FUNGAL INFECTION AND AFLATOXIN CONTAMINATION IN STORED NUTMEG ( ) KERNELSMyristica fragrans AT VARIOUS STAGES OF DELIVERY CHAIN IN NORTH SULAWESI PROVINCE OKKY SETYAWATI DHARMAPUTRA , SANTI AMBARWATI , INA RETNOWATI1,2* 1 1 and NIJMA NURFADILA1 1SEAMEO BIOTROP, Jalan Raya Tajur Km 6, Bogor 16134, Indonesia 2 16680, aDepartment of Biology, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor, Bogor Indonesi Received 22 January 2015/Accepted 21 May 2015 ABSTRACT Fragrant nutmeg ( ) is an important commodity that has been used in food and pharmaceutical Myristica fragrans industries, hence its quality should be monitored. The objectives of the research were obtain information on the (a) to postharvest handling of nutmeg investigate the occurrence of fungi (includ ) and aflatoxin kernels; (b) to ing A. flavus contamination in stored nutmeg oisture content and percentage of damaged kernels and (c) mto measure nutmeg kernels. Methods used in this study included survey, interviews and sample collection in the delivery chain. This study was ions conducted in April and May 2013, in three reg (North Minahasa, Siau Tagulandang Biaro (Sitaro) and Sangihe Talaud) and two cities (Bitung and Manado). The total number of nutmeg samples collected from kernels different point of the delivery chain was 76. It consisted of samples collected from farmers (25 samples), collectors s (22) and exporters (29). The results showed that , the moisture content of nutmeg kernels collected from North Sulawesi Province was not higher than the maximum limit of moisture content determined by Indonesian National Standard or SNI (10%). Nutmeg kernels collected from farmers and collectors had a high percentage of damaged kernels. and were the dominant fungi infecting nutmeg kernels collected from Aspergillus niger Endomyces fibuliger farmers and collectors, while was the dominant fungus found infecting nutmeg kernels stored by Eurotium repens the exporters. Aflatoxin B and total aflatoxin contents nutmeg kernels samples collected from farmer and exporter1 s s of were Based on statistical analysis using non-parametric analysis, the effect of delivery chain did not give relatively high. any significant difference on moisture content, percentage of damaged kernels, total fungal pulation and s nutmeg po total aflatoxin content. Our study suggested that methods the postharvest handling of nutmeg kernels conducted by farmers, collectors and exporters in North Sulawesi Province (North Minahasa, Sitaro and Sangihe Talaud regions), Bitung and Manado cities should be improved to minimize aflatoxin B contamination1 and total aflatoxin . Keywords: aflatoxin, delivery chain fungi, , North Sulawesi rovince, P , nutmeg kernelsMyristica fragrans INTRODUCTION Nutmeg is native to the Moluccas Islands of Indonesia, but nutmeg is also grown in nowadays Penang Island in Malaysia, in the Caribbean (particularly Grenada), in the southern state of Kerala in India and in Zanzibar Islands. Fragrant nutmeg ( ) is an important Myristica fragrans c o m m o d i t y w i d e l y u s e d i n f o o d a n d pharmaceutical industries, therefore , it is important monitor its quality (Direktorat to Jenderal Perkebunan 2012). Based on statistical data from the Directorate General of Estate Crops in Indonesia, area s planted with nutmeg were 75,062 ha . The in 2008 distribution areas covered 19 provinces. The largest plant area was in North ation of nutmeg Moluccas (33%), followed by N Aanggroe ceh D (23%), North Sulawesi arussalam or NAD (18%), Moluccas (12%), West Java (5%) and the rest (9%) in other provinces. Indonesia contributes 75% (8,943 tonnes) of nutmeg production in the world (Revitalisasi Perkebunan Pala Siau, Sulawesi Utara 2010). North Sulawesi Province is one of the most important nutmeg producing in . Nutmeg from provinces Indonesia* Corresponding author : okky@biotrop.org BIOTROPIA Vol. 22 No. 2, 2015: 129 - 139 129 DOI: 10.11598/btb.2015.22.458 mailto:okky@biotrop.org BIOTROPIA Vol. 22 No. 2, 2015 130 this is exported to t Netherlands, Italy, province he Japan and Vietnam. During and storage s, postharvest period nutmeg kernels can be infested by insects and c o l o n i z e d by m i c r o o r g a n i s m s. A m o ng microorganisms, fungi important are the most cause of stored foodstuff deterioration. Fungal infection in foodstuff can cause discolouration and mycotoxin contamination, as well as decrease in physical quality and nutritional content (Sauer et al. 1992). Aflatoxins are toxins produced by certain fungi, such as Aspergillus flavus A. and parasiticus are considered