THE IMPACT OF MONEY MARKET ON THE ECONOMIC GROWTH OF NIGERIAN (1986-2021)

BACTERIOLOGICAL ANALYSIS OF SORREL BEVERAGE (ZOBO) SOLD IN EMENE, ENUGU STATE. BY SAMUEL PRAISE CHIMAROKE MB/2020/833 DEPARTMENT OF MICROBIOLOGY FACULTY OF NATURAL SCIENCES CARITAS UNIVERSITY AMORJI-NIKE ENUGU STATE. AUGUST 2024 BACTERIOLOGICAL ANALYSIS OF SORREL BEVERAGE (ZOBO) SOLD IN EMENE, ENUGU STATE. BY SAMUEL PRAISE CHIMAROKE MB/2020/833 DEPARTMENT OF MICROBIOLOGY FACULTY OF NATURAL SCIENCE CARITAS UNIVERSITY AMORJI-NIKE ENUGU STATE. IN FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF BACHELOR OF SCIENCE (B.Sc.) DEGREE IN MICROBIOLOGY AUGUST, 2024. CERTIFICATION This is to certify that I, Samuel Praise Chimaroke, MB/2020/833 have carried out this research project titled “Bacteriological analysis of sorrel beverages (zobo drink) sold in Emene, Enugu state.” submitted it for the award of a Bachelor of Science (B.Sc.) degree in Microbiology, Faculty of Natural Sciences, Caritas University, Amorji-Nike Emene, Enugu State. The project is free of all plagiarism and has not been submitted elsewhere for award of any degree or diploma. SAMUEL PRAISE CHIMAROKE ...…………………………………………. (Student) Student’s Signature and Date APPROVAL This research Bacteriological Analysis Of Sorrel Beverage (Zobo) Sold In Emene, Enugu state has been examined and accepted as research done by the student in fulfillment of the requirements for the award of bachelor of science B.Sc. honour degree in Microbiology. Prof. S.C. Onuorah …………………… ……………….. (Supervisor) Signature Date Dr. P.N Amadi …………………… ……………….. (Head of Department) Signature Date ………………………. …………………… ……………….. (External Examiner) Signature Date ACKNOWLEDGEMENT First, I am most grateful to GOD ALMIGHTY for helping me to excel in my efforts to complete this project. My heartfelt gratitude goes to my project supervisor, for his valuable input, suggestions and guidance whilst working on this project. I am very thankful to my parents who provided all I needed to complete this project writing, regardless of life’s hardship. I express my thanks to all lecturers and friends for all the coordination, contribution and efforts devoted to the success of this project. ABSTRACT Zobo drinks are made from Hibiscus sabdariffa and have been shown to be very nutritive with medicinal properties. This work was done to determine the bacteriological quality of zobo drinks sold in Emene, Enugu state. Five zobo samples were purchased from different locations. Nutrient agar and streak plate method was employed for bacteria isolation. Different microscopic and biochemical test were employed to further identify the bacterial isolates. After the analysis the organisms were identified as Staphylococcus spp Corynebacterium spp, Klebsiella spp, Pseudomonas spp, Escherchia coli and Proteus spp. These organisms identified have been implicated in several gastrointestinal diseases such as dysentery, diarrhoea and many others. The result implies that precautions were not taken by the manufacturers and handlers of zobo drink hence the bacteriological contamination which could have been caused by the manufacturers and handlers themselves or gotten from the unhygienic environment or materials used in the production and packaging of the drinks. However, education of the manufacturers and handlers is necessary to avoid possible health hazard. TABLE OF CONTENTS Title page …………………………………………………………………….………..……i Certification………………………………………………………………………..……….………ii Approval ……………………………………………………………………………………....iii Acknowledgement ……………………………………………………………..……..……...…iv Abstract ……………………………………………………………………………...v Table of content……………………………………………………………………….………...vi CHAPTER 1 1.0 Introduction…………………………………………………………………………1 1.1 Aims and objectives …………………………………………………………….….3 CHAPTER 2 2.0 Literature review……………………………………………………………………13 2.1 Sorrel plant and its origin (Hibiscus sabdariffa L.)………………...………………14 2.2 Description of sorrel plant……………………………………………………….…15 2.3 Uses of sorrel plant (calyx, seed, and leaf)………………………..………………..17 2.4 Nutrient composition of the different parts of sorrel plant (calyx, seed and leaf)….20 2.5 Phytochemicals in plant foods………………………………………………….…..22 2.5.1 Antioxidants………………………………………………………………………..29 2.7 Antinutritional and food toxicant factors (phytate, tannins cyanide and trypsin inhibitor) …………………………………………………………………………………………………31 2.8 Antinutrient and food toxicant composition of the different parts of sorrel plant (calyx, seed and leaf) …………………………………………………………………………..33 CHAPTER 3 3.0 Materials and method used………………..………………………………………….38 3.1 Sample collection / description ……………………..…………..…………………….38 3.2 Agar used ………………………………………………………….…………….…..38 3.3 Reagents …………………………………….………………………………………..38 3.4 Equipment and glassware…………………………………….……………………..…38 3.5 Isolation / counts of bacteria …………………….………….…………………….….39 CHAPTER 4 Results………………………………………………………………………………………52 Discussions………………………………………………………………………………….53 CHAPTER 5 Conclusions………………………………………………………………………………….55 References …………………………………………………………………………………..56-60 LIST OF TABLES Table 1: The Total Bacterial Count of Zobo Drink Sample Table 2: Characteristics of Bacteria Isolated from Zobo drink Sample A Table 3: Characteristics of Bacteria Isolated from Zobo drink Sample B Table 4: Characteristics of Bacteria Isolated From Zobo Drink Sample C Table 5: Characteristics of Bacteria Isolated From Zobo Drink Sample D Table 6: Characteristics of Bacteria Isolated from Zobo drink Sample E Table 7: Characteristics of Bacteria Isolated From Zobo Drink Control Sample Table 8: Morphological characteristics of the bacterial isolates Table 9: Percentage of occurrence of the bacteria isolated from zobo drink samples CHAPTER ONE 1.0 INTRODUCTION 1.1 BACKGROUND OF THE STUDY In recent times, there has been an upsurge in the consumption of ready-to-eat food. This is probably due to their convenience. Ready-to-eat foods occur in varieties of food including fruits, fruit juices, nutritional drinks, snacks etc. Generally, they are food items consumed on purchase from vendor, hawkers and consumed immediately without any further preparation. Most Ready-to-eat foods are found in public places including markets, motor parks, and streets, outside schools, hospitals and even express way. Snacks are also vended along highways linking several geographical areas in the country (S.C. Izah, E.R. Aseiba, L.A. Orutugu LA,2015). The authors attributed this to high level of unemployment and failed family and community values. Typically, food is an essential resource required by the human body for the enhancement of normal functioning of the body. Food are classified based on readiness to consumption including fully processed Ready-to-eat to such as akara, fried groundnut, rice and bean, dish, fura, bread, zobo drink; and minimally processed ready-to-eat such as tomato, cabbage, ginger drink, lettuce, carrot, kunu zaki drink. Most snacks are purchased to quench hunger and or thirst. They are sometimes used as an appetizer. Some ready-to-eat foods such as fruit juice are mostly consumed due to their refreshing attributes, nutritive values and health benefits. Fruits are potential source of nutrients, micronutrients, vitamins and fiber for humans (A. Bukar, A. Uba, T.A. Oyeyi,2010). Most of these fruits include watermelon, paw-paw, and pineapples. Some of these fruits are processed into juice/wine such as pineapple juice, watermelon and paw-paw blend, orange, apple, pineapple. Other essential nutritional drinks consumed by a large number of the populace in Nigeria are kunu and Zobo (N.N. Odu, A.O. Adeniji,2013). Zobo is produced from the dried calyces of Hibiscus sabdariffa. H. sabdariffa which belongs to Malvaceae family is an annual erect bushy branched herb found in tropical and semi-tropical regions of the world mainly in West Africa and the East Indies. H. sabdariffa grows to about 3.5metres in height. As such can be classified as microphanerophytes (thus trees and shrubs whose height ranged from 2 – 8meters). H. sabdariffa has cylindrical and dark green to red colored stem, tap root system and the leaves are green to red in colour. The flower is pale yellow with fleshy red calyces (. S.O. Umeh, B.C. Okeke, J. Okpalla, G.C. Agu,2015). H. sabdariffa thrives well in loamy well drained soil with annual rainfall of about 1500- 2000mm3 and can tolerate floods, heavy winds and stagnant waters. Zobo is prepared with the addition of flavor such as orange and pineapple etc and preservatives such as garlic, ginger, mixture of garlic and ginger. This is because of its short shelf life of about 24 hours after production without refrigeration. H. sabdariffa is used to produce diverse food products and beverages in different countries of the World. In Nigeria two botanical varieties of H. sabdariffa exist including red/brown and green. The brown/red type is widespread in the northern guinea and Sudan savanna while the green type is common in Southern Guinea savanna. The calyces of the green variety of H. sabdariffa are used to cook soup, stew and sauces while the calyces of the red variety are used for the production of Zobo drink and soup. Hence, the plant is cultivated and used as vegetable for soup preparation especially in Western Nigeria and some special type of tea, dried calyces is used for the production of sorrel drink. Sorrel drink is known as Zobo or Yakwua or Zoborodo in Hausa, Iseipa (Yoruba) and Sorrel in English in Nigeria. H. sabariffa is known as bissap and drinks is prepared through aqueous extraction in a certain solid-to solvent ratios, which is popularly consumed during the month of Ramadan in Senegal. In other African countries, the beverage from H. sabariffa is called drink of the Pharaohs (Egypt), tea Karkade (Sudan), da Bilenni (Mali). The seed of H. sabariffa can be fermented to produce Bikalaga, fermented food in some African countries, such as Burkina Faso, Mali, Niger, Nigeria, Cameroon and Sudan among others, which are called Furundu (Sudan), dawadawa botso (Niger), datou (Mali), Mbuja (Cameroon) (C. Parkouda, B. Diawara, L.I.I. Ouoba,2008). Zobo is a major nutritional drink consumed by several socio-economic classes of people in Nigeria and some other West African countries. Due to ease of production and availability of raw material especially H. sabdariffa, they are source of livelihood to several families in both Northern and Southern Nigeria especially in the rural areas. Poor sanitary condition at the point of production and contaminated water could put consumers of these products into risk. The quality of zobo drink depends mainly on the physicochemical constituents of the raw materials, water used in their production and the hygienic condition of the processors. Water is a major is a major resource used in the production of these drink from their raw materials. Poor quality with regard to both physicochemical (colour, pH, turbidity, total suspended solids, total hardness, total alkalinity salinity, electrical conductivity), heavy metals (lead, cadmium, chromium, iron, zinc, copper, nickel, arsenic) and microbial (total heterotrophic bacteria, total fungi, total coliform and fecal coliforms) could also impact on the overall quality of the drink. The environment is which the drinks are processed could also influence the quality especially in the microbial perspectives. Zobo drink is a nutritional drink consumed by people in Nigeria. However, the consumption of local beverages could be a potential source of transfer of zoonotic and foodborne pathogens including staphylococcosis, Salmonellosis, Brucellosis, Tuberculosis, Shigellosis, Listeriosis, E. coli, infections etc.( G.A. Umaru, I.S. Tukur, U.A. Akensire, Z. Adamu, O.A. Bello, A.H.B. Shawulu, M. Audu,2013). Therefore, this paper reviews the quality of Zobo drink prepared and consumed in Emene, Enugu. 1.2 AIMS AND OBJECTIVES Aim: The aim of this project work is to analyze the bacteriological contamination of sorrel beverages (zobo drink) sold in Emene Enugu state. Objectives: 1. To assess the level of bacteriological contamination in the zobo samples. 2. To isolate the bacteria in the zobo samples. 3. To identify the bacteria isolated from the zobo samples. CHAPTER TWO LITERATURE REVIEW 2.0 LITERATURE REVIEW In developing countries Plant foods are the most important dietary source to meet the nutritional needs of the majority of people due to their availability, low cost and acceptability. The enormous potential of some plant foods still however remain available but not on used. Some commercial foods in Nigeria are becoming very expensive and out of the reach of many families, particularly poor families. Some locally available, cheap and nutritious food crops needs to be exploring in the production of derivable commercial products. This would expand the diet and help to reduce the problems malnutrition experienced by many people in different parts of the country (WHO, 2008a). Sorrel plant (Hibiscus sabdariffa) is an acid tasting herb of the Malvaceas family(McLean, 1973; James, 2003; Yadong et al., 2005). The plant is an annual crop widely grown in the northern part of Nigeria, with well nutritional value (Onyeke, 2001). It is one of the under-exploited food crops of Nigeria. Sorrel plant bears edible leafs, seeds and leaves. Food processing industry has potentials exploitation to the different parts of the plant (McLean, 1973; James, 2003). Little studies has however been carried out on the development of products from the calyx, seed and leaf of the sorrel plant. Already, there is a popular drink produced from sorrel calyx which is used for entertainment during ceremonies. This drink has been concentrated to produce bottled carbonated juice (Karkadeh juice) as reported by McLean (1973) in Sudan. Globally, many researchers have noted the food potentials of the different parts of sorrel plant (Mclean, 1973; Duke and Atchley, 1984; Nnam and Onyeke, 2003; Rich, 2004 and Yadong et al., 2005). The calyx could be processed to produce jam, marmalade, jelly, ices, ice cream, herbal tea, wine, juice, syrup, sauces, gelatin and butter. Sorrel calyx has some medicinal uses such as the treatment of bilious attacks, decreasing the viscosity of the blood, reducing blood pressure and as diuretic for stimulating intestinal peristalsis. It can also be used to treat cancer, cough, and scurvy (Yadong et al., 2005). 