Search results for: cacao

Authors: Ian Marc G. Cabugsa, Kim Ryan Won, Kareem Mamac, Manuel Dee, Merlita Garcia

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An optimized cacao fermentation technique was developed for the cacao farmers of Davao City Philippines. Cacao samples with weights ranging from 150-250 kilograms were collected from various cacao farms in Davao City and Zamboanga City Philippines. Different fermentation techniques were used starting with design of the sweat box, prefermentation conditionings, number of days for fermentation and number of turns. As the beans are being fermented, its temperature was regularly monitored using a digital thermometer. The resultant cacao beans were assessed using physical and chemical means. For the physical assessment, the bean cut test, bean count tests, and sensory test were used. Quantification of theobromine, caffeine, and antioxidants in the form of equivalent quercetin was used for chemical assessment. Both the theobromine and caffeine were analyzed using HPLC method while the antioxidant was analyzed spectrometrically. To come up with the best fermentation procedure, the different assessment were given priority coefficients wherein the physical tests – taste test, cut, and bean count tests were given priority over the results of the chemical test. The result of the study was an optimized fermentation protocol that is readily adaptable and transferable to any cacao cooperatives or groups in Mindanao or even Philippines as a whole.

Keywords: cacao, fermentation, HPLC, optimization, Philippines

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Authors: Atuhurra Jesse, N'guessan Yves-Roland Douha, Pabitra Lenka

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The detection of diseases within plants has attracted a lot of attention from computer vision enthusiasts. Despite the progress made to detect diseases in many plants, there remains a research gap to train image classifiers to detect the cacao swollen shoot virus disease or CSSVD for short, pertinent to cacao plants. This gap has mainly been due to the unavailability of high quality labeled training data. Moreover, institutions have been hesitant to share their data related to CSSVD. To fill these gaps, image classifiers to detect CSSVD-infected cacao plants are presented in this study. The classifiers are based on VGG16, ResNet50 and Vision Transformer (ViT). The image classifiers are evaluated on a recently released and publicly accessible KaraAgroAI Cocoa dataset. The best performing image classifier, based on ResNet50, achieves 95.39\% precision, 93.75\% recall, 94.34\% F1-score and 94\% accuracy on only 20 epochs. There is a +9.75\% improvement in recall when compared to previous works. These results indicate that the image classifiers learn to identify cacao plants infected with CSSVD.

Keywords: CSSVD, image classification, ResNet50, vision transformer, KaraAgroAI cocoa dataset

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Authors: Ian Marc G. Cabugsa, Kim Ryan A. Won

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Fast, reliable and simultaneous HPLC analysis of theobromine and caffeine in cacao and cocoa products was optimized in this study. The samples tested were raw, fermented, and roasted cacao beans as well as commercially available cocoa products. The HPLC analysis was carried out using step gradient solvent system with acetonitrile and water buffered with H3PO4 as the mobile phase. The HPLC system was optimized using 273 nm wavelength at 35 °C for the column temperature with a flow rate of 1.0 mL/min. Using this method, the theobromine percent recovery mean, Limit of Detection (LOD) and Limit of Quantification (LOQ) is 118.68(±3.38)%, 0.727 and 1.05 respectively. The percent recovery mean, LOD and LOQ for caffeine is 105.53(±3.25)%, 2.42 and 3.50 respectively. The inter-day and intra-day precision for theobromine is 4.31% and 4.48% respectively, while 7.02% and 7.03% was for caffeine respectively. Compared to the standard method in AOAC using methanol in isocratic solvent system, the results of the study produced lesser chromatogram noise with emphasis on theobromine and caffeine. The method is readily usable for cacao and cocoa substances analyses using HPLC with step gradient capability.

