Ibrahim Muhammad

COMPARATIVE STUDY OF THE BECTERIOLOGICAL QUALITY OF CABBAGE SOLD IN BUK RESTAURANTS AND FROM VENDORS WITHIN THE OLD AND NEW CAMPUS. BY KATAKO ABUBAKAR NAFISAT LSC/18/MCB/00291 A RESEARCH PROJECT SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY, FACULTY OF LIFE SCIENCES, COLLEGE OF NATURAL AND PHARMACEUTICAL SCIENCES, BAYERO UNIVERSITY KANO, IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF BACHELOR OF SCIENCE (B.Sc) DEGREE IN MICROBIOLOGY SUPERVISED BY PROF. BASHIR MUHAMMAD APRIL, 2024. DECLARATION I KATAKO ABUBAKAR NAFISAT with the registration number LSC/18/MCB/00291 Declare that this work was done and compiled by me. No part or whole work has been presented to any institution for degree or any institution. All sources of information are duly acknowledged. _______________________________ KATAKO ABUBAKAR NAFISAT LSC/18/MCB/00291 CERTIFICATION This is to certify that the Research work was carried out and compiled by KATAKO ABUBAKAR NAFISAT with the registration number (LSC/18/MCB/00291). SIGN----------------------------- ------------------------ EXTERNAL SUPERVISOR DATE SIGN:---------------------------------- ------------------------ DR. BASHIR MUHAMMAD DATE SUPERVISOR SIGN----------------------------------- ------------------------ DR. HABIBU ABDU USMAN DATE LEVEL COORDINATOR SIGN---------------------------------- ------------------------- PROFESSOR MUHAMMED YUSHAU DATE HEAD OF THE DEPARTMENT DEDICATION This Research project is dedicated to Almighty Allah (S.W.T) and to my beloved parents, my family members, and my friends, who have been my constant inspiration for the entirety of this program. For their unwavering care and support shown to me in the pursuit of my educational career, I say may Allah reward them abundantly. ACKNOWLEDGEMENTS Praises and adulations to the Almighty, the Everlasting, the All Knowing, the Most Gracious, and the Most Merciful. And praises to his Noble Prophet Muhammad (S.A.W), his family, his companions and whoever follows the path of righteousness. My appreciation and gratitude are due to Allah for his endless mercy, blessings, and for sparing my life. He made it possible for this work to see the light of day. I wish to express my gratitude to my institution based supervisor PROF. .B. MUHAMMED, for his relentless guidance and patience towards the completion of this research wok. His effort, time and energy has been immense, the completion of this work wouldn’t have been possible without my institutional based supervisor. I also want to acknowledge my lecturers and the staff of my department. They contributed effectively in various methods, via proper upbringing, consistent advice and support toward achieving success in my field of study. I sincerely appreciate the effort put in by my parents for, their prayer, advice, sacrifice, support, patience, love and financial assistance. And I also want to thank my friends for their patience, love and support. Finally I would like to thank my course mates and my colleagues for their kindness and overall attitude towards me, one which cannot be over-emphasized. I cannot conclude without acknowledge my unforgettable brother and sisters Yakubu Musa, Aisha Katako, Zainab Katako, Sadiq Katako, Hafsat Katako and Bilkisu Katako. TABLE OF CONTENTS TABLE OF CONTENTS DECLARATIONii CERTIFICATIONiii DEDICATIONiv ACKNOWLEDGEMENTSv TABLE OF CONTENTSvi ABSTRACTviii CHAPTER ONE1 1.1 INTRODUCTION1 1.2 STATEMENT OF RESEARCH PROBLEM4 1.3 JUSTIFICATION OF THE RESEARCH5 CHAPTER TWO7 2.0 Literature Review7 2.1 Microbial flora of RTE vegetables9 2.2 Factors affecting the microbiological quality of RTE vegetables9 2.3 Microbiological safety standards for RTE vegetables10 2.4 Poor quality Cabbage11 2.5 Source of contamination of Cabbage12 2.6 Consequences of Contaminated Cabbage13 2.7 Microorganisms Associated with Cabbage14 2.8 Effects of Consumption of Poor Hygienic RTE Vegetables15 2.9 Methods of washing and reducing microbial load from cabbage16 2.10 Mechanisms of using salt to wash vegetables17 2.11 Mechanisms of using vinegar to was vegetable (cabbage)18 CHAPTER THREE20 3.0 MATERIALS AND METHODS;20 3.2 Sample site.21 3.3 Sample collection21 3.4. Sample Preparation and Dilution21 3.5 Preparation of culture media21 3.5.1 Preparation of Nutrient Agar22 3.5.2 Preparation of MacConkey Agar:22 3.5.3 Preparation of Mannitol Salt agar23 3.5.4 Preparation of salmonella shigella agar23 3.5.5 Preparation of Eosin Methylene blue agar24 3.6 isolation of bacteria24 3.6.1 serial dilution25 3.6.2 Enumeration of Coliform Bacteria25 3.6.3 Aerobic Mesophilic Bacteria Count26 3.6.4 innoculation of the sample26 3.7 identification of the bacteria27 3.7.0 Characterization of Isolate and identification27 3.7.1 Gram staining and Microscopy27 3.7.2 Gram staining27 3.8 Biochemical tests27 3.8.1 Catalase test28 3.8.2 Coagulase test:28 3.8.3 Indole Tests:29 3.8.4 Methyl Red Test:29 3.8.5 Voges Proskauer Test:29 3.8.6 Citrate test30 3.9 Statistical Analysis30 CHAPTER FOUR31 RESULT31 CHAPTER FIVE42 DISCUSSION, CONCLUSION AND RECOMMENDATIONS42 5.1 Discussion42 5.2 Conclusion44 5.3 Recommendations44 REFERENCES45 ABSTRACT This research work entitle comparative study of the becteriological quality of cabbage sold in buk restaurants and from vendors within the old and new campus. The aim is to determine the bacteriological quality of cabbage sold in BUK restaurants and from vendors within the old and new campus. The researchers collected cabbage samples from restaurants and vendors across Bayero University Kano's campuses. After visually inspecting the cabbage, they washed it with sterile water and prepared a diluted solution for bacterial enrichment. Various culture media like MacConkey agar were then sterilized and used to isolate bacteria present on the cabbage. Serial dilutions of the enriched samples were plated on these media to separate individual bacterial colonies. Finally, Gram staining and specific biochemical tests like catalase or indole tests helped identify the types of bacteria present within the cabbage samples. Throughout this process, sterile techniques were strictly followed to prevent contamination of the samples. The findings raise significant public health concerns and necessitate stricter hygiene practices throughout the entire cabbage supply chain. The findings reveal a widespread presence of bacteria, including concerning pathogenic types, across a significant portion of the samples. This highlights a potential public health risk associated with consuming raw or undercooked cabbage. Based on the findings, the researcher recommend that, the University should develop stricter hygiene measures by collaborating with farms and vendors to improve hygiene practices throughout the cabbage supply chain and also the University should provide consumer education by developing educational campaigns to raise awareness about bacterial contamination risks and promote proper washing techniques (e.g., vinegar solution) for cabbage at home. CHAPTER ONE 1.1 INTRODUCTION Vegetables are plants rich in essential bioactive nutrients like minerals, fiber and vitamins (Conner, et al., 2017), they are consumed (raw or preheated) by ruminant animals and humans (de Evan, et al., 2019). Most vegetables are usually green plants e.g. spinach, lettuce, and pumpkin, while others such as cabbage, onion, mushroom and radish, are nongreen vegetable plants (Amao, 2018). Vegetables are very fragile in nature, therefore in order not to lose their unique nutrients, it is recommended that consumers eat them raw or slightlyheated, as too much heat resulting from over cooking, will destroy their nutrients (Chaturvedi, et al., 2013; Feng, et al., 2022). The health benefits derived from consuming fresh vegetables containing high fiber and vitamins content make them more popular for the people who care about boosting their immunity using proper diet especially in the post-COVID pandemic era (Chowdhury, 2020). Cabbage (Brassica oleraceaver-capitata) is an important vegetable known to mankind for over 4,000 years (Teshome, et al., 2018). It is a member of the mustard or cruciferous family (Brassicaceae), which includes mustard, rape, turnip, wasabi (Eutrema wasabi), radish, watercress, many oriental vegetables, and a very important model plant Arabidopsis thaliana (Shrestha, 2019). In terms of life cycle, cabbage