Development and application of an enzyme immunoassay for the detection of the mycotoxin Fumigaclavine A

The present study describes the development of specific polyclonal antibodies against fumigaclavine A (FuA) in rabbits, and the development of a highly sensitive enzyme immunoassay (EIA) for this mycotoxin. The Mannich condensation reaction with formaldehyde was used to conjugate FuA to keyhole limpet hemocyanin (KLH) as the immunogen, and to bovine serum albumine (BSA) for as the coating antigen. Conjugation of FuA to KLH with formaldehyde proved to be an effective approach for the preparation of immunogen for anti-FuA antibody production. A competitive indirect EIA was optimized using antiserum obtained from one rabbit. The EIA was very sensitive for FuA, with a 50% inhibition concentration (IC50) value of 3.3 ng/ml and a detection limit of the standard curve in buffer solutions of 0.5 ng/ml. The EIA was very specific for FuA with 1.3%, 12.6%, and 0.2% cross-reactivity with FuB, FuC, and FuD, respectively. IsoFuA and several other lysergic acid derivatives (ergonovine, ergotamine, and alpha-ergocryptine) were tested but did not cross-react in this assay. The EIA was applied to the analysis of FuA in silage and in tissue from the respiratory system of birds with aspergillosis. The detection limit for FuA in silage was at 10 ng/g, average recoveries from artificially contaminated control samples were 77.8%. None of 24 analyzed silage samples contained detectable amounts of FuA. Although the number of samples was limited, the results indicate that there is not a widespread problem of FuA in silage. The detection limit of the assay for FuA in tissue from the respiratory system of birds was at 1.5 ng/g, with average recoveries from artificially contaminated of 92.7%. FuA was found in 66% tissue samples of aspergillosis cases. Cultivation of fungal growth on respiratory tissue samples of aspergillosis cases on malt extract agar (MEA) yielded fungal material which was typical for A. fumigatus. When the mycelium was extracted and assayed by FuA EIA, high amounts of FuA was found (up to 8 mg/g mycelium). Further analysis of the mycelium extract by HPLC found FuA, FuC, and other unidentified compounds. Hydrolysis of FuC which have been isolated from mycelium extract of A. fumigatus gave a new fumigaclavine derivative. FuD is proposed as the name of this compound. Different specificity pattern of the FuA EIA and that of a previously developed ergonovine EIA were studied. In competitive indirect EIA, the anti-FuA antibody did detect neither ergonovine nor IsoFuA. Conversely, by competitive direct EIA format, the anti-ergonovine antibody was able to detect both FuA and IsoFuA. Using these assays in combination with HPLC separation of cheese extracts, a series of blue-veined cheeses from the German market were analyzed. None of 16 blue-veined cheese samples contained FuA. However, the presence of IsoFuA in blue-veined cheese samples was detected using the ergonovine EIA. This is the first description of antibodies against FuA and the first development of an EIA for FuA. This is also the first report demonstrating that FuA is correlated with aspergillosis in birds. However, the role of this mycotoxin in this disease remains to be clarified.Die vorliegende Arbeit beschreibt die Herstellung spezifischer Antikörper gegen