UNIVERSITY OF NIGERIA NSUKKA DEPARTMENT OF PURE AND

UNIVERSITY OF NIGERIA, NSUKKA DEPARTMENT OF PURE AND INDUSTRIAL CHEMISTRY TOPIC: COMPARATIVE STUDY OF THE MINERAL COMPOSITION OF RAW AND PROCESSED AFRICAN LOCUST BEANS SEED BY: BANKOLE MARY OLUWATOSIN PG/M. Sc/10/57583 SUPERVISOR: PROF C. O. B. OKOYE AUGUST 2014

OUTLINE �INTRODUCTION(Background- Objectives) �EXPERIMENTATION/ METHODOLOGY �RESULT AND DISCUSSION �CONCLUSION

LEGUMES AS SOURCES OF PLANT PROTEIN Nigeria, a country with a population of 140 million people with an annual budget running in excess of several trillions has several of her citizenry struggling with malnutrition. It becomes imperative to source for cheaper but quality food crops laden with nutritive and mineral elements. The high cost of animal protein has directed interest towards several leguminous seeds.

Among the plant species, legumes are considered as a major source of dietary protein. Food legumes offer a singular advantage of providing plant proteins with reduced cost of production, less difficulty of processing and yet, higher prospect of boosting energy efficiency than animal protein. One of the leguminous plants used by man in many African countries, is the African locust bean (Parkia biglobosa).

�Parkia AFRICAN LOCUST BEAN (Parkia biglobosa) biglobosa is popularly called “African locust bean” in English. In Nigeria, it is called “Iru” in Yoruba language, “Dawadawa” in Hausa language and “Ogiri Okpe” in Igbo language. Fermented Parkia biglobosa seeds are popular food condiment in Nigeria, and other West African countries.

However, the most popular form of consumption is in its traditional fermentation into ‘Iru’, a food condiment for seasoning traditional soups which also adds protein to the diet. It is known to contribute to calorie and protein intake. The raw African locust beans are unpalatable and nutritionally deficient, but when fermented into condiment, ‘Iru’ the chemical and nutritional characteristics of the seeds change.

Processed African locust beans are consumed mainly because it is rich in minerals and provide valuable protein. The protein and amino acid composition of Parkia biglobosa has been reported by several researchers like Ogbonnaya et al and Elemo et al. Oyenuga , Campbell-Platt and Odunfa also carried out some nutritional studies on African locust beans seed while Eka, Aderibigbe and Odunfa also examined some aspects of the processed seed.

Statement of Problems Many people have worked on raw and processed African locust beans but nobody has compared the minerals present in raw African locust bean, processed African locust beans and the water used in processing it. Also toxicants like cyanide and arsenic has not been tested for in the previous researches that have been done on raw and processed African locust beans.

Aim and Objectives of the Research project The objectives of the study are as follows: 1. To determine the mineral composition in the raw African locust bean seed. 2. To determine the mineral composition in the processed African locust bean seed. 3. To determine the mineral composition of the water that was used in cooking the raw African locust beans. 4. To compare the minerals concentration in the raw and processed African locust bean.

JUSTIFICATION The presence of heavy metals in raw and processed African locust bean necessitates this research work.

SAMPLE COLLECTION Four samples of raw African locust beans were purchased, three was processed by different individuals while I processed the last one. SAMPLE PREPARATION The raw African locust bean samples were broken in a big machine to remove the seeds for milling. The samples were grinded separately in the machine. The moisture content were determined by weighing 100 g of the samples into previously weighed evaporating dish. The dishes and content were placed in an air oven at temperature of 105° for 3 hours. They were cooled in a dessicator and weights taken. The process was repeated until a constant weight was obtained.

PROCESSING OF RAW AFRICAN LOCUST BEANS SEED The raw African locust bean was cooked for about 20 hours and left overnight before draining off the water. It was poured into a wooden mortar where I mashed it until the seed coats removed then washed with water. The seed was cooked again for 6 hours, water was drained off then wrapped with bags. It was left for four days so as to ferment to “Iru” (processed African locust bean).

