EXTRACTION OF OIL FROM MANGO SEED Nwaokobia Kingsley Graduate from the Department of Agricultural and Bio-Environmental Engineering Technology.

ABSTRACT The extraction of oil from mango seeds was investigated. Mango seeds were collected, dried and ground into powder. The mango oil was extracted using a Soxhlet apparatus. Hexane and Ethanol were used to extract the oil at varying time of extraction (4, 5, 6, 7 and 8 hours). The yield of oil was determined for each experiment. The effects of time and extraction solvent on the yield of oil was investigated by using a 22 factorial design. The effects of the extraction solvent on the physicochemical properties was also investigated. Oil extracted with hexane had better overall quality than the Ethanol extracts. Its acid value, saponification value, ester value, refractive index and specific gravity were 5.61 mgKOH/gOil, 207 mgKOH/gOil, 201.39 mgKOH/gOil, 1.443 and 0.909 respectively. Hexane always gave higher yield of oil than Ethanol. The results suggest that Hexane is a much better solvent for the extraction of mango oil.

i CHAPTER ONE

INTRODUCTION Background of study The mango fruit is a drupe or stone fruit which embeds a shell within its hardened endocarp (the inside layer of a fruit). Mango fruits are produced by mango trees (Mangifera Indica). This tree belongs to the Anarcardiaceae family, a family of fruit bearing trees, and is a distant relative of the Pistachio and Cashew trees. Mangoes are one of the most important fruits worldwide and they are grown in tropical and sub- tropical regions especially Asia (Fahimdanesh & Bahrami, 2013). They are consumed worldwide and are relished because of their sweet tasty flesh. The mango is aboriginal to the Indian subcontinent and Southeast Asia and it is cultivated in many tropical regions and distributed widely in the world. It is one of the most extensively exploited fruits for food, juice, flavor, fragrance and colour and is a common ingredient in new functional foods. Chutneys, jams, sauce, nectar, pickles and are also made from mangoes. Various parts of the tree are used in medicines and its leaves are ritually used as floral decorations at weddings and religious ceremonies (Kittiphoom, 2012).

Mango trees (Mangifera Indica) reach 35 - 40 m in height, with a crown radius of 10 m. The leaves are evergreen, alternate, simple, 15 - 35 cm long and 6 - 16 cm broad; when the leaves are young they are orange-pink, rapidly changing to a dark glossy red, and then dark green as they mature. The fruit takes from 3 - 6 months to ripen. The ripe fruit is variable in size and color, and may be yellow, orange, red or green when ripe, depending on the cultivar (Fahimdanesh & Bahrami, 2013). Nearly half of the world’s mangoes are produced in India, but the country accounts for a small percentage of the international trade because India consumes most of its own production (Sondhi, 2011). India is the largest producer of Mangoes in the world while China and Thailand are the second and third largest producers respectively. The mango is the national fruit of India, Pakistan and the Philippines and the tree is also the national tree of Bangladesh (Kittiphoom, 2012). Mango is rich in a variety of photochemical and nutrients. The fruit pulp is high in dietary fiber and in vitamin C and vitamin A. it contains essential vitamins and dietary minerals at good levels. The mango peel also contains essential nutrients. According to mango varieties, the seed (kernel and endocarp) represents from 10% to 25 % of the whole fruit weight. The kernel inside the seed represents from 45% to75% of the seed and about 20% of the whole fruit (Fahimdanesh & Bahrami, 2013). The kernel can be ground into kernel powder for oil extraction. The oil is semi-solid at room temperatures, but melts on contact with skin, making it appealing for baby creams, heat-care balms, hair products and other moisturizing products (Sivkishen, 2014). Also, the confectionary industry has developed an interest in the possibility of using mango kernel oil as a substitute for Cocoa (natural of the Cocoa bean used in production) which is very expensive.

Problem Statement The fruit of the Mango is never totally consumed or processed because the seed is protected by a hardened shell (endocarp). This seed is usually disposed of after consumption or industrial processing. Due to the large utilization of mango fruits, more than one million tons of mango seeds are being produced as waste annually (Fahimdanesh & Bahrami, 2013). If these seeds could be utilized one way or another, it would eliminate wastage and also birth the production of new products. The usefulness of the whole Mango kernel, especially the oil, in comparison with mango juice, is yet to gain rural and industrial attraction in African countries including Nigeria. This underutilization could be partly due to the limited knowledge of 1 the composition of the kernel oil and its toxicology status. Hence, this research work is necessary so as to provide more knowledge on the required experimental conditions for optimum production of mango kernel oil and on the properties of the oil. Aims and Objective of Study The aim of this work is to extract oil from mango seeds while varying experimental conditions and characterize the oil. The objective of this work includes: 1. An estimation of oil yield from mango seeds 2. The determination of the effect of the following on the yield a) Extraction solvent b) Time 3. The measurement of physiochemical properties of the extracted oil.

Method and Scope The method that will be employed in this work is the use of a Soxhlet Apparatus to extract mango oil from the seed kernel. This method involves drying the seed, separating the kernel from the endocarp, crushing the kernel and extracting the oil using a Soxhlet apparatus. The oil is then weighed and analyzed. The procedure is repeated at different times and extraction solvents. The extraction happens because the ground mango kernel releases oil into the solvent upon contact with the hot solvent. Hexane and ethanol will be used as extraction solvents. This work is limited to the extraction of mango oil from mango species found in the south west of Nigeria. Also, other methods of extraction will not be considered due to the time and economic constraints of this research.

Relevance of Research to the Society The following will be the relevance of this research to the Nigerian society:  To serve as a reference material for other studies and advancements in the area of mango oil extraction in Nigeria.

CHAPTER TWO LITERATURE REVIEW The Mango The mango (Mangifera Indica) is a luscious tropical fruit with a smooth green skin that develops patches of red, yellow, and/or gold as it ripens and orange-yellow flesh that is exceptionally juicy when ripe. The seed of the mango is larger than any other in the fruit kingdom – practically as long and wide as the entire length and width of the fruit. Almost flat, the seed is surrounded first by some fibrous matter, then by plump, fleshy fruit, whose taste is a spicy-sweet mélange of peach and pineapple. A member of the sumac family (Anarcardiaceae), the mango is cousin to pistachios and cashew nuts (Micheal, 2005).

Mangoes are one of the leading fruit crops of the world, ranking seventh among the top twenty fruits. In fact, more mangoes are consumed by more people in the world on a regular basis than apples. Mangoes come in more than a thousand varieties and vary in form from round to kidney-shaped, ranging from diminutive plum- sized to melon-sized fruits weighing up to 4 pounds. Most commercially grown varieties are about the size and 2 shape of a large avocado: 4 to 5 inches in length and about 8 ounces in weight. One exception is the manila mango, a pear-sized, golden yellow variety now beginning to appear in U.S. markets (Micheal, 2005).

The mango tree is a magnificent evergreen that grows up to 100 feet tall and produces beautiful, shiny, thick, pointed leaves 8 to 14 inches long. Symmetrical in shape, the mango tree is a beautiful ornamental value for its cooling shade as well as its fruit. Mango trees begin to produce fruit four to six years after planting and continue bearing fruit for about forty years. Tiny, delicate pinkish white flowers precede the fruit, which grows in clusters from long stems attached to the main branches. Each mango tree bears an average of 100 mangoes each year (Micheal, 2005).

The wild mango originated in the foothills of the Himalayas in India and Burma, and wild mango trees still grow in India and Southeast Asia today. However, the wild mango with its tiny size, fibrous texture, and unpleasant turpentine taste, bears little resemblance to the luscious cultivated mangoes we now enjoy. The cultivation of the mango began in Moghul, India, where the fruit is still considered sacred and is thought to have aphrodisiac effects. In the sixteenth century, a special technique employing grafting was developed for propagating the mango. This techniques and variations upon it are still used today because even cultivated mangoes will not develop true from seed but will revert back to their wild ancestry, producing a highly fibrous fruit that tastes like turpentine. The earliest mention of the mango tree, Mangifera Indica, which means “the great fruit bearer,” is found in Hindu scriptures dating back to 4000 BC. In Tamil, the language of south eastern India, the mango was given its original name, mancay or mangay, which the Portuguese later changed to manga. Legend relates that the Buddha, delighting in the mango, was given a whole grove of mango trees where he could rest whenever he wished. The mango’s association with the Buddha caused the mango tree to be held in awe as capable of granting wishes, and also to be revered in India as a symbol of love. In fact, in the oldest Sanskrit writings, the mango tree is central to a legend of undying love (Micheal, 2005).

