing and production technology

In animal production system, nutrition is a key factor in production of economically

healthy, high quality product. Feed is one of the major inputs in aquaculture

production and there is an increasing demand for quality feeds. Adoption of

appropriate processing technology is an important factor to determine the final quality

13
of feed. In feed formulation, ingredients are selected in correct amount to form a

single uniform mixture or pellets at possible low cost that will provide all the

nutritional requirements of the target fish (Vipincuma et al., 2019). The basic steps

involved in fish feed manufacturing are; selection of ingredients, grinding, feed

formulation, mixing, pelleting, drying, packing and storage

1. Selection of ingredients: The first operation in the feed processing plant involves

the selection of raw materials. The quality of feed ingredients both dry and liquid form

has an important impact on the quality of final feed. It should be fresh and free from

contaminants like sand, stones, and others earthen materials. The ingredients should

be examined for quality check and also for nutrient analysis (Vipincuma et al., 2019).

2. Grinding: Grinding or particle size reduction is a major step in feed manufacture.

Prior to use, ingredients must be powdered, in order to get uniform particle size. The

grinding of ingredients generally improves feed digestibility, acceptability, mixing

properties, pellet ability, and increases the bulk density of some ingredients. It is

accomplished through the use of many types of manual and mechanical operations

involving impact, attrition, and cutting. The most commonly used grinding machines

are hammer mill, pulverizers, flour mill etc. The powdered ingredients are then passed

through a standard mesh sieve to obtain a uniform particle size. Sieving the

ingredients helps in preparing feed pellets with uniform and attractive physical

appearance (Omola et al., 2022).

3. Feed formulation: In this process appropriate dietary ingredients are selected on

the basis of availability, chemical composition, nutritional value and, cost. The

14
ingredients are selected in appropriate amount and blended to produce a compounded

feed which is nutritionally balanced, palatable, pelletable, and easy for storage. The

important points to be considered for feed formulation are: nutritional requirements of

the species to be cultivated, feeding habits of the species, expected feed consumption

and type of feed processing required (Lovell, 2013).

4. Mixing: Feed mixing may include all possible combinations of solids and liquids.

Sieved ingredients were weighed and mixed in desired proportion according to the

formulation. Generally dried ingredients are mixed first followed by liquid materials.

Liquid materials such as fish oil may be added at the end and further homogenized.

Water is also added for increasing the moisture level may also be added. For proper

mixing of different feed ingredients into a homogenous mass, the mixing may be 20 to

30 minutes. Mixing can be done in batches or in continuous mixers. Batch mixing can

be done on an open flat surface with shovels or in any containers. Continuous mixers

are such that the material moves through the mixers as it is being mixed. The types of

mixers used are horizontal ribbon mixers, vertical mixers, and turbine mixers (Omola

et al., 2022).

5. Pelleting: It is the process of compacting of feeds by extruding individual

ingredients or mixtures of ingredients. Pelleting converts the homogenous mixture

into a quality feed, having physical characteristics that make them suitable for feeding.

Pelletization is mainly done using two types of machines namely extruder pelleting

and compressed pelleting (Omola et al., 2022).

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 i. Extruder pelleting technology

Fish feed extrusion process refers to cook the mixture of feed ingredients under high

temperature, pressure, and moisture by the means of an extruder within a short time.

The basic components in an extruder are, a barrel fitted with a die plate and a screw

shaft conveyor connected to a high speed motor (Vipincuma et al., 2019).

The most important operating parameters are the temperature, pressure, diameter of

the die apertures and shear rate.

ii. Compressed pelleting technology

The pelletizer works on the principle that the finely ground feed mixture is pelleted by

compression process. Compressed pelleting then involves exposing the mixture to

steam for 5-20 seconds obtaining 85o C and 16% moisture followed by forcing. the

mix through holes in metal die by the action of a roller located inside the die. This

process is also known as steam pelleting, due to the use of steam to precondition the

mix prior to compression. The combination of heat, moisture, and pressure in which

gelatinization of the starch occurs. As the pellets emerge to outside surface of the die,

they are cut off by a stationary adjustable knife to the desired length. Pellet quality is

influenced by the fat level, moisture, and humidity (Vipincuma et al., 2019).