Keywords: cacao, caffeine, HPLC, step gradient solvent system, theobromine

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Authors: Jeffrey A. Lavarias, Jessie C. Elauria, Arnold R. Elepano, Engelbert K. Peralta, Delfin C. Suministrado

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Shelling process is one of the primary processes and critical steps in the processing of chocolate or any product that is derived from cocoa beans. It affects the quality of the cocoa nibs in terms of flavor and purity. In the Philippines, small-scale food processor cannot really compete with large scale confectionery manufacturers because of lack of available postharvest facilities that are appropriate to their level of operation. The impact of this study is to provide the needed intervention that will pave the way for cacao farmers of engaging on the advantage of value-adding as way to maximize the economic potential of cacao. Thus, provision and availability of needed postharvest machines like mechanical cacao sheller will revolutionize the current state of cacao industry in the Philippines. A mechanical cacao sheller was developed, fabricated, and evaluated to establish optimum shelling conditions such as moisture content of cocoa beans, clearance where of cocoa beans passes through the breaker section and speed of the breaking mechanism on shelling recovery, shelling efficiency, shelling rate, energy utilization and large nib recovery; To establish the optimum level of shelling parameters of the mechanical sheller. These factors were statistically analyzed using design of experiment by Box and Behnken and Response Surface Methodology (RSM). By maximizing shelling recovery, shelling efficiency, shelling rate, large nib recovery and minimizing energy utilization, the optimum shelling conditions were established at moisture content, clearance and breaker speed of 6.5%, 3 millimeters and 1300 rpm, respectively. The optimum values for shelling recovery, shelling efficiency, shelling rate, large nib recovery and minimizing energy utilization were recorded at 86.51%, 99.19%, 21.85kg/hr, 89.75%, and 542.84W, respectively. Experimental values obtained using the optimum conditions were compared with predicted values using predictive models and were found in good agreement.

Keywords: cocoa beans, optimization, RSM, shelling parameters

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Authors: Marlene Yu Lilin Wätzold, Wisnu Harto Adiwijoyo, Meike Wollni

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Land-use conversion from tropical forests to cash crop production in the form of monocultures has drastic consequences for biodiversity. Meanwhile, high dependence on cash crop production is often associated with a decrease in other food crop production, thereby affecting household dietary diversity. Additionally, deforestation rates have been found to reduce households’ dietary diversity, as forests often offer various food sources. Agroforestry systems are seen as a potential solution to improve local biodiversity as well as provide a range of provisioning ecosystem services, such as timber and other food crops. While a number of studies have analyzed the effects of agroforestry on biodiversity, as well as household livelihood indicators, little is understood between potential trade-offs or synergies between the two. This interdisciplinary study aims to fill this gap by assessing cacao agroforestry’s role in enhancing local bird diversity, as well as farm household dietary diversity. Additionally, we will take a landscape perspective and investigate in what ways the landscape composition, such as the proximity to forests and forest patches, are able to contribute to the local bird diversity, as well as households’ dietary diversity. Our study will take place in two agro-ecological zones in Ghana, based on household surveys of 500 cacao farm households. Using a subsample of 120 cacao plots, we will assess the degree of shade tree diversity and density using drone flights and a computer vision tree detection algorithm. Bird density and diversity will be assessed using sound recordings that will be kept in the cacao plots for 24 hours. Landscape compositions will be assessed via remote sensing images. The results of our study are of high importance as they will allow us to understand the effects of agroforestry and landscape composition in improving simultaneous ecosystem services.

Keywords: agroforestry, biodiversity, landscape composition, nutrition

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Authors: Antonio B.Tangayan Jr., Hershey P. Mondejar, Pet Roey Pascual, Zeam Voltaire E. Amper