2.1 Sorrel plant and its origin (Hibiscus sabdariffa L.) Hibiscus has more than 300 species, which are distributed in tropical and subtropical regions around the world (Yadong et al., 2005). Most hibiscus species are used as ornamental plants and are believed to have some medicinal properties. One of the species is Hibiscus sabdariffa, commonly named as: “red sorrel” or “roselle”, “India sorrel”, and “Florida cranberry”. Sorrel belongs to the family of Malvaceae (James, 2003; Yadong et al., 2005). Many authors suggest different areas of origin of sorrel. McLean (1973) reported that sorrel originated from Turkey. Adamson, Long and Bagby (1979) suggested that it originated from India where it is cultivated annually and was introduced as a potential new crop for the Southern United States. Sorrel is currently grown in Florida for its edible fruit.Murdock, an African Archaeologist was the first to report on the origin of sorrel atNiger waterside, Centre for the cultivation of indigenous plants in about 4000 B.C. (McLean, 1973). It was reported that sorrel plant was singled out from okra, cotton and kenaf as distinct specie. In 1936, sorrel was specifically identified in an analysis of reports by a British Empire Agricultural Officer (Sammy, 1970). Uganda assessments and reports show that the original habitat of sorrel was West Africa and the plant was carried to India during the Muslim invasion in the 8th century AD. Later, it was carried to the West Indies during the slave trade and Jamaica in 1707 (McLean, 1973). It was from the West Indies during the Muslim invasion that sorrel was exposed to Florida. Sorrel was imported to Florida after an interval of 250 years. The plant was then transported to Queen Land and Australia. It was from Australia that sorrel was transported to California in 1896 and Philippines in 1905. In the 16th century, the plant was grown in England, Basle and Switzerland. Finally, it spread to other parts of the world (McLean, 1973). McLean (1973) reported that the first research worker to promote sorrel food potential was Wester in 1907 at the US Department of Agriculture, Washington D. C. He studied “its culture and uses”. About the same time, a systematic varietal study was carried out on the plant by Albert and Gabrielle. Howard in the year 1911 worked on Indian fibre plants at Pusa. Crane carried out a study on “roselle – a potentially important plant fibre” in 1949. McLean (1973) observed that information on the plant is not lacking; development of recipes and products on its edible parts is scanty in the literature. Sorrel plant has many names which originated from botanists working where it grows. Florida named the plant Florida cranberry. The Europeans named it sour, which was derived from the roots. In Guatemala, it is called Jamaica red rose. The French referred to the plant as Oscille and it is called Rouselle or Roselle by the English. The Spanish named it Rozella or Rossella. Its other French name is Guimauve de sabdariffa (Ketmic acid). The word sorrel came from German, meaning sour (James, 2003). According to Morton (1987), sorrel plant is relatively close to okra (Hibiscus esculentis), kenaf (Hibiscus cannabinus), cotton (Gossypium spp) and swanp or rose mallow (Hibiscus moschatus) and cranberry (Vaccinium spp.). 2.2 Description of sorrel plant Sorrel plant (Hibiscus sabdariffa) is an erect, bushy branched annual herbal plant. It grows in a warm and humid tropical and subtropical region. There are two major species of sorrel – Var altissima Wester and Var sabdariffa. Var altissima Wester is a tall vigorous unbranched plant, about 5m in height. It is not edible but the stem is used for the production of fibre (McLean, 1973). The second type, Var sabdariffa, is of two varieties. The varieties are differentiated by their calyces as follows: the red-calyx and the yellow-calyx varieties. The plants are grown for their edible calyces, seeds and leaves. The plants are about 2 to 5m tall. The stems are reddish in colour. The leaves are alternately arranged on the stem with green to red veins, which are divided into 3 to 7 lobes with errate margins. The flowers are red to yellow with a dark centre containing short – peduncles. The seeds are brownish in colour and enclosed in a capsule. It is difficult to remove the coat from the fruit (Morton, 1987; Yadong et al., 2005). 2.3 Uses of sorrel plant (calyx, seed, and leaf) All parts of the sorrel plant are useful (Adamson, Martin and Minton, 1975; Duke and Atchley, 1984; Nnam and Onyeke, 2003 Rich, 2004). In recent times, there is a global attention on the sorrel plant. This may be because of its nutritional, refreshing and medicinal uses. Currently in Nigerian, sorrel drinks are no longer limited to northern Nigeria from where it seems to have originated. Many people in Nigeria use it as a drink (Ekwuazi, 1995). There is more information on the medicinal uses of sorrel plant than on its nutritional uses. Yadong et al., (2005) reported that sorrel is used in many folk medicines as laxative to aid kidney functions, sores and wound healing. Duke and Atchley (1984) reported that sorrel is used in the treatment of hypertension, profuse menstruation, heat burn, remedy for headaches, snake bite, dysentery and eye diseases. One recent report on the sorrel plant by Yadong et al., (2005) stated that many parts of sorrel including calyces, seeds, leaves and roots are used for various food uses. The calyx: Red calyces are used for various food purposes such as juice, jam, jelly, syrup, gelatin, ice cream, tea, butter, flavors, color, beverages, spices, sauces, tarts and other deserts (Yadong et al., 2005). Duke (1983) reported that in Switzerland, sorrel calyces are used in jam, jellies, sauces and wines. The dried sorrel calyces are used in West India for tea, wine, jelly, marmalade, ice cream, sherbets, butter, pies, sauces, tarts and other deserts. It is also used for coloring and flavoring rum in the West Indies. In Mexico, it is used as a red beverage (Duke, 1983). The fresh calyces are eaten raw in salads. The calyx is cooked and used as flavoring in cake, jellies, soups, sauces, pickles and pudding (Rich, 2004). In Europe the calyces are used for beverages (McLean, 1973). The author further reported that both the French and English-speaking people use sorrel calyces as flavoring for jellies syrups and wine. In Nigeria (Yoruba States and parts of northern Nigeria), calyces are boiled and used for making soups in place of meat. It is eaten with yam or foo- foo. At Kazaure in Kano State of Nigeria, when a girl gets married, she must learn how to prepare sorrel juice. In Sudan, the drink is taken hot and Europeans drink it cold (Perry, 1971). In Arab and Senegal, the Moslem during the Ramadan fast consumes sorrel drink during the night. This is because it sustains them during the day-long fast (McLean, 1973). The researcher in her personal communication with Haljia Mohammed (2006) who produced and sold sorrel drinks, said that the Nigerian Moslem consume sorrel drink at night to sustain them during the day-long fast. In Swati, the calyces are used in the treatment of pneumonia (Duke, 1983). In Guinea, the calyces are used for treating fever, heart ailments, hypertension, neurosis, debility, cough, cancer, bilious condition, scurvy. It is also used as an antiseptic, astringent, cholagoggue, diuretic, emollient, purgative, digestive, sedative stomach and tonic (Rich, 2004; Yadong et al., 2005). Rich (2004) and Yadong et al. (2005) reported that the calyces are used in folk medicine as tonic tea for digestive and kidney function. It reduces blood pressure, bilious attacks, stimulates intestinal peristalsis. Purse glove (1975) reported that acid jellies extracted from the edible calyces are used to prevent scurvy. The seed: The seeds are one of the Africa’s food grains. Sorrel seeds are used in the production of oil, food condiment, foo-foo, coffee substitute, porridge, soups and sauces (Rich, 2004). In northern Nigeria, sorrel seeds are ground into a meal and used as a nourishing food for the invalid (McLean, 1973). In Ghana, the seed is boiled whole and in Uganda, it is crushed and boiled in water to a thin porridge and eaten with staple foods (MacLean, 1973). The extracted oil is used as a substitute for castor oil applied to Carmel sores (Dalziel, 1979). The seeds can be roasted and used as coffee substitute or roasted and ground into powder and used in oily soups and sauces (Rich, 2004). Tivs in Nigeria use sorrel seeds in making Ashwekon - a sour condiment. Adekunle (1995) and Ekwazi (1995) reported that where there is no locust bean, sorrel seeds can be used as condiment. In Sudan, it is used as seasoning after fermentation and in southern Sudan; it is ground into flour and eaten as foo-foo (McLean, 1973). McLean (1973) reported that during the revolt of the Mahdi in Sudan, the seeds were roasted and used as coffee. It was reported that sorrel seeds played a very big role in the production of drugs (Duke, 1983; Yadong et al., 2005). Taiwanese regard the seeds as diuretic, laxative and tonic (Duke, 1983). The seeds have properties similar to those of cotton seed. The oil from the seed is used as a substitute for crude castor oil (Rich, 2004; Yadong et al., 2005). In South America and the west Indies, the seed is used as a snakebite remedy (Duke, 1983). The leaf: Sorrel leaves are used in various food production processes such as in seasoning, salad substitute, flavouring, tea, soups, sauce, spices and pot herb (Rich, 2004; Yadong et al., 2005). In many parts of the world, the leaves are consumed as a green vegetable. In Shangaan, the tender young leaves are eaten raw in salads or cooked as green vegetable. The leaf is used in flavouring and added to curries as seasoning (Yadong et al., 2005). McLean (1973) reported that the leaves are used as seasoning in India. Sorrel leaves are used as vegetable in Ghana. McLean (1973) observed that sorrel leaves are pounded to prepare an infusion to preserve porridge in Ghana. In Banyoro of Uganda, the leaves are crushed and boiled in water to the consistency of a thin porridge, which is eaten as a sauce with staple foods (McLean, 1973). In the western part of Nigeria, the leaves are used as vegetable in cooking soup (Ekwuazi, 1995). In Senegal, the fresh leaves are pounded into puree and served over rice or fish. The flavour has led to its uses in salads in India (McLean, 1973). In Africa, especially in Guinea, the leaves are used for the treatment of abscess, tooth decay and as laxative (Rich, 2004). The Shangaan use the sorrel leaves in the form of tea for the treatment of hypertension and profuse menstruation (Faraja and Tarkhanib, 1999). Duke (1983) reported that in South America, the leaves gave a very fair inhibition, when tested for its antibacterial effect. The Zulu use a lotion of the leaf for the treatment of penile irritation of any sort, including venereal sores and urethritis. An infusion of the leaves is used as an injection in the urethra and vagina for gonorrhea. The leaves are very mucilaginous and are used as a tonic tea for digestive and kidney function (Rich, 2004). 2.4 Nutrient composition of the different parts of sorrel plant (calyx, seed and leaf) The calyx: Yadong et al. (2005) reported that fresh calyces are rich in riboflavin, ascorbic acid, niacin, carotene, calcium and iron. The red calyx contains antioxidants. Duke (1983) and Duke and Atchley (1984) reported that the fresh calyx per 100g sample contains 49 calories, 84.5% moisture, 1.9% protein, 0.1% fat, 12.3% total carbohydrate, 2.3% fiber, 12% ash, 1.72mg calcium, 57mg phosphorus, 2.9mg iron, 300μg β-carotene and 14mg ascorbic acid. The reports show that the inflorence, per 100g contains 44 calories, 86.2% moisture, 1.6% protein, 0.1% fat, 11.1% total carbohydrate, 2.5% fibre, 1.0% ash, 160mg phosphorus, 3.8mg iron, 28.5μg β-carotene, 0.04mg thiamin, 0.6mg riboflavin, 0.5mg niacin and 14mg ascorbic acid. Onyeke (2001) reported that sorrel calyx on wet (fresh) weight basis, per 100g sample contains 3.99% crude protein, 37.12% total carbohydrate, 1.66% fat, 1.26% crude fibre,1.76% ash, 77.95% moisture, 4.11mg calcium, 748.33mg iron, 20.08mg phosphorus, 28.95mg sodium, 1.55mg zinc, 0.89mg magnesium, 0.95μg copper, 220.92RE β-carotene, 15.56mg thiamin (Vit. B1), 17.38mg riboflavin (vit. B2) and 54.66mg ascorbic acid (vit. C). On dry weight basis, the calyces per 100g contains 8.02% crude protein, 69.70% total carbohydrate, 4.52%fat, 2.67% crude fibre, 5.74% ash, 11.93% moisture, 3.0mg calcium, 1.00mg magnesium, 833.0mg iron, 23.33mg phosphorus, 16.01mg sodium, 1.37mg zinc, 0.78μgcopper,285.29(RE) retinol equivalent, 24.67mg thiamin (vit. B1), 0.96mg riboflavin (vit. B2),56.83mg ascorbic acid (vit. C) (Nnam and Onyeke, 2003). McLean (1973) conducted a study on sorrel calyx (dry weight basis) reported for 100g sample, 13.28% moisture, 86.72% dry matter, 1.17% crude protein, 12.36% fat, 12.48% reducing sugar, 14.50% citric acid , 4.87% α-malic acid, 322mg phosphorus, 310mg magnesium and 101mg ascorbic acid. McLean (1973) study on the dry calyces showed 17.60% moisture, 83.60% dry matter, 0.36% fat, 11.65% crude protein, 20.75% crude fibre, 11.00% ash, 59.93% carbohydrate, 1473mg calcium, 237mg phosphorus, 41mg iron, 218μg