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Vol.

3/2007 ISSN 2345-1234

MALAYSIAN COCOA JOURNAL


Adviser Dato Dr. Azhar Ismail Editor Dr. Rosmin Kasran Vice Editor Dr. Lee Choon Hui Secretary Dr. Tan Chia Lock Editorial Committee Dr. Douglas Furtek Alias Awang Denamany, G. Suzannah Sharif Hii Ching Lik Harnie Harun Hj. Omar Hj. Tompang Ramle Kasin

Published by MALAYSIAN COCOA BOARD 5th & 6th Floor, Wisma SEDCO Lorong Plaza Wawasan, Off Coastal Highway 88999 Kota Kinabalu, Sabah, Malaysia. All rights reserved. No part of this publication may be reproduced in any form or by any means without permission in writing from Malaysian Cocoa Board

Malaysian Cocoa Journal Volume 3/2007

CONTENTS

AGRONOMY THE EFFECT OF VARIOUS RATES OF PHOSPHATE APPLICATION ON THE GROWTH OF COCOA SEEDLINGS AND ITS NUTRIENT UPTAKE IN RELATION TO CHEMICALLY AVAILABLE PHOSPHORUS IN THE SOIL AND AGE OF SEEDLING ............................ 1 Mohd.Yusoff, A.S., Ahmad Kamil, M.J. and Hamzah, D. ENTOMOLOGY EFFECT OF INSECTICIDES ON PARASITISM OF COCOA POD BORER EGGS BY TRICHOGRAMMATOIDEA BACTRAE FUMATA* NAGARAJA (HYMENOPTERA: TRICHOGRAMMATIDAE) ON COCOA IN MALAYSIA ........................................................................... 13 Azhar, I. and Long, G. E. PROCESSING AND PRODUCT DEVELOPMENT PHYSICAL STABILITY AND COLOR CHANGES OF MALAYSIAN COCOA BUTTER UPON STORAGE ............................................................................................................................................ 20 Asimah, H. EFFECT OF CARBON TO NITROGEN RATIOS (C/N) ON BENZALDEHYDE FORMATION FROM RHIZOPUS OLIGOSPORUS IN A SOLID-STATE FERMENTATION SYSTEM USING COCOA POD HUSKS AS SUBSTRATE ......................................................................... 26 Norliza, A.W. EFFECT OF FERMENTATION AND STORAGE ON MYCOTOXIGENIC FUNGI, OCHRATOXIN A AND AFLATOXIN B1 IN COCOA BEANS FROM SOUTH WESTERN NIGERIA ...................................................................................................................................... 35 Aroyeun, S.O., Adegoke, G.O., Varga, J., Kocsub, S., Pl, K. and Vgvolgyi, C. MINIMUM INHIBITORY CONCENTRATION OF COCOA TANNIN GEL TOOTHPASTE ON SELECTIVE MOUTH BACTERIA ......................................................................................................... 47 Azila, A.K. and Nur Azilah, A. MATHEMATICAL MODELING OF DIRECT SOLAR DRYING OF COCOA BEANS ............................. 52 Hii, C.L. THE EFFECTS OF COCOA BUTTER EQUIVALENT ADDITION TO THE COCOA BUTTERS MELTING AND CRYSTALLIZATION PROFILES .................................................................. 58 Fisal A., Rosinah, R. and Suzannah, S. TRANSFER OF TECHNOLOGY EVALUATION ON ADOPTION OF TRANSFER TECHNOLOGY IN THE SMALLHOLDER DEVELOPMENT PROGRAMME ................................................................................... 65 Mahmod, M.N. SHORT COMMUNICATION DEVELOPMENT AND EVALUATION OF SUITABLE ARTIFICIAL DIETS FOR COCOA POD BORER REARING ......................................................................................................... 71 Meriam M.Y., Furtek, D. and Henipa, J. STABILITY OF HIGH INTERNAL PHASE (HIP) COCOA BUTTER EMULSION .................................. 75 Samuel, Y.K.C. and Sarini, H.

Malaysian Cocoa Journal

Agronomy THE EFFECT OF VARIOUS RATES OF PHOSPHATE APPLICATION ON THE GROWTH OF COCOA SEEDLINGS AND ITS NUTRIENT UPTAKE IN RELATION TO CHEMICALLY AVAILABLE PHOSPHORUS IN THE SOIL AND AGE OF SEEDLING Mohd.Yusoff, A.S., Ahmad Kamil, M.J. and Hamzah, D. Malaysian Cocoa Board, Cocoa Research and Development Centre, Miles 10, Apas Road P.O. Box 60237, 91012 Tawau, Sabah, Malaysia Malaysian Cocoa J. 3: 1-12 (2007) ABSTRACT - A pot trial was conducted to evaluate the effect of various rates of phosphorus application on the growth of cocoa seedlings growing on soil from Table series with low P availability of 5 ppm and to determine the effect of phoshorus application on the nutrient contents of cocoa seedlings. Results from the trial conducted showed that the application of P to the soil significantly increased the number of leaves, height of plant, girth size and total dry matter of the plant components. Significant effect of P on the number of leaves, height and girth size was firstly observed for two and three months cocoa seedlings. The application of P at various rates significantly increased the dry matter of roots, stem and leaves for both six and nine months of cocoa seedlings. The magnitude of the growth and dry matter yield of seedlings responsed with the increasing of P applied. The highest rate of P application at 400 ppm gave the best growth and the highest dry matter yield of cocoa seedlings. This study revealed that higher rates of P application are essential during the initial stage of cocoa seedlings establishment. The application of P also significantly increased the P content in the leaves, stem and roots for both 6 and 9 month of cocoa seedlings. At the age of 6 months, P had resulted the significant increase in Ca and Mg in the roots and K in the stem. However, the application of P had decreased the N content in the stem. For the seedling of nine month-old, the significant uptake of Ca and Mg was observed in the leaves, Mg and K in the roots and K, Ca and Mg in the stem. The nutrient uptake was also influenced by the age of the seedlings especially for N, P, K and Mg in all plant components. Ca increased by age of plants for recently hardened leaves (leaf 1) and the youngest leaves of the oldest leaves (leaf 2). The total P uptake was significantly different for various rates of P application for seedlings at both ages of cocoa seedlings, where at the higher P rates, the total P uptake simultaneously increased. This study also showed that the dry matter yield of plant components have a linear and quadratic regression with various rates of P application for both ages of cocoa seedlings. However, the quadratic regression is suitable to use as it would explain the effect of various rates of P application as higher in R value. Key words: Phosphates, Rates, Growth, Nutrient, Cocoa INTRODUCTION Nutrients such as N, P, K, Ca and Mg are essential and they should be adequately supplied for the growth of plants. One of the essential elements is P which is an essential part of nucleic acids as well as other vital cellular components. In its many compounds phosphorous have roles in cell division, in stimulation of early root growth, in hastening plant maturity, in energy transformations within the cells, and in fruiting and seed production. In cocoa planting, P is one of the essential element which is necessarily to be applied for the growth and early pod bearing for most of Malaysian soils which are Ultisols and Oxisols (Ling, 1984). Other researches conducted have described the importance of P fixation in cocoa nutrition (Ahenkorah, 1968; Wessel, 1970). In Nigeria, Wessel (1970) has observed that P application had resulted good response to cocoa with soil which has P less than 10 ppm on the top of soil profiles. Another finding on phosphate study had also indicated that P was found as an essential element for the formation of flowers and cherelles in the fruit trees. It has been proven by Jadin (1976) who found that cocoa applied with phosphate and additional N fertilisers had improved the flowering and yield of cocoa. The same results were also observed for other crops such as tomato (Besford, 1979), coconut, coffee and citrus (De Guess, 1973). In addition, Ling and Mainstone (1982) reported that continuous application of P was essential for cocoa. Albeit, the P requirement in cocoa is rather low, but in the condition of low in

Malaysian Cocoa Journal

its availability would lead to weak growth and in the extreme of low in P the necrosis symptom would be exhibited by cocoa. In view of the important of P in cocoa planting in particular at the nursery stages, we had carried out extensive study with the objectives:- a) to determine the response of cocoa seedlings to the application of various rates of P, b) to know the effect of P to the major nutrient uptake, and c) to study the relationship of leaf positions to the nutrient uptake as affected by P application. This paper describes some results of the effect of P application on the growth of cocoa seedlings and its nutrient contents. MATERIALS AND METHODS A pot trial was carried out at the nursery house of plastic roofing system at Cocoa Research Centre, Department of Agriculture, Sabah. Soil from Table series at the depth 0f 0-15 cm with chemical and physical properties as shown in Table 1 was used in the study. The soil was dried, crushed and sieved to obtain the size of 2-5 mm. The soils of 17 kg was put into a big plastic bag and then inserted into a big plastic pale. The experiment was laid out in Randomized Complete Block Design with 4 replications. Fertiliser treatment was done by taking out 1 kg of soils and mixed thoroughly with monocalcium phosphate at the rates of 50 ppm, 100 ppm, 200 ppm and 400 ppm while the control no calcium monophosphate was applied and regarded as 0 ppm despite of the naturally phosphate content in the oil is 5 ppm. The soil then was put back into the container and incubated for about one week and watered at field capacity using deionized water. Three opened pollinated seeds of PA156 x SCA9 were sown in the soil and after 2 weeks later only one good seedling was left in the container. Other seedlings were removed. The seedlings were watered with deionized water at field capacity every day. All

treatments received the same amount of N and K in the form of Urea and KCl at equivalent rates of 2.5 g N and 3 g K2O. The amount of 113.3 g CaCO3 was also applied which is equivalent at the rate of 2000 kg/ha in all treatments. The growth parameters such as number of leaves, height of plants and girth size were recorded at 4 weekly intervals. At the age of 6 and 9 months, 6 plants from each treatment were harvested to measure the dry matter weight of plant components and nutrient contents. The soil from each treatment container was also taken for chemical analyses according to SIRIM standard. The nutrient contents as affected by leaf positions were also determined by sampling the leaves at 3 positions: a) position 1 - recently hardened leaves (leaves turning to brown after flushing - leaf 1), b) the youngest leaves from the oldest leaves -taken at the point of stem colour changing, brown to green - leaf 2, and c) other leaves - regardless of leaf positions. The objective is to determine the effect of position to the nutrient contents in which an indicator leaf could be marked or assigned. The soil samples were analysed for pH (1:2.5 soil:water extract), organic C, total N as outlined by Metson (1956), CEC and exchangeable Ca, Mg, K and Na in NH4OAc extract (SIRIM, 1980a) and available P by Bray and Kurtz No. 2., and mechanical analysis by the International pipette methods (Black, 1965). The components of cocoa seedling such as leaves, stem and roots were separated and ground using a stainless steel grinder with 1 mm sieve. The samples of plant components were then digested by wet ashing using H2SO4 - H2O for determination of total N, P, K Ca and Mg (SIRIM 1980b).

Malaysian Cocoa Journal

Table 1. Physical and chemical properties of soil from Table series Depths
(cm)

pH

C
(%)

Total N
(%)

P
(ppm)

Ex.K
(ppm)

Ex.Ca
(ppm)

Ex.Mg
(ppm)

CEC
% me

Ex.Al
%me

Silt
%

Clay
%

Coarse sand
%

Fine sand
%

0-15 15-30

4.6 4.6

1.50 0.73

0.14 0.10

3.00 5.00

0.16 0.08

3.29 2.21

0.43 0.23

10.79 9.44

0.08 0.14

23.00 23.00

69.99 70.00

5.00 4.00

3.00 3.00

RESULTS AND DISCUSSION Effect of various rates of P application on the growth of seedlings Tables 2, 3 and 4 show the effect of various P rates on the number of leaves, plant height and girth sizes of stem on the seedlings. P application had significant effect on the number of leaves, plant height and girth size for 2 months and 3 months and 4 months seedlings respectively. The results suggested that P plays the most important role in increasing the leaves numbers, plant height and girth size to the cocoa seedlings. The results also showed that at the highest rate of P application resulted better plant growth. Our findings are in agreement to the findings by Mainston et al. (1973) which found that application of P from CIRP increased the stem diameter, height of plants, fresh weight of stem and roots. They found that the effect of P application was the greatest when general cocoa development was good and was slight when development was poor.

Effect of various rates of P application on the plant dry matter yield Tables 4 and 5 show that P application significantly increased the dry matter yields of roots, stem and leaves at two stages of plant ages, 6 and 9 months. This indicates that P plays a major role on the growth of plants. This can be well explained by a regression analysis as shown in Tables 7 and 8, which shows that the increase in dry matter yield for all plant components is linearly and quadratically regressed with P application at different rates. Quadratic and linear regression was significant as resulted by the increase in P application for dry matter yield of stem and leaves while dry matter yield of roots is slightly affected by the increased rates of P application. Despite of slightly root increase as affected by the increased in P application, possibly roots had resulted the increase in the dry matter yield other plant component as efficiency in the nutrient uptake by roots. This results showed that at higher rates of P application, it considerably enhanced the plant growth and thus become important nutrient in the early establishment of cocoa seedlings.

Table 2. Effect of various rates of P application on the number of leaves Treatments (ppm P) 0 50 100 200 400 1 4.0a 3.9a 4.0a 4.0a 4.0a 2 6.6a 6.8ab 7.2c 7.4c 7.5c 3 10.0a 11.2b 11.8bc 12.1c 11.8bc Age of seedlings (months) 4 5 6 15.1a 20.3a 26.3a 17.1b 23.3b 29.0b c c 18.2 25.6 31.2c c c 19.2 26.3 32.5c c c 18.9 26.7 32.5c 7 33.4a 36.4b 37.3bc 39.8cd 41.0d 8 39.1a 41.0ab 43.6bc 46.0cd 47.1d 9 45.3a 48.8b 51.0b 51.3bc 53.5c

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD

Malaysian Cocoa Journal

Table 3. Effect of various rates of P application on the height of plant (cm) Treatments (ppm P) 0 50 100 200 400 1 12.7a 12.5a 12.6a 13.0a 12.5a 2 15.6a 15.4a 15.8a 16.0a 15.5a 3 24.3a 26.7bc 26.3b 28.3c 27.9bc Age of seedlings (months) 4 5 6 37.5a 52.4a 74.5a 41.5ab 60.4b 79.8b bc bc 45.5 63.3 84.4bc cd cd 49.1 67.7 85.0c d d 51.2 71.0 93.3d 7 96.6a 101.1ab 103.2ab 107.5bc 115.3c 8 110.2a 113.9ab 117.2a 117.9b 126.2b 9 124.7a 127.4a 129.8a 130.3a 141.7b

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 4. Effect of various rates of P application on the girth size (mm) Treatments (ppm) 0 50 100 200 400 1 10.7a 10.5a 10.7a 10.9a 10.4a 2 13.1a 13.1a 13.0a 13.5a 12.6a 3 19.6a 19.9a 20.2ab 20.8b 20.1b Age of seedlings (months) 4 5 6 24.7a 33.1a 39.2a 29.5ab 35.4b 42.7b bc c 30.8 36.7 43.1bc c c 33.2 37.1 44.0cd bc c 30.9 37.4 44.4d 7 43.0a 46.1b 46.9bc 47.2bc 48.2c 8 53.8a 56.4b 57.7b 58.0b 60.2c 9 59.7a 61.3a 63.8a 64.1b 65.6b

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 5. Effect of various P rates on the dry matter yield of plant components for 6 month-old seedlings Treatments (ppm) 0 50 100 200 400 Roots 8.6a 11.1b 11.1b 11.4b 10.8a Dry matter yield (g) Stem Leaves 12.2a 20.4a b 15.8 23.9b b 17.5 26.2c b 17.8 26.4cd c 20.3 28.0d Total 41.2a 50.8b 54.8c 55.6c 59.1d

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 6. Effect of various P rates on the dry matter yield of plant components for 9 month-old seedlings Treatments (ppm) 0 50 100 200 400 Roots 26.0a 30.3bc 32.7c 32.1bc 30.1b Dry matter yield (g) Stem Leaves 40.3a 47.4a ab 44.3 54.0b bc 47.2 55.8c cd 49.8 56.7cd d 54.2 62.8c Total 113.7a 128.6b 135.7c 138.6c 147.1d

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD

Malaysian Cocoa Journal

Table 7. Regression analysis as affected by P rates on dry matter weight of plant components for 6 monthold seedlings Plant components Leaves Stem Roots Total Model Linear Quadratic Linear Quadratic Linear1 Quadratic2 Linear Quadratic Equation Y=22.65 + 0.0155X Y=21.19 + 0.5X + 0.00008X2 Y=14.18 + 0.017X Y=13.06 + 0.407X-0.00006X2 Y=10.04 + 0.0037X Y=9.326 + 0.019X-0.00004X2 Y=46.87 + 0.0361X Y=43.58 + 0.1063X-0.0002X2 R2 0.547 0.711 0.700 0.802 0.163 0.366 0.637 0.815

X = ppm P applied 1 = not significant at =0.05 2 = only significant at =0.05, Others significant at =0.05 and =0.01 Table 8. Regression analysis as affected by P rates on dry matter weight of plant components for 9 monthold seedlings Plant components Leaves Stem Roots Total Model Linear Quadratic Linear Quadratic Linear1 Quadratic2 Linear Quadratic Equation Y=50.46 + 0.0325X Y=49.17 + 0.06X -0.00007X2 Y=42.36 + 0.0321X Y=40.86 + 0.06X-0.00008 X2 Y=29.40 + 0.0055X Y=27.01 + 0.057X-0.0001X2 Y=122.22 + 0.0702X Y=117.04 + 0.1805X-0.0003X2 R2 0.653 0.687 0.500 0.536 0.047 0.337 0.576 0.681

X = ppm P applied 1 = not significant at =0.05 2 = only significant at =0.05, Others significant at =0.05 and =0.01 Effect of various rates of P application on the nutrient content in the plant components Tables 9-18 show the effect of various rates of P application on the nutrient contents in the plant components. It shows that significant increased in P in all plant components at both ages of cocoa seedlings. The nutrients such as P and K significantly increased at 6 month-old seedlings and N and P at 9 month-old seedlings in leaf 1. Meanwhile, P, K, Ca and Mg increased significantly for seedlings of 6 months age and N, P and K at 9 month-old seedlings in leaf 2. For other leaves only P significantly increased at 6 month-old seedlings and P and K at 9 month-old seedlings. Significant increased in N, P, Ca and Mg was observed at 6 month-old seedlings while P, K and Mg at 9 month-old seedlings in the roots. Different nutrient uptakes in the respective plant components were observed as affected by P application. It is clear that P had stimulated and enhanced various nutrients uptake in the cocoa seedlings. Our finding is in agreement to Mainstone et al. (1973) who found that higher P application has resulted the increase in N, P, Mg and Ca in leaf.

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Table 9. Effect of various rates of P application on the nutrient content in the leaf 1 of 6 month-old seedlings Treatments (ppm) 0 50 100 200 400 N 1.91a 1.93a 1.86a 1.97a 1.87a P 0.10a 0.16b 0.16b 0.16b 0.22c Nutrient content (%) K 1.56a 1.45a 1.35a 1.53a 1.56b Ca 1.51a 1.51a 1.43a 1.51a 1.58a Mg 0.34a 0.38a 0.40a 0.39a 0.36a

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 10. Effect of various rates of P application on the nutrient content in the leaf 2 of 6 month-old seedlings Treatments (ppm) 0 50 100 200 400 N 2.00a 1.95a 1.93a 2.03a 1.93a P 0.15a 0.16a 0.15a 0.18ab 0.22b Nutrient content (%) K 1.51a 1.21a 1.43a 1.51a 1.58b Ca 1.06a 1.17a 1.26a 1.27a 1.47a Mg 0.34a 0.37ab 0.42a 0.39ab 0.38ab

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 11. Effect of various rates of P application on the nutrient content in other leaves of 6 month-old seedlings Treatments (ppm) 0 50 100 200 400 N 2.31a 2.38a 2.31a 2.33a 2.30a P 0.13a 0.19b 0.20bc 0.22c 0.25d Nutrient content (%) K 1.51a 1.21a 1.43a 1.51a 1.58b Ca 1.79a 1.75a 1.73a 1.80a 1.80a Mg 0.36a 0.39a 0.39a 0.40a 0.40a

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 12. Effect of various rates of P application on the nutrient content in other leaves of 6 month-old seedlings Treatments (ppm) 0 50 100 200 400 N 1.42c 1.21c 1.24c 1.17a 1.09a P 0.08a 0.11b 0.12bc 0.14c 0.20d Nutrient content (%) K 1.20a 1.42a 1.33a 1.41a 1.52a Ca 0.43a 0.47a 0.51a 0.58a 0.66a Mg 0.34a 0.39a 0.41a 0.47a 0.51a

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD

Malaysian Cocoa Journal

Table 13. Effect of various rates of P application on the nutrient content in the stem of 6 month-old seedlings Treatments (ppm) 0 50 100 200 400 N 1.26a 1.10a 1.10a 1.03a 1.02a Nutrient content (%) P K Ca 0.09a 1.56a 1.51a 0.25b 1.45a 1.51a b a 0.25 1.35 1.43a b a 0.24 1.53 1.51a c a 0.36 1.56 1.58a Mg 0.34a 0.38a 0.40a 0.39a 0.36a

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 14. Effect of various rates of P application on the nutrient content in the leaf 1 of month-old seedlings Treatments (ppm) 0 50 100 200 400 N 1.80a 2.03a 2.03a 2.07b 2.06b Nutrient content (%) P K Ca 0.08a 2.07a 0.46a 0.16b 2.17a 1.53a b a 0.15 1.83 0.71a b a 0.17 1.84 0.73a c a 0.20 2.11 0.73a Mg 0.34a 0.38a 0.36a 0.36a 0.37a

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 15. Effect of various rates of P application on the nutrient content in the leaf 2 of 9 month-old seedlings Treatments (ppm) 0 50 100 200 400 N 1.85a 1.96a 2.00b 2.07b 1.99b Nutrient content (%) P K Ca 0.07a 1.49bc 0.88a 0.10b 1.30a 0.90a b ab 0.12 1.38 1.03a b ab 0.13 1.37 1.12a c c 0.16 1.67 1.17a Mg 0.45a 0.48a 0.44a 0.44a 0.44a

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 16. Effect of various rates of P application on the nutrient content in the other leaves of 9 month-old seedlings Treatments (ppm) 3 50 100 200 400 N 2.03a 2.22a 2.21a 2.16a 2.13a Nutrient content (%) P K Ca 0.08a 1.67a 0.86a 0.14a 1.63a 1.05a b a 0.15 1.55 1.10a c a 0.17 1.62 1.12a d b 0.19 1.72 1.29a Mg 0.38a 0.42a 0.43b 0.41ab 0.42b

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD

Malaysian Cocoa Journal

Table 17. Effect of various rates of P application on the nutrient content in the roots of 9 month-old seedlings Treatments (ppm) 0 50 100 200 400 N 1.10a 1.04a 1.01a 0.99a 0.94a Nutrient content (%) P K Ca 0.05a 1.00a 0.37a 0.08b 1.16b 0.42a b b 0.08 1.16 0.43a c a 0.10 1.25 0.49a d b 0.14 1.43 0.52a Mg 0.23a 0.30b 0.31bc 0.33bc 0.36c

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 18. Effect of various rates of P application on the nutrient content in the stem of 9 month-old seedlings Treatments (ppm) 0 50 100 200 400 N 0.82a 0.80a 0.83a 0.82a 0.76a Nutrient content (%) P K Ca 0.04a 1.75a 0.71a 0.09a 2.01bc 1.19a b ab 0.11 1.93 0.90a c bc 0.14 1.99 1.03a d c 0.20 2.15 1.16a Mg 0.25a 0.37b 0.35b 0.38bc 0.41c

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Effect of seedling age on the nutrient content in various plant organs Table 19 shows the effect of seedling age on nutrient content in the plant parts. Age had significant effect on the nutrient uptake of certain plant organs. There was significant difference in the age for the uptake of P and Mg in leaf 2 and other leaves and roots. Meanwhile, the age had significant different for the uptake of N and K in leaf 2, other leaves, roots and stem, whereas the uptake of Ca in leaf 1 and leaf 2. The uptake of Ca in other leaves and roots had no significant effect as affected by the seedling age. Mg uptake in leaf 2, other leaves, roots and stem had significant different as affected by the seedling age. The effect of the seedling age on nutrient content in plant components exhibited either increasing trend or decreasing trend as shown in Table 19. This finding indicates that the age of seedlings often demonstrated more pronounced effect than nutritional effect. So different sampling times may give different results of leaf analysis. In this case, it is difficult to take leaves at different times as for fertiliser recommendation at young stages of cocoa.