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A study on in vitro larvicidal acitivity of different levels of Madre de Cacao (Gliricidia sepium) concentrated crude ethanolic extract (CCEE) against horn fly larvae (Haematobia irritans) was conducted. The air-dried leaves of Gliricidia sepium were infused in a 1:3 ratio (w/v) using ethanol as solvent and concentrated in a rotary evaporator (60°C). A total of 120 larvae of Haematobia irritans were exposed in various concentration: 200, 400, 800 and 1000 ppm. Based on the result after 5 hours of exposure, CCE G. sepium extract at 200 ppm showed less effect with 30% mortality compared to 400 ppm, 800 ppm and 1000 ppm with 70%, 83%, and 100% mortality, respectively. Findings also revealed that CCE of G. sepium extract at 1000 ppm, 800 ppm, and commercial larvicide were comparable in causing mortality of H. irritans larvae from the first hour up to the fifth hours of exposure. However, on the fifth hour, 400 ppm was also found to be effective. This suggests that the higher the concentration of CCE G. sepium extract and the longer the time of exposure, the higher is the percentage mortality of the larvae. Thus, CCE G. sepium extract can be used as an alternative for commercial larvicide.

Keywords: horn fly, in vitro, larvicidal, Madre de Cacao

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Authors: O. Rozita, N. M. Nik Aziz

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High-density planting is usually recommended for a small area of planting in order to increase production. The normal planting distance for cocoa (Theobroma cacao L.) in Malaysia is 3 m x 3 m. The study was conducted at Cocoa Research and Development Centre, Malaysia Cocoa Board, Jengka, Pahang with the objectives to evaluate the suitability of seven cocoa clones under four different planting densities and to study the interaction between cocoa clones and planting densities. The study was arranged in the split plot with randomized complete block design and replicated three times. The cocoa clone was assigned as the main plot and planting density was assigned as a subplot. The clones used in this study were PBC 123, PBC 112, MCBC4, MCBC 5, QH 1003, QH 22, and BAL 244. The planting distance were 3 m x 3 m (1000 stands/ha), 3 m x 1.5 m (2000 stands/ha), 3 m x 1 m (3000 stands/ha) and (1.5 m x 1.5 m) x 3 m (3333 stands/ha). Evaluation on yield performance was carried out for seven years. Clones of PBC 123, QH 1003, and QH 22 obtained the higher yield, meanwhile MCBC 4, MCBC 5, and BAL 244 obtained the lowest yield. In general, high-density planting can increase cocoa production with good management practices. Among the cocoa management practices, the selection of suitable clones with small branching habits and moderately vigorous and proper pruning activity were the most important factor in high-density planting.

Keywords: clones, management, planting density, Theobroma cacao, yield

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Authors: Alam Anshary, Flora Pasaru, Shahabuddin

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The Cocoa Pod Borer (CPB), Conopomorpha cramerella (Snell.) is present on most of the larger cocoa producing islands in Indonesia. Various control measures CPB has been carried out by the farmers, but the results have not been effective. This study aims to determine the effect of application of Beauveria bassiana treatments and pruning technique to the control of CPB in the cocoa plantation people. Research using completely randomized design with 4 treatments and 3 replications, treatment consists of B.bassiana, Pruning, B. bassiana+pruning (Bb + Pr), as well as the control. The results showed that the percentage of PBK attack on cocoa pods in treatment (Bb + Pr) 3.50% the lowest compared to other treatments. CPB attack percentage in treatment B.bassiana 6.15%; pruning 8.75%, and 15.20% control. Results of the analysis of production estimates, the known treatments (Bb + Pr) have the highest production (1.95 tonnes / ha). The model results estimated production is Y= 0,20999 + 0,53968X1 + 0,34298X2+ 0,31410X3 + 0,35629X4 + 0,08345X5 + 0,29732X6. Farm production costs consist of fixed costs and variable costs, fixed costs are costs incurred by the farmer that the size does not affect the results, such as taxes and depreciation of production equipment. Variable costs are costs incurred by farmers who used up in one year cocoa farming activities. The cost of production in farming cocoa without integrated techniques control of CPB is Rp. 9.205.550 million/ha, while the cost of production with integrated techniques control is Rp. 6.666.050 million/ha.

