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MICROBIAL PROFILE OF SUYA MEAT IN ENUGU STATE

IBE ROSEMARY NKECHINYERE

MB/2006/154

DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY

CARITAS UNIVERSITY

AMORJI NIKE ENUGU

AUGUST 2010.

MICROBIAL PROFILE OF SUYA MEAT IN ENUGU STATE

IBE ROSEMARY NKECHINYERE

MB/2006/154

A PROJECT SUBMITTED TO THE

DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY, FACULTY OF NATURAL SCIENCES,

CARITAS UNIVERSITY,

AMORJI –NIKE, ENUGU, ENUGU STATE.

IN PARTAL FULFILMENT OF THE REQUIREMENT OF THE AWARD OF BACHELOR OF SCIENCE (B.SC), DEREE IN MICROBIOLOGY AND BIOTECHNOLOGY.

AUGUST, 2010.

DEDICATION

I dedicate this work to God Almighty and to our Mother Mary for the grace and strength given to me to carry out this work successfully.

CERTIFICATION

This is to certify that the project title “Microbial Profile of Suya Meat,” was carried out by Ibe Rosemary Nkechinyere under the supervision of Ms Nmema E.E. and it is accepted in partial fulfillment of the Bachelor of Science (B.Sc) degree in of Microbiology and Biotechnology. The department recognizes that Ibe Rosemary Nkechinyere (MB/2006/154) bear full responsibility for the content of this work.

________________ _____________

Name and signature Date

________________ _____________

Miss Nmema E.E. Date

(Supervisor)

________________ _____________

Mr Amadi E.C. Date

(H.O.D.)

________________ _____________

Prof. Nduka Okafor Date

(Dean of Natural science)

___________________ ____________

External Examiner Date

ACKNOWLEDGEMENT

My profound gratitude goes to God the father, who has been a father to me; the son who has been a true friend and to the Holy Spirit who has been a brother and my companion throughout my years as an undergraduate.

My special thanks goes to my project supervisor Ms Nmema E.E., whose untiring support, suggestion, and guidance has led me this far and made this research writing worthwhile.

I am particularly grateful to Prof. Nduka Okafor (Dean of Natural Science), Mr Amadi E.C.(H.O.D) and to all my lecturers who have done a tremendous work in my academic pursuit, I pray that God Almighty will bless you all in million fold.

My sincere gratitude also goes to all the members of my family; Mr and Mrs Ibe who worked so hard for me both morally and financially, I pray that God will give them long lives and good health to enjoy the fruit of their labour. Also to my siblings especially to my dearest elder sister Mrs Ibeh Kelechi whose encouragement, financial support, and love have kept me going throughout my stay in school; and to my lovely brother Ikechukwu and sister’s Chika, Ogechi, Chiamaka and chichi. I will not forget my relatives’ aunty Elizabeth aunty Susan aunty Ann and to others, I want to say a big thank you for always being there for me, I love you all.

My appreciations also goes to my darling friends who have always being there to pray, finance and encourage me, when I feel that all hope was lost; they are Juliet, brother Christian, Everistus Gabriel, Charles Oche, sister Happiness, Chioma, Blessing, brother Kenneth Titilope,Nonye, Bolanle,to mention but a few.

I will not forget to thank myself for typing a beautiful work and all who assisted and corrected me in one the course of my typing.

ABSTRACT

Ten (10) samples of suya meat in Enugu were collected randomly and analyzed microbiologically and the isolates were identified as Staphylococcus aureus (35%) Escherichia coli (15%) Streptococcus species (15%) Pseudomanas (35%) . The most frequently isolated organism was Staphylococcus and Pseudomonas. The total viable bacterial counts ranged from 1.9x 3.8x cfu/g whereas, total coliform count ranged 1.1x -3.0x cfu/g on MacConkey and Nutrient agar respectively. The result revealed that the hygienic condition of the meat have fallen below acceptable standard for human comsumption.

