View with images and charts Reports On Bangladeshi Fruits 1.1 Fruits Due to geographical situation, Bangladesh does not possess rich mineral resources and consequently the economy mostly depends on agriculture. The cultivable land is not enough to grow the required foodstuff to the vast number of people in the country. Plant kingdom has supported to safeguard the survival of the human being on earth from the very emergence of the civilization. The four basic needs of human life, i.e. food, clothing, shelter and medicine are obtained from plant kingdom. A major part of the global energy requirement is supplied by the plant source as fuel. In the view of a botanist, a fruit is the ripened ovary and sometimes the fleshy enlarged floral parts of a plant or herb. When a fruit is a ripened ovary, it is known as true fruit and when it is enlarged fleshy floral part, it is known as false fruit. Botanical or nutritional point of view fruits may or may not be edible. On the other hand, in general sense, a fruit is the ripened ovary or its associated parts, which can be eaten raw. Fruits were the first food for human being on the earth. In ancient times people survived on roasted meats and fresh fruits. In course of time and with the progress of civilization people have changed their food habit. Nowadays cooked food as well as fresh fruits is taken as diet. The fruits because of their importance in various aspects of human life attract the researchers very much. Fruits have been used from ancient time to alleviate the suffering of human beings. At the beginning of nineteenth century when modern chemistry and pharmacy began to develop, the original impetus to the study of natural products chemistry utilizing fruits and medicinal plants started to develop. In the recent
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View with images and charts
Reports On Bangladeshi Fruits
1.1 Fruits
Due to geographical situation, Bangladesh does not possess rich mineral resources and consequently the economy mostly depends on agriculture. The cultivable land is not enough to grow the required foodstuff to the vast number of people in the country. Plant kingdom has supported to safeguard the survival of the human being on earth from the very emergence of the civilization. The four basic needs of human life, i.e. food, clothing, shelter and medicine are obtained from plant kingdom. A major part of the global energy requirement is supplied by the plant source as fuel.
In the view of a botanist, a fruit is the ripened ovary and sometimes the fleshy enlarged floral parts of a plant or herb. When a fruit is a ripened ovary, it is known as true fruit and when it is enlarged fleshy floral part, it is known as false fruit. Botanical or nutritional point of view fruits may or may not be edible. On the other hand, in general sense, a fruit is the ripened ovary or its associated parts, which can be eaten raw. Fruits were the first food for human being on the earth. In ancient times people survived on roasted meats and fresh fruits. In course of time and with the progress of civilization people have changed their food habit. Nowadays cooked food as well as fresh fruits is taken as diet.
The fruits because of their importance in various aspects of human life attract the researchers very much. Fruits have been used from ancient time to alleviate the suffering of human beings. At the beginning of nineteenth century when modern chemistry and pharmacy began to develop, the original impetus to the study of natural products chemistry utilizing fruits and medicinal plants started to develop. In the recent decades antibiotics, vitamins and hormones are the follow up results of researchers in the chemistry of natural products.
Fresh fruits are nutritious and delicious food all over the world. They are ready source of energy with the unique capacity of guarding against many diseases caused by deficiency of nutritional ingredients. This is because fruits are the sources of various vitamins, carbohydrates, proteins, fats, many essential minerals and enzymes. They are medicinally very important and easily digestible. Hence, fruits are extremely beneficial to the human body for their normal growth and healthy condition. Fruits in the daily diet have been strongly associated with reduced risk for some forms of cancer, heart disease, stroke and other chronic diseases.
Fats and oils are found widely distributed in nature, in both the plants and animal kingdoms. Vegetables fats and oils occur predominately in seeds and fruits, but they are also found in
the roots, branches, stems and leaves of plants. In some seeds, as for instance in most cereals, fat occurs almost exclusively in the germ (embryo).
The formation of fat in the plant is obscure. The carbohydrate matter which is synthesized by the plant from CO2 and H2O is apparently converted into fat through various metabolic pathways. In seeds, only a very small amount of fat is present after the fall of the flower. As the seeds ripen there is an increase in fat and decrease in carbohydrate. It has also been shown that during the later phase of the development the instauration of the fat increase while the fatty acid content decreases.
1.2 Carbohydrate in Fruits
The ultimate source of all carbohydrates is plants, which built them from carbon dioxide and water, by photosynthesis.
Light
xCO2 + xH2 O (CH2O) x + O2
Chlorophyll Carbohydrate
Carbohydrates constitute one of the most important groups of natural products. Earlier, Carbohydrates were defined as compounds containing carbon, hydrogen and oxygen, the latter two elements being present in the same ratio as in water i.e. they were regarded as the hydrates of carbon and thus corresponded to the formula Cx(H2O)y, i.e. glucose C6H12O. But it is found that certain carbohydrates do not correspond to this formula i.e. rhamnose, C6H12O5; while several compounds although not carbohydrates, correspond so this formula, i.e. acetic acid C2H4O2. Now-a-days carbohydrates are defined as the optically active polyhydroxyaldehydes or ketones or substances that can be hydrolyzed to either of them.
Carbohydrates exist in plants and fruits either as polymers or as free sugars. They serve as source of energy (e.g. sugar) and as store of energy (e.g. starch and glycogen). Certain carbohydrates (e.g. cellulose) support the plant tissues while some others (e.g. chitin) form the major constituent of the shells crabs and lobsters. Sugars make fruits sweet and yield alcohol on fermentation; cellulose materials such as cotton, linen, jute, straw, grass, wood etc. supply clothes, plastics, lacquers, paints and explosives; ribose and deoxyribose are components nucleic acids (RNA & DNA) which determine the human heredity1.
1.3 Free Sugars
Free sugars are white crystalline carbohydrates that are soluble in water and generally have a sweet taste. In other words, free sugar is a generic term that includes a class of water-soluble carbohydrates with various degree of sweetness. Sugars are classified as monosaccharide & oligosaccharides. The monosaccharides are polyhydroxyaldehydes or ketones, which cannot be hydrolyzed to simpler sugars, e.g. glucose, fructose etc. The oligosaccharides yield two to ten monosaccharide molecule on hydrolysis, e.g. sucrose, maltose etc. Sugars are further classified as reducing and non-reducing sugars. Reducing sugars are those carbohydrates, which reduce Fehling solution or Tollens’ reagent. All
monosaccharide and disaccharides other than sucrose are reducing sugars. Non-reducing sugars are those carbohydrates, which do not react with Fehling solution or Tollens’ reagent. The non-reducing sucrose must first be inverted during hydrolysis, i.e. converted into a mixture of the two reducing sugars, glucose and fructose by dilute acid.
C12H22O11 + H2O = C6H12O6 + C6H12O6
Glucose Fructose
1.4 Sources of Free Sugars
The presence of free sugars in fruits, vegetables and other plant fractions are known for a long time. Most of the fruits, especially which are sweet contains substantial amounts of free sugars. Vegetables are usually not sweet in taste. So these may be considered to contain only small amounts of free sugars. With the increasing importance of dietary fiber in human, the fruits and vegetables, the main source of dietary fiber, have become even more important. The study of dietary fiber will remain incomplete without proper evaluation the free sugar contents of the corresponding fruit. Some representative and systematic studies of free sugars and dietary fiber analysis have been reported which discussed here. Studies of the free sugar content of guava, mango and kamranga, which grow in many tropical countries, had been reported2,3. Glucose, Fructose and Sucrose were identified and quantified as free sugars in these fruits.
The free sugar content of the tropical and sub-tropical fruits like banana, jackfruit, lichi, mango, papaya, wax-jambu and melon had been reported4. Glucose and Fructose were identified and quantified in almost equal amounts in most of the cases. Sucrose was also identified in all cases except in papaya and wax-jambu.
The free sugar content of the Indian pineapple had been reported5 and glucose, fructose and sucrose were identified as free sugars by gas chromatographic analysis.
Glucose, fructose, sucrose and myo-inositol were identified6 in almond, pecan and macadamia nuts. Traces of sorbitol were also detected in pecans and almonds.
The sugar composition and components of prunus persica fruit were identified and analysed7 by HPLC. Sucrose was the major component of free sugar. The other main sugars were glucose, fructose, sorbitol and myo-inositol.
The rapeseed meal was extracted with aqueous 80% ethanol and eleven low-molecular weight carbohydrates were identified by chromatographic separation8. Identified Carbohydrates were stachyose, raffinose, di-galactosyl glycerol, melibiose, sucrose, myo-inositol, glucose, glactitol, galactose and fructose. In this report the sugars were identified and quantified as their trimethylsilyl derivatives by means of GLC.
A comparison of the sugar content of four fruits (guava, mango, yellow passion fruit and purple passion fruit) was determined9 by GLC and Nelson-Somogyi’s method. Total sugars as determined by Nelson-Somogyi’s method were only slightly lower than the total sugars determined by GLC in the same fruits.
Sugar composition of Carica papaya during fruit development was determined10 by GLC. Sucrose made up less than 18% of the total sugar content 110 days after anthesis and increased rapidly to make up 80% of the sugars about135 days after anthesis.