ASHING PROCEDURE FOR ANALYSIS OF SAMPLE 2 g of finely grounded raw African locust bean and processed African locust bean samples was weighed into a flask, 5 ml concentrated HNO 3 was added(as necessary) and boiled until digestion was completed. The solution was allowed to cool and was decanted into 100 ml volumetric flask with two 5 ml portion of deionized water. The solution was diluted to mark, cooled and mix thoroughly.

�A blank solution was also prepared. The solutions were poured into sample bottles for Atomic Absorption Spectrophotometry (AAS GBC quanta version model 2. 02) analysis for zinc, iron, manganese, calcium, magnesium, chromium, copper, lead, cadmium and nickel. The solution was used for determination of potassium and sodium using a flame emission photometer(P 7 P 7 Jenway Photometer).

20 mls molybdate ascorbic acid solution was mixed with 2 mls of the digested sample solution in a flask to determine the concentration of phosphorus. The solution was immersed in boiling water bath for 15 mins, then cooled under water jet until it had 20 -30 o. C.

After a colour development (blue colour), a portion was transferred into the absorption cell and the absorbance was read on a Jenway 6405 UV/visible spectrophotometer at 660 nm(wavelength of phosphorus). The procedure was repeated for all the samples. The absorbance of the blank solution containing 2 ml distilled water solution and 20 mls molybdate ascorbic acid solution was also read and extrapolated on the calibration graph.

DIGESTION PROCEDURE FOR ANALYSIS OF SAMPLE FOR ARSENIC AND SELENIUM. 2 g of finely grounded raw African locust bean and processed African locust bean samples was weighed into a Macro kjedahl flask. 20 ml of 1: 1 perchloric acid, nitric acid solution were added into all the flask samples and left overnight before heating in a fume cupboard until the brown fumes cleared off.

The solution was allowed to cool while the flask containing the raw and processed samples were filtered into a 100 ml volumetric flask using funnel and whatman 44 filter paper. This was then made up to the mark with distilled water. In the case of the water samples, 10 mls of the water samples were measured into a Macro kjedahl flask.

20 ml of 1: 1 perchloric acid, nitric acid solution were added into the water sample and left overnight. The mixture was heated on a heating mantle in a fume cupboard until the brown fumes cleared off. The solution was allowed to cool and poured into a 100 ml volumetric flask and made up to mark with distilled water

DETERMINATION OF ARSENIC USING TITRIMETRIC METHOD. 20 mls of the sample solutions was put in a 250 ml conical flask, 10 ml of distilled water was added, 1 g of sodium bicarbonate crystal and 1 ml of 1% starch solution were added and swirled carefully until the crystal dissolved. The solution was titrated slowly with 0. 02 N iodine solution contained in the burette until a permanent blue colour solution was formed signifying the end point.

DETERMINATION OF SELENIUM USING TITRIMETRIC METHOD. 40 mls of the sample solution was put in a 250 ml conical flask, 10 ml of 2% starch solution, 6 ml of 1: 1 hydrochloric acid and 0. 4 g of pure sodium bicarbonate was added. 10 ml of 10% potassium iodide solution was also added in a thin stream while swirling the solution. After 1 minute, the solution was titrated with 0. 1 N sodium thiosulphate contained in the burette until the colour changed from blue to dirty brown and finally violet red.

EXTRACTION PROCEDURE OF SAMPLE FOR CYANIDE DETERMINATION The finely grounded raw African locust bean and processed African locust bean was soaked in distilled water in a corked conical flask and allowed to stay for 48 hours. The mixture was filtered into 50 ml volumetric flask using funnel and whatman 44 filter paper.