Fumigaclavin A (FuA) und die Entwicklung eines hochempfindlichen Enzymimmuntests (EIA) für dieses Mykotoxin. Die Mannich-Kondensation mit Formaldehyd wurde verwendet, um Fumigaclavin A an keyhole limpet hemocaynin (KLH) als Immunogen und an bovines Serumalbumin (BSA) als Beschichtungsantigen zukoppeln. Die Kopplung von FuA an KLH mittels Formaldehyd erwies sich als effektiver Ansatz zur Herstellung eines Immunogens zur Gewinnung von Anti-FuA Antikörpern in Kaninchen. Ein kompetitiver indirekter EIA wurde erstellt und optimiert, unter Verwendung polykloner Antikörper eines Kaninchens. Der EIA war sehr empfindlich für FuA, mit einer 50%-Dosis von 3,3 ng/ml und einer Nachweisgrenze von 0,5 ng/ml (in Pufferlösung). Der EIA war relativ spezifisch für FuA, mit Kreuzreaktionen von 1,3%, 12,6% bzw. 0,2% Kreuzreaktion für FuB, FuC bzw. FuD. IsoFuA und einige andere getestete Lysergsäurederivate (Ergonovin, Ergotamin, und alpha- Ergokryptin) wurden ebefalls untersucht, zeigten aber keine Kreuzreaktion in diesem Testsystem. Der EIA wurde zur Untersuchung von FuA in Silage und in Gewebsproben (Repirationstrakt) von Vögeln mit Aspergillose eingesetzt. Die Nachweisgrenze für FuA in Silage betrug 10 ng/g, die durchschnittlichen Wiederfindungsraten für FuA in künstlich kontaminierten Kontrollproben lagen bei 77,8%. Keine der 24 untersuchten Proben enthielt nachweisbare Gehalte an FuA. Obwohl nur eine begrenzte Probenanzahl untersucht wurde, zeigen die Ergebnisse, dass FuA kein weit verbreitetes Problem in Silage darstellt. Die Nachweisgrenze für FuA in Gewebsproben des Repirationstrakts von Vögeln lag bei 1,5 ng/g, mit durchschnittlichen Wiederfindungsraten für FuA in künstlich kontaminierten Proben von 92,7%. FuA wurde in 66% der Proben von Vögeln mit Aspergillose nachgewiesen. Bei einer Kultivierung von Pilzisolaten des Atmungstraktes auf Malzextraktagar (MEA) entwickelte sich ein Mycel, das typisch für A. fumigatus war. Bei Extraktion des Mycels und Untersuchung im FuA-EIA wurden große Mengen an FuA (bis zu 8 mg/g Mycel) nachgewiesen. Bei einer weiteren Untersuchung des Mycelextrakts mittels HPLC wurden FuA, FuC und andere, nicht identifizierte Komponenten gefunden. Eine Hydrolyse von FuC, das aus dem Mycelextrakt von A. fumigatus gewonnen wurde, ergab ein neues Fumigaclavin-Derivat. FuD wird als Name für diese Verbindung vorgeschlagen. Zur Untersuchung von Blauschimmelkäse auf Clavin-Alkaloide wurden die unterschiedliche Spezifitäten des FuA-EIA und eines zuvor entwickelten Ergonovin-EIA ausgenutzt. Im kompetitiven indirekten EIA für FuA waren alle Proben negativ. Dagegen wurde bei Verwendung eines kompetitiven direkten EIA unter Verwendung von Anti-Ergonovin-Antikörpern hohe Meßwerte erzielt. Bei einer Untersuchung mit Hilfe dieser beiden Testsysteme und einer HPLCTrennung von Käse-Extrakten wurden Blauschimmelkäse des deutschen Marktes untersucht. Keine der 16 untersuchten Proben enthielt FuA, jedoch wurde die Anwesenheit von IsoFuA in Blauschimmelkäse nachgewiesen. Die vorliegende Arbeit beschreibt erstmals die die Entwicklung von Antikörpern gegen FuA und eines Enzymimmuntests für dieses Mykotoxin. Auch wird erstmalig eine Korrelation zwischen FuA-Gehalten in Gewebe des Repirationstrakts und der Aspergillose von Vögeln beschrieben. Die Rolle dieses Mykotoxins bei diesem Krankheitsbild bleibt allerdings noch zu klären

