ASSESSMENT OF MICROBIOLOGICAL SPOILAGE OF WATERMELON

ASSESSMENT OF MICROBIOLOGICAL SPOILAGE OF WATERMELON BY KILANI, HAMEEDAH MOTUNRAYO U2019/5555046 A RESEARCH PROJECT SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY FACULTY OF SCIENCE UNIVERSITY OF PORTHARCOURT RIVERS STATE, NIGERIA. DEPARTMENT OF MICROBIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENT OF THE AWARD OF BACHELOR OF SCIENCE (B.Sc.) IN MICROBIOLOGY NOVEMBER, 2024. DECLARATION I, KILANI, HAMEEDAH MOTUNRAYO with Matriculation Number U2019/5555046 declare that this project on ASSESSMENT OF MICROBIOLOGICAL SPOILAGE OF WATERMELON was carried out by me, that it is my original work and that it has not been submitted, either wholly or in part, for the award of degree in this or another institution. KILANI, HAMEEDAH MOTUNRAYO Signature Date ————------------- U2019/5555046 Confirmation by Supervisor DR SIXTUS .E. IBEKWE Signature Date ————-------------   CERTIFICATION The Board of Examiners hereby certified that this research project was carried out by KILANI, HAMEEDAH MOTUNRAYO with Matriculation Number U2019/5555046 and has been read and approved as meeting the requirements for the award of Bachelors of Science (B.Sc.) Degree, in the Department of Microbiology, Faculty of Science, University of Port Harcourt. DR SIXTUS .E. IBEKWE _____________ ____________ Supervisor Signature Date Prof. C. B. Chikere _____________ ____________ Head of Department Signature Date Microbiology Prof. N. C. Ehirim _____________ ____________ Dean of Faculty of Science Signature Date …………………… _____________ ____________ External Supervisor Signature Date   DEDICATION This work is dedicated to almighty Allah, to my parents and siblings for their unending support and assistance. ACKNOWLEDGEMENT My sincere gratitude goes to Almighty Allah for His grace and Protection upon my life and throughout my project practical and research work. I wish to Appreciate my project supervisor DR. SIXTUS IBEKWE for his guidance, recommendation and teachings during the practical . I also wish to Appreciate my HOD Prof Blaise Chickere, Prof Mathew Wegwu, Prof frank Peterside, Dr Josiah Hart. for your unending love and support throughout my stay in uniport I wish to appreciate my parent Prof and Mrs. Abdulrazaq Kilani for their unending love, care, moral, emotional and financial support, I also wish to Appreciate some important people in my life my dad prof Abdulrazaq Kilani for your words of encouragement always my sister Mudafiah ,my brothers Husayn and Abdulraziq ,Bro Idris, Farouk, , , for their unending love and support throughout my stay in Uniport. and my friends Barakah and Islamiyah. I also want to say a big thank you to the laboratory technologist MR AUSTIN and everyone I worked with for their utmost support, guidance understanding and willingness to impact knowledge throughout. ABSTRACT Watermelon spoilage is a common issue that leads to significant post-harvest losses, often resulting from microbial activity. This study aims to assess microbial spoilage in watermelon samples, focusing on the presence and impact of bacteria and fungi as primary spoilage agents. Samples of spoiled watermelon were collected from various locations and analyzed to identify the microbial communities responsible for deterioration. Both bacteriological and mycological examinations were conducted using selective media to isolate and identify bacteria and fungi associated with the spoilage process. Common bacterial isolates include staphylococcus and Pseudomonas spp., while fungal isolates often include candida species. The findings of this study highlight the importance of microbial contamination in watermelon spoilage and provide insights into potential prevention methods. Understanding the microbial profile of spoiled watermelons could aid in developing better storage and handling practices to minimize waste and enhance fruit quality .watermelon samples showing sign of spoilage were collected from locations Alakahia ,Rumualogu ,and choba the outer rind was disinfected with ethanol to focus on internal spoilage ,The flesh was homogenized using sterile tools, The portion of the homogenized sample were prepared for microbiological analysis. sterile media without any sample inoculation were incubated this ensured that any microbial growth observed in the test samples originated from the watermelon and not external contamination. .These control helped validate the finding and minimize errors,ensuring that the observed microbial growth was solely due to the spoilage of watermelon samples.   