DETERMINATION OF CYANIDE BY TITRATION WITH SILVER NITRATE. 25 mls of the sample filtrate were put in 250 mls conical flask. 75 mls of distilled water, 6 mls of 6 M ammonia solution, 2 mls of 10% potassium iodide solution and 3 drops of diphenylcarbazide indicator was added. The flask was placed on a sheet of black paper and titrated with standard 0. 05 M of Ag. NO 3 solution. The Ag. NO 3 solution was dropped wisely until permanent turbidity appeared.

DETERMINATION OF CYANIDE IN WATER SAMPLE BY TITRATION WITH SILVER NITRATE. 20 mls of distilled water was added to 10 mls of the water sample in a corked conical flask and left for 48 hours. 25 mls of the sample solution was put in 250 mls conical flask. 75 mls of distilled water was added with 6 mls of 6 M ammonia solution. 2 mls of 10% potassium iodide solution and 3 drops of diphenylcarbazide indicator was added. The flask was placed on a sheet of black paper and titrated with standard 0. 05 M of Ag. NO 3 solution. The Ag. NO 3 solution was dropped wisely until permanent turbidity appeared.

ASHING PROCEDURE FOR ANALYSIS OF WATER SAMPLE FOR MANGANESE, ZINC, IRON, SODIUM, POTASSIUM, PHOSPHORUS, CHROMIUM, COPPER, LEAD, CADMIUM AND NICKEL DETERMINATION. 15 mls of concentrated HNO 3 was added to 50 mls each of the water sample in a flask and boiled. The heating continued until it reduced to 20 mls. The solution was allowed to cool, poured into 100 ml volumetric flask made up to mark and mix thoroughly.

The procedure was repeated for each water sample and poured into sample bottles for Atomic Absorption Spectrophotometry (AAS) analysis for manganese, zinc, iron, copper, chromium, lead, cadmium and nickel. The same solution was used for determination of potassium and sodium using a flame emission photometer. The same solution was used for determination of phosphorus using UV/Visible spectrophotometer.

DETERMINATION OF CALCIUM AND MAGNESIUM IN THE WATER SAMPLES USING TITRIMETRIC METHOD 20 mls of the water sample was put in a 100 ml conical flask. 2 mls of ammonia buffer was added with 3 drops of Erichrome Black T indicator and mixed together. The colour changed to wine red. The solution was titrated against 0. 02 M EDTA (in burette) and swirled until the colour changed from wine red to permanent blue.

Total hardness = TV x N x 50 x 1000 Vol. of. sample taken TV= Titre value N=Normality of EDTA=0. 02 N Equivalent weight of Ca. CO 3 =50

RESULT The moisture content percentage for raw African locust beans was 7. 01 ± 0. 01 while that of the processed African locus beans was 37. 10± 0. 20. The increase in moisture content may be due to boiling and subsequent soaking in water. The concentration levels of the minerals in raw and processed African locust beans as well as the water for processing the beans are given below

TABLE 1: MINERAL CONCENTRATION IN RAW AFRICAN LOCUST BEAN SAMPLE (RALB) IN (mg/kg) S/N RALB Na Ca K Mg Mn Zn Fe P Cr As Se CN Cu Pb Cd Ni samples 1 A 210. 48 141. 33 2411. 26 398. 55 46. 21 28. 66 74. 68 946. 32 0. 086 0. 073 0. 050 0. 078 54. 33 ND 0. 411 ND 2 B 222. 44 146. 55 2427. 53 401. 26 45. 97 28. 49 72. 66 954. 33 0. 062 0. 051 0. 040 0. 073 51. 56 ND 0. 394 ND 3 C 235. 66 139. 84 2467. 44 411. 33 46. 04 28. 46 71. 38 952. 44 0. 050 0. 045 0. 030 0. 051 54. 22 ND 0. 388 ND Mean ± SD 222. 86± 142. 57± 3. 2435. 41± 28. 403. 71± 6. 74 46. 07± 0. 28. 54± 72. 91± 951. 03± 0. 07± 0. 056± 0. 04 ± 0. 07± 53. 37± ND 0. 40± ND 12. 6 53 91 13 0. 11 1. 67 4. 19 0. 02 0. 01 0. 02 1. 57 0. 02 210. 48 - 139. 84 - 2411. 26 - 398. 55 - 45. 97 - 28. 46 - 71. 38 - 946. 32 - 0. 050 - 0. 045 - 0. 030 - 0. 051 - 51. 56 - 235. 66 146. 55 2467. 44 411. 33 46. 21 28. 66 74. 68 954. 33 0. 086 0. 073 0. 050 0. 078 54. 33 214. 43 144. 52 2455. 67 394. 24 46. 14 28. 24 70. 85 951. 44 0. 055 0. 030 0. 043 52. 42 Range 4 D ND 0. 388 - ND 0. 411 ND 0. 408 ND