β-carotene,60μgthiamin,191μg riboflavin, 3708μg niacin, 37076μg tryptophan and 37mg ascorbic acid. McLean (1973) reported that fresh calyces contain 0.72% fat and dry calyces contain 5.58% fat. Onyeke (2001) reported that red variety of the calyces of sorrel plant contain red anthocyanin monoxide in the form of prisms to which is given the name hibiscin. Yadong et al., (2005) reported that calyces contain pectin which is good in making firm jelly. McLean (1973) reported that sorrel calyx from Guinea, on dry weight basis, contained 7.93% protein and the calyx from Ivory Coast contained 9.3% protein. Duke (1983) and Rich (2004) reported that the calyx contains gossypetin, anthocyanin, which decrease the viscosity of blood and stimulate intestinal peristalsis. The seed: Yadong et al. (2005) reported that sorrel seeds are high in proteins. Duke (1983) reported that sorrel seeds contain 7.6% moisture, 24.0% crude protein, 22.3% fat, 15.3% fibre, 23.8% N-free extract, 7.0% ash, 0.3% calcium, 0.6% phosphoric acid and 0.4% sodium. McLean (1973) reported the acid components of the seed lipids as 2.1% myristic, 35.2% palmitic, 2.0% palmitoleic, 3.4% stearic, 34.0% oleic, 14.4% linoleic, 4.5% fatty acid and 2.9% sterculic acid. Duke (1983) reported the sterols in the seed oil as 61.3% β-sitosterol, 16.5% campasterol, 5.1% cholesterol and 3.2% ergosterol. Rich (2004) reported that the seed yields 20% oil. Faraja and Takhanib (1999) reported that the seed oil in East Indies and Senegal contained 55.9% stearic acid, 9.1% palmitic, 32.6% oleic and 2.4% linoleic. The oil is used as remedy for eye diseases and dysentery. McLean (1973) reported that the seeds could be kept for over a year without variation in the oil or fatty acid content. The raw sorrel oil contains a reasonable amount of free fatty acid which is easily refinable by conventional method. This oil is claimed to have good cooking properties. McLean (1973) reported that the fat component of sorrel seed has a density of 0.923 at 15oC, iodine index of 107.5mg and saponification index of 109. The fatty acid component of the seeds fat is as follows: 18.6% palmitic, 1.2% stearic, 35.8% oleic, 38.4% linoleic and 6.0% epoxyeleic. Its density at 150C is 0.923, iodine index 107.5 and saponification index 109. This report also showed that the unsaponifiable fraction of the oil yielded β-sitosterol. Onyeke (2001) reported that on fresh weight basis, the seeds per 100g sample contained 12.48% crude protein, 56.17% carbohydrate, 12.55% fat, 1.62% crude fibre, 1.37% ash, 23.95% moisture, 7.97mg calcium, 2.52mg magnesium, 430.67mg iron, 43.24mg phosphorus, 18.36mg sodium, 15.7mg zinc, 0.54μg copper, 58.41 (RE) β- carotene (retinol equivalent), 6.36mg thiamin (vit. B1), 6.12 mg riboflavin (vit. B2) and 12.92 mg ascorbic acid. On dry weight basis, sorrel seeds per 100g sample contained 18.55% crude protein, 54.69 carbohydrate, 12.84% fat, 3.05% crude fibre, 4.65% ash, 13.32% moisture, 33.28mg calcium, 22.11mg magnesium, 547.33mg iron, 54.69mg phosphorus, 14.43mg sodium, 1.03mg zinc, 1.05μg copper, traces of β-carotene, thiamin (vit. B1), riboflavin (vit. B2) and 40.45mg ascorbic acid (Onyeke, 2001). The McLean's (1973) study in Africa on sorrel seeds showed 8.20% moisture, 91.80% dry matter, 17.43% fat, 21.35% crude protein, 11.98% crude fibre, 5.34% ash, 43.90% carbohydrate, 388mg calcium, 503mg phosphorus, 4.5mg iron, 120μg thiamin, 153mg riboflavin, 1525μg niacin and traces of ascorbic acid. McLean (1973) obtained the following results for sorrel seed in Nigeria: 7.40% moisture, 93.60% dry matter, 21.38% fat, 28.94% crude protein, 16.74% crude fibre, 5.40% ash and 25.50% carbohydrate. The leaf: A study conducted by Duke (1983) reported that the sorrel leaf per 100g sample contained 43% calories, 85.6% moisture, 3.3g protein, 0.3g fat, 9.3g total carbohydrate, 1.6g fibre, 1.6g ash, 213mg calcium, 93mg phosphorus, 4.8mg iron, 4135μg β-carotene, 0.17mg thiamin, 0.45mg riboflavin, 1.2mg niacin and 54mg ascorbic acid. Onyeke (2001) reported the following values for sorrel leaf on fresh weight basis per 100g sample: 1.46% crude protein, 32.57% carbohydrate, 1.44% fat, 2.31% crude fibre, 2.34% ash, 76.22% moisture, 12.10mg calcium, 2.24mg magnesium, 654mg iron, 23.64mg phosphorus, 35.54mg sodium, 0.69mg zinc, 0.14μg copper, 365.86 (RE) β-carotene, 16.03mg thiamin, 19.84mg riboflavin and 11.90mg ascorbic acid. On dry weight basis, Onyeke (2001) reported 4.87% crude protein, 65.56% carbohydrate, 4.82% fat, 4.50% crude fibre, 9.95% ash, 14.36% moisture, 22.15mg calcium, 28.24mg magnesium, 430.80mg iron,56.85mg phosphorus, 21.80mg sodium, 1.03mg zinc, 0.96μg copper, 15.74mg riboflavin, 16.82mg ascorbic acid and traces of β-carotene and thiamin. McLean (1973) reported the following nutrient composition for sorrel leaf on 100g sample: 86.20% moisture, 13.40% dry matter, 7.84% fat, 12.12% crude protein, 8.74% carbohydrate, 62.39% reducing sugar, 8.91% de-malic acid, 1290mg calcium, 280 mg phosphorus and 40mg iron. McLean (1973) also observed that sorrel leaves are rich in β-carotene. Faraja and Tarkhanib (1999) reported that the leaf of sorrel contains mucilage, which is used as a dressing for septic wounds. 2.5 Phytochemicals in plant foods Nutraceutical refers to foods or parts of foods that provide medical or health benefits, including prevention and treatment of disease (Young and Young, 2003). Phytochemicals and antioxidants are two specific types of nutraceutical (Cindy, 2000). Some researchers (Dresbach and Rossi, 2000; Cindy, 2000) reported that the word "Phyto” comes from the Greek word plant. Phytochemical means plant chemicals. Goldberg (1994) defined phytochemicals as plants manufactured chemicals that have multiple functions. They also report that there are over 900 different types of phytochemicals. Phytochemicals are found in foods and are identified as components of food, which are consumed on daily basis. There may be more than 100 different phytochemicals in one vegetable dish (Goldberg, 1994). Human beings have an immune system that protects them from illness. Plants naturally produce different phytochemicals to protect them from bacteria, viruses and fungi. Scientists have discovered that these same compounds can also work as powerful disease blockers in humans (Dresbach and Rossi, 2000). Goldberg (1994) reported that some phytochemicals in plants attract insects to encourage fertilization and others provide defenses against predators such as viruses and animals. Dresbach and Rossi (2000) revealed some different phytochemicals that have been identified in plant foods. They include flavonoids found in berries, citrus fruits, onions, apples, grapes and soybeans. Sulforaphane, indoles, and isothiocyanates found in cruciferous vegetables such as broccoli and cabbage. Protease inhibitors found in grains and beans. Allicin found in garlic. Lycopene found in high amounts in tomatoes and red peppers. Reveratrol found in high amounts in grapes. Dresbach and Rossi (2000) reported the following as types of phytochemicals allyl Sulfides found in allium (garlic, onions, chives and leeks). Lycopene found in solanaceous vegetables (tomatoes and peppers). It protects against heart disease, certain cancers and multitude of other disorders. Gingerols and flavonoids are found in herbs and spices (ginger, mint, rosemary, thyme). Monoterpenes (limonene) and carotenoids are found in citrus fruits (oranges, grapes and grape fruits. Cindy (2000) reported that fruits and vegetables that are bright in colour (yellow, red, green, blue and purple) usually have the most phytochemicals and nutrients. Goldberg (1994) reported that active phytochemical extracts from leaves, stems, roots, flower and fruits of plants may be classified into three main categories. The first are compounds such as pyrrolizidine alkaloids, nicotine and hydrazine derivatives that are highly toxic and have no discernible therapeutic use. The second are morphine, digitalis and vinca alkaloids. These may be toxic but can elicit useful physiologic responses for treatment of disease when used in controlled amounts or for defined clinical conditions. The third group may exhibit chemo preventive activity against diseases such as atherosclerosis, cancer and diverticular disease. Most active are high molecular weight fibres (celluloids, pectins, lignins) and low molecular weight compounds (carotenoids, dithiolthiones, flavonoids, indole, carbinols, isothiocyanates, mone triterpenoids, and thioalkyl derivatives). Moon and Micozzi (1989) and Goldberg (1994) reported that all plants are sources of high molecular weight fibre. Specific low molecular weights phytochemical with chemo preventive activity are contained within a variety of plants. Cruciferous plants, such as cabbage and broccoli are excellent sources of indoles, dithiolthiones, isothiocyanates and chlorophyllins. Legumes (soybeans, peanuts, beans and peas) contain flavonoids, isoflavonoids and other polyphenols that act as antioxidants and estrogenic agonists/antagonists. Citrus fruits and licorice root contain mono- and titerpenses that act as antioxidants, cholesterol synthesis inhibitor and stifle growth of rapidly dividing cells. Thicallyl derivatives are found in garlic, leeks, and onion. They prevent thrombi formation, decrease cholesterol synthesis and prevent DNA damage. Moon and Micozzi (1989) studies revealed that different types of phytochemicals work in different ways to prevent disease. Some phytochemicals have anti-inflammatory properties and can inhibit clotting that can lead to a heart attack. Other phytochemicals have been found to reduce the risk of heart disease by influencing the absorption of cholesterol in the body. Some act like scavengers and remove carcinogens (cancer causing compounds) from the cells. Role of phytochemicals the majority of phytochemicals that have chemo preventive activity have no clearly defined role as essential nutrients except for the vitamins (ascorbate, tocopherols). Phytochemical deficiencies have not been identified. Their low concentrations in the diet have been associated with increased risks for cancer, cardiovascular diseases and diabetes (Moon and Micozzi, 1989). Dresbach and Rossi (2000) revealed that phytochemicals have been recognized as non-nutritive plant chemicals that hold within them certain disease – preventing properties. They are not yet classified as nutrients. Their properties aid animal and humans by creating a preventive barrier against diseases and sickness. Many researchers have recognized that there are phytochemicals that are present not only in plants for their protection, but also aid the human life. Cindy (2000) observed that foods with phytochemical may provide protection from diseases such as cancer, diabetes, heart disease, and hypertension. Some of the common types of phytochemicals include carotenoids found in carrots and the yellow, red and orange pigments of plants and ellagic acid found in berries. Phytochemicals play a major role in the prevention of heart diseases, cancer and menopausal health symptoms. Consumption of varied diet from fruits, vegetables, grains; legumes and seeds have health benefits. Other foods that contain high amounts of phytochemicals include garlic, onion, broccoli, cabbage, carrots, tomatoes, vegetable oil, strawberries, lemons, and hot peppers (Rossi and Dresbach, 1980). Dresbach and Rossi (2000) reported that eating three additional servings daily of fruits and vegetables may reduce stroke rates by 22% and risk of bleeding stroke by 51%. Ronald and Ronald (1997) reported that Nicotine and Caffeine in food are under the food regulation which specified that all food shall contain less than 1.60mg of nicotine or nicotinamide 100g. Effect of phytochemicals Goldberg (1994) reported that most phytochemicals are non-nutritive substances that can be beneficial or physiologically or pharmacologically active. Depending on the quantities consumed, duration of consumption and the conditions of use, most phytochemicals have the potential capacity to produce cellular or tissue injury. Goldberg (1994) reported that due to the cardiovascular disease protection and anticarcinogenic effects of phytochemicals, the risk factors of cardiovascular disease may be ameliorated by lowering cholesterol formation, blocking platelet aggregation and altering prostalglandin formation. Anticancer activities include detoxification of enzymes and antioxidant capacity, inhibition of DNA adducts formation, altering hormone metabolism and modifying promotion and mechanisms of progression. Another USDA report on phytochemicals showed that coffee contained caffeine and some negative effects of coffee consumption on human diseases such as inflammation, diabetes, and cardiovascular disease. Lynn (2007) observed that drinking coffee causes indigestion, headaches and interferes with sleep. Coffee usually has two to three times the caffeine of tea. An eight-ounce cup of coffee contains around 135mg caffeine; tea contains only 30 to 40mg per cup. The caffeine is a problem when more than 5 to 6 cups of a caffeinated beverage is consumed at one time. Flavonoids Flavonoids are a large group of ubiquitous molecules synthesized by plants (Djebbar, Nassima, Dina, Meriem, Nadjet and Hania, 2009). Januel, Fay, Ruggiero, Layarde and Vericel (2003) reported that flavonoids possess antioxidant activities which prevent free damage to biological molecules like lipids, proteins and DNA which can cause man cardiovascular, neurodegenerative diseases, cancer and diabetes. The George Mateljan Foundation (2010) reported that flavonoids constitute the largest class of polyphenols, widely spread in the plant kingdom and common in human diet. Verena, Mario and Karl (2006) reported that flavonoids are polyphenols abundantly found in fruits, vegetables, and herbs (tea, ginger