TABLE OF CONTENT Title Page i Declaration ii Certification iv Dedication v Acknowledgement vi Abstract vii List of Tables ix List of Figures x Table of Content viii CHAPTER ONE: INTRODUCTION 1.1 Background to the study 1 1.2 Statement of the Problem 2 1.3 Aims and objective of the study 2 1.4 Significance of the study 2 CHAPTER TWO: LITERATURE REVIEW 2.1 General Overview of Watermelon (Citrullus lanatus) 3 2.1.1 Origin and early history of watermelon 3 2.1.2 Spread of watermelon to other part of the world 4 2.1.3 Early uses of watermelon 5 2.1.4 Nutritional importance of watermelon 7 2.1.5 Economic importance of watermelon in Nigeria 8 2.1.6 Fungal spoilage of watermelon 10 CHAPTER THREE: MATERIAL AND METHODS 3.1 Study area 11 3.2 Materials 11 3.3 Sample Collection 12 3.4 Preparation of Culture Media 12 3.5 Microbial Isolation and Culturing 13 3.5.2 Serial Dilution 13 3.5.3 Inoculation and Incubation Bacterial Isolation 13 3.5.4 Microbial Identification 14 3.5.5 Gram Staining for Bacteria 14 3.5.6 Biochemical Tests for Bacteria 14 3.5.7 Fungal Identification 15 3.5.8 Enumeration of Microbial Load 15 CHAPTER FOUR: RESULTS AND DISCUSSIONS 4.1 Results 16 4.2 Discussion 26 CHAPTER FIVE: SUMMARY CONCLUSION AND RECOMMENDATIONS 5.1 Summary of the findings 28 5.2 limitations of the study 28 5.3 Conclusion 29 5.4 Recommendation 39 References LIST OF TABLES Table 4.1.1: Total Heterotrophic bacteria count of spoilt watermelon samples gotten from Choba 16 Table 4.1.2: Staphylococcus count of spoilt watermelon samples from choba 16 Table 4.1.3: Pseudomonas count of spoilt watermelon samples 17 Table 4.1.4: Total Heterotrophic bacteria count of Spoilt Watermelon samples gotten from Alakahia 18 Table 4.1.5 Staphylococcus count of spoilt water melon samples 18 gotten from Alakahia Table 4.1.6 Pseudomonas count of spoilt water melon samples gotten from Alakahia 19 Table 4.1.7 Total Heterotrophic bacteria count of spoilt water melon samples l from Rumualogu 20 Table 4.1.8: staphylococcus count of spoilt watermelon samples from Rumualogu 20 Table 4.1.9: Pseudomonas count of spoilt watermelon samples from Rumualogu 21 Table 4.1.10 Morphological/Biochemical Characterization of Bacterial Isolates from spoilt watermelon samples studied 23 Table 4.1.11 Cultural characteristics and cell morphology of yeast isolate from spoilt watermelon 24   LIST OF FIGURES Figure 4.1: Percentage Occurrences of Bacteria Isolates from Spoilt watermelon samples 25 CHAPTER ONE INTRODUCTION 1.1 Background to the study The study is made to understand types and behavior of microorganisms associated with the spoilage of watermelon widely consumed as perishable fruit. However like many other fruit watermelon is susceptible to microbial spoilage ,especially during post harvest handling and storage leading to quality deteriration and food safety . Citrullus lanatus (watermelon) is a popular fruit due to its high-water content and nutritional benefits which include vitamins, mineral and antioxidant. However, it’s high moisture content and nutrient rich composition makes it highly susceptible to microbial contamination and spoilage. (Ali et al., 2019) yeasts, and mould. These microorganisms could lead to off flavour, odours, texture change and even potentially harmful substances, Bacteria such as Psedomonas spp, Lactic acid bacteria, Enterobacteriaceae. Yeast such as Candida and Saccharomyces Spp which can ferment sugar in watermelon leading to alcohol production and spoilage. Moulds such as Aspergillus and Penicillium spp which can grow on the surface of the fruit especially in humid conditions. Spoilage of watermelon not only leads to food waste but also poses a risk (kumar et al,.2020) to public health. Consumption of spoiled Watermelon can lead to food borne illness caused by pathogenic microorganisms or their toxins. 1.2 Statement of the Problem The economic loss triggered by farmers, dealer and sellers of watermelon as a result of spoilage microorganism and measures to be taken mitigate the effect of their microbial agents in the crux of the study(Ezugwu,I.,&Nwobodo,C.2021) 1.3 Aims and objective of the study The aim of the study is to isolate and identify the microbial spoilage organisms in spoilt watermelon sold in selected market within Obio Akpor LGA, Rivers State. The objective of the study is: 1. To identify and characterize the microbial spoilage species responsible for the spoilage of Watermelon 2. To provide insight into the spoilage mechanism of watermelon which can inform better handling, storage and preservation method in order to extend the shelf life of the fruit. 1.4 Significance of the study Studying the microbial spoilage in spoilt watermelon is crucial because it helps us understand the stuff that makes our watermelon go bad. By figuring out which germs are causing the spoilage, we can learn how to keep our watermelons fresh for longer. This knowledge can lead to better storage practices, improved food safety, and less wastage of delicious watermelons. CHAPTER TWO LITERATURE REVIEW 2.1General Overview of Watermelon (Citrullus lanatus) 2.1.1Origin and early history of watermelon Watermelon (Citrullus lanatus) is thought to have originated in Africa, particularly in the northeastern region, around present-day Sudan and southern Egypt. The wild ancestor of the modern watermelon, Citrullus colocynthis, was a drought-resistant plant that provided a source of water in arid regions. Archaeological evidence suggests that watermelon was domesticated over 4,000 years ago by ancient Egyptians, who cultivated it primarily for its water content rather than sweetness.(Renner et al.,2017) By the 10th century, watermelon had spread to southern Europe and the Middle East. From there, it traveled via trade and exploration to China, which is now the largest producer of watermelon globally. Watermelon was brought to the Americas by European colonists and enslaved Africans during the transatlantic slave trade in the 16th century, where it quickly became a popular crop, especially in the southern United States. Over the centuries, watermelon underwent selective breeding to enhance its sweetness, size, and flesh color, evolving into the modern varieties that are widely enjoyed today.