LIST OF TABLES

TABLE 1: Total viable and coliform counts ( )

TABLE 2: Characterization /identification of Isolate

TABLE 3: Frequency of occurrence of isolates

TABLE OF CONTENTS

Certification i

Dedication ii

Acknowledgement iii

Abstract

Tableofcontents iv

List of Tables v

CHAPTER ONE

1.0 Introduction 1

1.1 Objectives 4

CHAPTER TWO

2.0 Literature review 5

2.1Suya meat 5

2.2 Preparation of suya 5

2.3Preparation of meat

2.4 Meat spoilage

2.5 Factors that affect the growth of microorganisms in meat

2.5.1 Temperature

2.5.2 pH

2.5.3. Water availability

2.5.4 Nutrients

CHAPTER THREE

3.0 Materials and methods

3.1 List of reagents

3.2 List of glassware

3.3 Preparation of media

3.4 Sample collection

3.5 Preparations of samples

3.6 Determination of total viable counts

3.7 Characterization and Identification of Isolates

3.7.1 Gram reaction

3.7.2 Motility test

3.7.3 Catalase test

3.7.4 Coagulase test

3.7.5 Oxidase test

3.7.6 Urease test

3.7.7 Citrate test

3.7.8 Vogues Proskauer test

3.7.9 Indole test

3.7.10 Carbohydrate fermentation test

3.7.11 Methyl red test

CHAPTER FOUR

4.0 Results

CHAPTER FIVE

5.1 Discussion

5.2 Summary and Conclusion

5.3 Recommendations

References

Appendix

LIST OF TABLES

TABLE 1: Total viable and coliform counts ( )

TABLE 2: Characterization /identification of Isolate

TABLE 3: Frequency of occurrence of isolates

CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGRONUD OF THE STUDY

Meat is the flesh of animals which serves as food; it is obtained from sheep, cattle, goat and swine (Hamman, 1997). Meat is a major source of protein and have valuable qualities of vitamins for most people in many parts of the world, thus they are essential for the growth, repair and maintenance of body cells which is necessary for our everyday activities.

Meat could be traced back to human history, then when primitive men use raw flesh of dead animals, but as man developed, he domesticated as well as wild animals. Beef have been the major supply of meat in Nigeria as a result of extensive and semi-intensive cattle production system in Nigeria by Fulani and Hausa people of the northern Nigeria. (Umoh, 2004).

Suya meat is a boneless lean meat of mutton, beef, goat or chicken meat staked on sticks, coated with its sauces, oiled and then roasted over wood using a fire from charcoal. It is a popular, traditionally processed meat product that is served hot and sold along streets, at clubs, picnics centers, and restaurants and within institutions. Suya meat is one of the intermediate moisture products that are easy to prepare and highly relish which is a mass consuming fast food and its preparation and sales are usually not done with strict hygiene condition because they are still done locally.

Due to the chemical composition and characteristic, meat are highly perishable food which provides excellent source of growth of many hazardous microorganisms that can cause infection in human and also lead to meat spoilage and economic loss. The most important bacteria meat spoilage is caused by lactic acid bacteria which is physiologically related group of fastidious and ubiquitous gram-positive organisms, these includes many species such as Lactobacillus, Leuconostoc, Pediococcus and Streptococcus.

Since meat has a high nutritive value, microorganisms could easily grow on it. The possible sources of contamination are through slaughtering of sick animals, washing the meat with dirty water, handling by butchers, contamination by flies, processing close to sewage or refuse dumps environment, spices, transportation and use of contaminated equipment such as knife and other utensils. (Igyor and Uma, 2005).

The slaughtering process affords extensive contamination of sterile tissue with gram-negative enteric bacteria from animal intestine including Salmonella specie and Escherichia coli as well as contaminant such as gram-positive Lactic cocci associated with humans, animals and the environment. Enterococci and Clostridia have been isolated from lymph node of red meat animals (Lawries, 2000, Alexander et. al. 1998).

Microorganisms grow on meat causing visual, textural and organoleptic changes when they release metabolite (Jackson et. al. 2001). The smoke produced as a number of effects including preservative effect resulting from the deposition of organic compounds all presents in the smoked product (Suya meat). (Dineen et.al.1999). A preservative effect is also induced by the surface drying that occurs to the extent of 30% total weight loss in hot smoked product. Antioxidant effect is produced by the phenolic deposite unto the product.

The microbial load in meat and meat product increases as long as growth conditions are favorable. The factor influencing microbial growth includes acidity pH, temperature, water activity, gaseous requirement, nutrient and competition of microbes for the nutrient. Controlling these factors implies maintaining long shelf life of meat and meat product but proper preservation of meat could be achieved by the combination of two or more preservation method which includes drying, salting and high temperature (Nester e, al 2001).