root). Flavonoids are synthesized only in plants. They are adverse group of phytochemicals, exceeding four thousand in number (Subramani and Cosimir, 2002). Manthey (2000), Verena et al. (2006), Subramani and Casimir (2002) reported that from human nutrition perspective, flavonoids are important components of a healthy diet because of their antioxidant activity. Flavonoids types are quereetin, dihydroflavonols, anthocyanins, anthocyanidins, flavones, flavonols, isoflavones, flavanones, and flavanols. Most of the flavonoids present in fruit and vegetables are attached to sugars except for flavonoids that occur as monomers and as polymers named procyanidins. Flavonoids are different substances found in virtually all plants. They are responsible for many of the plant colours that dazzle with their brilliant shades of yellow, orange and red. They may be named after the unique plant that contains them (The George Mateljan Foundation, 2010). Roles Middleton and Kandaswami (1993) reported that flavonoid foods help protect blood vessels from rupture or leakage enhance the power of vitamin C, protect cell from oxygen damage and prevent excessive inflammation through the body. Verena et al. (2006) reported that flavonoids may delay or prevent the onset of cancer, coronary heart disease, antitumor and can also inhibit platelet aggregation. Subramani and Casimir (2002) reported in his studies that flavonoid intake reduced ovarian cancer risk all cardiovascular diseases. Neto (2007) reported that flavonoid present in fruit, fruit juice and vegetables prevent cancer, fight urinary infections and reduce breast cancer cells. Djebbar et al. (2009) reported that flavonoids from fruits and fruit juices have led to cardioprotective activity and can be the molecules for therapy against diseases. Flavonoids can act directly as antibiotics by disrupting the function of microorganisms like virus or bacteria (Middleton and Kandaswami, 1993). The George Metaljan Foundation (2010) reported that flavonoids protect cell structures, support vitamin C, control inflammation and act as antibiotic activity. Middleton and Kandaswami (1992) reported that excessive bruisability, nose bleeds, swelling after injury, hemorrhoids, frequent cold or infection are evidence of flavonoids deficiency. The George Metaljan Foundation (2010) reported that flavonoids help to prevent and treat allergy, asthma, atopic dermatitis, candida infection, cataracts, diabetes, Gout, Hemorrhoids, Muscular degeneration, migraine, periodontal disease, stomach ulcer and varicose veins. Sources Flavonoids are rich in apples, apricots, blue berries, pears, raspberries, strawberries, black beans, cabbage, onions, parsley, pinto beans, tomatoes (Neto, 2007). Middleton and Kandaswami (1992); Subramani and Casimir (2002) reported that all fruits, vegetables, herbs and spices contain flavonoids. Products made from these fruits, vegetable, herbs and spices also contain a wide variety of flavonoid. Carotene Carotene is polyunsaturated hydrocarbons containing forty carbon atoms per molecule, variable numbers of hydrogen atoms, and no other elements (Linus Pauling Institute 2009). Carotenoids are red, yellow and orange pigments, which are widely distributed in nature and are abundant in yellow – orange fruits and vegetables and dark green leafy vegetable. Linus Pauling Institute (2009) defines carotene as an orange photosynthetic pigment that is important for photosynthesis. Carotenes are all coloured to the human eye. Carotenes are found in plants in two primary forms designated by characters from the Greek alphabet, alpha – carotene (β-carotene). Carotene are fat soluble and insoluble in water. The pink colour is due to beta carotene. They absorb from the blue- green algae in diet (Ajila and Prasada, 2008). Actions View History Search (2010) reported that other carotene like Gamma – carotene (γ-carotene) delta-carotene (δ-carotene), epsilon-carotene (ε- carotene) and zeta carotene (ζ-carotene) exist. Β-carotene is an antioxidant and protects plant cells against the effects of ultraviolet light. Roles Higher dietary intake and blood serum levels of alpha-carotene are associated with significantly lower risk of death (The Alpha – Tocopherol, Beta-carotene Cancer Prevention study Group, 1994). Artemis and Gopalan (2003) reported that carotene prevent blindness, heart disease, cancers and degenerative eye disease. Sources Carotenes are rich in carrots, sweet potatoes, manger, cantaloupe melon, apricots, spinach, beet greens, kale, turnip greens, mustard greens, water cress, fresh thyme broccoli, parsley, romaine lettuce, rose hips, pumpkin, cassava, Ivy gaird, and wolfberries (Linus Pauling Institute, 2009; Ajila and Prasado, 2008; Artemis and Gopalan,2003).http://www.md- phc.com/nutrition/carotenoids and human health.htm reported that carotenes are contained in tomatoes, apricots, pink grape fruit, guava, skin of red grapes, papaya, watermelon, carrots, pumpkin, cabbage, lima beans, green beans, broccoli, brussels sprouts, kale, kiwi, lettuce, spinach, tomatoes, honeydew melon and oranges. Lycopene Lycopene is a highly unsaturated straight chain hydrocarbon with a total of 13 double bounds, 11 of which are conjugated. This unique nature of the lycopene molecule makes it very potent antioxidant (Rao, Gun and Rao, 2003). Lycopene is one of the major carotenoids in diets and found in tomatoes and tomato products. It accounts for about 50% of carotenoids in human serum. Among the common dietary carotenoids’ lycopene has the highest singlet oxygen quenching capacity in vitro. Other outstanding features are its high concentration in testes, adrenal gland and prostate (Gerster, 1997). Rao et al., (2003) define lycopene as a carotenoid phytonutrient. It is the most potent antioxidant naturally present in many fruits and vegetables. Bramley (2000) reported that lycopene is the red-coloured carotenoid predominantly found in tomato fruit, but in few other fruits or vegetables. Bramley (2000) defined lycopene as one of over 600 carotenoids found in nature. Lycopene accumulates in relatively few tissues and can easily be seen in ripe tomato fruit, watermelon and pink grapefruits. Roles Bramley (2000) reported that three research directions have been used to study the role of lycopene in humans: epidemiology (bioavailability), the effect of lycopene on tumor proliferation and biochemical/immunological mechanisms of lycopene action. Rao et.,al .(2003); Rao and Agarwal (1999) reported that lycopene reduces cancer, coronary heart disease and osteoporosis. Lycopene bears significant potential in the treatment and prevention of prostate cancer (Rao et al., 2003). Rao and Agarwal (1999); Giovannucci, Liu, Stampfer, Willett (2002) reported that studies on role of lycopene showed that lycopene associated with the prevention of prostate cancer. Lavecchia (1997) reported that the Mediterranean diet which is rich in vegetables and fruits, has been responsible for the lower cancer rate in that region. Rao and Agarwal (1999) reported that intake of lycopene reduced lung, prostate, stomach, breast, cervical and uterine cancers. Lycopene as a potent antioxidant plays an important protective role against coronary heart disease. Rao and Balachandran (2003), Rao and Heber (2001) reported in their epidemiological studies that lycopene resist the risk of coronary heart disease. The potent antioxidant properties of lycopene are the evidence for the role of oxidative stress in bone health (Rao et al., 2003). Iwasaki and Gagnon (1992) reported that male infertility can be as a result of oxidative stress. Human spermatozoa are particularly vulnerable to oxidative damage due to the abundance of unsaturated fatty acids in the sperm plasma membrane. Sources Bramley (2000) observed that humans are unable to synthesize carotenoids but can obtain lycopene from the diet. 85% of dietary lycopene come from tomatoes, fruit and tomato based products, watermelon, pink grapefruit, guava and papaya (Shi and Le Mageur, 2000). Alkaloids Alkaloids are group of naturally occurring chemical compounds which mostly contain basic nitrogen atoms. They include some related compounds with neutral and weakly acidic properties (Andreas, 2009). Robbert (1998) reported that some synthetic compounds of similar structure are attributed to alkaloids. Carbon, hydrogen and nitrogen, molecules of alkaloids may contain sulfur and chlorine, bromine or phosphorus. Dimitris, Gregory and Robert (1997) classified alkaloids into five major groups. True alkaloids contain nitrogen in the heterocycle and originated from amino acids egatropine, nicotine and morphine. Protoalkaloids contain nitrogen and originated from amino acids e.g. mescaline, adrenaline and ephedrine. Polyamine alkaloids are those derivatives of putrescine, spermidine and spermine. Peptide and cyclopeptide alkaloids and pseudoalkaloids do not originate from amino acids e.g. caffeine, theobromine and theophylline. Spiller (1997) reported that most alkaloids are poorly soluble in water but can dissolve in organic solvents like diethyl ether and chloroform. They have a bitter flavour taste. Chan, Chan, Tanlinson and Critchley (1993) reported that alkaloids produce neurologic, cardiovascular and gastrointestinal disturbances can lead to death. Some alkaloids may, however, display tumor inhibitory activity due to the interaculation of the alkaloids with the DNA (Roja and Rao, 2000). Roles Alkaloid containing plants were used by humans since ancient times for therapeutic and recreational purposes (Tadeusz, 2007). Alkaloids can be purified from crude extracts by acid-base extraction. Many alkaloids are toxic to other organisms. They often have pharmacological effects. They are used as medications, recreational drugs or in entheogenic rituals, examples are the local anesthetic and stimulant cocaine, the stimulant caffeine, nicotine and analgestic morphine or the antimalarial drug quinine (Okwu and Okwu,2004). Paul (2002) and Tadeusz (2007) reported that alkaloids help in protection and prevention of insects and chordate animals from eating plants. In medical use, it enhances the effect of the drug and reduces side effects. In agriculture, alkaloids are used as insecticides. It is used as psychoactive drugs, as stimulants (cocaine and cathinone) of the central nervous system. Sources Alkaloids are produced by a large variety of organisms, including bacteria, fungi, plants and animals. Alkaloids are found in leaves, fruits or seeds, root, raw plants in the form of salts of organic acids, and different tissues of the same plants (Manfred, 2002; Paul, 2002; Tadeusz, 2007). Caffeine Caffeine is a naturally occurring stimulant found in the leaves, seeds or fruit of over sixty plants around the world (Heaney, 2002). Ministry of Agriculture, Fisheries and Food (MAFF) (1998) reported that caffeine is part of a group of compounds known as methylxanthines. Caffeine and tea also contain other dimethyl an, theophylline which has similar properties to caffeine and theobromine whose pharmacological action is far less potent than caffeine and theophylline. Caffeine is the most frequently ingested pharmacologically active substance in the world. It is found in common beverages (tea, coffee, and soft drinks), cocoa or chocolate (Nawrot, Jordan, Eastwood, Rotstein, Hugenholtz and Feeley, 2003). Roles Smith (2002) reported that moderate use of caffeine is not likely to cause health problems. Thelle (1995) reported that caffeine per day does not affect cardiovascular health. Caffeine and Ibuprofen combination help in relieving headache but chronic headache sufferers should avoid caffeine because it might exacerbate symptoms (Yang, Maliakal and Meng 2002). Researchers agreed that moderate use of caffeine (300 milligrams or about three cups of coffee, per day) is not likely to cause health problem (Thelle, 1995; Yang et al.,2002; Heaney, 2002). Heaney (2002) reported that caffeine stimulates the nervous, cardiovascular systems, decreased fatigue. Smith and Haddad (2000); Juhn (2002) reported that caffeine intake increase the excretion of calcium, prevent osteoporosis and weight control. Sources Caffeine are rich in coffee, tea, leaves, beverage, herbs, cola or pepper type, chocolate and soft drink.(Thella,1995;Heaney,2002). Nicotine Nicotine is a highly addictive psychoactive chemical. Nicotine is a stimulant and one of the main factors leading to continued tobacco smoking (Hecht, 1999). Tobacco contains nicotine which is the single greatest cause of preventable death in the United State (Mokdad, Marks, Stroup, Gerberding, 2004). WHO (2008b) reported that tobacco caused 5.4 million deaths in 2004. The United States Centers for Disease Control and Prevention describes tobacco use as “the single most important preventable risk to human health in developed countries and an important cause of premature death worldwide. Feng, Hu, Hu, Tang (2006) reported that nicotine in smoke contain several carcinogenic pyrolytic products that bind to DNA and cause genetic mutations. He also responded that acrolein, which is abundant in tobacco smoke, also binds to DNA, causes mutation and cancer. Roles the increased risk of contracting disease is directly proportional to the length of time person continues to smoke as well as the amount smoked. If someone stops smoking, these chance gradually decrease as the damage to the body is repaired. Risks vary according to amount of tobacco smoked, with those who smoke more at greater risk. Light smoking is still a health risk. Doll, Peto, Boreham, Sutherland (2004) reported that nicotine in tobacco leads to diseases affecting the heart, lungs cancer, cancers of the larynx and mouth, pancreatic cancer and also reduced overall life expectation in regular smokers. Doll et al., (2004); Mokdad et al. (2004); Feng et al., (2006), WHO (2008b). In their studies reported the risks of nicotine intake in tobacco and smoking. The primary risks include many forms of cancer (bladder, larynx, head and neck, breast, esophagus, pancreas, stomach, myceloid leukemia, liver, cervical, colorectal, ovarian, adrenal gland, small intestine and various childhood cancers), cardiovascular, renal, influenza, infection, impotence, psychological, stress, cognitive function, miscarriages, decreases appetite and social and behavioural (divorce). Sources Nicotine are found in tobacco, smoke, cigarette, food chocolate, drink coffee, tea, wine (Doll et al., 2004; Feng et al., 2006). 