Keywords: cacao, cocoa pod borer, pruning, Beauveria bassiana, production costs

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Authors: B. Saripah, A. Alias

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Cocoa Theobroma cacao (Linnaeus) (Malvales: Sterculiaceae) is subjected to be infested by various numbers of insect pests, and Conopomorpha cramerella Snellen (Lepidoptera: Gracillariidae) is the most serious pest of cocoa in Malaysia. The pest was indigenous to the South East Asia. Several control measures have been implemented and the chemicals have been a major approach if not unilateral, in the management of CPB. Despite extensive use of insecticides, CPB continues to cause an unacceptable level of damage; thus, the combination of several control approaches should be sought. The study was commenced for 12 months at three blocks; Block 18C with complete management practices which include insecticide application, pruning, fertilization and frequent harvesting, Block 17C was treated with frequent harvesting at intervals of 7-8 days, and Block 19C was served as control block. The results showed that the mean numbers of CPB eggs were recorded higher in Block 17C compared with Block 18C in all sampling occasions. Block 18C shows the lowest mean number of CPB eggs in both sampling plots, outside and core plots and it was found significantly different (p ≤ 0. 05) compared to the other blocks. The mean number of CPB eggs was fluctuated throughout sampling occasions, the lowest mean number of eggs was recorded in January (17C) and November (18C), while the highest was recorded in April (17C) and December 2012 (18C). Frequent spraying with insecticides at the adjacent block (18C) helps in reducing CPB eggs in the control block (Block 19C), although there was no spraying was implemented Block 19C. In summary, the combination of complete management practices at Block 18C seems to have some effect on the CPB population at Blocks 17 and 19C because all blocks are adjacent to each other.

Keywords: cocoa, theobroma cacao, cocoa pod borer, conopomorpha cramerella

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Authors: Suhaida Salleh, Tee Yei Kheng

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Black pod is the most commonly destructive disease of cacao (Theobroma cacao) which cause major losses to global production of cocoa beans. The genus of Phytophthora is the important pathogen of this disease worldwide. The species of P. megakarya causes black pod disease in West Africa, whereas P. capsici and P. citrophthora cause the incident in Central and South America. In Malaysia, this disease is caused by P. palmivora which infect all stages of pod development including flower cushion, cherelle, immature and mature pods. This pathogen destroys up to 10% of trees yearly through stem cankers and causes 20 to 30% pod damages through black pod rot. Since P. palmivora has a high impact on cocoa yield, it is crucial to identify some of the abiotic factors that can constrain their growth. In an effort to evaluate the effect of different substrates and temperatures to the growth of P. palmivora, a laboratory study was done under a different range of temperatures. Different substrate for the growth of P. palmivora were used which are corn meal agar (CMA) media and detached pod of cocoa. An agar plug of seven days old of P. palmivora growth was transferred on both substrates and incubated at 24, 27, 30, 33 and 36ᵒC, respectively. The diameter of lesion on pod and the cultural growth of pathogen was recorded for 7 consecutive days. The optimum incubation temperature of P. palmivora on both substrates is at 27ᵒC. However, the growth tends to be inhibited as the temperature increases. No lesion developed on pod surface incubated at 36ᵒC and only a small lesion observed at 33ᵒC. The sporulation with the formation of white mycelial growth on pod surface was only visible at optimum temperature, 27ᵒC. On CMA, the pathogen grew over the entire range of temperatures tested. The study is, therefore, concluded that P. palmivora grow the best at temperature of 27ᵒC on both substrates and their growth begin to inhibit when the temperature rises to more than 27ᵒC. The growth pattern of this pathogen is similar on both pod surface and cultural media.

