Food Control 22 (2011) 1021e1026. Contents lists available at ScienceDirect. Food Control. journal homepage:

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1 Food Control 22 (2011) 1021e1026 Contents lists available at ScienceDirect Food Control journal homepage: The monitoring of semolina contamination by insect fragments using the light filth method in an Italian mill P. Trematerra a, *, V. Stejskal b, J. Hubert b a Department of Animal, Plant and Environmental Science, University of Molise, Via De Sanctis, I Campobasso, Italy b Crop Research Institute, Drnovska 507, Praha 6 Ruzyne, CZ , Czech Republic article info abstract Article history: Received 26 May 2010 Received in revised form 18 November 2010 Accepted 28 November 2010 Keywords: Light filth method Semolina IPM Flour mill Italy Although insect contamination decreases the quality and safety of cereal products there has been no quantitative or qualitative risk evaluation of semolina contamination by insect fragments in European mills. Monitoring of semolina contamination in Italian mills over one year (June 2008 to June 2009) enabled us to evaluate (i) the frequency, (ii) quantity, (iii) seasonal dynamics, and (iv) pest origin (e.g. pest species that are responsible for contamination) of contamination. Insect fragments and undamaged whole insect bodies were extracted from semolina samples. The frequency of fragments in samples decreased as follows: mandibles, legs, adult cuticle fragments. The amount of contamination ranged from 0 to 15 (median ¼ 2) insect fragments per 50 g of semolina. The level of contamination was below the Italian regulatory limit (e.g. 75 fragments/50 g of semolina). Internally feeding grain pests (Sitophilus spp., Rhyzopertha sp.) were mainly responsible for the contamination. Contamination by externally feeding pests (Tribolium spp., Cryptolestes spp., Oryzaephilus spp., Nemapogon sp., and psocids) and pests infesting wheat in the field (thrips and aphids) was less frequent. There was a seasonal increase in the number of fragments from April to June. The number of fragments decreased after mill fumigation for one month (September 2008). Although internally feeding grain pests arrive at the mills as a hidden infestation from the grain stores, these are the main cause of semolina contamination. The presence of whole insect bodies in semolina and a decrease in fragment numbers after fumigation indicated that mill infestation is partly responsible for insect fragment contamination in semolina. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction In recent years, the European food industry has faced the need to respond to requests for qualitative and food safety standards in alimentary products with respect to both nutritional characteristics and hygienic-sanitary aspects, including the elimination of insect infestation (Neethirajan, Karunakaran, Javas, & White, 2007; Stejskal & Tlustos, 2009). Various food products and food industry branches differ in their pest infestation risk. Bakeries, food and feed mills are among the most endangered food industry facilities with regards to insect infestation (Scirocchi et al., 2004; Stejskal & Verner, 1996; Trematerra & Catalano, 2009). Stored product pests have the capacity to infest a vast array of cereal products. Physical injury to the food product is not the only harmful effect caused by storage pests. The presence of an insect mite and physical and chemical rodent contaminants in food may not be acceptable not * Corresponding author. Department of Animal Plant and Environmental Science, University of Molise, Via De Sanctis, I Campobasso, Italy. address: trema@unimol.it (P. Trematerra). only for aesthetic but also medical reasons. The beetle Tribolium spp. excretes quinones of carcinogenic importance (El-Mofty, Khudoley, Sakr, & Fathala, 1992; Pappas & Wardrop, 1996). Stored product insects harbour pathogenic bacteria (Lakshmikantha, Subramanyam, McKinney, & Zurek, 2010a; Larson, Subramanyam, Zurek, & Herrman, 2008; Yezerski, Cussatt, Glick, & Evancho, 2005), for example Tribolium spp. is suggested as a vector of important pathogens, i.e Enterococcus faecalis OG1RF:pCF10 (Lakshmikantha, Subramanyam, & Zurek, 2010b). Flour mites produce allergens (Eaton et al., 1985; Stejskal & Hubert, 2008) and transmit fungi containing mycotoxins (Hubert et al., 2003). Customers are extremely sensitive to the unintentional presence of any living organism in their food. For example, finished pasta products are vulnerable to infestation by the rice weevil (Sitophilus oryzae) (Stejskal, Kucerová, & Lukás, 2004), which is considered by customers as aesthetically unacceptable. Insect fragments in flour and semolina are also unacceptable to consumers (Throne, Hallman, Johnson, & Follett, 2003; Toews et al., 2007). In the USA certain (non-zero) maximal acceptable levels of physical contaminants in food products have been established. The US Food and /$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi: /j.foodcont