is a short lived perennial crop, usually biennial. Cabbage grows best on well-drained fertilized soils with constant availability of adequate moisture and under moderate temperature and pH in the range 6.0 - 6.5. It is essential not to grow cabbage on the same field year after year because of accumulation of various pathogens, to which crops is highly susceptible (Tsoho and Salau, 2012). Cabbage contains calcium in the range of 22-150mg/100g. Its accumulated mineral source is at very high level of phosphorus, sulphur, chlorine, calcium, iron and potassium (Jahangir et al., 2009). Cabbage comprises potentially useful amount of copper, zinc and a number of other important minerals and trace elements. Cabbage has a lot of health benefits which includes prevention of oxidative stress, induction of detoxificative enzymes, and stimulation of immune system reduction of cancer cells and inhabits malignant transformation and carcinogenic mutation. It also plays an important role in the etipathology of many diseases such as vasospasm, atherosclerosis, cancer, heart attack, stroke and liver damage (Athar, et al., 2004a). Ready-to-eat (RTE) vegetables are those that are washed, cut, pre-packaged, and ready for consumption without the need for additional processing or cooking. These types of vegetables have become increasingly popular due to their convenience and health benefits. However, the microbiological quality of RTE vegetables can be a potential risk to human health as they can become contaminated with pathogenic microorganisms during the various stages of production, handling, storage, and transportation. Studies have shown that the contamination of RTE vegetables can be attributed to several factors such as poor hygiene practices, inadequate sanitation, improper storage temperature, and the use of contaminated water during cultivation and processing (Jay, et al 2005; Sagoo et al., 2013). The presence of pathogenic bacteria such as Salmonella, Listeria monocytogenes, Escherichia coli, and Shigella on RTE vegetables can cause foodborne illnesses such as diarrhea, vomiting, fever, and even death in severe cases (Alvarez-Ordóñez et al., 2011). In recent years, various interventions have been proposed to reduce the microbial contamination of RTE vegetables, including the use of disinfectants, irradiation, and biological control agents (Kumar and Sabina, 2017; Lopez-Galvez et al., 2018). However, it is essential to ensure that these interventions do not compromise the nutritional quality of the vegetables or lead to the development of antimicrobial resistant microorganisms. Ready-to-eat vegetables are widely consumed as a healthy and nutritious choice for meals. However, they are often associated with foodborne illness outbreaks. Microbial contamination of vegetables can occur at any stage from production to consumption. This necessitates the need for microbiological analysis to ensure the safety of ready-to-eat vegetables. In this article, we review studies on microbiological analysis of ready-to-eat vegetables. Ready-to-eat (RTE) vegetables are widely consumed because of their convenience, taste, and nutritional value. RTE vegetables are minimally processed, washed and packaged, and are predominantly consumed raw or with minimal cooking. However, these are also highly perishable in nature and hence vulnerable to poor hygiene practices during harvesting, processing, transportation, retail, and at the consumer level. Poor hygiene, contamination from water, soil, animals, and human handling are some of the factors that contribute to microbial contamination in RTE vegetables, and pose a potential risk to public health. Therefore, understanding the microbiological quality of RTE vegetables is critical in ensuring their safety for consumption. This literature review highlights scientific research reports on the microbiological quality of ready-to-eat vegetables. The microbiological quality of ready-to-eat vegetables is influenced by several factors, including the level of contamination on the raw materials, the conditions of processing, and storage. According to a study by Alizadeh et al. (2021), the most common microorganisms found in ready-to-eat vegetables are coliforms, Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Salmonella spp., and Listeria monocytogenes (L. monocytogenes). These microorganisms have been implicated in foodborne illness outbreaks associated with ready-to-eat vegetables. The presence of these microorganisms in ready-to-eat vegetables could be due to contamination from irrigation water, soil, or equipment used during transportation, storage, and processing. A study by Barreto et al. (2021) revealed that irrigation water was a significant source of microbial contamination in ready-to-eat vegetables. Several microbiological methods are used to analyze ready-to-eat vegetables, including culture-based and molecular techniques. The culture-based techniques involve the use of selective media to isolate and enumerate specific microorganisms. The molecular techniques involve the use of polymerase chain reaction (PCR) and next-generation sequencing (NGS) to identify and quantify microorganisms in samples. According to a study by Ferri et al. (2021), molecular techniques such as PCR and NGS are more sensitive compared to culture-based methods for the detection of microorganisms in ready-to-eat vegetables. PCR can detect low levels of microbial contamination, while NGS can identify multiple microorganisms in a single test. (RTE) vegetables are foods that are consumed without further cooking preparation, which makes them susceptible to contamination by pathogenic microorganisms. 1.2 STATEMENT OF RESEARCH PROBLEM Consumption of vegetable products has increased in recent years (Feng, et al., 2022), these vegetables have also become vehicles for the transmission of some kinds of pathogens when eaten raw from unhygienic preparation causing food poisoning (Chaturvedi, et al., 2013; Chowdhury, 2020). They are widely exposed to microbial contaminations through contact with water, soil, dust, and by handling at harvest or during postharvest processing. They therefore harbor both human and plant pathogens (Teshome, et al., 2018). Pathogenic bacteria that have been detected in fresh vegetables (Spinach and cabbage) are coliform bacteria, Escherichia coli, Staphylococcus aureus and Salmonella spp. (Tambekar and Mundhada, 2006). This study therefore aims to identify and compare the bacteriological load of cabbage sold in Bayero University Kano State, North-Western Nigeria. 1.3 JUSTIFICATION OF THE RESEARCH Cabbage are essential part of human diet and are highly consumed in every family. It is essential to health because of its high nutritional value. It provides nutrients such as vitamins and minerals and also is of medical important. The cabbage are reliable to contamination from various sources such as soil, man, water, air, and insects (Yong, 2014). Therefore, Isolation and identification of possible pathogenic bacteria from fresh vegetables are necessary to enlighten consumer and restaurants owners within Bayero university kano of various ways of hygienic practices that leads to reduction of microbial load and a determination of the student’s population in case of food borne outbreak in the campus (Anupama et al., 2020). Food borne diseases cause an estimated seventy-six million (76 million) cases of illnesses and three hundred and twenty-five thousand (325,000) hospitalisations and five thousand two hundred (5200) deaths annually in the United States (CDC, 2003). This research is critical to ensure the safety and quality of cabbage consumed by individuals in bayero university kano. By assessing the bacteriological quality and identifying potential sources of contamination ,this study can help inform food safety practices and improve the overall health as well as the well being of consumers and shows the effectiveness of the most common methods of washing and reducing microbial load on cabbage it also contribute to the existing knowledge on food safety and promotes awareness among both food establishments and consumers. AIM AND OBJECTIVE OF THE STUDY AIM, The aim of this work is to determine the bacteriological quality of cabbage sold in BUK restaurants and from vendors within the old and new campus. OBJECTIVE i. Isolation and identification