Reverence for the mango was extended beyond India when explorers carried the fruit to other tropical countries, including Thailand, Malaysia, Indonesia, and the Philippines, where it was cultivated successfully. As the mango adapted to these new locales, new varieties evolved and were praised with nicknames, such as the “apple of the tropics,” the “king of fruits,” and the “fruit of the gods.” Chinese travelers brought the mango home to China and the mango soon became a garden favorite. By the end of the seventh century BC, the mango had journeyed west to Baghdad, Persia and then North Africa. Mangoes were first recorded in Europe by Friar Jordanus in 1328, but Europeans did not delight in them as had the inhabitants of countries with tropical climates – a reaction that continues to this day, as the mango remains an infrequently eaten fruit in Europe. However, by the sixteenth century, Portuguese explorers carried the mango to East and West Africa and Brazil, and by the eighteenth century, the fruit had sailed to the West Indies (Micheal, 2005).

In the nineteenth century, the mango continued its travels west, arriving in Florida, Mexico and Hawaii. Mangoes were grown on the east coast of Florida by 1825. Today, India remains the world’s largest producer of mangoes, and the mango is India’s main fruit crop, far outnumbering all others. Mangoes are an excellent source of carotenes, vitamin C and copper, providing 184 percent of the daily value of vitamin A, 1% of the daily value of vitamin C, and 20% of the daily value of copper in one cup (165grams) of sliced fruit. They are a very good source of B vitamins, with one cup of sliced mango providing 17% of the daily value of vitamin B6, 9% of the daily value of folic acid. Mangoes are also a good source of vitamin E. they also contain a number of enzymes, including one similar to the papain in papayas that improve digestion (Micheal, 2005). 3 Mango tree plantation Ripe Mango fruits Longitudinal section of a Mango fruit

Overview of Mango Seed Kernel and Oil Mango Seed Kernel Mango seed is a single flat oblong seed that can be fibrous or hairy on the surface, depending on the cultivar. Inside the seed coat 1 - 2 mm thick is a thin lining covering a single embryo, 4 - 7 cm long, 3 - 4 cm wide, and 1 cm thick. Mango seed consists of a tenacious coat enclosing the kernel. The seed content of different varieties of mangoes and their ranges are shown the Table 2.1.

Table 2.1 Mango Seed Kernel composition ranges

Kernel Composition Values in percentage Seed in fruit 3 – 25 Kernel in seed 54 – 85 Moisture 33 – 86 Protein 4.0 – 8.1 Crude Fiber 1.7 – 7.6 Ash 1.0 – 3.7 Total carbohydrates 70 – 76 Phenolic compounds 0.1 – 6.4 Crude fat 3.7 – 12.6

Although mango seed kernels have a low content of protein, the quality of protein is good. Mango seed kernel is high in potassium, magnesium, phosphorus, calcium and sodium. Potassium is an essential nutrient and has an important role is the synthesis of amino acids and Proteins. Calcium and magnesium plays a significant role in photosynthesis, carbohydrate metabolism, nucleic acids and binding agents of cell walls. Calcium assists in teeth development. Magnesium is essential mineral for enzyme activity, like calcium and chloride; magnesium also plays a role in regulating the acid-alkaline balance in the body. Phosphorus is needed for bone growth, kidney function and cell growth. It also plays a role in maintaining the body’s acid-alkaline balance. The mango seed kernel is a nutritional promising seed. It also contains antioxidant vitamins such as C, E and A. Antioxidant vitamins have been reported to reduce oxidative processes which are known to be vital in the initiation of arthrosclerosis (Kittiphoom et al 2012). Although phenolic compounds act as anti-nutritive factors, they have recently become the subject of intense research because of their high antioxidant activity. Tannins, gallic acid, coumarins, caffeic acid, vanillin, mangiferin, ferulic acid and cinnamic acid have been identified in the MSK and analyzed for their antioxidant activity. (Preedy, 2011)

4 Mango Oil Mango oil is an oil that is obtained from the seed by extraction. The oil is semi-solid at room temperatures, but melts on contact with skin, making it appealing for baby creams, heat-care balms, hair products and other moisturizing products (Sivkishen, 2014). Also, the confectionary industry has developed an interest in the possibility of using mango kernel oil as a substitute for (natural fat of the Cocoa bean used in chocolate production) which is very expensive. The mango oil releases salicylic acid, a pain reliever used to make aspirin (Bird, 2009). Mango kernel oil has been used in the cosmetics industry as an ingredient in soaps, shampoos and lotions because it is a good source of phenolic compounds including microelements like selenium, copper and zinc .In addition, the extract of mango seed kernel exhibited the highest degree of free-radical scavenging and tyrosinase-inhibition activities compared with methyl gallate and phenolic compounds from the mango seed kernel and methyl gallate in emulsion affected the stability of the cosmetic emulsion systems. (Kittiphoom, 2013)

Composition of Mango Oil The major saturated fatty acids in mango seed kernels oil were stearic and palmitic acids and the main unsaturated fatty acids are oleic, linolenic and linoleic acids (Table 2.2). The comparison of the composition in fatty acids of mango seed kernel oil with that of vegetable oils indicates that this plant is rich in acids stearic and oleic. The composition of mango kernel fat shows a wide variation which may be attributed to differences in the variety and location. (Kittiphoom, 2012)

Table 2.2 Composition of Mango oil

Fatty acid Composition 3 – 18 24 – 57 Oleic acid 34 – 56 Linoleic acid 0 – 13 Linolenic acid 0.2 – 5.3 Saturated 27 – 75 Unsaturated 34.2 – 74.3 Physical and Chemical Properties of Mango Oil The physical properties of mango oil include specific gravity, total oil yield, refractive index, melting point and colour of mango seed kernel oil. The values of these properties are shown below.

Table 2.3 Physical properties of mango oil

Physical Properties Values Amount of oil (%) 12.5 0.2 Moisture of kernel content (%) 8.5 0.1 ± Specific gravity at 40oC 0.900 0.03 ± Refractive index at 40oC 1.443 0.01 ± Melting point (oC) 30.0 1.20 ± Colour Pale Yellow to Ivory cream ± 5 The chemical properties of mango oil include the free fatty acid composition, peroxide value, iodine number, saponification number and unsaponifiable matter. The chemical properties of oil are amongst the most important properties that determines the present condition of the oil. Free fatty acid and peroxide values are valuable measures of oil quality. The (IV) is the amount of iodine (in grams) necessary to saturate 100g of oil sample and is a measure of the amount of unsaturation in and oils. The saponification value is the milligrams of KOH necessary to saponify 1g of oil sample and shows the capacity of forming soaps of oil. The peroxide value (PV) is a measure of the extent of oxidation of a fat or oil. Unsaponifiable matter is the component of an oily mixture which fails to form soap when blended with NaOH. The unsaponifiable matter in vegetable oils is of great importance for oil characteristics and stability.

Table 2.4 Chemical properties of Mango oil

Chemical Properties Ethanol extract Hexane extract Hexane extract (Kittiphoom, (Kittiphoom, (Fahimdanesh, 2013) 2013) 2013) Acid Value (mg KOH/g oil) 27.55±0.55 0.10±0.012 1.50 0.20

Peroxide value 26.35±2.1 8.72±3.4 1.26± 0.25 Iodine number 59.17±2.3 38.5±3.9 55.15± 2.20 Saponification number 206.0±13.8 207.5±14.2 196.0± 6.5 Unsaponifiable matter (%) 2.98 ±0.15 ±

Mango seed processing Mango Drying of seeds and Grinding of Kernel Extraction of Oil from Seeds removal of kernels into Kernel powder the Kernel powder

Flowchart for mango seed processing The mango seed consists of the endocarp (a tenacious coat) and the kernel of the mango. The oil that is extracted is located in the kernel. The seed has to be processed before it is ready for oil extraction.

Drying and Grinding The mango pulp is peeled off the mango fruit. The seed is then dried either by sun drying or using a drying oven. The seeds can also be placed on rooftops in open sunlight for drying. After the seeds are properly dried, the kernels are separated from the endocarps. The kernels are then well ground into fine powder. This powder is properly stored in a bottle and refrigerated at around 4oC.

Mango Oil extraction The soxhlet extractor is the one of the approaches used for extraction of mango oil from the kernel powder. A soxhlet extractor consists of a round bottom flask fitted with a glass sample/siphon chamber in the neck of the flask. On top of the sample chamber is a standard water-cooled condenser. The solid sample is placed in a 6 cellulose or fibre glass porous holder called a thimble; the solvent is placed in the round bottom flask. Using a heating mantle around the flask, the solvent is vaporized, condensed, and drips or washes back down over the sample. Soluble analytes (substances undergoing analysis) are extracted and then siphoned back into the round bottom flask. This is a continuous extraction process as long as heat is applied. The extracted analyte concentrates in the round bottom flask. The soluble analyte in this experiment is the mango oil. The extraction solvent can be hexane, ethanol, petroleum ether or chloroform (Micheal, 2005).