2.5.1 Antioxidants: Antioxidants are compound in fruits and vegetables that may be helpful in avoiding chronic disease. They act as a defense system against oxidative damage in our bodies and may be helpful in avoiding chronic diseases and the effects of aging. Some examples of antioxidants are vitamins A, C, and E and beta-carotene (Cindy, 2002). Effects of antioxidants. Lynn (2007) reported 10 health benefits of drinking tea. He showed that tea contains antioxidants, protects the body from the revenges of aging and effects of pollution. Tea may reduce the risk of heart attack and stroke, unwanted blood clots formed from cholesterol. Blood platelets cause heart attack and stroke. Drinking tea protects and builds strong bones. Tea actually contains fluoride and tannins for healthier teeth and gums. It fights off infection (immune defenses), protects against cancer because of polyphenols and antioxidants found in tea. Caffeinated beverages keep the body hydrants including tea. Recent research showed that the caffeined beverages contributed to the fluid intakes. The only time the caffeine becomes a problem as far as fluid is concerned is when you drink more than five or six cups of a caffeinated beverage at one time (Lynn, 2007). Legumes (soybeans, peanuts, beans and peas) contain flavonoids, isoflavanoids and other polyphenols that act as antioxidants and antagonist. Citrus fruits and licorice roots contain mono and triterpenes that act as antioxidants. USDA reports showed that camellia sinensis contains high levels of polyphenols with strong antioxidant activity (e.g.catechins, gallaogatechin, epicatechin, epigallocatechin, gallate and epigallocatechin gallate). Antioxidant activity is believed to quench free radicle contributing to beneficial effects on several human chronic diseases (atherosclerosis, hypercholesterolemia, diabetes, obesity and cancer). The genus camellia belongs to the genus contain numerous phytochemical including tea catechuic camellia sinensis is the source for several (Lynn, 2007). Bagchi and Puri (1998) reported that the balance between the production of free radicals and the antioxidant defenses in the body has important health implications. If there are too many free radicals produced and too few antioxidants, “oxidative stress” develops to cause chronic damage. Dasgupta and De (2004) reported that free radicals precipitate cancer, atherosclerosis, and heart disease. Bagchi and Puri (1998) reported that free radicals precipitate acute respiratory distress syndrome, myocardial infarction, senile cataracts, cancer, atherosclerosis, cerebrovascular accidents, and rheumatoid arthritis. Aruonia (2003) and Coruh, Celep and Ozgokee, 2007) reported that ischemic heart disease, Alzheimer’s disease, Parkinson’s disease and aging process are attributable to free radicals. 2.7 Antinutritional and food toxicant factors (phytate, tannins cyanide and trypsin inhibitor) Plants contain a wide variety of chemical substances that are known to exert deleterious effects when ingested by man and animals. These substances are known as antinutrients. These antinutritional factors include tannins, phytates, goitrogens, trypsin inhibitors, haemagglutinin or lectins, cyanogen and α- galactosides of the oligosaccharide sugars. Phytate Phytates play a significant role in limiting the nutritive value of dry seeds by contact with minerals, proteins and other trace elements (Mba-Anyadioha, 2008). Nnam (1994) and Nnam and Onyeke (2003) reported that the importance of phytic acid in nutrition lies in its ability to chelate several mineral elements including calcium, iron, magnesium and zinc. They are phytate-mineral complexes. These reduce the availability in the intestinal tract. Glahn, Wortley, South and Miller (2002) and Matuschek and Svanbery (2002) reported that phytate in plant foods binds essential dietary minerals in the digestive tract and made them unavailable for absorption. It forms insoluble complexes with Cu², Zn², Fe³ and Ca². It also reduces the bioavailability of these essential minerals. Maerhav, Anaitai, Patti and Godfrey (1985) reported that phytates are chelate agents. They bind important divalent minerals (calcium, magnesium, cobalt, manganese, zinc, copper and iron) to form complex phytate. Matuschek and Svanbery (2002) reported that phytate myoinositol hexsokis phosphate was found in all the plant seeds. The mature legume and cereal grains had the highest concentration. It is uniformly distributed and associated with the protein complexes. It is contained in both the bran and germ of the dry seeds. Phytate is a source of phosphorus. Phytate hydrolyzed very rapidly in germinating seeds by phytates. Phytate is unavailable to humans due to lack of an endogenous enzyme system that can catalyze the hydrolysis of the molecules to its moieties. During digestion, they interfere with absorption of nutrients by reacting with metals to form insoluble metal complexes. Phytate reduces the bioavailability of dietary copper and other trace elements (Lopez, Lienhardt, Coudray and Ramsey, 2002). Glahn et al., (2002) reported that phytate is an antinutrient due to its adverse effects. It reduces the bioavailability of minerals and causes growth inhibition. Heaney, Weaver and Fitzsimmons (1991) reported that the safe level of phytate in soy bean phytate content: Effect on calcium absorption is 3.01mg/g. Some processing methods like fermentation (Omafuvbe, Falade, Osuntogun, Adeausi, 2004); germination (Roa, 2003), soaking (Obizoba and Amaechi, 1992) and other autolytic treatments may reduce or eliminate phytate in seeds. Tannins Tannins are acid substances found in plant seeds. Obiakor (2001) reported that tannins are polyphenolic compounds found in many plants identified in dry seeds. They are located mainly in the seed coat with low or negligible amount in the cotyledons (Roa, 2003). Reichert, Fleming and Schwab (1980) reported that 81.60% of the condensed tannin was located in the testa layer. About 15.10% was located in the pericarp. Only 57% of the tannins extractable from untreated seeds were accounted for by summing the tannins present in all components. This demonstrates the reactivity of tannins in the presence of water. Tannins have deleterious effects due to their strong interactions with protein. The resulting complexes are not readily digested by monogastrics. This lowers protein digestibility (Obizoba and Amaechi, 1992). Henry and Massey (2001) reported that tannins interact with proteins and enzymes to increase the excretion of proteins and essential amino acids. Santamaria, Elia, Serio and Todaro (1999) reported that low bioavailability of minerals in plant food was attributed to chelation of minerals with tannins. Gillooly, Bothwell, Torrance, Bezwoder, Macphil, Derman, Novelty, Morrall and Marvel (1984) reported that the presence of tannins in food bind nutrients such as iron and proteins to reduce their bio-availability. Schiavone, Guo, Tassone, Gasco, Hermandez, Denti, and Zoccarate (2007) reported the safe level of tannins in effects of a natural extract of chestnut wood on digestibility, performance traits and nitrogen balance of broiler chicks is 150-200mg/g. Several processing methods have been shown to eliminate tannins. Simple soaking of the seeds and air-drying destroyed or insolubilized of the tannins (Roa, 2003). They showed that the extractable tannins content in plant seeds was reduced by inhibiting water, HCL or NaOH solutions into the white seeds and storing the treated grains under CO2 atmosphere. Storage of acid- or water- treated seeds may insolubilize the tannins by protein-tannins extraction. Obiakor (2001) reported that decreases in tannins were functionable during fermentation processing. Cyanide Cyanide (cyanogenic compounds) is classified as antinutritional factor known as goitrogens. Goitrogens adversely affect iodine utilization by the thyroid gland (Portor, Baskin, 1996). Conn (1973) reported that more than 1000 species of plants contain cyanogenic glucosides and that hydrocyanic acid (HCN) is released when the tissues of the plants are crushed or otherwise disrupted. The release of the acid may be due to treatment with acid or the use of appropriate hydrolytic enzymes. Hydrocyanic acid has been incriminated in the etiology of tropical ataxic neuropathy, a group of nervous disorders diagnosed in patients who have been depending for a long time on a diet of cassava products (Oshuntokun, 1973). Oke (1974) reported the presence of cyanogenic glucosides, which are habitually consumed by man in maize, millet, sorghum, lima bean, kidney bean, sweet potato, yam, bamboo and sugar cane, kernel of almond, lemon and cassava among others. Lima beans and cassava contain a high concentration of 40 – 60mg HCN/100g, respectively. The most common cyanogenic glycosides in cereals are dhurrin. Oke (1969) reported that the safe level of cyanide in the role of hydrocyanide acid in nutrition is 35mg/100g. Processing like crushing of foods before soaking in water could detoxify the cyanogenic glucoside. This is accomplished through the action of the enzyme present in the plant tissue. It breaks down the glucosides to yield free hydrogen cyanide (Nnam,1994). Trypsin inhibitor Trypsin are mostly located in the outer layer of the cotyledon of legumes (Obiakor, 2001). However, the trypsin inhibitory activity varies from one seed to another. Harwood (1986) reported the safe level of trypsin inhibitor in effects of protease inhibitors on the Cebu’s monkey is 0.54mg/g. Adequate heat treatment has been shown to completely eliminate the presence of trypsin inhibitors in seeds (Nnam, 1994). Other processing methods like soaking in water through leaching (Obizoba & Amaechi, 1992) germination (Ologhobo and Fetuga, 1983) and fermentation (Omafuvbe et al., 2004) reduced the presence of trypsin inhibitors. 2.8 Antinutrient and food toxicant composition of the different parts of sorrel plant (calyx, seed and leaf) The calyx: Onyeke (2001) reported that sorrel calyx on wet (fresh) weight basis contained 0.43mg phytate, 5.40mg tannins and 0.05mg cyanide. On the dry weight basis, sorrel calyx contained traces of phytate, tannins and cyanide (Onyeke,2001). The seed: A study conducted by Onyeke (2001) reported that sorrel seed on wet (fresh) weight basis contained 0.44mg phytate, 4.70mg tannins and 0.58mg cyanide. On dry weight basis, the seed contained 0.30mg phytate, 1.33mg tannins and 0.77mg cyanide. The leaf: Nnam and Onyeke (2010) reported that on wet (fresh) weight basis the leaf contained traces of phytate, tannins and cyanide. Processing Technique processing of food is a technology, which is as old as history of man and food. Plant and animal foods require some degree of processing for maximum utilization of nutrient. Foods are processed to improve flavouring, stability, introduce functionality, improve nutritive value, palatability, storage keeping quality, and digestibility, decrease the possibility of toxicity and most cases reduce effect of antinutritional factors in plant foods (Obizoba and Amaechi,1992; Nnam;1994; Onyeke, 2001; Mba-Anyadioha, 2008; Nnam and Baiyeri,2008). Processing of food does not only improve palatability but also nutrient availability. However, Roa (2003) reported that in some instances processing methods may not decrease antinutrients but may lead to the formation of new ones. The antinutrients are shown to have deleterious effect on health of both man and animals. Processing of sorrel calyces, seeds and leaves is important due to the antinutrients they contain (Onyeke, 2001). The processing method that would be used in the product development includes milling, heat treatment, soaking, dehulling and fermentation. Heat treatment: Heat treatment is an act of cooking food by heat either by wet or dry method. Heat treatment makes food soft, palatable, kills contaminating bacteria and destroys antiphysiological substances present in the food (Onyeke, 2001). Nnam and Baiyeri (2008) noted that heat processing is the most encountered in food preparation and it has a very important effect on various food compounds and quality. Heat treatment involves boiling in water, autoclaving; pressure steaming, roasting (drying), frying, baking, microwave cooking, and extrusion cooking, among others. Heating leads to the gelatinization of starch and the denaturation of protein and to other interactions involving other ingredients in food. These changes alter the texture, taste, digestibility and acceptability of the products. Colour, flavour and aroma are developed in cooked, baked, fried, puffed, toasted and roasted products (Obiakor, 2001). Autoclave treatment has effect on tannins level by in seeds, and improves flavour and nutritive value changes during drying. Continuous heating decreases protein (Lapez et al.,2002).The loss of protein quality on continued heating is related to increasing maillad brawing causing lysine to be rendered unavailable (Nnam and Baiyeri, 2008) Cooking is an act of applying heat treatment on food. Cooking destroys contaminating bacteria. It can be done through the wet or dry method. By wet method, it involves boiling in water and pressure steaming. Dry method involves roasting, autoclaving