of microorganisms associated with cabbage sold within BUK old and new site. ii. Determination of aerobic mesophilic count associated with cabbage sold within BUK. iii. Determination of coliform bacteria in vegetable sample CHAPTER TWO 2.0 Literature Review Food is indispensable to the maintenance of life, but can also be responsible for ill health. The foods we eat are hardly sterile as they contain a lot of microorganisms if poorly prepared or handled. Some microorganisms in food are natural microflora while others are introduced into food materials through the process of harvesting, transportation, storage, processing, preparation, distribution and selling (Adams and Moss, 2008). Despite the increased knowledge and industrialization obtainable these days, food borne diseases are perhaps the most widespread health problems and important causes of reduced economic productivity (WHO, 1992) with an incidence in every person at least once a year (Whiteney et al., 1990). The available evidence indicates that biological contaminants are the major cause of food borne diseases (Adams and Moss, 2008). It is however concluded that food quality and safety are central issues in today’s food economy (Grunert, 2005). However, fruits and vegetables are amongst the highly nutritious foods providing vitamins and minerals and are highly recommended foods that are commonly consumed (Gupta and Rana, 2003; Isa et al., 2014). Increasing health awareness has led to consumption of raw foods in recent years, these foods get polluted through the production activities and vegetables get contaminated at each step from cultivation to consumers (Shobha, 2014). The presence of numerous genera of spoilage bacteria, yeast and moulds and occasional pathogens on fresh produce has been recognised for many years (Benchat, 1996). A wide variety of fresh fruits and vegetables have been associated with diseases caused by microbial pathogens they harbour (Linda, 1997; Eni et al., 2010). The safety of food and fresh produce is a global issue covering both the countries that import and supply them (Muhammad et al., 2016). Among the vegetable group of highest concern from microbiological safety identified by Mritunjay and Kumar (2015) are salad, cabbage and spinach. A study conducted by Heaton and Jones (2008) on microbial contamination identified fruits and vegetables consumption as a risk factor for infection with enteric pathogens. There have been several studies conducted to investigate the microbiological quality of ready-to-eat (RTE) vegetables. These studies have looked at both the levels of various microorganisms present in the vegetables as well as the prevalence of specific pathogens such as Listeria monocytogenes and Salmonella. One study conducted in China by Chen et al. (2017) found that out of 60 samples of RTE vegetables collected from various supermarkets and restaurants, 48.3% were contaminated with total viable counts (TVC) exceeding the permissible limit of 10^6 CFU/g. The study also found that 26.7% of the samples tested positive for the presence of Listeria monocytogenes. Similarly, a study conducted in India by Mahajan and Bajaj (2016) evaluated the microbial quality of RTE leafy vegetables sold in five different localities of Mumbai. The study found high levels of TVC in the samples tested, with almost all (98%) exceeding the permissible limit of 10^6 CFU/g. The study also found the presence of pathogenic bacteria such as Escherichia coli and Salmonella in some of the samples. Another study conducted in Egypt by Aboubakr et al. (2014) evaluated the microbiological quality of RTE salads sold in various food outlets in Alexandria. The study found that all samples were contaminated with various microorganisms, with TVC exceeding the permissible limit in 52.5% of the samples. The study also isolated several pathogenic bacteria such as E. coli and Staphylococcus aureus from the samples. These studies highlight the potential risks associated with consuming RTE vegetables and the need for proper sanitation and hygiene measures during their production and handling. 2.1 Microbial flora of RTE vegetables RTE vegetables are rich in natural microbiota comprising beneficial and pathogenic microorganisms. Several studies have reported the presence of microorganisms such as Staphylococcus aureus, Bacillus cereus, Escherichia coli, Salmonella spp., Listeria spp., and Campylobacter spp. in RTE vegetables (Beuchat, 2002; Heaton et al., 2001; Piras et al., 2011; Shukla et al., 2004). Ramirez et al. (2012) showed by culture-independent methods that RTE vegetables harbor unique and diverse bacterial communities that varied among different vegetables and geographic locations. Bacterial isolates from RTE vegetables have also been found to exhibit resistance to antibiotics, which poses an additional concern (Vaishali et al., 2016). 2.2 Factors affecting the microbiological quality of RTE vegetables The microbiological quality of RTE vegetables is influenced by various factors, including the type of vegetable, location, seasonality, and handling practices. For instance, tomatoes, lettuce, and spinach have been found to be highly contaminated with pathogenic microorganisms (Beuchat, 2002; Shukla et al., 2004). Seasonality also affects the microbial load of RTE vegetables, with higher loads during the growing seasons. Furthermore, poor hygiene practices during processing, transportation, and storage contribute to increased microbial contamination in RTE vegetables, leading to foodborne illnesses (Boor and Wiedmann, 2004; Leyer et al., 2020). 2.3 Microbiological safety standards for RTE vegetables Several countries have established microbiological safety standards and guidelines for RTE vegetables to ensure their safety for consumption. The European Union (EU) Commission Regulation No. 2073/2005 sets maximum limits for specific microorganisms such as Salmonella spp., Listeria monocytogenes, and E. coli in RTE vegetables (European Commission, 2005). Similarly, the US Food and Drug Administration (FDA) has set microbiological safety standards for fresh-cut produce, including ready-to-eat vegetables, to prevent foodborne contamination (FDA, 2021). This study assessed the microbiological quality of ready-to-eat vegetables in Jordan. The results showed that 57.6% of the samples tested positive for total coliform bacteria, while 11.8% tested positive for Escherichia coli (E. coli). The study concluded that ready-to-eat vegetables may pose a risk to public health due to their potential contamination with bacterial pathogens (Alvares et al., 2015). The study evaluated the microbiological quality and safety of fresh-cut vegetables sold in Spain. The results showed that 19.4% of the samples tested positive for total coliform bacteria, while 3.5% tested positive for E. coli. The study concluded that fresh-cut vegetables may be a potential source of foodborne illness if they are not properly handled and stored (Commane et al., 2017). The study investigated the microbiological quality of fresh vegetables and salads sold in the United Kingdom. The results showed that 63.5% of the samples tested positive for total bacteria, while 5.3% tested positive for E. coli. The study concluded that fresh vegetables and salads may pose a risk to public health due to their potential contamination with bacterial pathogens (Gorai et al., 2017). The study assessed the microbiological quality of fresh vegetables and fruits sold in various markets in Hyderabad, India. The results showed that 80% of the samples tested positive for total coliform bacteria, while 16.3% tested positive for E. coli. The study concluded that fresh vegetables and fruits sold in markets may be contaminated with bacterial pathogens and pose a risk to public health (Singh et al., 2018). The study evaluated the microbiological quality of ready-to-eat vegetables and assessed their potential health risks. The results showed that 55% of the samples tested positive for total coliform bacteria, while 7.5% tested positive for E. coli. The study concluded that ready-to-eat vegetables may be a potential source of foodborne illness if they are not properly handled and stored. Ready-to-eat vegetables (RTEV) are becoming increasingly popular due to their health benefits and convenience. However, concerns have been raised about the microbiological quality of these products. Several studies have been conducted to investigate the microbiological quality of RTEV, and the results have indicated the presence of various microorganisms, including pathogenic bacteria (Signh et al., 2018). 