M. Ottoboni

Mycotoxin Contamination in the EU Feed Supply Chain: A Focus on Cereal Byproducts

Mycotoxins represent a risk to the feed supply chain with an impact on economies and international trade. A high percentage of feed samples have been reported to be contaminated with more than one mycotoxin. In most cases, the concentrations were low enough to ensure compliance with the European Union (EU) guidance values or maximum admitted levels. However, mycotoxin co-contamination might still exert adverse effects on animals due to additive/synergistic interactions. Studies on the fate of mycotoxins during cereal processing, such as milling, production of ethanol fuels, and beer brewing, have shown that mycotoxins are concentrated into fractions that are commonly used as animal feed. Published data show a high variability in mycotoxin repartitioning, mainly due to the type of mycotoxins, the level and extent of fungal contamination, and a failure to understand the complexity of food processing technologies. Precise knowledge of mycotoxin repartitioning during technological processes is critical and may provide a sound technical basis for feed managers to conform to legislation requirements and reduce the risk of severe adverse market and trade repercussions. Regular, economical and straightforward feed testing is critical to reach a quick and accurate diagnosis of feed quality. The use of rapid methods represents a future challenge.

Nutritional composition of black soldier fly (Hermetia illucens) prepupae reared on different organic waste substrates

BACKGROUND Black soldier fly larvae are converters of organic waste into edible biomass, of which the composition may depend on the substrate. In this study, larvae were grown on four substrates: chicken feed, vegetable waste, biogas digestate, and restaurant waste. Samples of prepupae and substrates were freeze-dried and proximate, amino acid, fatty acid and mineral analyses were performed. RESULTS Protein content of prepupae varied between 399 and 431 g kg-1 dry matter (DM) among treatments. Differences in amino acid profile of prepupae were small. On the other hand, the ether extract (EE) and ash contents differed substantially. Prepupae reared on digestate were low in EE and high in ash (218 and 197 g kg-1 DM, respectively) compared to those reared on vegetable waste (371 and 96 g kg-1 DM, respectively), chicken feed (336 and 100 g kg-1 DM, respectively) and restaurant waste (386 and 27 g kg-1 DM, respectively). Prepupal fatty acid profiles were characterised by high levels of C12:0 in all treatments. CONCLUSION Since protein content and quality were high and comparable for prepupae reared on different substrates, black soldier fly could be an interesting protein source for animal feeds. However, differences in EE and ash content as a function of substrate should be considered.

A survey of the mycobiota associated with larvae of the black soldier fly (Hermetia illucens) reared for feed production

Feed security, feed quality and issues surrounding the safety of raw materials are always of interest to all livestock farmers, feed manufacturers and competent authorities. These concerns are even more important when alternative feed ingredients, new product developments and innovative feeding trends, like insect-meals, are considered. The black soldier fly (Hermetia illucens) is considered a good candidate to be used as feed ingredient for aquaculture and other farm animals, mainly as an alternative protein source. Data on transfer of contaminants from different substrates to the insects, as well as the possible occurrence of toxin-producing fungi in the gut of non-processed insects are very limited. Accordingly, we investigated the impact of the substrate/diet on the intestinal mycobiota of H. illucens larvae using culture-dependent approaches (microbiological analyses, molecular identification through the typing of isolates and the sequencing of the 26S rRNA D1/D2 domain) and amplicon-based next-generation sequencing (454 pyrosequencing). We fed five groups of H. illucens larvae at the third growing stage on two substrates: chicken feed and/or vegetable waste, provided at different timings. The obtained results indicated that Pichia was the most abundant genus associated with the larvae fed on vegetable waste, whereas Trichosporon, Rhodotorula and Geotrichum were the most abundant genera in the larvae fed on chicken feed only. Differences in the fungal communities were highlighted, suggesting that the type of substrate selects diverse yeast and mold genera, in particular vegetable waste is associated with a greater diversity of fungal species compared to chicken feed only. A further confirmation of the significant influence of diet on the mycobiota is the fact that no operational taxonomic unit common to all groups of larvae was detected. Finally, the killer phenotype of isolated yeasts was tested, showing the inhibitory activity of just one species against sensitive strains, out of the 11 tested species.

Computer image analysis: an additional tool for the identification of processed poultry and mammal protein containing bones.

