ABSTRACT
Analysis of proximate composition, mineral composition, heavy metals concentration, microbiological counts and identification of microbial contaminants were carried out in beef and Kilishi samples that were produced and collected from six production spots in Kaduna state, Nigeria viz: Sabon Gari, Tudun Wada, Zaria city, Malali, Anguwan Sarki and Kasuwa. Also, Kilishi that was prepared in the Laboratory that served as control was analyzed. Results of proximate composition showed that Kilishi is richer in nutrients. It showed that the profile proximate parameters in the study samples is protein > crude fat > ash > moisture > carbohydrate > crude fibre. The highest protein content found was in dried beef. From the study samples, Kasuwa Kilishi had the highest protein content (64.28%), Malali Kilishi had the highest moisture and fat contents (9.25 and 22.42% respectively), while Anguwan Sarki Kilishi had the highest ash content (9.42%). Also, Tudun Wada Kilishi had the highest crude fibre content (3.42%), Zaria City Kilishi had the highest carbohydrate content (8.32%), while the Laboratory Kilishi that served as control recorded the lowest percent moisture (7.22%) and had lower values of protein and carbohydrate. Results of mineral composition showed that Kilishi contained the highest contents of Na, P and K while the concentrations of Ca and Mg found were highest in dried beef. Kasuwa Kilishi had the highest contents of Na (8281.00) and Mg (474.55) compared to other Kilishi samples. The highest values of P (8915.00) and K (9610.00) obtained were from Malali Kilishi, Tudun Wada Kilishi had the highest Ca content (459.55), while the Laboratory Kilishi had the lowest mineral contents of Ca, Mg and P. The results revealed that Kilishi contained the highest concentrations of the heavy metals. The concentrations (mgkg-1) of Fe (142.18 – 192.80), Zn (80.09 – 135.43), Pb (1.83 – 13.75) and Ni (2.08 – 19.88) in this study exceeded their threshold effects levels
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(80.00, 50.00, 0.50 and 0.50 mgkg-1/day respectively). From the study samples, Co and Mn contents (0.00 – 0.68 and 10.95 – 19.50 respectively) exceeded their threshold effects levels (0.16 and 11.00 mgkg-1/day respectively); except in Kilishi samples from Sabon Gari and Zaria City were Co was below detection limits. However, Cd and Cr were below detection limits in all the samples, while Cu contents (2.84 – 7.58) in the entire sample were below the threshold effects levels 8.0 mgkg-1/day). The Laboratory Kilishi had Co and Mn contents that were below threshold effects levels. Tudun Wada Kilishi had the highest Fe value, Sabon Gari Kilishi had the highest Zn value, Zaria City Kilishi had the highest Pb value, while Sabon Gari Kilishi had the highest Ni value. Microbiologically (cfu/g), Staphylococcus and fungi counts in all the samples (1.56 × 105 – 3.63 × 107 and 1.01 × 104 – 2.02 × 105 respectively) were above WFP recommended limits 106 and 102 respectively. Coliforms counts (0.00 – 9.61 × 106) were above WFP threshold limits in some of the study samples. However, the Mesophillic aerobic counts (0.00 – 9.03 × 105) were below the threshold effects limits of 106 with Tudun Wada Kilishi having the highest value. Microbial contaminants of Staphylococcus aureus, Bacillus spp., Salmonella, Klebsiella, Escherichia coli, Aspergillus, Penicillium and Geotricum candidum were isolated in some samples with Staphylococcus aureus predominantly detected in all the samples. The Laboratory Kilishi had Mesophillic counts below detection limits, Coliform counts below the threshold effect value and Bacillus, Salmonella, E. coli, Aspergillus and Penicillium species not detected.