(Manniche,1989) 2.1.2 Spread of watermelon to other part of the world The spread of watermelon (Citrullus lanatus) from its African origin to other parts of the world followed a path shaped by trade, exploration, and migration. Here is an overview of its global spread: 1. Africa (Prehistoric – Ancient Times): Watermelon originated in northeastern Africa, where early varieties were domesticated in regions like Sudan and Egypt over 4,000 years ago. The fruit’s ability to store water made it valuable in arid regions, and it spread across Africa through indigenous cultivation. 2. Egypt and the Mediterranean (2000 BCE): Watermelon was cultivated by ancient Egyptians, with seeds and depictions found in tombs. From Egypt, trade routes facilitated the spread of watermelon to the Mediterranean, including Greece and Rome, where it was appreciated for its refreshing qualities.(Manniche ,1989) 3. Middle East and India (7th – 10th centuries): Watermelon was introduced to the Middle East through Arab traders. By the 10th century, it had spread to Persia, where it became a popular fruit. From there, it reached India, further expanding its presence in South Asia. 4. China (10th century): By the 10th century, watermelon had reached China via the Silk Road and trade routes. China became a major center of watermelon cultivation and remains the largest producer of watermelon globally today. 5. Southern Europe (11th – 13th centuries): Watermelon was introduced to southern Europe during the Moorish conquest of Spain. It spread throughout the Iberian Peninsula and Italy, eventually reaching France and other parts of Europe, where it was grown in warmer regions. 6. The Americas (16th century): Watermelon was brought to the Americas by European colonists and enslaved Africans during the 16th century. It first arrived in the Caribbean and then spread to South and North America, becoming a staple crop in the southern United States. It thrived in the warm, humid climates of the region. 7. Global Expansion (17th – 19th centuries): Through European colonization and trade, watermelon was introduced to regions across the world, including Southeast Asia, the Pacific Islands, and Australia. It became a popular fruit in tropic2 subtropical climates worldwide. Today, watermelon is grown in over 100 countries, with modern varieties bred for sweetness, size, and other desirable traits. The spread of watermelon illustrates its adaptability to different climates and its lasting appeal as a source of refreshment and nutrition. 2.1.3 Early uses of watermelon Early uses of watermelon varied depending on the region and time period, but the fruit's unique characteristics made it valuable in multiple ways: 1. Hydration: In its early domesticated forms, watermelon was primarily valued for its water content. In arid regions like northeastern Africa, where the fruit originated, it provided a critical source of hydration, especially during dry seasons when water was scarce. The ability to store water in the fruit made it essential in regions with limited water sources.(Paris,2015) 2. Food: Ancient Egyptians and other early civilizations ate the flesh of the watermelon, though early varieties were not as sweet as modern ones. The fruit was often used as a refreshing snack or included in meals, especially during the hot summer months. 3. Seeds: Watermelon seeds were also consumed as a food source. Roasting watermelon seeds was a common practice in many cultures, and they were considered a nutritious snack rich in fats and proteins. 4. Medicine: In some cultures, watermelon was used for medicinal purposes. The cooling and hydrating properties of the fruit were thought to help treat heatstroke and dehydration. In traditional African and Middle Eastern medicine, parts of the watermelon plant, including seeds and rind, were used to promote health and treat illnesses related to the digestive system.(Manniche,1989) 5. Ceremonial and Cultural Uses: Watermelon had symbolic significance in some ancient cultures. For instance, seeds of the fruit were found in Egyptian tombs, suggesting that it played a role in burial rites and was valued in the afterlife as a source of sustenance.(Chomicki&Renner ,2015) 6. Livestock Feed: In some regions, early varieties of watermelon, particularly those that were less sweet, were fed to livestock. The rind and flesh provided a source of moisture and nutrients for animals. These early uses demonstrate that watermelon was an important resource for both humans and animals, prized not only for its edible qualities but also for its utility in various aspects of daily life. 2.1.4 Nutritional importance of watermelon Watermelon is not only a refreshing fruit but also highly nutritious. Its key nutritional benefits include: 1. Hydration: Watermelon is about 90% water, making it excellent for hydration, especially in hot weather. 2. Low in Calories: It’s low in calories (about 30 calories per 100 grams), making it a great snack for those watching their weight. 