and frying. Cooking of food makes food soft, palatable and destroys anti-physiological substance present in many foods (Onyeke, 2001). Falade, Sowunmi, Oladipo, Tubosu and Adewusi (2003) reported that heat improves digestibility by destroying trypsin inhibitors and various toxin. The increased digestibility may be due to destruction of the heat labile protease inhibitors (Nnam, 1994). In Nigeria, the most common cooking method for preparing sorrel products are boiling, steaming and roasting. Boiling is mostly used because it does not require too much gadgets in the local communities. When sorrel calyx, seed and leaf were dried over airtight oven (autoclave) the fresh seed changed colour from greenish to dark brown (Onyeke, 2001). The change was attributed to chemical changes during drying Milling: Milling method of processing food aims at separating the endosperm, germ and bran from each other. Onyeke (2001) reported that milling reduced proximate composition and mineral contents of sorrel seed. Perdesea and Eggum (1983) reported that ash, fat and crude fibre were reduced to 40% of corresponding level in sorghum at an extraction rate of 64%. Milling at high extraction rate of 80% was recommended to avoid loss of nutritious embryo and endosperm. Tannins and phytate content of low tannins sorghum was significantly reduced by milling. However, leucine glutamic acid and proline of the sorghum grain increased after milling. The biological value and net protein utilization of the grain were not significantly affected by the degree of milling. Soaking: Soaking as a processing method softens the cotyledon and helps the seed to dehull quickly. Seeds soaked in water imbibe sufficient water to act as a solvent, reactant and medium for transport of nutrients during germination process. Nutrient content of food can be lost during soaking. Soaking leads to some loss of essential nutrients as minerals, proteinaceous bodies, pectic substances and other soluble solutes located in the bran of the seed (Obizoba & Amaechi, 1992). Nnam (1994) reported that trypsin inhibitors are located in the outer layer of the cotyledon of legume and can be lost during soaking. Osho (1989) reported that trypsin inhibitors reduced protein digestibility, thereby reducing the nutritive value of the seeds. Oguntimein (1993) reported that trypsin inhibitors, saponins, toxic acid and other nutritional stress factors are either inactivated, destroyed, reduced, or altered by proper heat treatment, especially when combined with other process such as soaking, germination and fermentation. Soaking reduces the levels of antinutritional factors (Obiakor, 2001). Soaking improved biological value of some legumes (Nnam, 1994). This was attributed to the leaching of some antinutrients in the soaking water. The better digestibility of soaked seeds may be due to leaching into the soaking water of certain antinutritional factors like phytate, trypsin inhibitors, tannins from seed (Oguntimein, 1993). Nnam and Onyeke (2003) observed that soaking decreased antinutritional factors in seeds. It reduced or eliminated the antinutritional factors such as tannins (Obizoba and Amaechi,1992) and phytate content of legumes (Nnam, 2000). Obizoba and Atii (1991) reported that soaking reduced cooking time and helps in the removal of seed-coat in the grains studied. Soaking offers other advantages in terms of the nutritional quality of the legume. Soaking soybean for 12 hours in water brought about significant improvement in in vitro digestibility (IVPD) of starch and protein (Matusdek and Suanberg,2002). The impact on protein digestibility was much when the seeds were soaked in mixed salt solution. Prolonged soaking initiates activation of certain enzymes, eventually leading to improvement in protein digestibility. FAO (1990) reported that soaking of cassava tuber in water improves detoxification as cells are broken by osmosis and fermentation, which facilitate hydrolysis. Soaking of baobab and rice in water, leach out coloring matter and substances that impart odor in the products (Nnam and Obiakor, 2003). Dehulling: Dehulling of seed involves the removal of seed-coat either manually or mechanically. Dehulling enhances texture, appearance, cooking time, digestibility of protein, nutritive value and colour of food. This may be due to the removal of antinutritional factors and toxic substances which abound in the seed coat. Oomah, Mazza and Kenaschuke (1996); Obiakor (2001) showed that dehulling is another way by which toxic materials can be removed, especially, if it is preceded by soaking and fermentation. Dehulling may lead to loss of many nutrients, especially those located in the testa. Some proteins, thiamin and calcium were lost in dry mechanical dehulled soybean seeds (Parsons, Hashimoto,Wedekind and Baker, 1991). Onyeke (2001) showed that dehulling of sorrel seeds resulted in loss of cyanide and phytate. Oomah and Mazza (1996) also reported that tannins levels are reduced after dehulling beans. Another report by Nnam and Onyeke (2010) showed that tannins (2.40mg) were not completely removed by dehulling. Oomah and Mazza (1996) reported that removal of hulls from flaxseed resulted in an increase in oil (237g/kg) and protein (135g/kg) contents and the concentration of carbohydrate was reduced by 24g/kg. Fermentation: Achinewhu (1986) defined fermentation as a microbial process where carbohydrates are broken down by metabolism with the production of acid. FAO/WHO (1996) defined fermentation as the biochemical modification of primary food product brought about by the action of microflora enzymes. Fermentation as a technique is a method of preparing and preserving food to produce new product. Fermentation is employed to improve the availability of amino acid and nutritive value of food. It is one of the traditional known methods of processing food (Obadina,Oyewole and Awojobi, 2008). Fermentation technique increases activity of the protease, lipase, amylase and pectinase enzymes (Nnam, 1994). Obizoba and Amaechi (1992); Obiakor (2001) reported that crude fibre decreases during fermentation because of enzyme activity in the media. Fermentation could be done commercially/industrially or traditionally. Fermentation is used to enhance the taste, shelf life, texture, colour, flavour, aroma, solubilization and digestibility of food. It improves nutritional quality, reduces cooking time and decreases toxic substances in food (FAO/ WHO, 1996; WHO, 1998). During fermentation proteins are hydrolyzed to amino acids. Galacto-oligosacchrides are hydrolyzed to simple sugars, minerals are made more available. The inedible foods are made edible (Odunfa,1985a; Obizoba and Amaechi, 1992; Mbata, Ikenebomeh and Ahonkhai, 2007). Nnam (1994) showed that minerals are known to be more available after fermentation of corn, cowpea and African yam-bean. Fermentations are used to decrease the carbohydrate for the synthesis of other compounds like protein, esters, alcohol, organic acids and their salts (Nnam, 1994; Nnam, 2004). Obiakor (2001) and Mbata et al. (2007) reported that fermentation decreases carbohydrate and has a sparing effect on other nutrients. The fermentation process may be complete oxidation in which sugars are converted to carbon dioxide and water. It may be partial lactic acid, butyric acid, alcohol aldehydes, hydrogen and some flavour compounds. Fermentation may impart new color, flavour, taste, odour and texture to a product (Nnam, 1994). Fermentation is used for the production of food condiment, gari, ogi and pito beer in Nigeria. Obiakor (2001) found that there were decreases in content of sulphur-containing amino-acid such as cysteine and methionine and the increase in protein content. Fermentation is one of the oldest methods of preserving food by man. It is used in producing fermented food condiment such as okpei, dawadawa and ogiri, which are used to improve flavor in food like soups. Odunfa (1985b) reported that fermented castor oil beans had decreased toxic substances. Omaffuvbe (1998) reported that fermented products are ground to make oily pastes which are used as flavouring condiment in soups and sauces. They produced pleasant, very strong putrid odour with some ammonical odour which give pleasant odour, flavour, colour, taste, and texture to a product (Nnam,1994; Omafuvbe,1998) when added to soups and sauces. These contributed to the protein and essential fatty acid intake. Obiakor (2001) reported that lipases hydrolyze fat to glycerol and fatty acids. The fatty acids are used in the synthesis of new lipids during the metabolic activities of the microflora. Aworth (1993) and Obiakor (2001) observed that fermentation increases minerals such as calcium and phosphorus during the hydrolysis of phylate. Aworth (1993) observed that fermentation increased β-carotene as well as B- complex vitamins, especially riboflavin. It increases protein quality, improves availability of various nutrients and removes antiphysiological factors. Omafuvbe et al. (2004) reported the effect of fermentation on the nutrient content of locust beans as follows: protein and fat were increased and the carbohydrate and crude fibre were reduced. Omafuvbe (1998) reported that fermentation of soybeans for varying periods (8, 12 and 16 hours) caused significant increases in the in vitro digestibility of protein. Obiakor (2001) reported increased riboflavin and nicotinic acid in fermented maize over 72-hour fermentation. Fermentation is a function of yeast, mould, bacteria or their combinations. Yeast fermentation is of primary importance in the production of oriental foods e.g. tempeh. Lactate fermentation is desirable for food preservation. Most lactate fermentation is initiated by leuconostoc mesenteroides and pediococeus. Lactobacillus plantarum and L. brevis normally succeed the spherical bacteria. The activities of these microflora lower pH to the desired level of about 3.6. Fermentation by lowering pH provides for more optimal conditions for enzymatic degradation of the phytate present in cereals in the form of complexes with protein or polyvalent cations such as iron, zinc, calcium and magnesium (Obiakor, 2001). Fermentation is the basis for the production of many indigenous beverages from sugary and starchy substrates. It is also used in the production of modern wine from various fruit juices and palm wine from palm sap obtained from palm trees and raffia palm. The modern beer, whisky, brandy and gin are products of controlled fermentation. Nnam (1994) reported that minerals are known to be more available after fermentation. FAO/WHO (1996) reported that fermentation has various effects on the nutrient quality of food. Microbial fermentation leads to a decrease in the level of carbohydrates and indigestible oligosaccharides in legumes. Obizoba and Amaechi (1992) reported that fermentation lowered the flatulence in baobab seeds. They also reported that the fermented product is ground to make oil pastes which are used as flavouring condiments in soups and sauces. CHAPTER THREE 3.0 MATERIALS AND METHODOLOGY 3.1 EQUIPMENTS AND GLASS WARES 1. Five different samples of Zobo. 2. Sterile containers or bottles 3. Pipettes (0.1,1.0, 5.0 and 10 mL) 4. Sterile Petri dishes 5. Media (Nutrient agar/ Eosin methylene blue agar, Lactose broth, MacConkey broth/ agar triple sugar iron agar Urea agar and Simmon's citrate agar) 6. Incubator 7. Autoclave or pressure cooker 8. Microscope, microscope slides and cover slips 9. Sterile loops and needles 10. Conical flasks 11. Peptone water 12. Cotton wool 13. Distilled water 14. pH meter 15. Thermometer 16. Aluminum foil 17. Test tubes 18. Test tubes racks 19. Spatula 20. Filter papers 21. Weighing balance 22. Durham's tubes 23. Protective clothes (Laboratory coat, nose/face mask, head covers and hand gloves) 24. Bijou bottles and masking tape 25. Refrigerator 3.2 Reagents Needed 1. Crystal violet, Lugol's iodine, acetone, ethanol, neutral red/ safranine 2. Hydrogen peroxide (3%) 3. Plasma (animal or human) 4. Sugars (glucose, sucrose, lactose, maltose and fructose) 5. Bromothymol blue/ Bromocresol purple indicators 6. Methyl red solution 7. Malachite green solution 8. Indole test (kovac's) reagent 9. Voges-Proskauer reagents 10. Oil immersion 11. Oxidase test reagent AGAR USED ➢ MCCONKEY AGAR ➢ NUTRIENT AGAR 3.3 METHODOLOGY 3.3.1 STUDY AREA This study was carried out in Emene Enugu, Enugu State, Nigeria. 3.3.2 SAMPLE COLLECTION Five zobo samples were purchased from different locations mentioned above and was transferred immediately to the Laboratory for bacteriological analysis. The samples collected were for bacteriological and biochemical analysis. 3.3.3 PREPARATION OF MEDIA Five different media were used in this study. These are Nutrient Agar, MacConkey Agar, Mannitol Salt Agar (MSA), Cetrimide Agar, Sabouraud Dextrose Agar (SDA). The media were prepared according to manufacturer’s procedure. Nutrient Agar 28 g of nutrient agar was transferred into a conical flask containing 100 ml of distilled water. The medium was then left to soak for 10 minutes before swirling to dissolve property. MacConkey Agar 53.5g of MacConkey Agar was suspended in 1000 millimeters distilled water. It was heated to boiling point to dissolve the medium completely and autoclaving at 1210C for 15 minutes. It was cooled at 45 – 500C before it was poured into a sterile petriplates Mannitol Salt Agar (MSA) Mannitol Salt Agar (111g) was suspended in 1000 millimeter of distilled water to prepare the medium. The medium was boiled to dissolve completely and then sterilized in an autoclave at 1210C for 15 minutes. It was cooled at 45 – 500C before it was poured into a sterile petri-plates. Cetrimide Agar 45.5g of Cetrimide Agar was suspended in 1000millimeter of distilled water. 