2.4 Poor quality Cabbage Ready-to-eat vegetables have gained popularity over the years due to their convenience and nutritional value. However, poor quality ready-to-eat vegetables can pose health risks to consumers. Below are some examples of poor quality ready-to-eat vegetables: Contamination with Pathogens: Ready-to-eat vegetables are often contaminated with pathogens such as Salmonella, Listeria, and E. coli. These pathogens can cause serious illnesses such as food poisoning, diarrhea, and gastroenteritis. A study conducted by Luo et al. (2015) found that 5 out of 10 samples of ready-to-eat vegetables tested positive for Salmonella. High Levels of Pesticides: Ready-to-eat vegetables are sometimes treated with pesticides to prevent pests and diseases. However, some farmers use high levels of pesticides, which can be harmful to consumers. A study by Islam et al. (2016) found that most of the samples of ready-to-eat vegetables tested contained high levels of pesticides, which can cause health problems such as cancer, neurological disorders, and birth defects. Poor Quality Packaging: Ready-to-eat vegetables are often packaged in plastic containers or bags, which can affect their quality. Poor quality packaging can lead to contamination, oxidation, and loss of nutrients. A study conducted by Chang et al. (2014) found that some ready-to-eat vegetables packed in plastic containers had low levels of antioxidants, which are important for good health. 2.5 Source of contamination of Cabbage Contaminated irrigation water: Irrigation water contaminated with human or animal waste can transfer pathogens such as Salmonella and E. coli to ready-to-eat vegetables. (Olaimat & Holley, 2012) Soil contamination: Soil can be a reservoir for various pathogens, including bacteria, viruses, and parasites, which can contaminate ready-to-eat vegetables during cultivation, harvesting, or processing. (Friedman, 2013) Harvesting equipment and workers: Harvesting equipment and workers can transfer pathogens such as Listeria, Salmonella, and E. coli to ready-to-eat vegetables during picking, sorting, and packaging. (Harris et al., 2012) Contaminated surface areas: Food processing and packing facilities can be a source of contamination if surfaces are not properly cleaned and sanitized from previous use. (Mukherjee et al., 2014) Cross-contamination during food preparation: Ready-to-eat vegetables can be contaminated if they come into contact with raw meat, poultry, or seafood during food preparation. (Buchholz & Davidson, 2012) Contamination during transportation and storage: Temperature abuse during transportation and storage of ready-to-eat vegetables can promote the growth of bacteria and increase the risk of foodborne illness. (Beuchat & Ryu, 2017) 2.6 Consequences of Contaminated Cabbage Contaminated ready-to-eat (RTE) vegetables can cause a range of negative consequences, including foodborne illness and economic losses for the food industry. Here are some of the consequences of contaminated RTE vegetables, supported by relevant citations and references: Foodborne illness: Consuming contaminated RTE vegetables can lead to foodborne illnesses, such as listeriosis, salmonellosis, and E. coli infections. These illnesses can cause symptoms ranging from mild to severe, and in some cases, can be life-threatening (FSA, 2020). Economic losses: The contamination of RTE vegetables can result in significant economic losses for the food industry due to recalls, legal actions, and damage to reputation and consumer trust. The costs of a recall can be substantial, with one estimate putting the average cost of a food recall at around $10 million (Buzby & Roberts, 2012). Public health concerns: Contamination of RTE vegetables can also raise public health concerns and result in negative media attention, leading to a loss of consumer confidence in the food industry. This loss of confidence can have long-term effects on sales and profits (Hayes, 2018). Regulatory action: Contaminated RTE vegetables can result in regulatory action, including fines, sanctions, and legal action, with potential long-term consequences for the food industry. The costs of such regulatory action can be significant, as are the requirements for compliance with regulations to prevent future contamination (FSA, 2020). 2.7 Microorganisms Associated with Cabbage Ready-to-eat (RTE) vegetables are commonly consumed as part of salads, sandwiches, and snacks. However, they have been identified as potential sources of foodborne illnesses caused by diverse pathogenic microorganisms. The following are some of the microorganisms associated with RTE vegetables: Salmonella: A gram-negative bacterium that is commonly associated with the contamination of RTE vegetables. It can lead to gastroenteritis, fever, and diarrhea when ingested by humans (Zhang et al., 2019). Listeria monocytogenes: A gram-positive bacterium that is known to survive under refrigeration and processing conditions used in the production of RTE vegetables. Listeriosis caused by this bacterium is a severe form of foodborne illness that can cause miscarriage, meningitis, and septicemia (Chen et al., 2021). Escherichia coli: A gram-negative bacterium that can cause severe diarrhea, kidney failure, and death, especially in vulnerable populations such as older adults, pregnant women, and young children (Azap et al., 2020). The presence of pathogenic E. coli strains, such as E. coli O157:H7, on RTE vegetables is a significant concern. Campylobacter: A gram-negative bacterium that is commonly found in poultry and can contaminate other food products, including RTE vegetables. Ingesting contaminated RTE vegetables can lead to Campylobacteriosis, a gastrointestinal illness that can cause diarrhea, fever, and cramps (Ge et al., 2020). Vibrio parahaemolyticus: A gram-negative bacterium that is associated with the consumption of raw or undercooked seafood and can also contaminate RTE vegetables. Ingesting contaminated RTE vegetables can lead to gastrointestinal illness characterized by diarrhea, cramps, and fever (Bafana et al., 2020). Staphylococcus aureus. Staphylococcus is a genus of Gram-positive bacteria in the family Staphylococcaceae from the order Bacillales. Under the microscope, they appear spherical, and form in grape-like clusters. was found in packaged salads and leafy vegetables was poor, with high levels of total plate count, E. coli, and Staphylococcus aureus. by Kaur et al. (2018) in India. 2.8 Effects of Consumption of Poor Hygienic RTE Vegetables Foodborne Infections: RTE vegetables can be contaminated with various pathogenic microorganisms, such as Salmonella, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Norovirus, which can cause foodborne infections. The symptoms of these infections include diarrhea, vomiting, nausea, abdominal pain, fever, headache, and dehydration. These infections can be more severe in immunocompromised individuals, pregnant women, and infants. (Abadias et al., 2014) Gastroenteritis: The consumption of contaminated RTE vegetables can lead to gastroenteritis, which is an inflammation of the gastrointestinal tract. It is characterized by symptoms such as diarrhea, abdominal pain, nausea, and vomiting. Gastroenteritis can be caused by various microorganisms, such as bacteria, viruses, and parasites, which can be transmitted through contaminated food or water. The risk of acquiring gastroenteritis is higher in individuals who have a weakened immune system, such as the elderly, young children, or those with chronic diseases. (Todd et al., 2018) Hepatitis A: Hepatitis A is a viral infection that affects the liver. It is transmitted through contaminated food or water, and is characterized by symptoms such as jaundice, fatigue, abdominal pain, nausea, and vomiting. The virus can survive on RTE vegetables, and can be transmitted if they are not properly washed or cooked. The risk of acquiring hepatitis A is higher in individuals who travel to regions with poor sanitation, or in individuals who have close contact with infected persons (CDC, 2021). 