dangerous . Aflatoxins due to associat with various diseases their ion in human and animals, such as aflatoxicoses and liver cancer. There are four naturally occurring aflatoxins in many stored commodities, i.e. aflatoxins B , B , G and G . The most common 1 2 1 2 and toxic aflatoxin is aflatoxin B (1 (AFB ) Basappa 1 2009). The European Union has determined Maximum Tolerable Limits (MTL) of B and AF 1 total aflatoxin in nutmeg kernels to be 5 and 10 ppb, respectively (FAO 2004). Since nutmeg is a m o n g e x p o r t i m p o r t a n t a g r i c u l t u r a l commodit for Indonesia, it is important to ies conform to nutmeg kernels importation rules the from countries. The first step in the importing postharvest handling is to monitor and improve the current procedure of nutmeg storage. kernels In relation to storage monitoring, several objectives in this study were set as follow : to (a) s obtain information on postharvest nutmeg handling methods to (b) ; investigate fungal population, fungal diversity (including Aspergillus flavus of stored ) and aflatoxin contamination nutmeg kernels collected from different points of the delivery chain in North Sulawesi Province and (c) to determine and percentage moisture content of damaged nutmeg kernels in the postharvest chain. MATERIALS AND METHODS Time and ocation of urveyL S s of nutmeg kernels wereSurvey and sampling conducted in April and May different 2013 at point of delivery chain s , i.e. at farmer, collector and exporter levels in five locations in North Sulawesi Province. The five locations chosen for the survey based on recommendation from the Agricultural Service of North Sulawesi Province were North Minahasa, Siau Tagulandang Biaro (Sitaro) and Sangihe Talaud regions; Bitung and Manado cities (Table 1). Interview using uestionnairesQ Interviews during the survey were conducted to collect information on postharvest nutmeg handling at different point the delivery chain s in (farmer collector ). The , and exporter levels que sti onnai re s on c ont aine d que sti ons postharvest handling carried out by procedures farmers, collectors , and exporters as well as problems encountered by them umber of . The n respondents from each level of delivery point was different depending on the conditions in the field during the survey. Sampling ethodM s The samples were collected from the places where the respondents obtained the nutmeg kernels. The number of samples nutmeg kernels at each level determined of delivery point was proportionally, based on the number of farmers, collectors and . exporters As much as shelled kernels 500 g of nutmeg and , in-shell kernels were1 000 g of nutmeg collected randomly from each respondent ach . E sample packed in a was clean plastic (polyethylene) and then was double packed in bag hermetic bags to minimize any changes to the nutmeg kernels samples due to long distance transportation between the location of sampling and laboratory in Bogor were, where the samples analyzed. Method for btaining orking amplesO W S The nutmeg shelled manually to in-shell s were obtain the skernel . Each sample was mixed homogeneously, then was into four parts. divided One part was used to determine the percentage of damaged nutmeg kernels and as a reserve sample, while the other three parts were ground using a Mill Powder Tech Model RT 04 The ground . nutmeg samples were then divided into eight parts to deter mine moisture content, fungal population, dominant fungal species infecting kernels aflatoxin ontent.and c Fungal infection and aflatoxin contamination in stored nutmeg ( ) Dharmaputra – et al. Myristica fragrans Deter m inatio n of Moi stur e Content, Percentage of Damaged Kernels, Fungal P A n Copulation and flatoxi ontent Moisture content of (based on wet nutmeg basis) w determinedas using the distillation method (SNI 1993). wereTwo replicates used for each sample. The percentage of damaged kernels was calculated from the weight of damaged the kernels ed by the and divid the weight of working sample damaged kernelfrom which the s were taken Damaged kernels included cracked, . broken, shriveled and mouldy kernels. wereFungi isolated using serial dilution the method followed by pour plate method with the Dichloran 18% Glycerol Agar (DG18) (Hocking & & Pitt 1980 Pitt Hocking 2009). Each fungal ; species identified according to Pitt and were Hocking (2009) as the main reference. Aflatoxin B (AFB ) and total aflatoxin1 1 contents were determined using HPLC with post-column derivatization (VICAM 2007). Data Collection and Analysis Data collection on nutmeg postharvest