The aims of this study were (1) to evaluate the potential of image analysis measurements, in combination with the official analytical methods for the detection of constituents of animal origin in feedstuffs, to distinguish between poultry versus mammals; and (2) to identify possible markers that can be used in routine analysis. For this purpose, 14 mammal and seven poultry samples and a total of 1081 bone fragment lacunae were analysed by combining the microscopic methods with computer image analysis. The distribution of 30 different measured size and shape bone lacunae variables were studied both within and between the two zoological classes. In all cases a considerable overlap between classes meant that classification of individual lacunae was problematic, though a clear separation in the means did allow successful classification of samples on the basis of averages. The variables most useful for classification were those related to size, lacuna area for example. The approach shows considerable promise but will need further study using a larger number of samples with a wider range.

Microscopy and image analysis based approaches for the species-specific identification of bovine and swine bone containing material

The aim of this study was to evaluate the potential of image analysis measurements in combination with the official analytical method for the detection of constituents of animal origin in feedstuffs, in distinguishing between bovine and swine (bone containing) material. Authentic samples of controlled origin containing bovine or swine meat and bone meals were analysed by the microscopic method, in accordance with the official analytical method. Sediment fractions of each sample were observed with a compound microscope at X40. A total of 362 bone fragment lacunae images were recorded and processed through image analysis software, deriving 30 geometric variables for each lacuna. Results indicated that not only were most variables significantly (P<0.001) different between bovine and swine samples, but also that two thirds of the same variables were bigger in bovine than in swine. This information, however, does not seem to be so effective in practice since bovine and swine features and measurements overlapped. It can be concluded that the microscopic method even when combined with image analysis does not fit all the requirements for accurately identifying prohibited ingredients of animal origin. A combined approach with other methods is therefore recommended.

Former food products safety: microbiological quality and computer vision evaluation of packaging remnants contamination

ABSTRACT The use of alternative feed ingredients in farm animal’s diets can be an interesting choice from several standpoints, including safety. In this respect, this study investigated the safety features of selected former food products (FFPs) intended for animal nutrition produced in the framework of the IZS PLV 06/14 RC project by an FFP processing plant. Six FFP samples, both mash and pelleted, were analysed for the enumeration of total viable count (TVC) (ISO 4833), Enterobacteriaceae (ISO 21528-1), Escherichia coli (ISO 16649-1), coagulase-positive Staphylococci (CPS) (ISO 6888), presumptive Bacillus cereus and its spores (ISO 7932), sulphite-reducing Clostridia (ISO 7937), yeasts and moulds (ISO 21527-1), and the presence in 25 g of Salmonella spp. (ISO 6579). On the same samples, the presence of undesired ingredients, which can be identified as remnants of packaging materials, was evaluated by two different methods: stereomicroscopy according to published methods; and stereomicroscopy coupled with a computer vision system (IRIS Visual Analyzer VA400). All FFPs analysed were safe from a microbiological point of view. TVC was limited and Salmonella was always absent. When remnants of packaging materials were considered, the contamination level was below 0.08% (w/w). Of note, packaging remnants were found mainly from the 1-mm sieve mesh fractions. Finally, the innovative computer vision system demonstrated the possibility of rapid detection for the presence of packaging remnants in FFPs when combined with a stereomicroscope. In conclusion, the FFPs analysed in the present study can be considered safe, even though some improvements in FFP processing in the feeding plant can be useful in further reducing their microbial loads and impurity. Graphical Abstract

Inclusion of Hermetia Illucens larvae or prepupae in an experimental extruded feed: process optimisation and impact on in vitro digestibility

Abstract This study investigated the effect of extrusion on digestibility of different blends containing Hermetia illucens (HI) larvae or prepupae. Five blends of HI larvae or prepupae and wheat flour, in a ratio of 25:75, with or without sunflower oil addition, have been formulated as follows: prepupae + wheat (no oil); prepupae + wheat (low oil); prepupae + wheat (medium oil); prepupae + wheat (high oil); larvae + wheat (no oil). Ether extract (EE) content in different blend was 31.5, 38.9, 46.3, 53.7 and 46.27 g kg−1 on wet basis (wb), respectively. Blends were homogeneous for moisture (238.9 g kg−1) and crude protein (112.6 g kg−1 wb). Feed blends were extruded by a co-rotating, conical twin-screw mini extruder and net torque value (NTV) was recorded as indicator of extrudability. The best performing blend was furtherly tested at four barrel temperatures (60, 70, 80 and 90 °C). In vitro organic matter digestibility (OMD) and in vitro crude protein digestibility (CPD) were measured to evaluate the effect of extrusion process on nutritional value. Increasing the blend EE content up to 53.74 g kg−1 wb, NTV was reduced by four times (<100 Ncm) compared to 31.5 and 38.9 g kg−1 wb EE blends. The best performing mixture was larvae + wheat (no oil). Extrusion process increased OMD but not CPD compared to unextruded control, while different extrusion temperature did not affect OMD nor CPD. Concluding, extrusion can contribute to increase OMD in insect containing feed blends. EE content in the blends is a key variable that should be defined in the process.