TABLE 2: MINERAL CONCENTRATION IN PROCESSED AFRICAN LOCUST BEAN SAMPLE (PALB) IN (mg/kg) S/ PALB N sample 1 Na Ca K Mg Mn A 235. 44 122. 68 2112. 32 455. 64 38. 44 2 B 231. 67 144. 66 2011. 54 468. 55 3 C 251. 33 145. 76 2027. 62 Mean 239. 48 137. 70 2050. 50 ±SD ± ± 13. 02 ± 54. 15 Fe P Cr As Se CN Cu Pb Cd Ni 24. 35 86. 75 1122. 41 0. 068 48. 66 0. 01 0. 431 ND 39. 84 24. 41 86. 88 1127. 33 0. 032 0. 046 0. 047 46. 52 0. 01 0. 427 ND 461. 44 38. 74 24. 74 86. 42 1114. 41 0. 037 0. 029 0. 030 45. 78 0. 02 0. 422 ND 461. 88 39. 01 24. 50 86. 69 1121. 39 0. 04± 0. 05± 46. 99± 1. 0. 02± 0. 43± 0. 00± ± 6. 47 ± ± ± 0. 24 ± 6. 53 0. 01 0. 02 50 0. 01 0. 00 0. 74 0. 22 ND 10. 44 Range 231. 67 4 D Zn 122. 68 2011. 54 455. 64 38. 44 24. 35 86. 42 1114. 41 0. 032 0. 029 0. 030 45. 78 - 0. 01 - 0. 422 - - - 48. 66 0. 02 - 251. 33 145. 76 2112. 32 468. 55 39. 84 24. 74 86. 88 1127. 33 0. 041 0. 068 240. 44 131. 33 2009. 65 474. 33 39. 33 24. 66 86. 33 1119. 24 0. 037 0. 020 0. 042 0. 022 0. 431 48. 72 0. 03 0. 412 ND

TABLE 3: MINERAL CONCENTRATION IN THE WATER SAMPLE IN (mg/kg) S/N RALB Na Ca K Mg Mn Zn Fe P Cr As Se CN Cu Pb Cd Ni samples 1 A 210. 48 141. 33 2411. 26 398. 55 46. 21 28. 66 74. 68 946. 32 0. 086 0. 073 0. 050 0. 078 54. 33 ND 0. 411 ND 2 B 222. 44 146. 55 2427. 53 401. 26 45. 97 28. 49 72. 66 954. 33 0. 062 0. 051 0. 040 0. 073 51. 56 ND 0. 394 ND 3 C 235. 66 139. 84 2467. 44 411. 33 46. 04 28. 46 71. 38 952. 44 0. 050 0. 045 0. 030 0. 051 54. 22 ND 0. 388 ND 222. 86± 142. 57± 2435. 41± 403. 71 46. 07 28. 54± 72. 91 951. 03 0. 07± 0. 056 0. 04 0. 07± 53. 37 ND 0. 40± ND 12. 6 3. 53 ± 6. 74 ± 0. 13 0. 11 0. 02 ± ± 0. 02 0. 01 Mean SD Range 4 D ± 28. 91 ± 1. 67 ± 4. 19 ± 0. 02 1. 57 210. 48 - 139. 84 - 2411. 26 - 398. 55 45. 97 28. 46 - 71. 38 946. 32 0. 050 0. 045 0. 030 0. 051 51. 56 235. 66 146. 55 2467. 44 - - 28. 66 - - - - 411. 33 46. 21 74. 68 954. 33 0. 086 0. 073 0. 050 0. 078 54. 33 0. 411 394. 24 46. 14 70. 85 951. 44 0. 055 0. 030 0. 043 52. 42 214. 43 144. 52 2455. 67 28. 24 ND ND 0. 388 0. 408 ND ND