10ml of glycerol was added and boiled to dissolve completely. The medium was sterilized by autoclaving at 1210C for 15minutes and cooled to 500C before being poured into sterile petri-dishes. Sterilization All glassware include pipettes, petri-dishes, flask, measuring cylinders and test tubes, distilled water, media, sample bottles and slides were sterilized at 1210C for 15 minutes at 15psi. Viable Count of Bacteria Nine (9) mm of the diluent (distilled water) was dispensed into test tubes and 1ml of the zobo sample to be analyzed were taken with the aid of 5ml sterile syringe and transferred to the first set of test tubes (dilution 10-1 ). The 10-2 dilution was prepared by transferring 1ml of dilution 10-1 into test tubes containing 9ml diluent. Similarly, 1ml of dilution 10-2 was dispensed into 9ml diluent to obtain dilution 10-3 . The same procedure was used to make serial dilution of 10-4 to 10- 6 . The pour plate method was used in determining the viable count of the various samples of zobo drink. Bacteria One millimeter (1ml) of the various dilutions were placed or transferred in the center of sterile Petri dishes using 5ml sterile syringe. Molten cool agar i.e. (Nutrient agar for bacteria count) 20ml was poured into the Petri dishes containing the inoculums (samples) and mixed well. After the solidification of the agar, the plates were inverted and incubated at 37ᴏ c for 24-48 hours. Different bacterial colonies present on the plates were counted and multiplied by the reciprocal of the appropriate dilution factor to obtain the viable count. Isolation of Bacteria and Maintenance of Pure Culture The pour plate method was used in isolating pure culture. Colonies that appeared on agar plate (Nutrient agar plates) were sub-cultured aseptically by streaking into newly prepared agar plates using sterile wire loop. The streaked plates for bacteria were incubated for 24hrs at 370C. Pure colonies of bacteria were inoculated into slants and incubated for 24hrs at 370C and kept in a refrigerator to keep the culture viable for further use. Identification of Bacteria Zobo Drink Samples Cultured isolates of bacteria cells were characterized and identified by using biochemical tests, as well as observing the characteristic colony morphology of bacteria colonies. Gram staining was also carried out on bacteria isolates to ascertain their response to gram staining. Morphological Characteristics The morphological characteristics of isolated bacteria colonies on growth media were observed after growth. Few of the morphological characteristics observed were those relating to bacteria colony shape, color, texture, elevation and margin. GRAM STAINING The Gram staining reaction was used to help identify isolates in culture (Gram positive or Gram- negative organisms). Using a sterile wire loop, a pure culture from a discrete colony on the culture plate were emulsified in a drop of normal saline on grease free clean microscope slides. PROCEDURE 1. The dried smear was fixed. 2. The fixed smear was covered with crystal violet stain for 60 seconds. 3. The stain was rapidly washed off with clean water. 4. The water was tipped off, and the smear covered with Lugol’s iodine for 60 seconds. 5. The iodine was washed off with clean water. 6. The smear was decolorized rapidly for few seconds using acetone and washed immediately with clean water. 7. The smear was covered with safranine for 60 seconds. 8. The stain was washed off with clean water 9. The back of the slide was washed clean and placed in a draining rack for the smear to air dry. 10. The smear was then examined microscopically with oil immersion objective to report the bacteria and cells. BIOCHEMICAL TEST Some biochemical tests used to identity bacteria isolates include; Catalase test, Coagulase test, Indole test, Oxidase test, Citrate utilization test, Methyl red test, Voges proskauer test and Sugar fermentation tests. These tests create a platform from which isolated bacteria colonies were identified. CATALASE TEST PRINCIPLE OF CATALASE TEST The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. The culture should not be more than 24 hours old. Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated as an end product of aerobic carbohydrate metabolism. PURPOSE OR USES OF CATALASE TEST The morphologically similar Enterococcus or Streptococcus (catalase negative) and Staphylococcus (catalase positive) can be differentiated using the catalase test. Also valuable in differentiating aerobic and obligate anaerobic bacteria. Semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis. It is used to differentiate aerotolerant strains of Clostridium, which are catalase negative, from Bacillus species, which are positive. Catalase test can be used as an aid to the identification of Enterobacteriaceae. PROCEDURE OF CATALASE TEST Tube Method 1. I Poured 1-2 ml of hydrogen peroxide solution into a test tube. 2. Using a sterile wooden stick or a glass rod, several colonies of the 18 to 24 hours test organism were taken and immersed in the hydrogen peroxide solution. 3. The result was Observed for immediate bubbling. Slide Method 1. a loop or sterile wooden stick was used to transfer a small amount of colony growth in the surface of a clean, dry glass slide. 2. a drop of 3% H O was placed in the glass slide. 3. The result was Observed for the evolution of oxygen bubbles. Result Interpretation of Catalase Test and Examples Positive: Copious bubbles produced, active bubbling Examples: Staphylococci, Micrococci, Listeria, Corynebacterium diphtheriae, Burkholderia cepacia, Nocardia, the family Enterobacteriaceae (Citrobacter, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella, Serratia), Pseudomonas, Mycobacterium tuberculosis, Aspergillus, Cryptococcus, and Rhodococcus equi. Negative: No or very few bubbles produced. Examples: Streptococcus and Enterococcus spp PRECAUTIONS OF CATALASE TEST The test organisms should not be taken from blood agar culture. Red Blood cells contain catalase and their presence will give a false positive test. Culture should be 18 to 24 hours old. Hydrogen peroxide must be fresh as it is very unstable. Iron wire loop should not be used. Some bacteria produce a peroxidase that catalyzes a breakdown of hydrogen peroxide causing the reaction to be weakly positive; (a few bubbles elaborated slowly). This should not be confused with a truly positive reaction. Do not add organism to reagent, particularly if iron-containing inoculating loops are used. Iron containing loops will cause false positive test results if exposed to hydrogen peroxide. COAGULASE TEST PRINCIPLE OF THE TEST S. aureus produces two types of coagulases: bound and free. Slide coagulase test is done to detect bound coagulase, whereas tube coagulase test is done to detect free coagulase. Both tests utilize rabbit plasma treated with anticoagulant to interrupt the normal clotting mechanism. Bound Coagulase It is also known as clumping factor. It is attached to bacterial cell wall and reacts directly with fibrinogen. This is shown by formation of visible mass. it doesn’t require coagulase reacting factor (CRF). Free Coagulase It is an extracellular enzyme (released from the cell). It converts fibrinogen to fibrin n by activity of coagulase reacting factor (CRF) in plasma. This is detected by appearance of fibrin clot in the tube coagulase test. It is usually recommended to do tube coagulase test on all ‘slide-coagulase-negative’ staphylococci. Procedure SLIDE TEST 1. Two separate drops of saline were placed on a slide. 2. Using a sterile inoculating loop, one or two colonies of organism were emulsified in one drop to make thick suspension of bacteria. 3. Then I added a loopful of plasma to both the suspension and saline drop and mix gently. 4. The result was observed for immediate coarse clumping of the mixture within 10-15 seconds. TUBE TEST 1. I diluted the plasma 1:10 with saline. 2. I took 2 test tubes and add 0.5 ml of diluted plasma to each. 3. I Inoculated a tube with bacterial colonies to make a cloudy suspension. Alternatively, add about 5 drops of thick 18-24 hours broth cultures. 4. Then I Incubated both tubes at 35 degrees Celsius for 1 to 4 hours in water bath. 5. Afterward, both tubes were examined for presence or absence of clots. RESULTS AND INTERPRETATION Slide Coagulase Test: The formation of clumps within 10-15 seconds is positive test result. Saline and plasma mixture should show no clumping. Tube Coagulase Test: A positive coagulase test is represented by any degree of clotting, from a loose clot suspended in plasma to a solid clot. If negative, the plasma remains a liquid. INDOLE TEST Indole Test In this test, 10 g of peptone and 8 g of sodium chloride were dissolved in 1 litter of distilled water. It was then heated and dispensed to the test tubes and sterilized. The isolates were inoculated into the test tubes and incubated at 37 oC for 2-5 days. About 2-3 drops of Kovac’s reagent was then added into the test tubes. The appearance of a red layer or red ring on the culture indicates a positive result. OXIDASE TEST The oxidase test detects the presence of a cytochrome oxidase system that will catalyse the transport of electrons between electron donors in the bacteria and a redox dye- tetramethyl-p- phenylene-diamine. The dye is reduced to deep purple color. This test is used to assist in the identification of Pseudomonas, Neisseria, Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella and Pasteurella, all of which produce the t h id View All Topics enzyme cytochrome oxidase. A number of reagents can be used for this test. Kovacs Oxidase Reagent: 1% tetra-methyl-p-phenylenediamine dihydrochloride, in water Gordon and McLeod’s Reagent: 1% dimethyl-p-phenylenediamine dihydrochloride, in water Gaby and Hadley (indophenol oxidase) Reagent: 1% α-naphthol in 95% ethanol 1% p-aminodimethylaniline HCL PRINCIPLE OF OXIDASE TEST Cytochrome containing organisms produce an intracellular oxidase enzyme. This oxidase enzyme catalyzes the oxidation of cytochrome c. Organisms which contain cytochrome c as part of their respiratory chain are oxidase-positive and turn the reagent blue/purple. Organisms lacking cytochrome c as part of their respiratory chain do not oxidize the reagent, leaving it colorless within the limits of the test, and are oxidase-negative. Oxidase positive bacteria possess cytochrome oxidase or indophenol oxidase (an iron containing haemoprotein). Both of these catalyse the transport of electrons from donor compounds (NADH) to electron acceptors (usually oxygen). The test reagent, N, N, N’, N’-tetramethyl-p-phenylenediamine dihydrochloride acts as an artificial electron acceptor for the enzyme oxidase. The oxidised reagent forms the colored compound indophenol blue. The cytochrome system is usually only present in aerobic organisms which are capable of utilising oxygen as the final hydrogen receptor. The end product of this metabolism is either water or hydrogen peroxide (broken down by catalase). PROCEDURE 1.1 g of oxidase reagent (tetra-methyl-p-phenylene diamine dehydrochloride) was dissolved in 10 ml of distilled water, which was added into filter paper in a petri dish. The isolated test culture was then smeared unto the filter paper using a glass rod. A positive result shows the development of dark purple color within 10 seconds. SUGAR FERMENTATION TEST PRINCIPLE OF THE TEST Different bacteria have different abilities to ferment specific carbohydrates – some can ferment one type of carbohydrate while others can’t ferment the same sugar. While fermenting carbohydrates, organic acids are produced as end products. These Table of Contents Objectives of Carbohydrate Fermentation Test Principle of Carbohydrate Fermentation Test Requirement for Carbohydrate Fermentation Test a. Culture Media b. Carbohydrates c. Reagents d. Equipment e. Test Organisms (Sample Bacteria) Procedure of Carbohydrate Fermentation Test Result Interpretation of Carbohydrate Fermentation Test Fermentation Pattern of Some Common Bacteria Precaution produced acids will decrease the pH of the medium. This decrease in pH causes the pH indicator present in the medium to change color. (The color change depends on the type of pH indicator used in the test.) If the bacteria release gases during carbohydrate fermentation, they will get trapped as an air bubble in the inverted Durham tube/fermentation tube submerged inside the fermentation broth. This color change indicates the occurrence of fermentation and the formation of an air bubble in Durham’s tube indicates the release of gas and hence, confirms that the test bacteria were able to use the specific carbohydrate used in the test medium. REQUIREMENTS FOR THE TEST a. Culture Media Carbohydrate Broth can be used for performing this test. The composition of the medium can be modified according to the test organism and its source or components available in the lab. In this article, we will be using a composition based on “Carbohydrate Consumption Broth Base” by HiMedia (M124) with a certain change in pH indicator and will add specific carbohydrates. Composition of Carbohydrate Broth Base per 990 Ml Proteose Peptone- 10.00 grams HM Peptone B (Beef extract)- 1.00 grams Sodium Chloride- 5.00 grams Phenol Red- 0.018 grams (Alternatively, Bromocresol purple 0.100 grams can be used) Preparation of Carbohydrate Broth I Measured the appropriate amount of media components and dissolve them in 990 mL of distilled water. I Added 10 grams of specific carbohydrates for the test and dissolve by shaking. Heat to boiling if needed for the complete dissolution of components. I Dispensed about 5 to 7 mL (or the volume required to submerge Durham’s tube) of the broth to test tubes. I Submerged a Durham tube in each tube completely and make sure there is no air bubble inside the submerged Durham tube. I Loosely put on the cap or cotton plug and autoclave the tubes for 15 