TABLE OF CONTENTS
TITLE PAGE Title page – – – – – – – – – i Declaration – – – – – – – – – ii Certification – – – – – – – – – iii Dedication – – – – – – – – – iv Acknowledgements – – – – – – – – v Abstract – – – – – – – – vi Table of contents – – – – – – – – viii List of tables – – – – – – – – – xiv List of figures – – – – – – – – – xv List of appendixes – – – – – – – – xvi List of acronyms and abbreviations – – – – – xvii 1.0 INTRODUCTION – – – – – – – 1
1.1 Background to Study – – – – – – 1
1.2 Statement of Research – – – – – – 2 1.3 Justification – – – – – – – – 3
1.4 Aim and Objectives – – – – – – – 3
1.5 Research Hypotheses- – – – – – – 4 2.0 LITERATURE REVIEW – – – – – – 5 2.1 Meat and its Composition – – – – – – 5 2.1.1 Water – – – – – – – – – 6
2.1.2 Protein – – – – – – – – 6
2.1.3 Fat – – – – – – – – – 7
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2.1.4 Organic substances – – – – – – – 7
2.1.5 Inorganic substances – – – – – – – 8
2.2 Beef Meat – – – – – – – – 8 2.3 Kilishi – – – – – – – – 9 2.3.1 Effects of packaging and storage on Kilishi and meat products – 10 2.3.2 Effects of ingredients on Kilishi and meat products – – – 11 2.4 Proximate Composition of Meat Products – – – 13 2.5 Mineral Composition of Meat Products – – – 14 2.5.1 Sodium (Na) – – – – – – – – 15 2.5.2 Calcium (Ca) – – – – – – – – 15 2.5.3 Magnesium (Mg) – – – – – – – 16 2.5.4 Phosphorus (P) – – – – – – – 17 2.5.5 Potassium (K) – – – – – – – – 17 2.6 Heavy Metals and and their Toxicity in Meat and Meat Products 18 2.6.1 Iron (Fe) – – – – – – – – 19 2.6.2 Copper (Cu) – – – – – – – – 20 2.6.3 Zinc (Zn) – – – – – – – – 21 2.6.4 Cobolt (Co) – – – – – – – – 22 2.6.5 Manganese (Mn) – – – – – – – 22 2.6.6 Lead (Pb) – – – – – – – – 23 2.6.7 Cadmium (Cd) – – – – – – – 24 2.6.8 Chromium (Cr) – – – – – – – 26 2.6.9 Nickel (Ni) – – – – – – – – 26
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2.7 Microbiological Effect of Meat and Meat Products – – 27 2.7.1 Mesophillic aerobic count – – – – – – 28 2.7.2 Staphylococcus – – – – – – – 29 2.7.3 Coliform – – – – – – – – 29 2.7.4 Fungi (moulds and yeasts) – – – – – – 30 3.0 METHODOLOGY – – – – – – – 32 3.1 Study Area – – – – – – – – 32 3.2 Location of the Experiment – – – – – – 35 3.3 Materials Procurements and Kilishi Preparations – – 35 3.3.1 Preparation of Kilishi by commercial producers – – – 35 3.3.2 Beef preparation – – – – – – – 36 3.3.3 Groundnut paste preparation – – – – – – 36 3.3.4 Ingredients preparation – – – – – – 37 3.3.5 Kilishi production, packaging and storage – – – – 37 3.4 Sample Labeling – – – – – – – 38 3.5 Determination of Proximate Parameters – – – – 38 3.5.1 Moisture content – – – – – – – 38 3.5.2 Crude fat – – – – – – – – 39 3.5.3 Crude protein – – – – – – – – 40 3.5.4 Ash content – – – – – – – – 43 3.5.5 Crude fibre – – – – – – – – 43 3.5.6 Carbohydrate content – – – – – – – 45 3.6 Determination of Mineral Contents and Heavy Metals – – 45 3.6.1 Preparation of reagents and aqueous stock solutions- – – 45
3.6.2 Digestion of sample – – – – – – – 48
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3.6.3 Preparation of calibration curve – – – – – 50 3.6.4 Instrumental parameters for atomic absorption spectroscopy (AAS) 50 3.6.5 Determination of sodium (Na) and potassium (K) – – – 50 3.6.6 Determination of phosphorus (P) – – – – – 51 3.6.7 Determination of heavy metals – – – – – 52 3.6.8 Calculation of metal concentration – – – – – 52 3.7 Microbiological Procedures – – – – – 53 3.7.1 Culture media preparation – – – – – – 52 3.7.2 Sample preparation and dilution – – – – – 53 3.7.3 Calculation of microbial count – – – – – 54 