Effect of replacement of soybean oil by Hermetia illucens fat on performance, digestibility, cecal microbiome, liver transcriptome and liver and plasma lipidomes of broilers.

BACKGROUND: In contrast to protein-rich insect meal, the feed potential of insect fat is generally less explored and knowledge about the suitability of insect fat as a fat source specifically in broiler diets is still limited. In view of this, the present study aimed to comprehensively investigate the effect of partial (50%) and complete replacement of soybean oil with insect fat from Hermetia illucens (HI) larvae in broiler diets on performance, fat digestibility, cecal microbiome, liver transcriptome and liver and plasma lipidomes. Thus, 100 male, 1-day-old Cobb 500 broilers were randomly assigned to three groups and fed three different diets with either 0 (group HI-0, n = 30), 2.5% (group HI-2.5, n = 35) or 5.0% (HI-5.0, n = 35) Hermetia illucens (HI) larvae fat for 35 d. RESULTS: Body weight gain, final body weight, feed intake, and feed:gain ratio during the whole period and apparent ileal digestibility coefficient for ether extract were not different between groups. Cecal microbial diversity did not differ between groups and taxonomic analysis revealed differences in the abundance of only four low-abundance bacterial taxa among groups; the abundances of phylum Actinobacteriota, class Coriobacteriia, order Coriobacteriales and family Eggerthellaceae were lower in group HI-5.0 compared to group HI-2.5 (P < 0.05). Concentrations of total and individual short-chain fatty acids in the cecal digesta were not different between the three groups. Liver transcriptomics revealed a total of 55 and 25 transcripts to be differentially expressed between groups HI-5.0 vs. HI-0 and groups HI-2.5 vs. HI-0, respectively (P < 0.05). The concentrations of most lipid classes, with the exception of phosphatidylethanolamine, phosphatidylglycerol and lysophosphatidylcholine in the liver and cholesterylester and ceramide in plasma (P < 0.05), and of the sum of all lipid classes were not different between groups. CONCLUSIONS: Partial and complete replacement of soybean oil with HI larvae fat in broiler diets had no effect on growth performance and only modest, but no adverse effects on the cecal microbiome and the metabolic health of broilers. This suggests that HI larvae fat can be used as an alternative fat source in broiler diets, thereby, making broiler production more sustainable.

Effect of Ecdysterone on the Hepatic Transcriptome and Lipid Metabolism in Lean and Obese Zucker Rats.

Conflicting reports exist with regard to the effect of ecdysterone, the predominating representative of steroid hormones in insects and plants, on hepatic and plasma lipid concentrations in different rodent models of obesity, fatty liver, and diabetes, indicating that the effect is dependent on the rodent model used. Here, the hypothesis was tested for the first time that ecdysterone causes lipid-lowering effects in genetically obese Zucker rats. To test this hypothesis, two groups of male obese Zucker rats (n = 8) were fed a nutrient-adequate diet supplemented without or with 0.5 g ecdysterone per kg diet. To study further if ecdysterone is capable of alleviating the strong lipid-synthetic activity in the liver of obese Zucker rats, the study included also two groups of male lean Zucker rats (n = 8) which also received either the ecdysterone-supplemented or the non-supplemented diet. While hepatic and plasma concentrations of triglycerides and cholesterol were markedly higher in the obese compared to the lean rats (p < 0.05), hepatic and plasma triglyceride and cholesterol concentrations did not differ between rats of the same genotype fed the diets without or with ecdysterone. In conclusion, the present study clearly shows that ecdysterone supplementation does not exhibit lipid-lowering actions in the liver and plasma of lean and obese Zucker rats.

Tandem mass tag-based proteomics for studying the effects of a biotechnologically produced oyster mushroom against hepatic steatosis in obese Zucker rats.