minutes at 121 C and 15 lbs. pressure. b. Carbohydrates Glucose, lactose, sucrose, maltose, mannitol, galactose, starch, rhamnose, aesculin, salicin, adonitol, dulcitol, sorbitol, cellobiose, xylose, mannose, trehalose, inositol, raffinose, melibiose, and cellulose are carbohydrates commonly used as substrate in the carbohydrate fermentation test. pH indicators Equipment Test tubes, weighing balance, inoculating loop, Durham tubes, Bunsen burner, autoclave and incubators, PPE and other general laboratory equipment. Test organisms TEST PROCEDURE This test was carried out using a media called triple sugar iron agar, containing dextrose (simple sugar), lactose and sucrose. The medium was prepared according to the manufacturer's directive which is to dissolve 65gm into 100ml of distilled water, after which 10ml of the prepared medium was added into test tubes, 3 drops of phenol red was added. Durham's tubes were inserted in an inverted position (for detection of gas production) making sure it touches the medium. The tubes were plugged with non-absorbent cotton-wool and sealed with aluminum foil before being sterilized with autoclave at 105°C for 15 minutes at 15Ib pressure. After sterilization, each tube were inoculated with specific colony and incubated for 24 hours at 37°C and unincubated tubes serve as control. Acid production was indicated by a change in colour from orange to yellow colour, indicating that acid has been produced and gas production was indicated by the presence of air spaces between the Durham tubes and the medium used presence of air bubbles also indicated presence of gas interring that gas has been produced. If no color change or air bubble is seen, re- incubate the tubes for the next 24 hours and observe for color change and air bubbles. Some may need incubation of 5 days or more. RESULTS AND INTERPRETATION OF SUGAR FERMENTATION TEST Positive fermentation is denoted by the color change of the media from reddish-orange to yellow. Negative fermentation is denoted by no color change of the medium (remains reddish-orange). (If another pH indicator is used, read the color change accordingly.) Gas production is indicated by the formation of an air bubble in Durham’s tube. PRECAUTION Be sure there is no air bubble in the inverted Durham tube. While inoculation, avoid touching (inoculating over) the Durham tube. Swirl the media to ensure proper distribution of the sample in the broth. CITRATE UTILIZATION TEST This test is used to determine the ability of the bacteria to use sodium citrate as the only source of carbon and inorganic ammonium hydrogen phosphate as a source of nitrogen. The citrate tests are only possible if the organisms are capable of fermenting the citrate. The process takes place via the enzyme known as citrase. The test is conducted in the medium of simmon citrate agar. PROCEDURE ➢ Consider a slant, Simmons citrate agar was injected gently on the slant by slightly touching the tip of the needle to the colony, which is 18-24 hours old. ➢ In the citrate medium, organisms require more time to grow. Therefore, the solution was incubated at 35˚C to 37˚C for 18-24 hours. Some organisms may also seem to require seven days of incubation. ➢ Observe the color change in the solution, the development of blue color indicates the alkylation process. INTERPRETATION The reagent used – bromothymol blue Positive result: growth on the medium indicates the change in color of the reagent from green to blue. Negative results: very less or no growth at all in the medium and color of the solution remains the same. List of some bacteria that gives a positive result for the citrate test ➢ Providencia ➢ Typhi and Para typhi A, salmonella ➢ Serratia marcescens ➢ Proteus mirabilis ➢ Enterobacter species List of some bacteria that gives negative result ➢ E. coli ➢ Salmonella typhi ➢ Yersinia enterocolitica MRVP TESTS The MRVP tests is actually two separate tests that uses one broth medium containing glucose to determine the possible types of glucose fermentation being conducted by a bacterial species. Some bacterial species may ferment glucose using mixed acid fermentation pathway (detected in the MR test or methyl red tests) while some other bacterial species may ferment glucose via the butanediol fermentation pathway ( detected in the VP test ). Since the type of fermentation conducted differs by bacterial species, testing the type of fermentation pathway is useful for characterizing bacteria and identifying them. LABORATORY INSTRUCTIONS ➢ I Obtained a MR-VP broth tube. ➢ I Used tape to label the tube with your name and the test media as well as the assigned organisms. MR-VP broth. ➢ I Incubated the inoculated tube in a test tube rack. ➢ After incubation, using a transfer pipette, ➢ I transferred half the inoculated MR-VP broth to a new and clean test tube. ➢ I Labelled this tests tube VP. ➢ The other half was left in the original MR-VP test tube. ➢ I Labelled this test tube MR METHYL RED TEST ➢ I Added 10 drops of methyl red reagent to MR tube. ➢ The color of the medium was examined. ➢ I Recorded the results. VOGES-PROSKAEUR TEST ➢ 15 drops of Barritts A reagent (alpha -naphthol) was added to the “VP” tube. ➢ 5 drops of Barritts B reagent (40%KOH) was added TO THE “VP” tube. ➢ I Held the test tube from the glass and mix the tube well by flicking it with your fingers. ➢ Let the test tube sit undisturbed in attest tube rack for 20 minutes. ➢ The color of the top layer of liquid was examined and the results recorded. MAINTENANCE OF THE STRAINS Distinct colonies from each of the samples were stored in nutrient agar at 4˚C and cryopreserved in glycerol nutrient broth in a deep freezer. CHAPTER FOUR 4.0 RESULTS The biochemical characteristics of the bacterial isolates are shown in Tables below, the isolates are Staphylococcus epidermidis, Corynebacterium spp, Klebsiella spp, Pseudomonas spp, Escherichia coli, and Proteus spp etc. Figure 1 shows the total count of bacterial Isolates from various samples. The morphological characteristics of bacterial isolates from the zobo samples are shown in Table 8. These morphological characteristics can be known by looking out for the form, elevation, texture, margin and pigmentation of the colonies seen on the plate. This aids in the identification and characterization of the colonies after which gram stain and biochemical tests are carried out to further identify the organisms. TABLE 1: The Total Bacterial Count of Zobo Drink Sample Zobo Drink Samples Dilutions Colonies On Plates Bacterial Count (Cfu/ml) A 10-6 86 8.6 X 105 B 10-6 69 6.9 X 105 C 10-6 63 6.3 X 105 D 10-6 88 8.8 X 105 E 10-6 54 5.4 X 105 CONTROL 10-6 41 4.1 X 105 Table 2: Characteristics of Bacteria Isolated from Zobo drink Sample A Iso: Isolate, G.S: Gram Stain, Cat: Catalase, Coa: Coagulase, Ind: Indole, M.R: Methyl red, V.P: Voges Proskauer, Mot: Motility, Cit: Citrate, Oxi: Oxidase, G: Glucose, S: Sucrose, L: Lactose, M: Maltose, Probable Org.: Probable organism, ND: Not determined, +: Positive, -: Negative, A: Acid production, S. epidermidis: Staphylococcus epidermidis, Cory. Spp: Corynebacterium spp, Pseudo spp.: Pseudomonas spp, E. coli: Escherichia coli. Table 3: Characteristics of Bacteria Isolated from Zobo drink Sample B Table 4: Characteristics of Bacteria Isolated From Zobo Drink Sample C Table 5: Characteristics of Bacteria Isolated From Zobo Drink Sample D Table 6: Characteristics of Bacteria Isolated from Zobo drink Sample E Table 7: Characteristics of Bacteria Isolated From Zobo Drink Control Sample Table 8: Morphological characteristics of the bacterial isolates ORGANISMS MORPHOLOGICAL CHARACTERISTICS Staphylococcus epidermidis Round, raised, smooth, shiny, with gray-white colour Corynebacterium spp. Round, convex, dull, with grayish colour Klebsiella spp. Round, raised, shiny, with pink colour Escherichia coli Very large, flat and milky colony, with a smooth edge Proteus spp. Large colorless colony with a swarming motion on the agar Surface Table 9: Percentage of occurrence of the bacteria isolated from zobo drink samples 4.1 DISCUSSIONS The result obtained from the analysis show that bacteria isolated from the Zobo sample include Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Escherichia coli, Bacillus subtillis, Enterobacter aerogenes, Enterobacter cloacae and Proteus vulgaris. This is in agreement with the findings of some earlier works carried out by F.A Akinyosoye, B.J. Akinyele, 2000 and W.Braide, I.J Odiong and S. Oranusi , 2012 where they identified microorganisms associated with the plant. Most strains of these isolates are toxic and their detection in zobo drink poses a serious health hazard to the consumers. These microorganisms have been implicated in outbreak of dysentery, diarrhoea and other gastrointestinal diseases. However, among the six samples analyzed, samples D, A and B has the highest bacterial load which might be as a result of inadequate ascetic condition during their preparation and processing. Escherichia coli that was isolated from the zobo drink is an indication of a feacally contaminated drink that must have been from the water (feacally contaminated) during the processing of the zobo drink. This is because most vendors are admitted to using water to dilute zobo syrup after boiling and this is a possible source of bacterial contamination to the already boiled zobo. Staphylococcus specie in zobo drink could possibly be through the processing methods which usually involve the use of hands since the organism is a common flora of the skin. Surprisingly, the control sample prepared under hygienic conditions was also contaminated. Presumably, the source of contamination of the control sample may have come from the raw materials (dried calyces and additives) which were purchased from the open market. One reason for this is the fact that Hibiscus sabdariffa calyces are displayed in large bowls and polyethylene bags for prospective consumers and in the process exposed to microbial contamination. Also, the calyces are usually boiled or soaked in hot water to extract the red pigments and raw spices (grounded) added to the drink after boiling might have served as sources of contamination. Besides, other sources of contamination might be the packaging materials or containers which are not properly washed and sterilized. This work corresponds to the work of previous author who reported that hawked zobo drink harbored bacteria (E. coli). suggested that the presence of microorganisms in zobo drinks produced with a boiling method is indicative of post-production contamination resulting from the addition of water, sugars, preservatives and other additives like pineapple flavoring. This work also corresponds to the work of previous authors, who reported that zobo drink harbored Proteus spp and Escherichia coli, which indicates the presence of contaminants from water and humans. Local nutritional drinks are consumed by a lot of people probably due to their medicinal and nutritional properties. Zobo is produced from the calyces of H. sabdariffa and is one of the local drinks consumed in Nigeria irrespective of the socio-economic status. Different products have been severally produced from H. sabdariffa. The zobo drink are sold in several public places in Nigeria including market, motor parks, streets, outside schools, hospitals and highway way due to their convenience and low cost. This paper reviews the quality of Zobo drink sold in Nigeria. The zobo has several health benefits due the presence of proximate, phytochemical, nutrients, minerals and vitamins found in the plant. However, there are challenged by low shelf life which causes deterioration in quality due to the presence of spoilage microorganisms. Despite the fact that most of the microorganisms found in Zobo drink are spoilage microbes, some are mainly contaminants from the environment including soil and water, processing utensils including spices and preservatives and hygienic conditions of both processors and marketers. Some of the microbes are of public health importance. The study also found that microbial density and diversity seldom exceed the acceptable and tolerable limit for aerobic viable microbes found in ready-to-eat food. The occurrence of coliform in Zobo drinks portent a potential risk to consumers. The risk of food borne diseases due to the presence of microbes found in the zobo drink could be reduced through the use of sterile materials for packaging, brief pasteurization of the product after packaging at a temperature ≤80 - ≥100°C, improving the hygienic conditions of zobo drink handlers since some microbes such as S. aureus in the product are attributed to handling processes (normal flora in skin and nasal cavity of humans), avoiding excessive talking during processing especially during packaging to avoid introduction of some species of Streptococcus (which are found in the buccal cavity of humans). CHAPTER FIVE 5.0 CONCLUSION The various bacteria genera isolated and identified from the samples includes; Staphylococcus, Corynebacterium, Klebsiella, Pseudomonas, Escherichia and Proteus. 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Microbiology, 2000, Book Abstract. p23. Braide .W., I.J Odiong and S. Oranusi Phytochemical and antibacterial properties of the seed of water melon (Citrullus lanatus). Prime Journal of microbiology Research (PJMR), 2012: 2(3): 99 – 104. APPENDIX Figure 1.0 Var altissima wester and Var sabdariffa – red variety (source Onyeke, 2013) Figure 2.0 Var sabdarifaa – yellow variety (source Onyeke, 2013) FIG 3.0 RSC – Red sorrel calyces (Source: Photo by the researcher at the Dept. of Home Science, Nutrition and Dietetics, University of Nigeria, Nsukka) FIG 4.0 RSS – Red sorrel seeds (Source: Photo by the researcher at the Dept. of Home Science, Nutrition and Dietetics, University of Nigeria, Nsukka) ~ 1 ~