Keywords: cocoa, Phytophthora palmivora, substrate, temperature

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Authors: Nasiruddin Khan

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The action of (-)-epicatechin, a cocoa (Theobroma cacao) flavanol that modulates redox/oxidative stress are contributed mainly to their antioxidant properties. The present study investigates the concentration and time dependent effect of (-)-epicatechin metabolites 3MeEc, 4MeEc, and 4SulEc on the production of ROS on BAEC using L-012, Lucigenin as chemiluminescence dye and XO/HX system. Our result demonstrates that 3MeEc shows significant (P <0.05) lowering effect of ROS production in BAEC with increasing concentration of metabolite while L-012 was used as chemiluminescence dye but not in the case of Lucigenin. In XO/HX system, using L-012 as chemiluminescence dye, 3MeEc and 4MeEc showed significant lowering effect on ROS production with increasing concentration from 100-500nM as compared to the positive control (SOD). When Lucigenin was used as chemiluminescence dye, 3MeEc exerted significant lowering effect with increasing concentration when compared to the positive control (SOD) whereas 4MeEc showed significant lowering effect in ROS production from 250 nM on as compared to positive control. For 4SulEc, a significant lowering effect of ROS production was only observed at 100 and 250 nM. Overall, although each metabolite shows considerable effect, 3MeEc exhibited more pronounced effect on decreasing the production of ROS as compared to other two metabolites.

Keywords: epicatechin metabolites, HO-1, Nrf2, ROS

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Authors: Guity Karim, Valiollah Ayareh

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Probiotic dairy products are those that contain biologically active components that may affect beneficially one or more target functions in the body, beyond their adequate nutritional effects. As far as chocolate milk is a popular dairy product in the country especially among children and youth, production of a symbiotic (probiotic + peribiotic) new product using chocolate milk, Bifidobacterium lactis (DSM, Netherland) and inulin (Bene, Belgium) would help to promote the nutritional and functional properties of this product. Bifidobacterium Lactis is used as a probiotic in a variety of foods, particularly dairy products like yogurt and as a probiotic bacterium has benefit effects on the human health. Inulin as a peribiotic agent is considered as functional food ingredient. Experimental studies have shown its use as bifidogenic agent. Chocolate milk with different percent of fat (1 and 2 percent), 6 % of sugar and 0.9 % cacao was made, sterilized (UHT) and supplemented with Bifidobacterium lactis and inulin (0.5 %) after cooling . A sample was made without inulin as a control. Bifidobacterium lactis population was enumerated at days 0, 4, 8 and 12 together with measurement of pH, acidity and viscosity of the samples. Also sensory property of the product was evaluated by a 15 panel testers. The number of live bacterial cells was maintained at the functional level of 106-108 cfu/ml after keeping for 12 days in refrigerated temperature (4°C). Coliforms were found to be absent in the products during the storage. Chocolate milk containing 1% fat and inulin has the best effect on the survival and number of B. lactis at day 8 and after that. Moreover, the addition of inulin did not affect the sensorial quality of the product. In this work, chocolate has been evaluated as a potential protective carrier for oral delivery of B. lactis and inulin.

Keywords: chocolate milk, synbiotic, bifidobacterium lactis, inulin

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Authors: Thavananthan Sivananthawerl

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Coconut palm is existing for more than 2000 years in Sri Lanka. However, cultivation on a large scale (plantation) began only in the 19th century. Due to different light perceptions during the growth stages of palm, there is a huge potential to grow crops in-between rows of coconut plants which are grown with wider, fixed spacing. Intercropping under coconut will have multiple benefits such as increasing soil fertility, increasing sunlight utilization, increasing total crop productivity, increasing income & profit, maximum use of resources, reducing the risk, and increasing food security. Growing potential annual, agricultural intercrops could be classified as ‘agri-silvicultural’ system. This is the best agri-silvicultural system that can be named under any perennial crop system in Sri Lanka. In the late 1970’s cassava, pepper and cacao are the major intercrops under the coconut plantations. At the early ages of the palm (<5 years) light-loving crops such as pineapple, passion, papaya, and cassava are recommended and preferred by the cultivators. In between 5-20 years of age, the availability of light is very low, and therefore shade tolerant/loving crops (pasture, yam, ginger) could be used as the intercrops. However, after 20 years of age (>20 years) canopy is getting small, and the light availability on the ground increases. So, light demanding crops such as pepper, banana, pineapple, betel, cassava, and seasonal crops could be grown successfully. Even though this is a sustainable system in several aspects, there are potential challenges ahead to the system. The major ones are land fragmentation and infrastructure development. The other factors are drought, lack of financial support, price instability of the intercrops, availability of improved planting materials, and development of dwarf varieties which reduces the light.