1.2 AIMS AND OBJECTIVES

This work is aimed at determining the microbial quality of suya meat sold in Enugu and has the following objectives:

1. To isolate, characterize and identify microbial species associated with Suya meat.

2. To establish the public health implication of consumption of Suya meat.

3. To offer useful information where necessary to the consuming public.

CHAPTER TWO

LITERARURE REVIEW

2.1 SUYA MEAT

Suya meat is a traditional stick meat product that is commonly produced by the Hausas in West Africa from beef, although chicken can also be used. It is produced from boneless meat hung on stick and spiced with peanut, cake, salt, vegetable oil and other flavor followed by roasting around a glowing charcoal fire. The smoke from the fire has a preservative effect on the Suya meat (Ogunbanwo et. al. 2004).

2.2 PREPARATION OF SUYA

Usually in Nigeria, skinless, boneless flesh of cattle or chicken is used for commercial preparation of suya meat. Finely grounded roasted peanut cake, red pepper, salt, grounded ginger, grounded garlic, chunked fresh tomatoes and minced fresh onions are required on suitable conditions depending on the quality of suya meat required to be prepared (Jay, 2000; Judge et. al.2000).

The process of preparation involves a few steps, first is the grounding of peanut. The shell and the skin are removed from the peanut before grinding into fine powder using mortar and pestle or crushed with a rolling pin. If the powder is oily; it is wrapped with an absorbent paper and squeezed for a minute or two. Next, the grinded pepper, garlic, ginger are stirred into the obtained peanut powder and mixed properly.

The meat is then cut into bit sizes or thin sliced, dipped and rolled in a bowl containing the mixed peanut-spice and allowed to coat completely. The minced or mutton meat are then kept for thirty minute or more for the peanut cake to stick to it after which the meat slices are threaded unto skewer and brushed with vegetable oil and roasted on the glowing charcoal fire for fifteen to twenty minute. It is finally removed from the skewer and served hot in a newspaper with sliced onions, tomatoes and cabbage (Judge et. al.2000).

2.3 MICROBIOLOGY OF MEAT.

Microorganisms destroy the inherent defense mechanism of the animal thereby subjecting the tissue to rapid decay as a result of its action (Lidwell et. al. 1996).

During slaughtering process, there is contamination of the sterile tissue with intestinal flora like gram-negative organisms which includes Escherichia coli as well as contaminants such as Pseudomonas specie and gram-positive lactic acid bacteria and Staphylococci specie associated with human, animals and their environment. Meat spoilage is usually associated with gram-negative Proteolitic bacteria which literally decompose the protein with production of offensive odour (Hamman, 1997).

The addition of salt and drying of fresh meat have been an effective means to control the meat micro flora and thus preserve the tissue for later consumption. The curing salt (sodium chloride, sodium nitrate and sodium nitrite) and subsequent proper handling methods, favours the growth of gram-positive bacteria, primarily Staphylococcus aureus while inhibiting the proliferation of gram-negative bacteria (Boles et. al. 2000).

There are also other types of common microorganisms apart from enteric organisms found in meat which are members of Micrococcaceae and Staphylococcaceae families. The predominant types are coagulase-negative Stapylococci that are salt tolerant and can also grow with or without oxygen. The most common strain belongs to the species of Stapylococcus carnosus, S. xylosus and S. kocuria variance by far. However, these organisms are harmless and do not present a microbial hazard. The most common lactic acid microorganisms found in fermented meat of various strains of Lactobacilli, Leuconostoc, Pediococci, Streptococci and Enterotocci (Lawries, 2001).

Bacillus species, Staphylococcus aureus, Staphylococcus epidermidis, Proteus species, Serratia species and Aspergillus species were isolated from suya meat samples collected from Enugu state (Chukwura and Mojekwu, 2002).

2.4 MEAT SPOILAGE

Meat is spoilt when it loses its nutritive value, texture and brings out offensive odour thus rendering it unfit for human consumption. A number of factors could cause meat spoilage (Nester et .al.2001).

Most deterioration or spoilage of meat could be caused by bacteria, yeast and mould. When meat is not properly handled, it leads to spoilage (microbial spoilage which makes it unfit for human consumption) (Sokari et. al. 1999).

2.5 FACTORS THAT AFFECTS THE GROWTH OF MICROORGANISMS ON MEAT.

A lot of factors affect the growth of microorganisms on meat. This factors includes temperature, pH, water availability, presence of nutrients, moisture, acidity (intrinsic factors), gaseous requirement, atmosphere of storage(extrinsic factors) (Nester et al; 2001).