Malaysian Cocoa Journal

Table 19. Effect of seedlings age on the nutrient content (%) in the plant components Treatments Leaf1 Nutrient N P K Ca Mg N P K Ca Mg N P K Ca Mg N P K Ca Mg Nutrient content (%) 6 month-old seedlings 9 month-old seedlings 1.90a 1.99b a 0.16 0.15a a 2.00 1.49b a 1.25 0.63b a 0.37 0.36a 1.97a 0.17a 1.50a 1.24a 0.38a 2.32a 0.19a 1.77a 1.13a 0.39a 1.10a 0.24a 2.25a 1.14a 0.45a 1.97a 0.11b 1.43a 1.02b 0.45b 2.15b 0.15b 1.64b 1.03a 0.41b 0.81b 0.12b 1.96b 1.00a 0.35b

Leaf2

Other leafs

Stem

N 1.22a 1.02b a P 0.13 0.10b a K 1.37 1.22b Ca 0.53a 0.45a a Mg 0.43 0.30b Means followed by the same letters in the same row are not significantly different at P<0.05 by LSD. Data are means for all rates of P treatments Effect of the leaf position on the nutrient content Tables 20 and 21 show that the effect of leaf position on the nutrient content. The leaf position has significant effect to the nutrient content in the leaves. Significant different can be seen for N, P, K and Ca for 6 and 9 month-old seedlings while Mg is not affected by the leaf position. From this result, it is difficult to sample the leaf to represent as an indicator leaf except for Mg at 6 and 9 month-old seedlings, either leaf 1, leaf 2 or other leaves can be taken as indicator leaf to indicate the nutrient status in the plants. Previous study on cocoa nutrient also found that leaf age and leaf position on a twig or branch are closely related. Preliminary findings from mature monocrop cocoa in a Dunlop estate showed significant changes in leaf nutrient level as affected by leaf age and position (Thong, 1976). Similar results have been reported by Wessel (1971). In general guideline for young cocoa in particular at the nursery stage, it is wise to sample all leaves to analyse the nutrient status in the plants. Soil analyses may offer better diagnostic tool to know the nutrient levels in the soil while nutrient deficiency in the plants can be detected through the visual nutrient deficiency symptoms rather than leaf analyses. Later, nutrient requirement can be adequately supplied to the plants.

Roots

Malaysian Cocoa Journal

Table 20. Effect of leaf position on the nutrient content (%) for 6 month-old seedlings Position Leaf1 Leaf2 Other leaves N 1.91a 1.97b 2.33c P 0.16a 0.17a 0.20b K 2.00a 1.50b 1.77b Ca 1.24a 1.24a 1.13b Mg 0.37a 0.38a 0.39a

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Table 21. Effect of leaf position on the nutrient content (%) for 9 month-old seedlings Position Leaf1 Leaf2 Other leaves N 2.00a 1.97b 2.16c P 0.15a 0.12a 0.15b K 2.00a 1.50b 1.64b Ca 0.63a 1.02a 1.08b Mg 0.36a 0.45a 0.41a

Means followed by the same letters in the column are not significantly different at P<0.05 by LSD Regression analysis on the effect of various rates of P application on the nutrient content Application of various P rates had significant either linear or quadratic regressions to some nutrient contents in the plant parts either positive or negative regression as shown in Table 22. This indicates that some nutrients increased with the increasing rates of P applied. In most cases P increased in all plant components as well as for Ca and Mg whereas K increased in the root The increase in P, Ca and Mg may be attributed from the absorption of these elements contain in monocalcium phosphate which consists of P, Ca and Mg elements. The increase of other elements may be the result of efficiency of roots to absorb other elements in the soils. N has negative regression with the increase of P application in the stem and roots. The negative effect of N may be attributed from the increase of activities for the formation of N and P bases in the plants such as nucleoprotein, chlorophyll and ATP and other compounds, thus N become less in the plants. As the rate of P used was restricted to 400 ppm it was predicted that some nutrient contents in the plant components might achieve the plateau point at higher rates of P application as it would reach the saturation point or at the amount which is adequately required by the plants for its growth especially those nutrients that have positively quadratic regression.

Table 22. Regression analysis on the effect of various rates of P application on the nutrient content (%) in plant components Plant components N ns *(-) **(-) Linear regression P K Ca ** ns * ** ns ** ** * * F-test Mg ns ** ** Quadratic regression N P K Ca ns ** ns ns **(-) ** ns ** **(-) ** ns ns Mg * ** **

Leaves Stem Root

Note: Linear regression : Quadratic regression : * - significant at = 0.05 ns - not significant

Y = a + bX Y = a + bX + cX2 ** - significant at = 0.01 (-) - negative regression, others positive

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Regression analysis: effect of various rates of P application to the chemical soil properties The application of P at various rates did not have any effect on the availability of other nutrients in the soil except the increase in P availability in the soils. The result indicated that higher P application resulted in

higher availability of P in the soil whereas other nutrients, C/N, CEC and pH were not affected by various rates of P application (Table 23). The availability of other nutrients are independence from P availability in the soil.

Table 23. Regression analysis on the effect of various rates of P application on soil chemical properties for soil polybags of 6 and 9 month-old seedlings Regression Linear Quadratic N ns ns P **(0.766/0.941) **(0.766/0.985) K ns ns Ca ns ns F-test Mg ns ns C% Ns Ns C/N ns ns CEC ns ns pH ns ns

* - significant at =0.05 ; the number in the parentheses is the R square at 6 and 9 month-old seedlings. ** - significant at =0.01 ns - not significant

RESULTS AND DISCUSSION P is an essential element for the growth of cocoa seedlings in particular for the soil with low availability of P. In our study, application of P on the soil from Table series which is low in P availability demonstrated good response to cocoa seedlings. One month application of P had no any distinct effect on the growth until after 2 months significant different for P application was observed for number of leaves, height of plants and girth size. The application of P increased P content in the plant as well as the other major nutrients. Higher rates in P application resulted better plant growth. This result indicated that at the early cocoa growing stage, high P was required to establish the plant. Leaf position had significant different in the nutrient content, therefore it is difficult to diagnose the nutrient level based on leaf analysis in the plant as a basis in fertiliser application for the seedlings. Only soil data may provide better indication of nutrient requirement for plants. In this study, there was no plateau point of P content as affected by various rates of P application. Higher rates of more than 400 ppm might be required to achieve the plateau point of P content. The study found that at 100 to 400 ppm of P application better plant growth was observed. In practical use, we recommend 100200 ppm for the P fertiliser application to the young cocoa seedlings.

ACKNOWLEDGMENT This project was funded by IRPA. The authors wished to thank to the Ministry of Science and Innovation for the funding. Thanks also due to the Director General of Malaysian Cocoa Board for the permission to publish this paper and Director of Biology for some meaningful advises for implementation of the project. The dedicated works by Mohd. Yusof Mohd. Yunus and Hajal Manggal are also appreciated. REFERENCES Ahenkorah, Y. (1968). Phosphorus retention capacities of some cocoa growing soils of Ghana and their relationship with soil properties. Soil Sci., 105: 24. Besford, R.T. (1979). Effect of phosphorus nutrition in peat on tomato plant growth and fruit development. Plant Soil 51: 341. Black, C.A. 1965. Method of Soil Analysis. Part I and II, American Society of Agronomy: Wisconsin. De Geus, J.G. (1973). Fertilizer Guide for the Tropics and Subtropics. Centre dEtude de lAzote, Zurich, Switzerland. Jadin, P. (1976), Relationships between the chemical potentials of the soils in Ivory Coast and the production of cocoa trees. Cafe,Cacao Th 20(4): 287.

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Ling, A.H. and B.J. Mainstone (1982). Phosphate requirement of cocoa on Malaysian inland soils. In Puspharajah, E. and Sharifuddin, H.S. Hamid, eds., Phosphorus and Potassium in the Tropics, Kuala Lumpur. Malaysian Soc. of Planters., Kuala Lumpur. Malaysian Soc. Soil Sci. pp. 365-381. Ling A.H. (1984). Cocoa nutrition and manuring on inland soils in Peninsular Malaysia. The Planter, Kuala Lumpur 60(694): 12 24. Mainstone, B.J., Thong, K.C. and Tan, K.S (1973). Effect of lime and rock phosphate addition upon growth of young cocoa. Proc. Conf. Fert. Chem. Trop. Soils. Kuala Lumpur, November 193. Malaysian Society of Soil Science. Metson, A.J. 1956. Method of chemical analysis for soil survey samples. Bull.12, Soil Bureau, Department of Scientific and Industrial Research.

SIRIM (1980a). Recommended methods for soil chemical analysis, MS 679: Part I to V, Malaysia. SIRIM (1980b). Recommended methods for plant chemical analysis, MS 678: Part I to VIII, Malaysia. Thong, K.C. (1976). Problems associated with leaf and soil analyses techniques in relation to cocoa. Proc. Sem. On Soil and Leaf Analytical and Interpretations MSSS, Serdang 1976. p. 96. Wessel, M. (1970). Fertiliser requirement of cacao in South-Western Nigeria. Communication No. 61 of Dept. of Agri. Res. of the Royal Tropical Institute, Netherland. Wessel, M. (1971). Fertiliser requirements of cacao (Theobromae cacao L.) in South Western Nigeria. Comm. 61; Royal Trop. Inst., Amsterdam. The Netherlands; pp.104.

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Entomology EFFECT OF INSECTICIDES ON PARASITISM OF COCOA POD BORER EGGS BY TRICHOGRAMMATOIDEA BACTRAE FUMATA* NAGARAJA (HYMENOPTERA: TRICHOGRAMMATIDAE) ON COCOA IN MALAYSIA Azhar, I.1 and Long, G. E.2 Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Malaysia 2 Department of Entomology, Washington State University, Pullman, WA 99164-6382, USA
1

Malaysian Cocoa J. 3: 13-19 (2007) ABSTRACT - Chemical field trials were conducted to determine the effects of cypermethrin and deltamethrin on parasitism of the cocoa pod borer (CPB), Conopomorpha cramerella (Snellen) by Trichogrammatoidea bactrae fumata Nagaraja (TBF). Cypermethrin resulted in higher TBF mortality (94.9 1.1%) than deltamethrin (88.3 1.7%). TBF mortality due to insecticidal sprays was lower in older parasitized eggs but was relatively high (>70%) regardless of the timing of spraying relative to age of parasitized eggs. Cypermethrin reduced CPB oviposition, TBF parasitization and emergence up to twice as much as deltamethrin did. The high mortality resulting from regular insecticide applications may have led to failure to recognize the potential of TBF as a biological control agent of CPB. Key words: Insecticide, Cocoa, Cocoa pod borer, Egg parasitoid INTRODUCTION The outbreak of the cocoa pod borer (CPB), Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae), on cocoa, Theobroma cacao L. (Sterculaceae), in Tawau, Sabah, in late 1980 was not anticipated by cocoa growers. The lack of comprehensive information about the biology and ecology of the CPB led to the institution of containment and eradication programs based mainly on rampasan and insecticides. While rampasan, or the removal of all cocoa pods >2.5 cm long, was aimed at creating a host-free period, it was however unsuccessful and labor intensive. Similarly, intensive insecticide spraying proved unsuccessful as the CPB continued to menace cocoa and to spread to other cocoa planting areas in the state. By 1983, the pest spread into cocoa areas of the neighboring state Sarawak and was discovered in Peninsular Malaysia in 1986 (Zam and Azhar, 1992). Currently, the pest has spread to all major cocoa growing areas in Malaysia, Indonesia and Papua New Guinea. Although current management programs recommended the use of integrated pest management (IPM) (Azhar, 1995), many growers, especially the larger cocoa estates, continue to rely on insecticides. Other components of IPM, including proper pruning and post-harvest management (Azhar, 1990), the use of resistant cocoa clones (Azhar, 1995), pod sleeving (Saripah and Azhar, 2003), sex pheromone for mating disruption and trapping Alias et al., (2004), as well as release of egg parasitoids (Azhar et al., 1999; Azhar et al., 2000), are less often used for reasons ranging from limited technical knowledge to the high cost of these components. Applications of insecticides require relatively little technical knowledge, are easy to schedule, and are relatively cheap. However, heavy reliance on insecticides creates instabilities in the relatively fragile cocoa agroecosystem that may lead to increasing environmental cost and resurgence of other pests by limiting effectiveness of natural enemies (Conway 1972; Azhar 1985). Two insecticides commonly used by cocoa growers are the synthetic parathyroid cypermethrin and deltamethrin (Tay et al., 1990) and their extensive use had resulted in the development of resistance in CPB in Sabah (Lee et al., 1996). An egg parasitoid, Trichogrammatoidea bactrae fumata Nagaraja (TBF), was discovered in association with CPB in 1982 (Lim, 1983). The use of TBF as a biological control agent of CPB through augmentative and inundative releases was made possible by rearing the parasitoids on eggs of the rice moth, Corcyra cephalonica (Stainton), (Lepidoptera: Pyralidae). TBF releases in Sabah have been limited to experimental plots in governmental research stations and large commercial plantations. Although TBF was effective in lowering pod damage in release plots (Lim and Chong, 1987), the practice was considered to be not cost-effective.

* Trichogrammatoidea bactrea fumata is now known as Trichogrammatoidea cojuangcoi (Alias et al 2004). 13

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Adult TBF may live up to 15 days (Lim, 1986), so are likely to come into contact insecticide residues repeatedly in frequently sprayed fields. Negative effects of insecticides on Trichogramma spp. and other egg parasitoids have been reported (e.g., Stern, 1963; Plewka et al., 1975, Navarajan et al., 1979, Bull and House, 1983; Tipping and Burbutis, 1983; Jacobs et al., 1984; Kao and Tzeng, 1986, Smilanick et al., 1996). There is therefore a need for information about the effects on TBF of insecticides commonly sprayed on cocoa. Studies were therefore made to ascertain the effect of the timing of insecticide application on TBF emergence and the effect of insecticides on the pattern of parasitism by TBF in the field. MATERIALS AND METHODS Study site Studies were conducted in Blocks 14 and 22 at the Cocoa Research Station, Department of Agriculture Sabah approximately 35 km northeast of Tawau, Sabah, Malaysia. Effect of the insecticides on parasitoid emergence Adult moths were obtained from pupae collected from dried leaves placed over harvested pod heaps to trap the emerging pre-pupae. Pupae were held in three large emergence cages (0.6 X 0.6 X 0.6 m) for the adults to emerge and mate. Two-day old moths (3 females:1 male) were released into an oviposition sleeve-cage containing a susceptible cocoa pod (Azhar and Long, 1996) and allowed to oviposit overnight. The oviposition cage consisted of muslin cloth supported by a wire frame to prevent the cloth from collapsing onto the pod. Each morning, the moths were transferred to another oviposition cage containing a fresh, susceptible pod. The moths were killed when their total egg production declined to below 10 per night. An excess of 1-day-old TBF adults (>1000 individuals) was released onto pods in the sleeve cages with fresh CPB eggs to allow the majority of the eggs to be parasitized. TBF were obtained from the Cocoa Research Station, Department of Agriculture Sabah where they were mass-reared using of the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae), eggs (Azhar and Long 1996). Prior to insecticide treatment, the pods were isolated by placing a band of grease (petroleum based) on the branch to each side of the pod stalk to prevent ants from attacking parasitized eggs. Pods were sprayed with deltamethrin and cypermethrin at recommended 14

rates (Table 1) on either 0, 2, 4 or 7 days after TBF parasitization (DAP) using a pneumatic knapsack sprayer. Control pods were sprayed with water. Parasitoid emergence and mortality were recorded. Table 1. Insecticides used in the study Application rate a.i. (%) (a.i./ha) Cypermethrin 5.66 EC 15.0 Deltamethrin 1.40 EC 4.5 a Both insecticides were applied on 18, 30 September, 10, 19 & 28 October 1989 Effect of insecticides on field parasitism Fifteen plots, each comprising of 6 trees, were established in each block. Insecticides were applied using a powerdriven mistblower capable of reaching higher pods in trees. Application rates and timing for each chemical treatment are shown in Table 1. Each treatment was replicated five times in a completely randomized design within each cocoa block. Water was sprayed as the control. About 12,500 TBF were released at four grid points every week in each block beginning in 1988. CPB egg densities and parasitism by TBF before, during and after treatment were determined by counting the number of eggs on susceptible pods on each tree at weekly intervals. Sampling was carried out over a period of 20 wk from the 1st-week of August to the 2nd-week of December 1989. Treatments were applied at weekly interval from 18 September and 1 November 1989. Statistical analyses Analysis of variance was performed using PROC GLM (SAS Institute 1988) to compare the effect of treatments on CPB oviposition, TBF parasitism and emergence relative to application times (i.e., before, during and after). Means were separated using Duncans multiple range test. Mean number of CPB eggs/pod, and percentages of parasitized CPB eggs and emergence of TBF from the parasitized eggs were transformed, using (x + 1) and arcsine (x) transformations respectively, to normalize the variances. Percent TBF mortality was adjusted using a formula modified after Immaraju et al. (1992): Adjusted % mortality = [((NB x C) - NA) / (NB x C)] x 100 where NB is the number of parasitized CPB eggs before treatment, NA is the number of TBF emerging after treatment, and C is the number of TBF emerging divided by the number of parasitized eggs in the control.

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RESULTS Effects of insecticide on parasitoid emergence TBF mortality within the CPB eggs following insecticide application decreased with increasing age of parasitized CPB eggs (Figure 1). Cypermethrin killed significantly more eggs (94.9 1.1%) on TBF than deltamethrin (88.3 1.7%) (Duncans multiple range test; P < 0.01) when TBF mortality was pooled over all ages of parasitized CPB eggs. It was more toxic than deltamethrin regardless of age of parasitized CPB eggs as indicated by the slope of the regression line for each insecticide (Figure 1). TBF mortality from both insecticide was significantly greater when the eggs were sprayed at 0 and 2 DAP than at 4 or 7 DAP (Table 2). However, regardless of the timing of these insecticides spraying in relation to the age of parasitized CPB eggs TBF mortality remained relatively high (>70%). Table 2. Mean adjusted mortalities of TBF treated with both cypermethrin and deltamethrin on different days after parasitization. DAP Percentage mortality 0 96.5 0.9a (84.7) 2 97.3 0.7a (85.2) 4 90.4 1.5b (77.9) 7 83.6 3.2c (72.1) Means followed by different letters are significantly different (P < 0.01) (Duncans multiple range test). Values in brackets are arcsine % transformed of mean adjusted values (%)

Effect of insecticides on TBF parasitism in the field Insecticide treatment significantly reduced both the percentage of TBF parasitization and emergence (P < 0.01) (Table 3). The effect of period of treatment was significant for all of the treatments on CPB oviposition, and TBF parasitization and emergence (P < 0.05) (Table 3). Over the treatment period (week 8-12), cypermethrin reduced CPB oviposition, and TBF parasitization and emergence by ca. 2, 1.6 and 1.3 times more than deltamethrin (Table 4). Although CPB oviposition and TBF emergence were significantly reduced in plots treated with deltamethrin during the treatment period (week 8-12), TBF parasitism levels were not distinguishable from levels before the treatment period (week 1-7) (Table 4). The negative impact of insecticide on CPB oviposition and TBF parasitization and emergence was generally observed in treated plots lasting to about four weeks after treatment, hereforth referred to as residual period (i.e. week 12-16; Figures 2, 3 and 4). During this residual period (about 4 weeks), the TBF emergence was severely depressed in cypermethrin treated plots. Interestingly, there was a resurgence of CPB oviposition in the insecticide treated plots after the residual period (>17th-week), while on the other hand, it continued to decline in the control plots (Figure 2). TBF parasitization and emergence generally recovered in all plots after the residual period (week 16 and after) and were noticeably higher in the deltamethrin treated plots, i.e. >15% and >23% respectively (Figures 3 and 4). DISCUSSION The results demonstrate that the recommended application rates for the insecticides to control the CPB are detrimental to TBF. These results are consistent

Figure 1. Relationship between mean adjusted mortality of TBF time of insecticide application 15

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Table 3. ANOVA for the effect of insecticide treatments on CPB oviposition, and TBF parasitism and emergencea. TBF TBF Eggs/pod Parasitism (%) Emergence (%) df F P F P F P Source of variationb Blk 1 5.97 0.0148 2.73 0.0988 12.22 0.0005 Trt 2 1.71 0.1814 5.38 0.0048 10.14 0.0001 Blk x Trt 2 1.30 0.2727 0.86 0.4254 1.79 0.1671 Time 2 17.58 0.0001 3.36 0.0356 10.11 0.0001 Blk x Time 2 2.81 0.0611 5.27 0.0054 11.10 0.0001 Trt x Time 4 0.81 0.5216 0.46 0.7673 0.34 0.8493 Blk x Trt x Time 4 0.78 0.5414 1.68 0.1542 1.13 0.3411 Error 571 a CPB oviposition is the (x + 1) transformed mean number of eggs/pod; TBF parasitism and emergence are the arcsine x transformed percentages. b Blk = Block; Trt = treatment; Table 4. Numbers of CPB eggs, TBF parasitization and TBF emergence before, during and after insecticide treatments in the field. Mn. no. of eggs/pod TBF parasitization (%) TBF emergence (%) Timing Cyper Delta Contr Cyper Delta Contr Cyper Delta Contr Before 0.73aA 0.72aA 0.81aA 5.7aA 5.9aA 7.2aA 26.5bA 26.6bA 41.9aA During 0.23bC 0.31bB 0.50aB 2.7bB 5.4bA 7.4aA 10.1bB 16.3bAB 30.2aAB After 0.54aB 0.67aAB 0.53aB 4.8aAB 4.9aA 7.6aA 12.1aB 13.2aB 22.7aB Means followed by the same letter are not significantly different (P>0.05, Duncan multiple range test). Lower case letters are for comparison of values within a treatment time (within row) while upper case letters are for values between treatment time (within column). Delta = Deltamethrin; Contr = Control; Cyper = Cypermethrin

Figure 2. Numbers of CPB eggs before, during and after insecticide treatments

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Figure 3. Effect of insecticide spraying on parasitism of CPB eggs by TBF

Figure 4. Effect of insecticide treatments on TBF emergence from parasitized CPB eggs (%)

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with other studies documenting the negative effects of insecticides on Trichogramma spp. (e.g., Stern, 1963; Navaranjan et al. 1979; Tipping and Burbutis, 1983; Bull and House, 1983; Kao and Tzeng, 1986) and other natural enemies (Smilanick et al., 1996; Murchie et al., 1997). Compared to deltamethrin, cypermethrin causes greater mortality of eggs parasitized by TBF. This is probably due to the higher rate of application and higher percentage of active ingredient compared to that of deltamethrin (Table 1). Despite the lower impact of deltamethrin, both insecticides cause significant mortality to TBF and could be due to the direct result of insecticides application. The insecticides might have killed the developing parasitoid larvae within the CPB eggs, hence reducing the TBF emergence. Although certain proportion of the parasitoid may emerge they may come into direct contact with insecticide residue causing mortality or reduce their fecundity. Another factor causing direct mortality to the TBF is the contact of the TBF adults with the insecticide residue especially lowers its effectiveness as a biological control agent. The mortality to TBF could be due to the direct or indirect result of insecticides application. Insecticide drift may also cause severe mortality to adult TBF and all developmental stages within parasitized eggs resulting in reduced performance of TBF. This is shown by the similar trend of reduced percentage of TBF emergence in the control compared to the two insecticide treatments (Figures 3 and 4). Direct mortality to adult parasitoids as a result of drift has been observed to severely limit the performance of released T. pretiosum Riley on cotton where 75% of caged parasitoids suffered mortality due to the drift of methyl parathion from neighboring sprayed fields (Stinner et al., 1974). In addition to direct mortality on TBF, the insecticide residues may have nonlethal but adverse effects on the later performance of the parasitoid, such as impairing adult searching capacity. Since fewer CPB eggs are parasitized by TBF and the reduced emergence after insecticide treatment, the potential of TBF as a biological control agent of CPB is not compatible with regular insecticide applications. In order to minimize the adverse impact of insecticides on TBF, the parasitoids could be released after the residual period. The recovery to near normal of TBF parasitism and emergence following the period supports this suggestion. This strategy is feasible where insecticides are applied at specific stages of crops phenology, such as the 4-6 sequential applications during the low crop seasons (Sidhu et al., 1987). 18

These data should permit the integration of parasitoid releases and insecticide applications. Although carefully-timed releases of TBF could be used to compliment the insecticide applications, further work is needed on levels of application of insecticide that are least detrimental to TBF and on the persistence of insecticide residues on cocoa. ACKNOWLEDGEMENTS We thank Roslan Saadi, Abd. Karim Ramli, Zailani Mohd Jamil, and Norhazazi Mohd Alimuddin (all formerly at MARDI Tawau) for their technical assistance. William J. Turner and Lynell K. Tanigoshi of the Department of Entomology, Washington State University (WSU), and Paul C. Schroeder of the Department of Zoology, WSU, kindly read and criticized the manuscript. This paper forms part of the MARDI-JPNS collaborative project on cocoa pod borer, and was supported in part by (1) the American Cocoa Research Institute (ACRI), and (2) the Washington State Universitys College of Agriculture and Home Economics Agricultural Research Center: Department of Entomology Project 0355 - Ecosystem Interactions of Economically-Important Insects. REFERENCRES Alias, A., Azhar, I. and Schilthuizen, M. (2004). Taxonomic status of Trichogrammatoidea bactrae fumata Nagaraja, an egg parasitoid of the cocoa pod borer, Conopomorpha cramerella (Snellen) in Malaysia.: A critical review. Malaysian Cocoa J. 1: 83-85. Azhar, I. (1985). Natural and cultural control in the management of selected cocoa pests in Malaysia, pp. 255-267. In B. S. Lee, W. H. Loke and K. L. Heong (eds.), Integrated pest management in Malaysia. Kuala Lumpur: Malaysian Plant Protection Society (MAPPS). Azhar, I. (1990). Postharvest management of cocoa pod borer. MARDI Res. J. 18: 71-80. Azhar, I. (1995). An overview on the management of key insect pests of cocoa with major emphasis on the cocoa pod borer, Conopomorpha cramerella. The Planters 71: 469-480. Azhar, I. and Long, G.E. (1996). Effect of cocoa pod age on distribution and egg parasitism of the cocoa pod borer in Malaysia. Entomol. Exp. Appl. 81: 81-89. Bull, D. L. and House, V. S. (1983). Effects of different insecticides on parasitism of host eggs by