The fat level of the mixture should be not less than 2-3% to lubricate the holes in the

die and to reduce the dustiness and not higher than 8-10% to avoid excessive

lubrication causing insufficient compression of the feed mixture. The moisture level is

also critical as the excessive moisture results in soft pellets and insufficient moisture

results in crumbly pellets (Omola et al., 2022).

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6. Drying: Immediately after pelleting the feed should be dried to reduce the moisture

content below 10%. This is essential for good shelf life of the feed. Different type of

dryers are used for drying feed pellets, like horizontal conveyer type, vertical hopper

type, hot air oven and fluid bed dryers. The ambient temperature used for drying feed

is at 65-75 oC. Higher temperature is not desirable (Omola et al., 2022).

7. Packing: The dried feed is cooled before packing. Good quality packing covers are

used to prevent damage to the feed quality on transportation and storage.

8. Storage: The feeds are composed of biological material and may degrade upon

prolonged storage. Therefor feeds should be stored in clean, cool and dry warehouses

storage areas away from direct sunlight. The deterioration may be due to oxidative

damages or microbial damages. Provisions should also be made to avoid infestations

from insects, pests, rodents etc. Storage condition accelerates the process of feed

deterioration (Vipincuma et al., 2019).

Temperature and humidity represents the major environmental factor that determines

the storage. Depending on the relative humidity stored feed will reach an equilibrium

with the atmospheric moisture content regardless of the original moisture content.

High moisture content favours the microbial growth. High temperature cause

oxidative damage to feed additives like vitamins thus lowering the nutritional quality

of feed and also accelerate deterioration process caused by microbes. Apart from these

factors light and oxygen also effect feed decay. The feed should be stored for

minimum time (Vipincuma et al., 2019).

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2.2 Compositions and microbial qualities of some fish feeds

The world population is on the rise, as well as the demand for aquatic food products

(fish). Production from capture fisheries at the global level is leveling off and most of

the main fishing arcas have reached their maximum potential. Sustaining fish supply

from capture fisheries will therefore, not be able to meet the growing global demand

for aquatic food (fish). At present, the aquaculture sector contributes a little over 40

million tonnes (excluding aquatic plants) to the world aquatic food production (Olin &

Paul, 2015).

As fish feed is very important for a successful aquaculture production, the natural

food nutrient available in the pond cannot give the desired fast growth within the

expected period. Growth, health and reproduction of fish and other aquatic animals are

primarily dependent upon adequate supply of nutrient, both in terms of quality and

quantity. Supplied inputs such as feeds and fertilizers have to be ensured, so that the

nutrients and energy requirements of the species under culture could be met and the

reproduction goals of the system achieved (Hassan, 2019),

Nettleton et al. (2017) reported that the nutrient profile of catfish showed that it is

highly nutritious, high in protein, low in fat and cholesterol and a good source of

certain vitamins and minerals. The residual low level of protein in the flesh of the fish

is passed unto the consumer which consumes a fish with poor quality nutrient in its

flesh and which makes the fish a poor source of quality animal protein that it is

supposed to be. When talking of quality of a fish feed it takes into consideration the

pellet firmness and quality, that is its durability in water as fish feed of low quality

18
disintegrates easily in the ponds thereby polluting the water and causing wastage of

the feed (Esuga, 2014). Fish feed should be packaged such that it will be free from of

mycotoxins and aflatoxins in storage and is also free from contaminants (Esuga, 2014).

Formulated feed plays a significant role in semi intensive fish farming where a high

stocking density of fish is maintained than the natural fertility of the water can support

(Jhingran, 2021). The use of commercial feeds with well-balanced nutritional profile

has become crucial for the success of aquaculture under intensive conditions (Abid &

Ahmed, 2019). Formulated feed is found to be useful in providing adequate nutrient

components and energy required for better fish growth and production and is known

to increase the carrying capacity of culture system thereby enhancing fish production

by several folds (Nazish & Mateen, 2011). The net production obtained when fed with

supplementary feed was 7.7 times greater as compared to the treatment without feed in

polyculture system (Kabir et al., 2019).

