Effects of aflatoxin B1 on metabolism- and immunity-related gene expression in Hermetia illucens L. (Diptera: Stratiomyidae).

Contamination of food products with mycotoxins such as aflatoxin B1 (AFB1) poses a severe risk to human health. Larvae of the black soldier fly (BSFL), Hermetia illucens (Diptera: Stratiomyidae), can successfully metabolize AFB1 without any negative consequences on their survival or growth. Organic waste streams contaminated with mycotoxins can be upcycled into protein-rich BSFL as an alternative feed for livestock and the left-over feed residue into nutrient-rich crop fertilizers. However, the underlying mechanisms that allow BSFL to metabolize AFB1 are unknown. In this study, five-day-old BSFL were fed with either a control or an AFB1-spiked (20 µg/kg) diet to elucidate the underlying mechanisms. Larval samples were collected at three timepoints (6 h, 24 h and 72 h) and subjected to RNA-Seq analysis to determine gene expression patterns. Provision of an AFB1-spiked diet resulted in an up-regulation of 357 and a down-regulation of 929 unique genes. Upregulated genes include multiple genes involved in AFB1 metabolism in other (insect) species. Downregulated genes were generally involved in the insects' growth, development, and immunity. BSFL possesses a diverse genetic arsenal that encodes for enzymes capable of metabolizing AFB1 without trade-offs on larval survival. In conclusion, the adverse impact of AFB1 exposure on immunity-related processes is observed in the transcriptomic response, and is indicative of a trade-off between detoxification and immune responses.

Comparison of commercial allergen ELISA kits for egg detection in food matrices.

Consumption of low levels of egg already can evoke harmful physiological responses in humans in those allergic to eggs. By detection of egg in food products, using Egg ELISA kits to determine its unintended presence, food producers can respond to avoid potential safety or quality risks of their products. Selection of an ELISA kit fit for the issue at hand is challenging due to, amongst others, lack of information on assay performances with specified matrices. In this study, performances of seven commercial egg ELISA kits are compared for nine different relevant matrices: cookie, chocolate, pasta, dressing, stock cube, wine, vegetable drink and milk, ice cream and meat/meat replacers. The presence of egg was unified for all ELISA kits to mg total egg protein kg-1 food product. In every matrix, kit performances for recovery, intra- and interassay were compared, and also processing is accounted for by determination of egg in incurred samples. All seven kits were able to detect egg qualitatively at the VITAL3 ED01 level of 0.2 mg total egg protein and the corresponding relevant portion size for each matrix. For quantitative results, each ELISA kit showed an increase in detected egg concentration with increased egg levels and performed within the set criteria for recovery for the cookie, chocolate, stock cube and wine. For pasta, vegetable drink and milk, ice cream, and salad dressing, recovery of egg was within the set criteria for at least 4 ELISA kits. Most challenging matrices were meat/meat replacers, showing high matrix effects which could not be explained by the possible egg presence in the cognate blank. Only one ELISA kit was able to recover egg within the set criteria for the meat/meat replacer matrix. Results enable food industry to choose for ELISA kits suitable for egg detection in the matrix of interest.

Bioconversion of Different Waste Streams of Animal and Vegetal Origin and Manure by Black Soldier Fly Larvae Hermetia illucens L. (Diptera: Stratiomyidae).

Black soldier fly larvae (BSFL) are considered a commercially viable solution for global organic waste problems. The objective of this study was to assess the feasibility of rearing BSFL on a wide range of low-value waste streams and its potential to transform them into high-quality animal feed and fertilizer. Six waste streams of different origins were selected and each tested in triplicate. Several parameters were analysed: growth performance, waste reduction index (WRI), conversion efficiency (ECI) and larval composition. Frass composition was also analysed. Larvae reared on fast food waste (FFW) had the highest ECI and WRI and the lowest values when reared on pig manure slurry mixed with silage grass (PMLSG) and slaughter waste (SW). The highest protein content was found for larvae reared on mushroom stems (MS) although this substrate had the lowest protein content. Moreover, the frass nutritional profile was proportionally related to the substrate's nutritional profile: the protein-rich substrate (SW) resulted in protein-rich frass and the low-protein substrate (MS) resulted in protein-poor frass. The same was true for the lipid content. In conclusion, this study showed that BSFL can be successfully reared on a wide range of waste streams that can affect the larval and frass chemical compositions.

Direct comparison of metabolic health effects of the flavonoids quercetin, hesperetin, epicatechin, apigenin and anthocyanins in high-fat-diet-fed mice.

Dietary flavonoid intake is associated with reduced risk of cardiovascular diseases, possibly by affecting metabolic health. The relative potency of different flavonoids in causing beneficial effects on energy and lipid metabolism has not been investigated. Effects of quercetin, hesperetin, epicatechin, apigenin and anthocyanins in mice fed a high-fat diet (HF) for 12 weeks were compared, relative to normal-fat diet. HF-induced body weight gain was significantly lowered by all flavonoids (17-29 %), but most by quercetin. Quercetin significantly lowered HF-induced hepatic lipid accumulation (71 %). Mesenteric adipose tissue weight and serum leptin levels were significantly lowered by quercetin, hesperetin and anthocyanins. Adipocyte cell size and adipose tissue inflammation were not affected. The effect on body weight and composition could not be explained by individual significant effects on energy intake, energy expenditure or activity. Lipid metabolism was not changed as measured by indirect calorimetry or expression of known lipid metabolic genes in liver and white adipose tissue. Hepatic expression of Cyp2b9 was strongly downregulated by all flavonoids. In conclusion, all flavonoids lowered parameters of HF-induced adiposity, with quercetin being most effective.