handling methods at farmer, collector and exporter levels were carried out by conducting inter views with far mers, collectors and exporters. Data of moisture content (MC), percentage of damaged kernels, fungal total population and total aflatoxin content were collected from farmers (25 samples), collectors (22 samples) and exporters (29 samples) at North Sulawesi Province. Total samples at farmer and collector levels were collected from North Minahasa, Sitaro and Sangihe Talaud regions. Meanwhile, total samples at exporter level were collected from Bitung and Manado cities. Data of MC, percentage of damaged nutmeg kernels, total fungal population and total aflatoxin content were analyzed with non- parametric one way Kruskal-Wallis test. 131 Tabl 1 , samplese Level of delivery chain sub-district origin of the sample and the number of nutmeg kernels at various stages of delivery chain in North Sulawesi Province Region/ City Level of delivery chain Sub-district origin of the sample Number of samples North Minahasa Farmer Kauditan 16 Collector Kauditan 17 Exporter - Siau Tagulandang Biaro (Sitaro) Farmer East Siau 1 West Siau 1 Collector East Siau 3 Exporter - Sangihe Talaud Farmer Kendahe 2 Central Tabukan 1 Tamako 1 East Tahuna 1 West Tahuna 1 Mangawito 1 Collector Kendahe 2 Exporter - Bitung Farmer - Collector - Exporter Halmahera Island 1 Lembeh Island 1 Kauditan 1 Some locations 1 Bitung 2 Manado Farmer - Collector - Exporter Siau 12 Tahuna 1 Manado 10 Total number of samples 76 RESULTS AND DISCUSSION Results of with ar mer s, Inter views F C Eollectors and xporters The total number of respondents was 26 (14 farmers, 8 collectors and 4 exporters). Interviews related to nutmeg postharvest handling at farmer and collector levels were conducted only in North Minahasa, Sitaro and Sangihe Talaud reg , ions while interviews at exporter level were conducted in Bitung and Manado cities. Method of Collecting Nutmeg At farmer level, farmers picked generally nutmeg fruits from the trees . They also (±50%) collected nutmeg fruits which had fallen on the ground . armers collected nutmeg (±50%) F also fruits that had fallen on the ground, because this method was easier to be conducted compared to harvesting nutmeg fruits directly from the trees. Price of nutmegs picked directly from the trees was similar to price of nutmegs that had fallen on the ground. ,Therefore farmers did not pay attention to the method of proper nutmeg postharvest handling. At collector level, a total of 57% of collectors bought nutmegs from farmers in three conditions, i.e. wet, semi-dry and dry conditions; for both shelled and in-shell .nutmeg s If a collector bough nutme st gin-shell , then a wooden stick was used for conducting the shelling process. As much as 12.5 and 87.5% of farmers sold nutmeg s in semi-dry and dry conditions respectively. In , general farmers sold nutmeg in dry condition, , s because price higher than sold in the was those semi-dry or wet conditions. At exporter level, up to 75% of exporters bought shelled nutmegs from collectors, while 25% of exporters bought a mixture of shelled and in-shell . nutmegs Most of exporters bought shelled s the weight of shelled nutmeg , because nutmegs was that of lighter than in-shell nutmegs, which could the costdecrease transportation . Method of Drying and Storing N s utmeg At the farmer level, 87.5% of farmers dried shelled and in-shell nutmegs using sun-drying method by spreading the nutmegs on tarpaulin placed on the ground. The dried nutmegs were subsequently stored in plastic bags. After 1 kg of nutmegs were collected and shelled, they were sold to collectors. In general, farmers did not do any attempts to sort dried nutmegs based on physical quality, because they wanted to sell dry nutmeg as soon as possibles . At the collector level, drying of nutmegs was conducted using two methods, i.e. sun-drying and smoke drying methods. In general, collectors used smoke drying method, especially during rainy season. The smoke drying method used coconut shells as fuel. Up to 57% of collectors dried both shelled and in-shell nutmegs using sun- drying method on tarpaulin placed on the ground. A total of 43% of collectors smoke-dried nutmegs on wire and wooden racks, followed by sun-drying method. For better result, collectors dried the nutmeg kernels using wire and wooden racks at elevated position ±1 m from the floor, to avoid contamination by animal faeces or dirt. In the night or when it rained, the semi-dried and wet nutmeg kernels were packed in the same plastic bags. As much as 71% of collectors sold nutmeg