Soxhlet apparatus There are several instrumental advances in solvent extraction that have made extraction a more efficient process. These advances generally use sealed vessels under elevated pressure to improve extraction efficiency and are classified as pressurized fluid (or pressurized solvent) extraction methods. One approach is the Accelerated Solvent Extraction system. This technique is used for extraction solid and semisolid samples, such as food, with liquid solvents. It uses conventional solvents and mixtures of solvents at elevated temperatures and pressure to increase the efficiency of the extraction process. Increased temperature, up to 200oC compared with 70-80oC normal boiling points of common solvents, accelerates the extraction rate while elevated pressure keeps the solvent liquid are temperatures above their normal boiling points, enabling safe and rapid extractions. Extraction times for many samples can be cut from hours using a conventional approach to minutes, and the amount of solvent used is greatly reduced. Another approach also using high pressure and temperature is that of microwave assisted extraction. The sample is heated with the extraction solvent in a sealed vessel by microwave energy. The temperature can be raised to about 150oC with the already described advantages of high temperature and high pressure. One limitation of microwave assisted extraction is that some solvents are “transparent” to microwave radiation and do not hear; pure nonpolar solvents such as the normal alkanes (e.g. hexane, which is used for extraction of mango oil) are examples of such transparent solvents (Micheal, 2005). Another approach is the use of supercritical fluid extraction (SFE). A supercritical fluid is a substance at a temperature and pressure above the critical point for the substance. Supercritical fluids are more dense and viscous than the gas phase of the substance but not as dense and viscous as the liquid phase. The relatively

7 high density (compared with the gas phase of a supercritical fluid allows these fluids to dissolve nonvolatile o organic molecules. Carbon dioxide, CO2 has a critical temperature of 31.3 C and a critical pressure of 72.9atm; this temperature and pressure are readily attainable, making supercritical CO2 easy to form. Supercritical CO2 dissolves many organic compounds, so it can replace a variety of common solvents; supercritical CO2 is used widely as a solvent for extraction. The advantages of using supercritical CO2 includes it low toxicity, low cost, nonflammability, and ease of disposal. Once the extraction is complete and the pressure returns to atmospheric pressure, the carbon dioxide immediately changes to a gas and escapes from the opened extraction vessel. The pure samples of extracted analytes are left behind (Micheal, 2005).

Analysis of Mango Oil There is no organized, foolproof scheme for the qualitative analysis of fats, and the problems of identifying individual fats and oils is quite complicated. This is particularly true in the case of mixtures and processed fats. Even with the most sophisticated instruments, it is quite possible to encounter mixtures that defy identification of the source of the oil. Admittedly, the availability of more specific, meaningful, and reliable instrumental techniques has simplified identification and detection of adulteration. Also chromatographic procedures and spectrophotometry are used most commonly in combination with the determination of physical and chemical constants (saponification value, iodine value, acid value, peroxide value and others) that are constant and typical for individual fats and oils.

The Acid Value of Oil During storage, fats may become rancid due to peroxide formation at their double bonds by atmospheric oxidation and their hydrolysis by microbes with the liberation of free acids. The amount of free acids associated with fat gives a fair indication of its quality and age (Nigam, 2007). The acid value is defined as the number of milligrams of KOH required to neutralize the free fatty acids present in 1g fat. The measure of fat acidity normally reflects the amount of fatty acids hydrolyzed from triacylglycerols. Free fatty acid (FFA) is the percentage by weight of a specified fatty acid (e.g., percent oleic acid). In addition to FFAs, acid phosphates and amino acids also can contribute to acidity. In samples containing no acids other than fatty acids, FFA and acid value may be converted from one to the other using a conversion factor equation.

%FFA as oleic × 1.99 = acid value

8 Acid value conversion factors for lauric and palmitic are 2.81 and 2.19, respectively (Nielsen et al 2010). FFA is calculated as free oleic acid on a percentage basis for most fats and oils sources, although for coconut and palm kernel oils it is usually calculated as lauric acid and for as palmitic acid. Free fatty acid is an important fat quality indicator during each stage of fats and oils processing. Crude vegetable oils may have abnormally high FFA levels if the seed has been field damaged or improperly stored. Seed and fruit enzyme lipases are activated by moisture, and hydrolysis is initiated, which increases the FFA content. Higher crude oil FFA levels equate to higher refining losses. FFA is the result of hydrolysis of the fat or oil. Moisture must be present for hydrolysis to develop. This reaction is accelerated with heat and pressure, as are most reactions (O'Brien, 2008).

Saponification Value of a Fat When fats and oils are heated with an alcoholic solution of KOH, free fatty acids and glycerol are liberated. The refluxing with an alkali causes the hydrolysis of glyceryl esters, yielding glycerol and potassium salts of fatty acids (soaps). Subsequently, the test sample as well as the control is titrated against HCl to determine the amount of KOH used in the saponification process. The saponification value is the number of milligrams of KOH required to neutralize the fatty acids resulting from the complete hydrolysis of 1g fat. It is a measure of the alkali-reactive groups in fats and oils and it was used to predict the type of glycerides in a sample. The saponification value gives an indication of the nature of fatty acids in the fat, since the longer the carbon chain is, the lesser is the amount of acid liberated per gram of fat hydrolyzed (Nigam, 2007). Hence, glycerides containing short-chain fatty acids have higher saponification values that those with longer chain fatty acids. The saponification value is an indication of the average molecular weight of fat. For pure fatty acids, the saponification value equals the acid value. The ester value is the difference between the saponification value and the acid value. In oils and fats, the ester value is a measure of the amount of glycerides present (Pomeranz, 2002). The saponification value, along with the iodine value determination, were useful screening tests both for quality control and for characterizing types of fats and oils. However, the results overlap too much to identify individual fats or oils; for example, both domestic vegetable oils and animal fats have saponification values in the 180 to 200 range. Saponification value analysis has been replaced almost exclusively in edible fats and oils processing by fatty acid composition analysis by gas/liquid chromatography (GLC) (O'Brien, 2008).

Iodine Value for a Lipid Sample The amount of iodine a fatty acid can take up indicates it degree of unsaturation, and is termed its iodine value. The iodine value is a chemical constant for a fat or oil. It is a valuable characteristic in fat analysis that measures unsaturation, but does not define the specific fatty acids. Iodine value analyses are very accurate and provide nearly theoretical values, except in the case of conjugated double bonds or when the double bond is near a carboxyl group. However, unless the history of fat or the type of fat in the product is known, an iodine value may be somewhat meaningless by itself. For example, a product prepared with a meat fat with consistency and performance characteristics similar to a -based product will have a considerably different iodine 9 value. Further, even vegetable oil-based product will have a considerably different iodine value. Further, even vegetable oil products with comparable functionality, but different source oils will not have like iodine values. Iodine value is a useful tool for process control and product specification. Iodine value is a measure of the unsaturation of fats and oils and is expressed as the number of centigrams of iodine absorbed per gram of sample. Iodine value of oil can be determined using Wijs reagent (iodine chloride), a Fourier transform-near infrared (FT-NIR) spectroscopy procedure, differential scanning calorimetry and other techniques (O'Brien, 2008).

Peroxide Value of Oil Oxidation of lipids is a major cause of their deterioration, and hydroperoxides formed by the reaction between oxygen and the unsaturated fatty acids are the primary products of this reaction. Hydroperoxides have no flavour or odour but break down rapidly to form aldehydes, which have a strong, disagreeable flavour and odour. The peroxide concentration, usually expressed as peroxide value, is a measure of oxidation or rancidity in its early stages. Peroxide value measures the concentration of substances (in terms of milliequivalents of peroxide per 1000 grams of sample) that oxidize potassium iodide to iodine (O'Brien, 2008). Peroxide value measures a transient product of oxidation, (i.e., after forming, peroxides and hydro peroxides break down to form other products). A low value may represent either the beginning of oxidation or advanced oxidation, which can be distinguished by measuring peroxide value over time or by using a procedure that measures secondary products of oxidation. For determination in foodstuffs, a disadvantage of this method is the 5g fat or oil sample size required; it is difficult to obtain sufficient quantities from foods low in fat. This method is empirical and any modifications may change results. Despite its drawbacks, peroxide value is one of the most common tests of lipid oxidation. High-quality, freshly deodourized fats and oil will have a peroxide value of zero. Peroxide values > 20 correspond to very poor quality fats and oils, which normally would have significant off flavours (Nielsen, 2010). High peroxide values usually mean poor flavour ratings, but a low peroxide value is not always an indication of a good flavour.