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Trichogramma pretiosum Riley. Southwest. Entomol. 8: 46-53. Conway, G. R. (1972). Ecological aspects of pest control in Malaysia, pp. 467-488. In M. T. Farver and J. P. Milten (eds.) The careless technology - ecology and international development. New York: Natural History Press. Immaraju, J. A., Paine, T. D., Bethke, J. A., Robb, K. L. and Newman, J. P. (1992). Western flower thrips (Thysanoptera: Thripidae) resistance to insecticides in coastal California greenhouses. J. Econ. Entomol. 85: 9-14. Kao, S. S. and Tzeng, C. C. (1986). Initial toxicity tests of 24 pesticides to adults of Trichogramma chilones Ishii, p. 197. Proceedings of the 2nd. International Conference of Plant Protection in the Tropics. Kuala Lumpur: Malaysian Plant Protection Society. Jacobs, R. J., Kouskolekas, C. A. and Gross, Jr., H. R. (1984). Responses of Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) to residues of permethrin and endosulfan. Environ. Entomol. 13: 355-358. Lee, C. T. (1996). Insecticides resistance in cocoa pod borer, Conopomorpha cramerella Snellen and the strategy to prevent its development. The Planters 72: 593-599. Lim, G. T. (1983). Trichogrammatoidea bactrae fumata Nagaraja (Hymenoptera: Trichogrammatidae) a new egg parasitoid of Acrocercops cramerella Snellen. MAPPS Newsl. 7 (Suppl.): 5-6. Lim, G. T. (1986). Biological studies on Trichogrammatoidea bactrae fumata Nagaraja in the laboratory. Zeit. Angew. Entomol. 101: 48-54. Lim, G. T. and Chong, T. C. (1987). Biological control of cocoa pod borer by periodic release of Trichogrammatoidea bactrae fumata Nagaraja in Sabah, Malaysia, pp. 71-80. In P. A. C. Ooi, G. C. Luz, K. C. Khoo, C. H. Teoh, M. Md. Jusoh, C. T. Ho and G. S. Lim [eds.], Management of the cocoa pod borer. Kuala Lumpur: Malaysian Plant Protection Society (MAPPS). Murchie, A. K., Williams, I. H. and Alford, D. V. (1997). Effects of commercial insecticide treatments to winter oilseed rape on parasitism of Ceutorhynchus assimilis Paykull (Coleoptera: Curculionidae) by Trichomalus perfectus (Walker) (Hymenoptera: Pteromalidae). Crop Protect. 16: 199-202. 19

Nagaraja, H., Easaw, P. T., Vanialingam, T., Salvador, A., Nunez, E. F. and Galbizo, T. C. (1983). An egg parasite of the cocoa pod borer Acrocercops cramerella (Snellen) recorded in the Philippines. The Planter 59: 355-357. Navarajan, P., Dass, R., Ahmad, R. and Parshad, B. (1979). Effect of some insecticides on parasitism by the parasitoid Trichogramma brasiliensis (Trichogrammatidae: Hymenoptera). Z. Angew. Entomol. 88: 399-403. Plewka, T., Kot, J. and Krukierek, T. (1975). Effects of insecticides on the longevity and fecundity of Trichogramma evanescens West. (Trichogrammatidae: Hymenoptera). Polish Ecological Studies, 1, 197-210. Sidhu, M. J., Sim, C. H. and Johney, K. V. (1987). Practical aspects of chemical spraying for cocoa pod borer management in Sabah), pp. 19-40. In P. A. C. Ooi, G. C. Luz, K. C. Khoo, C. H. Teoh, M. Md. Jusoh, C. T. Ho and G. S. Lim [eds.] Management of the cocoa pod borer. Kuala Lumpur: Malaysian Plant Protection Society (MAPPS). SAS Institute. (1988). SAS/STAT Users Guide, Rel. 6.03 edition. Cary, N.C: SAS Institute Inc.. Pp. 1028. Smilanick, J. M., Zalom, F. G. and Ehler, L. E. (1996). Effect of methamidophos residue on the pentatomid egg parasitoids Trissolcus basalis and T. utahensis (Hymenoptera: Scelionidae). Biol. Contr. 6: 193-201. Stern, V. M. 1963. The effects of various insecticides on Trichogramma semifumatum and certain predators in southern California. J. Econ. Entomol. 56: 348-350. Stinner, R. E., Ridgway, R. L., Coppedge, J. R., Morrison, R. K. and Dickerson, Jr., W. A. (1974). Parasitism of Heliothes eggs after field releases of Trichogramma pretiosum, in cotton. Environ. Entomol. 3: 497-500. Tay, E. B., Azhar, I. and Chan, H. H. (1990). Research and management of the cocoa pod borer. Proceedings of the MCGC-Malaysian Cocoa Board Workshop on Cocoa Agricultural Research 25-26 July, 1989. Kuala Lumpur: Malaysian Cocoa Growers Council, pp. 93-107. Tipping, P. W. and Burbutis, P. P. (1983). Some effects of pesticide residues on Trichogramma nubilale (Hymenoptera: Trichogrammatidae). J. Econ Entomol.76: 892-896. Zam, A. K. and Azhar, I. (1992). Cocoa pod borer on the move. MAPPS Newsl. 16: 35-36.

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Processing and Product Development PHYSICAL STABILITY AND COLOR CHANGES OF MALAYSIAN COCOA BUTTER UPON STORAGE Asimah, H. Malaysian Cocoa Board, Cocoa Downstream Research Center, Lot 3, Jln P/9B, Section 13, 43650 Bandar Baru Bangi, Selangor, Malaysia Malaysian Cocoa J. 3: 20-25 (2007) ABSTRACT - Stability of Malaysian cocoa butter upon storage was studied for duration of 18 months. Twenty five samples of pure prime pressed cocoa butter were collected from local grinders for the stability study. The samples were packed in corrugated paper boxes lined with LLDPE plastic bags with a thickness of 65.8 m. The GTR and WVTR properties of the LLDPE plastic material are 6.87 cc/mm2/day at 23C, r.h. 70% and 3.76 g/ m2/ day at 37.8C, r.h. 90 2% respectively. The samples were stored at 28 3C and r.h. 85 4 %. Free fatty acid of the cocoa butter reached 1.75% by the 14th month of storage and the peroxide value was almost 4mEq/kg throughout storage duration. However FFA and PV values were below upper limits specified in Malaysia. The color changes of the cocoa butter were not significantly affected upon storage. Key words: Cocoa butter, Physical stability, Storage. INTRODUCTION In Malaysia, cocoa grinder produces cocoa powder, liquor, cocoa cake and butter. Supply of these products depends on the demand and buyers specification. Two types of cocoa butter produced in the market are the pure prime pressed butter and the deodorized cocoa butter. Cocoa butter is used in chocolate manufacturing, medical, pharmaceutical, and toiletry productions. Types of butter used in manufacturing depend on the purpose and the functional properties of the product. Cocoa grinders and chocolate manufacturer have reported differences in the physical and chemical properties from different source of beans, and indicated their effect on the quality of the finished products. Kattenberg (1981) has noted that stability of cocoa butter depends on crystallization of butter, tempering procedure, and storage condition. Good quality cocoa butter is hard and brittle at room temperature. It has relatively short melting range, liquefying between 27C-35C and melt sharply just below body temperature and leaving no greasy film or sensation on the product. Malaysian cocoa butter is the hardness and saturated fat compared to other origins cocoa butter such as Ghana, Ivory Coast and Brazil (Chin, A. H. G., 1989). Packaging provides protection for the products and preserved quality to ensure customers need and satisfaction. For marketing of products, proper packaging and presentation are important. Packaging is also the communication link between the producer or manufacturer and the customer. Good packaging systems or methods will preserve quality and prolong the shelf life of the products. High requirement of protective packaging for foodstuffs is important in order to protect the products from humidity and temperature fluctuation, volatile compound, and improper handling during transportation and storage. High temperature and relative humidity may accelerate undesirable chemical, physical and microbiological changes and resulting in rancidity, aroma loss, changes in color and other quality deterioration. Cocoa butter is often stored to maintain a sufficient stock for factory operation and sometimes as stockpiles for hedging in the market. The duration of butter storage is influenced by factors such as the market price and demand. Sometimes cocoa butter stored for more than one year to maintain sufficient stock as buffer against price fluctuations. Therefore, the objective of this research was to study stability of pure prime cocoa butter upon storage.

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MATERIALS AND METHODS Materials Samples of commercial pure prime pressed cocoa butter were obtained from local cocoa grinders. All chemicals were analytical grades. Experimental design In this study a complete randomized design was employed. Approximately 5 kg of samples were packed in corrugated paper boxes lined with LLDPE (Linear Low Density Polyethylene). The samples in boxes were stored at 283C and r.h. 854 %. Samples were collected every two months and analyzed for the physical tests. All analyses were done in triplicate. Plastic material properties Plastic material was tested for water vapor transmission rate and gasses transmission rate for oxygen using GTR Toyosyeiki tester, and ASTM D3985-95 method. Average thickness of the plastic material was measured using a calibrated micrometer at a minimum of five points distributed over the entire test areas. Maximum, minimum and average values were recorded. FAME (fatty acid methyl ester) analysis Cocoa butter 0.1 ml was dissolved in 1 ml of n-hexane. Then 1ml of sodium methoxide was added and mixed with a vortex shaker to obtain the methyl esters of the fatty acids. After 30 min. 1 l of the clear supernatant was injected into silica capillary column SGE BPX 70, 60 cm x 0.32 m of the gas chromatograph (GC). The column was purchased from Servco Services Sdn. Bhd., Selangor. FAME was detected with Shimadzu17A gas chromatography, Flame Ionization Detector (FID). The carrier gas was helium with purge flow 3ml/min. Column oven condition is 90C (5min.) to 400C at 8C/min. FFA (free fatty acid) test This test was done according to method of SIRIM (MS1119: 1988). Cocoa butter were melted at 50C and filtered with filter paper Whatman no.4. Approximately 5g of butter was placed into 250ml conical flask and added with 50 ml of hot alcohol, which has been neutralized with 0.01N sodium hydroxide (NAOH) and gently heated. Five ml of thymol blue indicator was added (to the hot mixture),

and titrated with 0.1N NaOH to a khaki color (stable for 30s) end point. FFA as percentage of oleic acid was calculated. Peroxide value (AOAC, 1989) Cocoa butter was melt at 50C and filtered with filter paper (Whatman no.4). 5g of oil was put into a 250 ml conical flask and added with 30ml of acetic acidchloroform solution (mixture of 3 volume of acetic acid with 2 volume of chloroform). Saturated potassium iodide was added 0.5ml and shaked for 1 minute, and then 30ml of distilled water was added to the mixture. The mixture was titrated with 0.01N sodium thiosulphate solution with vigorous shaking until the yelllow color disappeard. Starch solution 0.5 ml was added later and titration was continued until the blue colour disappeard. A blank test was carried out in parrallel with the sample. Color measurement Color changes were determined by direct reading of the L, a and b value using Hunterlab Color Quest II, 45/0 spectrophotometer. The equipment was warm up for about 30 minutes and then calibrated. Approximately 52g of cocoa butter was placed into transmission cell and then placed on reflectance. Reading of L, a and b values were recorded using computer system attached to the colorimeter. Statistical analysis The data were analyzed by analysis of variance at 5% level (p<0.05) and the means separated by Duncans Multiple Range Test. The statistical analyses were done using SAS V6.12. RESULTS AND DISCUSSION The samples were packed in corrugated paper boxes lined with LLDPE plastic bags with a thickness of 65.8 m. Gas transmitted rate (GTR) and water vapor transmitted rate (WVTR) properties of the LLDPE plastic material are 6.87 cc/m2/day at 23C, r.h. 70% and 3.76 g/ m2/day at 37.8C, r.h. 90 2% respectively. This indicates that the packaging material used has good barrier against water vapor and gasses. Therefore it is able to maintain the quality of the product during storage and transportation.

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Table 1 shows the FAME percentage of fats. There is no significant variation in the composition of fats at initial and end of storage. The FAME profiles for both conditions are the same. This indicates there is not much loss in the unsaturated fatty acids. The free fatty acid for pure prime pressed cocoa butter increased steadily from 1.46 to 1.85 after 18 months of storage duration (Figure 1). Although the samples were protected with proper packaging and less exposure to light, air, humidity, temperature and other factors that may accelerate the hydrolysis of the

unsaturated lipid, it still deteriorates. Therefore like any other fat, cocoa butter can also deteriorate. Cocoa butter is one of the most stable lipids known due to the composition and structure of the cocoa butter gives it by nature an outstanding protection. Just over one third of all fatty acids present in the triacyl glycerols are unsaturated of which by far the largest part consists of oleic acid. Only about less than 10% are polyunsaturated linoleic acids, whereas the very unstable linolenic acid is virtually absent. In addition, almost all unsaturated fatty acids are located at the 2 position of the triglycerides, which allows for structural protection (Anon, 2001).

Table 1. FAME profile (%) of cocoa butter at beginning and end of storage FAME C12:0 C14:0 C15:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1n9t C18:1n9c C18:2n6t C18:2n6c C18:3n6G C18:3n63A C20:0 C20:1n9 C20:2 C20:3n6 C20:3n3 C22:0 C23:0 C24:0 C22:6n3 *Not significantly different at p<0.05. *Concentration at month 0 0.0075 0.1176 0.0223 25.475 0.2379 0.2755 0.0221 35.7032 0.0530 33.1918 0.0341 2.5397 0.0068 0.2299 1.2232 0.0510 0.0044 0.0199 0.0123 0.1980 0.4382 0.1085 0.0340 *Concentration at month 18 0.0075 0.1176 0.0223 25.875 0.2379 0.2755 0.0221 36.2032 0.0530 32.4918 0.0341 2.1397 0.0068 0.2299 1.2232 0.0510 0.0044 0.0199 0.0123 0.1980 0.4382 0.3085 0.0340

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Figure 1. Free fatty acid of cocoa butter during storage [S1: sample 1, S2: sample 2, S3: sample 3, S4: sample 4, S5: sample 5. Samples 1-5: pure prime pressed cocoa butter] The initial peroxide value for pure prime pressed cocoa butter is about 3.2mEq/kg (Figure 2). The peroxide values increased to 4.0 mEq/kg by 10th month of storage. There was no significant different (p< 0.05) among the samples upon storage duration. Therefore the finding indicated that Malaysian cocoa butter undergo oxidation upon storage and the peroxide value was at the maximum level allowed in Malaysian standard. In order to reduce the peroxide value it is advisable to keep the butter in modified packaging condition or under vacuum packing to prevent oxidation.

Figure 2. Peroxide value of cocoa butter during storage [S1: sample 1, S2: sample 2, S3: sample 3, S4: sample 4, S5: sample 5. Samples 1-5: pure prime pressed cocoa butter]

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Changes in color of the samples are measured in term of CIELAB (L, a, b) values. L indicates the lightness of the samples ranging from 0 to 100, a values indicates the redness of the sample and b values indicates the yellowness of the samples. The finding of the study showed that there was no

significant (p<0.05) changes in L, a and b values in all samples tested throughout the storage duration (Figures 3, 4 and 5). We can conclude that the color changes are insignificant because the samples were kept in boxes and not exposure to light.

Figure 3. L value intensity of cocoa butter [S1: sample 1, S2: sample 2, S3: sample 3, S4: sample 4, S5: sample 5. Samples 1-5: pure prime pressed cocoa butter]

Figure 4. a value intensity of cocoa butter [ S1: sample 1, S2: sample 2, S3: sample 3, S4: sample 4, S5: sample 5. Samples 1-5: pure prime pressed cocoa butter]

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Figure 5. b value intensity of cocoa butter [S1: sample 1, S2: sample 2, S3: sample 3, S4: sample 4, S5: sample5. Samples 1-5: pure prime pressed cocoa butter] CONCLUSION Malaysian pure prime pressed cocoa butter packed in corrugated boxes lined with low density polyethylene plastic material with thickness of 65.8 m and stored at temperature 28.3C and r.h. 85.4 % still undergo quality changes with minimal deterioration in free fatty acid and peroxide value. However the free fatty acid and peroxide values are below the acceptable limit given by Malaysian specification. No changes in color of the cocoa butter noted. Therefore pure prime cocoa butter is still in a good condition and acceptable after 18 months of storage. REFERENCES AOAC. 1989. Official Methods of Analyses of the Association of Official Analytical Chemists. 14th Edition, Washington D.C. Anon, 2001. The De Zaan Cocoa Products Manual. ADM Cocoa publication B.V. Chin, A.H.G. 1989. Cocoa butter characteristics of Malaysian clonal cocoa. MARDI Res. J. 17(1): 37-44 Kattenberg, H.R. 1981. The Quality of Cocoa Butter. Manufac. Confect. 67: 115. SIRIM 1988. Methods of analyses For Malaysian Cocoa Butter and Malaysian Cocoa Powder. Pub MS 1119.UDC543.663.918. Malaysia.

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EFFECT OF CARBON TO NITROGEN RATIOS (C/N) ON BENZALDEHYDE FORMATION FROM RHIZOPUS OLIGOSPORUS IN A SOLID-STATE FERMENTATION SYSTEM USING COCOA POD HUSKS AS SUBSTRATE Norliza, A.W. Malaysian Cocoa Board, Cocoa Downstream Research Center, Lot 3, Jln P/9B, Section 13 43650 Bandar Baru Bangi, Selangor, Malaysia Malaysian Cocoa J. 3: 26-34 (2007) ABSTRACT - Studies on cultivation of Rhizopus oligosporus in a solid state fermentation system (SSF) for the production of a flavoring compound, i.e. benzaldehyde were undertaken. The optimized carbon and nitrogen sources were determined upon screening process on glucose, fructose, lactose, maltose, sucrose and starch (carbon sources) and L-phenylalanine, aspartame, yeast extract, soy peptone and ammonium nitrate (nitrogen sources) as well. Each of the sources was embedded in cocoa pod husks (CPH) which has resulted in the largest production of 1.10 x 106 benzaldehyde abundance with 11.82 gl-1 mycelial growths for glucose and 1.11 x 105 benzaldehyde abundance with 15.85 gl-1 mycelial growths for aspartame. Furthermore, glucose and aspartame were mixed together in specific proportions namely, 1/1, 1/10, 1/20, 10/1 and 20/1 in order to see the affect of C/N ratio upon mycelial growth and benzaldehyde formation. Benzaldehyde production was the highest at C/N ratio (glucose/ aspartame) 1/20, i.e. 1.92 x 106 abundance with 25.70 gl-1 mycelial growth at the optimized condition of 24C after 96 hours fermentation. This mixture has indeed boosted the production as much as 42% as compared when glucose and aspartame were utilized separately. A large content of aspartame in C/N ratio, i.e. 1/20 has contributed in more compounds produced followed by 1/10, 1/1, and the least produced were from 10/1 and 20/1. The application of principal component analysis (PCA) method has proven to be idealistic in the acknowledgement of 1/20 sample as an optimum C/N ratio for the highest benzaldehyde production. Key words: Cocoa pod husks, C/N ratios, Benzaldehyde, Rhizopus oligosporus, Solid-state fermentation system INTRODUCTION Cocoa pod husks (CPH), an agro-waste of cocoa cultivation are usually left under the trees after pod breaking. Cocoa pod husks contained crude protein (5.7-9.7%), pectin (5.3-7.1%), nitrogen-free extract (44.2-51.3%), crude fiber (33.2-39.4%), and ash (8.810.2%). A hot-water-soluble polysaccharide, extracted from CPH in 2% yield, was shown to be composed mainly of L-rhamnose, L-arabinose, D-galactose, and D-mannose, together with small amounts of glucose, xylose, and an unidentified pentose (Blakemore et al., 1966). However, CPH are usually characterized by a lack of important physical, nutritional or sensory characteristics, which are needed if it is to be used as a source of nutrient (Zarkadas et al., 1995). Benzaldehyde is an aromatic flavor compound consisting of a benzene ring substituted with a carbonyl group containing one hydrogen atom (Figure 1). Aromatic compounds such as benzaldehyde and vanillin represent a very large market in the flavor industry. The consumer preference for products made with natural ingredients has directed research towards the exploitation of microbial biosynthetic pathways to produce natural flavors. Natural benzaldehyde is tied up as a glycoside (amygdalin) in the stones of apricots, peaches, cherries and in almonds and can be released by enzymatic hydrolysis. A drawback is that toxic byproducts such as hydrocyanic acid may be formed during the extraction process. Microbial production of benzaldehyde from phenylalanine has been reported in a submerged culture fermentation system (SmF) using various kind of microorganisms such as Lactobacillus plantarum (Nierop Groot and Jan, 1998), Polyporus tuberaster (Kawabe and Morita, 1994) and Phanerochaete chrysosporium (Jensen et al., 1994). Among bacteria, benzaldehyde formation has been reported for a strain of Pseudomonas putida in the mandelic acid pathway (Tsou et al., 1990) while incubation of a cell extract of Lactobacillus plantarum with phenylalanine revealed that benzaldehyde was

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indeed formed (Nierop Groot and Jan, 1998). Benzaldehyde production via solid-state fermentation (SSF) system has not yet been reported. However,

benzaldehyde was reported to be produced by a locally isolated Penicillium diversum cultivated in a palm kernel cake of SSF system (Sue Yie and Omar, 2000).

Figure 1. Molecular structure of benzaldehyde Moreover, Rhizopus spp. are microscopic filamentous fungi of the order Mucorales. They belong to the Phycomycetes (primitive fungi), subclass Zygomycetes (Onions et al., 1981; Fassatiova, 1986). Several Rhizopus species are of considerable interest for the food industry. They have been used in SSF system for several centuries, especially in Asia (China, Korea, Japan, Indonesia, Malaysia, Singapore, Java, etc.) for preparing many fermented foodstuffs (Hesseltine, 1965). Rhizopus not only enhances the digestibility and protein content of foodstuffs (Soccol et al., 1992), but also prevents the formation of toxic substances such as aflatoxin B1 (Ko, 1988). Therefore, an attempt has been made in utilizing CPH, which will be embedded with carbon and nitrogen sources for the formation of benzaldehyde in a SSF system. SSF can be defined as a method of culturing microorganisms (particularly fungus) on and/or within the particles of a solid matrix to produce biomass, enzymes, organic acids or special secondary metabolites such as mycotoxins and flavors (Bajracharya and Mudgett, 1980). The supplementation of nutrients (e.g. phosphorus, nitrogen and salts) is essential in order to convert a raw substrate into a form suitable for use (Raimbault, 1998). Earlier work (Norliza, 2003) showed the addition of Lphenylalanine, a precursor for the synthesis of benzaldehyde by Rhizopus oligosporus in a SSF system has given rise to the benzaldehyde production of about 38.69 mgg-1 substrate. The aim of this work was to study the effectiveness of CPH as an inert substrate, which were embedded with carbon and nitrogen sources for the production of volatile fragrance compounds particularly benzaldehyde. MATERIALS AND METHODS Preparation of cocoa pod husks (CPH) Cocoa pod husks from unidentified clones of CPH were collected from the Cocoa Research & Development Center, Hilir Perak, Malaysia. The CPH were then cleaned and sun dried before ground to a particle size of 1.00 mm by using Rotor Beater Mills. Preparation of Rhizopus oligosporus inoculum Rhizopus oligosporus strain NRRL 2710 was obtained from American Type Culture Collection (ATCC) and maintained on slants of potato dextrose agar (PDA) at 4C. For inoculums preparation, the organism was grown on malt extract agar plate at 24C for 7 days for complete sporulation. Twenty five ml sterile water was added to the plate and the spores were scraped with an inoculating loop in a Laminar flow hood. The spore suspension was used as the inoculums for subsequent fermentation (Tunga et al., 1998). Culturing of R.oligosporus NRRL 2710 The inoculums of R. oligosporus NRRL 2710 was inoculated into erlenmeyer flasks (250 ml) which contained 10 g of sterilized ground CPH with C/N ratio (w/w) namely, 1/20, 1/10, 1/1, 10/1 and 20/1, separately. Initially, the substrates were sterilized at 121C for 20 min and left to cool. The sterilized

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substrates were then added with 100% inoculums (1x10 5 spores/ml) and thoroughly mixed with sterilized glass rod prior to statically incubated at 24C. After 96 hours of fermentation, the mycelium was then harvested and dried at 105C for 3 hours and weighed (Yonekichi et al., 1977). Effect of carbon sources To find a suitable carbon source for the benzaldehyde production, fungus was cultivated in the medium (10 gm of CPH) which has been prepared earlier containing 2% w/w (0.2 g) of; (i) glucose, (ii) fructose, (iii) lactose, (iv) maltose, (v) sucrose, and (vi) starch, respectively inside the erlenmeyer flask. Sampling for the mycelial growth and benzaldehyde formation was done at the fourth day of fermentation. Effect of nitrogen sources To find a suitable nitrogen source for the benzaldehyde production, fungus was cultivated in the medium (10 gm of CPH) which has been prepared earlier containing 2% w/w (0.2 g) of; (i) L-phenylalanine, (ii) aspartame, (iii) yeast extract, (iv) soy peptone and (v) ammonium nitrate, respectively inside the erlenmeyer flask. Sampling for the mycelial growth and benzaldehyde formation was also being done at the fourth day of fermentation. SPME procedures The SPME device was purchased from Supelco Co. (Bellefonte, PA), as was the fused silica fiber coated with polydimethyl-siloxane/divinylbenzene (75 m PDMS/DVB). For this work, this type of fiber was used as it showed the greatest response for all compounds at optimal SPME sampling conditions 30 min of extraction time at 40C (Antonio et al., 2006). Instrumental analysis Analyses were performed using a Hewlett-Packard 6890 Gas Chromatograph, equipped with an HP-5MS, film thickness (0.25 m) capillary column, linked to an HP 5972A mass spectrometer system. The temperature of the injection block was 240C; the oven temperature program was 45C, rising at 7C/min to a final temperature of 200C. Peak areas and retention times were calculated using Hewlett-Packard

software. Helium was used as carrier gas at a flow rate of 1.00 ml/min. The sampling procedure involved placing 10 ml of the solution into a 20 ml vial and sealing with a screwtop septum-containing cap. The SPME needle was then inserted through the septum and suspended in the headspace of the vial to adsorb the analytes. The vial was placed in a water bath maintained at 40C for 30 min, where it equilibrated; for thermal adsorption, the SPME was allowed to remain in the injector for 15 min, splitless injection mode was used, the split valve being opened after 2 min (Garcia et al., 2004). Data analysis All data were obtained from triplicate assays and the reported values are means standard deviation. For SPME-GC/MS results, no external or internal standards were used in the present work. Results were described in terms of relative (%) areas and were visually reproducible. Since relative area was used no attempt to use any statistical method was made. The results described in the present work were taken from one chromatogram of each sample; thereby chromatographic data must be regarded qualitatively. RESULTS & DISCUSSION Effect of carbon and nitrogen sources on benzaldehyde production Benzaldehyde productions were the highest in the glucose and aspartame medium among those tested, 1.10 x 106 and 1.11 x 106, respectively (Table 1). Although mycelial growth was the highest in starch (14.64 gl-1) compared to 11.82 gl-1 in the sucrose medium, the benzaldehyde production of starch medium was the lowest (5.22 x 10 5). The same phenomenon can be seen when the fungus were grown in the nitrogen sources where the highest benzaldehyde abundance did not necessarily came from the maximum mycelial growth. In the case of nitrogen sources, aspartame was the largest producer of benzaldehyde (1.1 x 106) followed by L-phenylalanine (9.39 x 105) but yeast extract was the one with the highest mycelial growth (17.56 gl-1) compared to 15.85 gl-1 for aspartame.