2 1022 P. Trematerra et al. / Food Control 22 (2011) 1021e1026 Drug Administration (FDA, 1988) has established a level of detection of 75 insect fragments per 50 g of wheat, but this level is often higher than US mills will tolerate (Flinn & Hagstrum, 2001). In Canada, the number of fragments in wheat flour should be less than twenty in three test samples of 50 g each (Bhuvaneswaria et al., in press). Similarly, there is zero or near zero tolerance to insect fragments in food in most European countries. It is well accepted that millers must establish and regularly operate an efficient pest control programme, based on regular pest monitoring, to decrease populations of living arthropods in mills (Trematerra & Gentile, 2008). After pest control intervention it is necessary to monitor foodstuffs for the levels of immobile insect stages and contaminates; i.e. dead bodies, their fragments, eggs and faeces (Perez-Mendoza, Throne, Dowell, & Baker, 2003; Singh, Jayas, Paliwal, & White, 2009). This is because arthropod fragments and eggs easily escape routine visual control because of their microscopic size (Kucerova & Stejskal, 2002; 2008; 2009). There are various methods available for the detection of insect eggs and fragments in food materials (Neethirajan et al., 2007). Detection of dead pest bodies, eggs and fragments in the whole grain is mainly based on sieving methods (Hubert, Nesvorna, & Stejskal, 2009). Less frequently filth flotation, NRA, X-rays, acoustic, immunochemical methods, image analysis and other laboratory techniques are employed (Krizkova-Kudlikova, Stejskal, & Hubert, 2007; Lukás, Kucerová, & Stejskal, 2009; Neethirajan et al., 2007; Perez- Mendoza, Throne, Maghirang, Dowell, & Baker, 2005). The filth flotation techniques were originally developed for the identification of pest fragments in flour (Brader et al., 2002; Dimov, Silveira, Elian, & Penteado, 2004; Locatelli, Moroni, & Daolio, 1993). However, the light filth method was recently adopted for semolina (Trematerra & Catalano, 2009), frozen mushrooms or vegetables (Baldassari, Francia, & Baronio, 2004; Martini, Marzullo, & Baronio, 2009), pepper and cumin (Graciano, Atui, & Dimov, 2006). Although powerful methods for the detection of pests and fragments are available, there is little if any published information on the actual spectra and annual dynamics of insect fragments in milling cereal products. The main reason is that most mill companies consider data on pest infestation and especially on fragment contamination of finished cereal products as market-sensitive and therefore strictly confidential. The study was based on one year of observations in Italian mill during which more than 250 semolina samples were analysed by filth flotation to detect the occurrence of insect fragments occurrence. The aim of the work was to evaluate (i) the frequency of various types of insect fragments in semolina, (ii) the quantity of insect fragments in semolina samples, (iii) the seasonal dynamics of the fragments in semolina samples, and finally (iv) the spectra of pest species in order to identify the main contributors to the fragments in semolina produced by a commercial Italian mill over a period of one year. The general aim of the work was to demonstrate that mill pest infestation, as described by Trematerra and Catalano (2009), may have a negative impact on contamination and reduce the quality of some of the finished milling products in Italy. 2. Material and methods 2.1. Sample site and sampling The observations were carried out at an industrial semolina mill located in the Apulia region, South Italy. The mill was a large building of 18,000 m 3, with seven floors processing 500 tons of Triticum durum Desf. (hard wheat) per day. Sampling was carried out over 12 consecutive months, from June 2008 to July 2009, focusing on four cells of the structure (identified by the numbers 1, 2, 3, and 4). The analysed semolina was obtained from national and international grinding wheats, taken individually or as a mixture. Semolina was collected weekly and each time one of the four monitored cells was filled with new semolina. The number of samples was as follows: 71, 62, 63 and 61 for cells 1, 2, 3 and 4 respectively, giving 257 samples in total. The differences in the numbers of fragments among the monitored cells were compared by KruskaleWallis test using XLSTAT 2007 (Addinsoft USA, New York, NY, USA). General fumigation of the mill was carried out in the second week of August, using sulphuryl fluoride with 62 h of exposure. As a result of this fumigation treatment, the mill suspended its activities for about 10 days, the period considered necessary for the toxic effects of the gas to decay Sample processing Solid impurities were isolated, identified and analysed using the official light filth method as described in the Decree of the Italian Politics Agricultural Office, 12 January 1999, Official Methods of Cereals Analysis e Supplement No. 5 Determination of solid impurities in flour and transformed products and Identification of substances of biological origin and mineral extraneous substances in cereal flours (Anonymous, 1999). The apparatus was constructed according to recommendation of Glaze and Bryce (1994). A quantity of semolina varying from 1.5 to 2.5 kg was taken for each sample, from which laboratory specimens weighing at least 600 g were obtained according to the norms in force. These samples were homogenized inside their containers using a spatula; 50 g of semolina was collected from these samples at different points, weighed for analysis, and introduced into a flask through a glass funnel for dusts. The sample was submitted to acetic-nitric digestion until ebullition, and