Factorial Experimental Design In statistics, a full factorial experiment is an experiment whose design consists of two or more factors, each with discrete possible values or "levels", and whose experimental units take on all possible combinations of these levels across all such factors. A full factorial design may also be called a fully crossed design. Such an experiment allows the investigator to study the effect of each factor on the response variable, as well as the effects of interactions between factors on the response variable. (Batra, 1984)

For the vast majority of factorial experiments, each factor has only two levels. For example, with two factors each taking two levels, a factorial experiment would have four treatment combinations in total, and is usually called a 2×2 factorial design.

If the number of combinations in a full factorial design is too high to be logistically feasible, a fractional factorial design may be done, in which some of the possible combinations (usually at least half) are omitted.

10 Implementation The notation used to denote factorial experiments conveys a lot of information. When a design is denoted a 23 factorial, this identifies the number of factors (3); how many levels each factor has (2); and how many experimental conditions there are in the design (23=8).

The notation system of factorial design can be understood by considering some basic examples below:

 2×2 is a 2 by 2 factorial design that denotes an experiment with two factors each having two levels. This experiment will have 4 total conditions.

 2×4×4 is a 2 by 4 by 4 factorial design that denotes an experiment with 3 factors with the first factor having 2 levels, the second factor having 4 levels and the third factor having 4 levels. This experiment will have 32 total conditions.

Table 2.5 Notation System for a 2x2 Factorial Experiment.

A B (1) - - a + - b - + ab + +

A = A effect B = B effect

To save space, the points in a two-level factorial experiment are often abbreviated with strings of plus and minus signs. The strings have as many symbols as factors, and their values dictate the level of each factor: conventionally, - for the first (or low) level, and for the second (or high) level. The points in this experiment can thus be represented as shown above.

The factorial points can also be abbreviated by (1), a, b, and ab, where the presence of a letter indicates that the specified factor is at its high (or second) level and the absence of a letter indicates that the specified factor is at its low (or first) level (for example, "a" indicates that factor A is on its high setting, while all other factors are at their low (or first) setting). (1), this is used to indicate that all factors are at their lowest (or first) values.

Analysis of a Factorial Matrix A typical factorial matrix is a 2 × 2 factorial design. The two factors are type of training and presentation rate. Each factor has two levels. Table 2.6 2 by 2 factorial experiment design.

11 The subject is told to memorize the words by rote (repetition) and by imagery. Two different presentation rates are used. 4 experimental conditions are possible due to the design.

Main Effects A main effect is the overall effect of an independent variable. E.g. main effect of “type of training,” compares data in shaded cells (A2B1 and A2B2) with data in non-shaded cells (A1B1 and A1B2). This is therefore a comparison between all the subjects which received imagery training irrespective of the presentation rate and the subjects which received rote training irrespective of the presentation rate. Likewise, the main effect of presentation rate compares data in shaded cells with data in non-shaded cells. Table 2.7 2 by 2 factorial experiment design with notations

To calculate the main effect, the [marginal] means of each row and column are calculated. Table 2.8 2 by 2 factorial experiment design with calculated marginal means.

The main effect is the calculated by subtracting one marginal mean from another marginal mean.

Main effect of imagery is 20 – 15 = 5 words Hence Imagery produces better recall than rote by 5 words.

Main effect of rate is 14.5 – 20.5 = -6 words 2-sec produces a worse recall than 4-sec by 6 words. These subtractions must be carried out consistently in one direction.

12 Simple Main Effects These involve calculating the effect of moving from one cell to another at one level of the independent variable. For example, the simple main effect of training at 4 seconds is like moving from imagery at 4 sec to rote at 4 sec. it is subtracting 18 from 23 or 23 from 18. The answer is -5 if you move from imagery to rote. This implies that a subject remembers 5 less words using rote than imagery using a 4 sec/word presentation rate. In this example, the independent variable being considered is presentation rate while the level being considered is 4 sec/word. The simple main effect can also be calculated for training at 2 sec, rate at imagery and rate at rote.

Interactions Another example is considered here. The independent variables are course emphasis and student majors. This factorial matrix has no main effects because the marginal means are equal. Table 2.9 Factorial matrix with no main effects.

An interaction is when the size (value) or direction of the simple main effect [on the dependent variable] of independent variable 1 changes at different levels of the independent variable 2. In this 2 by 2 table, there are 4 simple main effects Major at lab: 80 to 70; SME = -10 Major at lecture: 70 to 80; SME = 10 Emphasis at science: 80 to 70; SME = -10 Emphasis at humanities: 70 to 80; SME = 10. Where SME denotes Simple main effect

Since the simple main effects of both “major” and “emphasis” change in direction, it can be said that there is an interaction. It is possible for no interaction to exist. This occurs when the simple main effects that are being compared are the same. From the interaction calculations, it can be said that humanities majors do better at lecture than lab and science majors do better at lab than lectures. Note: When the data is plotted, data with interaction will not have lines parallel to each other.

13 CHAPTER THREE MATERIALS AND METHODS In this chapter, the materials and methods used in the experiment are described in appropriate sections.

Raw materials and reagents 1. Ripe mangoes: The mangoes were obtained from an open market in Ojure, Ogun state. Nigeria. 2. Absolute ethanol 3. Hexane 4. 0.1M Potassium hydroxide (KOH) 5. Phenolphthalein Indicator 6. 0.5M alcoholic Potassium hydroxide (KOH) 7. 0.5M Hydrochloric acid

Equipment 1. Plastic food packs. 2. 200ml and 500ml-Beakers. 3. Mortar Pestle. 4. Drying Oven. 5. Blender. 6. Small transparent containers for samples. 7. Weighing Balance. 8. Thermometer. 9. Heating Mantle. 10. 150mm Diameter Filter paper. 11. 200ml-Soxhlet Apparatus. 12. 250ml and 500ml-Round bottom flask. 13. Reflux Condenser. 14. Spatula. 15. Burette. 16. Pipette. 17. Scientific 300037 Programmable Refractometer. 14 18. 250ml-Conical flask. 19. Pipette sucker

Methods Flowchart of process REMOVAL DRYING OF GRINDING OF OF PULP SEEDS KERNEL

PHYSICOCHEMICAL EXTRACTION ANALYSIS OFOIL OF OIL Figure 3.1: Flowchart of process Removal of pulp: The mangoes were dispersed among students for consumption of the pulp and returning of the seed kernel. Drying: The seed kernels were dried in a drying oven at 1000C for 8 hours. The seeds were then cracked open and the kernel was removed. The kernels that were still moist were then further dried.

Drying oven

Grinding: All the kernels were ground using a mortar pestle. Then the ground kernels were further ground using a kitchen blender to make the particle size of the kernel powder less than 2000 microns. The kernel powder was then stored in a plastic container in a cool and dry place.

Mortar pestle

15 Kitchen blender

Extraction of Oil: Oil was extracted from the kernel using a soxhlet apparatus and a heating mantle. The soxhlet apparatus is made up of a condenser, an extractor and a flask (round bottom or flat bottom) which is being heated. As the diagram shows, the kernel powder encapsulated in filter paper was placed in the extractor. The organic solvent used for extraction was placed in the flask. The solvent vaporizes and rises to the condenser where it is condensed. The condensed organic solvent then drips on the kernel powder. The kernel powder upon contact with hot organic solvent begins to secret oil which is soluble in the organic solvent. When the oil and solvent mixture has risen high enough (higher than the siphon tube), the mixture flows through the siphon tube back into the flask.

Soxhlet apparatus Heating mantle

The oil is then recovered from the organic solvent by heating the mixture with a condenser coupled to the flask. The organic solvent vapourizes and is then condensed back into another container.

Extraction of Oil from the Mango Kernel Powder Determining the effects of time and choice of organic solvent on the yield of oil from the kernel powder. The experiment was carried 10 times at different conditions.