There is a growing interest among the farmers to opt for pelleted feed over traditional

feeds such as mash for fish culture which are comparatively superior to mash feed

since, loss of nutrients out of leaching and wastage of feed is less. Moreover, as the

feed wastage is less, physico-chemical and biological conditions of the aquaculture

system under application of pellet feed might be better because of less decomposition

and organic loading. Feed comprises about 60% of the total cost of any aquaculture

venture. The global demand for formulated feeds for fish was estimated to be 29.3

million tonnes in 2008 and is expected to grow with increasing aquaculture (FAO,

2018).

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Microbiological parameters are important to determine the safety and quality of feeds.

Feed may be contaminated during processing, storage, transport or handling.

Contaminated feed frequently causes disease and for that reason, it is necessary to

establish surveillance programs for microbiological feed hazards (Radanov et al.,

2016). Feed ingredients must be safe and suitable for storage and Consumption. Even

a single unsafe component may jeopardize the quality of compound feed. Some

microorganisms introduced during storage, primarily moulds, can negatively affect

feed quality including reducing dry matter and nutrients, causing musty or sour odours,

and producing toxins (Meeusen, 2017).

Nwabueze and Nwabueze (2011) reported on the microbial flora of fish feeds sold in

Asaba, Southern Nigeria. Three different particle sizes each of Coppens (6mm, 4.5

and 3mm), Dizengolf (10mm, 4.5mm and 2mm) and Durante (6mm, 3mm and 2mm)

were purchased and used in the study. Out of the nine sizes used, Escherichia coli and

Staphylococcus aureus were found in eight. Four of the samples had both E. coli and S.

aureus. Bacterial counts from Coppens feed was significantly higher (P<0.05) than

bacterial counts from Dizengolf and Durante fish feeds which had counts not

significantly different (P>0.05) from each other. No fungus was found. Results

obtained show that fish feeds sold in Asaba may have microbial flora with bacteria as

the dominant microorganism. Storage conditions and unhygienic handling during

storage, probably introduced bacterial flora in the fish feeds.

Sanal et al. (2018) reported on evaluation of the quality of commercial fish feeds in

India with respect to microbiological parameters. The TPC ranged from 2.0 × 102

20
to3.13 × 104 CFUg-1 in different feeds. Presence of E. coli was detected in one of the

feeds with 1.15 × 102 CFUg-1. Coliform bacteria were not detected in any of the feeds.

Enterobacteriaceae was present in three feeds in the range of 5.45 × 102 to 1.58 × 103

CFUg-1. Yeast and mould count ranged from 101 to 1.68 × 104 CFUg-1 in the feeds

analyzed. The results obtained from the present study indicate that the feeds were

contaminated with microorganisms.

Bello et al. (2016) reported on proximate composition of some commercial fish feeds

available in Maiduguri, Borno State, Nigeria. The fish feed investigated have different

sizes of 2.0, 3.0, 4.5, 6.0, 8.0 and 9.0mm. The results revealed that moisture contents

were not declared by most manufacturing company. Protein content in CF ranged

between 1.3 and 7.5; 6.2 and 7.5 in EF, 1.2 and 4.2 in MF, 1.9 and 5.0 in VF in all the

sizes while lipid content ranged between 0.7 and 1.5 in CF, 0.0 and 1.0 in EF, 0.6 and

2.0 in MF; VF 8.0 and 9.0 % respectively, while UF had no written specification;

therefore, the difference cannot be quantified.

Omola et al. (2022) reported on proximate analysis of some selected fish feeds

marketed within Kano metropolis, Nigeria. The result of the proximate investigation

showed the following composition: 7.28-11.16 % ash, 6.10-12.26 % moisture, 11.13-

14.29 % lipid, 3.01-5.61 % fibre, 37.45- 44.30 % protein and 20.26-27.70 %

carbohydrate. There was no critical distinction (p>0.05) noticed among the values of

all the proximate compositions of the fish feeds studied. However, a few values

exceeded the permissible limit, whereas the majority fell within the limits set by

relevant regulatory agencies.