Light microscopy with a differential staining technique for the characterization and discrimination of insects versus marine arthropods in processed animal proteins

ABSTRACT The aim of this study was to evaluate the use of light microscopy with differential staining techniques for the discrimination of insect material from marine arthropods – classified as fishmeal. Specifically, three samples of single-species insect material, Hermetia illucens (HI), Bombyx mori (BM) and Tenebrio molitor (TM), and two samples of marine arthropods, shrimp material and krill, were analysed and compared after staining by two reagents to enhance fragment identification. Alizarin Red (AR) and Chlorazol Black (CB), which react respectively with calcium salts and chitin, were tested for their potential efficacy in distinguishing between insect and marine materials. Results indicated that AR failed to stain HI, BM and TM materials. By contrast, the three insect species materials tested were stained by CB. When shrimp fragments and krill were considered, AR and CB stained marine materials reddish-pink and light blue to black, respectively. By combining these results, it can be suggested that CB staining may efficiently be used to mark insect materials; AR does stain shrimp fragments but does not stain the tested insect material, indicating a possible approach for discriminating between insects and marine arthropods. However, since the present study was performed on pure materials and a small set of samples, possible implementation of this technique still needs to be confirmed in complex matrices such as compound feed.

Nutritional evaluation of former food products (ex-food) intended for pig nutrition

ABSTRACT Ex-food or former food products (FFPs) have been proposed as one of the categories with great promise as alternative feed ingredients. FFPs’ nutritional potential is not yet fully exploited. Therefore, the aim of this study was to perform a nutritional evaluation of selected FFPs. In particular, six samples of mixed FFPs, all based on bakery products, were analysed for moisture, crude protein, ether extract, crude fibre, neutral detergent fibre, acid detergent fibre, starch and ash. Nitrogen-free extractives and non-structural carbohydrate were also determined. Based on FFPs’ composition data, estimation of digestible energy and metabolisable energy values for pigs were calculated. Further, the in vitro digestibility values of FFPs were investigated using a multi-step enzymatic technique. A wheat sample was included as a control feed ingredient in the study. All data were reported on dry matter basis. FFPs have shown a nutrient composition comparable with cereal grains. In the tested FFPs, the average protein content was 10.0% and the average starch content was 52.4%. Nitrogen-free extractive ranged from 61.2% to 74.7%, whereas non-structural carbohydrate ranged from 58.5% to 79.3%. Compared with wheat, FFPs were characterised by a relative high fat content, averaging about 10.1%. The relatively high nitrogen-free extractive/non-structural carbohydrate/starch and fat concentration designated FFPs as valuable energy sources. Digestible energy and metabolisable energy averages were 17.2 and 16.9 MJ kg–1, respectively. The average in vitro digestibility value of FFPs samples was 88.2% ± 5.8%, comparable with that of wheat (90.6% ± 1.6%). FFPs are a fat-fortified version of common cereals grains. The high energy content and digestibility values elect FFPs as promising non-traditional ingredients for swine.

Combining E-Nose and Lateral Flow Immunoassays (LFIAs) for Rapid Occurrence/Co-Occurrence Aflatoxin and Fumonisin Detection in Maize

The aim of this study was to evaluate the potential use of an e-nose in combination with lateral flow immunoassays for rapid aflatoxin and fumonisin occurrence/co-occurrence detection in maize samples. For this purpose, 161 samples of corn have been used. Below the regulatory limits, single-contaminated, and co-contaminated samples were classified according to the detection ranges established for commercial lateral flow immunoassays (LFIAs) for mycotoxin determination. Correspondence between methods was evaluated by discriminant function analysis (DFA) procedures using IBM SPSS Statistics 22. Stepwise variable selection was done to select the e-nose sensors for classifying samples by DFA. The overall leave-out-one cross-validated percentage of samples correctly classified by the eight-variate DFA model for aflatoxin was 81%. The overall leave-out-one cross-validated percentage of samples correctly classified by the seven-variate DFA model for fumonisin was 85%. The overall leave-out-one cross-validated percentage of samples correctly classified by the nine-variate DFA model for the three classes of contamination (below the regulatory limits, single-contaminated, co-contaminated) was 65%. Therefore, even though an exhaustive evaluation will require a larger dataset to perform a validation procedure, an electronic nose (e-nose) seems to be a promising rapid/screening method to detect contamination by aflatoxin, fumonisin, or both in maize kernel stocks.