3. Rich in Vitamins • Vitamin C: Helps boost the immune system and promotes skin health. • Vitamin A: Important for eye health and skin health. 4. Antioxidants: • Lycopene: A powerful antioxidant that gives watermelon its red color and has been linked to heart health and reduced cancer risk. • Cucurbitacin E: Another antioxidant with anti-inflammatory properties. 5. Minerals: • Potassium: Supports heart health, regulates blood pressure, and aids in muscle function. • Magnesium: Helps with muscle and nerve function, blood sugar regulation, and bone health. 6. Amino Acids: • Citrulline: May improve exercise performance and reduce muscle soreness. 7. Fiber: Watermelon contains small amounts of dietary fiber, which is good for digestion. Incorporating watermelon into the diet provides a sweet, hydrating, and nutrient-packed boost. 2.1.5 Economic importance of watermelon in Nigeria Watermelon (Citrullus lanatus) plays a significant economic role in Nigeria for several reasons: 1. Income Generation for Farmers: Watermelon farming provides a source of livelihood for many Nigerian farmers, especially in regions like the North where the climate supports large-scale production. The crop is relatively easy to grow, and its high demand makes it a profitable venture.(Eke-Okoro et al.,2019) 2. Employment Opportunities: The watermelon value chain—from cultivation, harvesting, transport, and marketing to street vendors—creates various job opportunities. This contributes to reducing unemployment, especially in rural areas.(ojo &Adeoye 2020) 3. Health and Nutrition: Watermelon is a rich source of vitamins, antioxidants, and hydration. Its health benefits make it a popular fruit, contributing to the overall health and well-being of Nigerians. Increased demand for nutritious fruits can spur growth in local production, further stimulating the agricultural sector.(Ajewole&Adeogun,2018) 4. Export Potential: Although local consumption is high, watermelon has the potential for export, especially to neighboring countries. This could enhance Nigeria’s non-oil export earnings and diversify its revenue base.(Fakayode et al.,2019) 5. Agro-Processing Industry: Watermelon can be processed into juice, snacks, and other products. This creates opportunities for agro-processing industries and value-added products, boosting the economy through domestic processing and possibly export of processed goods. 6. Urban and Rural Market Growth: The high demand for watermelon in urban markets like Lagos and Port Harcourt boosts local economies. The vibrant trade of watermelon across both small and large markets encourages the movement of goods and enhances economic activity. Watermelon thus holds substantial economic importance in Nigeria, contributing to agriculture, employment, trade, and health. 2.1.6 Fungal spoilage of watermelon Fungal spoilage in watermelon can occur due to various types of molds. It is essential to store watermelon in cool dry place and avoid any damage to fruit skins ,as this can lead to fungal growth .Watermelon (Citrillus lantatus) is a nutrient rich fruit popular for his high water content and refreshing flavour. However, its perishability renders it vulnerable to spoilage by various microorganism, particularly fungi. Fungal spoilage not only led to economic losses but can also poses health risk due to potential production of mycotoxins (Onyeaka et al.,2020) Aspergillus and penicillum are the most common fungal species associated with fungal spoilage. Aspergillus SPP causes spoilage under warm and humid condition studies by (Onyeaka et al.,2020) found that Aspergillus thrives during both pre harvest and post-harvest stages and the fungi can produce harmful aflatoxins.(Sharma et al.,2020) Penicilluum species causes postharvest decay of watermelon .the typical blue or green molds that forms on watermelon skin is a sign of penicillium infection ,which can result from improper storage conditions such as high humidity or low temperatures (Gonzalez et al.,2021) penicillium also produce mycotoxins like patulin which are toxic to human if ingested the research by fanaei and Rezaee(2021) shows that fungal growth could be minimized by storing watermelons at temperatures between 10C and 15C CHAPTER THREE MATERIAL AND METHODS 3.1 Study area The watermelon samples were collected from Choba Alakahia, and rumualogu in ObioAkpor LGA. They were all collected from local markets and then transported into the Laboratory Choba market is located close to the university of Port Harcourt and it is known for its vibrant trade in agricultural products, including a wide variety of fruits Alakahia market is also a popular market in Obio Akpor which is known to sell variety of fruits. Rumualogu market is another market in Obio Akpor which is a well-known market for variety of goods, fresh products and local products. 3.2 Materials The following materials were used in the study: Watermelon Samples: Spoilt watermelon samples collected from local markets or farms. Sterile Equipment: Petri dishes, test tubes, beakers, forceps, scalpels, and swabs. Media: Nutrient agar, Citrimide Agar and Potato Dextrose Agar (PDA) for bacterial and fungal isolation. Reagents: Distilled water, ethanol (70% for sterilization), saline solution, and staining reagents such as crystal violet, iodine, and safranin for Gram staining. Laboratory Equipment: Incubator, autoclave, microscope, colony counter, and refrigerator. 