Experiment Run 1: Extracting with Hexane for 4hours  200ml of hexane was measured in a measuring cylinder and then poured into a round bottom flask and then cocked.  The round bottom flask was placed on a heating mantle. 16  It was then uncorked and the extractor of the soxhlet was placed on it.  A condenser was placed on the extractor and properly connected to a tap.  A piece of filter paper was rolled and used to make a cone.  30g of the kernel powder was weighed and poured into the cone.  A small rope was attached to the cone to ensure that it can be pulled out without spilling. The cone was lowered into the extractor.  The heating mantle was switched on and set to a temperature of 700C. The extraction was left to continue for 4 hours.  After 4 hours, the heating mantle was switched off and the apparatus was allowed to cool.  The condenser was removed and dismantled. The cone was removed from the extractor and any liquid left in the extractor was poured back into the round bottom flask.  A condenser was then connected to the round bottom flask which still sat on the heating mantle.  A conical flask was attached to the other end of the condenser to collect the hexane that vapourizes from the mixture in the round bottom flask.  The heating mantle was switched on and set to 700C. The hexane was allowed to vapourize till very little hexane was left.  The residual solvent was removed by placing the oil in a drying oven at 700C for 1 hour.  The total yield of oil was expressed in percentage

Experiment Run 2: Extracting with Hexane for 5hours  200ml of hexane was measured in a measuring cylinder and then poured into a round bottom flask and then cocked.  The round bottom flask was placed on a heating mantle.  It was then uncorked and the extractor of the soxhlet was placed on it.  A condenser was placed on the extractor and properly connected to a tap.  A piece of filter paper was rolled and used to make a cone.  30g of the kernel powder was weighed and poured into the cone.  A small rope was attached to the cone to ensure that it can be pulled out without spilling. The cone was lowered into the extractor.  The heating mantle was switched on and set to a temperature of 700C. The extraction was left to continue for 5 hours.  After 5 hours, the heating mantle was switched off and the apparatus was allowed to cool.  The condenser was removed and dismantled. The cone was removed from the extractor and any liquid left in the extractor was poured back into the round bottom flask.  A condenser was then connected to the round bottom flask which still sat on the heating mantle.  A conical flask was attached to the other end of the condenser to collect the hexane that vapourizes from the mixture in the round bottom flask.  The heating mantle was switched on and set to 700C. The hexane was allowed to vapourize till very little hexane was left.  The residual solvent was removed by placing the oil in a drying oven at 700C for 1 hour.  The total yield of oil was expressed in percentage

17 Experiment Run 3: Extracting with Hexane for 6hours  200ml of hexane was measured in a measuring cylinder and then poured into a round bottom flask and then cocked.  The round bottom flask was placed on a heating mantle.  It was then uncorked and the extractor of the soxhlet was placed on it.  A condenser was placed on the extractor and properly connected to a tap.  A piece of filter paper was rolled and used to make a cone.  30g of the kernel powder was weighed and poured into the cone.  A small rope was attached to the cone to ensure that it can be pulled out without spilling. The cone was lowered into the extractor.  The heating mantle was switched on and set to a temperature of 700C. The extraction was left to continue for 6 hours.  After 6 hours, the heating mantle was switched off and the apparatus was allowed to cool.  The condenser was removed and dismantled. The cone was removed from the extractor and any liquid left in the extractor was poured back into the round bottom flask.  A condenser was then connected to the round bottom flask which still sat on the heating mantle.  A conical flask was attached to the other end of the condenser to collect the hexane that vapourizes from the mixture in the round bottom flask.  The heating mantle was switched on and set to 700C. The hexane was allowed to vapourize till very little hexane was left.  The residual solvent was removed by placing the oil in a drying oven at 700C for 1 hour.  The total yield of oil was expressed in percentage

Experiment Run 4: Extracting with Hexane for 7hours  200ml of hexane was measured in a measuring cylinder and then poured into a round bottom flask and then cocked.  The round bottom flask was placed on a heating mantle.  It was then uncorked and the extractor of the soxhlet was placed on it.  A condenser was placed on the extractor and properly connected to a tap.  A piece of filter paper was rolled and used to make a cone.  30g of the kernel powder was weighed and poured into the cone.  A small rope was attached to the cone to ensure that it can be pulled out without spilling. The cone was lowered into the extractor.  The heating mantle was switched on and set to a temperature of 700C. The extraction was left to continue for 7 hours.  After 7 hours, the heating mantle was switched off and the apparatus was allowed to cool.  The condenser was removed and dismantled. The cone was removed from the extractor and any liquid left in the extractor was poured back into the round bottom flask.  A condenser was then connected to the round bottom flask which still sat on the heating mantle.  A conical flask was attached to the other end of the condenser to collect the hexane that vapourizes from the mixture in the round bottom flask.  The heating mantle was switched on and set to 700C. The hexane was allowed to vapourize till very little hexane was left. 18  The residual solvent was removed by placing the oil in a drying oven at 700C for 1 hour.  The total yield of oil was expressed in percentage

Experiment Run 5: Extracting with Hexane for 8hours  200ml of hexane was measured in a measuring cylinder and then poured into a round bottom flask and then cocked.  The round bottom flask was placed on a heating mantle.  It was then uncorked and the extractor of the soxhlet was placed on it.  A condenser was placed on the extractor and properly connected to a tap.  A piece of filter paper was rolled and used to make a cone.  30g of the kernel powder was weighed and poured into the cone.  A small rope was attached to the cone to ensure that it can be pulled out without spilling. The cone was lowered into the extractor.  The heating mantle was switched on and set to a temperature of 700C. The extraction was left to continue for 8 hours.  After 8 hours, the heating mantle was switched off and the apparatus was allowed to cool.  The condenser was removed and dismantled. The cone was removed from the extractor and any liquid left in the extractor was poured back into the round bottom flask.  A condenser was then connected to the round bottom flask which still sat on the heating mantle.  A conical flask was attached to the other end of the condenser to collect the hexane that vapourizes from the mixture in the round bottom flask.  The heating mantle was switched on and set to 700C. The hexane was allowed to vapourize till very little hexane was left.  The residual solvent was removed by placing the oil in a drying oven at 700C for 1 hour.  The total yield of oil was expressed in percentage.

Experiment Run 6: Extracting with Ethanol for 4hours  200ml of Ethanol was measured in a measuring cylinder and then poured into a round bottom flask and then cocked.  The round bottom flask was placed on a heating mantle.  It was then uncorked and the extractor of the soxhlet was placed on it.  A condenser was placed on the extractor and properly connected to a tap.  A piece of filter paper was rolled and used to make a cone.  30g of the kernel powder was weighed and poured into the cone.  A small rope was attached to the cone to ensure that it can be pulled out without spilling. The cone was lowered into the extractor.  The heating mantle was switched on and set to a temperature of 800C. The extraction was left to continue for 4 hours.  After 4 hours, the heating mantle was switched off and the apparatus was allowed to cool.  The condenser was removed and dismantled. The cone was removed from the extractor and any liquid left in the extractor was poured back into the round bottom flask.  A condenser was then connected to the round bottom flask which still sat on the heating mantle.

19  A conical flask was attached to the other end of the condenser to collect the Ethanol that vapourizes from the mixture in the round bottom flask.  The heating mantle was switched on and set to 800C. The Ethanol was allowed to vapourize till very little Ethanol was left.  The residual solvent was removed by placing the oil in a drying oven at 800C for 1 hour.  The total yield of oil was expressed in percentage.

Experiment Run 7: Extracting with Ethanol for 5hours  200ml of Ethanol was measured in a measuring cylinder and then poured into a round bottom flask and then cocked.  The round bottom flask was placed on a heating mantle.  It was then uncorked and the extractor of the soxhlet was placed on it.  A condenser was placed on the extractor and properly connected to a tap.  A piece of filter paper was rolled and used to make a cone.  30g of the kernel powder was weighed and poured into the cone.  A small rope was attached to the cone to ensure that it can be pulled out without spilling. The cone was lowered into the extractor.  The heating mantle was switched on and set to a temperature of 800C. The extraction was left to continue for 5 hours.  After 5 hours, the heating mantle was switched off and the apparatus was allowed to cool.  The condenser was removed and dismantled. The cone was removed from the extractor and any liquid left in the extractor was poured back into the round bottom flask.  A condenser was then connected to the round bottom flask which still sat on the heating mantle.  A conical flask was attached to the other end of the condenser to collect the Ethanol that vapourizes from the mixture in the round bottom flask.  The heating mantle was switched on and set to 800C. The Ethanol was allowed to vapourize till very little Ethanol was left.  The residual solvent was removed by placing the oil in a drying oven at 800C for 1 hour.  The total yield of oil was expressed in percentage.