kernels to exporters, while 29% of nutmeg kernels were sold to large traders, without being sorted. Before being sold, the nutmeg kernels were stored for about a month. During storage, 71% of collectors did not monitor nutmeg kernels for aflatoxin contamination, due to lack of aflatoxin detection equipment, although they knew the impact of aflatoxin on human and animal health. At the exporter level, 75% of exporters grouped the nutmeg kernels manually based on qualities. The exporters usually stored the sorted nu tm e g ker n e ls i n gu nn y a nd p la sti c (polypropylene) bags. Those were exporters aware of potential aflatoxin contamination and the impact on human and animal health. They monitored the nutmeg kernels for aflatoxin contamination using a long wave ultraviolet lamp, in which aflatoxin contaminated nutmeg kernels would produce blue green yellow fluorescence and be removed prior to export. Source and umber of amplesN S The total number of samples nutmeg kernels collected from different points in the delivery chain was 76 consisted of nutmeg kernels . This samples from farmers (25 samples), collectors (22 samples) and exporters (29 samples) (Table 1). 132 BIOTROPIA Vol. 22 No. 2, 2015 Moisture Content The moisture content (MC) of foodstuff at the beginning of storage is one of the important factors influencing the quality of foodstuff during storage. A high initial MC value at the beginning of storage provides an opportunity for growth and development of spoilage and mycotoxigenic fungi. According to Indonesian National Standard or SNI, maximum moisture content value for nutmeg kernels and their processed products should be 10% (SNI 01-0006-1993). The range and mean MC values of nutmeg kernels collected from farmers, collectors and exporters in North Minahasa, Siau Tagulandang Biaro (Sitaro) and Sangihe Talaud regions; Bitung and Manado cities are presented in Table 2. The highest and the lowest MC values of nutmeg kernels collected from farmers in North Minahasa region were 18.00 and 8.00%, respectively. For collectors, those MC values were 15.50 and 7.50%, respectively. The mean MC value of nutmeg kernels collected from farmers was similar to that collected from collectors, i.e. 10.88 and 11.07%, respectively. The mean MC value of nutmeg kernels collected from farmers and collectors were higher than the maximum limit determined by . The high MC value of SNI nutmeg kernels collected from farmers was probably due to the short period of drying after the harvesting process, low intensity of sun light during the sun-drying process, limited storage space and short duration of storage. The highest and lowest MC values of nutmeg kernels from farmers in Sitaro region were 10.50 and 10.00%, respectively, while the highest and lowest MC values of nutmeg kernels from collectors were 11.50 and 6.50%, respectively. The MC values of nutmeg kernels from farmers in Sitaro region exceeded the maximum limit determined by . SNI In Sangihe Talaud region the range and mean of MC values obtained from the farmers' and collectors' samples were lower than the maximum limit determined by . The highest and SNI lowest MC values of nutmeg kernels from farmers were 9.00 and 6.98%, respectively; while the highest and lowest MC values of nutmeg kernels from collectors were 8.00 and 7.99%, respectively. The range of MC values of nutmeg kernels collected from exporters in Manado (7.00 - 11.50%) was wider than that collected from Bitung (7.50 - 10.00). The mean MC value of nutmeg kernels from exporters in Bitung (8.75%) and Manado (9.48%) cities were lower than the maximum limit determined by . SNI 133 Tab 2 , undamaged and damaged kernelsle Range and mean of moisture content of nutmeg collected from farmers, collectors and exporters in North Sulawesi Province Region / City Level of delivery chain Range (mean) of moisture content (% wet basis) Range (mean) of undamaged kernels (%) Range (mean) of damaged kernels (%) North Minahasa Farmer 8.00 – 18.00 (10.88) 0 – 88 (22.78) 12.00 –100 (77.22) Collector 7.50 – 15.50 (11.07) 0 – 81.42 (21.20) 18.58 – 100 (76.12) Exporter - - - Siau Tagulandang Biaro (Sitaro) Farmer 10.00 – 10.50 (10.25) 52.91 – 61.88 (57.40) 38.13 – 47.09 (42.61) Collector 6.50 – 11.50 (8.83) 0 – 47.85 (15.95) 52.15 – 100 (84.05) Exporter - Sangihe Talaud Farmer 6.98 – 9.00 (7.85) 0 – 61.54 (36.71) 38.46 – 100 (63.29) Collector 7.99 – 8.00 (8.00) 0 – 58.78 (29.39) 41.22 – 100 (70.61) Exporter - - - Bitung Farmer - - - Collector - - - Exporter 7.50 – 10.00 (8.75) 0 – 75.31 (35.25) 24.69 – 100 (64.75) Manado Farmer - - - Collector - - - Exporter 7.00 – 11.50 (9.48) 0 – 71.33 (26.42) 28.67 – 100 (73.58) Fungal infection and aflatoxin contamination in stored nutmeg ( ) Dharmaputra – et al. Myristica fragrans The mean MC value of nutmeg kernels collected from farmers and collectors in North Minahasa region were higher than that collected from other delivery chains in North Sulawesi Province. Overall, the highest mean MC value was recorded from nutmeg kernels collected from collectors in North Minahasa region, followed by those collected from farmers in Sitaro region, from exporters in Manado city, from collectors in Sitaro region, from exporters in Bitung city and from farmers and collectors in Sangihe Talaud region. Nutmeg kernels are very hygroscopic, therefore, storage at semi-dried condition caused the kernels to absorb moisture which led to initiation of mould growth, spoilage and mycotoxin contamination. Based on non-parametric statistical analysis, d significantly influenceelivery chain did not moisture content (MC) value kernelss of nutmeg ( ).Table 3 Percentage of U Damaged ndamaged and Kernels Damage to nutmeg kernels may contribute to infection by mycotoxigenic fungi such as A. flavus and increase the chances for af latoxin contamination during the postharvest storage. The r nge of damaged nutmeg kernels percentage a in North Minahasa region from farmers and collectors were 12.00 – 100 and 18.6 – 100%, respectively. In Sitaro region, the range of damaged nutmeg kernels from farmers and collectors were 38.1 – 47.1% and 52.2 – 100%, respectively. Means of damaged nutmeg kernels percentage from farmers and collectors were 42.6 and 84.1%, respectively. In Sangihe Talaud region, the mean of damaged nutmeg kernels percentage collected from farmers (63.29%) was lower than that collected from collectors (70.6%). In Bitung city the percentage of damaged nutmeg kerne s collected from exporters was l 64.8%. In Manado city the percentage was 73.6% (Table ). Means of damaged nutmeg kernels 2 percentage collected from farmers and collectors in North Sulawesi P were higher than rovince those collected from exporters. This might be due to improper sorting regimes used by farmers and collectors. In addition, many farmers and collectors still manually shelled the nutmegs using wooden stick, which can increase the percentage of damaged nutmeg kernels. Based on statistical analysis using non- parametric analysis, elivery chain did not d significantly influence the percentage of damaged nutmeg Table 3kernels ( ). Fungal Population Diversity and Dominance The highest diversity of fungal species was found in nutmegs collected from farmers and collectors in North Minahasa region, 13 and i.e. 12 species, while the lowest number respectively, of fungal species were observed in samples obtained from farmers in Sitaro region (7 species) and those obtained from collectors in Sangihe Talaud region (2 species; Tables 4, 5, 6). Aspergillus flavus was found in 56% samples from farmers and in 53% samples from collectors in North Minahasa region. Fungal population isolated from samples from farmers was more contaminated than those from collectors (Table 5). The dominant fungi found in nutmeg samples from farmers was (81%) Penicillium citrinum followed by (69%) and A. niger Eurotium repens (63%). Three dominant fungi found in nutmegs collected from collectors were Endomyces fibuliger (76%), (76%) and (76%) which A. niger P. citrinum may have been caused by high moisture content. 134 Table 3 The effect of nutmeg delivery chain on moisture content (MC), percentage of damaged nutmeg kernels, fungal total population and total aflatoxin Level of delivery chain MC (%) Percentage of damaged kernels (%) Fungal total population (cfu/g wet basis) Total aflatoxin content (ppb) Farmer 9.98 ± 2.62 a 70.55±29.66 a 3.9x105±1.7x106 a 141.10±392.11 a Collector 10.49 ± 2.52 a 76.70±32.39 a 1.3x106±3.2x106 a 2.15±4.54 a Exporter 9.33 ± 1.13 a 71.75±30.27 a 9.9x103±1.1x104 a 50.63±213.43 a Note: Means in the same group followed by the same letter in a column are not significantly different at 5% level BIOTROPIA Vol. 22 No. 2, 2015 No was isolated from nutmeg samples A. flavus obtained from collector in the Sitaro region. At farmer level, A. niger, A. penicillioides, Eurotium repens P. citrinum and were isolated from all samples, dominated by Only a A. penicillioides. small number of nutmeg samples were colonized by in the Sangihe Talaud region. No A. flavus nutmeg samples from collectors appeared to contain . All samples of nutmegs A. flavus obtained from farmers and collectors in all sampling