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Table 1 shows that glucose (carbon source), L-phenylalanine and aspartame (nitrogen source) produced significant abundance of benzaldehyde after 96 hours fermentation. In R.oligosporus, swelling of the spores and subsequent emergence of germ tubes are triggered by glucose (Medwid and Grant, 1984). This explained the huge amount of benzaldehyde produced when glucose was embedded with CPH for fermentation process. Utilization of phenylalanine in the benzaldehyde formation has been widely reported for cultures of Ischnoderma benzoinum (Fabre et al., 1996; Krings et al., 1996), Polyporus tuberaster (Kawabe and Morita, 1994) and Phanerochaete chrysosporium (Jensen et al., 1994). Moreover, immobilization of the white-rot fungus Bjerkandera

adusta resulted in an increased production of benzaldehyde in a medium containing L-phenylalanine (Lapadatescu et al., 1997). Aspartame (N-L-aspartyl-L-phenylalanine methyl ester), in the presence of ascorbic acid and a transition metal catalyst, such as Cu(II) or Fe(III), has been reported to produce benzaldehyde via a free radical attack on the aspartame (Lawrence and Yuan, 1996). From the experimentation, aspartame, which comprised of Lphenylalanine, has been highly converted to benzaldehyde more than L-phenylalanine itself. This maybe due to enzymatic reaction in the aspartame metabolic pathway which was made easier with higher formation of phenylpyruvic acid prior to conversion to benzaldehyde (Masja and Jan, 1998).

Table 1. Effect of carbon and nitrogen sources on the mycelial growth and benzaldehdye production by R.oligosporus NRRL 2710 in static flask cultures* No. Carbon & Nitrogen sources Dry cell weight (g l-1) Benzaldehyde (abundance) 1.1 x 106 1.34 x 105 8.53 x 105 2.37 x 105 4.66 x 105 5.15 x 104 6.99 x 105 1.53 x 105 6.35 x 105 3.21 x 105 5.22 x 105 8.36 x 104 9.39 x 105 1.05 x 105 1.11 x 106 1.66 x 105 7.16 x 105 7.20 x 104 5.70 x 105 6.48 x 104 3.38 x 105 1.27 x 105

Carbon sources (2% w/w) Glucose 11.82 0.62 Fructose 11.58 0.28 Lactose 9.33 0.33 Maltose 12.27 0.27 Sucrose 12.73 0.21 Starch 14.65 0.32 Nitrogen sources (1% w/w) 1. L-phenylalanine 16.74 0.48 2. Aspartame 15.85 0.89 3. Yeast extract 17.56 0.34 4. Soy peptone 3.41 0.99 5. Ammonium nitrate 2.43 0.50 *Fermentations were carried out in triplicate for 96 hours at 24C 1. 2. 3. 4. 5. 6. Effect of C/N ratio on benzaldehyde production Among the 11 carbon and nitrogen sources examined, glucose and aspartame were favorable to the benzaldehyde production (Table 1). Therefore, investigations of C/N ratio (mass ratio) on benzaldehyde production using glucose and aspartame have been carried out. As shown in Figure 3, the 1/20

ratio (high concentration ratio of nitrogen to carbon source) was preferred for both optimal mycelial growth (25.70 gl-1) and benzaldehyde production (1.92 x 106). Germination of R.oligosporus spores has previously been shown to be optimal at 37C and at pH 4 and was stimulated by glucose and L-alanine (De Reu et al., 1995). This is in line with previous

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study by Kawabe and Morita (1994) who have stated that benzaldehyde and benzyl alcohol, which are secondary metabolites of L-phenylalanine, were not produced in greater amounts as the concentration of nitrogen decreased. Poor growth and poor benzaldehyde productions were obtained at the 1/1, 10/1 and 20/1 C/N ratios; approximately 8.92 gl-1 with abundance of 3.73 x 105, 8.33 gl-1 with abundance of 3.14 x 105

and 5.97 gl-1 with abundance of 2.83 x 105, respectively whereas moderate growth and benzaldehyde production was obtained from 1/10 ratio at 16.16 gl-1 with abundance of 6.38 x 105, respectively (Figure 3). The result showed that mycelial growth was stimulated by about fivefold when 20 part of aspartame (w/w) was added to the medium. This is parallel with benzaldehyde productions which also increased by about fivefold as compared to the 1/1, 10/1 and 20/1 ratios, respectively.

Figure 3. The optimal glucose and aspartame concentration on mycelial growth and benzaldehyde production by R.oligosporus NRRL 2710 in static flask cultures

Principal component analysis Principal component analysis (PCA) was carried out as a new methodology to study relationships among volatile compounds produced from each of C/N ratio which were determined earlier by PDMS/DVB coupled with SPME/GC-MS identification. Results indicated that only two principal components were necessary to explain 72% of total variance. Principal component 1 (PCA 1) retained the 55%, whereas principal component 2 (PCA 2) the other 17%. The

score of samples using the first two PCAs, produced a sample distribution according to their characteristics. Three types of C/N ratio, namely 1/1, 20/1 and 10/1 groups had the lowest scores on PCA 1, whereas the 1/20 and 1/10 groups showed the highest and intermediate score values, respectively on the same component. In the PCA loading plot, from PCA 1, 20 compounds were selected because of their relation with the differences among C/N ratio. In Table 3 details on compounds selected are shown.

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Table 3. Total area counts of identified enriched fermented CPH volatile compounds extracted for 30 min by SPME with polydimethyl siloxane/ divinylbenzene (PDMS/DVB) 65 m Compounds 1/1 10/1 ND ND ND ND cyclopropane, 1,2-dimethyl-, trans cis-2-Pentene 1-pentene cyclohexane, 1-(cyclohexylmethyl)8.18 x 103 ND ND 9.74 x 103 ND ND 2.57 x 104 ND ND ND 1.19 x 104 ND 1/10

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No. Abundance [C/N ratio] 1/20

Retention Time (min)

20/1 ND 1.17 x 104 ND ND

1. 2. 3. 4.

31
formic acid, methyl ester benzenepropanoic acid, methyl ester ND ND 3.73 x 105 1.16 x 104 ND ND ND benzaldehyde phenylacetaldehyde nonyl aldehyde pyrazine, 2-ethyl-5-methylsilane, diethoxymethyloctadecylND 3.09 x 103 6.38 x 105 1.53 x 104 2.82 x 103 ND ND

5. 6. 7. 8. 9. 10. 11. 12. 13. 2-heptanol 1H-Indol-5-ol (s)-3-ethyl-4-methylpentanol-1 linalool oxide cis-linalool oxide trans-linalool oxide benzeneethanol linalool z-pyranic oxide 2,6-bis (1,1-dimethylethyl) phenol 5.01 x 104 ND 1.11 x 104 7.34 x 104 5.35 x 104 ND ND ND 8.37 x 103 1.37 x 105 ND ND ND 3.80 x 105 3.54 x 105 1.82 x 104 2.01 x 104 1.42 x 104

6.95 x 104 ND 3.13 x 104 ND 4.72 x 105 4.44 x 105 2.56 x 104 2.80 x 104 4.06 x 104 ND 1.61 x 104 1.92 x 106 1.19 x 104 1.05 x 104 1.85 x 104 ND

2.23 x 104 ND ND ND 1.21 x 104 8.42 x 103 ND ND ND 7.23 x 105 ND 3.14 x 105 ND ND ND 6.13 x 104

5.32 x 104 2.59 x 104 1.12 x 104 1.09 x 105 7.62 x 104 ND ND ND ND ND ND 2.83 x 105 ND ND ND ND

14. 15.

16. 17. 18.

19.

20.

Aliphatic hydrocarbons 6.52 6.53 6.71 22.97 Alcohols 6.86 7.29 9.83 11.03 11.54 11.93 12.56 13.98 21.08 Esters 3.28 16.26 Aldehydes 8.33 10.35 12.28 Nitrogen compound 9.71 Other 7.39

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The largest positive contribution was due to benzaldehdye, which was found in all ratios, showing the highest content in C/N ratio 1/20 sample according to its highest content of aspartame which happened to be the precursor of benzaldehyde formation (Lawrence and Yuan, 1996). Other important compounds detected in 1/20 sample are cis-linalool oxide and trans-linalool oxide which were present at moderate abundance, 4.72 x 105 and 4.44 x 105, respectively. Compounds 2-heptanol, 2,6-bis (1,1dimethylethyl) phenol, (s)-3-ethyl-4-methylpentanol1, linalool z-pyranic oxide, benzeneethanol, pyrazine,

2-ethyl-5-methyl-, benzenepropanoic acid, methyl ester, phenylacetaldehyde, 1-pentene and nonyl aldehyde were present at moderate level; 6.95 x 104, 4.06 x 104, 3.14 x 104, 2.80 x 104, 2.56 x 104, 1.85 x 104, 1.61 x 104, 1.19 x 104, 1.19 x 104 and 1.05 x 104, respectively. The location of 1/10 sample in Figure 4 is slightly lower than 1/20 indicates less substances identified in the sample. It was stated that compounds (s)-3-ethyl-4-methylpentanol-1 and Pyrazine, 2-ethyl5-methyl- were not detected in 1/10 sample. Reduction of volatile compounds occurred along the descent of ratios down the PCA 1 as shown in Figure 4.

Figure 4. Score plot showing the five types of C/N ratio (glucose/aspartame) on the PC1 and PC2 dimensions.

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CONCLUSION Cocoa pod husk (CPH) can be used as an inert support in relation to volatile compounds production particularly benzaldehyde via solid state fermentation system (SSF). Cocoa pod husks need to be supplemented with C/N ratio, 1/20 glucose/aspartame at 24C after 96 hours of fermentation in order to obtain the highest abundance of benzaldehyde production and R.oligosporus growth. Solid-state fermentation provides a potential way to convert the CPH into various potential value-added products such as fungal bioinoculants. In this case, 13 compounds of interest including benzaldehyde were detected from C/N ratio 1/20, 11 from 1/10 sample, 9 from 1/1 sample, and 6 and 7 compounds from 10/1 and 20/1 samples, respectively. These volatile components comprise the main constituents of essential oils. The PCA result as shown in Figure 4 has also perceived 1/20 sample as an optimal C/N ratio in contributing large abundance of significant volatile compounds. ACKNOWLEDGEMENT Financial support from IRPA (Intensification of Research in Priority Areas) is gratefully acknowledged (Project No. 03-04-07-1202 EA10405). REFERENCES Antonio, J., Maria P.A., Gabriel, B. and Mariono, U. (2006). Application of solid-phase microextraction to virgin olive oil quality control. J. Chromatography A. 1121: 140-144. Bajracharya, R. and Mudgett, R.E. (1980). Solid substrate fermentation of alfalfa for enhanced protein recovery. Biotechnol. Bioeng. 21: 551-560. Blakemore, W.R., Dewar, E.T. and Hodge, R.A. (1966). Polysaccharides of the cocoa pod husks. J. Sci. Food Agric. 17: 558-561. De Reu, J.C., Rombouts, F.M. and Nout, M.J.R. (1995). Influence of acidity and initial substrate temperature on germination of Rhizopus oligosporus sporangiospores during tempe manufacture. J. Appl. Bacteriol. 78: 200-208.

Fabre, C.E., Blanc, P.J. and Goma, G. (1998). Production of 2-phenylethyl alcohol by Kluyvermyces marxianus. Biotechnol. Prog. 14: 270-274. Fassatiova, O. (1986). Moulds and filamentous fungi in technical microbiology. Prog. Ind. Microbiol. 22: 1-233. Garcia, M.E., Ansorena, D., Astiasaran, I. and Ruiz, J. (2004). Study of the effect of different fiber coatings and extraction conditions on dry cured ham volatile compounds extracted by solidphase microextraction (SPME). J. Talanta. 64: 458-466. Hesseltine, C.W. (1965). A millennium of fungi, food and fermentation. Mycologia. 57: 149-197. Jensen, K.A., Evans, K.M.C., Kirk, T.K. and Hammel, K.E. (1994). Biosynthetic pathway for veratryl alcohol in the ligninolytic fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 60: 709-714. Kawabe, T. and Morita, H. (1994). Production of benzaldehyde and benzyl alcohol by the mushroom Polyporus tuberaster K2606. J. Agric. Food Chem. 42: 2556-2560. Ko, S.D. (1988). Self protection of fermented foods against aflatoxin. Proceedings of fat oils by immobilized fungus at constant water concentration. J. Ferment. Technol. 66: 567575. Krings, U., Hinz, M. and Berger, R.G. (1996). Degradation of [2H] phenylalanine by the basidiomycete Ischnoderma benzoinum. J. Biotechnol. 51: 123-129. Lapadatescu, C., Ferron, G., Vergoignan, C., Djian, A., Durand, A. and Bonnarme, P. (1997). Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens. Appl. Microbiol. Biotechnol. 47: 708-714. Lawrence, G.D. and Yuan, D. (1996). Benzaldehyde formation from aspartame in the presence of ascorbic acid and transition metal catalyst. J. Agric. Food Chem. 44: 3461-3466.

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Masja, N.N.G. and Jan, A.M.B. (1998). Conversion of phenylalanine to benzaldehyde initiated by an aminotransferase in Lactobacillus plantarum. Appl. Environ. Microbiol. 64(8): 3009-3013. Medwid, R.D. and Grant, D.W. (1984). Germination of Rhizopus oligosporus sporangiospores. Appl. Environ. Microbiol. 48: 1067-1071. Nierop Groot, M.N. and Jan, A.M. (1998). Conversion of phenylalanine to benzaldehyde initiated by an aminotransferase in Lactobacillus plantarum. Appl. Environ. Microbiol. 64: 3009-3013. Norliza, A.W. (2003). The production of benzaldehyde by local isolated fungus in a solid state fermentation system. Masters of Science Thesis. Univ. Sains Malaysia. Onions, A.H.S., Allsopp, D. and Eggins, H.O.W. (1981). Smiths introduction to industrial mycology, 7th Ed. Edward Arnold, London. Raimbault, M. (1998). General and microbiological aspects of solid substrate fermentation. Elect. J. Biotech. 1(3): 1-21. Soccol, C.R., Cabrero, M.A., Roussos, S. and Raimbault, M. (1992). Selection of Rhizopus for growing on raw cassava. In: Guerrero R (Ed.) Proceedings of The VI International Symposium on Microbial Ecology, 6-11 th September 1992, Barcelona, p 32.

Sue Yie, L. and Che Omar, I. (2000). Optimization of a solid state fermentation for benzaldehyde production by a locally isolated, Penicillium diversum using palm kernel cake (PKC) as substrate. Pakistan J. Biol. Sci. 3(10): 1752-1754. Tsou, A.Y., Ransom, S.C. and Gerlt, J.A. (1990). Mandelic acid pathway of Pseudomonas putida: sequence relationships involving mandelic acid racemase, (S)-mandelic acid dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli. Biochemistry. 29: 9856-9862. Tunga, R., Banerjee, R. and Bhattacharyya, B.C. (1998). Optimizing some factors affecting protease production under solid state fermentation. Bioproc. Engin. 19: 187-190. Yonekichi, S., Tae, H.L and Hideo, S. (1977). On the convenient method for glucosamine estimation in koji. Agric. Biol. Chem. 619-624. Zarkadas, C.G., Yu, Z. and Burrows, V.D. (1995). Assessment of the protein quality of two new Canadian-developed oat cultivars by amino acid analysis. J. Agric. Food Chem. 43: 422-428.

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EFFECT OF FERMENTATION AND STORAGE ON MYCOTOXIGENIC FUNGI, OCHRATOXIN A AND AFLATOXIN B1 IN COCOA BEANS FROM SOUTH WESTERN NIGERIA Aroyeun, S.O.*1, Adegoke, G.O.2, Varga, J3., Kocsub, S.3, Pl, K.3 and Vgvolgyi, C.3 1 Cocoa Research Institute of Nigeria, PMB, 5244, Ibadan, Oyo state, Nigeria 2 University of Ibadan, Faculty of Technology, Department of Food Technology, Nigeria 3 University of Szeged, Faculty of Sciences, Department of Microbiology, Hungary. Malaysian Cocoa J. 3: 35-46 (2007) ABSTRACT - The mycobiota of cocoa bean samples from five major cocoa producing states of Nigeria were examined with emphasis on the presence of potential mycotoxin producing species. The mycobiota of cocoa beans at 3, 4, 5, and 6 days from different fermentation methods (basket, tray, heap and sack), and from cocoa bean samples collected from warehouses located in the southwestern part of Nigeria were also examined. Aspergillus was the most prevalent genus identified in this study at all stages of fermentation and storage , contributing to > 98% of total fungi isolated. Penicillium and Zygomycetes isolates were also detected. A. niger, A. flavus and A. tamarii isolates were detected in a majority of the samples tested. A. flavus and A. tamarii isolates were more common during fermentation of cocoa beans. A. ochraceus was not detected during the fermentation process. The frequency of occurrence of A. niger, A. flavus, A .tamarii and A. fumigatus during fermentation were 31.3%, 62.5%, 68.8% and 12.5% of the samples respectively. The method of fermentation affected type of mycotoxigenic fungi as well as capacity to produce mycotoxins. Aflatoxin B1 was more frequently detected in beans fermented with heap method (> 1ng/ml) than other methods of fermentation examined. Eleven out of fourteen Aspergillus flavus isolates were able to produce AFB1 in the range of 0.6 - >1ng/ml. Significantly lower numbers of fungi were detected on the fifth day of fermentation in all methods Seven A. niger isolates produced a fluorescent metabolites similar to AFB1 on thin layer chromatography plates and confirmation using Enzyme Linked Immunosorbent Assay (ELISA) showed AFB1 in the range of 0.11ng/ml and >1ng/ml while none was able to produce Ochratoxin A maybe the conditions were not favourable for toxin production. Some isolates of black Aspergillus niger detected during storage at the warehouse produced up to 3.4ng/ml of Ochratoxin A. This study showed that fermentation of cocoa beans does not constitute enough risks for mycotoxin contamination because OTA was not detected and the AFB1 was lower than the EU regulatory levels. Storage at the warehouses involved detection of Ochratoxin A at levels up to 3.72 ng/ml higherthan 2ng/ml regulatory limits for cocoa and cocoa products. There was biodeterioration of cocoa bean qualities as a result of mycotoxigenic fungi contamination. Key words: Cocoa beans; Detection, Mycotoxigenic; Fermentation, Nigeria *Corresponding author: Tel: +2348059152795, email address:aroyeun2000@yahoo.co.uk INTRODUCTION Fungal contamination and mycotoxin production in food is a great threat to the safety and good health of consumers (Varga et al., 2000, Bankole et al., 2004). Mould growth can occur during fruit development, harvest and storage. Evidence abound in the literature that people of West Africa are experiencing heavy dietary exposure to food-borne mycotoxins (Bankole and Adebanjo, 2003). According to the World Development Report, diseases caused by mycotoxins lead to reduced life expectancy in developing countries (Miller, 1996). The West African countries have tropical climate with an all year round high ambient temperature and relative humidity that provide favourable condition for the growth of mycotoxigenic moulds. The sub-region also has poorly developed processing facilities, storage, transportation and skilled human resources. Many of the moulds capable of producing mycotoxins (mycotoxigenic fungi) are also frequent contaminants of other agricultural commodities (seeds, grains, fruits and vegetables). Moulds which are of importance in commodities because of potential mycotoxin production include members of the genera Aspergillus, Penicillium, Fusarium, Alternaria and Cladosporium. These

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organisms are capable of growing on a variety of substrates and under a diverse condition of moisture, pH, and temperature. Thus, most types of seeds, grains, fruits and vegetables are susceptible to fungal invasion before and during harvest, transport or storage. If mould growth occurs there is a concomitant possibility of mycotoxin production. Fungi that invade agricultural commodities can be divided into three groups (Christensen, 1965). These include: (a) field fungi, which invade commodity in the field before harvest and include species of Alternaria, Fusarium and Cladosporium; (b) storage fungi, which predominate in the commodities during post harvest and storage and consist primarily of Aspergillus spp and Penicillium spp; and (c) advanced decay fungi, such as Fusarium and Chaetomium, which grow after considerable deterioration must have occurred. Nigeria is the fourth largest producer of cocoa beans after Cote DIvoire, Ghana, and Indonesia. Cocoa is cultivated in many parts of Nigeria, the largest cocoa bean producing areas being located in the southwestern part of the country in Ondo, Osun, Kwara, Oyo, Ekiti and Ogun states. The production of cocoa beans is in the hands of the farmers who have different methods of fermenting their beans (Aroyeun et al., 2006). Fermentation, an important process in the post harvest processing of cocoa bean is responsible for the generation of good flavour notes observed after drying or roasting of cocoa bean (Rohan, 1969; Ziegleder and Biehl, 1988). Good flavour characteristics are developed as a result of good fermentation methods. Many different fermentation methods are currently being used by farmers in Nigeria and there are diverse reasons for the choice of methods. Farmers use basket methods of fermentation based on seasonal reduction in cocoa beans and as such fermented beans obtained are not similar in quality when compared with other methods involving larger quantities such as heap, tray and sweatboxes. The types of fermentation methods determine cocoa bean sensory qualities such as acidity, bitterness and astringency, mouldiness, slatiness, flavour development, % brown beans and purple beans. The purposes of fermentation according to Frazier and Westhoff (1978) involved removal of the adhering pulp from the bean, killing the embryo in the seed and providing aroma, flavour and developing brown coloration in the bean.

Cocoa of commercial qualities should conform to some international standards in relation to moisture and butter fat contents, bean sizes and absence of mouldy or germinated beans (Aroyeun et al., 2006). In Nigeria, most farmers use heap and basket methods of fermentation. Tray methods have been recommended to farmers by the CRIN while sack method has been used in the Southeastern zone of Nigeria by some farmers. Mould growth and mycotoxin contamination can occur on cocoa beans during fermentation and drying or when beans get wet during transport and storage at the warehouse (Probert, 2003). Cocoa farmers in developing countries, Nigeria in particular, have not yet adopted the recommended methods of growing, harvesting, drying and storing of cocoa beans designed to reduce mould growth and mycotoxin formation (Aroyeun et al., 2006). These methods are expensive and impracticable for immediate application in the developing countries including Nigeria (Detroy et al., 1971). Since Nigeria, as a cocoa producing country, aims to produce beans with lower levels of mouldiness during harvest and storage, this study was carried out to establish the effect of fermentation and storage on mycotoxigenic fungi, Ochratoxin A, and Aflatoxin B1 in cocoa beans obtained from south western part of Nigeria. MATERIALS AND METHODS Collection of samples Altogether sixty two cocoa bean samples were examined, among which thirty two samples were collected from five cocoa producing states in southwestern Nigeria: Ondo, Osun, Kwara, Ogun and Ekiti states. Samplings were also carried out from cocoa beans fermented for 3, 4, 5 and 6 days by applying the basket, heap, tray and sack methods of fermentation at the Cocoa Research Institute of Nigeria as practiced by farmers visited at different states. Fermentation processes were as described earlier (Aroyeun et al., 2006). Since the time for sample collection was September 2005, which was a rainy season, farmers confirmed that drying took a longer time than usual due to low sunlight. Moisture contents of cocoa bean were recorded.