3.3 Sample Collection Spoilt watermelon samples were collected from various local markets in Port Harcourt (Choba, Alakahia and rumalogu). A total of (9) watermelon samples showing visible signs of spoilage were obtained. The samples were placed in sterile plastic bags and transported to the laboratory for immediate analysis. The spoilage characteristics included soft rot, discoloration, and slimy texture. 3.4 Preparation of Culture Media Culture media used for the microbial isolation were prepared as follows: Nutrient Agar: Prepared by dissolving nutrient agar powder in distilled water and sterilized by autoclaving at 121°C for 15 minutes. Potato Dextrose Agar (PDA): PDA was used for fungal isolation. It was prepared by dissolving PDA powder in distilled water, followed by autoclaving. Mannitol salt agar: is a selective and differential medium used primarily for the isolation and identification of Staphylococcus species. It contains a high concentration of salt (7.5% NaCl), which inhibits the growth of most bacteria except for Staphylococcus species, which are salt-tolerant. Cetrimide Agar Is a selective medium used for the isolation and identification of Pseudomonas aeruginosa from clinical and non-clinical specimens. The medium contains cetrimide (a quaternary ammonium compound), which inhibits the growth of most bacteria except for Pseudomonas species. Pseudomonas aeruginosa produces a distinctive green or blue-green pigment called pyocyanin, which helps in its identification on this medium. 3.5 Microbial Isolation and Culturing Preparation of Watermelon Extract Each watermelon sample was surface-sterilized using 70% ethanol. A small portion (about 10g) of the watermelon flesh was cut using a sterile scalpel. The sample was homogenized in 90 ml of sterile saline solution to prepare a 1:10 dilution. 3.5.2 Serial Dilution A serial dilution of the watermelon extract was carried out up to a 10⁻⁶ dilution using sterile saline to ensure proper isolation of microorganisms. 3.5.3 Inoculation and Incubation Bacterial Isolation A 1 ml aliquot from each dilution was spread on nutrient agar, Mannitol salt and cetrimide agar The plates were incubated at 37°C for 24-48 hours for bacterial growth. Fungal Isolation: A 1 ml aliquot from each dilution was inoculated on Potato Dextrose Agar (PDA) plates. The plates were incubated at room temperature (25-28°C) for 5-7 days for fungal growth. 3.5.4 Microbial Identification Morphological Identification Bacterial Colonies: After incubation, the bacterial colonies were observed for morphology, including size, shape, color, and texture. Fungal Colonies: Fungal colonies were identified based on their color, surface texture, and the presence of spore structures under a microscope. 3.5.5 Gram Staining for Bacteria Gram staining was performed to differentiate between Gram-positive and Gram-negative bacteria. The procedure involved: Heat-fixing bacterial smear onto a microscope slide. Staining with crystal violet for 1 minute, followed by iodine for 1 minute. Decolorizing with ethanol and counterstaining with safranin for 30 seconds. Observing the slides under a microscope for Gram reaction. 3.5.6 Biochemical Tests for Bacteria Several biochemical tests were performed to confirm bacterial identity: Catalase Test: A drop of hydrogen peroxide was added to bacterial colonies. The production of bubbles indicated a positive result. Oxidase Test: Bacteria were tested using oxidase reagent to check for the presence of cytochrome oxidase enzymes. Coagulase Test: This test was performed to differentiate between coagulase-positive Staphylococcus species and coagulase-negative bacteria. 3.5.7 Fungal Identification Fungal identification was done using lactophenol cotton blue staining for spore observation under a microscope. The spores were compared with reference literature for identification. 3.5.8 Enumeration of Microbial Load The microbial load in spoilt watermelon was determined using the colony-forming unit (CFU) count: For bacteria, plates with 30-300 colonies were counted, and the CFU/mL was calculated using the formula: [CFU/mL =\frac (\text (Number of colonies) \times \text (Dilution factor (text (Volume plated)] For fungi, the total number of fungal colonies was also recorded, and the fungal load was calculated similarly.   CHAPTER FOUR RESULTS AND DISCUSSIONS 4.1 Results Table 4.1.1: Total Heterotrophic bacteria count of spoilt watermelon samples gotten from choba Total Heterotrophic Bacteria Counts Sample/Time Dilution Average Count CFU/g Log CFU/g Wmc1 10-5 90 9.0x107 7.954 Wmc2 10-5 96 9.6 x107 7.982 Wmc3 10-5 64 6.4×107 7.806 WMC – Watermelon Samples in Choba 4.1.2: Staphylococcus count of spoilt watermelon samples from choba Table 4.1.2: Staphylococcus count of spoilt watermelon samples Staphylococcus Counts Sample/Time Dilution Average Count CFU/g Log CFU/g Wmc1 10-3 15 1.5×105 5.176 Wmc2 10-3 77 7.7×105 5.886 Wmc3 10-3 19 1.9×105 5.278 WMC – Watermelon Samples in Choba Pseudomomas count of spoilt watermelon samples from choba able 4.1.3: pseudomomas count of spoilt watermelon samples Pseudomomas Counts Sample/Time Dilution Average Count CFU/g Log