Samp DISCUSSION Na Ca K Mg Mn Zn Fe P Cr As Se CN Cu Pb Cd Ni ND 0. 40± ND les RAL B PAL B Mea 222. 8 142. 2435. 4 403. 71 46. 07 28. 54 72. 91 951. 03 0. 07 0. 04± 0. 07 53. 37 n ± 6± 57± ± 6. 74 ± 0. 11 ± 1. 67 ± 4. 19 ± ± 0. 01 ± 1. 57 SD 3. 53 1 0. 02 12. 6 1± 28. 9 ± 0. 13 Mea 239. 4 137. 2050. 5 461. 88 39. 01 n±S 8 ± 70± ± 6. 47 D 10. 44 13. 0 ± 54. 15 0. 02 86. 69 1121. 3 0. 04 0. 05± 0. 05 46. 99 0. 02± 0. 0. 43± 0. 0 ± 0. 74 ± 0. 22 ± 0. 24 9± 6. 53 ± ± 0. 02 ± 1. 50 01 0. 02 Mea 0. 40± 0. 60 0. 29± 0. 88± 0. 0. 03± 1. 09± 0. 14± 0. 64± 0. 0. 01 0. 04 n±S 13 0 24. 50 ± ± 0. 01 0. 02 0± 0. 00 2 wate r D 0. 13 ± 0. 04 0. 05 0. 01 0. 12 0. 03 03 ±ND ND 0. 02 0. 30± ± 0. 06 1 1 ND ND ND

The data were analyzed statistically to determine whethere was significant difference between the concentration level of minerals in raw and processed African locust beans. The mean concentration reported in the raw and processed African locust bean in this work is high compared to what Ijarotimi et al [2012] and Makanjuola reported in their work but less than the concentration reported by Bello et al [2008] in calcium, magnesium, potassium, sodium, manganese, iron, zinc and copper.

The mean concentration level of calcium was not significantly different (p>0. 05). The reasons for some minerals increasing in the processed African locust beans are not clearly known. The processing (cooking and fermentation) had no significant influence on the level of the calcium in the raw and processed African locust since the concentration was significantly very low. Arsenic, Nickel and Cyanide occur naturally in the soil through volcanic emission, weathering of soil and rocks.

The presence of heavy metals in the African locust bean can also be attributed to the presence of contaminants in the soil where they were planted. Air pollution from industries, waste incineration, oil refineries can also contribute to the release of toxic contaminants into the food. Lead, cadmium and arsenic can be released from fumes of vehicle exhaust. The mean concentration of the minerals in the water samples were all within the maximum acceptable concentration by World Health Organization [63] except arsenic which was higher than the acceptable concentration of 0. 01 mg/l.

CONCLUSION This work has shown that the raw and processed African locust bean is a very good source of food which has all the minerals needed by the body. The increase in moisture content may be as a result of metabolic activities of microorganisms during the fermentation period. The chemical composition indicates that it is a good source of macro and micro nutrient. The processing of using safe methods is important because of toxicant already present in the soil. Activities such as boiling, soaking, subsequent fermentation can lead to increase in some of the mineral composition.

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