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Different methods of fermentation were described as follows: Basket Method: The quantity used was 6.4kg. The basket was lined with plantain leaves before filling with the wet cocoa beans. The cocoa beans were later covered with plantain leaves to prevent dissipation of heat. During the 6-day fermentation, the beans were stirred and turned to aerate them and create a homogenous distribution of heat within the fermenting mass. The depth of the basket was 40cm with 38cm diameter. The basket was placed on a sloping tray lined in fresh banana leaves to allow the sweatings to drain freely. After filling, the basket was covered with fresh plantain leaves to retain some of the heat of the fermentation and to keep out rainwater and other unwelcome intruders. After, 48 hours, the basket was emptied and the beans were thoroughly mixed. The basket was then refilled and all the banana leaves were replaced with new ones. Sack Method: This method involved the use of Bagco super sack containing 93.0kg of wet cocoa beans. In this method, there were no plantain leaves to line the bag because the material was able to produce enough heat that the plantain leaves were supposed to produce. The depth of the beans was 100cm from the top. A single turn between 24 and 36 hours from the start of fermentation was sufficient to ensure uniformity. The turning was carried out with a turning stick from the top to the bottom of the sack. The use of sack in this process was considered as modified packaging (Aroyeun et al., 2006). Heap Method: This method is commonly used in Nigeria amongst cocoa farmers. It involves collection of cocoa beans and piling them in a heap containing between 100kg and 200kg. In this method tarpaulin was used to cover the beans to maintain constant temperature in the fermentation. Constant turning was ensured at 24 hours interval to allow aeration and homogenous distribution of heat. In this experiment 53.0kg of beans were used with depth of 60cm. Tray method: This consists of a wooden tray with dimension of 30 x 20 x 10cm3 lined with net to allow free flow of sweatings. The trays were lined with plantain leaves before filling with wet cocoa beans, after which the banana leaves were used as a top cover. The depth of the beans was about 40cm from the top

of the rectangular wooden tray. The weight used in this experiment was 93.0kg. Fermentation proceeded undisturbed for 6 days and at 3, 4, 5 and 6 days and at each fermenting day, triplicate samples of 1.0g of cocoa beans were taken dried and analyzed. Another set of cocoa bean samples were obtained from 14 warehouses in Lagos, Oyo, Ogun and Ondo States. The ambient temperature and relative humidity values at the sampling points were taken with thermohydrograph. All samples were kept in a cool dry place until analysis (3-5C). The location of the warehouses were close to the point of shipment. Determination of mycobiota of the samples Fungi were assessed by directly plating duplicate samples of weighed cocoa beans (approximately 1.0g) using Dichloran Rose Bengal Chloramphenicol medium (King et al.,1979). Plates were incubated in triplicates at 28C for 7-10 days. Penicillium and Aspergillus isolates were picked off onto Czapek Dox agar (CZA) and malt extract agar (MEA) plates for further identification to species. Fungal isolates were identified using macro- and micromorphological criteria. Aspergillus isolates were identified according to Raper and Fennell (1965). Examination of mycotoxin production A. flavus, A. tamarii and A. niger isolates were cultivated in YES (2% yeast extract, 15% sucrose) media for 10 days at 25C in the dark. The Toxiklon OTA and aflatoxin enzyme immunoassay kits (Agricultural Biotechnology Centre, Gdll, Hungary) were applied for the detection of OTA and aflatoxin production in the isolates as described previously (Tren et al., 1996). The detection limit of the ELISA technique is 0.01 ngL-1, with standard deviation of 25%. Screening for Aflatoxin B1 Cultures on Yeast Extract Sucrose (YES) were extracted with chloroform (1 : 1, v/v) for 45min on a wrist action shaker. The chloroform extract was dried over anhydrous sodium tetraoxosulphate (VI) and after evaporation in vacuo, the residue was taken up in a small volume of benzene. Aliquots of the various extracts were examined by thin layer chromatography (tlc) on plate coated to 500m thickness with silica gel G or F-HR and developed in methanol: chloroform

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(1:9). Chromatography plates were examined under long wave u.v. light (365nm) for evidence of fluorescent compounds. Confirmatory tests for aflatoxin were made by derivatization with trifluroacetic acid (Stack and Pohland, 1975). Screening for Ochratoxin A Ochratoxin A was identified under UV light (360nm) as bluish - green fluorescent spots with the same mobility as that of an Ochratoxin A standard (Makor Chemicals, Jerusalem, Israel). The fluorescence of Ochratoxin A spots from both the standards and the extracts changed to deep blue on treatment of chromatogram with NaHCO3 (5% NaHCO3 in 17 % (ethanol). Inoculation of sound cocoa beans with mycotoxigenic fungi Triplicate samples of sound, sterilized, fermented and dried cocoa beans (6g) were inoculated with conidial suspensions of A.flavus, A.tamarii and A.niger isolates incubated for 7days at 28C in Yeast Extract Sucrose (YES) broth. The inoculated cocoa beans were later dried at 60C for 2 hours and reserved for further analysis. Chemical analysis Triplicate samples of mouldy beans inoculated with A.flavus, A.tamarii, and A.niger were analyzed for proximate composition in order to determine the effects of fungal contamination on biodeterioration of cocoa beans according to AOAC, 2000. Healthy cocoa beans served as the control. HPLC analysis For HPLC analysis, 10-ml quantities of YES (2% yeast extract, 15% sucrose) medium contained in 30-ml vials were inoculated with 0.1ml of dense conidial suspensions of the strains. The vials were incubated as slants at 30C for 10 days in the dark. After incubation, 1ml chloroform was added and they were centrifuged at 4000g for 10 minutes. The chloroform extracts were evaporated and ochratoxin A was redissolved in appropriate amounts of the mobile phase (57% acetonitriles, 41%, H2O, 2% acetic acid and detected on a. HPLC isocratic system with C18 column (Microsorb MY, VARIAN, 250 x 4.bmm, 5 l particle size) and a fluorescence detector (excitation 330nm, emission 460nm), with a loop of 50l. The mobile phase was pumped at 1ml/min. Extracts were

considered positive if they yielded a peak at a retention time identical to that of standard OTA. The standard solution was prepared as described by AOAC (2000) using OTA obtained from sigma chemical Co. (St. Louis, MO. USA). Immunochemical detection of Ochratoxin A and AFB1 For immunochemical screening, 10ml-quantities of YES (2% yeast extract, 15% sucrose) medium in 100ml Erlenmeyer flasks were inoculated with 0.1ml of dense conidial suspensions of the strains and incubated at 30C for 10 days in the dark. 1-ml portions of these media were then mixed with 1ml chloroform and centrifuged at 4000 x g for 10min. The chloroform phase was transferred to a clean tube, vortexed with 1ml of 0.13M NaHCO3 (pH 8.3) and centrifuged at 4000 x g for 10 minutes. From appropriate dilutions made with 0.13M NaHCO 3 , 50l aliquots were applied to microtiter plates coated with a specific monoclonal antibody preparations developed against each of OTA and AFB1 (Toxiclon ochratoxin A and aflatoxin B1 enzyme immunoassay for the quantitative analysis of OTA and AFB 1 ; Agricultural Biotechnology Centre, Goddolo, Hungary). Direct competitive ELISA tests were performed as described earlier (Gyongyosi et al., 1995). All trials were replicated thrice. Data analysis To establish the presence of similar species in the warehouse, Sorensens Index of similarity was used with formula: S=2c/a+b.100 (Wagner 1977) where a: the number of species at one site; b: number of species at the other site; and c: common species to both sites. All other data generated were subjected to ANOVA using SPSS (1999) for windows. RESULTS AND DISCUSSION Mycobiota of cocoa beans from different states The cocoa bean samples collected from five states in the southwestern part of Nigeria were contaminated by different fungal species (Table 1). Aspergillus niger, A.tamarii and A. flavus isolates were detected in samples from Ondo, Kwara, Osun and Ekiti states while sample from Ogun state contains only A.tamarii

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(100%). The cocoa bean samples from Kwara, Osun and Ondo states showed lower occurrence 35-43% of A.tamarii. Penicillium isolates and isolates belonging to Zygomycetes (mainly Rhizopus isolates) were also recorded. Aspergillus fumigatus was present in samples from Kwara, Osun and Ekiti states at frequency of occurrence of 28.57%, 14.29%, and 66.67% respectively (Table 1). The fungi encountered most frequently were A. flavus, A. tamarii and A. niger, in agreement with earlier studies where A. tamarii, A. flavus, A. niger, Penicillium species and Zygomycetes were considered as major contaminating microorganisms of cocoa beans (ICCO, 1993). Earlier reports have also indicated the presence of isolates of A. flavus, A. niger, A. ochraceus and A. fumigatus as the most common Aspergillus species in soybean and other oil seeds (e.g cotton, peanut seeds and their products; (Mazen et al., 1990; Sabah, 1992; Kady and Youssef , 1993). Although cocoa beans contain cocoa butter up to 50%, the detection of similar species as reported for other oily seeds (El-Kady and Youssef, 1993) in this study may be due to environment, handling and processing method such as fermentation and drying. Zygomycetes were also detected in samples from Ondo state, Osun state and Ogun state at 28.57%, 28.57% and 100% respectively (Table 1). Some members of the Zygomycetes are unavoidably present during fermentation of oily seeds as reported by El-Kady and Youssef (1993). In comparison with other crops of tropical origin, the mycobiota of cocoa beans was also similar to that of green coffee beans, with the exception that A. ochraceus isolates could not be recovered in this study (Martins et al., 2003; Tren et al., 1997). Mycobiota of cocoa beans from warehouses The frequency of occurrence of A. tamarii and A. niger isolates was higher than that of other species in samples collected from warehouses (Table 2). The incidence of A. niger in cocoa beans increased during storage, while those of other fungi decreased slightly in freshly collected cocoa beans in comparison to their occurrence during fermentation (Table 3). Aspergillus fumigatus was present in the warehouse and in samples from the states; the high temperature favourable for its growth being a thermotolerant organism has probably enhanced its presence. Indices of similarity, which permitted a comparison of the different warehouses by species composition, showed that the two sites most different in their fungal flora over the

entire sampling period were Lagos-Ogun and OgunOndo from the state categories, and Kwara-Ogun for the warehouse categories, so the heavily contaminated and the lightly contaminated areas showed the greatest differences in their species composition (Table 5). Temperature, location and methods of cocoa bean fermentation and storage must have accounted for these differences in their fungal flora. As for those places where the index of similarity is greater than 50% such as the similarity in species composition of Lagos-Oyo, Lagos-Ondo and Oyo-Ondo, there is the possibility that samples analyzed might have come from farmers in same region since most of the cocoa beans were collected at different centers before transferring them to Lagos where the beans can be shipped to the consuming countries. Mycobiota of cocoa beans using different fermentation methods In the fermentation process, the incidence of A. flavus and A. tamarii was higher than found during storage (Table 3). On the third day of fermentation A. flavus and A. tamarii dominated in the samples, with the exception of the sack method of fermentation. Aspergillus tamarii was also found in the third and the fourth day in heap and basket fermentation. On the other hand, A. niger was most frequently isolated from cocoa beans subjected to tray fermentation (Table 4). The frequency of mycotoxigenic moulds contamination was lowest on the fifth day of fermentation in all the methods of fermentation examined. The most frequently isolated species were A. niger, A. flavus and A. tamarii in all methods. Aspergillus tamarii and A. flavus were found to be more common during fermentation of cocoa beans. This is in agreement with previous studies (ICCO, 1993). A. ochraceus was not detected during the fermentation process. However, significant differences could not be observed between the mycobiota of the cocoa beans subjected to different types of fermentation. The predominance of Aspergillus niger strain as contaminants of cocoa bean at all stages from the filed to post-harvest storage reflects earlier observation (ICCO, 1993) on the mycoflora of cocoa beans in tropical countries. The relatively primitive methods used for primary storage, sun drying and subsequent bulk storage of cocoa beans provide many opportunities for fungal contamination for cocoa and cocoa products.

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Table1: Mycotoxigenic fungi detected in five states in the southwestern part of Nigeria
States ONDO KWARA OSUN OGUN EKITI Fungal species detected Aspergillus niger 6/14 (43%), Aspergillus tamarii 5/14 (36%), Aspergillus flavus 5/14 (36%), Penicillium sp. 1/14 (7%), Zygomycetes 4/14 (29%) Aspergillus tamarii 3/7 (43%), Aspergillus flavus 4/7 (57%), Aspergillus niger (3/7), Penicillium sp. 0/7 (0%), Aspergillus fumigatus 2/7 (29%) Aspergillus flavus 4/7 (57%), Aspergillus fumigatus 1/7 (14%), Aspergillus tamarii, 4/7, (57%), Aspergillus niger 3/7 (43%), Penicillium sp. 2/7 (29%), Zygomycetes 2/7 (29%). Aspergillus tamarii, 1/1 (100%), Penicillium sp. 1/1 (100%), Zygomycetes 1/1 (100%), Aspergillus fumigatus 2/3 (67%), Aspergillus flavus 1/3(33%), Aspergillus niger 1/)3 (33%), Penicillium 0/3(0%), Aspergillus tamarii 1/3 (33%)

Table 2: Mycotoxigenic isolates from selected warehouses in the southwestern part of Nigeria
Location of warehouses LAGOS OYO OGUN ONDO Fungal species Aspergillus tamarii (3/9), Aspergillus fumigatus (1/9), Aspergillus niger (3/9), Aspergillus flavus (1/9), Zygomycetes (1/9) Aspergillus niger (1/1), Aspergillus flavus (1/1), Aspergillus tamarii (1/1), Penicillium sp. (1/1) Aspergillus niger (1/1) Aspergillus niger (3/3), Aspergillus fumigatus (2/3), Aspergillus tamarii (2/3), Penicillium sp. (1/3)

Table 3: Frequency of occurrence of moulds isolated from cocoa bean samples at different points
A. niger A. flavus A. tamari A. fumigatus Penicillium sp. Zygomycetes Fresh beans before fermentation 12/29 (41.3%) 13/29 (44.8%) 13/29 (44.8%) 3/29 (10.3%) 4/29 (13.8%) 7/29 (24.1%) Fermentation 5/16 (31.3%) 10/16 (62.5%) 11/16 (68.8%) 2/16 (12.5%) 0/16 (0%) 2/16 (12.5%) Warehouse 8/14 2/14 6/14 3/14 2/14 1/14 (57.1%) (14.3%)) (42.9%) (21.4%) (14.3%) (7.1%) Fermentation 5/16 (31.3%) 10/16 (62.5%) 11/16 (68.8%) 2/16 (12.5%) 0/16 (0%) 2/16 (12.5%)

Table 4: Effect of fermentation days on distribution of mycotoxigenic fungi


Fermentation type/day Heap/3 Heap/4 Heap/5 Heap/6 Sack/3 Sack/4 Sack/5 Sack/6 Basket/3 Basket/4 Basket/5 Basket/6 Tray/3 Tray/4 Tray/5 Tray/6 Total positive A. tamarii + + + + + + + + + + + 11 A. flavus + + + + + + + + + + 10 A. niger + + + + + 5 A. fumigatus + + 2 Zygomycetes + + 2 Penicillium sp. 0

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Table 5: Indices of similarity of warehouses with respect to mycotoxigenic fungi occurrence


Warehouse Lagos-Oyo Lagos-Ogun Lagos-Ondo Oyo-Ondo Oyo-Ondo Ogun-Ondo A 6 6 6 4 4 1 B 4 1 5 1 5 5 C 3 1 4 1 3 1 Index of similarity 60 28.6 72.7 40 66.7 33

a-Number of species at one Warehouse; b- Number of species at the other Warehouse, c-Number of species common to both. Index of Similarity, S=2c/a+b .100 Biodeterioration of cocoa bean One of the reasons for which fungal contamination is detested in agricultural produce, is the biodeterioration of produce caused by storage fungi. According to (Table 6), A. niger, which was the predominant Aspergillus strains identified caused a more loss in protein, % ash, and % fat than other species identified during fermentation. The effect of fungal contamination on % moisture content was also significant. Significant differences were observed between control sample (non-contaminated sample) and samples from which mycotoxigenic fungi were detected. Storage fungi have been reported in earlier studies to cause deterioration in quantity and quality of stored produce (Airede, 2005). Such deterioration, according to (Airede, 1999), can be manifested in the form of loss in germinative capacity, discoloration of beans and in biochemical changes such as increases in free fatty acid (FFA), decreases in protein and other desirable nutritional qualities. Commercially, the quality of cocoa beans is determined by cocoa butter content (55 - 58%) (Aroyeun et al., 2006). This study established the reduction in the quantity of cocoa butter in all the contaminated cocoa beans. The % cocoa butter of control sample was significantly different (P < 0.05) from all other cocoa beans contaminated with mycotoxigenic mould. Reduction of protein levels of cocoa beans as reported in this study from (15.3 to 5.2%) indicated that the mycotoxigenic fungi identified must have possessed enzymes capable of digesting protein and were thus able to utilize the form of organic protein present. Airede, (2005), reported biodeterioration of quality in palm kernel as a result of presence of mycotoxigenic fungi. Concentration of AFB1 and OTA The results presented in Table 7 shows that aflatoxin B1 was detected at levels lower than 5ng/ml in 100% of the cocoa samples analyzed. Only one out of 13 A. niger isolates produced Aflatoxin B 1 >1ng/ml (7.65%). Six out of thirteen isolates of A.niger produced aflatoxin B1 at levels of 0.11-0.59ng/ml while one out of ten isolates of Aspergillus tamarii examined was able to produce AFB1 >1ng/ml. Ninety percent A. tamarii isolates produced AFB1 in the range of 0.01-0.059ng/ml (40%) and 0.11-0.59ng/ml (50%). 64.3% of Aspergillus flavus produced detectable amount of AFB1 (9 out of 14 isolates). This finding conformed with previous reports of Bankole et al., (2004) and other authors (Airede, 1995; 2005) who established that A. flavus produced AFB1 toxin. Many of the aflatoxigenic strains came from basket fermentation (three isolates), Ondo State (2 isolates) and warehouse (9 isolates). All the Aspergillus fumigatus strains produced far lower AFB1 when compared to other aflatoxigenic strains. Various fluorescent metabolites were produced also by other isolates tested but no attempt was made to characterize these compounds (Figure 1 and 2). The ochratoxigenic isolates of A. niger from the warehouses were able to produce OTA within the range of 3.013.72ng/ml (Table 8) while majority had less OTA concentration. Many previous reports have indicated low ochratoxin A production by strains of black Aspergillus (Abarca et al., 1994; Chelkowski, et al., 1987; Teren et al., 1997). This results is an indication that strains capable of producing OTA as discovered in the study are low producers. Hence the OTA levels of most of the samples were below the EU regulatory limit of 2ng/ml.

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Table 6: Effects of fungal contamination on % nutritional composition of cocoa bean


Fungi Control A.flavus A.tamarii A.niger % Moisture Content 5.3b 10.45a 10.63a 11.1a % Crude Protein 15.3a 8.2b 5.4c 5.2c %Ash 7.3a 6.3b 6.0b 5.9b % Fat 56a 28.1b 20.7c 18.8c % Carbohydrate 16.1c 46.95b 57.27a 59.0a

a,b,c: Values along the same columns with same superscripts are not significantly different at p < 0.05 Table 7: Distribution of aflatoxin B1 by fungi isolated from cocoa beans
Fungi A. niger 1/3 A. fumigatus A. tamari A. flavus 4/10 1/3 1/3 5/10 3/14 2/14 1/10 9/14 0.01-0.059 5/13 Aflatoxin B1 concentration (ng/ml) 0.06-0.1 0.11-0.59 1/13 6/13 0.6-1.0 >1 1/13

Table 8: Ochratoxin A in cocoa beans sampled from different warehouses based on thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA)
Locations Lagos Sample code WAH1 WAH2 WAH3 WAH4 WAH5 WAH6 WAH7 WAH8 WAH9 WAH10 WAH11 WAH12 WAH13 WAH14 Colour under TLC + ve + ve + ve + ve - ve + ve - ve - ve - ve - ve + ve - ve - ve + ve HPLC (ng/ml) 1.69 3.01 3.40 0.06 1.33 1.02 1.50 1.10 0.36 0.71 LOD LOD LOD 3.72 ELISA (ng/ml) 1.81 2.99 3.30 0.53 1.50 0.06 1.25 1.05 0.61 0.81 0.08 0.72 1.11 3.2

Oyo Ogun Ondo

+ve- positive, -ve- negative; LOD- limit of detection (0.05ng/ml)

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Table 9: Ochratoxin A contents of cocoa beans sampled from selected southwestern states of Nigeria
States Ondo Sample code OF1 OF2 OF3 OF4 OF5 OF6 OF7 OF8 OF9 OF10 OF11 OF12 OF13 OF14 KW1 KW2 KW3 KW4 KW5 KW6 KW7 EK1 EK2 EK3 OS1 OS2 OS3 OS4 OS5 OS6 OS7 OG1 Colour under TLC - ve - ve - ve - ve - ve + ve + ve + ve - ve - ve - ve - ve - ve - ve + ve + ve + ve - ve - ve - ve - ve - ve - ve - ve - ve - ve + ve + ve - ve + ve + ve + ve HPLC (ng/ml) LOD LOD LOD LOD LOD 2.50 2.20 LOD LOD LOD LOD LOD LOD LOD 1.20 0.06 2.70 LOD LOD LOD LOD LOD LOD LOD LOD LOD LOD LOD LOD LOD LOD LOD ELISA (ng/ml) 0.03 0.10 0.73 1.10 1.02 0.11 0.53 0.30 1.40 0.66 0.08 0.13 0.02 0.68 1.40 0.31 0.70 0.29 0.16 0.16 0.30 0.07 0.05 0.40 0.25 0.37 1.08 1.50 1.33 2.21 1.11 1.21

Kwara

Ekiti Osun

Ogun

-ve- negative,

+ve; LOD- limit of detection (0.05ng/ml)

Figure 1. Thin Layer Chromatography of A. tamarii isolate examined (lane 10) (1 l of extract applied) with bluish fluorescent indicating positive aflatoxin B1 (Lane 16) and negative OTA (lane 17).

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Figure 2. Thin Layer Chromatography separation of A. flavus (2, 4, 5, 8, 10 and 15) and Aspergillus niger isolates (5 and 16) showing production of fluorescent metabolites with same Rf as the standards as the aflatoxin B1 (lane 16) and ochratoxin A (lane 20). According to Table 8, highest ochratoxin A contents up to 3.72ng/ml) were recorded at the Ondo warehouse while lower values in the range of <LOD 2.7ng/ml were detected in some states in the southwestern part of Nigeria (Table 9). The high relative humidity at the warehouse (>70%) might have accounted for higher moisture content which might have resulted in the high ochratoxin A incidence. Other reasons for the levels of both AFB1 and OTA in this study might be related to rewetting of the beans after drying during transportation from the farm to the warehouse and inadequate drying of the beans probably due to inadequate sunlight. Moisture content of cocoa beans greater than 8% is also likely to result in mould growth and subsequently, production of mycotoxin (Aroyeun et al., 2006). Moreover, the period of sample collection was at the peak of rainfall (September), thus the traditional sun drying employed by the predominantly resource-poor Nigerian farmers may be difficult and takes longer time. Consequently, farmers are forced to load cocoa bean into stores with high moisture content which favour mould invasion and mycotoxin contamination. This possibility is high as dry cocoa bean is hygroscopic in nature. Aflatoxin B1 detected in all the samples examined were far lower than the maximum regulatory limit 20g/kg permitted in Nigerian foods. The percentage of samples with ochratoxin A levels lower than the EU limit was also significant. (ICCO, 1993). Although, generally, the incidence of aflatoxin obtained in this work was low (less than 5ng/ml), further possibilities of higher quantity is imminent when there are conditions favourable to mould growth and mycotoxin production. High incidence of aflatoxin can occur at high temperature which ranged from about 36 42C. Coincidentally the fermentation temperature of cocoa bean was within this range which indicated that occurrence of A. flavus is unavoidable but not necessarily aflatoxin production. Good hygiene practices (GHP) and avoidance of contamination during handling and storage can reduce aflatoxin B1 in the cocoa samples. This report does not conform with El-Kady and Youssef (1993) who obtained a high incidence of aflatoxin in soybean seeds at temperature of 36 42C. The difference in these two findings might be as a result of substrate and other specific experimental conditions (Edds, 1979).