Quercetin tests negative for genotoxicity in transcriptome analyses of liver and small intestine of mice.

Given the positive results of quercetin in in vitro genotoxicity studies, the in vivo genotoxic properties of this important dietary flavonoid warrant testing, especially considering possible high intake via widely available food supplements. Here, this was done by transcriptome analyses of the most relevant tissues, liver and small intestine, of quercetin supplemented mice. Quercetin (0.33%) supplemented to a high-fat diet was administered to mice during 12 weeks. Serum alanine aminotransferase and aspartate aminotransferase levels revealed no indications for hepatotoxicity. Microarray pathway analysis of liver and small intestine showed no regulation of genotoxicity related pathways. Analysis of DNA damage related genes also did not point at genotoxicity. Furthermore, a published classifier set of transcripts for identifying genotoxic compounds did not indicate genotoxicity. Only two transcripts of the classifier set were regulated, but in the opposite direction compared with the genotoxic compounds 2-acetylaminofluorene (2-AAF) and aflatoxin B1 (AFB1). Based on the weight of evidence of three different types of analysis, we conclude that supplementation with quercetin at ~350 mg/kg bw/day for 12 weeks in mice showed no up-regulation of genotoxicity related pathways in liver and small intestine.

Thermoneutrality results in prominent diet-induced body weight differences in C57BL/6J mice, not paralleled by diet-induced metabolic differences.

SCOPE: Mice are usually housed at 20-24 °C. At thermoneutrality (28 °C) larger diet-induced differences in obesity are seen. We tested whether this leads to large differences in metabolic health parameters. METHODS AND RESULTS: We performed a 14-wk dietary intervention in C57BL/6J mice at 28 °C and assessed adiposity and metabolic health parameters for a semipurified low fat (10 energy%) diet and a moderate high fat (30 energy%) diet. A large and significant diet-induced differential increase in body weight, adipose tissue mass, adipocyte size, serum leptin level, and, to some extent, cholesterol level was observed. No adipose tissue inflammation was seen. No differential effect of the diets on serum glucose, free fatty acids, triacylglycerides, insulin, adiponectin, resistin, PAI-1, MMP-9, sVCAM-1, sICAM-1, sE-selectin, IL-6, ApoE, fibrinogen levels, or HOMA index was observed. Also in muscle no differential effect on mitochondrial density, mitochondrial respiratory control ratio, or mRNA expression of metabolic genes was found. Finally, in liver no differential effect on weight, triacylglycerides level, aconitase/citrate synthase activity ratio was seen. CONCLUSION: Low fat diet and moderate high fat diet induce prominent body weight differences at thermoneutrality, which is not paralleled by metabolic differences. Our data rather suggest that thermoneutrality alters metabolic homeostasis.

Quercetin induces hepatic lipid omega-oxidation and lowers serum lipid levels in mice.

Elevated circulating lipid levels are known risk factors for cardiovascular diseases (CVD). In order to examine the effects of quercetin on lipid metabolism, mice received a mild-high-fat diet without (control) or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Gas chromatography and (1)H nuclear magnetic resonance were used to quantitatively measure serum lipid profiles. Whole genome microarray analysis of liver tissue was used to identify possible mechanisms underlying altered circulating lipid levels. Body weight, energy intake and hepatic lipid accumulation did not differ significantly between the quercetin and the control group. In serum of quercetin-fed mice, triglycerides (TG) were decreased with 14% (p<0.001) and total poly unsaturated fatty acids (PUFA) were increased with 13% (p<0.01). Palmitic acid, oleic acid, and linoleic acid were all decreased by 9-15% (p<0.05) in quercetin-fed mice. Both palmitic acid and oleic acid can be oxidized by omega (ω)-oxidation. Gene expression profiling showed that quercetin increased hepatic lipid metabolism, especially ω-oxidation. At the gene level, this was reflected by the up-regulation of cytochrome P450 (Cyp) 4a10, Cyp4a14, Cyp4a31 and Acyl-CoA thioesterase 3 (Acot3). Two relevant regulators, cytochrome P450 oxidoreductase (Por, rate limiting for cytochrome P450s) and the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3) were also up-regulated in the quercetin-fed mice. We conclude that quercetin intake increased hepatic lipid ω-oxidation and lowered corresponding circulating lipid levels, which may contribute to potential beneficial effects on CVD.

Interference of flavonoids with enzymatic assays for the determination of free fatty acid and triglyceride levels.

Flavonoids are bioactive food compounds with potential lipid-lowering effects. Commercially available enzymatic assays are widely used to determine free fatty acid (FFA) and triglyceride (TG) levels both in vivo in plasma or serum and in vitro in cell culture medium or cell lysate. However, we have observed that various flavonoids interfere with peroxidases used in these enzymatic assays, resulting in incorrect lower FFA and TG levels than actually present. Furthermore, addition of isorhamnetin or the major metabolite of the flavonoid quercetin in human and rat plasma, quercetin-3-O-glucuronide, to murine serum also resulted in a significant reduction of the detected TG levels, while a trend was seen for FFA levels. It is concluded that when applying these assays, vigilance is needed and alternative analytical methods, directly assessing FFA or TG levels, should be used for studying the biological effects of flavonoids on FFA and TG levels.