sites contained A. niger. The total fungal population and diversity of samples from exporters were lower in Bitung city than in Manado city (Table 7). In Bitung city, A. flavus was only isolated from 2% of samples. The dominant fungi were xerophilic spoilage fungi such as (all samples) followed by E. repens E. chevalieri (83%). In Manado city, much higher number of samples contained (39%). A. flavus Again, the dominant fungal population isolated were (87%), followed by (70%) E. repens A. niger and (65%).A. penicillioides Aspergillus flavus was found in 56% samples from farmers and in 53% samples from collectors in North Minahasa region. Fungal population isolated from samples from farmers was more contaminated than those from collectors (Table 5). The dominant fungi found in nutmeg samples from farmers was (81%) Penicillium citrinum followed by (69%) and A. niger Eurotium repens (63%). Three dominant fungi found in nutmegs collected from collectors were Endomyces fibuliger (76%), (76%) and (76%) which A. niger P. citrinum may have been caused by high moisture content. No was isolated from nutmeg samples A. flavus obtained from collector in the Sitaro region. At farmer level, A. niger, A. penicillioides, Eurotium repens P. citrinum and were isolated from all 135 Table 4 Fungal populations and diversity in nutmeg samples obtained from farmers collectors in North Minahasa and region No Fungi Number (%) samples infected by fungi Range (mean) of fungal population in nutmeg (cfu/g wet basis) Farmer Collector Farmer Collector 1. Aspergillus flavus 9 (56) 9 (53) 0.5 x 10 – 8.8 x 103 (1.5 x 103) 0.7 x 10 – 5.2 x 103 (7.8 x 102) 2. A. niger 11 (69) 13 (76) 0.2 x 10 – 2.2 x 104 (4.3 x 103) 0.1 x 102 – 8.8 x 103 (2 x 103) 3. A. ochraceus - 1 (6) - 0.7 x 10 (0.7 x 10) 4. A. penicillioides 2 (13) 3 (18) 2.2 x 102 – 1.3 x 103 (7.8 x 102) 4.5 x 10 – 2.7 x 102 (1.7 x 102) 5. A. tamarii 8 (50) 4 (24) 0.8 x 10 – 1.2 x 103 (3.3 x 102) 1.5 x 102 – 7.3 x 102 (3.7 x 102) 6. A. sydowii 2 (13) 1 (6) 1.2 x 10 – 1.8 x 103 (9.2 x 102) 8.2 x 102 (8.2 x 102) 7. A. wentii 1 (6) - 1.2 x 102 (1.2 x 102) - 8. Endomyces fibuliger 7 (44) 13 (76) 5.2 x 10 – 8.4 x 106 (1.3 x 106) 1.5 x 10 – 1.1 x 107 (2.2 x 106) 9. Eurotium chevalieri 5 (31) 3 (18) 1.5 x 10 – 0.4 x 10 4 (1.2 x 103) 3.3 x 10 – 7.3 x 10 3 (2.6 x 103) 10. E. repens 10 (63) 9 (53) 0.5 x 10 – 6.8 x 103 (1.7 x 10 3) 1.3 x 10 – 3.2 x 10 3 (9.0 x 102) 11. E. rubrum - 7 (41) - 0.2 x 10 – 3.5 x 10 3 (9.4 x 10 2) 12. Penicillium citrinum 13 (81) 13 (76) 0.3 x 10 – 0.6 x 10 5 (6.3 x 103) 1.3 x 10 – 0.4 x 10 4 (7.8 x 102) 13. Rhizopus sp. 2 (13) - 2.5 x 10 2 – 7.2 x 10 2 (4.8 x 10 2) - 14. Syncephalastrum racemosum - 1 (6) - 0.2 x 10 3 (0.2 x 10 3) 15. Trichoderma sp. 2 (13) - 0.1 x 102 – 1 x 103 (5.1 x 102) - Notes: Number of samples collected from farmers: 16 Number of samples collected from collectors: 17 Fungal infection and aflatoxin contamination in stored nutmeg ( ) Dharmaputra – et al. Myristica fragrans samples, dominated by Only a A. penicillioides. small number of nutmeg samples were colonized by in the Sangihe Talaud region. No A. flavus nutmeg samples from collectors appeared to contain . All samples of nutmegs A. flavus obtained from farmers and collectors in all sampling sites contained A. niger. The total fungal population and diversity of samples from exporters were lower in Bitung city than in Manado city (Table 7). In Bitung city, A. flavus was only isolated from 2% of samples. The dominant fungi were xerophilic spoilage fungi such as (all samples) followed by E. repens E. chevalieri (83%). In Manado city, much higher number of samples contained (39%). A. flavus Again, the dominant fungal population isolated were (87%), followed by (70%) E. repens A. niger and (65%).A. penicillioides Nutmegs imported from India, Sri Lanka, Indonesia and Brazil were infected by , A. niger A. flavus Rhizopus stolonifer and . The dominant fungi in these samples were (Mandel 2005). A. flavus The water availability of semi-dried and damaged nutmeg kernels provided environmental conditions which are conducive to xerophilic and xerotolerant fungi, including mycotoxigenic species. The boundary conditions for growth and my c o t ox i n p r o d u c t i o n su g g e s t e d t h a t aflatoxigenic and ochratoxigenic fungi may be able to th ve under storage conditions (Lacey & ri Magan 1991; Sanchis & Magan 2004; Magan & Aldred 2007). 