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CONCLUSIONS In this study, it was established that the bulk of contamination of cocoa beans by mycotoxigenic fungi was the warehouse where conditions of high relative humidity of the environment enhanced the increase in moisture content, which resulted in mycotoxin production. All the methods of fermentation examined did not significantly differ in terms of mycotoxigenic fungi. This study was able to establish that the optimum numbers of days for effective fermentation irrespective of the method was five days where the lowest numbers of mycotoxigenic fungi were recorded. Although aflatoxin B1 and ochratoxin A were detected in some isolated Aspergillus strains at state levels, fermentation stages and at warehouses, their concentrations were still within regulatory limits of the European Union (EU). Several A. flavus and A. niger isolates were found to be able to produce aflatoxin and ochratoxin, respectively, indicating that these species may be responsible for mycotoxin contamination of cocoa beans in Nigeria. The introduction of Good Agricultural practices (GAP) can be used to reduce this fungal contamination to the barest minimum in order to safeguard the safety of the consumers of cocoa beans. Apart from this, prompt and adequate drying of cocoa beans must be given priority. Low storage moisture content must be maintained. Cocoa beans should be stored at moisture content below 8%. Temperature of storage at the warehouse need to be lowered to 22 2C. After drying, cocoa beans taken to the Warehouse should not be put in an environment where rewetting is inevitable because dry cocoa beans is hygroscopic in nature and there could be moisture pick-up which could enhance mould growth and subsequent mycotoxin production. Further studies are in progress to develop a method for lowering mycotoxin levels in cocoa products using essential oil of Aframomum danielli. REFERENCES Abaraca, M.L., Bragulat, M.R., Castella, G. and Cabanes, F.J. (1994). Ochratoxin A production by strains of Aspergillus niger var. niger. Appl.Environ.Microbiol. 60: 2650-2652

Airede, C.E. (1999). Reduction of Post Harvest Losses in the Oil Palm Industry. In Proceedings of the 23 rd Annual Conference of the Nigerian Institute of Food Science and Technology, Abuja (E. Elemo ed ) pp 245 - 246. Airede C.E. (2005). Potential Mycotoxin Contamination of Oil Palm Kernels and By Products. Paper presented at Mycotoxin Workshop in collaboration with International Atomic Energy Agency with NAFDAC. March, 2005. AOAC (2000). Official Methods of Analysis, 19th edition. Washington D.C. Association of Analytical Chemists. Aroyeun, S. O., Ogunbayo, J. O. and Olaiya, A. O. (2006). Effect of modified packaging and storage time of cocoa pods on the commercial quality of cocoa beans. Br Food J. 108: 141-151 Bankole S.A., Ogunsanwo, B.M and Mabekoje, O.O. (2004). Natural occurrence of moulds and aflatoxin B1 in melon seeds from markets in Nigeria. Food and Chemical Toxicol. 42: 1309-1314. Bankole, S.A., Adebanjo, A. (2003). Mycotoxin contamination of food in West Africa: current situation and possibilities of controlling it. African Journal of Biotechnology 2 (9): 254-263. Chelkowski, J.R.A. Samson, Wicwiorowska, M. and Golinski, P. (1987). Ochratoxin A formation by isolated strains of the conidial stage of Aspergillus glaucus Link exGrey (Eurotium herbatorium Wiggers Link exGrey) from cereal grains. Die Nahrung 31: 267-270 Christensen, C. M. (1965). Fungi in cereal grains and their products. pp 9-14. In G.N. Wogan (ed.) Mycotoxins in Foodstuffs, M.I.T. Press, Cambridge, M.A. Detroy, R. W., L.illehoj, E. B. and Ciegler, A. (1971). Aflatoxin and related compounds. In Ciegler A, Kadis S, Ajl SJ (eds) Microbial toxins, Vol. VI: Fungal toxins. Academic Press, New York, 3-178 Edds, G.T. (1979). Aflatoxins. In Conference on Mycotoxins in Animal Feeds and Grains Related to Animal Health (SHIMODA, W., Editor). Food and Drug Administration Rockville, Maryland.

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El-Kady, I. A. and Youssef, M.S. (1993). Survey of mycoflora and mycotoxins in Egyptian soybean seeds. J Basic Microbiol 33: 371-378. Frazier, W.C. and Westhoff, D.C. (1978). Food Microbiology, MacGraw Hill Book Company. 3rd Ed. Pg. 398. Gyongyosi, H.A., Barna-Vetro, J., Solti, L. (1995). A new monoclonal antibody detecting ochratoxin A at pictogram level. Lett Appl. Microbiol 22: 103-105 ICCO (International Cocoa Organization) (1993) Are mycotoxins a problem to the chocolate industry?
http://www.icco.org/questions/mycotoxin.htm

King, A., Douglas, J. R., Hocking, A.D. and Pitt, J.I. (1979). Dichloran Rose Bengal medium for enumeration and isolation of molds from foods. Appl Environ Microbiol 37: 959-964 Martins, M.L., Martins, H.M. and Gimeno, A. (2003). Incidence of microflora and of ochratoxin A in green coffee beans (Coffea arabica). Food Addit Contam 20: 1127-1131 Mazen, M.B., El Kady, M., Sabah, I.A. and Saber, M. (1990). Survey of the mycoflora and mycotoxins of cotton seed and seed products in Egypt. Mycopathologia 110: 133-138 Miller, J.D. (1996). Global significance of mycotoxins and phycotoxins. In Cardwell, K.F. (Ed.), Proceedings of the workshop on Mycotoxins in Food in Africa, November 6-10, 1995. International Institute of Tropical Agriculture, Cotonou, Benin, pp.18-22. Probert, J. (2003). Whats in store for coffee and cocoa? In: African Farming March/April, 2004. Raper, K.B. and Fennel, D.I. (1965). The Genus Aspergillus. Williams and Wilkins, Balitimore

Rohan, T.A. (1969). The flavour of chocolate, its precursors and a study of their reaction. Gordian 69: 443-447, 500-501, 542-544, 587-590. Sabah, S. M. (1992). Fungal contamination, natural occurrence of mycotoxins and resistance for aflatoxigenic accumulation of some broad bean (Vicia faba L.) cultivars. J. Basic Microbiol 4: 249-258. Stack, M.E., and Pohland, A.E. (1975). Collaborative study of a method for chemical confirmation of the identity of aflatoxin. Journal of Association Official Analytical Chemists 58: 110-113. Tren, J., Varga, J., Hamari, Z., Rinyu, E. and Kevei, F. (1996). Immunochemical detection of Ochratoxin A in black Aspergillus strains. Mycopathologia 134: 171-176 Tren, J., Palgyi, A. and Varga, J. (1997). Isolation of ochratoxin producing Aspergilli from green coffee beans of different origin. Cereal Res Commun 25: 303-304 Varga, J., Rig, K., Tren, J. and Mesterhzy, . (2000). Recent advances in Ochratoxin research I. Production, detection and occurrence of ochratoxins. Cereal Res Commun 29: 85-92 Wagner-Merner, D.T. (1977). Arenicolous fungi from the South and Central gulf coast of Florida. Nova Hedwigia 23: 915-922 Ziegleder, G. and Biehl, B. (1988). Analysis of Cocoa Flavour Components and Flavour Precursors. In Analysis of Nonalcoholic Beverages; Linskens, H.F., Jackson, J.F., Eds.; Springer; Berlin 331-393.

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MINIMUM INHIBITORY CONCENTRATION OF COCOA TANNIN GEL TOOTHPASTE ON SELECTIVE MOUTH BACTERIA Azila, A.K. and Nur Azilah, A. Malaysian Cocoa Board, Cocoa Downstream Research Center, Lot 3, Jln P/9B, Section 13 43650 Bandar Baru Bangi, Selangor, Malaysia Malaysian Cocoa J. 3: 47-51 (2007) ABSTRACT - Tannin extract has the ability to inhibit the plaque growth on tooth surface and at the same time improving the gingival health. Specifically, tannin that extracted from cocoa materials has this ability on selective major plaque bacteria, i.e. Streptococcus mutans (S. mutans) and Streptococcus sanguis (S. sanguis), in-vitro. This study measures the ability of cocoa tannin gel toothpaste to inhibit the mentioned bacteria by determination of minimum inhibitory concentration (MIC), using agar dilution method. The active ingredient in gel toothpaste was extracted with deionised water and diluted up to final dilution of 1:1024. Two ml of the dilution was poured into agar up to final dilution of 1:10240. The selective bacteria were then streaked on the agar and incubated for 72 hours prior to MIC determination. MIC is the lowest concentration of preparation which did not allow visible growth of the organism. The cocoa tannin gel toothpaste showed an inhibition of the S. mutans at 0.015 mg/L, whereas 0.290 mg/L for S. sanguis. Key words: Tannin, Gel toothpaste, Plaque bacteria, Cosmetics, Cocoa INTRODUCTION Tooth decay is caused by the Streptococcus mutans (S. mutans) bacteria due to its ability producing an enzyme, called glucosyltranferase, on its surface that cause polymerization of glucose, and at the same time adheres along with the bacteria tightly to the tooth enamel, which leads to the formation of plaque. Plaque is a sticky compound of bacteria and food substances adhering to the teeth. The bacteria associated with dental decay are also commonly found in mouth even when active dental decay is absent. Damage to the tooth begins when bacterial plaque comes in contact with foods high in sugar and carbohydrates. The bacteria ingest, break down the sugar and produce acids that attack the enamel which leads to tooth decay, if not stopped. Dental or tooth decay is an actively destructive process as a result of interaction between tooth, plaque and food (Figure 1).

Figure 1: Interaction Leads to Tooth Decay Plaque formation is initiated by weak attachment of streptococcal cells to salivary glycoprotein forming or pellicle on the surface of the teeth where it was then formed in three stages (Susan, 2004). Initially, a group of bacteria or colony, categorized as primary colonizers, was formed shortly after brushing, i.e. Streptococcus mutans, Streptococcus sanguis, Actinomyces viscous and small colony of Lactobacillus and Veinolla. These bacteria can easily be removed by brushing and application of 47 antibacterial agent in toothpaste or mouthwash. In the next three days, accumulation of these bacteria provides a suitable environment for the growth of second colonizers, i.e. Fusobacterium nucleatum, Prevotella intermedia and Capnocytopga sp. Later, tertiary colonizers (Porphyromonas gingivalis, Actinobacillus sp., etc.) are the main oral microorganisms that can lead to dental caries. At this stage, tooth brushing is inadequate to prevent teeth from the tooth decay and periodontal disease (Pader, 1988).

Malaysian Cocoa Journal

S. mutans is the bacterium that is primarily implicated in the development of tooth decay which has the ability to survive and even grow under conditions hostile to other bacteria. Thus, application of toothpaste with the active ingredient, such as fluoride and triclosan that can inhibit the plaque formation is advantageous to the cosmetic industry which can prevent tooth decay by retarding the growth of S. mutans. Cocoa tannin is one of the active ingredients that disrupted the cell wall of the said bacteria and therefore inhibit the formation of plaque. It is recommended to inhibit the plaque formation at its earliest stage by brushing and prolong the inhibition by the usage of antibacterial properties toothpaste. Refined and unrefined cocoa may also be added at levels up to 25% of the active components in toothpaste (Shuch, 2003), which is the antimicrobial substance (i.e. tannin or polyphenols). According to Strlfors (1966), tannin can inhibit tooth caries in animal even at low concentration. Specifically, catechin, a type of polyphenols (group of tannin that discovered in cocoa), has the ability to inhibit the growth of bacteria related to tooth decay i.e. S. mutans (Christine, 1998) and other sources of tannin, such as tea extract, also proven to have the ability in reducing tooth decay (Yu, 1995). However, formulation of

toothpaste comprising tannin from cocoa materials has not been conducted, and is not available yet in the market. In this study, the ability of cocoa tannin gel toothpaste to inhibit the mentioned bacteria was determined using an agar dilution method. The minimum inhibitory concentration (MIC) was taken at the lowest concentration of preparation which did not allow visible growth of the organism on series of agar plates (agar dilution) consist of the antimicrobial agents, which in this study is the cocoa tannin gel toothpaste. Determination of MIC using agar dilution method has been proven to be easier compared to other method such as broth dilution or well diffusion. This method is also claimed to be more reproducible than the other former techniques (Moran, 1988). MATERIALS AND METHODS Gel toothpaste formulation The gel toothpaste formulation was described in details elsewhere as well as the extraction and purification of cocoa tannin from cocoa powder prior to incorporation into the gel toothpaste formulation (Azila, 2006). The ingredient of gel toothpaste formulation for this study is listed in Table 1 below.

Table 1: Gel Toothpaste Ingredients Material Ingredients Abrasive Sodium bicarbonate Thickener/binder Xanthan gum & Carbomer1 Preservative Sodium benzoate Surfactant Sodium lauryl ether sulphate (SLES) Humectants Sorbitol & Glycerin Water Purified deionised water Active agent Cocoa tannin extract Flavouring Peppermint oil & spearmint oil Colouring Blue or green Sweetener Sodium saccharin pH modifier To pH 7
1 acrylates/C10-30 Alkyl Acrylate Crosspolymer

Six samples of different cocoa tannin extract content were prepared with a sample as a control. The samples were named with F114 for gel toothpaste of tannin extract from 0.5g cocoa powder, F115 (from 1g cocoa powder), F116 (from 3g cocoa powder), F117 (from 5g cocoa powder), F118 (from 7g cocoa powder) and F119 (from 10g cocoa powder). Two commercial toothpastes, namely Market A and Market 48

B, were also used as a check. Market A toothpaste contained natural green tea essence (gel form) in addition of fluoride as active agent, while Market B is an ordinary fluoride toothpaste. Bacteria strains Two bacteria species were selected for this study, i.e. Streptococcus mutans and Streptococcus sanguis.

Malaysian Cocoa Journal

These bacteria were purchased from Institute of Medical Research (IMR), Kuala Lumpur, in freeze dried form and grown in Nutrient Broth and Mueller Hinton Broth (MH Broth) for comparison. Both bacteria grew successfully in both broths. These facultative bacteria were grown for 48 hours in incubator and their growth were indicated by the sediment of colloid at the bottom of the flask. Minimum Inhibitory Concentration (MIC) Ten grams of formulated cocoa tannin gel toothpaste was mixed with 10mL of distilled water and made into 1:1024 dilution series with distilled water. Two millilitres of each of the dilution was added into Mueller Hinton agar which has been prepared earlier. Each mixture was autoclaved at 121C for 15 minutes before poured onto plates giving a range of dilution from up Growing the bacteria in broth

to 1:10240 with 3 replicates and left for 24 hours at room temperature to set. Using an inoculator, each plate was inoculated with both organisms and incubated for 72 hours at 37C. Figure 2 shows the summary of the MIC method. This method was carried out to determine the MIC of cocoa tannin gel toothpaste against selected bacteria strains, i.e. S. mutans and S. sanguis. In this study, the scale below was applied to interpret the inhibition of the bacterial strains on the agar plates. Scale; 1: 2: 3: 4: 5: no colony on streak line, colony grow on the 1/4 of the streak line, colony grow on the half of the streak line, colony grow on the 3/4 of the streak line, full colony on the streak line. Toothpaste stock solution/ standard on MH agar

Mueller Hinton broth + distilled water Boil to dissolve completely Sterile by autoclaving at 121C for 15 minutes Left 48 hours in incubator Organism
MH agar preparation

10 g toothpaste + 10 mL of distilled water Mix 5 minutes with rotary mixer Centrifuge 2000 g Supernatant diluted with distilled water up to final dilution 1:1024 2 mL of each solution + 18 mL of MH agar Mixed well Pour to plates of 1:10240 Left 24 hours for observation @ Troom Add bacteria using inoculator Incubate 72 hrs at 37C MIC determination

Mueller Hinton agar + distilled water Boil to dissolve completely Sterile by autoclaving at 121C for 15 minutes

Figure 2: Summary of Minimum Inhibitory Concentration (MIC) Method 49

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RESULTS AND DISCUSSION Table 2 below represented the data obtained by the agar dilution method, where it is divided into two section, i.e. I) Streptococcus mutans and II) Streptococcus sanguis. The first column is the name of the samples while the second column is the amount of cocoa powder used to extract cocoa tannin prior to incorporation into the gel toothpaste. Second column represents the dilution of the toothpaste where the data in the column is in the average as the streak was made in triplicate. The next column is the concentration of tannin (mg/L) in the plate agar. Two types of commercial toothpastes (different brand) as mentioned earlier, where Market A is toothpaste with green tea essence, which contain tannin and combined with fluoride, while Market B is toothpaste ordinary toothpaste with fluoride as the active ingredient, were tested against the selective bacterial strains. In Table 2, both of the commercial toothpaste indicates an inhibition of S. mutans effectively. However, the inhibition of Market B toothpaste against S. sanguis is not very effective at dilution 1:64 compared to Market A toothpaste due to the absence of natural green tea extract, which is high content in tannin, with the combination of fluoride. According to the Yu (1995), the combination of tannin and fluoride (Ta-F) enhance the acid resistance of tooth enamel, where the tannin coagulate the proteins in dentine to make them more insoluble and at the same time fluoride react with calcium to form CaF2 for blocking up the dentinal tubes simultaneously, thus reducing the tooth decay more effectively. Fluoride alone as active ingredient in preventing tooth decay is said to be less effective as it fails to act like the combination of Ta-F in dentine, where the S. sanguis exhibited the growth of its population in petri dish containing Market B toothpaste. As indicated in Table 2, S. mutans growth was effectively inhibited by cocoa tannin gel toothpaste, in-vitro. Although the result showed four values of greater than 1.00 but less than 1.50, i.e. at 3.520 mg/ L tannin, 2.050 mg/L tannin, 1.460 mg/L tannin and 0.059 mg/L tannin, these values however can be omitted as isolated cases, where it might be indicating the contamination (EUCAST, 2000). Thus, at low

concentration of cocoa tannin, this plaque bacterium can be overcome at MIC of 0.015 mg/L. In addition, other ingredient in the gel toothpaste, specifically sodium bicarbonate which acts as an abrasive in the toothpaste formulation (Table 1), also have the ability to stop the growth of S. mutans on tooth surface (Kelly, 2000). Combination of this abrasive with cocoa tannin in gel toothpaste formulation is advantageous as it can enhance the reduction of plaque growth. S. sanguis was inhibited at 0.29mg/L as shown in Table 2. The results were expected to behave like S. mutans pattern; however, it turns to be unexpected where the inhibition was less effective with the increasing amount of tannin concentration. The ineffectiveness was might be due to the regrowth of susceptible organisms following deterioration of the active agent as the incubation of the organism might be prolonged (EUCAST, 2000). These two bacteria, i.e. S. mutans and S. sanguis, have the ability to polymerize the glucose, by producing glucosyltranferase, for bacteria adherence on tooth surface (Pader, 1988). According to Yu (1995), the glucosyltranferase or water insoluble glucan is inhibited by the presence of tannin, where it also prevents the aggregation of S. mutans. Therefore, S. mutans, was the major primary plaque, must be inhibited and further eliminating the presence of S. sanguis will help to prevent tooth caries. CONCLUSION The MIC of cocoa tannin gel toothpaste is 0.015 mg/ L where it can inhibit the S. mutans growth. However, the inhibition on S. sanguis is less effective compared to the inhibition on S. mutans. Nevertheless, the inhibition on S. mutans by cocoa tannin gel toothpaste is adequate to promote good tooth health with proper teeth brushing. ACKNOWLEDGEMENT Research supported by Intensive Research in Priority Area (IRPA) of Ministry of Sciences, Technology and Innovation Malaysia (Project No. 01-04-07EA10405).

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Table 2: Minimum Inhibitory Concentration (MIC) of coaoa tannin gel toothpaste against S. mutans and S. sanguis
I) Streptococcus mutans Sample Market A Market B Control F114 F115 F116 F117 F118 F119 Market A Market B Control F114 F115 F116 F117 F118 F119 Cocoa powder (g) 0.00 0.50 1.00 3.00 5.00 7.00 10.00 0.00 0.50 1.00 3.00 5.00 7.00 10.00 1:2 Tannin (mg/L) 1: 4 Tannin (mg/L) 1:16 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.00 3.20 2.20 Tannin (mg/L) 9.380 18.750 56.250 93.750 131.200 187.500 9.380 18.750 56.250 93.750 131.250 187.500 1:64 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.47 1.00 1.00 1.00 1.03 1.47 3.17 2.93 Tannin (mg/L) 2.340 4.690 14.060 23.440 32.810 46.880 2.340 4.690 14.060 23.440 32.810 46.880 1:256 1.00 1.00 1.00 1.01 1.00 1.17 1.00 1.00 1.00 1.00 1.93 1.00 1.00 1.03 3.42 1.30 3.10 2.40 Tannin (mg/L) 0.059 1.170 3.520 5.860 8.200 11.720 0.059 1.170 3.520 5.860 8.200 11.720 1:1024 1.00 1.00 1.00 1.00 1.00 1.00 1.07 1.13 1.00 1.00 1.80 1.00 1.02 1.00 2.75 1.32 3.60 1.60 Tannin (mg/L) 0.015 0.290 0.880 1.460 2.050 2.930 0.015 0.290 0.880 1.460 2.050 2.930

1.00 1.00 1.00 1.00 150.000 1.00 300.000 1.00 900.000 1.00 1500.000 1.00 2100.000 1.00 3000.000 1.00 1.00 1.00 1.00 150.000 1.00 300.000 1.00 900.000 3.00 1500.000 1.10 2100.000 1.63 3000.000

1.00 1.00 1.00 1.00 37.500 1.00 75.000 1.00 225.000 1.00 375.000 1.00 525.000 1.00 750.000 1.00 1.00 1.00 1.15 37.500 1.00 75.000 1.05 225.000 4.83 375.000 2.50 525.000 2.17 750.000

II) Streptococcus sanguis

REFERENCES Azila, A. K. (2006). Research Report on Formulation of Cocoa Based Toothpaste. (IRPA NO: 0304-07-1303-EA10405). Cocoa Downstream Research Centre, Malaysian Cocoa Board. Christine, D.W. (1998). Functional Foods for Health. http://www.ag.uiuc.edu Kelly, J.S. and Kristin, R.T. (2000). Caries Prevention News: Sodium bicarbonate and hydrogen peroxides; the effect on the growth of Streptococcus mutans. Journal of Dental Hygiene 79: 7. Moran, J., Addy, M. and Wade, W. (1988). Determination of minimum inhibitory concentrations of commercial toothpastes using agar dilution method. J. Dent. 16: 27-31. Pader, M. (1988). Oral Hygiene Products & Practice. U.S.A: Marcel Dekker Inc.

Shuch, D.J. & Curatola, G.P. (2003). Dental Formulation. U.S. Patent; 6,503,483. Susan, K.H. (2004). Microbiology Dental Plaque. http://www.dent.ucla.edu Yu, H., Oho, T. and Xu, L. X. (1995). Effects of several tea components on resistance of human tooth enamel. Journal of Dentistry 23: 101-105. Strlfors, A. (1966). Inhibition of hamster caries by cocoa; the effect of whole & defatted cocoa, and the absence of activity in cocoa fat. Arch. Oral Biol. 11: 149-161. EUCAST (2000). Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution method. European Committee for Antimicrobial Susceptibility Testing (EUCAST) Definitive Document (E.Def 3.1). European Society of Clinical Microbiology & Infectious Diseases (ESCMID) 6: 509-515.

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MATHEMATICAL MODELING OF DIRECT SOLAR DRYING OF COCOA BEANS Hii C. L. School of Chemical and Environmental Engineering, Faculty of Engineering and Computer Science, University of Nottingham, Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia Malaysian Cocoa J. 3: 52-57 (2007) ABSTRACT - In this paper attempt was made to model the direct solar drying of cocoa beans using different mathematical models such as Lewis, Henderson and Pabis, Page, Logarithmic, Two-term model and Wang and Singh models. Drying experiment was conducted in Malaysia using cocoa beans prepared from 5-day fermentation technique and a direct solar drier was used. The performance of these models was compared using the determination of coefficient (R2), reduced chi-square ( 2) and root mean square error (RMSE). Results showed that the logarithmic model was found satisfactory in modeling the drying curve of the solar drying process. The effective diffusivity of the drying process was also determined. Key words: Cocoa, Solar drying, Modeling, Regression, Diffusivity INTRODUCTION Cocoa are planted mostly by smallholders in Malaysia, which currently has cocoa planting areas of 25,000 ha, and is about 76% of the nation planting areas (Anon, 2005). In a smallholder farm, fresh cocoa beans are fermented in wooden boxes for five days and sun dried until it reaches the safe moisture level of 7.5%. The bigger holding will use hot air dryer to dry the bigger volume of beans but this technique tends to produce beans of weaker product quality such as high acidity, insufficient browning, smoke contamination and case hardening (Mc Donald et al., 1981). Sun drying is still the most widely used method by cocoa smallholders due to its simplicity, low cost set up and requires only direct sunlight which is renewable and abundant. It was reported that sun dried cocoa beans have better flavour quality and less acidic due to its gentle drying process (Jinap, 1995). However, it has various inherent disadvantages such as unsuitable to be used during rainy season, contamination by rain or dusts, needs frequent supervision and etc. In addition to that, the cocoa harvesting season in Malaysia usually coincides with the rainy season. Therefore, a direct solar drier was proposed to the smallholders with the ultimate goal of product protection. Mathematical modeling of the cocoa drying process has been reported in literatures mostly in the area of hot air drying (Nganhou et al., 1992; Wan Daud et al., 1994; Fotso et al., 1994; Talib et al., 1995). However, there is no report at all on modeling of solar drying of cocoa beans through intensive literature review. Therefore, the presence study was carried out to model the drying kinetics of cocoa in direct solar drying and select the best model for this process. MATERIALS AND METHODS Fresh cocoa beans were harvested from Sg. Ruan, Pahang and fermented using wooden boxes for five days. The beans were turned every two days to ensure uniformity during fermentation. Upon fermentation, the beans were spread on a perforated wooden platform inside a direct solar dryer (Figure 1). The drying mechanism is mainly through direct exposure of the product to sunlight which penetrates through the transparent enclosure. The capacity of the dryer was 20 kg of fermented cocoa beans. Drying started from 8 am to 6 pm daily and mixed manually every hour to ensure uniformity.