CFUg Wmc1 10-3 NG NG - Wmc2 10-3 NG NG - Wmc3 10-3 16 1.6×105 5.204 WMC - Watermelon samples in Choba, NG - no growth   Table 4.1.4: Total Heterotrophic bacteria count of Spoilt Watermelon samples gotten from Alakahia Total Heterotrophic Bacteria Counts Sample/Time Dilution Average Count CFU/g Log CFU/g WmA1 10-7 64 6.4x108 8.806 WmA2 10-7 63 6.3x108 8.799 WmA3 10-7 52 5.2x108 8.716 WMA -Watermelon samples in Alakahia 4.1.5 Staphylococcus count of spoilt water melon samples gotten from Alakahia Table 4.1.5 Staphylococcus count of spoilt watermelon gotten from Alakahia Staphylococcus Counts Sample/Time Dilution Average Count CFU/g Log CFU/g WMA1 10-4 72 7.2×106 6.857 WMA2 10-4 85 8.5×106 6.929 WmA3 10-4 99 9.9×106 6.995 WMA- Watermelon samples in Alakahia 4.1.6 pseudomomas count of spoilt water melon samples gotten from Alakahia Table 4.1.6 pseudomomas count of spoilt watermelon gotten from Alakahia Pseudomomas Counts Sample/Time Dilution Average Count CFU/g Log CFU/g WmA1 10-4 NG Ng NG WMA2 10-4 NG NG NG WMA3 10-4 NG NG NG WMA-Watermelon samples in Alakahia ,NG -NO GROWTH 4.1.7 Total Heterotrophic bacteria count of spoilt water melon samples l from Rumualogu Table 4.1.7: Total Heterotrophic bacteria count of spoilt watermelon samples from Rumualogu Total Heterotrophic Bacteria Counts Sample/Time Dilution Average Count CFU/g Log CFU/g WMR1 10-7 67 6.7 x108 8.826 WMR2 10-7 104 1.04x108 8.017 WMR3 10-7 75 7.5x108 8.875 WMR- watermelon in Rumualogu 4.1.8: staphylococcus count of spoilt watermelon samples from Rumualogu Table 4.1.8: Staphylococcus count of spoilt watermelon samples from Rumualogu Staphylococcus Counts Sample/Time Dilution Average Count CFU/g Log CFU/g WMR1 10-4 57 5.7×105 5.755 WmR2 10-4 33 3.3×105 5.518 WmR3 10-4 30 3.0×105 5.477 WMR-Watermelon in Rumualogu 4.1.9: Pseudomomas ount of spoilt watermelon samples from Rumualogu Table 4.1.9: pseudomomas count of spoilt watermelon samples from Rumualogu Pseudomomas Counts Sample/Time Dilution Average Count CFU/g Log CFU/g WMR1 10-4 NG NG NG WmR2 10-4 NG NG NG WmR3 10-4 71 7.1×104 4.851 WMR-Watermelon samples in Rumualogu 4.1.10 Morphological/Biochemical Characterization of Bacterial Isolates from spoilt watermelon samples studied The morphological characterization of the norganisms isolated from the spoilt watermelon samples studied on are as detailed in Tables 4.21 to 4.30. The predominant genus isolated were Staphylococcus, pseudomomas, Escherichia coli, Table 4.1.10: Morphological/Biochemical Characterization of Bacterial Isolates from water samples studied Isolate code gramreaction cellmorphology oxidase indole citrate motility MR VP H2S gas slant Butt Glucose lactose Maltose Fructose Coagulase Catalase Tentative identity NA(WMR1) + Cocci - - + - - + - - A A A A A A + - Staphylococcus MSA(WMR1) + Cocci - - + - - + - - A A A A A A - + Staphylococcus CA(WMR1) - Rod - + + + - + - - B A A A A A - + psedomonas NA(WMR2) + Rod + - - + + - - + A A A A A A - + Escherichia coli MSA(WMR2) + Cocci - - + - + + - - A A A A A A - + Staphylococus NA(WMA1) + Cocci - - + - + - - - A A A A A A - + Staphylococcus NA(WMA2) + Rod - - - + + - - + A A A A A A - + Staphylococcus MSA(WMC1) + Cocci - - - + + - - + A A A A A A - + Staphylococcus NA(WMC2) + Rod - + - + + - - + A A A A A A - + Escherichia Coli MSA(WMC3) + Rod + - - + + - - + A A B A A A + + Staphylococcus CA(WMC2) + Rod + + + + - + - - B A A A A A - + Pseudomonas NA(WMC3) + Cocci - - + - - + - - A A A A A A _ + Staphylococcus NA -NUTRIENT AGAR, MSA-MANITOL SALT AGAR, CA-CITRIMIDE AGAR,,WMC- WATERMELON SAMPLE IN CHOBA ,WMR-WATERMELON SAMPLE IN RUMUALOGU, WMA-WATERMELON SAMPLE IN ALAKAHI Table 4.1.11 Cultural characteristics and cell morphology of yeast isolate from spoilt watermelon Isolate code cultural characteristics cell morphology Tentative Identity WMC1 Shiny Smooth Cream coloured Round to oval it has Candida Tropicalis Colonies Psedophyta WMC2 Shiny Smooth Cream coloured Round to oval it has Candida Tropicalis Colonies psedophyta WMC3 Shiny Smooth Cream coloured Round to oval it has Candida Tropicalis Colonies psedophyta WMA1 Shiny Smooth Cream coloured Round to oval it has Candida Tropicalis Colonies Psedophyta WMA2 Shiny Smooth Cream coloured Round to oval it has Candida Tropicalis Colonies Psedophyta WMA3 Shiny Smooth Cream coloured Round to oval it has Candida Tropicalis Colonies psedophyta WMR1 Shiny Smooth Cream coloured Round to oval it has Candida Tropicalis Colonies psedophyta WMR2 Shiny Smooth Cream coloured Round to oval it has Candida Tropicalis Colonies psedophyta WMR3 Shiny Smooth Cream coloured Round to oval it has Candida Tropicalis Colonies psedophyta WMC - Watermelon Samples in Choba, WMA - Watermelon Samples in Alakahia, WMR – Watermelon Samples in Rumualogu, Table 4.1.12 Percentage Occurrence of Bacterial Isolates from spoilt watermelon samples studied. Organisms Frequency of Occurrence % Occurrence Staphylococcus sp. 7 59 Escherichia coli 3 25 Pseudomomas sp. 2 16 Figure 4.1: Percentage Occurrences of Bacteria Isolates from Spoilt watermelon samples 4.2 Discussion This study examined the microbial spoilage in spoilt watermelon sample from three different location in Obio Akpor .The findings reveals notable variation in microbial spoilage organism these result provide