Hepatic steatosis is a very common response to liver injury and often attributed to metabolic disorders. Prior studies have demonstrated the efficacy of a biotechnologically produced oyster mushroom (Pleurotus sajor-caju, PSC) in alleviating hepatic steatosis in obese Zucker rats. This study aims to elucidate molecular events underlying the anti-steatotic effects of PSC. Tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS/MS was used to quantify and compare proteins in the livers of lean Zucker rats fed a control diet (LC), obese Zucker rats fed the same control diet (OC) and obese Zucker rats fed the control diet supplemented with 5% PSC (OPSC) for 4 weeks. Using this technique 3128 proteins could be quantified, out of which 108 were differentially abundant between the OPSC and OC group. Functional enrichment analysis of the up-regulated proteins showed that these proteins were mainly involved in metabolic processes, while the down-regulated proteins were involved in inflammatory processes. Results from proteomic analysis were successfully validated for two up-regulated (carbonic anhydrase 3, regucalcin) and two down-regulated (cadherin-17, ceruloplasmin) proteins by means of immunoblotting. SIGNIFICANCE: Valorization of low-grade agricultural waste by edible fungi, such as the mushroom Pleurotus sajor-caju (PSC), represents a promising strategy for the production of protein rich biomass since they boast of a unique enzyme system that has the ability to recover nutrients and energy from biodegradable waste. Herein, we describe the metabolic effects of PSC feeding using a combined quantitative proteomics and bioinformatics approach. In total, 108 proteins were identified to be regulated by PSC feeding in the liver of the obese rats. Complementary usage of a bioinformatics approach allowed us to decipher the mechanisms underlying the recently observed lipid-lowering and anti-inflammatory activity of PSC feeding in obese Zucker rats, namely a reduction of fatty acid synthesis, an improvement of hepatoprotective mechanisms and an enhancement of anti-inflammatory effects.

Influence of a Biotechnologically Produced Oyster Mushroom (Pleurotus sajor-caju) on the Gut Microbiota and Microbial Metabolites in Obese Zucker Rats.

Mushrooms are a rich source of dietary fiber. This study aimed to characterize the modulation of colonic microbiota in Zucker rats after supplementing their diet with a biotechnologically produced oyster mushroom (Pleurotus sajor-caju). Microbiota composition and short chain fatty acids (SCFAs) in the colon and bile acids in the plasma of the rats were analyzed to assess the effects of P. sajor-caju supplementation on the microbiota in the colon and its interplay with the host in the event of hepatic steatosis. Microbiota profiles were distinctly modulated by P. sajor-caju supplementation between the obese control rats and the obese rats fed the 5% P. sajor-caju-supplemented diet. P. sajor-caju enhanced the growth of SCFAs-producing bacterial genera, including Faecalibaculum, Bifidobacterium, Roseburia, and Blautia, and decreased the relative abundance of the pathogenic genus Escherichia-Shigella. This was also accompanied by distinct changes in the concentrations of bile acids in the plasma and concentrations of SCFAs in the colon, supporting the initial potentiality of P. sajor-caju as a prebiotic in cases of hepatic steatosis and liver inflammation.

Characterization of the Nutritional Composition of a Biotechnologically Produced Oyster Mushroom and its Physiological Effects in Obese Zucker Rats.

SCOPE: Sustainable protein sources are needed to meet the increasing protein demands of a continuously growing world population. This study is focused on the biotechnological production of a protein rich oyster mushroom (Pleurotus sajor-caju; PSC) by valorization of an agricultural side stream and the evaluation of the physiological effects of PSC in a rat model of metabolic syndrome. METHODS AND RESULTS: PSC is produced via submerged cultivation in a 150 L bioreactor that utilizes isomaltulose molasses as its sole carbon source, and is further analyzed for its nutritional composition. A feeding trial is performed using Zucker rats which are fed a 5% PSC supplemented diet, for 4 weeks. Biochemical analyses reveal a significant reduction of the liver lipid concentrations and liver inflammation in the PSC fed obese rats in comparison to the obese rats from the control group. Hepatic qPCR analyses, differential transcript profiling, and enzyme activity measurements reveal a number of altered pathways that may be responsible for these anti-steatotic and anti-inflammatory effects of the mushroom. CONCLUSION: Bioconversion of a low quality agricultural side stream to an improved protein source is performed by submerged cultured PSC, and the obtained mycelium shows strong anti-steatotic and anti-inflammatory effects.

Branched-Chain Fatty Acids as Mediators of the Activation of Hepatic Peroxisome Proliferator-Activated Receptor Alpha by a Fungal Lipid Extract.