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Figure 1. The direct solar drier used in experimentation Samples were taken every 24 hours (100 gm) before mixing for moisture content analyses. Moisture content of the beans was determined according to Association of Official Analytical Chemists (AOAC, 1990). The ambient temperature was recorded using a mercury glass thermometer placed at a shaded area about 10 feet away from the trial site. The experiments were repeated three times to obtain an average set of results. Drying was terminated when the moisture content reached 7.5%. The following mathematical models are used to describe the sun drying process (Table 1). In this model, the moisture ratio (MR) was simplified to M/M0 where subscript 0 denotes initial condition. The coefficient of determination of linear regression (R2) was one of the primary criterions for selecting the best model. In addition to that, statistical parameters such as reduced chi-square (2) and root mean square error (RMSE) were used to determine the fitness. Regression analysis was performed using Microsoft Excel and Matlab software.

Table 1. Mathematical Models Used to describe the solar drying Process Model name Model equation Lewis Henderson and Pabis Page Logarithmic Two term model

Model Constants k a, k k, n a, k, c a, b, c, d a, b

Wang and Smith Note: MR = Moisture content at time t / Initial moisture content Constants k, n, a, b, c and d need to be determined from regression analysis

MR = exp MR = aexp(-kt) n MR = exp(-kt ) MR = aexp(-kt) + c MR = aexp(-kt) + cexp(-dt) MR = 1 + at + bt 2


(-kt)

In general, the higher R2 values and the lower 2 and RMSE values show the best fitting parameters. The parameters are described below: Chi-square:

i=1

(MRexp,i MRpre,i)2 Nz
1/2 N

................ (1)

1 Root mean square error: RMSE = N

i=1

(MRpre,i MRexp,i)

................ (2)

Where, N = number of observations, z = number of constants MRexp,i = ith experimental data, MRpre,i = ith predicted data

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RESULTS AND DISCUSSION Drying curve The variation of ambient air temperatures during the experimental periods is as shown in Figure 2. In average, the temperatures recorded ranged from 26.6C to 34.1C with the highest temperature being recorded at 3 pm. The temperatures recorded were all well below the temperature (60C) detrimental to cocoa beans quality (Mc Donald et al., 1981). During the course of experimentation the weather was quite sunny with occasional overcast days. The

drying curve of the solar drying process is as shown in Figure 3. The initial moisture content was 38.3% and it took 144 hours for the beans to reach below the safe moisture content of 7.5%. Figure 4 shows the drying rate of the solar drying process. The drying rate initially increased for the first 24 hours and gradually decreased but with a slightly higher drying rate towards the last day. This observation is due to the unpredictable weather condition (e.g overcast day, rainy or sunny day) and hence the drying rate was influenced.

Figure 2. Average ambient temperature profile throughout the 6-day drying period

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Figure 3. Comparison of the experimental and predicted moisture content values using the logarithmic model

Figure 4. Drying rates of the solar drying of cocoa beans

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Mathematical modeling The solar drying curve was fitted with six mathematical models and summary of the results of fitting is as shown in Table 2. It can be seen that only three models were able to provide a good fit with R2 greater than 0.95. Generally, 2 values were between 0.5614 and 18.9538 while for RMSE were between 0.5664 and 3.6795, respectively. It is quite clear that the Logarithmic model gave the highest R2 (0.9979), the lowest 2 (0.5614) and RMSE (0.5664). The Henderson and Pabis model was the least fitted with the lowest R2

(0.9196) value and the highest 2 (18.9538) and RMSE values (3.6795), respectively. Based on these results, the logarithmic model was selected and the comparison of the experimental and predicted moisture content values can again be seen in Figure 3 (As appeared in previous section). Effective diffusivity The effective diffusivity of the cocoa beans can be estimated based on the analytical solution of one-dimensional Ficks law of diffusion which can be simplified to a straight line equation (Doymaz, 2005):

In (MR) = In

2Deff t
r2

.............. (3)

Table 2. Summary of regression analysis for various models and parameters Model name Model constants R2 2 Lewis Henderson and Pabis Page Logarithmic k = 0.0137 a = 0.7769 k = 0.0112 k = 0.06750 n = 0.06637 a = 0.8462 k = 0.0286 c = 0.1756 a = 0.2631 b = 103.375 c = 0.7421 d = 0.0116 a = -0.0174 b = 0.0001 0.9425 0.9196 0.9878 0.9979 11.6944 18.9538 1.8805 0.5614

RMSE 3.1660 3.6795 1.1590 0.5664

Two term model

0.9790

5.5331

1.5399

Wang and Smith

0.9247

12.1920

2.9510

Note: R2 = coefficient of determination of linear regression 2 = Chi-square (Equation1) RMSE = Root mean square error (Equation 2) The effective diffusivity (Deff) was calculated by plotting ln MR versus time and the slope of this straight line was used to determine Deff. The value was found to be 2.2974 x 10-08 m2/s. There is no reported value of Deff for cocoa beans in the area of solar and sun drying. However, various diffusivities of other agricultural products are as shown in Table 3 and the range recorded is within 10-09 to 10-11 m2/s (Doymaz, 2005).

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Table 3. Effective diffusivities of various agricultural products Products Effective diffusivity, Deff (m2/s) Apricot Grape Prune Fig CONCLUSION Various mathematical models tested showed that the logarithmic model best fitted the drying data with the highest R2 (0.9979), the lowest 2 (0.5614) and RMSE (0.5664). Therefore, this model can be used to describe the thin layer drying kinetics of the cocoa solar drying process. The effective diffusivity value was estimated by Ficks diffusion model and was found to be 2.2974 x 10-08 m2/s. REFERENCES Anon (2005). Malaysian Cocoa Monitor. Kota Kinabalu, Malaysian Cocoa Board. AOAC (1990). Offical Methods of Analysis. 15th edition, Washington DC, Association of Official Analytical Chemist. Doymaz, I. (2005). Sun drying of figs: an experimental study. Journal of Food Engineering. 71: 403-407. 8.90 x 10-10 -1.30 x 10-09 7.91 x 10-10 - 2.50 x 10-09 4.30 x 10-10 - 7.60 x 10-10 7.77 x 10-10 - 2.45 x 10-09 Fotso, J., Lecomte, D., Pomathios, L. and Nganhou, J. (1994). Convective drying of cocoa beans: Drying curves for various external conditions. Drying 94: 937-994. Jinap, S. (1995). Organic acids in cocoa beans - A review. Asean Food Journal. 9(1): 3-12. Mc Donald, C.R., Lass, R.A., and Lopez, A.S.F. (1981). Cocoa drying-A review. Cocoa Growers Bulletin. 31(7): 5-41. Nganhou, J., Lecomte, D., and Dumargue, P. (1992). Heat and mass transfer through a thick bed of cocoa beans under stationary and transient inlet conditions. Drying 92: 1515-1523. Talib, M.Z.M., Daud, W.R.W., and Ibrahim, M.H. (1995). Moisture desorption isotherms of cocoa beans. Trans. ASAE. 38(4): 1153-1155. Wan Daud, W.R., Meor Talib, M.Z., and Hakimi Ibrahim, M. (1994). Drying of cocoa beans. Drying 94: 1129-1135.

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THE EFFECTS OF COCOA BUTTER EQUIVALENT ADDITION TO THE COCOA BUTTERS MELTING AND CRYSTALLIZATION PROFILES Fisal A., Rosinah, R. and Suzannah, S. Malaysian Cocoa Board, Cocoa Downstream Research Center, Lot 3, Jln P/9B, Section 13 43650 Bandar Baru Bangi, Selangor, Malaysia Malaysian Cocoa J. 3: 58-64 (2007) ABSTRACT - Thermal profile of cocoa butter (CB) added with 1 - 10 % of cocoa butter equivalent (CBE) was examined with Differential Scanning Calorimetry (DSC). The thermal profiles provide valuable information such as melting characteristics and enthalphy. The series of CB mixtures were subjected to the cooling and heating cycles that permitted cocoa butter to crystallize into polymorph II (PII) and polymorph I (PI). The thermal profile indicates that the addition of CBE will increase the rate of polymorph transformation of PI to PII. The curve for polymorph PII and PI were clearly observed and are well defined but slowly diminished when CBE were added up to 4 %. However, at more than 5 % CBE addition, there was no clear and welldefined border of PII and PI. Mathematical model was constructed to understand the effects of CBE addition to CB. Based on the regression programme (stepwise) the best possible model were not ideal (R2 = 0.72). This indicated that data collected were able to describe the relationship but this is not sufficient to produce the mathematical model. The results obtained showed that DSC is an efficient method for detecting CBE addition to CB. The sensitivity of DSC to subtle changes in chemical composition of the samples, make DSC an attractive possibility for development as a quality control procedure. Key words: Cocoa butter, Cocoa butter equivalent, Regression, Thermal profiles, DSC. Abbreviations: CB - Cocoa Butter; CBE - Cocoa Butter Equivalent; DSC - Differential Scanning Calorimetery INTRODUCTION The new draft by the European Commission relating to cocoa and chocolate will allow the addition of vegetable fats other than cocoa butter (CB) of up to 5 % into chocolate (Simoneau et al., 1999; Storgaard, 2000). The foreign fat added must be totally compatible with cocoa butter. Cocoa butter equivalent (CBE) is a fat produced to imitate cocoa butter and the major triglycerides of CBE are very similar to CB. Many researches have been carried out using the various components in CB to detect CBE addition to CB. Some of the most common components that were analysed to detect the addition of foreign fat are the triglycerides composition (Simoneau et al., 1999; Geeraert and Sandra, 1987), steryl ester (Gordan and Griffith, 1992; Freega et al., 1993) and sterol (Crews et al., 1997). In this study, DSC was used as a mean to explore and detect CBE addition in CB mixtures by monitoring the changes in CB thermal profile. Differential Scanning Calorimetery (DSC) are widely used and recognized as important equipment in fats and oils industry. DSC has been used to detect eutectic problems in fat blending (Md. Ali and Dimick, 1994), hardness of cocoa butter from different 58 geographic regions (Chaiseri and Dimick, 1989) and characteristics of seed crystals from different origin (Chaiseri and Dimick, 1995a, 1995b). DSC eliminate the use of chlorinated chemicals such as chloroform and dichloromethane. This will reduce the possibilities of chemical exposure to handlers and reduce the expenditure for disposal of chemical waste. Furthermore it provides easy, fast and reliable results. The objectives of this study were to explore the potential of DSC to detect CBE addition to CB and effect on the CB thermal and crystallization profiles. MATERIALS AND METHODS Materials Cocoa butter and cocoa butter equivalent (CBE) were obtained from KL Kepong Cocoa Products Sdn. Bhd. and International Speciality Fat Sdn. Bhd., respectively. CBE was added into cocoa butter system at 1% level up to 9%. Tempering method The CB and CB mixtures were tempered according to Chaiseri and Dimick (1989). This tempering method will induce CB to crystallize in PII and PI. Five grams of CB mixtures were melted at 60C for 2 min, quenched to 0C and held for 2 min. Analysis was

Malaysian Cocoa Journal

conducted immediately by heating the sample from 0 C to 50 C at 20 C/min. The hardest CB will have the largest area under polymorph II. Fatty acids composition Fatty acid methyl esters (FAME) was prepared by dissolving 0.05 g of the CB CBE mixtures in 0.95 ml hexane (Fischer Chemicals, U.K) to which was added 0.05 ml of 1 M sodium methoxide. Approximately 1 l of FAME/hexane solution was injected into a DB 23 capillary column (Agilent Technologies, U.S.A) installed in a Shimadzu GC 17A (Shimadzu Corp, Japan) Gas Chromatography with FID detector. Helium was used as the carrier gas. The injector temperature was set at 260C, column oven was programmed from 180C to 210 C at 3C / min and the detector was set at 300C. The composition of each individual fatty acid was identified as percentage of area under the respective fatty acids. Peaks identification was based on the retention times of standards. Statistical analysis Data were analyzed by using analysis of variances. The relationships of variables in thermal and crystallization profiles of DSC were analyzed by stepwise regression. Both statistical analyses were analyzed by using SAS package V6.12 (SAS Institute, Cary, NC). RESULTS AND DISCUSSION Fatty acid composition The fatty acid composition of CB and CBE are showed in Table 1. CBE contain higher level of saturated fatty acid (palmitic acid and stearic acid) compared to palm stearine and palm olein. Composition of fatty acids in CBE is more similar to CB. CBE contained lower level of stearic acid compared to CB but higher than palm stearine and palm olein. CBE also contained higher level of palmitic acid compared to CB, which is typical for CBE produced from palm oil.

Oleic acid content in CBE was similar to palm stearine and cocoa butter but lower than palm olein. Meanwhile, linoleic acid content in CBE is similar with CB but lower than palm stearine and palm olein. Table 1. Comparison of fatty acid composition (% area) of CB, CBE, palm stearine and palm olein Fatty acid (%) Lauric acid (C 12) Myristic acid (C 14) Palmitic acid (C 16) Stearic acid (C 18) Oleic acid (C 18:1) Linoleic acid (C 18:2) Palm Stearine (Deffense 1985) Palm Olein (Deffense 1985) -

Cocoa butter

CBE

0.12

0.09

0.54

30.7

39.88

66.2

39.4

42.5

33.54

4.8

4.3

20.7

22.54

24.6

42.3

2.9

2.01

5.0

11.3

Variables measured in thermal and crystallization of CB at different CBE addition The variables that were used to describe the changing of thermal and crystallization profile are presented in Figure 1 and Figure 2.

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Figure 1. The variables measured in thermal profile of CB *Abbreviations: I Peak 1 II Peak 2 III Offset IV Onset V DH1 VI DH2 Peak of the low melting fraction Peak of the high melting fraction Offset of the high melting fraction Onset of the low melting fraction Enthalphy of the low melting fraction Enthalphy of the high melting fraction

Figure 2. The variables measured in crystallization profile of CB *Abbreviations: i OnsetC ii PeakC iii OffsetC iv DHC Onset of crystallization Peak of crystallization Offset of crystallization Crystallization enthalphy

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Behaviors of CB thermal profile of CB at different CBE addition Graph A in Figure 3 showed the typical CB melting profile. Two prominent peaks, PI and PII were well separated. Peak 1 of the thermal profile was at 22.70C, Peak 2 at 26.76C with enthalphy of 69.02 J/g (Table 2). This thermal profile is often associated with hard cocoa butter, where the height of PIIs peak is almost identical to peak I. According to Chaiseri and Dimick (1989), this behaviour indicates that the hard cocoa butter had a faster transformation rate. Graph B (Figure 3), showed the effects of CBE addition (3 %) on the CB thermal profile. The two peaks are still visible but not well separated and the depth of the valley that separated the two fractions is shallower. Peak 1 of the thermal profile was at 22.95C

and Peak 2 at 26.80C. The melting enthalphy also increased to 66.79C from 65.87C. At 5 % CBE addition (Graph C; Figure 3), only one peak was detected. The melting profile was derivatized to enhance the identification of the two fractions. The derivative graph can be seen in Figure 3 as the thick line. The derivative graph also indicate only one peak exist and no valley was identified. The other effect of 5 % CBE addition is the offset temperature decreased as the CBE addition increased; i.e from 16.24 C to 14.55 (p<0.05). CBE addition showed no significant effect on the enthalphy although the enthalphy showed slight increase from 65.87 J/g to 69.02 J/g.

a.

b.

c.

Figure 3. The thermal profile of CB with different level of CBE addition shown in the thin line; (a) CB before CBE addition, (b) CB after 3 % CBE addition, (c) CB after 5 % CBE addition. The thick line shows the derivative of the thermal profile.

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Behavior of CB crystallization profile of CB at different CBE addition The addition of CBE increased the onset, offset and peak temperature of the crystallization profile. The onset temperature of crystallization of CB was 16.16C but increased to 17.52C after 9% CBE addition (Table 3). Meanwhile the peak of crystallization of CB increased from 11.08C to 18.81C and at 9% CBE addition. However, offset temperature of CB crystallization profile decreased from 4.83C to 4.61C. However the addition of CBE to CB did not affect the enthalphy (H) of crystallization significantly. The enthalphy of crystallization decreased from - 50.92 J/g to - 44.25 J/g at 9 % CBE addition (p < 0.05). At 5% CBE addition to CB, the enthalphy of crystallization decreased to 50.65 J/ g. This observation showed that CBE addition to CB did not affect the crystallization behavior significantly. Regression analysis on variables measured in thermal and crystallization profile of CB at different CBE addition The best mathematical model to describe the effects of CBE addition is; % CBE = 17.7815 + 0.4120 Peak 1 0.9655 Peak 2 1.4104 Onset + 0.6941 Offset. The parameters are significant (p<0.05) but not ideal due to low R2 (0.72). The model showed that, the

addition of CBE has positive effects on Peak 1 and offset. However the model showed negative effects on Peak 2 and Onset. The mathematical model also indicates; the CBE addition did not change the melting enthalphy significantly. These results imply that, the addition of CBE will decrease the onset temperature but will increase the offset temperature. CBE addition to CB also increased the transformation rate of PI to PII. This indicates, the addition of CBE will promote CB to rapid nucleation and growth. The addition of CBE to CB will increase the proportion of saturated fatty acid because CBE is high in saturated fat and low in unsaturated fatty acid especially linolenic acid. High content of saturated fatty acids are associated with rapid nucleating crystals and faster crystals growth. Chaiseri and Dimick (1995a, b) reported that CB rich in saturated fatty acids like Malaysian CB showed rapid nucleating and high crystals growth rate. This observation implied that the addition of CBE could be detected by monitoring its thermal profiles. CONCLUSION The DSC method is sensitive enough to detect changes in cocoa butter with CBE. This method can identify CBE addition more than 4 % by visually monitoring the changes in the temperature variable of the thermal profile thermo gram. However, further studies are required on different cocoa butters to determine the robustness of this formula.

62

Table 2. The effects of CBE addition to the CB thermal profile Level of CBE additions in cocoa butter (%) Parameter 0 1 2 3 4 5 6 7
un
a

8
un
a

9
un
a

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Peak 1 (C)
ab a b a ab a a a

22.70 26.05
a

ab

23.32 26.64 15.38 b 31.78 a 65.63 a 39.75 a 25.88


a a a a a

22.28 26.80 14.97 b 32.23a 66.79a 39.72a 27.07 0.68a 1.47a 1.58a un un 0.66a un un 26.39 69.02 69.28 40.63 a un un un 69.84 un un
a

22.95 26.76 14.82 b 32.5a 67.02 a 69.02ab 69.28ab 69.84ab 33.28abc 33.64ab 34.07a 14.55cde 14.41 de 14.22de 13.85 e 34.11a 70.31 a un 70.31 un un
a

22.79 26.16 26.09 26.21 25.88

un

un

Peak 2 (C) 15.59ab 32.23 a 66.38 a 40.35 a 26.03


a

26.76

25.84a 15.00bcd 34.10a 69.30ab un 69.30a un un

Onset (C)

16.24a

Offset (C)

31.49a

DH (J/g)

65.87a

DH1 (J/g)

38.67a

DH2 (J/g) 0.65a 1.58a 1.54a 0.65a

27.21

RaDh2Dh1 (J/g) (Dh2 / Dh1)

0.70 a

63
11.68a 5.42a 5.07a 4.34a 11.50a 11.86a 11.13a 4.44a 11.40a 4.73a

RaDh1Dh2 (J/g) (Dh1 / Dh2)

1.42 a

* un = undetected * Data in different column with different superscripts are significantly different (P < 0.05).

Table 3. The effects of CBE addition to the CB crystallization profile Level of CBE additions in cocoa butter (%) Parameter 0 1 2 3 4 5 6 OnsetC (C) 16.16b 16.57ab 16.37ab 17.05a 16.63ab 17.06a 17.14a 10.67a 3.83a -48.68ab

7 17.39a 11.4a 4.18a -50.45ab

8 17.46a 11.07a 4.09a -50.78ab

9 17.52a 18.81a 4.61a -44.25b

PeakC (C)

11.08a

OffsetC (C)

4.83a

DHC (J/g) -50.92a -51.35a -50.41a -50.50a -49.38a -50.65ab * Data in different column with different superscripts are significantly different (P < 0.05).

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Table 4. Stepwise regressions on thermal profile of CB at different CBE addition Step Regression equation
1 2 3 %CBE = %CBE = %CBE = 26.5057 14.4357 7.3513 17.7815 (8.9494) (0.0580) - 1.5994ONSET + 0.5564PEAK1 + 0.3659PEAK1 - 1.6446ONSET - 1.6143ONSET

R2
0.5733 0.6854 0.7146 + 0.6947 OFFSET (0.2909) (0.0248)

Pr > F
< 0.0001 < 0.0001 < 0.0001

+ 0.3437 OFFSET - 1.4104 ONSET (0.2466) (< 0.0001)

%CBE =

+ 0.4120PEAK1 (0.2043) (0.0546)

- 0.9655PEAK2 (0.5716) (0.1036)

0.7439

< 0.0001

* Figures in the first row parentheses are the standard error of the respective parameters. * The P values of these variables are in the second row parentheses. REFERENCES Chaiseri, S. and Dimick, P.S. (1989). Lipid and Hardness Characteristics of Cocoa Butters from Different Geographic Regions. J. Am. Oil Chem. Soc. 66 (11): 1771-1776 Chaiseri, S. and Dimick, P.S. (1995a). Dynamic Crystallization of Cocoa Butter. 1. Characterization of Simple Lipids in Rapid- and Slow- Nucleating Cocoa Butters and Their Seed Crystals. J. Am. Oil Chem. Soc. 72: 1491-1496. Chaiseri, S. and Dimick, P.S. (1995b). Dynamic Crystallization of Cocoa Butter. II. Morphological, Thermal and Chemical Characteristics During Crystal Growth. J. Am. Oil Chem. Soc. 72: 1497-1504 Crews, C., Calvett, S.R. and Brereton, P. (1997). The analysis of sterol degradation products to detect vegetable fats in chocolate. J. Am. Oil Chem. Soc. 74 (10): 1276-1280. Deffense, E. (1985). Fractionation of palm oil. J. Am. Oil Chem. Soc. 62 (2): 376 -385. Frega, N., Bocci, F., Giovannoni, G. & Lerker, G. (1993). High resolution GC of unsaponifiable matter and sterol fraction in vegetable oil. Chromatographia 36: 215-217. Geeraert. E. & Sandra, P. (1987). Capillary GC of triglycerides in fats and oils using a high temperature phenylmethylsilicone stationery phase. Part II. Analysis of chocolate fats. J. Am. Oil Chem. Soc. 64 (1): 100-103. Gordan, MH. and Griffith, R. (1992). Steryl ester analysis as an aid to the identification of oils in blends. Food Chemistry. 43: 71-78. Md. Ali, A.R. and Dimick, P.S. (1994). Melting and Solidification Characteristics of Confectionery Fats: Lauric Hard Butter, Anhydrous Milk and Cocoa Butter blends. J. Am. Oil Chem. Soc. 71 (8): 803-806. Simoneau, C., Hannaert, P. and Anklam, E. (1999). Detection and quantification of cocoa butter equivalents in chocolate model system: analysis of triglyceride profiles by high resolution GC. Food Chemistry. 65: 111-116. Storgaard, S. (2000). New vegetable fats. Sweetec. 4.