Several low molecular weight carbohydrates were determined11 from the seeds of mung bean and chick bean. During germination, due mainly to their use as an easily available source of germinating energy, there was a rapid decrease of the raffinose family oligosaccharides in mung bean and a somewhat slower decrease in chick pea. Some growing fruits contain substantial amount of starch up to their maturity but the amount drastically falls below 1- 2 % on ripening12. The presence of stachyose, raffinose, sucrose and various monosaccharides in rapeseed meal was reported13 by chromatographic technique. A series of low molecular weight components were isolated14 form the dehulled and fat free meal of Brassica Compestris using chromatography on a carbon celite column, and carbohydrates composition of Brassica napus was reported15.
The low molecular carbohydrates from amlaki (Emblica officinalis) were analyzed. The presence of less than 1% of D-glucose, D–fructose and myo-inositol in the relative proportion of 1:1:3 was reported16.The low molecular weight components of seven Bangladeshi fruits such as mango, pineapple, guava, hogplum, kamranga, latkan and lukluki have been analysed17. The low-molecular weight sugars were isolated by extraction with aqueous 80% ethanol. It was found that the fruits contained glucose and fructose as the major sugar constituents along with myo-inositol.
Free sugar of some common fruits like, litchi, horbori, amloki, bangi, tarmuj, jamrul, kalojam, jalpai, karamcha and papaya have been reported18. Glucose and fructose were present as major sugar constituents including small amounts of sucrose in some of the fruits. Low molecular weight components were determined19 in stored tubers of three potato cultivars grown at four localities. Glucose (0.7-1.5%) and sucrose (0.7-1.2%) were the major components followed by fructose (0.1-0.8%), and myo-inositol (0.1-0.2%). In some samples negligible amount of galactose, maltose, melibiose, and raffinose were detected.
Glycerol, erythritol, threitol, arabinitol, xylitol, glucose, fructose and sucrose were and other free sugars present in varying amounts in the bank, stem and leaf in pigeon-pen (Cajanus cajon) plant20. Some low-molecular extractives from the source Pinus Silvestris were isolated21,22. In the hydrophilic extract, mainly carbohydrates and related cyclitols were found. The components included glucose, fructose, sucrose and shikimic acid in a total yield of 1.5-2.5 %.
Low molecular carbohydrates in the vegetables (E. Bean, L. Finger, Papaya, B. Gourd, Brinjal, W. Gourd and G. Banana) were analyzed23. Glucose was the main the constituent of the total polysaccharide of the vegetables but galactose was the major component of the soluble DF. The total free sugar, reducing sugar and non-reducing sugar of some Bangladeshi local fruits (Dab, Tarmuj, Komla, Malta, Pineapple, Wax apple, Blackberry, Burmese grape) were estimated24 by chemical method.
Glucose and fructose were the only two free sugars identified and quantified25. The total free sugars of litchi, mango, guava and pineapple were 11.3%, 10.1%, 6.0% and 8.8% respectively. Free sugars of some local vegetables (potol, karela, fulkopy, badhakopy, chichinga, gajor, shalgom, chalkumra) were identified and quantified26. Glucose content in any vegetables was 2-3 times more than the fructose content.
1.5 Fatty Acids
Fats are esters of long chain fatty acids and alcohols. The ester linkages of the fats are cleaved by NaOH to yield glycerol and sodium salts of long chain fatty acids (soaps).
CH2-O-CO-R1 CH2OH R1COONa
CH-O-CO-R2 + 3 NaOH CHOH + R2COONa
CH2-O-CO-R3 CH2OH R3COONa
Fat Glycerol Soap
The backbone of these compounds contains from 4 to more than 20 carbon atoms. Most natural sources of these compounds have an even number of carbon atoms because the biosynthetic pathway builds the backbone two carbons at a time. Fatty acid chains may contain one or more double bonds at specific positions (unsaturated and polyunsaturated), or they may be fully saturated. The physical and chemical properties of a fat depend on the composition of the fatty acid mixture. Animal fats tend to have a larger proportion of long chain saturated acids and are solids at room temperature. Fats from plant sources contain a higher proportion of unsaturated acids and are often liquids at room temperature due to hydrogen bonding. These fatty materials may influence the handling of the plant tissues as well as any chemical treatment done on it. Therefore, the study of fatty acids and the major constituent of all fatty matters are important. Poly unsaturated fats are usually of vegetable origin. Crisco is an example of a vegetable-derived, unsaturated fatty acid that has been hydrogenated to form a solid material. Fats are used in cooking because they are very high boiling compounds. The great numbers of the naturally occurring fatty acid belong to a few homologous series. The general formula of the fatty acids is CnH2nO2 may represent the series to which stearic acid belongs. As, however, their functional group is the carboxyl group, ─COOH, they are more conveniently expressed as CnH2n+1COOH, since these show the nature of the functional group.
1.6 Sources of Fatty Acids
The fatty acids occur in nature usually have straight chains and contain even number of carbon atoms. Fatty acids occur in plants in bound27 form as fats or lipids. Fats are the triglycerides of fatty acids of the same type or of the different types and yield fatty acids upon hydrolysis. Lipids are defined by their special solubility properties and are comprised of different kinds of compounds. These lipids comprise up to 7%27 of the dry weight in leaves in higher plants, about 1-5% in stems of green plant and are important as membrane constructs in the chloroplasts and mitochondria.
Lipids also occur in considerable amounts in the seeds or fruits of a number of plants. Although numerous fatty acids are now known in plants, the palmitic acid (C-16) is the major saturated acid21 in leaf and also occurs in varying quantities in some seed oils. Stark acid (C-18) is the major saturated acid in seed fats of a number of plant families27. Unsaturated acids (mainly C-16 and C-18) are widespread in both leaf and seed oils. A number of rare fatty acids (e.g.erucic and sterculic acid) are found in seed oils of a few plants.
Eight vegetables namely, Tricosanthes dioica, Daucus carota, Brassica campestris var. turnip, Brassica oliracea var. botrytis, Brassica olracea var. capitata, Momordica charantia, Benincaca cerifera and Trichosanthes anguina were analyzed28 for fatty acids
content and composition. Lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, arachidic acid and behenic acid were present in varying amounts (3.68-34 mg / 100 g in fresh vegetables) in the lipid part of the vegetables.
The constituent of fatty acids of some of oils were analyzed29 by gas chromatographic techniques. The major portions are short as well as long chain-saturated fatty acids like capric acid, lauric acid, myristic acid, palmotelic acid and palmitic acid. Few unsaturated fatty acids like arachidic acid, linolenic acid and oleic acid were also identified in coconut oil. The major proportion of long chain saturated fatty acid with small proportion of unsaturated one like palmitic acid and linoleic acid, respectively were identified in palm oil.
1.7 Scientific Classification
Kingdom: Plantae
Division: Magnoliophyta
Class: Eudicotyledoneae
Subclass: Rosidae
(Unranked): Eurosids I
Order: Oxalidales
Family: Elaeocarpaceae
Genus:ElaeocarpusSpecies: Elaeocarpus robustus
Binomial Name – Elaeocarpus robustus
Table 1: Scientific, English and Local Names of the Native Olive
Local Name English Name Scientific Name
Jalpai Olive Elaeocarpus robustus
1.8 General Description of Elaeocarpus
Elaeocarpus30, 31, 32 is a genus of tropical and subtropical evergreen trees and shrubs. The approximately 350 species are distributed from Madagascar in the west through India, Southeast Asia, Malaysia, southern China, and Japan, through Australia to New Zealand, Fiji, and Hawaii in the east. The islands of Borneo and New Guinea have the greatest concentration of species. These trees are well-known for their attractive, pearl-like fruit which are often colorful. Many species are threatened, in particular by habitat loss.
Olives in our country are not almost same to the foreign olives. There are many varieties of olives all over the world. Our native olive is Elaeocarpus robustus. Elaeocarpus robustus L. (Fam. Elaeocarpaceae) is a well-known evergreen 25 m tall fruit tree. It is native to Bangladesh and India. The olive tree has been cultivated for olive oil, fine wood, olive leaf and olive fruits. The native olive plant is believed to have originated in Australia; however, it is well grown in Bangladesh. It is cultivated in all districts of Bangladesh and occurs wild in the evergreen forest of Sylhet and Chittagong. The importance33 of fleshy sour fruits having citric acid occupies an important position in tropical countries since they provide needed vitamin-C in diets.
The native olive fruit has several uses as food adjuncts for human being. The fleshy ripe fruit is delicious, which is eaten raw or cooked and pickled. The plant is also important for its therapeutic uses. Leaves are used in rheumatism and as an antidote to poison and are considered as a cure for gonorrhea. Fruit is tonic, emmenagogue, appetizer, useful in biliousness, liver complaints, scabies, burning of the eyes, carries of the teeth, toothache etc. and prescribed in dysentery and diarrhea33.