The study aimed to test the hypothesis that monomethyl branched-chain fatty acids (BCFAs) and a lipid extract of Conidiobolus heterosporus (CHLE), rich in monomethyl BCFAs, are able to activate the nuclear transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha). Rat Fao cells were incubated with the monomethyl BCFAs 12-methyltridecanoic acid (MTriA), 12-methyltetradecanoic acid (MTA), isopalmitic acid (IPA) and 14-methylhexadecanoic acid (MHD), and the direct activation of PPARalpha was evaluated by reporter gene assay using a PPARalpha responsive reporter gene. Furthermore, Fao cells were incubated with different concentrations of the CHLE and PPARalpha activation was also evaluated by using the reporter gene assay, and by determining the mRNA concentrations of selected PPARalpha target genes by real-time RT-PCR. The reporter gene assay revealed that IPA and the CHLE, but not MTriA, MHD and MTA, activate the PPARalpha responsive reporter gene. CHLE dose-dependently increased mRNA concentrations of the PPARalpha target genes acyl-CoA oxidase (ACOX1), cytochrome P450 4A1 (CYP4A1), carnitine palmitoyltransferase 1A (CPT1A) and solute carrier family 22 (organic cation/carnitine transporter), member 5 (SLC22A5). In conclusion, the monomethyl BCFA IPA is a potent PPARalpha activator. CHLE activates PPARalpha-dependent gene expression in Fao cells, an effect that is possibly mediated by IPA.

Tenebrio molitor Larvae Meal Affects the Cecal Microbiota of Growing Pigs.

The hypothesis tested was that dietary inclusion of insect meal (IM) causes an alteration in the cecal microbiota composition and its fermentation activity of growing pigs. Five-week-old male crossbred pigs were randomly assigned to three groups of 10 pigs each, and fed isonitrogenous diets either without (CON) or with 5% IM (IM5) or 10% IM (IM10) from Tenebrio molitor larvae for four weeks. The relative abundance of the phylum Bacteroidetes was lower in group IM10 than in group CON (p < 0.05), whereas the relative abundance of Firmicutes and the Firmicutes:Bacteroidetes-ratio tended to be higher in groups IM10 and IM5 than in group CON (p < 0.1). The relative abundance of the Proteobacteria tended to be higher in group IM10 than in groups CON and IM5 (p < 0.1). The concentrations of the total short-chain fatty acids in the cecal digesta did not differ between the three groups, but the concentrations of the branched-chain fatty acids in the cecal digesta were higher in group IM5 and IM10 than in group CON (p < 0.05). The present study shows for the first time that the replacement of soybean meal by Tenebrio molitor larvae meal causes a shift of the cecal microbial community and its fermentation activity in growing pigs.

Supplementation of Sulfur-Containing Amino Acids or Essential Amino Acids Does Not Reverse the Hepatic Lipid-Lowering Effect of a Protein-Rich Insect Meal in Obese Zucker Rats.

The present study tested the hypothesis that the liver lipid-lowering effect of insect meal (IM) is caused by its low methionine concentration. A total of fifty, male obese Zucker rats were randomly assigned to five groups of 10 rats each (casein (C), IM, IM + Met, IM + Cys, and IM + EAA). While group C received a diet with casein, the IM-fed groups received a diet with IM as the protein source. In groups IM + Met, IM + Cys and IM + EAA, the diets were additionally supplemented with methionine, cysteine and essential amino acids (EAA), respectively. Hepatic concentrations of triacylglycerols and cholesterol, and hepatic mRNA levels and activities of lipogenic and cholesterogenic enzymes were markedly lower in the IM-fed groups than in group C (p < 0.05). All of these parameters either did not differ across the IM-fed groups or were only slightly higher in groups IM + Met, IM + Cys and IM+EAA than in the group IM. In conclusion, the results indicate that a difference in the amino acid composition between IM and casein, a low concentration of methionine in IM and a reduced cysteine synthesis secondary to a decreased methionine availability resulting from feeding IM are not causative for the lipid-lowering effect of IM.

Effect of pH on solubility of white Mineral Trioxide Aggregate and Biodentine: An in vitro study.

Background. The aim of this study was to evaluate the effect of acidic, neutral and alkaline environments on the solubility of white mineral trioxide aggregate (WMTA) and Biodentine (BD). Methods. Thirty-nine ring molds were randomly divided into three groups of A, B, and C (n = 12) with pH values of 7.4, 4.4 and 10.4, respectively, and an empty mold was used as a control. Each group was further divided into two subgroups (1 and 2) according to the material studied. The samples in groups A, B and C were transferred into synthetic tissue fluid buffered at pH values of 7.4, 4.4 and 10.4, respectively, and kept in an incubator at 37°C with 100% humidity. Daily solubility at 1-, 2-, 5-, 14-, 21-, and 30-day intervals and cumulative solubility up to 5-, 14-, and 30-day intervals were calculated. Statistical analysis was carried out with independent-samples t-test, two-way ANOVA and post hoc Tukey tests using SPSS 18. Statistical significance was set at P<0.05. Results. Both WMTA and BD exhibited the highest solubility in acidic pH with 5.4235±0.1834 and 10.7516±0.0639 mean cumulative solubility values at 30-day interval, respectively. At all exposure times, BD was significantly more soluble than WMTA (P<0.001). Conclusion. Acidic periapical environment jeopardized the solubility of both WMTA and BD, affecting their sealing characteristics in clinical applications like perforation repair procedures and blunderbuss canals.