Experiment Run 8: Extracting with Ethanol for 6hours  200ml of Ethanol was measured in a measuring cylinder and then poured into a round bottom flask and then cocked.  The round bottom flask was placed on a heating mantle.  It was then uncorked and the extractor of the soxhlet was placed on it.  A condenser was placed on the extractor and properly connected to a tap.  A piece of filter paper was rolled and used to make a cone.  30g of the kernel powder was weighed and poured into the cone.  A small rope was attached to the cone to ensure that it can be pulled out without spilling. The cone was lowered into the extractor.  The heating mantle was switched on and set to a temperature of 800C. The extraction was left to continue for 6 hours.  After 6 hours, the heating mantle was switched off and the apparatus was allowed to cool. 20  The condenser was removed and dismantled. The cone was removed from the extractor and any liquid left in the extractor was poured back into the round bottom flask.  A condenser was then connected to the round bottom flask which still sat on the heating mantle.  A conical flask was attached to the other end of the condenser to collect the Ethanol that vapourizes from the mixture in the round bottom flask.  The heating mantle was switched on and set to 800C. The Ethanol was allowed to vapourize till very little Ethanol was left.  The residual solvent was removed by placing the oil in a drying oven at 800C for 1 hour.  The total yield of oil was expressed in percentage.

Experiment Run 9: Extracting with Ethanol for 7hours  200ml of Ethanol was measured in a measuring cylinder and then poured into a round bottom flask and then cocked.  The round bottom flask was placed on a heating mantle.  It was then uncorked and the extractor of the soxhlet was placed on it.  A condenser was placed on the extractor and properly connected to a tap.  A piece of filter paper was rolled and used to make a cone.  30g of the kernel powder was weighed and poured into the cone.  A small rope was attached to the cone to ensure that it can be pulled out without spilling. The cone was lowered into the extractor.  The heating mantle was switched on and set to a temperature of 800C. The extraction was left to continue for 7 hours.  After 7 hours, the heating mantle was switched off and the apparatus was allowed to cool.  The condenser was removed and dismantled. The cone was removed from the extractor and any liquid left in the extractor was poured back into the round bottom flask.  A condenser was then connected to the round bottom flask which still sat on the heating mantle.  A conical flask was attached to the other end of the condenser to collect the Ethanol that vapourizes from the mixture in the round bottom flask.  The heating mantle was switched on and set to 800C. The Ethanol was allowed to vapourize till very little Ethanol was left.  The residual solvent was removed by placing the oil in a drying oven at 800C for 1 hour.  The total yield of oil was expressed in percentage.

Experiment Run 10: Extracting with Ethanol for 8hours  200ml of Ethanol was measured in a measuring cylinder and then poured into a round bottom flask and then cocked.  The round bottom flask was placed on a heating mantle.  It was then uncorked and the extractor of the soxhlet was placed on it.  A condenser was placed on the extractor and properly connected to a tap.  A piece of filter paper was rolled and used to make a cone.  30g of the kernel powder was weighed and poured into the cone.

21  A small rope was attached to the cone to ensure that it can be pulled out without spilling. The cone was lowered into the extractor.  The heating mantle was switched on and set to a temperature of 800C. The extraction was left to continue for 8 hours.  After 8 hours, the heating mantle was switched off and the apparatus was allowed to cool.  The condenser was removed and dismantled. The cone was removed from the extractor and any liquid left in the extractor was poured back into the round bottom flask.  A condenser was then connected to the round bottom flask which still sat on the heating mantle.  A conical flask was attached to the other end of the condenser to collect the Ethanol that vapourizes from the mixture in the round bottom flask.  The heating mantle was switched on and set to 800C. The Ethanol was allowed to vapourize till very little Ethanol was left.  The residual solvent was removed by placing the oil in a drying oven at 800C for 1 hour.  The total yield of oil was expressed in percentage.

Physical and Chemical Analysis of the Mango Oil Specific Gravity The specific gravity – SG - is a dimensionless unit defined as the ratio of density of the mango oil to the density of water at a specified temperature. This was done by measuring the density of mango oil in reference to the density of distilled water at 200C using a specific gravity bottle.

Specific gravity bottle

Procedure: I. The specific gravity bottle was weighed while empty. It was then filled with mango oil and weighed again. II. The difference in weights was divided by an equal volume of water to obtain the specific gravity of the mango oil.

Refractive Index The refractive index of a substance measures how the substance affects light traveling through it. It is equal to the speed of light in a vacuum divided by the speed of light in that substance. When light travels between two materials with different refractive indexes, it bends at the boundary between them. The refractive index test was carried out using a programmable Refractometer.

22 Procedure: I. The apparatus was standardized using pure distilled water whose refractive index at 200C is 1.3330. II. A drop of the sample was inserted into the machine. After about 1-2 minute(s) the machine read off the refractive index.

Refractometer

Saponification Value Saponification value is the number of mg of potassium hydroxide (KOH) required to saponify the esters in 1g of a sample; and to neutralize the free acids. It also indicates the amount of average molecular weight of triglycerides contained in the oil.

Procedure: I. 1g of oil was weighed into 250ml dry round bottom flask. II. 50ml of 0.5ml alcoholic potassium hydroxide was added to the oil. Porous bits were added to ensure uniform heating. III. The reflux condenser was setup and the contents of the round bottom flask was refluxed for about 1hr, after refluxing the mixture is allowed to cool and is then titrated against standard hydrochloric acid and the titre values are recorded. Similarly, IV. 50ml of the same alcoholic KOH, blank (no oil added) was refluxed in a round bottom flask for 1hr, cooled and titrated against standard 0.5N HCL. V. The titre value was recorded and the titre value gotten what then used to determine the saponification value. The saponification value is calculated mathematically as: Saponification value = (3.2) × × . Where, Z = volume of HCL required to neutralize excess alkali (ml) Z = (X – Y) ml X = titre value of HCL against oil and KOH after reflux (ml) Y = titre value of HCL against KOH alone after reflux (ml)

Acid Value Acid value (or "neutralization number" or "acid number" or "acidity") is the mass of potassium hydroxide (KOH) in milligrams that is required to neutralize one gram of chemical substance. The acid number is a measure of the amount of carboxylic acid groups in a chemical compound, such as a fatty acid, or in a

23 mixture of compounds. In a typical procedure, a known amount of sample dissolved in organic solvent is titrated with a solution of potassium hydroxide with known concentration and with phenolphthalein as a color indicator.

Procedure: I. 1g of the oil sample was weighed into 250ml conical flask. 95% alcohol (neutral alcohol) was prepared by diluting methanol with sodium hydroxide (5ml NaOH + 95ml ethanol = 100ml neutral alcohol). II. 50ml of neutral alcohol and 50ml benzene were added to the oil in the flask. The contents of the flask were shaken well to dissolve. III. The contents of the flask were shaken well to dissolve. The contents were then titrated against 0.1N potassium hydroxide solution using phenolphthalein as indicator. IV. The end point was the appearance of a pale permanent pink colour and the titre value were recorded. The acid value is calculated mathematically as: Acid value = (3.1) × × . Where, V = volume of KOH required to neutralize the solution (ml) N = strength of OH W = weight of oil used (g). The number 56.1 is the atomic weight of potassium hydroxide (KOH)

Ester Value Ester value was obtained by subtracting the acid value from the saponification value. Ester value represents the number of milligrams of potassium hydroxide required to saponify the esters present in 1g of the oil.

Safety Precautions  Zero error of the measuring balance was avoided when measurement was carried out.  The mango seeds were dried immediately after consumption of the pulp to ensure that there is no spoilage.  Special care was taken while using the heating mantle to avoid any unsafe conditions.  All the glasswares were handled with care to avoid damage of glassware.  During the experiment, all fans were switched off to avoid inaccuracy in measurements.

24 CHAPTER FOUR

RESULTS AND DISCUSSION OF RESULTS

In this chapter the result of the experiments carried out in the laboratory are outlined and described in various sections.

Results Determination of yield of oil For the two extraction solvents, hexane and ethanol, the yield of oil is shown below. (Detailed calculations are shown in appendix C)

Table 4.1 Yield of oil from hexane extraction Yield(%) using Amount of oil Equation of No of Hours extracted (g) Yield (%) Trendline 4 1.20 4.000 4.007 5 2.00 6.667 6.685 6 3.00 10.000 10.042 7 3.70 12.333 12.414 8 3.90 13.000 13.139

Table 4.2 Yield of oil from ethanol extraction Amount of oil Yield(%) using Equation of No of Hours extracted (g) Yield (%) Trendline 4 0.6 2.000 1.981 5 1 3.333 3.293 6 1.7 5.667 5.590 7 2.2 7.333 7.199 8 2.5 8.333 8.112

Factorial Experiment Design Using Manual Method Where A = Time (- = 4hours, + = 8hours) B = Organic solvent (- = Hexane, + = Ethanol)

25 Table 4.3 Manual factorial experiment results (Detailed calculation is shown in appendix C) Interaction Main Effects Effects RUN A` B AB Yield (%) 1 - - + 4.00 5 + - - 13.00 6 - + - 2.00 10 + + + 8.33 Absolute value of effect 7.665 3.335 1.335

From the above table, it is shown that factor ‘A’ which is time produces the greatest effect in the yield of mango, 7.665.