21
Uddin et al. (2019) reported on microbiological analysis of fish feeds with the

demonstration of the antibiotic susceptibility of the isolates and the antibacterial

activity of the feeds. The higher counts of TVBC, Escherichia coli, and

Staphylococcus aureus were recorded as 2.9 × 106 cfu/g, 3.3 × 105 cfu/g and 1.1 x 106

cfu/g respectively. On the other hand, Salmonella spp., Shigella spp., Vibrio spp. and

Pseudomonas spp. were also detected in several samples. Fungal count was also

noticed in all samples with a maximum load of 2.8 x 106 cfu/g and a minimum load of

2 × 104 cfu/g. All isolates found from fish feed samples were 100% sensitive against

gentamycin and ceftriaxone. The bacterial isolates also showed varying degree of

resistance against other antibiotics tested in this study.

22
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Collection of samples

Seven (7) samples of fish feed sizes were randomly purchased from different sellers

around Relief market Owerri, Imo State. The samples were packed in different sterile

nylons, labeled and were taken to the laboratory for microbiological assessment.

3.2 Sterilization of materials

The method described by Uddin et al. (2019) was adopted in the sterilization of the

media and glasswares that will be used in this study. All the glass wares used for the

experiment were sterilized using the laboratory autoclave at a temperature of 121 0C

for 15 minutes at 15psi. Wire loop was sterilized over burning flame and allowed till

its red-hot, while glass spreader was sterilized by dipping into 70 % ethanol and

passing over Bunsen flame. The media used in this study; nutrient agar, triple sugar

iron agar, Simmon’ s citrate agar, peptone water, eosin methyle and Sabouraud

dextrose agar were prepared according to manufacturer’ s instructions and sterilized

using the autoclave at a temperature of 121 0C at 15psi for 15 minutes and were

allowed to cool to a temperature of 45 0C and about 20 milliliters was poured into

sterile petri-dishes. The plates were allowed to cool and set for inoculation.

23
3.3 Proximate compositions of the fish feed

Proximate compositions of the different commercial fish feeds were analyzed

according to standard procedures given by Association of Official Analytical Chemist

(AOAC) (2015). The proximate composition of the fish feeds were analyzed in

triplicate for moisture, protein, ash, carbohydrate, fat and oil and ash contents.

3.3.1 Ash content determination

The method described by AOAC (2015) was adopted. 2.0g of the sample was weighed

out using digital electronic balance in a crucible which was washed and dried in an

oven, cooled and weighed. The porcelain crucible containing the sample was heated

on a Bunsen flame inside a furnace cupboard until no fume was observed. The

crucible with the residue was transferred to a preheated mantle furnace at 600 oC for 2

hours until complete ashing and constant weights were achieved with intermittent

cooling and weighing. The percentage of the ash was calculated.

3.3.2 Nitrogen and crude protein determination

The method described by AOAC (2015) was adopted. 2.0g of the sample was weighed

into a Kjelhadh flask. Five gram of anhydrous sodium sulphate and one gram of

Copper sulphate were added (as catalyst). Then 25 millilitre of concentrated H2SO4

was added to the sample and moved into a furnace cupboard and heated for 1 hour

using Thermo regulated heating mantle at temperature of 250C with intermittent

shaking till green color emerged. It was filtered to remove impurities. The filtrate was

reheated gently until green color disappeared and was allowed to cool. The digest was

24
transferred with several washings into a 250 millilitres volumetric flask and made up

to the mark with distilled water. It was however distilled using distillation apparatus.

100 millilitres capacity conical flask containing 5milliliter of boric indicator was

placed under the liquid. 10 millilitre of the digest was measured and transferred to the

cap of the Kjeldah distiller, followed by addition of 10 millilitres of 40% 0.1 mole

NaOH solutions. The solution was shaken thoroughly for 15 minutes to collect enough

Ammonium sulphate, evidenced by colorless solution. The receiving flask was

removed and the tip of the condenser was washed down into the flask. The solution

was titrated in the receiving flask using 0.01 ml HCL until blue colour appeared.

3.3.3 Crude fibre determination

The method described by AOAC (2015) was adopted. 2.0g of the sample was weighed

out using digital electronic balance and boiled with 200 ml 0f 1.25 % H2SO4 for 30

minutes in a flask using water bath. It was filtered using Whatmann 54 filter paper

through a funnel. The residue was washed with hot water to remove acid from it. The

residue was then transferred to another beaker and boiled for 30 minutes in a water

bath using 200 ml of 1.25% NaOH. It was again filtered and progressively washed

with boiled water. The residue was also transferred to a crucible and dried in the oven

to a constant mass. It was at this stage incinerated using mantle furnace, cooled and

reweighed. The percentage of the fibre was calculated.