3.7.4 Total Mesophillic aerobic counts – – – – – 54 3.7.5 Enumeration of Staphylococcus – – – – – 54 3.7.5 Enumeration of Coliform – – – – – – 55 3.7.5 Enumeration of fungi – – – – – – – 55 3.7.5 Detection of Salmonella – – – – – – – 55 3.7.5 Identification of bacteria species (Gram stain test) – – – 56 3.7.5 Biochemical identification of bacteria species – – – 56 3.7.5 Identification of fungi species (Lactophenol cotton blue stain test) 59 3.8 Environmental Conditions of Kilishi Production Spots – – 60 3.9 Statistical Analysis – – – – – – – 61 4.0 RESULTS/DATA PRESENTATION – – – – 62 4.1 Proximate Parameters in Dried Beef and its Kilishi Samples – 62 4.2 Mineral Content in Dried Beef and its Kilishi Samples – – 64 4.3 Heavy Metals Concentrations in Dried Beef and its Kilishi Samples 70
4.4 Microbial Counts in Dried Beef and its Kilishi Samples – 72
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4.5 Microorganisms Isolates in Dried Beef and its Kilishi Samples 74 5.0 DISCUSSION – – – – – – 76 5.1 Proximate Parameters in Dried Beef and its Kilishi Samples – 76 5.1.1 Moisture content – – – – – – – 76 5.1.2 Crude fat – – – – – – – – 76 5.1.3 Crude protein – – – – – – – – 77 5.1.4 Ash content – – – – – – – – 77 5.1.5 Crude fibre – – – – – – – – 78 5.1.6 Carbohydrate content – – – – – – – 79 5.2 Mineral Composition in Dried Beef and its Kilishi Samples – 79 5.2.1 Sodium (Na) – – – – – – – – 79 5.2.2 Calcium (Ca) – – – – – – – – 80 5.2.3 Magnesium (Mg) – – – – – – – 80 5.2.4 Phosphorus (P) – – – – – – – 81 5.2.5 Potassium (K) – – – – – – – – 81 5.3 Heavy Metals Concentrations in Dried Beef and its Kilishi Samples 82 5.3.1 Iron (Fe) – – – – – – – – 82 5.3.2 Copper (Cu) – – – – – – – – 82 5.3.3 Zinc (Zn) – – – – – – – – 83 5.3.4 Cobolt (Co) – – – – – – – – 83 5.3.5 Manganese (Mn) – – – – – – – 84 5.3.6 Lead (Pb) – – – – – – – – 85 5.3.7 Cadmium (Cd) – – – – – – – 85 5.3.8 Chromium (Cr) – – – – – – – 86
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5.3.9 Nickel (Ni) – – – – – – – – 86 5.4 Microbial Count in Dried Beef and its Kilishi Samples – – 87 5.4.1 Mesophillic aerobic plate counts – – – – – 87 5.4.2 Staphylococcus counts – – – – – – 88 5.4.3 Coliform counts – – – – – – – 88 5.4.4 Fungi (moulds and yeasts) counts – – – – – 89 5.5 Microorganisms Isolates in Dried Beef and its Kilishi Samples 89 6.0 CONCLUSIONS AND RECOMMENDATIONS – – 92 6.1 Conclusions – – – – – – – – 92 6.2 Recommendations – – – – – – – 95 REFERENCES – – – – – – – – 96 APPENDIXES – – – – – – – – 110
CHAPTER ONE
I.0 INTRODUCTION I.1 Background Meat and meat products are important for human diet because they provide the needed proteins, minerals and trace elements. Meat and meat products are extremely perishable mainly because preservation app.liances are hard to come by and where they are available, erratic power supply in the country is a main problem. Many methods of preserving meat have been used throughout history such as the use of high temperature (e.g. canning), low temperature (e.g. chilling), freezing and pasteurization, drying (e.g. hot air drying, wind and sun drying), smoking, use of radiation and the use of chemical preservatives (Alonge and Hiko, 1981). As a means of preserving meat, the early Fulani and Hausa herdsmen of Northern Nigeria and the Sahelian Africa have introduced Kilishi (Alonge and Hiko, 1981). The use of seasonings such as salt, spices, herbs and fermented sauces had been employed to enhance the acceptability of traditionally processed meat product like Kilishi. Study has revealed that Kilishi has a shelf life of 12 months at room temperature (Igene et al., 1990).