Factorial Design of Experiment Using Minitab Table 4.4 Minitab Factorial Experiment results RunOrder Blocks CenterPt Solvent Time Yield (% w/w) 1 1 1 Hexane 4 4 2 1 1 Hexane 8 13 3 1 1 Ethanol 4 2 4 1 1 Ethanol 8 8.333

Table 4.5 Absolute effect of main effects from Minitab Term Absolute effect from Minitab Time (A) 7.665 Extraction solvent (B) 3.335 Time and Solvent (AB) 1.335

Physicochemical Properties Table 4.6 Physicochemical properties of extracted Mango oil

Acid Saponification Ester Specific Oil value(mgKOH/gOil) value(mgKOH/gOil) value(mgKOH/gOil) Refractive gravity @ Sample index 200C Extracted with Hexane 5.61 207.0 201.39 1.443 0.909 Extracted with Ethanol 30.30 205.0 174.70 1.451 0.900

26 Table 4.7 Comparison of mango oil yield with literature.

Extraction Solvent (8hours) Yield (% w/w) Hexane extract 13.00 Ethanol extract 8.33 Hexane extract (Nzikuo et al 2010) 13.00 Ethanol extract (Kittiphoom and Sutasinee et al 2013) 6.96

Table 4.8 Comparison of Physicochemical properties of extracted oil with literature values

Saponification Acid value value(mgKOH Ester value Specific (mgKOH/gOil) /gOil) (mgKOH/gOil) Refractive gravity @ Oil Sample index 200C

Hexane extract 5.61 207.00 201.39 1.443 0.909 Hexane extract (Nzikuo et al 2010) 5.35 207.5 202.15 1.433±0.02 0.900±0.03

Ethanol extract 30.30 205.00 174.95 1.451 0.900 Ethanol extract (Kittiphoom and Sutasinee et al 2013) 27.55±0.55 206±13.8 178.45±13.25 1.444±0.01 0.903

14.00

Y 12.00 I 10.00 E 8.00 Yield vs Time L 6.00 D ( 4.00 % ) 2.00

0.00 0 2 4 6 8 10 TIME (HOURS) Figure 4.1 Plot of yield against time for the extraction of oil using Hexane.

27 9.00 8.00 7.00 Y 6.00 I 5.00 Yield vs… E 4.00 L 3.00 D 2.00 ( % 1.00 ) 0.00 0 2 4 6 8 10 TIME (HOURS) Figure 4.2 Plot of yield against time for the extraction of oil using Ethanol.

14.00

12.00 Y I 10.00 E 8.00

L 6.00 Hexane D ( 4.00 Ethanol % ) 2.00

0.00 0 2 4 6 8 10 TIME (HOURS)

Figure 4.3 Comparison of the Yield from Hexane and Ethanol extractions

Main Effects Plot for Yield (% w/w) Data Means

Time Solvent 11

10

9

8 n a

e 7 M

6

5

4

3

4 8 Hexane Ethanol

28 Figure 4.4 Main effects plot for mango oil yield

Interaction Plot for Yield (% w/w) Data Means

Hexane Ethanol Time 12 4 8 9

Time 6

3

Solvent 12 Hexane Ethanol 9

Solvent 6

3

4 8 Figure 4.5 Full interaction plot matrix for mango oil yield

10

8

6

Effect 4

2

0 Time (A) Extraction Time and solvent (B) Solvent (AB) Figure 4.6 Bar chart of the effects Discussion of Results The Effect of the Extraction Solvent and Time on the Yield of oil The effect of the time of extraction on the yield is clearly shown in Table 4.1 and 4.2 using hexane and ethanol as extraction solvents respectively. These results have been illustrated in Figure 4.1 and 4.2. It was observed that the yield of oil increased as the time of extraction increases from 2 to 8 hours. This was so because enough time is required for the oil to break away from the kernel powder. The longer the time of extraction, the longer the kernel powder stays in contact with the extraction solvent. The rate of increase in yield is much faster at lower extraction times than at higher extraction times. This is so because as the time of extraction increases, the amount of oil that can be extracted from the kernel powder reduces until no more oil can be extracted. The yields from the two solvent were compared in Figure 4.3. It was observed that Hexane gave more yield than ethanol at the different extraction times. This is because hexane is a very non-polar solvent which easily extracts the non-polar mango oil; while Ethanol which has both polar and non-polar parts doesn’t extract as much oil. Mango oil, being a non-polar solvent, extracted better in the hexane extractions because in solvent 29 dissolution, “like dissolves like”. A polar solvent dissolves polar solvents easily and doesn’t mix with non- polar solvent. Ethanol can only dissolve oil because it has a non-polar part. Hexane is still considered as a better extraction solvent because it has a lower boiling point (680C) than ethanol (780C). This implies that Hexane needs lesser energy than Ethanol to extract. Hexane is easier to recover after the extraction. Ethanol requires a higher temperature than Hexane and such temperature might damage the oil being extracted. From Table 4.7, it is shown that the yield from the Hexane extraction tallies with literature values while that of Ethanol is very close.

The Factorial Experiment Design for the effects of various factors on the oil yield In determining the oil yield, a factorial experiment was designed using 4 runs (i.e. 22) where the 2 main effects are: (A = time) and (B = extraction solvent) was put into consideration at their highest and lowest values. (I.e. time - = 4hours and + = 8hours, extraction solvent - = Ethanol and + = Hexane). The absolute value of effect of all main effects and interaction effects were determined and the factor A, time, was determined as the major factor affecting the oil yield with absolute value of 7.665. Thus, increase in time produces an increase in the yield of mango oil and also the factor B, extraction solvent, had a great effect on the oil yield with absolute effect of 3.335. The manual calculation of the factorial experimental results were compared with the factorial experimental design calculation using a software called “Minitab,” as shown in Table 4.3 and 4.5. Figure 4.4 shows the main effect plot for the mango oil yield (using data means). The time plot shows that the yield of oil increases with time. The solvent plot shows that the yield of oil decreases when the extraction solvent is changed from hexane to ethanol. The interaction plot for the mango oil yield shown in Figure 4.5 basically compares the relative strength of the effects across the factors. The two plots show how “time” and “extraction solvent” interact. The two factors differ in their effect on the yield. The yield increases with time and reduces when the solvent is switched from Hexane to Ethanol. From the plot, it can be seen that the yield increases with time irrespective of the solvent used. It can also be seen that Hexane gave a much higher yield than Ethanol at 8hours but their yields are closer at 4hours. This is because the extraction solvent had not contacted the kernel powder enough at 4hours.

Physicochemical properties of mango oil The acid value of mango oil is the mass of KOH in milligrams that is required to neutralize 1g of the mango oil. Basically, the acid value is used to quantify the amount of acid (free fatty acids, acid phosphates or amino acids) present in a sample. For oils, it is a measure of the free fatty acid content. Free fatty acids exist in an oil because of the hydrolysis of fatty acids from triacylglycerols. This reaction is accelerated with heat and pressure. From Table 4.8, it is shown that the Ethanol extract has a higher acid value than the Hexane extract. This is because more fatty acids are hydrolyzed during the Ethanol extraction than during the Hexane extraction. Since the hydrolysis is sped up by heat, the Ethanol extraction which requires a higher temperature than that of Hexane would cause more fatty acids to hydrolyze. Hence, using Ethanol as an extraction solvent yields an oil with high acid content. The acid value of both extracts agrees with literature as shown in Table 4.8 Refractive index is the ratio of the speed of light in a vacuum to that in the oil under examination which is related to the degree of saturation and the ratio of cis/trans double bonds, and can also provide hints on the oxidative damage. The refractive index increased from 1.443 to 1.451 for the Hexane and Ethanol extracts 30 respectively. Basically, the refractive index is used for rapid sorting of fats and oils of suspected adulteration. Hence the refractive index of the Ethanol extract was higher than the Hexane extract because the mango oil continues to be adulterated as the heating temperature increases. This is due to the high boiling point of Ethanol (780C). Both refractive index values of the mango oil falls within the literature range of values as shown in Table 4.8. The saponification value of mango oil is the number of milligrams of KOH required to saponify 1g of mango oil. It is a measure of the chain lengths of the fatty acid presents. The saponification value of the two extracts differs by a negligible amount. Hence, the saponification value of mango oil is not dependent on the extraction solvent used (Hexane or Ethanol). A high saponification value may suggest use of the oil in the soap industry. Therefore, mango oil has a very high chance of being used for the manufacturing of soap. Both saponification values of the mango oil falls within the literature range of values as shown in Table 4.8. The ester values decreased from 201.39 to 174.95 for the Hexane and Ethanol extracts respectively. The higher the ester value, the more the palatability of the oil. Hence the Hexane extracts is more palatable and will therefore have better taste (Olaniyan & Oje, 2007). Both ester values fall within the literature range of ester values as shown in Table 4.8. Specific gravity of the mango oil is the density of the mango oil relative to the density of water. Both specific gravity values fall within the literature range of values as shown in Table 4.8.