2.5.1 TEMPERATURE

Microorganisms have optimum, minimum and maximum temperature that they can grow. Listeria monocytogenes have been found to grow at C and even survive freezing. This ability of growing at low temperature provides opportunity for proliferation in contaminated meat products (Fraizer and Westhoff, 2000).Pseudomonas specie grow at temperature less than 20 and so are found.(Dineen et al;1999).

Psychrophiles have temperature optimal between C and C mesophiles between C and C and thermophile from C or C (Fararatti, 2000).

Examples of psychrophiles are

1. Achromobacter

2. Alcaligens

3. Pseudomonas

4. Streptococcus

5. Salmonella

6. Most yeast

7. Mould.

Examples of mesophiles are Bacillus strearothermophiles (Evans and Niren, 1980).

2.5.2 pH

Most bacteria grow optimally at above pH 7 and not well below pH 4 or above pH 9 (Abdul Raouf et al; 1995). But the pH of maximal growth is determined by the simultaneous operation of variables other than the degree of acidity or alkalinity itself proteolitic enzyme operate best near neutral pH 7.

2.5.3 WATER AVAILABILITY

Water is required by microorganism so reducing water below the optimum level which prolong shelf life of meat. If meat is stored at a relative humidity below 95% moisture will be lost from the surface. Since most spoilage bacteria can grow only on the surface, drying the meat. Surface will not favor their growth but staphylococcus aureus can grow in meat with 0.86 which is lower than that of other spoilage bacteria (Nester et al; 2001). Moulds are able to grow in drier conditions than bacteria so that desiccation has a selective effect on microbial growth. Xerphilic fungi have been found to grow at a low temperature 0.6 (Evans and Niren, 1999).

2.5.4 NUTRIENTS

Microorganisms depend on nutrients from meat product or meat for survival. Meat contains protein, phosphorus, fat and vitamins, which support the growth of microorganism. Pseudomonas aeruginosa synthesize it vitamins and so cause spoilage even in a medium without vitamin. Staphylococcus aureus require about 6.5% of sodium chloride for growth and is usually found in salty meat product (Boles et al; 2000).

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 MATERIALS

3.1.1 CHEMICAL AND REAGENTS

(1) Crystal violet

(2) Iodine

(3)Acetone (alcohol)

(4)Safranine

(5) Oil immersion

(6) Normal saline

(7) Hydrogen peroxide

(8) Distilled water

(9)Oxidase reagent

(10) Christensen’s urea broth

(11) Simon’s citrate agar

(12) Acetymythyl carbinol

(13) Buttered glucose broth

(14) Nepthol

(15) Sodium hydroxide

(16) Glucose phosphate peptone

(17) Methyl red

(18) Peptone water

(19) Tryptophan

(20) Kovac’s reagent

(21) Sugar solution

(22) Glucose

(23) Sucrose

(24) Lactose

(25) Mannitol

3.1.2 GLASS WARES AND EQUIPMENTS

(1) Cover slip

(2) Glass slide

(3) Rack

(4) Glass rod

(5) Filter paper

(6) Bijou bottle

(7) Wire loop

(8) Incubator

(9) Test tube

(10) Durham tube

(11) Autoclave

(12) Bunsen burner

(13) Weighing balance

(14) Pasteur’s pipette

(15) Sieve

(16) Petri-dishes

(17) Conical flasks

(18) Mortal pestle

3.2 SAMPLES COLLECTION

Twenty skewers of suya meat were obtained randomly from suya vendors at popular suya spots in Enugu. The samples were immediately wrapped in sterile aluminum foil to prevent contamination and then transported to the laboratory for microbial analysis without delay.

3.3 PRETREATMENT OF SAMPLES

A suya piece from each sample was removed from the skewers, and mashed in a sterile laboratory type mortar and pestle. 1g of the mashed suya meat was weighed and then aseptically introduced into 9ml of sterile distilled water, properly shaken and sieved before a twofold dilution was performed.

3.4 DETERMINATION OF TOTAL VIABLE COUNT AND COLIFORM COUNT

A two -fold serial dilution was made for the suya meat samples in appropriate dilution tubes. The media of choice are MacConkey agar and nutrient agar. The MacConkey agar is a differential medium used in the differentiation of lactose fermenters from non lactose fermenters. 1ml of each dilution was pipetted and plated on nutrient agar and MacConkey agar using the spread method. Incubation was c for 24hours.Developed colonies were counted to obtain total viable count and coliform counts respectively. Discrete colonies were purified by subculturing into nutrient agar plate and were subsequently identified using standard methods. (Bichanan and Gibbo, 1974,).