the impurities present were separated for flotation with alcohol and gasoline in a Wildman trap flask and collected on a paper filter through deep bed filtration with a Buchner funnel. The material gathered on the filter was observed under a dissection microscope and, in many cases, impurities were pulled out and placed on a slide with Faure liquid and observed under a compound microscope for identification Identification of insect fragments According to regulations, the isolated and identified fragments were classified into different categories: whole insects (adult and/ or larvae); cephalic capsules of insects; fragments of arthropods; moth scales; hair of mammals (rodents, man, other); textile fibres; and other fragments (metal, plastics, glass, combustibles). However in this study we focused on the quantification of the fragments of insect origin. 3. Results 3.1. The numbers of fragments in semolina Infestation was found in 73% of semolina samples; however 22% of the samples contained just one fragment (see Fig. 1A). The maximum number of fragments was 15 and was found in two samples. Generally there were no significant differences in the number of fragments between the sampled cells (KruskaleWallis test K (3, 253) ¼ 3.5, P ¼ 0.316). The distribution of fragments in separate cells was similar with means of 2.1e2.5 fragments and medians of 1 or 2 fragments per 50 g semolina sample (Fig. 1B). Natural textile fibres, crystalline particles and combusted particles were also detected, while rodent hairs were never found.

3 P. Trematerra et al. / Food Control 22 (2011) 1021e Fig. 1. The distribution of insect fragments in semolina samples obtained from the mill from July 2008 to June The numbers of fragments are related to 50 g of semolina; A - frequency histogram; B - scatterplots of fragment distribution in four sampled cells. During the period of study the number of fragments fluctuated, with a median of 0e5 fragments per 50 g of semolina (Fig. 2). After fumigation the median decreased from 2 to 0 fragments per 50 g of semolina. There was a remarkable increase in the median from April to June The identification of insects in the fragments According to the results obtained during our analyses, the fragments of insects identified through the light filth method had different origins (Fig. 3). The most frequently occurring fragments were mandibles, legs and adult cuticular fragments (Fig. 4). Numerous fragments came from both immature and adult insects infesting wheat during the field growing stage. They belonged to phytophagous insects that are active on the plants or on the ears of wheat: the legs and adults of aphids, and the head, legs and distal abdominal portions of immature and adult Thysanoptera. Many other fragments belonged to specimens infesting post-harvest cereals: mainly the head, mandibles and cuticular fragments of larvae, elytra and legs of adults, but also whole larvae and adults; there were also internally feeding insects, mainly Sitophilus spp. and Rhyzopertha dominica, which are active in grains during storage but in some circumstances are able to begin their infestations in the field. Represented in smaller quantity were externally feeding insects, for example fragments of cuticle, legs, mandibles and also adults and larvae of Tribolium spp., Cryptolestes spp., Oryzaephilus spp. and Nemapogon granellus, which are able to colonize the stored cereals and the machinery in the mills in which dust, cereal debris and flour residues are present. Finally, there were also some fragments associated with environmental pests like flies and structural pests such as Psocoptera. 4. Discussion For targeted monitoring, intervention and prevention, pest control operators and food sanitation technicians need to answer two basic questions: First, what is the real current risk for the occurrence of insect fragments in the final milling products for particular pest species? Second, what are the main entrance routes for insect fragments? The data from this study answer both questions. We found that all the number of fragments in samples of semolina were below the limits specified by FDA (FDA, 1988), even

4 1024 P. Trematerra et al. / Food Control 22 (2011) 1021e1026 Fig. 2. The temporal fluctuation of insect fragments in the samples. The fumigation of the mill is marked by an arrow. To describe the changes of fragment numbers, the medians of the number of insect fragments were calculated per all samples sampled during the month period. though the median infestation was two fragments per 50 g of semolina. This confirmed the acceptable quality of semolina, as reported previously (Germinara, de Cristofaro, & Rotundo, 2008; Rotundo, Cristofaro, de Cristofaro, & Chierchia, 1995; Trematerra & Catalano, 2009). However, there have been situations documented in Italy in which the infestation exceeded these limits, i.e. 3% of flour samples contained more than 75 insect fragments (Locatelli et al., 1993). Our data also show that the insect fragments and related Fig. 3. Some insect fragments recovered from semolina samples. Thysanoptera: head (1), abdomen (2), median leg (3); R. dominica head and mouth parts (4); Tribolium spp., mandible of adult (5); S. oryzae, mandible of adult (6); unidentified cuticular fragments (7 and 8); R. dominica: pronotum fragment (9), antenna (10). The scale is 100 mm.