136 Tabl and Siau Tagulandang e 5 Fungal population and diversity in nutmeg samples obtained from farmers collectors in Biaro (Sitaro) region No Fungi Number (%) samples infected by fungi Range (mean) of fungal population in nutmeg (cfu/g wet basis) Farmer Collector Farmer Collector 1. Aspergillus niger 2 (100) 2 (67) 0.2 x 10 – 2.5 x 10 (1.3 x 10) 5.5 x 102 – 4.2 x 104 (2.1 x 104) 2. A. penicillioides 2 (100) 1 (33) 1.8 x 10 – 2.0 x 103 (0.1 x 104) 3.7 x 102 (3.7 x 102) 3. Endomyces fibuliger 1 (50) - 0.5 x 10 (0.5 x 10) - 4. Eurotium chevalieri 1 (50) 1 (33) 6.7 x 10 (6.7 x 10) 2.8 x 10 (2.8 x 10) 5. E. repens 2 (100) 1 (33) 0.2 x 10 – 3.3 x 102 (1.7 x 102) 1.8 x 10 (1.8 x 10) 6. E. rubrum 1 (50) 1 (33) 1.7 x 10 (1.7 x 10) 0.3 x 10 (0.3 x 10) 7. Penicillium citrinum 2 (100) - 0.2 x 10 – 0.8 x 10 (0.5 x 10) - Notes: Number of samples collected from farmers: 2 Number of samples collected from collectors: 3 Tab and Sangihe Talaud ionle 6 Fungal population and diversity in nutmeg samples obtained from farmers collectors in reg No Fungi Number (%) samples Infected by fungi Range (mean) of fungal population in nutmeg (cfu/g wet basis) Farmer Collector Farmer Collector 1. Aspergillus flavus 1 (14) - 0.2 x 10 (0.2 x 10) - 2. A. niger 7 (100) 2 (100) 0.7 x 10 – 2.4 x 105 (5.1 x 104) 4.8 x 10 – 0.1 x 105 (5.4 x 103) 3. A. penicillioides 3 (43) - 0.1 x 102 – 3.7 x 102 (1.3 x 102) - 4. Endomyces fibuliger 1 (14) - 1.5 x 105 (1.5 x 105) - 5. Eurotium repens 4 (57) - 0.5 x 10 – 8.3 x 102 (2.2 x 102) - 6. E. rubrum 3 (43) - 0.3 x 10 – 0.5 x 103 (1.7 x 102) - 7. Fusarium solani 1 (14) - 5.2 x 104 (5.2 x 104) - 8. Penicillium citrinum 3 (43) 1 (50) 0.3 x 102 – 1.8 x 104 (6.8 x 103) 4.3 x 102 (4.3 x 102) Notes: Number of samples collected from farmers: 7 Number of samples collected from collectors: 2 BIOTROPIA Vol. 22 No. 2, 2015 Based on statistical analysis using non- parametric analysis, elivery chain did not d significantly influence the total fungal population ( ).Table 3 Aflatoxin T A ContentB and otal flatoxin s1 The range of AFB and total aflatoxin content 1 in nutmeg samples from farmers in North Sulawesi Province were 0.40 – 1,632.19 ppb and 0.58 – 1,831.48 ppb, respectively. Survey conducted at farmer level provided information a that postharvest handling was not conducted properly, i.e. mixed nutmeg picked the farmers s from the tree th fell on the ground s with ose ; consequently AFB and total t the aflatoxin conten1 in these samples (Table 8)were high . According to Horn (2003) soil serves as a reservoir for and that A. flavus A. parasiticus produce aflatoxins in agricultural commodities. Aflatoxigenic fungi reside in soil as conidia, sclerotia and hyphae, while act as primary inocula for directly infect peanuts (and possibly nutmeg ing fruit whic fell on the ground). Rh ange of AFB 1 and total aflatoxin in nutmeg samples content obtained from collectors in North Sulawesi Province were 0.11 – 14.59 ppb and 0.11 – 16.65 ppb, respectively. Sun-drying method was faster and more effective than smoke-drying method. However, if the weather is extreme, sun-drying method is not recommended, because it could reduce the quality of atsiri oil in nutmeg kernels. Therefore, many collectors used smoke-drying method, because the temperature can be controlled. smoke-drying The weakness using method the of long drying duration, includes need which may allow colonization of spoilage and mycotoxigenic fungi. Based on statistical analysis using non- parametric analysis elivery chain did not , d 137 Table 7 Fungal population and diversity in nutmeg samples collected from exporters in citBitung and Manado ies No. Fungi Bitung city Manado city Number (%) samples infected by fungi Range (mean) of fungal population in nutmeg (cfu/g wet basis) Number (%) samples infected by fungi Range (mean) of fungal population in nutmeg 1. Aspergillus flavus 2 (33) 0.5 x 10 – 7.7 x 102 (3.9 x 102) 9 (39) 0.1 x 102 – 5.3 x 102 (1.3 x 102) 2. A. niger 3 (50) 0.3 x 10 – 1.7 x 102 (7.1 x 10) 16 (70) 0.7 x 10 – 0.4 x 105 (3.1 x 103) 3. A. penicillioides 2 (33) 0.5 x 102 – 3.5 x 103 (1.8 x 103) 15 (65) 1.3 x 102 – 2.9 x 104 (6.6 x 103) 4. A. sydowii 1 (17) 0.5 x 10 (0.5 x 10) - - 5. A. tamarii 1 (17) 0.2 x 103 (0.2 x 103) 1 (4) 2.2 x 102 (2.2 x 102) 6. A. versicolor - - 2 (9) 4.5 x 10 2 – 1.2 x 10 3 (8.1 x 102) 7. Endomyces fibuliger 2 (33) 1.7 x 10 2 – 6.7 x 10 2 (4.2 x 102) 4 (17) 2.8 x 10 2 – 5.3 x 10 3 (1.7 x 103) 8. Eurotium chevalieri 5 (83) 0.2 x 10 – 2.5 x 10 3 (5.4 x 10 2) 5 (22) 0.5 x 10 2 – 3.7 x 10 3 (1.1 x 103) 9. Eurotium repens 6 (100) 0.1 x 10 2 – 8.8 x 10 2 (3.6 x 10 2) 20 (87) 2.5 x 10 2 – 1.8 x 10 4 (0.5 x 104) 10. E. rubrum 1 (17) 6.8 x 10 2 (6.8 x 10 2) 15 (65) 2.8 x 10 – 0.2 x 10 4 (5.2 x 10 2) 11. Paecilomyces variotii - - 1 (4) 