3.5 PROCEDURE FOR CULTURING THE PLATE

The samples were cultured aseptically with wire loop on the prepared plates i.e. MacConkey and Nutrient agar plates and incubated at 37 c between 18hour and 24hours. Then, the plates were read for growth of organisms.

3.6 PROCEDURE FOR IDENTIFICATION OF THE ORGANISMS

The isolates were characterized and identified based on their cultural characteristic and biochemical reaction as follows:

3.6.1 GRAM REACTION

It was carried out to differentiate gram position from gram-negative organisms. Staphylococcus aureus and Escherichia coli were used as control organisms.

METHOD:

A wire loop was sterilized in Bunsen burner and allowed to cool then a loopful of growth was collected from the agar plate and apply on a clean grease free slide then a drop of normal saline added, emulsfied and heat fixed by passing over a flame three times. The smear was flooded with crystal violet for 30-60seconds and then covered with iodine for 30-60secods and then washed off; it was decolorized with acetone until no colour runs off the slide and rinsed immediately. The slide was covered with safranin for 1minute and then washed off with clean water. The slide was kept in a rack to air dry after wiping the back with cotton wool.

The smear was then examined microscopically under oil immersion at 40x objective lens. Gram –positive bacteria appeared dark purple while gram-negative bacteria appeared red.

3.6.2 MOTILITY TEST

Motility test was aimed at identifying motile bacteria.

METHODS

A drop of normal saline was placed on a sterile slide and colony of test organism was suspended and emulsified and then covered with a cover slip. The slide was examined microscopically using 10x and 40x objective lens, movement in different directions gave a positive test.

3.6.3 CATALASE TEST

This was used to differentiate those bacteria that produce enzyme catalase from those that do not. Staphylococcus aureus and Escherichia coli were positive and negative controls respectively.

METHOD

Three milliliters (3ml) of hydrogen peroxide solution was poured into a sterile test tube, then a sterile glass rod was used to collect several colonies of the test organisms and inoculate in the hydrogen peroxide solution. It was observed for immediate active bubbling for positive test.

3.6.4 COAGULASE TEST

This was used to identify Staphylococcus aureus which produces the coagulase enzyme which cause plasma to clot by converting fibrinogen to fibrin. The slide method was used.

METHOD

A drop of sterile distilled water was placed on each end of a sterile slide. Then a colony of the test organism was emulsified on each spot to make two thick suspensions. A loopful of plasma was added to one of the suspensions and mixed gently. The slide was checked for clumping or cloting of the organisms within 10seconds. Plasma is not added to the second suspension which serves as control.

3.6.5 OXIDASE TEST

This was carried out to identify bacterial species that will produce the cytochromeoxidase enzyme. Pseudomonas aeruginosa and Escherichia coli were employed as positive and negative controls respectively.

METHOD:

A piece of filer paper was placed in a clean Petri dishes and 2-3 drops of fresh or nascent oxidase reagent was added. A colony of test organism was collected using a glass rod and smeared on the filter paper and observed. Blue-purple color within few seconds showed a positive test.

3.6.6 UREASE TEST

This test was aimed at identifying Enterobacteria that produce urease enzyme, which hydrolyze urea to give ammonia and carbondioxide. Proteus and Salmonella were used as control positively and negatively respectively.

METHOD:

The test organism was heavily inoculated onto Christensens urea broth in bijou bottle using sterile wire loop and incubated at 35 C- C for 3-24hours and examined, thereafter a pink color in the medium showed positive test.

3.6.7 CITRATE TEST

This test is based on the ability of an organism to use citrate as it source of carbon. It was used to identify the Enterobacteria.

METHOD:

Simon’s citrate agar medium was prepared in a slant bijou bottles, then a sterile wire loop was used to inoculate the test organism onto the slant medium and incubated at C for 48hours after which it was examined for color formation. A bright blue color in the medium gave a positive citrate test. Klebsiella pneumonia and Escherichia coli were employed as positive and negative controls respectively.

3.6.8 VOGUES PROSKAUER TEST

This test was used to identify members of the Enterobactiaceae that produce acetymythylcarbinol (acetone) a natural product formed from pyruvic acid in the course of glucose fermentation.

METHOD:

Buffered glucose broth was inoculated with the test organism and incubated at C for 3days. Three milliliters (3ml) of nephtol was then added followed by 3ml of sodium hydroxide solution, mixed well and allowed to stand for 1hour at room temperature. The formation of a pink color in the medium within 1hour indicates a positive result. Klebsiella pneumonia and Escherichia coli were used as positive and negative controls respectively.