Hazard Analysis Critical Control Point (HACCP) sebagai jaminan keamanan produk Sarang Burung Walet Tujuan Ekspor ke Tiongkok

ABSTRACT   Edible Bird Nest (EBN) is a food product of animal origin that obtains many benefits for Indonesia's export commodities. EBN contains many nutrients in which it is widely utilized in the health sector. EBN products have been exported to various countries and one of them is China. EBN products exported to China have potential harms such as physical, biological, and chemical hazards that pose risks to human health. Therefore, every product of animal origin needs food safety assurance. Hazard Analysis Critical Control Point (HACCP) is a food safety system developed to identify, evaluate, and control food safety hazards. HACCP is a system developed to prevent or reduce hazards to an acceptable extent during the globally adopted production. Through the implementation of a food safety assurance system in the EBN, it is expected to lower the risk of food hazards. This paper discussed HACCP in ensuring food safety of animal origin, especially Edible Bird Nest to fulfill the export requirements of Edible Bird Nest to China. Keywords: Animal-origin Food Safety, HACCP, Edible Bird Nes

Salbutamol Residue in Plasma and Urine of Balinese Calves after Single-Dose Administration

Salbutamol, a short-acting β2-adrenergic receptor agonist, due to illegal use in animal feed, is very dangerous for the safety of animal origin products. The purpose of this study is to determine the residue of salbutamol in plasma and urine of balinese calf after a single-dose administration. Three calves were given of feed fortification of salbutamol (10 mg/kg body weight). The salbutamol concentrations were measured in the plasma and urine. Samples were extracted with perchloric acid and purified by cation exchange solid phase extraction (SPE), then analyzed by High-Performance Liquid Chromatography (HPLC) with a photodiode array detector using an RP C18 column with a mixed mobile phase of purified water pH 3.0 (adjusted with phosphoric acid) and acetonitrile (90:10, v/v). The highest salbutamol concentration in the plasma sample was 0.28±0.15 μg/mL at 12 hours and decreased after 24 hours after feeding. The highest concentration of salbutamol in urine was 15.85±4.42 μg/mL at 18 hours and decreased gradually starting at 24 hours. This result concluded that salbutamol residues mostly excreted in the urine, and the highest salbutamol residues in plasma samples formed faster but also decreased more rapidly than residues formed in urine. The residue formed in plasma is lower than that in the urine

Karakteristik, Pengetahuan, Sikap dan Praktik Petugas Karantina Hewan dalam Pengendalian Bruselosis di Sulawesi Selatan

Praktik atau perilaku petugas karantina hewan dalam pengendalian bruselosis dipengaruhi oleh faktor internal berupa karakteristik individu yang bersifat khas dan dipengaruhi oleh faktor eksternal berupa lingkungan, sosial dan budaya. Tujuan penelitian ini adalah mengidentifikasi karakteristik individu petugas karantina hewan dan menganalisis pola hubungan karakteristik, pengetahuan, dansikap terhadap praktik petugas karantina hewan dalam pengendalian bruselosis di Sulawesi Selatan. Desain penelitian yang digunakan adalah cross sectional study. Metode pengumpulan data melalui wawancara dan pengamatan terhadap 51 orang petugas karantina hewan di dua Unit Pelaksana Teknis Badan Karantina Pertanian di Sulawesi Selatan. Pengumpulan data menggunakan kuisioner terstruktur, dan dianalisis menggunakan path analysis. Hasil studi menunjukkan bahwa karakteristik petugas karantina hewan sebagian besar berusia antara 30-45 tahun, telah bekerja sebagai PNS maupun bekerja di tempat yang sekarang kurang dari lima tahun, pendidikannya SMA/sederajat. Tidak semua petugas karantina hewan adalah pejabat fungsional dan mayoritas belum pernah mengikuti pelatihan terkait bruselosis. Terdapat hubungan yang nyata antara pendidikan dan pengetahuan, pengetahuan dan sikap, serta sikap dan praktik. Pendidikan formal berperan penting dalam terbentuknya pengetahuan, sikap, dan praktik petugas karantina hewan dalam pengendalian bruselosis. Sehingga upaya peningkatan pendidikan formal pada petugas karantina hewan perlu dilakukan

Analisis Kadar Nitrit pada Sarang Burung Walet Asal Pulau Sumatera Menggunakan Metode Kromameter