CHAPTER FIVE

CONCLUSIONS AND RECOMMENDATIONS

Conclusions A study of the effect of extraction solvent and time on the yield and physicochemical properties of mango oil was carried out in this research work. The findings in this work can be summarized in the following statements. 1. The yield of mango oil depended on the two main factors (Extraction solvent and time of extraction). 2. The time factor was the major determinant of the mango oil yield. 3. Hexane gave a higher yield than Ethanol at all the extraction times. 4. The acid value and refractive index of the mango oil from Ethanol extraction were higher than those from the Hexane extraction. 5. The ester value from the Hexane extract was higher than that from the Ethanol extract. 6. Hexane extraction gives higher yield and better quality than Ethanol extraction.

Recommendations In the course of this study, various challenges were encountered. Thus for the future improvements on the extraction of mango oil in this study, the following recommendations have been made:

1. A milling machine should be available within the experimental environment so as to reduce impurities and contamination of the paste. 2. New and well calibrated heating mantles should be made available to increase accuracy and ease of soxhlet extraction.

31 3. Water supply should be consistent in all parts of the experimental environment to reduce congestion of work in a small part of the environment. 4. Gas chromatography for free fatty acid analysis should be available within the experimental environment so that analysis can be easily monitored.

BIBLIOGRAPHY 1. Batra P. K. and Jaggi, S. (1984). Indian Agricultural Statistics Research Institute. New Delhi. 2. Bird, S. R. (2009). A Healing Grove: African Tree Remedies and Tituals for the Body and Spirit. Chicago Review Press. 3. Fahimdanesh, M., & Bahrami, M. E. (2013). Evaluation of Physicochemical Properties of Iranian Mango Seed. International Food Research Journal. IPCBEE vol.53 (2013) © (2013) IACSIT Press, Singapore . 4. Kittiphoom, S. (2012). Utilization of Mango seed. International Food Research Journal 19(4): 1325- 1335 (2012). 5. Kittiphoom, S., & Sutasinee, S. (2013). Mango Seed Kernel Oil and its Physicochemical Properties. International Food Research Journal 20(3): 1145-1149 (2013). 6. Nielsen, S. S. (2010). Food Analysis. Springer Science and Business Media. 7. Nigam. (2007). Lab Manual in Biochemistry: Immunology and Biotechnology. McGraw Hill Education, New York 8. O'Brien, R. D. (2008). Fats and oils: Formulating and processing for applications. (3rd, Ed.) CRC press. 9. Olaniyan, & Oje. (2007). Quality Characteristics of recovered from Shea Kernel through dry Extraction Process. 10. Pomeranz, Y., & Meloan, C. E. (2002). Food Analysis: Theory and Practice. Springer Science and Business Media.

11. Preedy, V. R., Vatson, R. R., & Patel, V. B. (2011). Nuts and Seeds in Health and Disease Prevention. Academic Press. 12. Sivkishen. (2014). Kingdom of Shiva. Partridge India. 14. Sondhi, A. (2011). Wonders of India Trees. TERI press.

32 APPENDIX A: EXPERIMENTAL RUNS

Weight of Volume of Weight kernel Extraction Solvent(ml) of Yield(% RUN powder(g) Solvent Time Temperature oil(g) w/w)

1 30 Hexane 200 4 65 1.20 4.00

2 30 Hexane 200 5 65 2.00 6.67

3 30 Hexane 200 6 65 3.00 10.00

4 30 Hexane 200 7 65 3.70 12.33

5 30 Hexane 200 8 65 3.90 13.00

6 30 Ethanol 200 4 75 0.60 2.00

7 30 Ethanol 200 5 75 1.00 3.33

8 30 Ethanol 200 6 75 1.70 5.67

9 30 Ethanol 200 7 75 2.20 7.33

10 30 Ethanol 200 8 75 2.50 8.33 Observation: As the solvent contacted the kernel powder in the soxhlet, the colour of the liquid began to change. During the Hexane extraction, Hexane changed in colour from colourless to light yellow. During the Ethanol extraction, Ethanol also changed to light brown.

APPENDIX B: FORMULAE

1. Yield of oil =

2. Effect of factorial experiment = × 100% ∑ ∑ 3. The acid value is calculated mathematically as: − Acid value = × × .

Where, V, volume of KOH required to neutralize the solution (ml) N = strength of OH W = weight of oil used (g). The number 56.1 is the atomic weight of potassium hydroxide (KOH) 33 4. The saponification value is calculated mathematically as: Saponification value = × × . Where, Z = volume of HCL required to neutralize excess alkali (ml) Z = (X – Y) ml X = titre value of HCL against oil and KOH after reflux (ml) Y = titre value of HCL against KOH alone after reflux (ml) 5. Ester value = Saponification value – Acid value

APPENDIX C: CALCULATIONS

1. PERCENTAGE YIELD OF MANGO OIL Yield of oil =

a. RUN 1 × 100% Time = 4hours Solvent = Hexane Temperature = 650C Yield of oil = . b. RUN 2 × 100 = 4.00% Time = 5 hours Solvent = Hexane Temperature = 650C Yield of oil = c. RUN 3 × 100 = 6.67% Time = 6 hours Solvent = Hexane Temperature = 650C Yield of oil = d. RUN 4 × 100 = 10.00% Time = 7 hours Solvent = Hexane Temperature = 650C Yield of oil = . e. RUN 5 × 100 = 12.33% Time = 8 hours Solvent = Hexane Temperature = 650C Yield of oil = . f. RUN 6 × 100 = 13.00% 34 Time = 4 hours Solvent = Ethanol Temperature = 750C Yield of oil = . g. RUN 7 × 100 = 2.00% Time = 5 hours Solvent = Ethanol Temperature = 750C Yield of oil = h. RUN 8 × 100 = 3.33% Time = 6 hours Solvent = Ethanol Temperature = 750C Yield of oil = . i. RUN 9 × 100 = 5.67% Time = 7 hours Solvent = Ethanol Temperature = 750C Yield of oil = . j. RUN 10 × 100 = 7.33% Time = 4 hours Solvent = Ethanol Temperature = 750C Yield of oil = . × 100 = 8.33%

2. ABSOLUTE VALUE OF EFFECT OF FACTORS

Effect of factorial experiment = ∑ ∑ A (Time) = −

B (Solvent) = × 13 + 8.33 − 4 − 2 = 7.665

AB (Time and Solvent)× (8.33 =+ 2 − 13 − 4) = 3.335

3. ACID VALUE × (4 + 8.33 − 13 − 2) = 1.335 Hexane extracted

Acid value = × × .

35 Where, V = 1.0ml N = 0.1 W = 1.0g

Acid value = . × . × . Ethanol extracted . = 5.61 /

Acid value = × × . Where, V = 5.4ml N = 0.1 W = 1.0g

Acid value = . × . × . 4. SAPONIFICATION. VALUE= 30.30 / Hexane extracted:

Saponification value = × × . Where, Z = (X-Y) ml X = 38ml Y = 30.6ml Z = 7.4ml N = 0.5N W = 1g

Saponification value = . × . × . Ethanol extracted: = 207.0 /

Saponification value = × × . Where, Z = (X-Y) ml X = 38ml Y = 30.7ml Z = 7.3ml N = 0.5N

36 W = 1g

Saponification value = . × . × . 5. ESTER VALUE = 205.0 / Hexane extract: Saponification value – Acid value = (207 – 5.61) = 201.39 mgKOH/gOil Ethanol extract: Saponification value – Acid value = (205 – 30.3) = 174.70 mgKOH/gOil

6. SPECIFIC GRAVITY Hexane extract Mass of oil = 45.45g Volume of specific gravity bottle = 50cm3 Density of oil = (45.45/50)g/cm3 = 0.909g/cm3 Density of water @200C = 1g/cm3 Specific gravity = 0.909

Ethanol extract Mass of oil = 45g Volume of specific gravity bottle = 50cm3 Density of oil = (45/50)g/cm3 = 0.900g/cm3 Density of water @200C = 1g/cm3 Specific gravity = 0.900

37