Keywords: coconut cultivation, agri-silviculture, intercrop, sunlight, annuals, sustainability

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Authors: Aka S. Koffi, N’Goran Yao, Philippe Bastide, Denis Bruneau, Diby Kadjo

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Cocoa beans (Theobroma cocoa L.) are the main components for chocolate manufacturing. The beans must be correctly fermented at first. Traditional process to perform the first fermentation (lactic fermentation) often consists in confining cacao beans using banana leaves or a fermentation basket, both of them leading to a poor product thermal insulation and to an inability to mix the product. Box fermenter reduces this loss by using a wood with large thickness (e>3cm), but mixing to homogenize the product is still hard to perform. Automatic fermenters are not rentable for most of producers. Heat (T>45°C) and acidity produced during the fermentation by microbiology activity of yeasts and bacteria are enabling the emergence of potential flavor and taste of future chocolate. In this study, a cylindro-rotative fermenter (FCR-V1) has been built and coconut fibers were used in its structure to confine heat. An axis of rotation (360°) has been integrated to facilitate the turning and homogenization of beans in the fermenter. This axis permits to put fermenter in a vertical position during the anaerobic alcoholic phase of fermentation, and horizontally during acetic phase to take advantage of the mid height filling. For circulation of air flow during turning in acetic phase, two woven rattan with grid have been made, one for the top and second for the bottom of the fermenter. In order to reduce air flow during acetic phase, two airtight covers are put on each grid cover. The efficiency of the turning by this kind of rotation, coupled with homogenization of the temperature, caused by the horizontal position in the acetic phase of the fermenter, contribute to having a good proportion of well-fermented beans (83.23%). In addition, beans’pH values ranged between 4.5 and 5.5. These values are ideal for enzymatic activity in the production of the aromatic compounds inside beans. The regularity of mass loss during all fermentation makes it possible to predict the drying surface corresponding to the amount being fermented.

Keywords: cocoa fermentation, fermenter, microbial activity, temperature, turning

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Authors: Benoit Atge, Alice Dhersin, Oscar Da Silva Cacao, Beatrice Martinez, Dominique Ducint, Catherine Verdun-Esquer, Isabelle Baldi, Mathieu Molimard, Antoine Villa, Mireille Canal-Raffin

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Introduction: Healthcare workers' exposure to antineoplastic drugs (AD) is a burning issue for occupational medicine practitioners. Biological monitoring of occupational exposure (BMOE) is an essential tool for assessing AD contamination of healthcare workers. In addition to BMOE, surface sampling is a useful tool in order to understand how workers get contaminated, to identify sources of environmental contamination, to verify the effectiveness of surface decontamination way and to ensure monitoring of these surfaces. The objective of this work was to develop a complete tool including a kit for surface sampling and a quantification analytical method for AD traces detection. The development was realized with the three following criteria: the kit capacity to sample in every professional environment (healthcare services, veterinaries, etc.), the detection of very low AD traces with a validated analytical method and the easiness of the sampling kit use regardless of the person in charge of sampling. Material and method: AD mostly used in term of quantity and frequency have been identified by an analysis of the literature and consumptions of different hospitals, veterinary services, and home care settings. The kind of adsorbent device, surface moistening solution and mix of solvents for the extraction of AD from the adsorbent device have been tested for a maximal yield. The AD quantification was achieved by an ultra high-performance liquid chromatography method coupled with tandem mass spectrometry (UHPLC-MS/MS). Results: With their high frequencies of use and their good reflect of the diverse activities through healthcare, 15 AD (cyclophosphamide, ifosfamide, doxorubicin, daunorubicin, epirubicin, 5-FU, dacarbazin, etoposide, pemetrexed, vincristine, cytarabine, methothrexate, paclitaxel, gemcitabine, mitomycin C) were selected. The analytical method was optimized and adapted to obtain high sensitivity with very low limits of quantification (25 to 5000ng/mL), equivalent or lowest that those previously published (for 13/15 AD). The sampling kit is easy to use, provided with a didactic support (online video and protocol paper). It showed its effectiveness without inter-individual variation (n=5/person; n= 5 persons; p=0,85; ANOVA) regardless of the person in charge of sampling. Conclusion: This validated tool (sampling kit + analytical method) is very sensitive, easy to use and very didactic in order to control the chemical risk brought by AD. Moreover, BMOE permits a focal prevention. Used in routine, this tool is available for every intervention of occupational health.