Kadar nitrit dalam sarang burung walet (SBW) telah menjadi perhatian dalam beberapa tahun terakhir. SBW yang diekspor dari Indonesia ke Negara Tiongkok harus memenuhi standar kadar nitrit (NO2), yaitu maksimum 30 ppm. Dinamika perkembangan teknologi dan jaman saat ini menuntut instrumen pengujian kadar nitrit secara akurat, diantaranya menggunakan spektrofotometer dan kromameter. Penelitian ini mengkaji kadar nitrit pada SBW bersih yang telah dilakukan pencucian asal Pulau Sumatera dengan menggunakan metode spektrofotometer dan mengevaluasi warna menggunakan kromameter berbasis sistem CIE pada parameter L*, a*, b*, C*, dan h*. Jumlah sampel ditentukan secara purposif dari rumah burung walet (RBW). Sebanyak 18 sampel SBW berasal dari berbagai wilayah di Sumatera. Sampel SBW diuji kadar nitritnya menggunakan spektrofotometer di Balai Besar Uji Standar Karantina Pertanian (BBUSKP) Jakarta dan kromameter diuji di laboratorium Ilmu Teknologi Pangan IPB, Bogor. Hasil kadar nitrit pada SBW menunjukkan bahwa persentase kadar nitrit di bawah 30 ppm adalah 72,22%. Nilai rata-rata L* pada grup A (kadar nitrit >30 ppm) dan B (kadar nitrit <30 ppm) secara berurutan adalah sebesar 67,65±1,97 dan 68,47±5,25. Hasil analisis statistik dengan uji-t menunjukkan tidak terdapat perbedaan yang signifikan (p>0,05) antara nilai L*, a*, b*, C* dan *h pada kedua grup. Metode kromameter tidak dapat digunakan sebagai metode tunggal dalam mengukur kadar nitrit pada SBW serta tidak dapat membedakan secara signifikan warna SBW yang berasal dari RBW yang berbeda

Postmortem Changes in pH, Color, Drip Loss, and Non-Protein Nitrogen in Beef Liver and Lungs During Storage in Refrigerator

Beef offal are consumed by people in some countries specifically in Asia.  Beef liver and lungs are favorite food which are used as meat in traditional food.  The objective of this study was to determine the postmortem changes in pH, color, drip loss, and non-protein nitrogen (NPN) content in beef liver and lungs during storage in refrigerator (3-4 ºC) until 5 d (120 h) after slaughter.  The beef liver and lungs were collected from the abattoir and transported in cool box (<7 ºC) to the laboratory within 3 hours.  The samples size of beef liver and lungs were 20 for each observation time.  In the laboratory the beef liver and lungs were measured directly for pH value, color (L*, a*, and b*), drip loss, and NPN content at 4 h postmortem (pm) and afterwards every beef liver sample was sliced into 5 pieces of 100-120 g and stored in chiller of 3-4 ºC.  The measurement of pH, color (L*, a*, and b* values), drip loss, and NPN content were conducted at 4 h, 24 h, 48 h, 72 h, 96 h, and 120 h postmortem.  Data were analyzed descriptively and by comparing the 95% confidence interval of mean of each observation.  The results showed that pH, color, drip loss, and NPN content in beef lungs were higher than the values in beef liver.  The pH of beef liver and lungs declines until 96 h pm and 48 h pm, respectively.  The L*, a*, and b* values of beef liver and lungs increased in general during storage.  Drip loss and NPN in beef liver and lungs tended to increase significantly during storage.   From this study the pH value and NPN can be used to determine the freshness of beef liver and lungs

POLA PEMELIHARAAN BURUNG WALET PADA PULAU-PULAU UTAMA PENGHASIL SARANG BURUNG WALET DI INDONESIA

Burung walet di Indonesia umumnya dibudidayakan pada rumah burung walet (RBW) secara tradisional dengan pola pemeliharaan tertentu. Pola pemeliharaan menjadi salah satu faktor pendukung bagi burung walet untuk memproduksi sarang burung walet (SBW) dengan kualitas baik secara berkelanjutan. Penelitian ini bertujuan untuk menganalisis pola pemeliharaan burung walet di RBW di pulau-pulau utama penghasil SBW di Indonesia. Suatu survei terhadap total 44 RBW di pulau Jawa, Sumatera, Sulawesi, dan Kalimantan telah dilakukan untuk mengetahui pola pemeliharaan di masing-masing pulau tersebut. Data dikumpulkan melalui wawancara secara langsung dengan menggunakan kuesioner terstruktur. Pertanyaan dalam kuesioner terdiri atas karakteristik bangunan, kebersihan, sumber makanan dan udara, dan lingkungan RBW. Hasil penelitian menunjukkan bahwa bangunan RBW umumnya bertingkat, dengan atap beton, dinding bata semen, lantai plester semen, dan sirip kayu. Rumah burung walet dibersihkan dibersihkan dengan cara digores/disapu dalam waktu kurang dari dua bulan. Kotoran burung walet umumnya digunakan untuk kebutuhan sendiri. Rumah burung walet umumnya tidak menyediakan bahan untuk menarik serangga atau pakan tambahan. Pakan burung walet umumnyaHymenoptera , dan sumber airnya adalah kolam di dalam gedung RBW. Lingkungan RBW merupakan kawasan pemukiman dan dekat dengan jalan raya. Pembinaan dan pemantauan terhadap pola pemeliharaan burung walet perlu terus dilakukan untuk mendapatkan SBW yang berkualitas baik.