Substrates and oxygen dependent citric acid production by Yarrowia lipolytica: insights through transcriptome and fluxome analyses.

BACKGROUND: Unlike the well-studied backer yeast where catabolite repression represents a burden for mixed substrate fermentation, Yarrowia lipolytica, an oleaginous yeast, is recognized for its potential to produce single cell oils and citric acid from different feedstocks. These versatilities of Y. lipolytica with regards to substrate utilization make it an attractive host for biorefinery application. However, to develop a commercial process for the production of citric acid by Y. lipolytica, it is necessary to better understand the primary metabolism and its regulation, especially for growth on mixed substrate. RESULTS: Controlling the dissolved oxygen concentration (pO2) in Y. lipolytica cultures enhanced citric acid production significantly in cultures grown on glucose in mono- or dual substrate fermentations, whereas with glycerol as mono-substrate no significant effect of pO2 was found on citrate production. Growth on mixed substrate with glucose and glycerol revealed a relative preference of glycerol utilization by Y. lipolytica. Under optimized conditions with pO2 control, the citric acid titer on glucose in mono- or in dual substrate cultures was 55 and 50 g/L (with productivity of 0.6 g/L*h in both cultures), respectively, compared to a maximum of 18 g/L (0.2 g/L*h) with glycerol in monosubstrate culture. Additionally, in dual substrate fermentation, glycerol limitation was found to trigger citrate consumption despite the presence of enough glucose in pO2-limited culture. The metabolic behavior of this yeast on different substrates was investigated at transcriptomic and 13C-based fluxomics levels. CONCLUSION: Upregulation of most of the genes of the pentose phosphate pathway was found in cultures with highest citrate production with glucose in mono- or in dual substrate fermentation with pO2 control. The activation of the glyoxylate cycle in the oxygen limited cultures and the imbalance caused by glycerol limitation might be the reason for the re-consumption of citrate in dual substrate fermentations. This study provides interesting targets for metabolic engineering of this industrial yeast.

Metabolic network analysis and experimental study of lipid production in Rhodosporidium toruloides grown on single and mixed substrates.

BACKGROUND: Microbial lipids (triacylglycerols, TAG) have received large attention for a sustainable production of oleochemicals and biofuels. Rhodosporidium toruloides can accumulate lipids up to 70% of its cell mass under certain conditions. However, our understanding of lipid production in this yeast is still much limited, especially for growth with mixed substrates at the level of metabolic network. In this work, the potentials of several important carbon sources for TAG production in R.toruloides are first comparatively studied in silico by means of elementary mode analysis followed by experimental validation. RESULTS: A simplified metabolic network of R.toruloides was reconstructed based on a combination of genome and proteome annotations. Optimal metabolic space was studied using elementary mode analysis for growth on glycerol, glucose, xylose and arabinose or in mixtures. The in silico model predictions of growth and lipid production are in agreement with experimental results. Both the in silico and experimental studies revealed that glycerol is an attractive substrate for lipid synthesis in R. toruloides either alone or in blend with sugars. A lipid yield as high as 0.53 (C-mol TAG/C-mol) has been experimentally obtained for growth on glycerol, compared to a theoretical maximum of 0.63 (C-mol TAG/C-mol). The lipid yield on glucose is much lower (0.29 (experimental) vs. 0.58 (predicted) C-mol TAG/C-mol). The blend of glucose with glycerol decreased the lipid yield on substrate but can significantly increase the overall volumetric productivity. Experimental studies revealed catabolite repression of glycerol by the presence of glucose for the first time. Significant influence of oxygen concentration on the yield and composition of lipids were observed which have not been quantitatively studied before. CONCLUSIONS: This study provides for the first time a simplified metabolic model of R.toruloides and its detailed in silico analysis for growth on different carbon sources for their potential of TAG synthesis. Experimental studies revealed the phenomenon of catabolite repression of glycerol by glucose and the importance of oxygen supply on the yield and composition of lipids. More systematic studies are needed to understand the mechanisms which should help to further optimize the lipid production in this strain of industrial interest.