Olives are a naturally bitter fruit that is typically subjected to fermentation or cured with lye or brine to make it more palatable. Green olives are typically washed thoroughly in water to remove oleuropein, a bitter carbohydrate34. Sometimes they are also soaked in a solution of food grade sodium hydroxide in order to accelerate the process. The green fruits are eaten fresh and also used in making soup, chutney, jelly and jams. Elaeocarpus robustus tree produce fine textured, moderately hard and strong wood which takes good finish and fitting with good working properties. The swan wood has been used better in parquet flooring. It is also used as suitable wood in making small furniture and musical instruments. Wood has several important industrial uses as fuel and to prepare some form of essential equipments such as match splints and boxes, mathematical instruments, packing cases and boxes
Olive trees like hot weather34 and temperatures below 140 C may injure even a mature tree. They tolerate drought well. They show a marked preference for calcareous soils, flourishing best on limestone slopes and crags, and coastal climate conditions. They grow in any light soil, even on clay if well drained, but in rich soils, they are predisposed to disease and produce poorer oil than in poorer soil. They are commonly grown from seeds, which are recalcitrant and difficult to germinate even after a short period of storage. The species is predominantly cross-pollinated leading to high seedling variability. Because of seed propagation, the plant qualities vary widely among the individuals33.Soup of the fruit is also given for stimulating secretion from the test buds. Etanolic extract of leaves are diuretic and cardiovascular stimulant34.
Considerable research supports the health-giving benefits of consuming olives, olive leaf and olive oil. Olive leaves are used in medicinal teas. Olives are now being looked at for use as a renewable energy source, using waste produced from the olive plants as an energy source that produces 2.5 times the energy generated by burning the same amount of wood. The smoke released has no negative impact on neighbours or the environment, and the ash left in the stove can be used for fertilizing gardens and plants. The process has been patented in the Middle East and the US35.
1.10 Strive of the Work
In the present world, natural resources are contributing a significant role to the economic upliftment of a country and thus paved the way for the development in the fields of
education and technology, which ultimately brought the special prosperity. Therefore, it is inevitable that major efforts should be concentrated on the proper utilization of natural resources. In general, natural resources may be broadly classified into agricultural and mineral resources. With a view to achieving the maximum utilization it is imperative to make a thorough survey of the agricultural and mineral resources.
Fruits as well as plant kingdom are directly associated with the lives and livings of human being and a little known about the chemical composition of these fruits, studies on the isolation, identification, quantification and characterization of the medicinally active compounds from them are very important for the well-being of human society.
Figure 2: Laboratory work during sample preparation
Fruits are the important source of nutrient and energy. In primitive days people used to take fresh fruits from their instinct and without knowing their nutritional value but in modern days with the advancement of food science, people know the nutritional value of fruits and their uses is on increase. Fruits are delicious and nutritious food in terms of calories, vitamins, minerals and other nutrients. Nevertheless, most of fruits of our country are seasonal fruits. Olive is one of them. The olives that are grown in our country are different from alien olives. Vast research has not been done in our native olive as to why I took native olive as my subject of research.
Elaeocarpus robustus locally known as Jalpai, is a well known fruit that yields during November-December. Green fruits are sour and cooked by the rural people to make various types of soups, chutneys, jellies and jams. In addition, it has a medicinal value. The fruit pulp is rich in vitamin C and citric acid.
Carbohydrates are the principal primary metabolite widely distributed in nature. The major constituent of Elaeocarpus robustus fruit is carbohydrate. In the present investigation on the carbohydrates of this fruit was undertaken.
Fatty acids, important for various purposes occur in plants, fruits and animals. The fatty tissues of animal contain large amounts of long chain saturated fatty acids. Plants contain higher proportion of unsaturated fatty acids. These fatty acids may influence the handling of the plant tissues as well as any chemical treatment done on it. Therefore, the study of fatty acids as well as the major constituents of all fatty matters is very important.
According to the data of the nutritional values, an extended and reliable analysis has been carried out to understand the composition and presence of several kinds of nutrients. The following experiments have been done related to this analysis.
1. Determination of moisture content
2. Determination of ash content
3. Analysis of free sugar
(i) Identification of free sugar by PC
(ii) Quantification of free sugar by chemical methods
(iii) Identification as well as quantification of free sugars by GC
4. Identification and quantification of fatty acids by GC.
The edible part of olive is skin and pulp. So all the cases, seeds were omitted for different type of analysis.
2.1 Materials and Methods
All the chemicals and reagents used in the experiments were analytical grade and procured from Sigma, E. Merck (Germany) and BDH (England).
2.2 Solvents
Solvents used in different experiments were ethanol, methanol, n-hexane, dichloromethane (DCM), petroleum ether etc.
2.3 Solid Reagents
Anhydrous sodium sulphate was freed from interfering organic substances and moisture by heating at 4000 C for at least 4 hours. Pure silica sand was also used.
2.4 Liquid Reagents
Borontrifluoride– Methanol complex solution of‘Merck Schuchardt’ was kept refrigerator and a definite amount of the solution was withdrawn for each hydrolysis.
2.5 Standard Reference
Tetramethylsilane, (CH3)4Si, also called TMS is used universally as standard reference substance for sugar analysis.
2.6 Distillation of Solvents
Analytical grade solvents (ethanol, DCM, petroleum ether etc.) were distilled before use. Petroleum ether (b. p. 400 – 600 C & 600 – 800 C) was obtained by distillation.
2.7 Evaporation
All evaporations were carried out under reduced pressure at bath temperature not exceeding 400 C to avoid decomposition of the samples.
2.8 Electric Oven
All glass apparatus were dried and anhydrous sodium sulphate was stored inside an oven (Memmerat)
2.9 Freeze-Drying
Freeze drying were carried out by HETOSIC CD 52 (Hetolab Equipment,Denmark) freeze-dryer. Aqueous extracts and fractions were first frozen in round bottomed flasks in an ethanol freezer (Hetofrig cd 5, Hetobirkero, Denmark) at -30 0c to -40 0C and finally the materials were subjected to freeze-drying operation.
2.10 Standard Solutions
2.10.1 Standard Derivatives of Free Sugars
Standard derivative mixtures of sugar solutions of ‘Sigma’ were prepared. These sugar mixtures were injected in GC.
2.10.2 Standard Derivatives of Fatty Acids
Standard derivative mixtures of fatty acid solutions of ‘Sigma’ were prepared. These fatty acid mixtures were injected in GC as the standard acid mixture.
2.10.3 Preparation of Methanolic 0.5 M KOH Solution
Methanolic KOH solution was prepared for hydrolysis of the oil. 7.1311 g KOH was taken in a 250 mL and made it up to the mark to prepare 0.5 M KOH in MeOH was prepared.
2.11 Refluxing
For the esterification of fatty acids a refluxing system was employed. The system consists of a condenser and a pear shaped flask being placed on a hot water bath.
2.12 Determination of Ash Content
The most widely used methods are based on the fact that minerals are not destroyed by heating, and that they have a low volatility compared to other food components. The three main types of analytical procedure used to determine the ash content of foods are based on this principle: dry ashing, wet ashing and low temperature plasma dry ashing. The method chosen for a particular analysis depends on the reason for carrying out the analysis, the type of food analyzed and the equipment available.
Typically, samples of 1-10g are used in the analysis of ash content. Solid foods are finely ground and then carefully mixed to facilitate the choice of a representative sample. Before carrying out an ash analysis, samples that are high in moisture are often dried to prevent spattering during ashing. High fat samples are usually defatted by solvent extraction, as this facilitates the release of the moisture and prevents spattering. Other possible problems include contamination of samples by minerals in grinders, glassware or crucibles, which come into contact with the sample during the analysis. For the same reason, it is recommended to use deionized water when preparing samples36.
There are a number of different types of crucible available for ashing food samples, including quartz, Pyrex, porcelain, steel and platinum. Selection of an appropriate crucible depends on the sample being analyzed and the furnace temperature used. The most widely used crucibles are made from porcelain because it is relatively inexpensive to purchase, can be used up to high temperatures (< 1200oC) and are easy to clean. Porcelain crucibles are resistant to acids but can be corroded by alkaline samples, and therefore different types of crucible should be used to analyze this type of sample. In addition, porcelain crucibles are prone to cracking if they experience rapid temperature changes. A number of dry ashing methods have been officially recognized for the determination of the ash content of various foods (AOAC37 Official Methods of Analysis). Typically, a sample is held at 500-600 ºC for 24 hours.
2.13 Chromatographic Method
The present experiments were done by two types chromatographic methods.
2.13.1 Paper Chromatography (PC)
Paper chromatography38 is an analytical technique for separating and identifying mixtures that are or can be coloured, especially pigments. This can also be used in secondary or primary colours in ink experiments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography , involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.