ARTIKEL REVIEW : BAKTERI NITRITASI DAN PERANANNYA DALAM KEBERADAAN NITRIT PADA SARANG BURUNG WALET

Edible bird nest is a high-value export commodity. The industry of edible bird nests encounters various challenges regarding food safety demands for consumers, especially related to the quality of edible bird nests and compliance of nitrite below 30 ppm for the export commodity to China. The purpose of this paper is to obtain information on nitrate content in edible bird nests, the impact of nitrite on consumers and mechanism of nitrite, nitrification processes and mechanisms of nitrification in nature, types of nitrifying bacteria, the nitrification process, and the role of nitrifying bacteria in the edible bird nests, and also nitrite testing methods. This paper shows the nitrite content in edible bird nests at various levels. Nitrite is toxic and dangerous. Nitrite can cause methemoglobinemia, impaired oxygen flow, and difficulty breathing. Hygiene conditions and the environment of the swallow’s house can affect the amount of nitrite in the edible bird nest. Alteration in nitrite can occur through changes in nitrogen in the air to nitrite. Nitrite forming in edible bird nests is a natural process of shift nitrogen in the swallow's house environment and influenced by nitrite-producing bacteria were found in swallow's houses and converting nitrate to nitrite. Nitrification bacteria are bacteria that important role in increasing organic content and the availability of nutrients in the soil by providing nitrate. There are a few bacteria nitrification find in nature and edible bird nests such as Nitrosomonas Sp, Nitrobacter Sp, Nitrospina Sp, Nitrosococcus Sp, Nitrocystis Sp, and Bradyrhizobium japonicum

Identifikasi Penambahan Daging Babi pada Pangan Berbahan Dasar Daging Sapi Menggunakan ELISA dan qPCR

Penelitian ini bertujuan untuk mengidentifikasi dan menganalisis penambahan daging babi ternak dan babi hutan baik yang mentah (raw) maupun yang diolah (cooked) di dalam pangan asal hewan berbahan dasar daging sapi menggunakan metode uji enzyme-linked immunosorbent assay (ELISA)  dan real-time PCR (qPCR). Sebanyak 40 sampel yang terdiri dari 20 daging babi ternak dan 20 daging babi hutan dihomogenisasi dengan daging sapi dalam bentuk mentah maupun olahan (bakso). Konsentrasi setiap daging babi ternak dan babi hutan dalam bentuk mentah dan olahan bakso pada campuran daging sapi antara lain 2%, 1%, 0.5%, 0.25%, dan 0.125%. Hasil uji ELISA pada penelitian ini menunjukkan bahwa uji ini mampu mengidentifikasi adanya penambahan daging babi ternak dan babi hutan dalam pangan asal hewan baik dalam bentuk mentah maupun olahan bakso hingga konsentrasi 0.25%. Hasil uji t terhadap nilai optical density (OD) uji ELISA menunjukkan bahwa tidak terdapat perbedaan dalam mendeteksi spesies babi ternak dengan babi hutan (p≥0.05), tetapi terdapat perbedaan pada sampel bentuk mentah dengan bakso (p<0.05). Hasil uji qPCR mampu mengidentifikasi adanya penambahan daging babi ternak dan babi hutan dalam pangan asal hewan baik dalam bentuk mentah maupun olahan bakso hingga konsentrasi 0.125%. Hasil uji t  terhadap nilai cycle threshold (Ct) uji qPCR menunjukkan bahwa tidak terdapat perbedaan dalam mendeteksi spesies babi ternak dengan babi hutan (p≥0.05), tetapi terdapat perbedaan pada sampel bentuk mentah dengan bakso (p<0.05). Metode ELISA dan qPCR dapat dijadikan sebagai metode pengujian untuk mengidentifikasi pencampuran spesies yang tidak dikehendaki, khususnya daging babi pada pangan asal hewan berbahan dasar daging sapi yang beredar di masyarakat