4.5 x 102 (4.5 x 102) 12. Penicillium citrinum 4 (67) 0.5 x 10 – 9.3 x 102 (2.7 x 102) 11 (48) 1.8 x 10 – 1.3 x 103 (2.4 x 102) Note : in Bitung city 6s Number of samples collected from exporters : in Manado city 23 Number of samples collected from exporters : Fungal infection and aflatoxin contamination in stored nutmeg ( ) Dharmaputra – et al. Myristica fragrans (cfu/g wet basis) significantly influence total aflatoxin content of nutmeg ( ).Table 3 Several exporters in North Sulawesi Province possess facilities for drying, shelling, sorting and storage facilities. Thus, aflatoxin contamination could be minimized. In this research, we also collected a sample of sorted nutmeg kernels visually assessed using the long wave ultraviolet lamp. This potentially contaminated sample from an exporter contained a total aflatoxin content of 0.18 – 1,112.58 ppb. Tabata (1993) reported et al. that aflatoxin was found in 3,054 foodstuff and their product samples, especially nutmeg samples. The highest aflatoxin contamination was found in nutmeg (80%), while AFB was also found in 1 pistachio nuts (1,382 ppb). Takahashi (1993) reported that in 1986 until 1991, as much as 29 (43%) of 67 nutmeg samples collected from Japan, were contaminated by aflatoxin. According to Okano (2012) the distribution of et al. aflatoxigenic fungi in 25 imported Indonesian nutmeg samples were contaminated with aflatoxins B or B and G. The incidence of aflatoxigenic fungi in the samples contaminated with high levels of aflatoxin was significantly higher than that in the samples with low levels of the toxins (r = 0.752). The toxin production of isolates from the samples in cultures of Yeast Extract Sucrose broth was examined by means of TLC and HPLC analyses. The ability of isolates to produce aflatoxins did not correlate with the contamination levels of aflatoxin in the samples. Overall, the postharvest handling procedures need to be standardized to minimize aflatoxin contamination. CONCLUSIONS Generally, moisture content (MC) of nutmeg samples collected from North Sulawesi Province was below the maximum recommended limit of Indonesian National Standard or SNI. Nutmeg samples collected from farmers and collectors generally had a higher percentage of damaged kernels. and were the A niger E. fibuligerspergillus 138 Tabl B e 8 Aflatoxin and total aflatoxin contents in nutmeg collected from farmers, collectors and exporters in North 1 Sulawesi Province Region/City Level of delivery chain Number of samples Number (%) samples contaminated by aflatoxin Range (mean) of AFB1 content in contaminated samples (ppb) Range (mean) of total aflatoxin content in contaminated samples (ppb) North Minahasa Farmer 16 6 (37.50) 0.40 – 762.24 (128.52) 0.58 – 910.48 (153.28) Collector 17 5 (29.41) 0.19 – 14.59 (4.70) 0.19 – 16.65 (5.67) Exporter - - - - Siau Tagulandang Biaro (Sitaro) Farmer 2 2 (100) 1.03 – 1.44 (1.23) 1.44 – 1.55 (1.49) Collector 3 3 (100) 0.11 – 0.69 (0.40) 0.11 – 1.34 (0.58) Exporter - - - - Sangihe Talaud Farmer 7 7 (100) 1.65 – 1 632.19 (335.92) 1.65 – 1831.48 (371.99) Collector 2 2 (100) 3.28 – 13.94 (8.61) 3.28 – 13.94 (8.61) Exporter - - - - Bitung Farmer - - - - Collector - - - - Exporter 6 3 (50) 1.40 – 799.25 (267.74) 1.40 – 1112.58 (372.18) Manado Farmer - - - - Collector - - - - Exporter 23 15 (65.22) 0.10 – 266.72 (18.37) 0.18 – 334.49 (20.69) BIOTROPIA Vol. 22 No. 2, 2015 dominant fungi in nutmeg kernels from farmers and collectors, while was the dominant E. repens species in samples obtained from nutmeg exporters in North Sulawesi province. Aflatoxin B and total aflatoxin contents in nutmeg samples 1 collected from farmers and exporters were relatively high. Although based on statistical analysis using non-parametric analysis, elivery d chain did not significantly influence moisture content ( ) nutmeg MC , percentage of damaged kernels, total fungal population and total aflatoxin content of nutmeg, the method of nutmeg postharvest handling especially at farmer and , c o l l e c t o r l e ve l s s h o u l d b e c o n d u c t e d a p p r o p r i a t e l y t o m i n i m i z e a f l a t o x i n contamination. ACKNOWLEDGEMENTS The authors would like to acknowledge SEAMEO BIOTROP for providing financial support through DIPA 2013. Thanks due to were the Indonesian Government's Office of Plantation Crop of North Sulawesi Province in Manado and to CV Multi Rempah Sulawesi for their information and cooperation during the survey; to Mrs Ratnaningsih, Mr Edi Suryadi and Mr. Iswadi for their technical support. 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