5 P. Trematerra et al. / Food Control 22 (2011) 1021e Fig. 4. The insect fragments contaminating the semolina; the total number of identified fragments are columns, the numbers above the columns are perceptual proportion from the all insect fragments. contaminants are everywhere, so zero tolerance is unrealistic. The temporal distribution of the fragments in the analysed samples showed an increasing trend from April to June (Fig. 2). This coincided with a decrease in grain infestation (cf. Stejskal et al., 2003; Aspaly, Stejskal, Pekár, & Hubert, 2007) and influenced the quality of the semolina. The fragment numbers decreased in the month after the mill was fumigated (September 2008); after fumigation the median insect fragments decreased from 2 to 0 fragments per 50 g of semolina. However, fragments were still present (see Fig. 2). The speculation is that the internally feeding species were not affected by the mill fumigation. From a managerial point of view, the filth test is therefore not suitable for quantification of the effect of the fumigation treatment and should be combined with other monitoring methods (i.e. traps) (Trematerra & Gentile, 2008). There are three possible routes for semolina pest infestation and contamination: insects come from the contaminated stores inside kernels and are turned into fragments during the milling process (Athanassiou, Kavallieratos, Palyvos, Sciarretta, & Trematerra, 2005) (route 1), insects actively migrate into the mill and semolina from the surrounding resources (cf. Campbell & Arbogast, 2004) (route 2) or insects migrate into the semolina from the uncelaned debris in mills and stores (Kucerova et al., 2003; Trematerra & Fiorilli, 2000) (route 3). Hidden infestations of grain kernels from the stores (i.e. route 1) are usually considered as the main source of insect fragments in flour and semolina since it has been documented that internally feeding pests such as grain weevils (Sitophillus spp.) and the lesser grain borer (Rhyzopertha sp.) are the most important contaminators in the USA (Flinn & Hagstrum, 2001; Perez-Mendoza et al., 2003). The rationale behind the technical difficulties in preventing contamination by internally feeding pests is that the immature stages and pre-emergent adults cannot be removed from the grain by cleaning before milling. The results of the filth tests showed that the fragments in semolina were mainly derived from internally feeding pests such as Sitophilus spp. and Rhyzopertha dominica entering semolina via route 1. This finding regarding route of infestation was consistent with common expectations and knowledge. However, by finding whole larvae and adults in semolina in this study (see Fig. 4), we have demonstrated that the infestation and contamination of the finished semolina product may be a result of migrating pests and routes 2 and 3. However, there was low correlation between the secondary pest species found in the semolina samples by filth flotation and that previously found in monitoring traps in the same mill (Trematerra, Gentile, Brunetti, Collins, & Chambers, 2007). Unexpectedly, there were also fragments of field pests (aphids and thysanoptera) in the finished semolina products. Infestation of wheat by these pests clearly originates from the preharvest period as previously documented by several authors (Dean & Luuring, 1970; Kakol & Kucharczyk, 2004). Currently we have no clear explanation as to how fragments of field pests could pass through the cleaning and milling process unless there is technological and cleaning malpractice. Usually, field pest fragments and externally feeding insects can be removed from grain entering the mill by cleaning. The entrance of kernels with hidden infestations may be prevented to a certain degree by using impact machinery (entoleters) and aspirators before the milling process. However, low throughput entoleter rates sometimes slow down the milling process. For this reason bypassing impact machines is not uncommon (Plarre & Reichmuth, 2000). 5. Conclusion Our study revealed an alarmingly high frequency of contamination of semolina by insect fragments. However, the contaminant numbers did not exceed critical hygienic levels such is i.e. 75 insect fragments per 50 g of wheat (FDA,1988). In spite of that, the obtained data show that the insect fragments and related contaminants are everywhere, so zero tolerance is unrealistic. The insect fragments entered the semolina as contamination from the field (field pests), contamination during the storage of the grain (fragments of stored product pests) and finally as contamination in the mill during semolina processing (whole larvae and adults). Although internally feeding grain pests were the main cause of semolina contamination, the presence of whole insect bodies in semolina along with a decrease in fragment numbers in semolina after fumigation indicated that pest infestation of the internal mill structure is partly responsible for semolina insect fragment contamination. Acknowledgement The authors thank to anonymous referees for valuable comments of manuscript. Jan Hubert and Vaclav Stejskal were supported by the Czech Ministry of Agriculture, grant no

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