3.3.4 Crude fat determination

The method described by AOAC (2015) was adopted. This involves the use of

methanol, the wet ground mustard seed samples are dissolved in the methanol to give
25
a single phase, miscible with water. Additionally, chloroform is added to give a

separation of the phases and then centrifuged to separate the solvents. The residue left

behind after the chloroform layer containing the dissolved fat is removed and

reweighed. The percentage of the fat was calculated.

3.3.5 Determination of moisture content

The method described by AOAC (2015) was adopted. A crucible was properly
0
washed and allowed to dry in an air oven at 110 C for 10 minutes to a constant

weight. Then the crucible was allowed to cool in desiccators for 30 minutes and was

labelled and weighed (W1). 2.0g of the sample was accurately weighed (W2). The

crucible containing the sample was placed in an oven maintained at 1030 C for 14

hours. It was then removed and allowed in the dessicator then finally weighed (W3).

The percentage moisture content was calculated.

3.3.6 Determination of Carbohydrate

The method described by AOAC (2015) was adopted. The total carbohydrate content

was determined by difference method. The sum of the percentage moisture, ash, crude

lipid, crude protein and crude fibre was subtracted from 100%. Carbohydrate = 100-

(% moisture + % ash + % protein + % lipids + % fibre).

3.4 Microbiological analysis

The method described by Ubeibi (2017) was adopted in the determination of the

microbial quality of the fish feeds. Five (5g) grams each of the fish feeds were

weighed into a sterile beaker and 45 ml of sterile water was added. This was
26
thoroughly homogenized in the beaker for 90 seconds. The ten-fold serial dilution was

prepared in 9 ml of solvent using 1 ml sterile pipette. At the end of the serial dilution,

0.1 ml of 10-5 dilution was aseptically inoculated onto sterile plates of nutrient agar,

eosin methylene blue agar, Salmonella-Shigella agar and Sabouraud dextrose agar

media for enumeration of microorganisms.

3.5 Microbial plate count

After the incubation of the different plates, the different colonies formed on the media

were counted using the digital colony counter. The total population of the colonies

was expressed as colony forming unit per gram (cfu/g).

3.6 Colonial morphology identification

The method described by Ubeibi (2017) was adopted in the colonial morphology

identification. Presumptive identification of the colonies were done by observing their

individual shape, colour, elevation, edge, surface, consistency and appearance on the

media used for isolation. Colonies with characteristic metallic sheen on EMB agar and

lactose fermenters on MacConkey agar were noted. The colonies were preserved in

sterile agar slants in test tubes. Purified colonies were further characterized using

Gram stain and biochemical tests.

3.7 Purification and preservation of isolates

Bacterial isolates were picked up with a sterile wire loop based on their morphological

appearances. The picked colonies were subcultured onto freshly prepared nutrient agar

plates to obtain pure cultures. They were further incubated for 24h at 37 0C. After
27
incubation, pure cultures were stored in McCartney bottle in a refrigerator at 4 0C.

Fungal isolates were subcultured onto freshly prepared Sabouraud dextrose medium.

3.8 Gram staining

The Gram staining technique described by Uddin et al. (2019) was adopted in the

determination of the gram reactions of the bacterial isolates. A smear of the colony

from pure culture was made on a clean grease-free glass slides to be stained. The

smears were allowed to air dry and later heat fixed. Crystal violet was added to the

slide and allowed for 1 minute. The slide was rinsed with a gentle stream of water for

a maximum of 5 seconds. Gram's iodine was added for 1 minute, then the slide was

rinsed again with water. The slide was rinsed with acid alcohol for 3 seconds and with

water. The secondary stain, safranin, was added to the slide and allowed for 1 minute.

The slide was rinsed with gentle stream of water for a maximum of 5 seconds. The

stained slides were allowed to air dry and were viewed under a microscope using x40

and x100 objective lenses. Gram positive bacteria retained the primary stain (crystal

violet) and appeared purple under the microscope. Gram negative, lost the primary

stain and took the secondary stain, causing it to appear pink when viewed under a

microscope.