The pollution of the environment with heavy metals has become a worldwide problem and of scientific concern because the metals are indigestible and most of them have toxic effects, which constitute a potential danger to humanity (Khan et al., 1996). Heavy metal pollution is a serious threat to the high demand of Kilishi because of the toxic effect which they impact in the Kilishi, bioaccumulation and biomagnifications in
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the food chain (Demirezen and Uruc, 2006). In addition, the microbiological pollutants acquired through the food chain of Kilishi products poses serious danger of diseases to humans. Toxicity is realized when these heavy metal levels are higher than the recommended limit which varies for individual elements in meat and meat products. There are over ten (10) heavy metals including cobalt (Co), lead (Pb), mercury (Hg), arsenic (As), thallium (Tl), nickel (Ni), manganese (Mn), zinc (Zn), cobalt (Co), cadmium (Cd) and chromium (Cr) that have particular significance in ecotoxicology, since they are highly persistent (Storelli et al.,2005). The levels of metals such as Mn, Zn, Co and Cr are toxic beyond a certain limit, whereas Pb, Ni and Cd are toxic even in trace amounts (Bury et al., 2003; Fernandes et al., 2008). Therefore, this study determined the mineral contents, heavy metals levels and microbiological contamination in Kilishi from different production spots in Kaduna state, Nigeria to establish if the levels were within the recommended limits by World Health Organization. The quality of the meat used for the production of the Kilishi samples was also investigated. 1.2 Statement of the Problem
The traditional method of Kilishi preparation in Kaduna State, Nigeria has the risk of exposure to heavy metal pollutants from the environment through vehicle emission, dirty slaughter places and dust. The producers of these meat products prepare it in open markets and even on roadsides. This may result in contamination and thus bioaccumulation of heavy metals in people who consume these products. The analysis of heavy metals that include iron, copper, zinc, cobalt, manganese, lead, cadmium, chromium and nickel, is therefore justified to provide precautions towards the use of the
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Kilishi products, as well as provide a basis to improve on its quality. This study will also serve to sensitize government authorities such as National Environmental Management Authority (NEMA) towards identifying sources of heavy metals pollution in the local places of Kilishi production and for systemic toxic heavy metals and microbiological control before and after the production of Kilishi. 1.3 Justification With the increased awareness and consumption of the Kilishi, this study was conducted in view of the scarcity of information of the nutritive quality and levels of heavy metal contamination in Kilishi popularly consumed in selected areas in Kaduna state of Nigeria. 1.4 Aim and Objectives The aim of this research is to determine the proximate composition, mineral content, heavy metals composition and microbial contaminations of Kilishi samples, differently prepared from the same beef source, at some selected Kilishi selling spots in Kaduna State of Nigeria. This aim would be achieved through the following objectives:
i. To prepare Kilishi by commercial producers, from the same beef source, at six-selected Kilishi selling spots in Kaduna State of Nigeria viz: Kasuwan Mata-Sabon Gari in Zaria, Yan Awaki-Tudun Wada in Zaria, Durumi Maigarke-Zaria City in Zaria, Raba Road-Malali in Kaduna, Sultan Bello Road-Anguwan Sarki in Kaduna and Kasuwa-Jos Road in Kaduna.
ii. To prepare Kilishi and raw beef in the oven as control samples, from the same beef source, in the laboratory.
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iii. To determine the proximate composition (moisture content, ash content, fat content, protein content, fibre content and carbohydrate) in the prepared samples.
iv. To determine the mineral content (Na, Ca, Mg, P and K) in the samples.
v. To determine selected heavy metals (Fe, Cu, Zn, Co, Mn, Pb, Cd, Cr and Ni) level in the samples.
vi. To determine the microbial quality (Mesophillic aerobic plate count, Staphylococcus count, Coliforms count and fungal count) and identify some selected bacteria and fungi species in the samples.
vii. To compare the results obtained of Kilishi prepared by Commercial producers from their respective production spots with values of the control samples prepared from the laboratory.
viii. To compare the results obtained with WHO and FAO standard limits for meat and meat products.
1.5 Hypothesis For this research, it is hypothesized that the levels of heavy metals concentrations and microbiological contamination in Kilishi meat products produced in six selected spots in Kaduna State, Nigeria do not differ significantly from the recommended limits specified by the International standards of World Health Organization (WHO) and Food and Agricultural Organization (FAO).
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