Paper chromatography
Chromatography jar
Figure 3: A paper chromatogram
A small concentrated spot of solution that contains the sample of the solute is applied to a strip of chromatography paper about two centimetres away from the base of the plate, usually using a capillary tube for maximum precision. This sample is absorbed onto the paper and may form interactions with it. Any substance that reacts or bonds with the paper cannot be measured using this technique. The paper is then dipped into a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container.
The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper; this can also be explained as differential adsorption of the solute components into the solvent. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibres in the paper. The more soluble the component the further it goes. Paper chromatography takes anywhere from several minutes to several hours.
In some cases, paper chromatography does not separate pigments completely; this occurs when two substances appear to have the same values in a particular solvent. In these cases, two-way chromatography is used to separate the multiple-pigment spots.
(1) Ascending paper chromatography
In this method, the solvent is in pool at the bottom of the vessel in which the paper is supported.
(2) Descending paper chromatography
In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper. The liquid moves down by capillary action as well as by the gravitational force, thus this method is also known as the gravitational method. In this case, the flow is more rapid as compared to the ascending method, and the chromatography is completed more quickly. The apparatus needed for this case is more sophisticated. The developing solvent is placed in a trough at the top which is usually made up of an inert material. The paper is then suspended in the solvent. Substances that cannot be separated by ascending method can sometimes be separated by the descending method.
(3) Rƒ value
Rƒ value may be defined as the ratio of the distance travelled by the substance to the distance travelled by the solvent. Rƒ values are usually expressed as a fraction of two decimal places but it was suggested by Smith that a percentage figure should be used instead.
Distance (cm) traveled by solute
Rf =
Distance (cm) traveled by solvent
Usually, the Rf value is constant for any given compound and it corresponds to a physical property of that compound.
In the present experiment, descending paper chromatographic method was applied.
2.13.2 Gas Chromatography
Gas-liquid chromatography38 (GLC), or simply gas chromatography (GC), is a common type of chromatography used in analytic chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture. In general, substances that vaporize below ca. 300 °C (and therefore are stable up to that temperature) can be measured quantitatively. The samples are also required to be salt-free; they should not contain ions. Very minute amounts of a substance can be measured, but it is often required that the sample must be measured in comparison to a sample containing the pure, suspected substance.
In gas chromatography, the moving phase (or "mobile phase") is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column. The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, and the temperature. The instrument used to perform gas chromatography is called a gas chromatograph (or "aerograph", "gas separator").
In a GC analysis, a known volume of gaseous or liquid analyte is injected into the "entrance" (head) of the column, usually using a micro syringe. As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times (retention time).
A detector39 is used to monitor the outlet stream from the column; thus, the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified (qualitatively) by the order in which they emerge (elute) from the column and by the retention time of the analyte in the column. Before starting, the detector and chart recorder must be zero.
Fig 4: A gas chromatograph with a headspace sampler
The column(s) in a GC are contained in an oven, the temperature of which is precisely controlled electronically. The rate at which a sample passes through the column is directly proportional to the temperature of the column. The higher the column temperature, the faster the sample moves through the column. However, the faster a sample moves through the column, the less it interacts with the stationary phase, and the less the analytes are separated. In general, the column temperature is selected to compromise between the length of the analysis and the level of separation.
Generally chromatographic data is presented as a graph of detector response (y-axis) against retention time (x-axis), which is called a chromatogram. This provides a spectrum of peaks
for a sample representing the analytes present in a sample eluting from the column at different times. Retention time can be used to identify analytes if the method conditions are constant. In addition, the pattern of peaks will be constant for a sample under constant conditions and can identify complex mixtures of analytes.
Quantification is possible in GC methods by analyzing the peak area or peak height. A larger peak area indicates a larger amount of analyte present in the sample. The area under a peak is proportional to the amount of analyte present in the chromatogram. By calculating the area of the peak using the mathematical function of integration, the concentration of an analyte in the original sample can be determined. Concentration can also be calculated using a calibration curve created by finding the response for a series of concentrations of analyte, or by determining the relative response factor of an analyte. The relative response factor is the expected ratio of an analyte to an internal standard (or external standard) and is calculated by finding the response of a known amount of analyte and a constant amount of internal standard (a chemical added to the sample at a constant concentration, with a distinct retention time to the analyte). In most modern GC-MS systems, computer software is used to draw and integrate peaks, and match MS spectra to library spectra.
In the present experiment, free sugars and fatty acids were analyzed with the help of GC.
3.1 Collection of Native Olives
Fruits being good quality, green and fresh were procured from a local market of Dhaka city in 2009. Only fresh fruits in prime condition can produce a good quality dried product. Wilted ones were not used. One moldy bean may give a bad flavor to all the lot. Carefully sorted, discarding any bruised or undesirable products and then washed carefully and thoroughly in cool water it was kept pieces uniform in size so they dry in same rate. A fruit slicer or food processor can be used. As many fruits as can be dried at one time were prepared. Holding fruits, even in the refrigerator, after washing and preparation for drying will result in loss of quality and nutrients. Only the edible parts of the fruits were taken for analytical purposes. In the present work, the seeds of the fruits were manually removed from the edible parts.
3.2 Determination of Moisture Content
Fruits can be preserved by drying. The longer the drying time, the less flavorful and the less tender the products are. The drying time can be hastened by drying small, uniformly cut pieces. Fruits are dried until brittle. At this stage, only 10 percent moisture remains and no microorganisms can grow. Controlled Moisture (CM) in fruits combine superior performance and concentrated nutrients with vivid color and exceptional flavor. CM fruits deliver better performance in frozen, fresh and refrigerated foods because they have less water content. Pizza crusts and dough formulae stay crisper, sauces, dips and spreads stay true to their original flavor and consistency; and dishes maintain appealing texture without being soggy. With lower moisture and higher solid content, CM fruits are nutritionally dense. In fact, it takes 33% less CM fruits in application to deliver one fruits serving as compared to fresh and individually quick frozen fruits40.
A known amount of fresh fruit was taken in a dry, cleaned and weighed watch glass. Then it was dried at 1050 C in an electric oven. The watch glass was cooled in a desicator and
weighed. The drying and cooling were repeated until a constant weight was obtained. The moisture content was calculated and presented in table 3.
Table 3: Relative Amount of Moisture Content
No of observation
Weight of samples
(g)
Weight of samples after 72 hours heating in oven (g)
Weight of
Moisture(g)
% of
Moisture
Mean % of
Moisture
1. 1.0212 0.1614 0.8598 84.20
84.132. 1.0231 0.1575 0.8656 84.60
3. 1.0006 0.1642 0.8364 83.60
3.3 Determination of Ash content
Recently, analytical instruments have been developed to dry ash samples based on microwave heating. These devices can be programmed to initially remove most of the moisture (using a relatively low heat) and then convert the sample to ash (using a relatively high heat). Microwave instruments greatly reduce the time required to carry out an ash analysis, with the analysis time often being less than an hour. The major disadvantage is that it is not possible to simultaneously analyze as many samples as in a muffle furnace40.
The ash content is the percentage of inorganic residue remaining after ignition of sample. A weighed quantity of sample of Elaeocarpus robustus was taken in a crucible. The contents were heated carefully to the ignition point and allowed to burn spontaneously. When burning was completed, the crucible heated at a dull–red heat in a muffle- furnace at 600 0 C for three hours. The crucible was then cooled and weighed with accuracy.
3.3.1 Calculation of Ash
% of ash =
Where, M1=Initial weight of sample in g and M2 = Weight of the ash in g
The results of the determination of the amount of ash content of the Elaeocarpus robustus have been calculated and given in the following table 4.
Table 4: The Relative Amount of Ash Content
Weight of empty crucible (g )
Weight of
Crucible +sample ( g )
Weight of
Sample
M1 ( g )
Weight of
Crucible + ash
( g )
Weight of ash
M2 ( g )
% of ash
27.4863 28.0454 1.2910 27.5343 0.0480 3.72
3.4 Extractions
10 pieces of Elaeocarpus robustus fruits were taken and weighed that was found to be 303.55 g. After slicing and chopping into the fruits, the edible parts (259.19 g) were extracted with aqueous 70 – 80 % ethanol (900 mL) by refluxing for 45 minutes on a metal heater. The insoluble residue was then filtered off, air-dried, powered and re-extracted with aqueous 70 – 80 % ethanol (500 mL X 2) by refluxing for 45 minutes on a metal heater. In each case, the volume of ethanol to be added was calculated by taking into consideration. The ethanolic extracts were then combined and the ethanol was removed by evaporation with added water. The resulting aqueous solution was partitioned with dichloromethane (2 X 200 mL) in a separatory funnel. The amount of aqueous layer and organic layer were found to be 388 mL and 274 mL respectively. The aqueous layer was taken and water was liberated under reduced pressure. This layer contained the low molecular weight carbohydrates. The aqueous layer was divided into two parts. One i.e. 20 mL was concentrated and freeze-dried. Another friction i.e. 40 mL was taken into quantitative study for free sugar analysis by chemical method.