3.9 Biochemical tests with the bacterial isolates

The method described by Ubeibi (2017) was adopted in the biochemical

characterization of the bacterial isolates. The biochemical tests carried out will include;

catalase, oxidase, coagulase, citrate utilization, indole production, sugar fermentation

and motility test.

28
3.7.1 Catalase test

Three milliliter (3 ml) of 3% hydrogen peroxide was poured in a test tube. A colony of

test organism was taken with sterile wooden or glass rod and immersed into hydrogen

peroxide solution. Generation of bubbles indicated oxygen production. If bubbles

were produced, the organism was catalase-positive. However, if bubbles were not

produced, the organism was catalase-negative.

3.7.2 Oxidase test

A piece of filter paper was placed in Petri-dish and 3 drops of freshly prepared

oxidase reagent were added. Using a sterile glass rod, a colony of test organisms was

removed from a culture plate and smeared on the filter paper. Oxidase-positive

organisms gave blue color within 5-10 seconds, and in oxidase-negative organisms,

color did not change.

3.7.3 Citrate test

A bacterial colony was inoculated in Simmons citrate agar and incubated at 35 °C to

37 °C for 18 to 24 h. Thereafter, development of blue color was observed. Citrate

positive showed that growth was visible on the slant surface and the medium became

an intense blue while citrate negative showed trace or no growth was visible and no

color change occurred.

3.7.4 Indole test

Test bacterial colony were inoculated in peptone water and incubated at 37 °C for 24-

28 h. Thereafter, 0.5 ml of Kovac’ s reagent was added. Positive test showed pink
29
colored ring was observed after addition of reagent. Negative test showed no color

change after reagent addition.

3.7.5 Motility test

The semi-solid agar of nutrient agar used for this study. The media was prepared in

slants and the organisms were inoculated by stabbing technique. Zig-zag growth along

the line of stabs indicated a positive result while none indicated a negative result.

3.7.6 Coagulase test

A drop of distilled water was placed on each end of a slide for each of the test

organisms. Thereafter a colony of each of the test organism was emulsified in each of

the drops to make two thick suspensions. A loopful of plasma was then-added to one

of the suspension and mixed gently for each of the test organism. Clumping within 10

seconds was an indication of positive test while none was an indication of a negative

test.

3.7.7 Sugar fermentation test

Each colony of the different test organisms were inoculated onto sterile agar slopes of

triple sugar iron agar using stab inoculation. After this, the inoculated, agar slopes

were incubated at 37 °C for 24 hours. The different colors of the slopes and butts in

addition to the presence of gas production and hydrogen sulphide (H2S) blackening

was an indication of the type of bacteria present.

30
3.9 Identification of the fungal isolates

The method described by Uddin et al. (2019) was adopted in the identification of the

fungal isolates. The fungal isolates were identified by morphological characteristics

on Sabouraud dextrose agar (SDA) and microscopic examination after lactophenol

cotton blue staining technique. Each of the fungal isolates were separately collected

with a sterile wooden stick and teased out on a drop of lactophenol cotton blue stain

on a clean glass slide. The wet mount preparations were then viewed under the

microscope for branched and unbranched hyphae.

31
 CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 RESULTS

Table 4.1 showed the proximate compositions of the fish feed used in this study. The

proximate compositions were; moisture (6.10% to 8.07%), ash (7.85% to 11.16%),

fats and oil (11.13% to 14.29%), protein (39.45% to 44.65%), fibre (3.01% to 4.99%)

and carbohydrate (20.26% to 27.70%).

32
Table 4.1: Proximate compositions of the fish feed samples

Samples/Sizes Proximate compositions/Results (%)

Moisture Ash Fats/oils Protein Fibre CHO

9 mm 6.10 7.85 11.13 39.45 5.01 30.46

6 mm 8.07 11.16 14.29 43.21 3.01 20.26

5 mm 7.24 9.64 13.22 40.48 4.62 24.80

4 mm 7.23 8.56 13.09 44.65 4.99 21.48

0.8 – 1.2 mm 8.00 10.64 13.09 44.32 3.44

20.51

Key: % = Percent CHO = Carbohydrate mm = Millimeter

33
Table 4.2 showed the microbial load of the fish feed samples used in this study. Total

viable bacterial counts ranged from 6.0 x 103 cfu/g to 3.4 x 104 cfu/g while total

fungal counts ranged from 1.0 x 103 cfu/g to 4.4 x 104 cfu/g.