2.9 Methods of washing and reducing microbial load from cabbage There are several methods commonly used to wash and reduce microbial load from vegetables like cabbage: 1. Rinsing with water: This is the most basic method, which helps remove surface dirt and some microbes. Its important to use clean, potable water for rinsing. 2. Soaking: Soaking cabbage in water for few minutes can help loosen dirt and microbes attached to the surface. Adding small amount of vinegar to water can help enhance microbial reduction. 3. Vegetable wash: Commercial vegetable wash solutions are availabe that claim to effectively remove pesticides, wax, and microbes from produce. These solutions usually diluted in water and the vegetables are soaked or sprayed with the solution before rinsing. 4. Blanching: Blanching involves briefly immersing vegetables in boiling water followed by rapid cooling. This method not only helps in reducing microbial load but also inactivates enzymes that can cause spoilage. 5. Use of brushes: Brushes designed specifically for cleaning vegetables can be used to scrub the surface of cabbage, helping to remove dirt and microbes. 6. Ultrasonic washing: Some advanced methods involving using ultrasonic wasves to dislodge microbes from the surface of vegetables. 7. Ozone treatment: Ozone is a powerful disinfectant and can be used to was vegetables. Ozone treatment involving bubbling ozone gas through water in which the vegetables are immersed, effectively killing microbes on the surface. It is important to note that while these methods can help reduce microbial contamination, thorough cooking of vegetables is still necessary to ensure safety, especially for vulnerable populations such as pregnant women, young children, the elderly, and those with compromise immune systems (Azap et al., 2020). 2.10 Mechanisms of using salt to wash vegetables Using salt to wash cabbage can help reduce microbial load through several mechanisms: 1. Osmosis: Salt draws water out of microbial cells through osmosis, causing them to dehydrate and die. This process disrupts the microbial cells’ structure and function, leading to their demise. 2. Antimicrobial properties: Salt has natural antimicrobial properties that can inhibit the growth of bacteria, fungi, and other microbes on the surface of cabbage. It creats a hostile environment for microbial growth, reducing their number. 3. Detachment: Salt can help loosen dirt, debris, and microbes attached to the surface of cabbage, making it easier to rinse them away during washing. 4. Preservation: Salt can act as a perservative, extending the shelf life of cabbage by inhibiting the growth of spoilage microorganisms. This is particularly useful in traditional methods of fermenting cabbage to make sauerkraut or kimchi, where salt helps create an environment conducive to beneficial fermentation bacteria while inhibiting harmful microbes. Using salt to wash cabbage is an effective method for reducing microbial contamination and preserving its freshness. The standard measurement or concentration of using salt to wash cabbage is one table spoon to one cup of water or 30g to I liter of water However, it is essential to rinse the cabbage thoroughly after salt treatment to remove excess salt before consumption. 2.11 Mechanisms of using vinegar to was vegetable (cabbage) Using vinegar to was cabbage can be effective in reducing microbial load through several mechanisms: 1. Acid pH: Vinegar is acidic, typically with a pH between 2.0 and 3.5, depending on the type. The acidic nature of vinegar creates an environment that is hostile to many microbes, inhibiting their growth and killing them. 2. Antimicrobial properties: Acetic acid, the main component of vinegar, has antimicrobial properties that can help kill bacteria, fungi, and other microorganisms present on the surface of cabbage. It disrupts the microbial cells’ structure and fuctions, leading to their inactivation. 3. Detachment: Vinegar can effectively decontaminate the surface of cabbage from pathogenic bacteria such as E. coli, Salmonella, and Listeria, reducing the risk of food borne illnesses. 4. Preservation: Vinegar can act as a preservative, extending the shelf life of cabbage by inhibiting the growth of spoilage microorganisms. This is particularly useful in pickling or fermenting cabbage to make dishes like coleslaw or sauerkraut, where vinegar contributes to the preservation process. Using vinegar to wash cabbage is a simple and effective method for reducing microbial contamination and enhancing food safety. The ratio of using vinegar is one part ot three parts of water, It is important to dilute vinegar with water before use, as the high acidity of undiluted vinegar may affect the taste or texture of the cabbage (Azap et al., 2020). CHAPTER THREE 3.0 MATERIALS AND METHODS; Cabbage (Brassica oleraceaver-capitata ) Sterile Macconkay agar Sterile Salmonella and Shigella agar Sterile Mannitol Salt agar Sterile nutrient agar Sterile EMB agar Sterile distilled water Sterile inoculating loops Sterile durham tubes Test tubes Sterile pipettes Disinfectants (ethanol) Biochemical reagents for identification ( indole, methyl red, Voges-Proskauer, citrate utilization) Bunsen burner or other sterilization equipment Microscope Autoclave or pressure cooker for sterilization pH meter Laminar flow hood (optional but recommended for aseptic work) Refrigerator for sample storage Laboratory glassware ( Petri dishes, conical flasks) Pipettes and pipette tips Incubator set to appropriate temperature for bacterial growth. 3.2 Sample site. The cabbage were purchased from restaurants in bayero university new campus ( Donkens, chips and chicken and Alkhairat) and from restaurant in Bayero university old campus ( chinkenzza and ) and from vegetable vendors within Bayero university kano old and new campus. 3.3 Sample collection Ten (10) samples of cabbage from 5 sample site were purchased from different restaurants at Bayero University Kano and 2 samples were purchased from vegetable vendor from each of the campus. The samples were appropriately labelled and placed in sterile sealed container in an insulated ice box to maintain the freshness and integrity of the vegetables and then transported to the Department of Microbiology laboratory to be analyzed within one hour of receipt (Eni et al., 2010). 3.4. Sample Preparation and Dilution Upon arrival at the laboratory, the samples were visually inspected for signs of physical damage, spoilage, or microbial contamination. Only intact and visually healthy cabbage specimen were selected for further analysis. The samples were then washed with sterile distilled water to remove any surface debris or contaminants and allow to dry for 2-3 Minutes. One gram (1g) of each sample was weighed into 9ml of peptone water and incubated for 18-24hrs at 37°C (Enrichment). 3.5 Preparation of culture media The culture media used for the isolation of the various organisms were prepared according to the manufacturers’ instructions and sterilized by autoclaving at 1210C for 15 minutes (Cheesebrough, 2005). The required amount was prepared according to the manufacturers’ instruction and specifications of the powdered media (Cheesebrough, 2005). 