3.4.1 Extraction Scheme of Elaeocarpus Robustus
Elaeocarpus robustus (259.19 g)
Extracted with C2H5OH by boiling 45 minutes
Residue Ethanol extract
Added distilled H2O
Water extract
Partitioned with DCM
Aqueous layer (60 mL) Organic layer
Divided into two parts
Freeze-dried (20 mL) aqueous part (40 mL)
Scheme 1: Extraction of Elaeocarpus Robustus
3.5 Analysis of Free Sugars
3.5.1 Identification and Detection of Free Sugars by Paper Chromatography
3.5.1.1 Preparation of Reagents for the Development of Paper Chromatograms
(i). Silver nitrate solution:
Saturated aqueous solution of silver nitrate (1 mL) was mixed with acetone (200 mL) and water was added drop wise until the precipitate formed was re-dissolved.
(ii). Sodium hydroxide solution:
Aqueous alcoholic sodium hydroxide solution (2%) was prepared by dissolving sodium hydroxide (4 g) in water (100 mL) then diluted with ethanol (200 mL).
(iii). Sodium thiosulphate solution:
Aqueous sodium thiosulphate solution (10 %) was prepared by dissolving sodium thiosulphate (40 g) in water (400 mL).
3.5.1.2 Development of Paper Chromatograms
The irrigated papers were dried in the air and the sugars were located on paper by dipping in or spraying with one of the following reagent systems:
(a) The paper was dipped in (i), dried and soaked with dip (ii).The developed chromatograms were then washed with dip (iii), followed by water and finally dried in the air.
(b) An alcoholic (0.1 M) solution of p-anisidine and phthalic acid, followed by heating at 1000 C for 10 minutes.
(c) An alcoholic (1 %) solution of oxalate followed by heating at 1000C for 10 minutes.
3.5.1.3 Detection of Sugars
The dried DCM – extracted aqueous layer was dissolved in water. It was spotted on Whatmann No. 1 paper separately, the paper was allowed to run 24 hours in the solvent system consisting of n- Butanol : ethanol : water (40: 11 : 19). After removing the papers from the tank, the chromatograms were developed.
Black spots are observed and marked by encircling with a pencil and labeling them.
HOCH2(CHOH)4CHO + AgNO3 Ag (s) + HOCH2(CHOH)4COONa
3.5.1.4 Calculation of Rf Values of Free Sugars by PC
Rf value of Glucose =
= 0.41
Rf value of Galactose =
= 0.45
Rf value of Arabinose =
= 0.50
Table 5: Identification of Sugar Components by Paper Chromatography
Sugar components Rf value
Glucose 0.41
Galactose 0.45
Arabinose 0.50
3.5.2 Quantification of Free Sugars by Chemical Method
The reagents used in this work were made by the following procedures:
A. R copper pentahydrate (34.64 g) crystals were taken in a beaker and distilled water (containing a few drops of dilute sulphuric acid) was added to it. The solution was then diluted to 500 mL.
Pure sodium hydroxide (60 g) and pure Rochelle salt (sodium potassium tartrate) (173 g) was taken in a beaker and water was added. Then the solution was filtered through a sintered glass funnel and made up the filtrate and washings to 500 mL. Then these two solutions were kept separately in tightly stopper bottles and mixed exactly equal volumes immediately before use.
3.5.2.3 0.5 M Sodium Hydroxide Solution
Solid Sodium hydroxide (5.23 g) was taken in a volumetric flask (250 mL) and made upto the mark with distilled water.
3.5.2.4 0.5 M Hydrochloric Acid
The supplied concentrated hydrochloric acid was 12 M (36 %, density-1.18 g/mL). 4.20 mL of this acid was taken in a volumetric flask (100 mL) and made upto the mark with distilled water.
3.5.2.5 Methylene Blue Indicator
3.5.2.6 Preparation of Standard Glucose Solution
Dry A.R glucose (1.26 g) was taken by accurate weighing in a volumetric flask (100 mL). Then the glucose was dissolved by distilled water and made upto the mark with distilled water.
3.5.2.7 Standardization of Fehling Solution with Standard Glucose Solution
To standardize the Fehling solution weighed out accurately 0.5 g of A.R glucose. Then it was dissolved in water and diluted to 100 ml in a volumetric flask. 10.0 ml of supplied Fehling solution was transferred in to a 250 ml conical flask and diluted with 100 ml of water. It was heated to boiling and then added glucose solution from a burette until the blue colour of the solution just disappeared. This gave an approximate value of the volume of glucose solution required. To obtain the exact volume, repeated the titration and added so much of glucose solution that 0.5 to 1.0 ml required to completed the reduction. The liquid was heated to boiling, maintained the gentle boiling for two minutes added 3 to 5 drops of ethylene blue indicator. The titration was completed in one minute by adding the glucose solution drop wise until the methylene blue colour just disappeared and it was repeated until the constant values were obtained41. The result was presented in table-6.
Table 6: Standardization of Fehling Solution with Standard Glucose Solution
Weight of A.R glucose was taken = 0.5304 g
No of Observation
Volume of Fehling Solution (mL)
Initial Burette reading (mL)
Final burette
reading (mL)
Difference
(mL)
Mean (mL)
1
20
0.0 18.60 18.60
18.602 18.60 37.20 18.60
3 0.00 18.60 18.60
3.5.2.8 Calculation100 mL standard glucose solution was contained 0.5304 g glucose (as prepared).
18.6 mL standard glucose solution ≡20 mL Fehling solution
0.5304 ×18.60
= g glucose
100
=0.0986 g glucose.
20 mL Fehling’s solution ≡ 0.0986 g of glucose.
3.5.2.9 Determination of Reducing Sugars
The supplied sample was taken in a burette (50.00 mL). Fehling’s solution (20.0 mL) was taken in a conical flask (250 ml) and an equal amount of water was added to it. The solution was heated to boil for about 2 minutes. Then the sample was added slowly to the solution from burette until the blue colour of the solution just disappeared. The solution was then
boiled for another 2 minutes. Then 2- 4 drops of methylene blue indicator was added to the solution and titration was continued for the complete disappearance of the blue color of the solution. The titration was repeated until consistent values were obtained.
Table 7: Determination of the Reducing Sugars
No. of Observation
Vol. of Fehling Solution /(mL)
Initial Burette Reading/ (mL)
Final burette
Reading/(mL)
Difference
(mL)
Mean (mL)
1
20
0.00 14.00 14.00
14.002 14.00 28.00 14.00
3 0.00 14.00 14.00
3.5.2.10 Calculation of Reducing Sugars
20 mL Fehling’s solution = 0.0986 g glucose.
So, 14 mL extract contains 0.0986 g glucose.
100 mL extract contains 0.7043 g glucose.
i.e. 40 mL extract contains 0.7043 g glucose.
60 mL extract contains 1.0565 g glucose.
So, 259.19 g olive sample contains 1.0565 g glucose.
100 g olive sample contains 0.4076 g glucose.
Therefore, the amount of reducing sugar is 0.4076 g / 100 g.
3.5.2.11 Determination of Total Free Sugars
A known amount (25 mL) of sample solution was taken in a 100 mL volumetric flask. Diluted the sample with distilled water and made up to the mark of the volumetric flask. Then exactly 25 mL solution was taken from this diluted solution and added dilute HCl solution (0.5 M, 20 mL). The solution was heated about half an hour on a boiling water bath. Cooled the solution and neutralized by adding with dilute NaOH Solution (0.5M). This solution was transferred quantitatively in 100 mL volumetric flask and made up to the mark with distilled water.
Measured amount of Fehling solution (20 mL) was titrated with the sample solution as described in sec. 3.5.2.7 and then the content of free sugar present in the sample was determined and the result was given in table 8.
Table 8: Determination of the Total Free Sugars Content
No of Observation
Volume of Fehling Solution
(ml)
Initial Burette Reading
(ml)
Final burette
Reading
(ml)
Difference
(ml)
Mean (ml)
1
20
0.00 28.90 28.90
28.902 28.90 47.80 28.90
3 0.00 28.90 28.90
3.5.2.12 Calculation of Total Free Sugars
28.90 mL hydrolysed extract ≡20 mL Fehling’s solution ≡0.0986 g glucose
100 mL hydrolysed extract contains 0.3412g glucose
100 mL hydrolysed extract contains 25 mL original extract, so 25 mL extract contains 0.3412 g of glucose.
100 mL hydrolysed extract contains 1.3648 g glucose.
i.e. 100 mL hydrolyzed extract contains 1.3648 g total sugars.
So, 40 mL olive extract contains 1.3648 g total sugars.
60 mL olive extract contains 2.0472 g total sugars.
So, 259.19 g olive sample contains 2.0472 g total sugars.
100 g olive sample contains 0.7899 g total sugars.
Reducing sugar obtained 0.4076 g, so inverted sugar obtained =0.7899 - 0.4076 = 0.3823 g.
Now 360 g inverted sugar = 342 g non–reducing sugar.
0.3823 g inverted sugar = 0.3632 g non–reducing sugar.