3.6.9 INDOLE TEST

This test was carried out for indole production by test organism which is important in identifying enterobacteria.

METHOD:

A sterile wire loop was used to inoculate a colony of test organism into 2ml of peptone water containing tryptophan. The tube was stoppered and incubated at 35⁰C -37⁰C for 24hours, after which kovac’s reagent was added to the medium. Observation of red coloration on the surface layer within 10minutes showed a positive result.

3.6.10 CARBOHYDRATE FERMENTATION TEST

This test is used to determine the ability of bacteria to utilize different sugars. Examples are mannitol, glucose, lactose and sucrose.

METHOD:

The four sugar solutions were prepared and poured into test tubes well stopped with Durham tube for gas collection. The sugar was autoclaved after which a loopful of test organisms was introduced into the sugar solution (Buchana, R. E., and Caibbons N. E. 1994). A change in color from pink to yellow shows fermentation and collection of gas bubbles in the Durham tube shows gas production of positive test. A control was set up without the organism inoculated.

3.6.11 METHYL RED TEST

This was carried out to identify Enterobacteria based on the ability to produce and maintain stable acid end product from glucose fermentation Escherichia coli was used as positive control.

METHOD

Glucose phosphate peptone water was used for inoculation of test organisms and incubated for 48 hours at c after which few drops of methyl red solution was added to the culture and read immediately. Formation of red color immediately showed a positive test.

CHAPTER FOUR

4.0 RESULTS

Suya samples collected randomly were carefully analyzed, the charaterization and identification result is presented on table 2. The isolates were identified as Staphylococcus 35%, Pseudomonas 35%, Streptococcus 15%, and Escherichia coli 15% The most frequently isolated organism was Staphylococcus and Pseudomonas species. The total viable count ranged from 1.9x – 3.8x . whereas total coliform count ranged from 1.1x -3.0x as shown in table 1.

TABLE 1: TOTAL VIABLE AND COLIFORM COUNTS (10³ CFU/G).

S/N	MAcCONKEY AGAR (coliform x10³)	NUTRIENTAGAR (total viable countx10³)
1	1.1	1.9
2	1.2	2.0
3	1.3	2.1
4	1.4	2.2
5	1.5	2.3
6	2.0	2.4
7	2.3	2.5
8	2.6	3.2
9	2.9	3.4
10	3.0	3.8