3.5.1 Preparation of Nutrient Agar Nutrient agar was prepared according to the guidelines outlined by the American Society for Microbiology (ASM). The following steps were followed to prepare1 liter of nutrient agar: 28 grams of nutrient agar powder were weighed and added to1 liter of distilled water in a heat-resistant glass flask .The mixture was stirred to ensure complete dissolution of the agar powder. The prepared nutrient agar solution was then autoclaved at 121°C for 15 minutes to sterilize the medium and eliminate any microbial contaminants. After autoclaving, the agar solution was cooled to approximately 45-50°C in a water bath. Subsequently, the molten agar was poured into sterile Petri dishes under aseptic conditions and allowed to solidify at room temperature. The prepared nutrient agar plates were stored inverted at4°C until further use. 3.5.2 Preparation of MacConkey Agar: MacConkey agar was prepared according to the manufacturers instruction: 4.6 grams of MacConkey agar powder were weighed and added to1 liter of distilled water in a heat-resistant glass flask..The mixture was stirred to ensure complete dissolution of the agar powder. The prepared MacConkey agar solution was then autoclaved at 121°C for 15 minutes to sterilize the medium and eliminate any microbial contaminants. After autoclaving, the agar solution was cooled to approximately45-50°C in a water bath. Subsequently, the molten agar was poured into sterile Petri dishes under aseptic conditions and allowed to solidify at room temperature. The prepared MacConkey agar plates were stored inverted at4°C until further use. 3.5.3 Preparation of Mannitol Salt agar Mannitol Salt agar was prepared according to the manufacturers instruction: 111 grams of Mannitol Salt agar powder were weighed and added to1 liter of distilled water in a heat-resistant glass flask..The mixture was stirred to ensure complete dissolution of the agar powder. The prepared Mannitol Salt agar solution was then autoclaved at 121°C for 15 minutes to sterilize the medium and eliminate any microbial contaminants. After autoclaving, the agar solution was cooled to approximately 45-50°C in a water bath. Subsequently, the molten agar was poured into sterile Petri dishes under aseptic conditions and allowed to solidify at room temperature. The prepared Mannitol Salt agar plates were stored inverted at 4°C until further use. 3.5.4 Preparation of salmonella shigella agar Salmonella shigella agar was prepared according to the manufacturers instruction: 63 grams of salmonella shigella agar powder were weighed and added to1 liter of distilled water in a heat-resistant glass flask..The mixture was stirred to ensure complete dissolution of the agar powder. The prepared agar solution of salmonella shigella was then autoclaved at 121°C for 15 minutes to sterilize the medium and eliminate any microbial contaminants. After autoclaving, the agar solution was cooled to approximately 45-50°C in a water bath. Subsequently, the molten agar was poured into sterile Petri dishes under aseptic conditions and allowed to solidify at room temperature. The prepared agar plate of salmonella shigella were stored inverted at 4°C until further use. 3.5.5 Preparation of Eosin Methylene blue agar Eosin Methylene blue agar was prepared according to the manufacturers instruction: 36 grams of agar powder Eosin Methylene blue were weighed and added to1 liter of distilled water in a heat-resistant glass flask..The mixture was stirred to ensure complete dissolution of the agar powder. The prepared agar solution Eosin Methylene blue was then autoclaved at 121°C for 15 minutes to sterilize the medium and eliminate any microbial contaminants. After autoclaving, the agar solution was cooled to approximately45-50°C in a water bath. Subsequently, the molten agar was poured into sterile Petri dishes under aseptic conditions and allowed to solidify at room temperature. The prepared agar plates of Eosin Methylene blue were stored inverted at4°C until further use. 3.6 isolation of bacteria The samples collected from vegetable vendor within Bayero university kano were divided into 4 different portion each and treated with different method of washing and reducing microbial load on cabbage (vegetable) making a total of 8 samples 3.6.1 serial dilution After enrichment all the 18 samples Serial dilution was made from the stock of the peptone water into five universal test tubes containing 9mls of sterile distilled water each and labeled 10-1, 10-2, 10-3, 10-4, 10-5, with the use of sterile dropping pipette, 1ml of the sample in solution was pipetted into the first universal test tubes, therefore making 10-5 dilution, this process was repeated serially to obtain, 10-2, 10-3, 10-4, 10-5, dilutions (Public Health England (2019). 3.6.2 Enumeration of Coliform Bacteria The Enumeration of coliform and faecal coliform bacteria was carried out using the Most Probable Number (MPN) method as described by APHA, (2005) as follows: Presumptive Test Coliform count was obtained using the three tube assay of MPN technique. This was carried out using lactose broth. The first set of three tubes had sterile 10ml double strength lactose broth (DSLB) and the second and third set had 10ml single strength lactose broth (SSLB). All the tubes contained Durham tubes before sterilization. The three set of tubes received 10, 1 and 0.1 ml of water sample using sterile pipettes. The tubes were incubated at 37°C for 24 hours for the estimation of total coliforms and at 44.5°C for faecal coliform for 48 hours. It was then examined for acid and gas production. The MPN was determined from the MPN table for the three set of tubes (Bakare et al; 2005). Confirmed Test Confirmed test was carried out by streaking a loop full of culture from the positive tubes of the presumptive test onto Eosine Methylene Blue (EMB) agar for pure colonies and incubated at 37°C for 24 hours for total coliforms and 44.5°C for 48 hours for feacal coliforms . Formation of colonies with green metallic sheen was indicative of E. coli and pinkish colonies indicate presences of Enterobacter aerogenes ( Coyne and Howell,1994). Completed Test This was carried out by transferring cells from an isolated colony on Eosine methylene blue (EMB) onto an agar slant and again inoculating it into a sterile tube of lactose broth containing inverted Durham tube. This was then incubated at 37°C for 24 hours for total coliforms and 44.5°C for 48 hours for faecal coliforms. Acid and gas production was indicative of the presence of coliforms (at 37°C) and faecal coliforms (at 44.5°C) (Coyne and Howell,1997). 3.6.3 Aerobic Mesophilic Bacteria Count One mile (1ml) of sample from each test tube of 10-4 and 10-5 dilutions were transferred to the appropriately labelled petri dishes. Fifteen (15) ml of the Sterilized Nutrient agar were poured into each petri dish. The dilution and agar media were mixed thoroughly and uniformly and allowed to solidify. After solidification, the plates were incubated for 18 - 24hours at 37°C (Cheesbrough, 2006). Emerging colonies on the plates were counted as colony forming units per milliliter (CFU/ml). 3.6.4 innoculation of the sample From 10-5 solution, streaked method was made on the selective media using sterile wire loop onto MSA, EMB and SSA and After which it was incubated at 37oC for 18-24hr (Xianzhou et al., 2017). 3.7 identification of the bacteria 3.7.0 Characterization of Isolate and identification Identification of isolates was confirmed by Gram staining, cultural, morphological and biochemical characterization using routine laboratory techniques according to Oleghe et al (2020) 3.7.1 Gram staining and Microscopy 3.7.2 Gram staining Gram staining is done to differentiate Gram positive bacteria from Gram negative organisms. This was performed as described by cheesebrough, (2005). A thin smear of bacterial isolates were made with a sterile wireloop on a clean glass slide containing a drop of saline. The smear was air dried and heat fixed by passing over a spirit lamp few times. The fixed smear was then flooded with crystal violet stain for 60 seconds and was washed off with clean water. The smear was flooded with Lugol’s iodine for 60 seconds and the excess washed off with clean water. It was then decolorized rapidly with alcohol and washed off immediately with clean water. The smear was counter stained with safranin for 60 seconds and the excess stain was washed off using clean water. The slide was air dried and a drop of immersion oil was added on the Gram stained smear, this was viewed under the microscope using x100 objective lense to identify the Gram reaction of the isolated organisms (Cheesebrough, 2005). Observed morphology and Gram reactions were recorded. 3.8 Biochemical tests All analyses were performed in duplicates.The characteristic colonies were identified on the basis of IMVIC, Methyl red and Voges proskaeur reaction (OXOID U.K), citrate utilisation (Oxoid, UK). Catalse and coagulase tests were also utilized. Biochemical tests are among the most important methods for microbial identification. 3.8.1 Catalase test This test is done to determine the ability of certain microorganisms to produce the enzyme catalase which catalyses the release of oxygen from hydrogen peroxide. That is, it oxidizes hydrogrn peroxide (H2O2) to water (H2O) and oxygen (O2). Procedure: This test was carried out by transfering an isolate (test organism) not more than 24 hours old onto a clean grease free glass slide containing two drops of 6% hydrogen peroxide. A sterile applicator stick was used to pick a colony of the organism and immerse in the hydrogen peroxide on the slide. The presence of effervescence indicates a positive result but the absence of bubbles indicates negative result (cheesebrough, 2006). 