Total sugars = (reducing sugar + non – reducing sugar)
= 0.4076 g + 0.3632g = 0.7708 g.
Therefore, the amount of total free sugars is 0.7708 g /100 g.
10 pieces of fruits contain 0.7708 g total free sugars; so a piece fruit contains 0.077 g total free sugars.
3.5.2.13 Determination of Non-Reducing Sugars
The value of non-reducing sugar was determined by subtracting the value of reducing sugar from the total free sugar and the results of the olive were given in table –9.
Table 9: Content of Total, Reducing and Non-Reducing Free Sugars
Fresh weight basis (%)
Total Sugars
(%)
Reducing Sugars
(%)
Non reducing sugars
(%)
0.77 0.41
0.36
3.5.3 Identification and Quantification of Free Sugars by Gas Liquid Chromatography
All sugars analyses were performed on GLC following Sweely et.al42. Evaporations of GLC samples were carried out under reduced pressure at below 400 C. For gas chromatographic analysis a PYE UNICAM 4500 (FID detector) GLC analyzer connected with a LKB 2220 recording integrator was used. Separations were performed on (i) CP Sil 5 WCOT quartz capillary column at 1850 -2100 C, 20 C per minute for trimethylsilyl derivatives and (ii) CP Sil 88 WCOT quartz capillary column at 1700 – 2200 C, 40 C per minute for alditol acetates.
3.5.3.1 Procedure of TMS- Derivatives for GC – MS Analysis
(ii) 20 µL of filtrate was taken to a pear shaped flask
(iii) It was evaporated to dryness with addition of 1 mL MeOH (3 times)
(iv) Added 1mL dry pyridine and vortex / sonication for 1 minute
(v) It was added silylating reagent to the dry extract
(vi) It was shaken well or vortex to mix and incubate at 700 C for 45 minutes
(vii) It was evaporated into dryness until pyridine free
(viii) 1 mL n–Hexane was added to dry mass vortex, filtered and then transferred into a vial
(ix) Ready for injection on GC- MS.
3.5.3.2 Gas Chromatographs Identification and Determination
1.0 µL sample was injected through a filter into the injector of GC-MS at the same condition the sugar standard mixture solutions were injected. Comparing the retention times of the different peaks in the chromatogram of the sample with those of standards, different sugars were identified.
3.5.3.3 Identification and Quantification Scheme of Free Sugars by GC
Freeze dried of Elaeocarpus robustus (from section 3.4.1)
Added n-hexane
20 µL was taken in a pear shaped flask
Evaporated to dryness with addition of 1 mL MeOH (3 times)
Added 1mL dry pyridine
Vortex / sonication for 1 minute
Added silylating reagent to the dry extract
Shaken well or vortex
Incubated at 700 C for 45 minutes
Evaporation into dryness until pyridine free
Added 1 mL n–hexane
Filtered and then transferred into a vial
Sent for GC
Scheme 2: Free Sugars analyses by GC
Table 10: Standard Retention Time of Different Methyl Esters of Different Sugars from Gas Chromatograms
Standard Retention Times
/minutes
Sugars
4.37
6.85
8.44
10.08
11.16
Rhamnose
Arabinose
Xylose
Mannose
Galactose
12.00 Glucose
3.5.3.4 Calculation of the Relative Percentage of Different Type of Sugars Area of the peak
Relative % of sugar = X 100
Total areas of the peaks
From the Gas Chromatograms three sugars were identified. They were arabinose, galactose and glucose. From the analyzed fruits, the relative proportion and relative percentage of sugars are given below:
Total areas of the peaks of free sugars = 4572 + 5820 + 23931
= 34323.
Relative percentage of arabinose = X 100
= 13.33 %.
Relative percentage of galactose = X 100
= 16.95 %.
Relative percentage of glucose = X 100
= 69.72 %.
Relative proportion =13.33 + 16.95 + 69.72
=100.
Table 11: Relative Proportion of Identified Free Sugars
Retention Times
( min) of sugars
Areas of sugars
Identified sugars Relative %
6.12 4572 Arabinose 13.33
11.10 5820 Galactose 16.95
12.02 23931 Glucose 69.72
Figure 5: Relative Percentage of Identified Free Sugars
3.6 Isolation, Identification and Quantification of Fatty Acids by GLC
This experiment introduces a procedure that is used routinely for fat analysis in which non volatile fatty acids are chemically converted to the corresponding volatile methyl esters. The resulting volatile mixture can be analyzed by gas chromatography. Any sample that can be vaporized (or the components could assume a vapor pressure of at least few mm of Hg) without thermal decomposition at the operating temperature, could be analyzed by GC. At present due to various reasons, including difficult instrumentation and lack of high temperatures materials, the separating temperature is generally limited to about 450oC. Samples that cannot be vaporized are converted into volatile derivatives (for example, fatty acids converted into methyl esters) and then subjected to GC analysis.
3.6.1 Analysis of Fatty Acids
The dried DCM extracted-aqueous layer (100.0mg) of fruit sample was dissolved in hexane (50.0 mL) and extracted with 5% sodium bicarbonate solution (25.0 mL X 2). The mixture was taken in a separatory funnel and shaken vigorously and allowed to stand for overnight. Two layers were obtained. The lower layer (aqueous) was separated and taken for the analysis of free fatty acid (FFA). The upper layer was separated and taken for the analysis of bound fatty acid (BFA)43.
3.6.2 Isolation of FFA
The lower part was acidified (pH 2.5) by 2M sulphuric acid. The mixture was then extracted with hexane (25.0 mL X 3). The hexane fraction was dried over anhydrous sodium sulphate, filtered and the filtrate was evaporated to dryness. Now the saponified materials obtained
from the hexane extract was taken in a pear shaped flask and 2.0 mL of borontrifluoride-methanol (BF3-MeOH) complex was added and the mixture was refluxed on a boiling water bath for 6 to 10 min. The mixture was then evaporated in a rotavapor to dryness and transferred in a small separatory funnel containing a little water (6.0 mL). The mixture was shaken vigorously and then extracted with hexane. The aqueous layer was discarded. The hexane part containing the methyl esters of fatty acids was made free from water by adding anhydrous sodium sulphate. The solution was filtered and the filtrate was concentrated for the analysis of free fatty acids by GLC (Shimadzu 9A, Column-BP-50, Detector-FID, 170C-1 min/4C-270C-30 min)43.
3.6.3 Isolation of BFA
The upper part was taken in a pear shaped flask; methanolic sodium hydroxide (0.5 M, 10.0 mL) was added to it and shaken well. The mixture was refluxed for 30 min in a boiling water bath. Then the mixture was evaporated to dryness by means of a rotavapor. A little water was added to the mixture and transferred to a seperatory funnel to settle down. The non-saponified materials were separated from the saponified portion (aqueous layer) by extraction with hexane. The aqueous layer containing fatty acids (as salts) was acidified by adding sulphuric acid and to pH 2.5. The mixture was extracted with hexane. The hexane part was taken in a conical flask and made from water by adding anhydrous sodium sulphate and then filtered. The filtrate contained saponified materials.
Now the saponified materials obtained from the hexane extract was taken in a pear shaped flask and 2.0 mL of borontrifluoride-methanol (BF3-MeOH) complex was added and the mixture was refluxed on a boiling water bath for 20 min. The mixture was then evaporated in a rotavapor to dryness and transferred in a small separatory funnel containing a little water (6.0 mL). The mixture was shaken vigorously and then extracted with hexane. The aqueous layer was discarded. The hexane part containing the methyl esters of fatty acids was made free from water by adding anhydrous sodium sulphate. The solution was filtered and the filtrate was concentrated for the analysis of bound fatty acids by GLC (Shimadzu 9A, Column-BP-50, Detector-FID, 170C-1 min/4C-270C-30 min)43.
3.6.4 Isolation Scheme for Fatty Acids Analyses
DCM extracted dried aqueous layer of Elaeocarpus robustus
Added 5 % of NaHCO3
Mixture (shaken vigorously)
Transferred in separatory funnel
Separated
Organic layer Aqueous layer
(Unreacted fatty material)
BFA
Added 10 mL of 0.05 M NaOH
Refluxed 30 minutes
Na-salt of FFA +Glycerol
Evaporation under reduced pressure with added distilled water
Transferred in a separatory funnel by adding hexane
Aqueous part taken
Added 0.2M H2SO4
Transferred in a separatory funnel
Taken Hexane part in a beaker
Added Na2SO4 & filtered
Filtrate (Dried & weighed which is total fatty acid)
Refluxed 20 minutes & evaporated
Added 1 mg of Benzoic acid & 2 mL BF3 – MeOH complex
Transferred in separatory funnel with added 6 mL – distilled water
Added hexane with shaken & hexane part taken
Added Na2SO4 & filter
Concentrated & transferred into vial
Sent for GLC
2nd part (Aqueous layer)
Aqueous part (Na- salt of fatty acid)
2 M H2SO4 added to control pH 2.5
Hexane is added and shaken
Transferred in a separatory funnel
Taken hexane part into a beaker
Anhydrous Na2SO4 was added & filtered
Transferred filtrate to the weighed pear shaped flask and dried under reduced pressure
Again weighed which was only FFA
Added 1 mg of C6H5COOH & 2 mL of BF3 -MeOH complex
Reflux on boiling water bath, minimum 30 minutes
Dried by rotavapor & transferred in a separatory funnel
Added 6 mL of distilled water, shaken & haxen added
Taken hexane part in beaker which is FFA of methyl ester
Added anhydrous Na2SO4, filter & concentrated by rotavapor
Transferred in a vial & sent for GLC
Scheme 3: Fatty Acids Analysis
3.6.5 Gas Chromatograms Identification of Fatty Acids
1.0 µL sample was injected through a filter into the injector of GC-MS and at the same condition; the fatty acid standard mixture solutions were injected. Comparing the retention times of the different peaks in the chromatogram of the sample with those of standards, different fatty acids were identified.