insight to various spoilage organisms. Microbial count :The total heterotrophic microbial count deducted in table 4.1 indicate that the bacterial load in choba (WMA1,WMA2,WMA2) ranged from 6.4×107 to 9.6×107 which indicated that spoilage was well established and may include other factor contributing to degradation and odour while the staphylococcus count in choba indicated that it range from 1.9×105 to 7.7×105 suggest that occurred through contamination from human contact which could occur due harvesting, transport and sale. The pseudomonas count just indicated 1.6×105 which shows inconsistent growth appearing only in some selected samples suggesting that it is not a primary spoilage organism for watermelon under the condition studied for samples in Choba. while total heterotrophic bacteria in Alakahia indicated from 5.2×108 to 6.4×108 which shows high bacteria load which includes factors contributing to degradation and odour, staphylococcus count indicated from 7.2×107 to 9.9×106 which has a high staphylococcus count which indicated potentially poor hygiene ,contamination from human which can occur during harvesting ,transport or sale .The result for pseudomonas (WMA1,WMA2,WMA3) indicated no growth indicating that pseudomonas was absent or above detectable levels in the samples. The total heterotrophic count in Rumualogu (WMR1,WMR2,WMR3) range from 1.04×108 to 7.5×108 indicate that spoilage was well established and may include various bacteria contributing to degradation and odour. The staphylococcus count in rumualogu samples from 3.0×105 to 5.7 ×105 indicates significant contamination from staphylococcus which could imply that handling practices across these location are source of contamination. The result emphasize on the importance of proper handling and storage practice to reduce microbial contamination and spoilage in watermelon .staphylococcus is a common spoilage organism across locations ,indicating that hygiene practices need improvement to minimize contamination .The absence or low presence of pseudomonas suggest it is less significant in watermelon spoilage under the condition you studied .The total heterotrophic bacteria count indicate spoilage and reflect the rapid bacterial growth that occur in exposed produce stressing the need for efficient processing and sale times to reduce spoilage rate . The biochemical characteristics of the bacteria isolates in Table 4.3 the bacterial isolates displayed a range of biochemical activities including Catalase, oxidase, indole among others the presence of catalase positive in the sample indicates that the catalase enzyme break down hydrogen peroxide into water and oxygen which can be confirmed by bubble formation when hydrogen peroxide is added. The presence of catalase positive suggest common spoilage bacteria such as Staphylococcus The indication of oxidase positive bacteria indicate that the presence of microorganism that produce the oxidase enzyme, which plays a role in the electron transport chain and it is involved in energy production, the presence of oxidase positive indicate species of Pseudomonas that can thrive in aerobic condition and contribute to watermelon spoilage. The biochemical test result indicated that spoilage in watermelon samples is primarily driven by a mix of aerobic and anaerobic bacteria capable of producing enzyme that degrade proteins ,carbohydrates, and other CHAPTER FIVE SUMMARY CONCLUSION AND RECOMMENDATIONS 5.1 Summary of the findings The study helps to identifies key bacterial and fungal species associated with watermelon spoilage. Common bacterial genera include Pseudomonas, while fungal species can include Candida tropicalis Each of these microbes contributes to spoilage through the production of enzymes that break down watermelon’s sugars, proteins, and other components Microbial spoilage is primarily driven by microbial enzyme activity, which degrades the fruit’s structural integrity, resulting in softening, off-odours, and changes in colour and texture. Bacteria are often involved in early spoilage stages, while fungi contribute more significantly as spoilage organism especially when the watermelon has a high sugar content. Different Factors such as temperature, humidity, and handling practices can significantly impact the rate of spoilage. High temperatures and high humidity can accelerate microbial growth, especially in damaged or bruised fruits. 5.2 limitations of the study When studying for the Spoilage organism accessing the few locations selected may not capture the full diversity of Microbial community. Different Factors such as temperature, humidity, and handling conditions can vary significantly and are challenging to control. These environmental differences may influence microbial growth rates, affecting the consistency and reproducibility of findings. The way the samples are stored before analysis could affect microbial activity, making it challenging to draw consistent conclusions about spoilage patterns.