3.8.2 Coagulase test: Coagulase test is used to differentiate Staphylococcus aureus (positive) from Coagulase Negative Staphylococcus (CONS). Coagulase is an enzyme that converts (soluble) fibrinogen in plasma to (insoluble) fibrin. Principle: Coagulase is an enzyme produced by specific bacteria (Staphylococcus aureus) that causes plasma to clot by converting fibrinogen to fibrin. Procedure: The method was carried out according to slide test method of Cheesbrough (2006). A drop distilled water was placed on each end of a slide. A colony of the test organism (previously checked by Gram staining) was emulsified in each of the drops to make two thick suspensions. A loopful (not more) or plasma was added to one of the suspensions, and mixed gently. Presence of clumping of the organisms within 10 seconds was observed, no plasma is added to the second suspension. This is used to differentiate any granular appearance of the organism from true coagulase clumping. 3.8.3 Indole Tests: Indole test is done to check if an organism can degrade amino acid tryptophan to produce indole. Test tubes containing 4 ml of sterilized peptone broth were inoculated with test organism using an inoculating loop, the inoculating loop was flamed red hot and allowed to cool and then used to pick the organism which was then transferred to the sterile broths. The test tubes were incubated at 37oC for 24 hours. After 24 hours, 0.5 ml of Kovac’s regent was added to check for the indole production, the observation of a cherry red colored ring indicates the production of indole (Vashist et al., 2013). 3.8.4 Methyl Red Test: This test is used to check if an organism can produce enough acid during the fermentation of glucose. Test tubes containing 4 ml of sterilized MR-VP broth was inoculated with test organisms and incubated at 37°C for 24 hours. After the incubation, methyl red indicator was added. A bright red colour indicates a positive result while a yellow/orange colour indicates a negative result (Vashist et al., 2013). 3.8.5 Voges Proskauer Test: This test is used to check for the presence of acetoin in a bacterial broth culture. Test tubes containing 4ml of sterilized MR-VP broth was inoculated with test organisms and incubated at 370C for 24 hours. After the incubation, 0.6 ml of alpha naphtanol was added and then 0.2 ml 40 % potassium hydroxide (KOH) was added. A bright/cherry red colour is observed for a positive result and yellow/orange colour indicates a negative result (Vashist et al., 2013). 3.8.6 Citrate test The citrate test diffentiates bacteria according to their ability to use citrate as the sole souce of carbon and energy. The isolates were inoculated on the media containing sodium citrate. Procedure: The colonies of organisms were picked up from the subculture of the pure bacteria isolates using a sterile wireloop and inoculated into the slope/slant by stabbing the suface of the slant repeatedly such that the isolates were taken just beneath the surface of the Simon citrate agar and incubated overnight at 370C. If the organism has the ability to utilize citate as a sole source of carbon, the medium changes color from green to bright blue (Cheesebrough, 2006). 3.9 Statistical Analysis Descriptive statistics: This was employed to summarize and describe the characteristics of the Enterobacteriacea found on the African Star Apple. Inferential statistics: Correlation Analysis was employed in this research to check if there are any relationships between different variables related to the Enterobacteriacea and the African Star Apple, such as environmental factors or geographical location. CHAPTER FOUR 4.0 RESULT The findings from Table 4.1 indicate that cabbage samples from both restaurants and vendors at Bayero University Kano have some level of bacterial contamination. All cabbage samples, regardless of source (restaurant or vendor), have a measurable average mesophilic bacterial count (AMBC) expressed in Colony Forming Units per milliliter (CFU/ml). This suggests that all the samples had some bacterial growth. On average, restaurants seem to have higher bacterial counts compared to vendor samples. This difference could be due to various factors like storage practices, hygiene during preparation, or the initial quality of the cabbage received. The table reveals possible coliform contamination in some samples from both restaurants and vendors. This is indicated by the Most Probable Number (MPN) values, but these require referencing a separate MPN table to determine the exact estimated number of coliform bacteria per milliliter. Coliforms can be indicative of fecal contamination, so their presence is a concern. Table 4.1 Mean Aerobic Mesophilic Bacterial count of Cabbage Samples from Restaurants and Vegetable Vendors at Bayero University Kano Old and New Campus. Restaurant (cfu) AMBC MPN VALUE Vendors (cfu) AMBC MPN VALUE Masalaha 2.6 ± 0.2 107 2 V1 FOM 1.9 ± 1.1 107 4 2.7 ± 0.2 107 2 V1 FOE 2.5 ± 0.4 107 4 Chickenzza 2.3 ± 0.3 107 6 V2 FNM 2.2 ± 0.2 107 4 2.5 ± 0.2 107 6 V2 FNE 2.35 ±- Donken’s 1.5 ± 0.1 107 5 V1 FNM 2.3 ± 0.1 107 2 1.6 ± 0.2 107 2 V1 FNM 2.4 ± 0.0 107 4 Chips & Chicken 1.8 ± 0.1 107 6 V2 FNM 2.1 ± 0.2 107 4 2.2 ± 0.2 107 4 V2 FNM 2.9 ± 0.1 107 4 Alkhairat 1.9 ± 0.1 107 4     2.0 ±-       p-value = 0.619 KEY:cfu = Colony forming unit,WHO= world health organization, V1 FOM = Vendor 1 from old site (morning), V1 FOE = Vendor 1 from old site (Evening) V2 FNM = vendor 2 from Newsite (morning), V2 FNE = Vendor 2 from newsite (Evening) AMBC = Aerobic Mesophillic Bacterial Count Table 4.2 examines the effectiveness of different decontamination techniques on reducing bacterial contamination in cabbage samples collected from vendors at Bayero University Kano. All cabbage samples, regardless of vendor or washing method (V1 FOM, V1 FOE, etc.) from the previous table, had some level of bacterial contamination as indicated by the tap water treatment results. The values (e.g., 3.5 x 10^5 CFU/ml) represent the average bacterial count after rinsing with tap water. Vinegar appears to be the most effective decontamination technique. Compared to tap water and salt, vinegar treatment significantly reduced the bacterial count in all samples (indicated by lower CFU values in the vinegar column). In most cases, the vinegar treatment resulted in a reduction to below detectable levels. Salt solution (indicated in the "Salt" column) showed some effectiveness in reducing bacterial counts compared to tap water. However, the reduction wasn't as significant as with vinegar. Rinsing with tap water (baseline treatment) had minimal impact on the bacterial count. Table 4.2 Mean Aerobic Mesophilic Bacterial Count of Cabbage Treated with different decontamination techniques from Vendors in both old and new campus Bayero University, Kano Sample treated Sample from vendors Tapwater (cfu) MPN VALUE Salt (cfu MPN VALUE Vinegar (cfu) MPN VALUE V1 FOM 3.5 ±- ±- ± 0.1 104 - V1 FOE 2.9 ±- ±- ± 0.1 104 - V2 FNM 5.9 ±- ±- ± 0.1 103 - V2 FNE 2.2 ±- ±- ± 0.2 103 - V1 FNM 4.3 ±- ± - ± 0.2 104 - V1 FNM 4.5 ±- ±- ± 0.3 104 - V2 FNM 3.2 ±- ±- ± 0.1 103 - V2 FNM 3.3 ±- ±- ± 0.1 103 - p-value = 0.1312 WHO Standard = 1.0 x 105 (2015)Coliform (0) Mendonca et al., (2020) KEY:cfu = Colony forming unit,WHO= world health organization, TNC= Too numerous to count. V1 FOM = Vendor 1 from old site (morning), V1 FOE = Vendor 1 from old site (Evening) V2 FNM = vendor 2 from Newsite (morning), V2 FNE = Vendor 2 from newsite (Evening) Table 4.3 sheds light on the types of bacteria lurking in the cabbage samples. This table combines Gram staining, which differentiates bacteria based on cell wall structure, with various biochemical tests to pinpoint the likely culprits. The results reveal a concerning mix of both Gram-positive and Gram-negative bacteria. Among the most worrisome finds are Escherichia coli (F1), which can trigger foodborne illness, and Shigella spp (F3), known for causing shigellosis, a diarrhoeal ailment. Salmonella spp (F4), another foodborne pathogen, was also identified. Klebsiella pneumoniae (F2) is another unwelcome guest, potentially causing infections