34
Table 4.2: Microbial load of the fish feed samples

Samples/Sizes TVBC (cfu/g) TFC (cfu/g)

Blue crown

9 mm 1.0 x 104 1.0 x 104

6 mm 6.0 x 103 4.4 x 104

5 mm 1.0 x 104 4.0 x 103

Ecofort

6 mm 1.0 x 104 1.0 x 103

4 mm 3.4 x 104 1.2 x 104

5 mm 1.0 x 104 4.0 x 103

Coppens

0.8 mm – 1.2 mm 7.0 x 103 3.0 x 103

Key: mm = Millimeter TVBC = Total viable bacterial counts

TFC = Total fungal counts cfu/g = Colony forming unit per gram

35
Table 4.3 showed the cultural morphology and biochemical characteristics of the

bacterial isolates from the fish feed samples. They were; Staphylococcus, Micrococcus,

Bacillus and Corynebacterium species.

36
Table 4.2: Cultural morphology and biochemical characteristics of the bacterial isolates from the fish feed samples

Morphological Characteristics Gram reaction Oxidase test Indole test Spore test Catalase test Citrate test Coaguase test Motility test S FT
S B G H2S Possible bacteria

Milkish, flat, rhizoid-like Gram positive rods - - + + - - - Y Y + - Bacillus species

dry-surface colonies in short chains

Milkish, raised, Gram positive rod - - - + - - - No Reaction - - Corynebacterium species

non-mucoid colonies

Yellowish, raised, non- mucoid Gram positive cocci - - - + - + - No Reaction - - Staphylococcus species
colonies in clusters

Milkish, raised, non-mucoid Gram positive cocci - - - + - - - No Reaction - - Micrococcus species

circular colonies of about in pairs
3mm in size

Key: - = Negative + = Positive S = color of slope B = color of butt G = Gas production H2S = Hydrogen sulphide production (blackening) R = Reddish coloration (alkaline
production) Y= Yellow coloration (Acidic production) SFT= Sugar fermentation test

30
Table 4.4 showed the frequency and percentage occurrence of the bacterial isolates

from the different fish feed samples. Bacillus species had the highest occurrence

7(41.2%) followed by Micrococcus species 5(29.4%) while Staphylococcus species

had the least occurrence 2(11.8%).

31
Table 4.4: Frequency and percentage occurrence of the bacterial isolates from

the different fish feed samples

Bacterial isolates Frequency Percentage occurrence

Bacillus species 7 41.2

Corynebacterium species 3 17.6

Staphylococcus species 2 11.8

Micrococcus species 5 29.4

Total 17 100.0

32
Table 4.5 showed the cultural morphology and microscopic characteristics of the

fungal isolates from the different fish feed samples. They were; Aspergillus,

Penicillium, Mucor and Kluyveromyce species.

33
Table 4.6 showed the frequency and percentage occurrence of the fungal isolates from

the different fish feed samples. Kluyveromyces species had the highest occurrence

7(41.2%) followed by Mucor species 5(29.4%) while Penicillium species had the least

occurrence 2(11.8%).

34
Table 4.6: Frequency and percentage occurrence of the fungal isolates from the

different fish feed samples

Fungal isolates Frequency Percentage occurrence

Kluyveromyce species 7 41.2

Aspergillus species 3 17.6

Penicillium species 2 11.8

Mucor species 5 29.4

Total 17 100.0

35
4.2 DISCUSSION

Fish feed is the significant primary step in the aquaculture generation chain. This

study evaluated the proximate and microbial qualities of different fish feeds sold in

Owerri Metropolis, Imo State. Proximate compositions of the fish feed were; moisture

(6.10% to 8.07%), ash (7.85% to 11.16%), fats and oil (11.13% to 14.29%), protein

(39.45% to 44.65%), fibre (3.01% to 4.99%) and carbohydrate (20.26% to 27.70%).

Most of the results were slightly higher than the range of values of (6.87-8.10%)

reported by Ayuba and Iorkohol (2012), and (5.12 - 8.26%) reported by Modupe et al.