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Transfer of Technology EVALUATION ON ADOPTION OF TRANSFER TECHNOLOGY IN THE SMALLHOLDER DEVELOPMENT PROGRAMME Mahmod, M.N. Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Sabah, Malaysia Malaysian Cocoa J. 3: 65-70 (2007) ABSTRACT - In 1996, Malaysian Cocoa Board (MCB) initiated the Smallholder Development Programme. The purpose of this programme is to rehabilitate unproductive cocoa fields and to increase cocoa smallholders productivity in Malaysia. Proper rehabilitating techniques were used such as selecting high yielding disease resistance clones, side grafting techniques, fertilizers application, pests and diseases control and provided advisory services to smallholders involved with the rehabilitation areas. Also, an effective technology transfer package was introduced to smallholder to ensure success in raising productivity with the correct use of technology and standard crop management practices. In 2004, evaluations on adoption of transfer technology in the Smallholder Development programme were conducted by MCB. The evaluations were made on the level of technology transfer accepted and adopted by smallholder. The level of technology acceptance measured the smallholders perception towards the short courses and practical trainings in the Smallholder Development Programme. The level of technology adopted measured the smalllholders ability to practice the recommended technology after completing the Programme. Key words: Technology transfer, Cocoa rehabilitation technology, Smallholder development INTRODUCTION Malaysian Cocoa Board (MCB) in collaboration with the Department of Agriculture had set up Smallholder Development Programme in 1996. The objective of this programme is to increase smallholders yield from 0.5 tonnes to 1.5 tonnes per hectare. In the implementation of this programme, potential smallholder farms in the cocoa growing areas were selected and rehabilitated for 3 years. The smallholders were directly involved in the programme, starting from rehabilitation or replanting of cocoa trees, attending short courses organized by MCB to marketing of cocoa beans. They were exposed to effective cultural practices through short courses on efficient cocoa rehabilitating techniques, selection and introduction of high yielding disease resistance clone, side-grafting, fertilizers application, pests and diseases control techniques. In 2004, MCB conducted a study on the effectiveness of technology transfer in the Smallholder Development Programme. This study evaluated the effectiveness of technology transfer among cocoa smallholders in Malaysia in particular to identify the level of technology transfer accepted and adopted by cocoa smallholders involved in the programme. Information on the smallholder's background, farm management, yield and level of cocoa technology adopted and smallholders acceptance to technology transfer were collected and analyzed. METHODOLOGY This study focused on the smallholders participated in the Smallholder Development Programme. A stratified random sampling of 253 respondents was made comprising of smallholder who had completed the programme. Altogether 203 smallholders or 80 percent of the participants selected were from Peninsular Malaysia (73), Sabah (110) and Sarawak (20). A preset questionnaire was used to obtain information on the smallholders background, farm activities, smallholders perception towards shortcourses and presentation materials used in the Smallholder Development Programme. Enumerators also made farm visits to evaluate the farm activities practiced by the smallholder. The Level of Technology Awareness was one of the approaches used by the extension agencies such as the Department of Agriculture and FAMA to measure the effectiveness of transfer technology. Rahim Sail et al. (RRIM, 1985) in doing research on

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the extension activities to rubber smallholder used this analysis to determine the level of acceptance of rubber technology by smallholder. The same methodology was also used to determine the level of technology TPT or TPT a b c d e f g h = = = = = = = = = Percentage <60 61 - 80 >81 RESULTS Analysis 1: Level of smallholders acceptance to technology transfer This study evaluated the smallholders acceptance level toward the technology transfer in the Smallholder Development Programme. Weighted points of low to high (0 5) were awarded to the smallholder who accepted the recommended technology. The recommended technologies were side grafting, pruning, fertilizer application, chemical spraying application, pest control, disease control, cocoa processing and identification of quality beans technology. Each technology was evaluated based on the smallholders' understanding, skill, interest and motivation after they had attended the short courses and practical training organized by MCB. The score of less than 60 percent was counted as low technology transfer acceptance level, score of 60 to 80 percent was counted as medium technology transfer acceptance level and the score of more than 80 percent was considered as high technology transfer acceptance level. 66 (a + b + c + d + e + f + g + h) Total technology recommended = Total technology adopted Total technology recommended

transfer awareness and adopted by cocoa smallholder. The level of technology transfer awareness and adopted is measured using the following formula:

100

100

Side grafting technology Pruning technology Fertilizer application technology Chemical spray application technology Pest control technology Disease control technology Cocoa processing technology Identification of quality beans technology Application of Technology Transfer Low Application/Input Medium Application/input High Application/Input

In Table 1, about 39.9% of the smallholder involved with the Smallholder Development Programme had achieved more than 80% effectiveness transfer of technology. According to study area, majority of the smallholders in Perak and Johor achieved high technology transfer acceptance level of 71.4% and 50.0% respectively. Smallholders in Pahang, Sabah and Sarawak were mostly in the medium technology transfer acceptance level of 60.7%, 53.6% and 55.0% respectively. Overall, majority or 54.2% of the smallholders effectiveness of technology transfer was in the medium technology acceptance level. As shown in Table 2, smallholder had high technology transfer acceptance level towards pruning (84.7%). Other technologies such as side grafting (79.3%), spraying chemical application (76.1%), identifying quality beans (75.6%), processing of beans (75.4%), fertilizers application (74.8%), pest control (74.3%) and disease control (73.0%) were in the medium technology transfer acceptance level.

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Table 1. Farmers acceptance to technology transfer % <40 41 - 60 61 - 80 81 - 90 >90 TOTAL Pahang Nos. % 0 0.0 2 3.6 34 60.7 12 21.4 8 14.3 56 Perak Nos. % 0 0.0 0 0.0 2 28.6 4 57.1 1 14.3 7 Johor Nos. % 0 0.0 1 10.0 4 40.0 1 10.0 4 40.0 10 Sabah Nos. % 0 0.0 9 8.2 59 53.6 32 29.1 10 9.1 110 Sarawak Nos. % 0 0.0 0 0.0 11 55.0 5 25.0 4 20.0 20 Total Nos. % 0 0.0 12 5.9 110 54.2 54 26.6 27 13.3 203

Table 2. Evaluation on farmers acceptance to technology transfer Technologies Side-grafting Pruning Apply Fertilizer Spraying Chemical Pest Control Disease Control Processing Bean Identify Quality Beans Average Pahang % 78.9 80.0 63.9 67.4 67.7 65.8 77.6 77.1 78.7 Perak % 82.0 84.0 84.0 80.0 74.0 71.0 88.0 88.0 81.7 Johor % 86.6 97.1 86.6 82.9 80.0 80.0 80.0 80.0 85.1 Sabah % 77.4 86.5 78.3 79.5 76.7 75.7 70.6 71.5 77.1 Sarawak % 86.0 84.0 81.0 77.0 78.0 77.0 87.0 86.0 82.0 Average % 79.3 84.7 74.8 76.1 74.3 73.0 75.4 75.6 76.8

In Figure 1, smallholders in Johor had highest technology transfer acceptance level of 85.1%. This contributed to pruning (97.1%), side grafting and fertilizer application (86.6%). Secondly, was Sarawak with technology transfer acceptance level of 82.0%. Sarawak smallholders had high technologies acceptance towards processing of dry beans (87.0%),

side grafting and identification of beans (86.0%). Thirdly, was Perak with technology acceptance level of 81.7%. Perak smallholders had high technology acceptance towards processing of beans and identifying quality beans (88.0%), pruning (84.0%) and fertilizer application (84.0%).

Figure 1. Evaluation on smallholders acceptance to technology transfer 67

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Analysis 2: Level of technology transfer adopted In this study, one point (1) was awarded to the smallholder who adopted the recommended technology and no point (0) was given if the smallholder did not adopt this technology. A total of 8 points were awarded or 100 percent if the smallholders applied all the 8 technologies. The total percentage score was based on the technology adopted by the smallholder. The score of more than 80 percent is counted as high technology transfer adopted level. The score of less than 60 80 percent is counted as medium technology transfer adopted level and less than 60 percent is counted as low technology transfer adopted level. In Table 3, a total of 104 smallholder or 51.2 percent of the respondents were categorized in the high technology transfer adopted level. Smallholder in Perak, Sarawak, Pahang, Johor and Sabah had high transfer technology adopted level of 85.7 percent, 65.0 percent, 50.0 percent, 50.0 percent and 47.3 percent respectively.

In the application of technologies, Table 4 and Figure 2 showed that most of the smallholder had high level of technology transfer adoption on pruning, identification of quality beans and side grafting technologies at 100.0 percent, 85.2 percent and 82.8 percent respectively. Smallholders had medium level of technology transfer adoption on spraying chemical (78.3%), pest and disease control (72.4%) and fertilizers application (63.5%). However, majority of the smallholders of the study area especially in Johor and Sarawak had high level of technology transfer adoption. Study areas in Pahang, Perak and Sabah had medium level of technology transfer adopted. Pahang had a low level of technology transfer adoption toward disease control and fertilizer application. Perak had low level of technology transfer adoption toward pest and disease control. Sabah had a low adoption level on processing technique. In this case, although they have the knowledge to process cocoa beans but preferred to sell wet beans due to small quantity produced and need fast cash return.

Table 3. Farmers level of technology transfer adoption % <40 41 - 60 61 - 80 81 - 90 >90 TOTAL Pahang Nos. % 12 21.4 7 12.5 9 16.1 15 26.8 13 23.2 56 Perak Nos. % 0 0.0 0 0.0 1 14.3 2 28.6 4 57.1 7 Johor Nos. % 0 0.0 3 30.0 2 20.0 0 0.0 5 50.0 10 Sabah Nos. % 13 11.8 3 2.7 42 38.2 36 32.7 16 14.6 110 Sarawak Nos. % 1 5.0 3 15.0 3 15.0 4 20.0 9 45.0 20 Total Nos. % 26 12.8 16 7.9 57 28.1 57 28.1 47 23.1 203

Table 4. Farmers level of technology transfer adoption by technology and area Technologies Side grafting Pruning Fertilizer application Spraying chemical Pest control Disease control Processing Identify quality bean Average Pahang % 92.8 100.0 48.2 66.1 58.9 64.3 100.0 73.2 75.4 Perak % 100.0 100.0 60.0 70.0 50.0 50.0 100.0 70.0 75.0 Johor % 85.7 100.0 85.7 85.7 85.7 85.7 100.0 100.0 91.1 Sabah % 72.7 100.0 70.0 86.4 81.8 80.0 15.5 90.0 74.5 Sarawak % 100.0 100.0 65.0 70.0 65.0 60.0 95.0 95.0 81.3 Average % 82.8 100.0 63.5 78.3 72.4 72.4 53.7 85.2 76.0

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Figure 2. Smallholders adopted transfer technology In Figure 3, with the exception of processing technology, the trend of technology transfer acceptance and adoption level was similar. However, it was found that the technology transfer adoption level was higher in side grafting, pruning, chemical spraying and identifying beans technologies as compared to the awareness level. Smallholders had highly accepted side grafting and pruning technologies due to effective extension approach, required no or less labour assistance and incur low cost to practice these technologies. The technology level for fertilizers application, pest and disease control were lowly adopted because of high cost chemical inputs and lack of labour assistance. CONCLUSION This study is to evaluate the technology transfer of Smallholder Development Programme by determining the transfer technology awareness level and transfer technology adopted level. In this study, two surveys were conducted, that is during the respondent participated in the programme and after they had completed the programme. It was found that both analyses showed a similar trend but had a higher percentage transfer technology adopted level on side 69 grafting, pruning, chemical spraying and identification of quality beans. By evaluating the transfer technology awareness and transfer technology adopted levels in the Smallholder Development Programme, it can measure the effectiveness of these courses or modules to educate smallholder to rehabilitate their farms. However, there are many factors that can influence the effectiveness of technology transfer. In this study, the main factors influencing the technology transfer in the Smallholder Development Programme were smallholders age, educational status, smallholders background and competition from other commodity. At the time of study, the average age of the smallholders participated in the Smallholder Development programme was 57 years old. The average age of the smallholders in Pahang, Perak, Johor, Sabah and Sarawak were 59 years, 60 years, 65 years, 59 years and 54 years old respectively. In relation to the smallholders educational status, about 35.0 percent of the smallholders did not have formal education. Majority or 61.6 percent of the smallholders had primary and secondary schooling. The smallholders after participating the programme had to manage the farm on their own. Smallholder

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Figure 3. Relationship between smallholders awareness and adopted technology especially in Pahang, Perak and Sarawak had difficulty to maintain and continue applying fertilizer and chemical inputs because of the high cost incurred. Result of the study varies according to areas due to the influence of other commodity in the area REFERENCES Anon (1992). Impak Pemindahan Teknologi Koko Di Kalangan Pekebun Kecil Di Malaysia. Ministry of Agriculture Malaysia and Agricultural University Malaysia. Mahmod, M.N., Masarudin, M.Y. and Omar, H.T. (1998). The cocoa smallholder development programme: a model of technology transfer an early progress. Paper presented at Malaysian International Cocoa Conference 1998 organized by Malaysian Cocoa Board in collaboration with the Cocoa Manufacturers group of Federation of Malaysian Manufacturers and Malaysian Tourism Promotional Board. 26-27 Nov. 1998, Sunway Pyramid Convention Centre Kuala Lumpur. Masarudin, M.Y. and Omar, H.T. (1998). Smallholder Development Programme The Programme and Progress in Rehabilitation of Cocoa Smallholder. Paper Presented at workshop on increasing productivity in cocoa cultivation through rehabilitation organised by Malaysian Cocoa Board, 3 Oct. 1998, Tawau, Sabah. such as palm oil. The study areas in Sabah such as Ranau and Tenom had shown that the farmers were motivated to continue planting cocoa while smallholder in Lahad Datu and Tawau showed more inclined to plant palm oil. This case is similar in Perak, as half of the participants had planted palm oil.

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Short Communication DEVELOPMENT AND EVALUATION OF SUITABLE ARTIFICIAL DIETS FOR COCOA POD BORER REARING Meriam, M.Y.1, Furtek, D.2 and Henipa J.1 Malaysian Cocoa Board, Cocoa Research and Development Centre, Miles 10, Apas Road P. O. Box 60237, 91012 Tawau, Sabah, Malaysia 2 Malaysian Cocoa Board, Cocoa Biotechnology Research Centre, Commercial Zone 1, Norowot Road, 88460 Kota Kinabalu Industrial Park, Sabah, Malaysia
1

Malaysian Cocoa J. 3: 71-74 (2007) ABSTRACT - Many species of insects have been successfully reared on artificial diets. However, no suitable artificial diet has been developed for cocoa pod borer (CPB). In this study, 172 artificial diets were evaluated. Artificial diet number 141 was the best diet in terms of duration of larva survival; larval survive up to 46 days. On the other hand, artificial diets number 63 and 64 were the best in terms of maximum number of larvae that survived, the average maximum numbers of larvae surviving were 6.40 and 6.08, respectively. No larvae pupated and no adult moths were obtained Key words: Cocoa, Cocoa pod borer, Artificial diet

Cocoa Pod Borer (CPB), Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae) is the most destructive pest attacking cocoa in the Southeast Asian countries, especially in Malaysia, Indonesia and the Philippines (Soekadar, 2000). In Sabah, East Malaysia an outbreak of the pest was first confirmed in Tawau, Quoin Hill area, in late 1980 covering 5,000 ha of cocoa. In Peninsular Malaysia, it was first confirmed attacking 800 ha of cocoa in Malacca in late September 1986 (Ling et al., 1987). Infestation by this pest, if left uncontrolled, may result in total yield loss (Azhar 1986a,b,c). Artificial diets have been developed for many insect pests and have been vital for developing control measures. However, before our study there has been no report CPB being successfully reared artificially in the laboratory. There is no media formulated or developed for CPB or any other member of the Gracillariidae family of Lepidoptera. Successful mass rearing of the cocoa pod borer from egg to adult in the laboratory would facilitate its use in testing and evaluation of insecticides, bio-control agents, B.t. toxin and semio-chemicals and their subsequent development for pest management.

The research was conducted at the Cocoa Research and Development Centre, Mile 10, Tawau. Laboratory was maintained at 25 to 30 1C and 70 - 90 10 % relative humidity. The experimental design was complete randomized design with three replicates. Media were poured into three different Petri dishes (9 cm in diameter) then kept in the dark. One hundred and seventy two (172) artificial diets were tested on the growth on the CPB. Preparation of the media for rearing the cocoa pod borer Blended agar powder, casein hydrosylate, woody plant medium, wheat germ, phytagel and soya protein. Add cholesterol, primrose oil, and flax seed oil. Add with the cocoa pulp juice. Adjust the pH to 6.5 with KOH. Autoclave the diet for 20 minutes at 121C. Cooled at 60C. Dissolved sucrose and glucose in 100 ml distilled water and filter sterile with the Kao & Michasyluk vitamins. Add into the media. Pour the media into the Petri dish.

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Table 1. Basic ingredient of an artificial diet for rearing the cocoa pod borer Compound Agar powder Woody plant medium Sucrose Casein hydrosylate Cholesterol Primrose oil Flax seed oil Pulp juice of cocoa Phytagel Kao & Michasyluk vitamins Glucose Soya protein Wheat germ Quantity 10.00 g 0.72 g 4.50 g 10.50 g 0.15 g 0.75 g 0.75 g 293 ml 0.75 g 3.00 ml 1.5 g 5.00 g 5.00 g

Experimental insects Male and female adults were collected from nearby cocoa field and were caged together in plastic aquarium tank. After 24 hours, fresh cocoa pods were hung with a wire string inside the cage. CPB eggs were collected after 24 hours. Ten eggs were placed on the media surface. There were three replicates for each diet. Data recording CPB egg hatchability and larvae growth were monitored daily. Among, the 172 artificial diets tested, no. 141 was the best for CPB rearing based on the maximum days of larvae survival. In this artificial diet, CPB larvae survived up to 44 days. Figure 1 shows the larvae on diet 141 from 1 to 44 days. No larval pupated and no adults were obtained, however.

Artificial diet number 141 was the best in terms of day of larva survival. Larvae could survive up to 46 days. Diets must be further refined to permit development from egg to larva to pupa to adult to egg again. This is our long term goal but our immediate goal is only to keep larvae alive long enough to evaluate the effect of the various insecticides compounds. ACKNOWLEDGEMENT The authors would like to acknowledge the Director General of the Malaysian Cocoa Board for his permission to publish this paper. The project was funded with IRPA grant, 01-04-07-0530.

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1 day

3 days

5 days

7 days

9 days

11 days

13 days

15 days

17 days

19 days

21 days

23 days

25 days

26 days

27 days

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34 days

44 days

Figure 1: CPB larvae from 1 to 44 days on artificial diet. Magnification of the larvae size is 20 x 0.75. REFERENCES Azhar I. (1986a). A threat of cocoa pod borer, (Conopomorpha cramerella), infestation to the Malaysian cocoa industry. I. On the biology and damage. Teknologi Koko Kelapa MARDI 2: 53 - 60. Azhar I. (1986b). A threat of cocoa pod borer, (Conopomorpha cramerella), infestation to the Malaysian cocoa industry. II. Control tactics. Teknologi Koko Kelapa MARDI 2: 61-69. Azhar I. (1986c). A threat of cocoa pod borer, (Conopomorpha cramerella), infestation to the Malaysian cocoa industry. III. Integrated pest management and contingency action plans. Teknologi Koko Kelapa MARDI 2: 71 - 76. Ling A.H., Yew C. C. and Koh T.S. (1987). Experience in handling the cocoa pod borer outbreak in Peninsular Malaysia. Proc. Sym Management of cocoa pod borer, Kuala Lumpur, Malaysia. Soekadar W. (2000). The use of entomopathogenic fungus (Beauveria bassiana) to control cocoa pod borer (Conopomorpha cramerella) in the field. Proc. INCOPED 3rd Int. Seminar on cocoa pest and disease, 16-17 October, Kota Kinabalu, Sabah, Malaysia.

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STABILITY OF HIGH INTERNAL PHASE (HIP) COCOA BUTTER EMULSION Samuel, Y.K.C. and Sarini, H. Malaysian Cocoa Board, Cocoa Downstream Research Center, Lot 3, Jln P/9B, Section 13 43650 Bandar Baru Bangi, Selangor, Malaysia Malaysian Cocoa J. 3: 75-77 (2007) ABSTRACT - Stability of high internal phase (HIP) cocoa butter emulsion was carried out by a microprocessor controlled centrifuge that detects the de-mixing processes of disperse systems during centrifugation over the whole sample height. Result showed that the formulated high internal phase (HIP) cocoa butter emulsion was able to withstand a 1,200 g of centrifugal force for 78,000 s without significant de-mixing processes detected and this can be translated into 3 years of stability in normal storage condition on shelf. Key words: Cocoa butter emulsion, Emulsion stability, Shelf life

High Internal Phase (HIP) cocoa butter emulsion is a pre-emulsified concentrates of emollients and moisturizers. Development of HIP cocoa butter emulsion appears as a foundation technology, which can be readily added with other additives by simple mixing in room temperature to produce a wide range of cream/ lotion based products. However, settling (sedimentation) and creaming (flotation) of the emulsions components are the common problem, which leading to the separation of two phases (Cooper and Gunn, 1975). The rate of sedimentation or creaming can be approximately described by Strokes Law, which describes the factors influencing the velocity of a dispersed phase droplets, moving under force of gravity, in a continuous liquid medium. The equation describing Stokes Law is given below (Knowlton, 2005; LUM, 2004): V = (2/9) (d2/) xg and where xg = 2 (R/9.81) [1] [2]

d xg R

= = = = =

particle diameter (m) viscosity of liquid (mPa s) multiples of gravity force (m/s2) angular velocity (1/s) rotor position (m)

V = de-mixing velocity = difference of densities (g/cm3)

The greater the de-mixing velocity, the more likely that dispersed phase droplet-droplet collisions will occur, which give rise to emulsion instability. From equation [1] and [2], de-mixing velocity of the emulsion can be increased by increasing the gravity force by centrifugation with appropriate angular velocity, and, therefore, the stability of the emulsion determination can be accelerated by detecting the clarification speed and its standard deviation should the de-mixing processes occurred. The course of creaming of the emulsion is easily observed since the whole test time is shown by the software. The transmission rises continuously from the bottom of the sample cell should the creaming phenomenon occur, which means it forms a particle free phase under a barely transparent flotation. A typical transmission profiles for creaming was as in Figure 1 (LUM, 2005). The objective of this paper was to determine the stability of the HIP cocoa butter emulsion developed by MCB using centrifugation method.

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Figure 1: Typical transmission profiles (The transmission rises continuously from the bottom of the cell, which means it form a particle free phase under a barely transparent flotation) An amount of 0.5ml HIP cocoa butter emulsion was filled in the standard plastic measurement cells provided by LumiFuge and subjected to centrifugation force of 1,200 g for 78,000 s at 30C using Lumifuge 116. Microprocessor controlled high optical resolution sensors coupled with the NIR (near infrared) transmission were used to detect the de-mixing processes of dispersed system during centrifugation over the whole sample height. During the centrifugation, transmission profiles were recorded every 306 seconds and up to 255 transmission profiles recorded to show the kinetics of the separation process along the sample height of 27 mm. The transmission profiles of the HIP cocoa butter emulsion, red in the beginning, green towards the end of the measurement was shown in Figure 2, showing no de-mixing processes were detected after 78,000 s at centrifugal force of 1,200 g.

Figure 2: Transmissions profiles from Separation Analyser LUMiFuge 116. (centrifuged at 1,200 g for 78,000 s at 30C. Transmission profiles were recorded every 306 seconds and up to 255 transmission profiles recorded to show the kinetics of the separation process along the sample height of 27 mm) 76

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By taking into account a linear dependency between the de-mixing velocity and the applied relative centrifugal force (xg); and the assumption of one month shelf life at gravity corresponds to 2,592,000 s [3,600 s (1hour) x 24 (1day) x 30 (1 month)] (LumiFuge, 2004), the HIP cocoa butter emulsion that could withstand 1,200 g of centrifugal force for 78,000 s without separation detected, are therefore, can be translated into 3 years (1,200 g x 78,000 s = 93,600,000 s = 36.11 months 3 years) stability at gravity. HIP cocoa butter emulsion exhibits considerably stable behavior for use as a preemulsified concentrates of emollients, and moisturizers, that is then readily incorporated into formulation at room temperature by simple mixing during the finishing step to produce a wide range of products. It is therefore, can reduce the stressful work of formulating or reformulating should there is a products range widening or a changes in formulations.

ACKNOWLEDGEMENTS Research project is supported by Intensive Research in Priority Area (IRPA) of Ministry of Sciences, Technology and Environment of Malaysia. REFERENCES Cooper, J.W. and Gunn, C. (1975). Chapter 11: Emulsions and Creams. In: Dispensing for Pharmaceutical Student 12th Edition. London: Pitman Medical Publishing Co. Ltd. Knowlton, J. (2005). Module 12: Emulsions. Society of Cosmetic Scientist. LUM (2004). Stability Analyser LUMiFuge Manual. Berlin: L.U.M. GmbH. LUM (2005). Stability of pharmaceuticals and cosmetics. LUMiFuge 114 Application.

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Aims & Scope Malaysian Cocoa Journal aims To create and maintain an active communication network for the exchange of information among scientists and the cocoa industry To support the research and professional activities of researchers in cocoa through publications To publish original research on cocoa and thereby added to the existing database of scientific knowledge. The Journal will be published once a year. The journal cover original scientific contributions dealing with cocoa, cocoa products and by-products as well as articles dealing with cocoa extension, licensing, marketing and other activities relating to the cocoa industry. Manuscript Preparation Layout of papers Full papers, figures, and tables must be typed double spacing on A4 paper (21.0 cm x 29.7 cm) with margins of at least 4 cm on the left, 2 cm on the right, 4 cm on the top and 4 cm at the bottom. Abstract Each full paper (research) must include an informative summary not exceeding 250 words. It should contain all essential information regarding objectives, materials and methods, results and conclusions, but excluding figures, sectional headings, tables and references. Full paper A paper which reports the results of research should be divided into the following sections: (i) Abstract, (ii) Introduction, (iii) Materials and Methods, (iv) Results, (v) Discussions, (vi) Conclusions, (vii) Acknowledgements and (viii) References. No full paper should exceed 3,000 words (except for review papers), figures and graphs. Each table, figure or graph is considered being equal to the number of words, which could have been typed in the equivalent space. Any contribution in excess of this length may not be published in the Journal. Papers are considered to have been submitted in the final form after they have been fully checked for typographical and other errors. The full paper should be in English. Manuscripts are to be written using Times New Romans, font size 10. A full guideline for manuscript writing is obtainable upon request. Submission of Manuscripts Send three of copies of the manuscript, disk in a heavy-paper envelope, enclosing the copies and figures in cardboard, if necessary, to prevent the photographs from being bent. Place photographs and transparencies in a separate heavy-paper envelope. Manuscripts must be accompanied by a covering letter to the following address, The Editor Malaysian Cocoa Journal, Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Sabah, Malaysia

MALAYSIAN COCOA BOARD


5th & 6th Floor, Wisma SEDCO, Lorong Plaza Wawasan, Off Coastal Highway Locked Bag 211, 88999 Kota Kinabalu, Sabah, Malaysia. Tel: 088 - 234 477 Fax: 088 - 253 037 / 239 575 http://www.koko.gov.my mcb_enquiry@koko.gov.my