3.6.6 Calculation of the Relative Percentage of Different Fatty Acids
Area of the peak
Relative percentage of fatty acid = X 100
Total area of the peaks
Table 12: Standard Retention Time (RT) of Different Methyl Esters of Different Fatty Acids from GC chromatograms.
Standard Retention times (min) Fatty acids
2.18 Caprylic acid
3.65 Capric acid
6.27 Myristic acid
9.43 Lauric acid
12.29 Palmitic acid
12.64 Palmitoleic acid
15.26 Oleic acid
15.65 Stearic acid
18.42 Arachidic acid
21.15 Behenic acid
24.92 Lignoceric acid
3.6.7 Calculation of Relative Percentage of FFA
From all the analyzed olive relative proportion and relative percentage of free fatty acids in olive is shown below-
In the present investigation, oleic, arachidic, behenic, and lignoceric acids were identified as free fatty acids.
Total areas of all the peaks of free fatty acids = 3533+2982+2788+1831
= 11134.
Relative percentage of Oleic acid = X 100
= 31.73%.
Relative percentage of Arachidic acid = X 100
= 26.78%.
Relative percentage of Behenic acid = X 100
= 25.04%.
Relative percentage of Lignoceric acid = X 100
= 16.45%.
Relative proportion = 31.73+26.78 +25.04+16.45
= 100.
The same calculation procedure was followed for all.
Table 13: Relative Amounts of Free Fatty Acids Elaeocarpus Robustus
Retention times (minutes)
Fatty acids Areas Relative percentages
(%)
13.23 Oleic acid 3533 31.73
16.14 Arachidic acid 2982 26.78
18.82 Behenic acid 2788 25.04
21.38 Lignoceric acid 1831 16.45
Figure 6: Relative Percentages of Free Fatty Acids in Elaeocarpus Robustus
Table 14: Relative Proportions of Bound Fatty Acids in Elaeocarpus Robustus
Retention Times
(minutes)
Fatty acids Areas Relative percentages
(%)
8.75 Myristic acid 17372 50.94
12.60 Palmitoleic acid 10339 30.03
15.07 Oleic acid 4907 14.38
15.45 Stearic acid 1484 4.35
Figure 7: Relative Percentages of BFAs in Elaeocarpus Robustus
4.1 The Moisture Content
In the present investigation, the moisture content of Elaeocarpus robustus was found to be 84.13%, which is very important for our digestion and all other mechanistic process of our body. Large moisture content of the fruits indicated that even after processing of food substantial amount of water is coming to the body fluid from the fruits. A healthier body needs a sufficient amount of water. Water refreshes our blood circulation. The relative recommended44 moisture level in fruits helps to digest food properly. This water part has several minerals and nutrients that help to develop the resistant power of diseases inside our body.
The edible portions of fresh fruits are generally juicy and the juiciness in many of the cases characterizes the fruits themselves, so it is very likely that the water content of the fruits should be high. These expectations were already been reported18,24 in most of the cases of tropical and subtropical fruits. Olives contain considerable percentage of water. From the moisture content, it can be said that olive covers a wide range of water intake inside our body.
Generally the higher the water content the lower the calorie content45 of each fruit. This high water content can help replenish fluid balance and make fresh and ready for exercise. It is also hard to overeat with fruit portions as the large water and fibre content causes the stomach to fill up quickly. For this reason, it may be a good idea to eat a piece of fruit before each meal.
Measurement of oil and moisture in olives provides information that enables maximization of yield through growth of olive varieties that yield high oil levels, harvesting the crop at the optimal time, and optimization of the oil extraction process. Since farmers often receive payment on the basis of moisture and oil content and not simply weight, both the olive producers and the olive processors profit from being able to make instantaneous on-line or at-line measurements of these constituents.
4.2 The Ash Content
Ash is the inorganic residue remaining after the water and organic matter have been removed by heating in the presence of oxidizing agents, which provides a measure of the total amount of minerals within a food. The amounts of ash content of the olives that are locally used and conventional dietary intake, taken by the people of Bangladesh are very important. Ash content measurement has been done to determine the important minerals in those olives sample. Using ashes later on helped to proceed on the iron content analysis of those samples. For the analysis of iron as an important nutrient of human health, the ash content of the sample of olive showed noticeable presence46. In the present experiment, the ash content of the sample was found to be 3.72 % that is good agreeable to the reported value46. Ash contents of fresh foods rarely exceed 5%, although some processed foods can have ash contents as high as 12 %, e.g., dried beef 47.
The measurement of ash content is the total amount of minerals present within a food, whereas the mineral content is a measurement of the amount of specific inorganic components present within a food, such as Ca, Na, K, Fe etc. Determination of the ash and mineral content of food is important for a number of reasons:
Nutritional labelling: The concentration and type of minerals present must often be stipulated on the label of a food.
Quality: The quality of foods depends on the concentration and type of minerals they contain, including their taste, appearance, texture and stability.
Microbiological stability: High mineral contents are sometimes used to retard the growth of certain microorganisms.
Nutrition: Some minerals are essential to a healthy diet (e.g. Ca, K, P, Na and Fe) whereas others can be toxic (e.g. Pb, Hg, Cd and Al)
Processing: It is often important to know the mineral content of foods during processing because this affects the physicochemical properties of foods.
4.3 Identification of Free Sugars by PC
In the paper chromatograms, the identified free sugars were found to be glucose, galactose and arabinose. The Rf values of glucose, galactose and arabinose were found to be 0.41, 0.45 and 0.50 respectively which are agreeable to the literature value. If Rƒ value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. If R ƒ
value = 1 then the solute has no affinity48 for the stationary phase and travels with the solvent front. The difference in Rf values required for two substances to be separated depends on the size of the spots and the length of the solvent flow.
4.4 Determination of Free Sugars by Chemical Method
Total free sugars of the fruits normally present the form either of reducing or in the form of non-reducing sugars. Fruits might be considered as one of the main sources of energy for human being and other animal due to presence of substantial amount of free sugars. In present experiment the amount of reducing sugars, non-reducing sugars and total free sugars were determined by chemical method. The total free sugars, reducing sugars and non-reducing sugars were given in table-9. 0.77 g total free sugar per 100 g fruit was found in the present investigation. This value is somewhat lower than the reported18 value. The above variations might be due to differences in fruit season, cultivars, storage time and the procedures of analyses used. In fruits, mainly glucose and fructose were identified as reducing sugars and in most of the cases, sucrose was identified, as non-reducing sugar18,24. During hydrolysis sucrose present in the fruit juice was broken down to equimolecular proportion of glucose and fructose.
It was observed that reducing sugar was higher in proportion than non-reducing sugar. Thus, the fruits that contain higher proportion of reducing sugar produced energy faster24
than the fruits, which contain higher proportion of non-reducing sugar. Because the sugars present in the form of non-reducing sugars like sucrose need to be hydrolyzed before producing energy.
In the native olive fruit, the free sugar was found to be less than one in the present investigation. Therefore, it is recommended that who are facing the problem of excess blood sugar (problem in diabetics) might take native olives as diet. Excess blood sugar increases free radicals, which is associated with blocked arteries, arterial damage, aging and heart disease.
4.5 Identification and Quantification of Free Sugars by GLC
Glucose, galactose and arabinose were the three free sugars identified and quantified by GLC. The relative proportion of glucose, galactose and arabinose were found to be 69.72 %, 16.95 % and 13.33 % respectively. Other components from the gas chromatogram were not detected.
As most fruit calories come from natural sugars, it is great idea to determine sugars for the general people who can make decision which fruits they take as diet.
4.6 Identification and Quantification of Fatty Acids
In the present study, oleic acid, arachidic acid, behenic acid and lignoceric acid were identified and quantified as free fatty acids. The relative proportions of the oleic acid, arachidic acid, behenic acid and lignoceric acid were found to be 31.73 %, 26.78 %, 25.04 % and 16.45 % respectively. Myristic acid, palmitoleic acid, oleic acid and stearic acid were identified and quantified as bound fatty acids. The relative compositions of bound fatty acids were found to be 50.94 %, 30.03 %, 14.38 % and 4.35 % respectively.