TABLE 4: CHARACTERISATION/IDENTIFICATION OF ISOLATES

ISOLATE

GRAM REACTION

CELLULAR ARRANGEMENT

IND

C-T

V-P

MOT

MAN

CAT

LAC

GLU

SUC

PROBABLE ORGANISM

S₁

+VE

-VE

+VE

A/G

Staphylococcus

S₂

+VE

Cocci in Chains

-VE

Streptococcus

S₃

-VE

+VE

-VE

+VE

-VE

+VE

-VE

+VE

-VE

+VE

A/G

E. coli

S₄

-VE

Rods

-VE

+VE

-VE

+VE

-VE

+VE

-VE

Pseudomonas

S₅

-VE

+VE

-VE

+VE

-VE

+VE

-VE

+VE

A/G

E. coli

S₆

+VE

-VE

+VE

A/G

Staphylococcus

S₇

-VE

Rods

-VE

+VE

-VE

+VE

-VE

Pseudomonas

S₈

+VE

-VE

+VE

A/G

Staphylococcus

S₉

-VE

Rods

+VE

-VE

+VE

-VE

+VE

-VE

Pseudomonas

S₁₀

+VE

Cocci in Chains

-VE

Streptococcus

S₁₁

-VE

+VE

A/G

-VE

E. coli

S₁₂

+VE

-VE

+VE

A/G

Staphylococcus

S₁₃

+VE

Cocci in Chains

-VE

Streptococcus

S₁₄

-VE

Rods

-VE

+VE

-VE

+VE

-VE

Pseudomonas

S₁₅

+VE

-VE

+VE

A/G

Staphylococcus

S₁₆

-VE

Rods

-VE

+VE

A/G

Pseudomonas

S₁₇

-VE

Rods

-VE

+VE

-VE

Pseudomonas

S₁₈

-VE

+VE

-VE

+VE

-VE

+VE

-VE

+VE

A/G

E. coli

S₁₉

+VE

Cocci in Chains

-VE

Streptococcus

S₂₀

+VE

-VE

+VE

A/G

Staphylococcus

KEY

+ positive

Negative

NR Not relevant

D Different strain given different result

CT → Citrate test

VP → Voges proskauer

OX→ Oxidase

CAT→ Catalase

MOT→ Motility

MR →Methyl red

MAN→ Mannitol

SUC→ Sucrose

LAC →Lactose

A/G →Acid and gas

GLU→Glucose

4.3 TABLE 3 FREQENCY OF OCCURRENCE OF ISOLATE

ISOTATE FREQENCE PERCENTAGE%

Staphylococcus 6 35

Escherichia coli 4 16

Pseudomonas 6 35

Streptococcus 4 15

Total 20 100

CHAPTER FIVE

5.0 DISCUSSION, SUMMARY, CONCLUSION AND RECOMMENDATIONS.

5.1 DISCUSSION

Meat basically contains all the nutrients necessary for microbial growth and metabolism, making it susceptible to microbial contamination. In view of the material quality of meat and meat products must be ascertained to ensure safety from infection after consumption of such products and to promote quality assurance.

Laboratory analysis carried out on suya meat was collected randomly suya vendors in Enugu, Enugu state. Some microorganisms were isolated, in which the result was in consonance with the literature of Chukwura and Majekwu(2002) which stated microbiological analysis of meat samples in Awka urban Anambra state, indicated contaminated of meat sample with various bacteria species including Staphylococcus aureus, and some enteric bacteria Gilbert Harrison(2001)also affirm that meat contains certain amount of salt by so permit the growth of Staphylococcus aureus whereas, the presence of some members of the enteric bacteriacea family is due to contamination from intestine slaughtered animals.

Four organisms were isolated from the suya sample in view of the of the unhygienic condition of meat handling in Nigeria, the organisms isolated could always be suspected in connection with meat contamination and spoilage. The organisms include Staphylococcus specie, Streptococcus specie, Escherichia coli Pseudomonas specie.

The presence of Staphylococcus specie affirms the cross contamination through processing (Gilbert and Harisson 2001).Since it is normal flora of the skin. Raw meat is usually carried on the body by butcher in Nigeria due to lack of education (Dada et.al; 1993). Confirmed that coliform often results from the water used for washing the meat which of course is always contaminated.

Also presence of Escherichia coli probably may arose from the use of non –portable water during washing of raw meat (Umor,2004).

The meat also showed presence of pseudomonas aeruginosa, which usually occurs around soil, vegetation and even surface (Field, 2002).

On the whole, the critical points of contamination of suya meat are roasting, handling and reheating. So control contamination of contamination can be achieved if aseptic techniques of suya preparation process are observed.

5.2 SUMMARY AND CONCLUSION

Suya meat constitutes a great source of protein which is needed for body building and repair of worn out tissue in human. Advances in the microbial quality of suya meat is very important and adequate steps must be taken to prevent contamination and spoilage by microorganisms.

The organisms isolated from the suya meat indicate that the standards of preparation and preservation have not improved much over the years and facilities used for preparation are not sterile. Aseptic techniques are not adequately employed in the meat industries as so to reduce microbial load of meat and its products for safety consumption by consumers and thus prevent food-borne diseases or infections.

5.3 RECOMMENDATIONS

Quality control unit should established in meat processing industries in Nigeria and Hazard Critical Control Point ( HACCP) concept should be applicable to the processing and renderings.

REFERENCES

Abdul, U.M., Beuchat, C.R., and Ammar, M. S. (1993).Survival and growth of Escherichia coli in ground roast beef as affected by pH, acidulates and temperature. Journal of applied and environmental microbiology 59(8): 2364-2368.

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APPENDIX 1

Preparation of Media and Reagents

The following media were used

Nutrient Agar (NA)

The medium was used for the enumeration of bacteria cells and so to maintain pure. Nutrient agar is a general medium. It was therefore, used here on the assumption that as many organism as are on the samples will grow. COMPOSITION

Nutrient Agar Gram per. Litre

Lab. Lemco powder 1.0

Yeast extract 2.0

Sodium chloride 15.0

Agar 5.0

Pepton 5.0

pH 7.4

Twenty three grams of nutrient agar powder in one liter of distilled water contained in a sterile conical flask and plug the mouth of the flask with non absorbent curtain cover neck of flask with foil paper and tight firmly with a rope at the neck of the flask. Then I mixed by shaking it and bring it boil to dissolve completely and autoclave at c for 15mins, was allowed to cool at 45 and mixed well before dispense aseptically in 20ml volume into Petri dishes. The medium was allowed to solidify on these plates and were used thereafter.