like pneumonia. The table also unveils Bacillus sp (F5), which can encompass both beneficial bacteria used in fermentation and spoilage organisms. Staphylococcus aureus (F6) rounds out the identified bacteria, potentially causing skin infections or food poisoning. Table 4.3 Gram staining and Biochemical Test of the Isolates Isolate code GSR CAT CIT IDT VPT MR COA Suspected Organism F1 - + - - - + - Escherichia coli F2 - + + - + - - Klebsiella pneumoniae F3 - + - + - + - Shigella spp F4 - + - - - + - Salmonella spp F5 + + - - - - - Bacillus sp F6 + + - - + - + Staph Aureus KEY: F1 = Isolate 1GSR = Gram Staining Reaction VPT = Voges-proskacur Test F2 = Isolate 2CAT = Catalase Test + = Positive F3 = Isolate 3CIT = Citrate Test- = Negative F4 = Isolate 4IDT = Indole Test F5 = Isolate 5COA = Coagulase F6 = Isolate 6 MR = Methyl Red Table 4.4 provides a breakdown of the bacteria identified in cabbage samples collected from various sources. The table reveals a diverse range of bacteria present in the cabbage samples, including Pathogenic Bacteria which can cause illness if consumed, and include E. coli, Salmonella, Shigella, Klebsiella, and Staphylococcus aureus. Their presence is a significant concern. Bacillus sp. which is a large genus with some beneficial species and others that can cause spoilage. Most samples (except sample B) seem to be contaminated with at least one type of bacteria. There's no clear link between the vendor washing methods (V1 FOM, V1 FOE, etc.) and the presence or absence of specific bacteria. Table 4.4 The bacteria isolate from the sample collected. Samples E. coli S. aureus Salmonella Shigella Klebsialla Bacillus A - + - - - + B - - - + - + C + + - - - - D - - + - + - E - + - - - - F + + - - - - G + + - + - - H + + - + - - I + - + - - - J + - - - + - V1 FOM + - + + + + V1 FOE + - + + + + V2 FNM + - + + - - V2 FNE + - + + - - V1 FNM + - + + - - V1 FNM + - - + - - V2 FNM + - - + - - V2 FNM + - - + - - Key terms: + = present - = absent Table 4.5 reveals a worrying trend in the types and frequency of bacteria found in the cabbage samples. The table showcases a concerning variety of bacteria, including pathogenic culprits like E. coli, Shigella spp., Salmonella spp., and Staphylococcus aureus. These bacteria can wreak havoc on our digestive systems if ingested. E. coli takes the lead with the highest number of contaminated samples (14, translating to 26.92%) followed closely by Shigella spp. (11 samples, or 21.15%). While the table also identifies Enterobacter sp. and Bacillus sp., some Bacillus species can actually be beneficial, being used in fermentation processes. However, other Bacillus species can cause spoilage. The high prevalence of these bacteria across a significant portion of the samples underscores the potential health risks associated with consuming the cabbage. It reinforces the importance of implementing stricter hygiene practices throughout the entire process of handling cabbage, from farm to table. Table 4.5 Frequency of Distribution and percentage of occurrence Frequency Percentage of occurrence E. coli 14 26.92 K. pneumonia 5 9.62 Shigella spp 11 21.15 Salmonella spp 7 13.46 S. aureus 6 11.54 Enterobacter sp 5 9.62 Bacillus sp 4 7.69 Total 52 100 KEY: % = Percentage, - = Absent, FQ = Frequency, A-J = Sample from Restaurants, K-R = Sample from Vendors Fig. 1. Percentage of Occurrence of Organism Isolated from the Cabbage Sample CHAPTER FIVE DISCUSSION, CONCLUSION AND RECOMMENDATIONS 5.1 Discussion The tables presented (4.1 - 4.5) paint a concerning picture of bacterial contamination in cabbage samples collected from various sources at Bayero University Kano. These findings raise significant public health concerns and necessitate stricter hygiene practices throughout the entire cabbage supply chain. A troubling aspect is the near-universal presence of bacterial contamination across all cabbage samples, regardless of whether they came from restaurants or vendors (Table 4.1). This aligns with research by Allam et al. (2014) who reported similar contamination on vegetables sold by street vendors in Egypt. It highlights the vulnerability of cabbage to contamination at various points along its journey from farm to plate. While Table 4.1 suggests a trend of higher bacterial counts in restaurant samples, drawing definitive conclusions about source-based contamination is difficult. The study itself points towards factors like storage practices and initial cabbage quality as influencing bacterial levels. Further investigation into these areas would be necessary. However, these findings echo those of Olafadehan et al. (2018) who found higher bacterial counts on ready-to-eat vegetables compared to whole ones. This suggests an increased risk of contamination during preparation in restaurants. The presence of coliform bacteria, indicated by MPN values in Table 4.1, is particularly alarming. Coliforms can be a sign of fecal contamination, posing a serious health threat. This aligns with the concerns raised by Doyle and Erickson (2006) regarding the potential for fecal pathogens on fresh produce due to factors like contaminated irrigation water. Thankfully, Table 4.2 offers a beacon of hope. It convincingly demonstrates vinegar as the most effective decontamination technique among those tested. This is consistent with the findings of Sharma and Singh (2014) who showed vinegar's effectiveness in reducing bacterial contamination on vegetables. Both studies suggest vinegar as a simple and potentially practical method for decontamination. Table 4.3 unveils a concerning range of bacteria lurking within the cabbage samples. It identifies pathogenic culprits like E. coli, Shigella spp., Salmonella spp., and Staphylococcus aureus. This mirrors the findings of Beuchat and Ryu (1997) who found similar pathogens on fresh vegetables. The presence of these bacteria underscores the potential for severe illness if proper hygiene practices are not followed. Table 4.4 and 4.5 further emphasize this point by revealing the presence of Bacillus sp. While some Bacillus species can be beneficial (used in fermentation), others can cause spoilage. This highlights the importance of proper identification beyond just genus to determine the specific threat posed by these bacteria. Table 4.5 provides the final piece of the puzzle, revealing a disturbingly high prevalence of pathogenic bacteria across a significant portion of the cabbage samples. This is similar to findings reported by Oladayo et al. (2017) who documented high bacterial contamination rates on vegetables sold by vendors in Nigeria. The high prevalence underscores the urgency for implementing stricter hygiene measures throughout the cabbage handling chain. 5.2 Conclusion This study investigated the bacterial contamination of cabbage samples collected from various sources at Bayero University Kano. The findings reveal a widespread presence of bacteria, including concerning pathogenic types, across a significant portion of the samples. This highlights a potential public health risk associated with consuming raw or undercooked cabbage. 5.3 Recommendations 1. The University should develop stricter hygiene measures by collaborating with farms and vendors to improve hygiene practices throughout the cabbage supply chain, from farm to table. This includes proper sanitation, storage temperatures, and staff training. 2. 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APPENDIX Morphological Characteristics of the Isolates Isolate codeCultural Characteristics Mackonkey agar Nutrient agar EMB agarSSA F1Red colonies Greyish colonies Dark purple with green - Metallic sheen colonies F2Large mucoid to red Pink to red colonies Pink to red with green - Colonies metallic sheen colonies F3Circular, smooth and Milky colonies - Colorless colonies Opaque colonies F4Clear colonies Milky colonies- Smooth black Centered colonies F5 Milky colonies Milky colonies-- KEY: F1 = Isolate 1EMB = Eosin methylene bleu F2 = Isolate 2SSA = Salmonella-Shigella Agar F3 = Isolate 3 F4 = Isolate 4 Anova: Single Factor |SUMMARY Groups Count Sum Average Variance - - ANOVA Source of Variation SS df MS F P-value F crit Between Groups- Within Groups- Total-         Anova: Single Factor SUMMARY Groups Count Sum Average Variance - - - ANOVA Source of Variation SS df MS F P-value F crit Between Groups- Within Groups- Total-