(jones et al.,2021). The study primarily focused on identifying microbial species through culturing technique ,which may have overlooked certain microranism that are viable but non culturable (VBNC).These organisms could still play a significant role in spoilage but require advanced molecular method like next generation sequencing to be accurately detected (Smith&Allen,2020) 5.3 Conclusion The study on microbial spoilage in watermelons concludes that spoilage is primarily driven by a variety of microorganisms, including bacteria and fungi, which degrade the fruit’s structure and nutritional content. Key spoilage organisms identified include species from genera such as Pseudomononas each contributing to texture changes, off-odours, and colour shifts in different stages of spoilage. The research identified bacteria and fungi responsible for watermelon spoilage ,revealing specific microbial communities that thrive under particular conditions such as temperature, humidity, and handling practices were found to significantly impact spoilage rates, with higher temperatures and damaged fruits accelerating microbial growth. Proper storage, such as refrigeration and minimizing fruit damage, can reduce the rate of spoilage and extend watermelon shelf life. (kumar &sharma 2020) However, due to limitations like restricted sample locations and standard identification methods, further studies with more diverse sampling and advanced molecular techniques are recommended for a comprehensive understanding of spoilage processes. These findings provide practical insights into spoilage prevention and highlight the importance of safe handling to mitigate health risks associated with microbial spoilage in watermelons.(Ahmed et al.,2021) Preventive measures ,such as post -harvest handling ,cooling and the use of natural antimicrobial agents could effectively inhiibit spoilage microorganism.Techniques like Modified atmosphere Packaging and the application of bio preservative have shown promise in extending shelf life by slowing microbial growth (Garrido et al.,2018) 5.4 Recommendation 1. Improved Storage Conditions: Store watermelons at cooler temperatures (e.g., refrigeration) to slow microbial growth. Avoid storing them in high-humidity environments, as this can promote faster spoilage. 2. Minimize Handling and Damage: Prevent bruising and cuts during transport and handling to reduce entry points for microorganisms. Encourage gentle handling practices from harvest to storage to maintain the fruit’s integrity. 3. Regular Quality Checks: Implement routine checks for spoilage signs, especially for watermelons stored in bulk, to identify and remove any spoiled fruits promptly. This can help prevent cross-contamination and further spoilage in other fruits. 4. Education on Spoilage Prevention: Train farmers, vendors, and consumers on safe handling and proper storage practices to reduce microbial contamination risks.   REFERENCES Ahmed, M., Zhang, Y., & Xi, C. (2021). Postharvest preservation techniques and their impact on the shelf life of fresh watermelon. Journal of Food Quality and Safety, 5(3), 201-215. Ajewole, O. C., & Adeogun, B. E. (2018). “Economic analysis of watermelon production in selected local government areas of Niger State, Nigeria.” Journal of Agronomy and Food Research, 15(2), 95-103. Ali, A., Abdul, A. H., & Shahbaz, M. (2019). “Post-harvest deterioration and microbial spoilage of fruits and vegetables.” Journal of Food Science and Nutrition, 7(4), 501–509. Chomicki, G., & Renner, S. S. (2015). “Watermelon origin solved with molecular phylogenetics including Linnaean material: Another example of museomics.” New Phytologist, 205(2), 526-532. Devi, P., & Kumar, R. (2018). “Mycotoxin contamination in fruits and their health impacts.” Journal of Food Safety and Health, 6(1), 215–223. Eke-Okoro, O. N., Ojiako, F. O., & Ogbonnaya, C. (2019). “Agricultural crop diversification and income growth among Nigerian rural farmers.” International Journal of Agricultural Economics, 4(3), 112-119. Ezugwu, I., & Nwobodo, C. (2021). “Challenges in reducing post-harvest losses of fruits in Nigeria.” African Journal of Agricultural Science, 15(9), 98–107. Ezugwu, I., & Nwobodo, C. (2021). “Challenges in reducing post-harvest losses of fruits in Nigeria.” African Journal of Agricultural Science, 15(9), 98–10 Garrido, D., Hernández, M., & Ferrer, C. (2018). Impact of modified atmosphere packaging on microbial spoilage in fruits. International Journal of Food Microbiology, 265, 111-119. Jones, A., Patel, N., & Roberts, C. (2021). Regional variability in microbial spoilage of fruits and vegetables. Agricultural Microbiology, 48(2), 156-164. Kumar, R., & Sharma, M. (2020). Microbial ecology and spoilage organisms in fresh produce. Food Microbiology, 88, 103412 Kumar, R., Singh, S., & Rajeshwari, S. (2020). “Microbial spoilage and its impact on food quality.” Food Research International, 8(2), 317–323. Manniche, L. (1989). An Ancient Egyptian Herbal. University of Texas Press. Ojo, M. A., & Adeoye, I. B. (2020). “Analysis of income and employment generation from watermelon production in Kwara State, Nigeria.” Journal of Agricultural Economics and Extension Research, 9(2), 203-214. Renner, S. S., Scarcelli, N., & Teshome, A. (2017). “Wild relatives of watermelon (Citrullus lanatus) in Africa and genetic insights into its domestication.” American Journal of Botany, 104(4), 536–541. Smith, D., & Allen, B. (2020). Limitations of culturing techniques in microbiology: Exploring VBNC organisms. Microbial Ecology, 80(4), 905-914.