(2012). Dietary moisture level is anticipated to influence the texture and palatability of

fish feeds and therefore increased moisture level may enhance feeding and improve

growth performance.

Ash content of the selected fish feeds are in agreement with the findings of Ayuba and

Iorkohol, (2012), who recorded 5.33 - 9.45% ash content in some fish feeds. However,

Al-Mahmud et al. (2012) reported 14.79 -18.84%. The ash content obtained in this

study falls within the range of FAO recommendation of (˂14) (FAO, 2011). Ash

content determination is important because the amount of minerals determine physico-

chemical properties of feeds, as well as retards the growth of microorganisms. The

values of the lipid content of all the feeds are higher than the recommended range of

5.7 – 8.0 by FAO (2011). Hassan (2001) asserted that lipids are primarily included in

formulated diet to maximize their protein sparing effect. These lipids may also have

been incorporated into the feeds by the producers in order to enhance the energy levels

of the feeds.

36
Crude protein is one of the foremost critical supplements to evaluate in a prospective

feed due to the reality that it is one of the foremost expensive to supply and its

insufficiency can have an extreme impact on the growth performance of fish

(Mahaesar et al., 2010). The findings from this study are in line with the report of

Ayuba and Iorkohol (2012) who reported 43.75 - 52.65%, Al-Mahmud et al. (2012)

and Modupe et al. (2012); reported 51.32 - 65.34% and 57.20 - 62.61% crude protein

respectively. The high protein level could be a deliberate act by the producers in order

to ensure rapid growth performance of the fish.

The microbial load of the fish feed samples showed that total viable bacterial counts

ranged from 6.0 x 103 cfu/g to 3.4 x 104 cfu/g while total fungal counts ranged from

1.0 x 103 cfu/g to 4.4 x 104 cfu/g. This is in line with the findings of Otoikhian (2012)

who reported mean total viable count of fish and animal feeds to range from 1.0 x 104

cfu/g to 1.5 x 107 cfu/g. Similarly, Uddin et al. (2019) reported microbial load ranging

from 2.9 x 106 cfu/g, 3.3 x 105 cfu/g and 1.1 x 106 cfu/g with fish feeds sold in Dhaka

city, Bangladesh.

The bacterial isolates from the fish feed samples were; Staphylococcus, Micrococcus,

Bacillus and Corynebacterium species. Bacillus species had the highest occurrence

7(41.2%) followed by Micrococcus species 5(29.4%) while Staphylococcus species

had the least occurrence 2(11.8%). Otoikhian (2012) reported the isolation of

Salmonella species, Escherichia coli, Bacillus species and Staphylococcus species

from animal and fish feeds. In another study, Uddin et al. (2019) reported microbial

37
the isolation of Vibrio, Salmonella, Pseudomonas, Shigella species, Escherichia coli

and Staphylococcus aureus from fish feeds sold in Dhaka city, Bangladesh.

The fungal isolates from the different fish feed samples were; Aspergillus, Penicillium,

Mucor and Kluyveromyce species. Kluyveromyces species had the highest occurrence

7(41.2%) followed by Mucor species 5(29.4%) while Penicillium species had the least

occurrence 2(11.8%). The presence of these organisms in fish feeds may also arise

from poor hygienic and sanitary practices employed in the manufacturing, processing

and packaging of fish feed. Therefore, there should be proper storage conditions and

hygienic handling during storage so as to reduce microbial contamination of fish feeds.

38
 CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATIONS

5.1 CONCLUSION

The result of the proximate compositions of the different fish feed samples showed

that most of the fish feeds had nutrients within the acceptable limit for growth and

performance of fish. The microbial quality evaluation revealed the presence of

opportunistic pathogens which could affect the health and quality of fish fed with the

fish feeds. Considering the importance of fish feed in aquaculture, government should

enforce strict compliance and further studies need to be carried out to assess other

feeds with comparable rate of growth performance and digestibility.

39
5.2 RECOMMENDATIONS

1. Since fish feeds are important part of aquaculture, fish feed sellers should

handle them with care to avoid contaminations.

2. Government should enforce strict compliance and further studies need to be

carried out to assess other feeds with comparable rate of growth performance

and digestibility.

3. There should be proper storage conditions and hygienic handling during

storage so as to reduce microbial contamination of fish feeds

40
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