MACCONKEY AGAR

This medium was used primarily to differentiate Lactose fermenters from non lactose fermenters and also to suppress swaming activity of proteus and other spreading organisms.

COMPOSITION

MacConkey agar Gram. per litre

Pepton 20.0

Lactose 10.0

Bile salts 5.0

Sodium chloride 5.0

Neutral red 0.075

Agar 12.0

Distilled water 100ml

PH 7.6

PREPARATION

Twenty eight grams of MacConkey agar powder was weighed out in one liter of distilled water contained in a sterile conical flask and plug the mouth of the flask with non absorbent curtain cover neck of flask with foil paper and tight firmly with a rope at the neck of the flask then mixed by shaking it and bring it boil to dissolve completely and then autoclave at c for 15mins, was allowed to cool at 45 and mixed well before dispense aseptically in 20ml volume into Petri dishes. Prior to incubation, the surface of the agar was dried by partial exposure at 37 . The appearance of the plate was clear pink/red. The media was preserved in the fridge.

(b) Nutrient agar is used at a concentration of 2.8g in every 100ml of distilled water.

Christensens urea broth

Urea broth base 95ml

Steril urea solution40% w/v 5ml

Christensens is used test if an organism is positive or negativeusing bacteria species.

Simon Citrate Agar (SCA)

This medium was used for the differentiation of Enterobacteriaceae based on the utilization of citrate as the sole source of carbon.

COMPOSITION

Magnesium sulphate 0.2g

Sodium ammonium sulphate 0.8g

Ammonium dihydrogen sulphate 0.2g

Sodium citrate tribasic 2.9g

Sodium chloride 5.0g

Bromothy molblue 0.08g

Agar 15g

Distilled water 1000ml

pH 6.9

PREPERATION

The powered urea agar was used and was prepared as directed by the manufacturer. 2.4g of urea agar was suspended in 95ml of distilled water and was dissolved by boiling. The medium was sterilized by autoclaving at 115 C for20minutes. The medium was cooled to about 50 C and 40% w/v sterie urea solution was added. The medium was dispensed into bijou bottle and were allowed to solidify in slant position. They were therefore used.

APPENDIX 2

Preparation of reagents.

(1)Methyl red solution

To make 50ml

Methyl red (pH indicator) 0.05g

Ethanol (ethyl alcohol) absolute 28ml

Distilled water 22ml

(a) The methyl red was weighed on a piece of paper (pre weighed) dissolved in ethanol and water.

(b) It was transferred to a clean brown bottle and the bottle labeled.

(1) Crystal violet gram stain

To make 1litre

Crystal violet 20g

Ammonium chloride 9g

Ethanol or methanol absolute 95ml

Distilled water 1litre

(2) Peptone water

To make 65bottle

Peptone 2g

Sodium chloride 1g

Distilled water 200ml

pH 7.6

(a) The peptone water and salt was dissolved water and dispense in 3ml amounts in screw cap bottles (Bijou bottle).

(b) It sterilize by autoclaving (with caps loosened) at 121 c for 15 minutes and allowed to cool with the cap tighten and the bottle was labeled.

Oxidase Reagent

To make 10ml:

Tetramethyl-p- phenylenediamine 0.1g

Dihydrochloride

Distilled water 10ml

The chemical was dissolved in distilled water and was used immediately.

Acetone–alcohol decolorizer:

To make 1 litre:

Acetone 500ml

Ethanol or methanol,absolute 475ml

Distilled water 25ml

(a) The distilled water was mixed with ethanol and was transferred into a srew cap bottle of 1 litre capacity.

The acetone was measured and added immediately to the alcohol solution and mixed well.

(b) The bottle was labeled and indicated highly inflammable then store in a safe place at room temperature.

Glucose phosphate peptone water:

To make about 50 bottles :

Peptone 0.5g

Glucose 0.5g

di-potassium hydrogen phosphate 0.5g

distilled water 100ml

The peptone and phosphate salt were dissolved in water by steaming,and allowed to cool, filtered and the pH was adjusted to 7.5.

(a) The glucose was added, mixed well and dispensed in 2ml amounts in small screw -cap tubes or bottles .

(b) It was sterilize by autoclaving (with cap loosened) at 11 C for 10minutes and allowed to cool,then tighten the container tops and label.

Thursday, March 16, 2017

MICROBIAL PROFILE OF SUYA MEAT IN ENUGU STATE