Fatty acids can be bound or attached to other molecules, such as in triglycerides or phospholipids. When they are not attached to other molecules, they are known as free
fatty acids. The uncombined fatty acids49 or free fatty acids may come from the breakdown of a triglyceride into its components (fatty acids and glycerol). However as fats are insoluble in water they must be bound to appropriate regions in the plasma protein albumin for transport around the body. The levels of free fatty acid in the blood are limited by the number of albumin binding sites available. Free fatty acids are an important source of fuel for many tissues since they can yield relatively large quantities of ATP. Many cell types can use either glucose or fatty acids for this purpose.
Saturated and unsaturated are the two types of fatty acids. Saturated50 fatty acid tends to increase blood cholesterol levels. Most saturated fats tend to be solid at room temperature with the exception of tropical oils. It is found mostly in meat and dairy products as well as some vegetable oils. Butter is high in saturated fat, while margarine tends to have more unsaturated fat. Saturated fats are a major risk factor for heart attacks and strokes. Diets high in saturated fat50 have been correlated with an increased incidence of atherosclerosis and coronary heart disease. Monounsaturated51 fatty acid tends to lower LDL cholesterol. It is found in both plant and animal products. Such as olive oil, canola oil, peanut oil and in some plant foods such as avocado. In the present investigation oleic acid and palmitoleic acid were identified and quantified as monounsaturated fatty acids.
Poly unsaturated52 fatty acid tends to lower blood cholesterol level. It is found mostly in plant sources (Safflower, Sunflower, Soya bean, Corn and Cottonseed). Although polyunsaturated fats are protective against cardiac arrhythmias a study of post menopausal woman with a relatively low fat intake showed that poly unsaturated fat was positively associated with progression of coronary atherosclerosis, where as monounsaturated fat was not. This probably is an indication of the greater vulnerability of polyunsaturated fats to lipid per oxidation, against which vitamin E has been shown to be protective. Although unsaturated fats are healthier than saturated fats, the Food and Drug Administration53recommendations stated that the amount of unsaturated fat consumed should not exceed 30% of one's daily caloric intake.
Fatty acids are essential parts of all body tissues, where they are a major part of the phospholipid component of cell membranes. Saturated fatty acids have been suggested to be the preferred fuel for the heart54. Fatty acids are used as a source of fuel during energy expenditure, and heavy exercise is associated with decreases in the plasma concentrations of all free fatty acids. In light exercise, fat metabolism may be controlled to favor adipose tissue lipolysis and extraction of free fatty acids from the circulation by muscle, whereas in heavy exercise, adipose tissue lipolysis is inhibited and hydrolysis of muscle triacylglycerols may play a more important part55.In the absence of sufficient dietary fat, the body is apparently capable of synthesizing the saturated fatty acids that it needs from carbohydrates, and these saturated fatty acids are principally the same ones that are present in dietary fats of animal origin.
Dietary stearic acid decreases plasma and liver cholesterol concentrations by reducing intestinal cholesterol absorption. Recent data from studies with hamsters, which have a lipoprotein cholesterol response to dietary saturated fat that is similar to that of humans, suggest that reduced cholesterol absorption by dietary stearic acid is due, at least in part, to reduced cholesterol solubility and further suggest that stearic acid may alter the microflora populations that synthesize secondary bile acids56. Commercially, behenic acid is often used to make hair conditioners and moisturizers for their smoothing properties. Because of its low bioavailability and very long chain length compared with other fatty acids, the effect of dietary behenic acid (behenate) on serum lipid concentrations in
humans is assumed to be neutral. In healthy subjects, although myristic acid is hypercholesterolemic, it increased both LDL and HDL cholesterol concentrations compared with oleic acid 57. Oleic and monounsaturated fatty acid levels in the membranes of red blood cells have been associated with increased risk of breast cancer. Oleic acid may be responsible for the hypotensive (blood pressure reducing) effects ofolive oil. Oleic acid may hinder the progression of Adrenoleukodystrophy, a fatal disease that affects the brain and adrenal glands. Oleic acid may help boost memory58.
4.7 Conclusion
The seed-free edible parts of native olives were taken for the present study where moisture content, ash content, total free sugars and fatty acids were analyzed by conventional methods. All estimations were conducted on fresh weight basis considering the presence of water content of the fruits materials. The water content was found to be 84.13 % which showed little amounts of dry materials. Due to large percentage of water the calories present in the fruits usually low. The ash content was found to be 3.72% which showed46 the presence of inorganic materials such as Fe, Ca, K, P, Na etc. With the help of ash content one can analyze the quality of food. Glucose, galactose and arabinose were identified by paper chromatography and gas liquid chromatography. In GLC the relative amount of glucose, galactose and arabinose were found to be 69.72%, 16.95% and 13.33% respectively. The total free sugars were estimated by chemical methods where it was found to be 0.77 g / 100 g. A piece of fruit contains 0.077 g of free sugars. Portion size can vary between different fruits; therefore total free sugars were calculated from an average piece of fruit. The sweetness of fruit depends on the proportion of free sugars present in it. Higher the free sugars sweeter the fruits are. The total free sugars either in the form of reducing sugars or in the form of non-reducing sugars presents in the fruits and these include a class of water soluble carbohydrates with various degree of sweetness. Reducing sugars as well as non-reducing sugars were found to be 0.41 % and 0.36 % respectively. Reducing sugars gives energy quicker24 than non-reducing sugars. As the native olives contained a little free sugar, it might be a good choice for diabetic patients and other patients who are suffering from high blood-sugar problems. A number of free fatty acids and bound fatty acids were detected where the individual fatty acids were identified and quantified as their methyl esters by GLC. Myristic, oleic, palmitoleic, arachidic, behenic, lignoceric and stearic acids were identified and quantified. Among them only oleic acid and palmitoleic acid were found as monounsaturated acids. Foods containing monounsaturated fatty acids lower low-density lipoprotein (LDL) cholesterol, while possibly raising high-density lipoprotein (HDL) cholesterol58. However, their true ability to raise HDL is still in debate. In children, consumption59 of monounsaturated oils is associated with healthier serum lipid profiles. The relative percentages of the oleic acid, arachidic acid, behenic acid and lignoceric acid as free fatty acids were found to be 31.73 %, 26.78 %, 25.04 % and 16.45 % respectively. The relative compositions of bound fatty acids i.e. myristic acid, palmitoleic acid, oleic acid and stearic acid were found to be 50.94 %, 30.03 %, 14.38 % and 4.35 % respectively.
Having many varieties and species, olives in our country are not alike to the alien olives. Our native olive tree i.e. Elaecarpus robustus L. (Fam. Elaeocarpaceae), native to Bangladesh and India is a well-known evergreen 25 metres tall tree which gives fruits, timber and fuel etc. The fleshy sour fruits having citric acid and vitamin C are used in making soup, chutney, jelly and jam which is very delicious.
Myristic acid
Palmitoleic acid
Oleic acidStearic acid
In the present study moisture content, ash content, total free sugars analysis and fatty acids analysis were carried out and determined. By using analytical grade reagents standard methods were applied all the experiments. Dry matter and ash content of the fruits were determined by standard conventional methods. For paper chromatography Whatman no. 1 papers were used. All sugars analyses were performed on GLC by Sweely et al. By Morrison et al. fatty acids analyses were performed.
Native olive fruits washing properly in cool water to free from dirt were carefully sorted and chopped by using a good fruit slicer which gave uniform pieces in sizes. Pre-weighed edible portion of fruit samples were dried to constant mass in an electrical oven at a temperature of 1050 C. The water content was found to be 84.13 %. Sample taken in a crucible was heated at a dull-red heat in a muffle-furnace at 6000 C. The ash content was found to be 3.72 %.
Extracting with C2H5OH the sliced edible parts of fruit samples were partitioned with DCM in a separatory funnel after removing C2H5OH. DCM- extracted aqueous layer was taken for free sugars analyses and fatty acids analyses. In paper chromatography, glucose, galactose and arabinose were detected as free sugars in where Rf values were found to be 0.41, 0.45 and 0.50 respectively. In chemical method the Fehling solution was standardized with standard glucose solution. The total free sugars were determined by standard Fehling solution. The total free sugars were found to be 0.77 %. Again, preparing TMS-derivatives the free sugars were identified and determined by GC. The relative proportion of glucose, galactose and arabinose were found to be 69.72%, 16.95 % and 13.33 % respectively.
Free fatty acids and bound fatty acids were identified and quantified by preparing methylesters. The relative amounts of oleic acid, arachidic acid, behenic acid and lignoceric acid as free fatty acids were found to be 31.73 %, 26.78 %, 25.04 % and 16.45 %. The relative proportions of myristic acid, palmitoleic acid, oleic acid and stearic acid as bound fatty acids were found to be 50.94 %, 30.03 %, 14.38 % and 4.35 % respectively.
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