Keywords: surface contamination, sampling kit, analytical method, sensitivity

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Authors: Clara Tramuta, Lucia Decastelli, Elisa Barcucci, Sandra Fragassi, Samantha Lupi, Enrico Arletti, Melissa Bizzarri, Daniela Manila Bianchi

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Introdution: Food allergies occurs, in the Western World, 2% of adults and up to 8% of children. The protection of allergic consumers is guaranted, in Eurrope, by Regulation (EU) No 1169/2011 of the European Parliament which governs the consumer's right to information and identifies 14 food allergens to be mandatory indicated on the label. Among these, mustard is a popular spice added to enhance the flavour and taste of foods. It is frequently present as an ingredient in spice blends, marinades, salad dressings, sausages, and other products. Hypersensitivity to mustard is a public health problem since the ingestion of even low amounts can trigger severe allergic reactions. In order to protect the allergic consumer, high performance methods are required for the detection of allergenic ingredients. Food safety laboratories rely on validated methods that detect hidden allergens in food to ensure the safety and health of allergic consumers. Here we present the test results for the validation and accreditation of a Real time PCR assay (RT-PCR: SPECIALfinder MC Mustard, Generon), for the detection of mustard traces in food. Materials and Methods. The method was tested on five classes of food matrices: bakery and pastry products (chocolate cookies), meats (ragù), ready-to-eat (mixed salad), dairy products (yogurt), grains, and milling products (rice and barley flour). Blank samples were spiked starting with the mustard samples (Sinapis Alba), lyophilized and stored at -18 °C, at a concentration of 1000 ppm. Serial dilutions were then prepared to a final concentration of 0.5 ppm, using the DNA extracted by ION Force FAST (Generon) from the blank samples. The Real Time PCR reaction was performed by RT-PCR SPECIALfinder MC Mustard (Generon), using CFX96 System (BioRad). Results. Real Time PCR showed a limit of detection (LOD) of 0.5 ppm in grains and milling products, ready-to-eat, meats, bakery, pastry products, and dairy products (range Ct 25-34). To determine the exclusivity parameter of the method, the ragù matrix was contaminated with Prunus dulcis (almonds), peanut (Arachis hypogaea), Glycine max (soy), Apium graveolens (celery), Allium cepa (onion), Pisum sativum (peas), Daucus carota (carrots), and Theobroma cacao (cocoa) and no cross-reactions were observed. Discussion. In terms of sensitivity, the Real Time PCR confirmed, even in complex matrix, a LOD of 0.5 ppm in five classes of food matrices tested; these values are compatible with the current regulatory situation that does not consider, at international level, to establish a quantitative criterion for the allergen considered in this study. The Real Time PCR SPECIALfinder kit for the detection of mustard proved to be easy to use and particularly appreciated for the rapid response times considering that the amplification and detection phase has a duration of less than 50 minutes. Method accuracy was rated satisfactory for sensitivity (100%) and specificity (100%) and was fully validated and accreditated. It was found adequate for the needs of the laboratory as it met the purpose for which it was applied. This study was funded in part within a project of the Italian Ministry of Health (IZS PLV 02/19 RC).

Keywords: allergens, food, mustard, real time PCR

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